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Plant_Mol_Biol-4-1-2238787

Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana

Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) in two heterologous systems, i.e. the yeast Saccharomycescerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2β rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Δ) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2β under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2β in plant stress responses. Introduction During their life plants are often challenged with various types of abiotic stresses. Proper physiological and biochemical responses to such stresses are controlled by an array of stress-dependent signal transduction pathways (Xiong et al. 2002). Several signaling molecules and second messengers including calcium ions (Ca2+; Knight et al. 1996; Knight et al. 1997) and inositol 1,4,5-trisphosphate (IP3; Takahashi et al. 2001) have been demonstrated to be important elements of signaling pathways that link the perception of stress to various types of downstream responses. As one of the central molecules in cell signaling, IP3 is subject to phosphorylation and dephosphorylation by specific inositol phosphate kinases and phosphatases, respectively, which helps to control intracellular IP3 concentration (Pattni and Banting 2004). In higher plants, over-expression of an inositol polyphosphate 5-phosphatase (Ins5Pase), possibly leading to intracellular degradation of IP3, modulated stress and ABA signal transduction (Perera et al. 2002; Burnette et al. 2003). Likewise, mutation of an inositol polyphosphate 5-phosphatase gene (CVP2) results in increased sensitivity to ABA and vein patterning defects in cotyledons (Carland and Nelson 2004). IP3 can also be hydrolyzed by FRY1-like inositol polyphosphate 1-phosphatases (Ins1Pases; Xiong et al. 2002). The fry1 mutant of Arabidopsis accumulated around ten-fold more IP3 than the corresponding wild type, exhibited hypersensitivity towards ABA, and was compromised in tolerance to freezing, drought and salt stresses (Xiong et al. 2001). These data demonstrate the importance of inositol polyphosphate phosphatases in IP3 metabolism and IP3-mediated stress signal transduction. However, evidence for a role of inositol polyphosphate kinases in relation to stress tolerance is currently lacking in plants. Inositol 1,4,5-trisphosphate 3-kinase (IP3K) and more generally inositol polyphosphate kinases play an essential role in cellular signal transduction and maintenance of Ca2+ homeostasis by phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4; Communi et al. 1995). Both IP3 and IP4 are second messengers responsible for Ca2+ mobilization from intracellular stores (Berridge 1997). IP4 itself is also able to regulate cytosolic Ca2+ concentration by promoting Ca2+ sequestration (Hill et al. 1988). In Arabidopsis thaliana, IP3K is encoded by two distinct genes, AtIpk2α and AtIpk2β, which are ubiquitously expressed in mature tissues (Stevenson-Paulik et al. 2002; Xia et al. 2003). Both of the Arabidopsis IP3Ks use IP3 as a substrate and display dual-specificity inositol polyphosphate 6-/3-kinase activities that successively phosphorylate IP3 to generate inositol 1,3,4,5,6-pentakisphosphate (IP5) predominantly via an inositol 1,4,5,6-tetrakisphosphate (IP4) intermediate (Stevenson-Paulik et al. 2002; Xia et al. 2003). Recently it was reported that AtIpk2α plays a role in pollen germination and root growth (Xu et al. 2005), while AtIpk2β functions in axillary shoot branching through the auxin signaling pathway (Zhang et al. 2007) and phytate synthesis (Stevenson-Paulik et al. 2005). Although AtIpk2β has well been characterized at the biochemical level and has been shown to participate in plant development, it remained unclear whether it is also involved in stress signaling. In this study we demonstrate that AtIpk2β restores the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Δ) deficient for the ARG82/IPK2 gene that encodes inositol polyphosphate multikinase. We also show that constitutive expression of AtIpk2β in transgenic tobacco enhances its tolerance towards various abiotic stresses. Thus, inositol polyphosphate kinase encoded by AtIpk2β very probably plays an important role in signaling pathways controlling the cellular response to abiotic stresses. Materials and methods Expression of AtIpk2β in yeast mutant arg82Δ The S. cerevisiaearg82/Ipk2 deletion strain (derived from BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR173c::kanMX4) was obtained from EUROSCARF (http://www.web.uni-frankfurt.de/fb15/mikro/euroscarf/). In this strain, the ARG82/IPK2 allele is disrupted through insertion of the kanamycin resistance gene, kanMX4. The AtIpk2β open reading frame was cloned into yeast expression plasmid pYX212. The resulting plasmid, pYX212-AtIpk2β, was used to transform the yeast mutant. The wild type strain BY4741 was used as control. Ten-fold serial dilutions (starting at OD600 = 1.0) of each strain were plated on YPD medium (1% yeast extract, 2% peptone, 2% glucose) supplemented with either NaCl (0.4 or 0.8 M) or mannitol (0.4 or 0.8 M), and incubated at 30°C for 48 h. For growth at 37°C, yeast cells were grown on media containing no extra NaCl or mannitol. Each experiment was repeated at least three times. For growth in liquid YPD medium, cells were cultured at 30°C for three days to saturation (OD600 = 12.0). Five μl of the cell culture were used to inoculate 3 ml of synthetic defined minimal medium supplemented with different concentrations of NaCl or mannitol. Cell density was measured at A600. Quantitative real-time PCR analysis of AtIpk2β expression in Arabidopsis Ten-day-old Arabidopsis seedlings grown on MS medium were used for different treatments as described previously (Shi and Zhu 2002). RNA was extracted with TRIZOL Reagent (Invitrogen) and reverse transcription of RNA was carried out using M-MLV RTase Synthesis Kit (TaKaRa). Quantitative real-time PCR was performed using the Rotor-Gene 3000 Series real time DNA amplification system under the following conditions: 95°C for 10 s; 40 cycles of 95°C for 5 s, 60°C for 20 s. AtIpk2β-specific primers were used (Table 1). Melting curve analysis was included to verify specificity of the DNA amplification. Expression of the reference gene Actin, which served as quantifying control, was monitored using gene-specific forward and reverse primers (Table 1). Data analysis was performed with Rotor-Gene software 6.0. Table 1Gene-specific primers used in this studyPrimer namePrimer sequenceFor yeast expression vectorpYX-F5′-CTCCCATGGTAATGCTCAAAGTCCCTG-3′pYX-R5′-GATTGTCGACCTAGCGCCCGTTCTC-3′For real-time RT-PCRAtIPK2β-F 5′-CCACGGTTTCGTTGGGGTTC-3′AtIPK2β-R 5′-GTTACAACCGCCATAAACCTCTG-3′AtActin2-F 5′-CATCCTCCGTCTTGACCTTGC-3′AtActin2-R 5′-CAAACGAGGGCTGGAACAAG-3′For RT-PCRNtActin-F 5′-TTACGCCCTTCCTCATGCAATT-3′NtActin-R 5′-GGCGCCACCACCTTGATCTTC-3′AY562132-F5′-GTAGCATTGTTGGTGGTGGTGTG-3′AY562132-R5′- ACCGTGGAGCAGTCAATGGAAG-3′AY554169-F5′- ACAGTCGTGAGCATTCCCAAC-3′AY554169-R5′-CCAAACCTTCTGTGCTACCTC-3′AY554170-F5′-CCGGAGTGAAGGGGATGG-3′AY554170-R5′-CAAGCAATGTGAATGGTATGTGAG-3′NtERD10B-F5′-CAATTTAGTGCAGGCCAGGC-3′NtERD10B-R5′-GGTCCATGGTGGCCAGGAAG-3′NtERD10C-F5′-GGGTAGCGCAAACGTGGAG-3′NtERD10C-R5′-CTTTTCCCTCAGCCTCGTGC-3′ Vector construction and plant transformation To create a Cauliflower Mosaic Virus (CaMV) 35S plant expression construct, the 903 bp long AtIpk2β cDNA containing the complete open reading frame was inserted via XbaI–SalI sites into the binary vector pBinAR-HPT. The resulting construct was introduced into Agrobacterium tumefaciens (strain LBA4404). Tobacco (Nicotiana tabacum cv. SR-1) leaf disks were infected with the transformed Agrobacteria. After two days of co-cultivation, the explants were transferred to regeneration medium containing 1-mg/l BAP, 0.1-mg/l NAA, 50-mg/l hygromycin and 200-mg/l ampicillin. Regenerated shoots were separated from the calli and transferred onto rooting medium containing MS salts, 0.1-mg/l NAA and 50-mg/l hygromycin. Rooted shoots were transplanted into soil. Seeds were harvested and homozygous plants were screened on a 12-h light/12-h dark photoperiod. Salt stress tolerance tests Wild type and homozygous T2 transgenic seeds were surface sterilized with a solution of 10% commercial bleach (0.525% sodium hypochlorite) for 10 min, and washed three times with sterile water. For germination assays, seeds were plated on MS medium supplemented with different concentrations of NaCl (0, 100 and 200 mM). For root growth measurements, 10-day-old seedlings cultured on solid MS medium were transferred to MS medium supplemented with different concentrations of NaCl. Plates were oriented vertically with seedlings kept upside down. Three replicates were performed for each experiment. Root length was recorded after seven days of treatment. To monitor stress effects on the growth of wild type and AtIpk2β expressing plants, 15-day-old seedlings were transferred to MS medium supplemented with 0, 100, 200 or 300 mM NaCl, and cultured at 25 ± 2°C with cool white fluorescent light (∼125 μmol m−2 s−1) under short day condition (12-h light/12-h dark). For salt stress treatments and ion measurements, wild type and transgenic seedlings were transferred to pots containing soil. Plants were watered bi-weekly with 1/8 concentrated MS salt solution supplemented with or without 200-mM NaCl and grown at 25 ± 2°C with cool white fluorescent light (∼125 μmol m−2 s−1), 70% humidity, under long-day conditions (16-h light/8-h dark) for 6 weeks. Mature plants treated in the same way were also used to determine cellular Na+ concentrations. The third leaf from the top of each plant was collected and dried at 90°C for 24 h, and the dry weight of each sample was measured. The dried leaf material was immersed in 0.1-M HAc, followed by water bath incubation at 90°C for 2.5 h, and analyzed for Na+ content using atomic absorption spectrophotometry. For hydroponic salt tolerance experiments, wild type plants and two independent transgenic lines (T2 generation) were grown hydroponically in Hoagland solution. Plants were grown at 25 ± 2°C with cool white fluorescent light (∼125 μmol m−2 s−1), 70% humidity, under long-day conditions (16-h light/8-h dark). The nutrient solution was replaced weekly. Oxidative stress experiments Malondialdehyde (MDA) concentration was determined as a biomarker for oxidative stress. To measure MDA concentration, leaf samples from plants grown at 200-mM NaCl were ground in 5 ml of 0.1% trichlor acetic acid (TCA) and mixed with 5 ml of 0.5% thiobarbituric acid. The samples were then boiled for 10 min, cooled down to room temperature and centrifuged at 1,200 × g. The cleared supernatant was analyzed by monitoring the difference in absorbance at A532 and A600. For oxidative damage experiments, leaf disks of 1.5-cm diameter were excised from healthy and fully expanded leaves of two-month-old tobacco plants. The disks were floated in a 10-ml solution with different concentrations of H2O2 or water (control) for 72 h. Chlorophyll a and b concentrations were measured as described previously (Lichtenthaler 1987). The treatment was carried out with continuous white light at 25 ± 2°C. The experiment was repeated at least three times. Enzyme assays and protein determination Enzyme assays were carried out spectrophotometrically using a nucleic acid and protein analyzer (Du 640, Beckman Coulter). One-month-old seedlings were treated with H2O or 1% H2O2 for 12 h, and ground in extraction buffer (1 mM ascorbate in 50-mM potassium phosphate buffer, pH 7.8) at a ratio of 0.1 g leaf material/1 ml ice-cold extraction buffer. The slurry was transferred to a centrifuge tube and centrifuged at 20,000 × g at 4°C for 20 min. The supernatant was used for enzyme activity assays and total protein determination as described previously (Badawi 2004; Bradford 1976). Ascorbate peroxidase (APX) activity was determined by measuring the rate of ascorbate oxidation at A290 in a 100-μl-reaction mixture (1-mM EDTA in 50-mM potassium phosphate buffer, pH 7.8, 0.2-mM ascorbate, 20-mM H2O2 and 5-μl soluble protein extract). APX activity is expressed as micromoles of ascorbate oxidized per minute per milligram of protein. Catalase (CAT) activity was measured by monitoring the decrease in absorbance at A240 caused by the disappearance of H2O2 in a 100-μl-reaction mixture (1-mM EDTA in 50-mM potassium phosphate buffer, pH 7.8, 20-mM H2O2 and 5-μl protein extract). The activity of superoxide dismutase (SOD) was measured at A560, in a 3-ml reaction mixture (13.05-mM dl-methionine, 0.1-μM EDTA, 0.075-mM Nitro Blue tetrazolium chloride and 2-μM riboflavin in 50-mM phosphate buffered saline, pH 7.8, and 100-μl protein extract). Whole plant drought tolerance test Wild type and transgenic plant seeds were surface sterilized as described above. Seedlings germinated on MS medium were transplanted to soil. After two months, healthy plants of the same size and age were pooled into two groups. For the first group (12 individual plants, each grown in a 6.5-inch pot), plants were irrigated with 60 g/l PEG-6000 to simulate drought stress. After 21 days, the plants were photographed. Simultaneously, plants of the second group were subjected to drought stress by omitting watering. After three weeks, the plants were re-hydrated and observed for recovery. Photos were taken 21 days after initiation of the stress treatment. The relative humidity was maintained at ∼60%. The experiments were repeated twice with three replicates in each experiment. Freezing tolerance test Three-week-old seedlings of wild type and transgenic plants grown on MS medium were cultured at 4°C for one day under long day condition (16-h light/8-h dark). After cold acclimation, the plants were left at −20°C for 1 or 2 h, transferred immediately to 4°C for another 12 h (overnight). Subsequently, plants were kept in the greenhouse at 25°C and observed further. Photographs were taken seven days after initiation of the recovery growth in the greenhouse. Determination of proline concentration Four-week-old seedlings grown on MS medium were transferred onto filter paper in a Petri dish saturated with 100- or 200-mM NaCl, incubated at 25 ± 2°C for 24 h under continuous white light. After the treatment, proline content was determined as described previously (Bates and Waldren 1973). Western blot analysis Leaves from either Arabidopsis or tobacco plants were ground in liquid N2 and suspended in 1.5 ml of 30 mM potassium phosphate buffer (pH 7.5) containing 40-mM NaCl and 3-mM dithiothreitol (Powell 1986). Homogenates were centrifuged for 5 min at 12,000 × g. Supernatants were transferred to new tubes, and protein concentrations were determined as described previously (Bradford 1976). A 10-μg sample of total protein was mixed with loading buffer, boiled for 3 min, and size-fractionated through electrophoresis in a 12.5% SDS polyacrylamide gel. Separated proteins were transferred to a PVDF membrane (Millipore Corporation) according to the manufacturer’s instructions. A polyclonal antibody against AtIpk2β protein purified from recombinant E. coli was raised in rabbits. Antiserum was purified by IgG affinity purification. The anti-AtIpk2β antiserum was used at a 1:5,000 dilution in TBST. Peroxidase-labeled affinity purified secondary antibody isolated from a pool of serum from goats immunized with purified rabbit IgG was used at a 1:1,000 dilution in TBST (KPL Corporation). Stress gene expression analysis using reverse transcription PCR Three-week-old tobacco seedlings grown on MS agar plates were treated with 300-mM NaCl for 5 h. Total RNA was isolated with TRIZOL Reagent (Invitrogen) and used for reverse transcription (RT) to generate first-strand cDNA. RT was performed on 2-μg total RNA per reaction using ReverTra Ace (TOYOBO). A portion of the resulting cDNA was then subjected to PCR amplification using gene specific primers (Table 1). Expression level of the Nicotiana tabacum ACTIN gene, which served as reference, was monitored with NtActin forward and reverse primers (Table 1). Statistical analysis For statistical analyses, the Student’s t-test was used to generate every P-value. The alpha level was 0.05. The tests were one-tailed. The data were normalized and all samples were normally distributed with homogeneity of variance. Results Yeast complementation with AtIpk2β In S. cerevisiae, the ARG82/IPK2 gene encodes an inositol polyphosphate kinase (Odom et al. 2000). The S. cerevisiaearg82/Ipk2 (arg82Δ) mutant strain that lacks Ipk2 activity has slowed growth at 30°C and is unable to grow at 37°C (Odom et al. 2000). We and others have reported previously that AtIpk2β rescues its growth defects at 37°C (Stevenson-Paulik et al. 2002; Xia et al. 2003). This result led us to postulate that AtIpk2β may also be able to rescue its growth defects when other abiotic stresses were imposed. We observed that under adverse growth conditions such as high salt concentration or upon osmotic stress treatment, growth of the arg82Δ mutant was severely impaired when compared to that of the wild type (Fig. 1a). To test whether AtIpk2β restores the growth defect of the mutant strain under these conditions, we transformed it with the ArabidopsisAtIpk2β cDNA. Expression of AtIpk2β almost completely restored growth of the arg82Δ mutant under both kinds of stresses (Fig. 1a). It also restored growth at 37°C (Fig. 1a), as reported earlier (Stevenson-Paulik et al. 2002; Xia et al. 2003). We also performed experiments using liquid media and observed again, that AtIpk2β restores growth under conditions of abiotic stress (Fig. 1b). Fig. 1Expression of AtIpk2β in S. cerevisiaearg82Δ. (a) AtIpk2β rescues growth defects of the yeast arg82Δ mutant under high salt or mannitol stress, and at 37°C. Ten-fold serial dilutions of the strain were plated and incubated at 30°C for 48 h on YPD medium supplemented with either 0.4- or 0.8-M NaCl, or with 0.4- or 0.8-M mannitol. Plates incubated at 37°C contained YPD medium without extra NaCl or mannitol. WT, wild type; arg82Δ, arg82Δ mutant; arg82Δ + AtIpk2ß, arg82Δ mutant harbouring plasmid pYX212-AtIpk2β. (b) Liquid yeast cultures. Cells were grown for three days to saturation in YPD medium (OD600 = 12.0). Five μl of cell culture were then used to inoculate 3 ml of defined synthetic minimal medium supplemented with different concentrations of NaCl or mannitol. Cell density was determined at various time points as absorbance at 600 nm. Cells were grown at 30°C. WT, wild type; arg82Δ, arg82Δ mutant; arg82Δ + AtIpk2ß, arg82Δ mutant harbouring pYX212-AtIpk2β; arg82Δ + vector, arg82Δ mutant harbouring pYX212 AtIpk2β is abiotic stress regulated in Arabidopsis We were interested to know whether different kinds of stresses such as salinity, cold and drought, or treatment with ABA would affect AtIpk2β transcript levels in Arabidopsis plants. To this end we performed quantitative real-time PCR (qRT-PCR). In ten-day-old seedlings grown on MS medium, AtIpk2β transcript level increased slightly upon exposure to cold or drought stress, but decreased in response to salt or ABA treatment (Fig. S1A in Supplementary Material). These results showed that AtIpk2β is a stress responsive gene potentially involved in different abiotic stress- and ABA-triggered signaling pathways in plants. We further tested AtIpk2β protein level by Western blot analysis. Fourteen-day-old Arabidopsis seedlings grown on MS medium were subjected to different stress treatments for 24 h. In seedlings treated with 500-mM mannitol, AtIpk2β protein level increased slightly, however no significant changes in protein level were detected in seedlings subjected to cold or NaCl stress (Fig. S1B in Supplementary Material). Constitutive expression of AtIpk2β in transgenic tobacco To test whether constitutive expression of AtIpk2β affects abiotic stress tolerance in plants, we introduced its open reading frame (Fig. 2a) into the genome of tobacco (Nicotiana tabacum cv. SR-1) by Agrobacterium tumefaciens-mediated transformation. Expression of AtIpk2β in the transgenic plant was controlled by the Cauliflower Mosaic Virus (CaMV) 35S promoter. Forty-six independent transgenic plants (T0 generation) were obtained; six lines were grown to produce seeds. Transgenic plants homozygous for the 35S:AtIpk2β transgene were obtained and three lines (S13, S14 and S15) were chosen for further experiments. Constitutive expression of AtIpk2β did not affect overall plant morphology although the transgenic plants grew slightly faster than wild type plants under normal growth condition. Western blot analyses were performed to test for the presence of AtIpk2β protein. A 33-kDa band corresponding to AtIpk2β was identified in Arabidopsis plants; similarly, 33-kDa AtIpk2β protein was detected in the transgenic but not the wild type tobacco lines (Fig. 2b), indicating that it was successfully expressed in the genetically modified plants. Fig. 2Construct used for tobacco transformation and Western blot analysis of AtIpk2β in wild type and transgenic tobacco plants. (a) Schematic map of the binary construct used for tobacco transformation. Expression of AtIpk2β is driven by the Cauliflower Mosaic Virus 35S promoter. Ocs, ocs terminator; RB and LB, right and left border of T-DNA, respectively. (b) Western blot analysis. Lane 1, wild type Arabidopsis; lane 2, wild type tobacco; lanes 3–7, transgenic AtIpk2β expressing tobacco lines S11, S13, S14, S15, and S18. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal loading of protein Expression of AtIpk2β increases salt tolerance of transgenic tobacco plants We first studied the salt tolerance of AtIpk2β expressing plants during germination and early stages of seedling development. Seeds of wild type and the three homozygous transgenic lines S13, S14 and S15 were germinated on MS medium containing different concentrations of NaCl. Germination rates of wild type and transgenic seeds were similar when cultured on normal MS medium (Fig. 3a). However, germination of wild type seeds was significantly impaired on MS medium supplemented with 100- or 200-mM NaCl, whereas germination of transgenic seeds was less strongly affected. After eight days on high salt MS medium (100-mM NaCl), only ∼28% of the wild type seeds, but more than 70% of the seeds obtained from AtIpk2β expressing plants germinated (Fig. 3b). When sown on MS medium supplemented with 200-mM NaCl, only ∼7% of the wild type seeds, but ∼30% of the transgenic seeds germinated after 18 days (Fig. 3c). At low salt concentration (50-mM NaCl), wild type and transgenic seeds displayed similar germination rates (data now shown). At 150-mM NaCl, both wild type and transgenic seeds germinated. However, development of wild type seedlings was impaired; plants produced smaller leaves and less root biomass than did transgenic seedlings (Fig. 3d). To further test the effect of salt on growth, 15-day-old wild type and transgenic seedlings were transferred to MS medium supplemented with NaCl at different concentrations. After two additional months, strong growth retardation was observed in wild type seedlings. Inhibition of growth was less evident in transgenic lines (Fig. 3e). A root-bending assay has previously been used to test the effect of elevated salt concentration on root growth (An et al. 2007; Zhu et al. 1998). At 50-mM NaCl on MS medium, root growth of both wild type and transgenic lines was accelerated, but it was inhibited at increasing NaCl concentrations. At 150-mM NaCl, inhibition of root growth was significantly more pronounced in wild type than in transgenic plants (Fig. 3f, g). Fig. 3Germination rate and early seedling development of transgenic plants. (a–c) Percentage of germinating seeds of wild type (WT) and transgenic T2 plants (S13, S15) grown on MS medium supplemented with 0-, 100- or 200-mM NaCl. Results are presented as means and standard errors from three independent experiments (≥100 seeds of each line were sown for each experiment). (d) Wild type (WT) and transgenic seeds (S13, S15) geminated on MS medium supplemented with 50 or 150-mM NaCl. Photo was taken three weeks after seeds were sown. (e) Wild type (WT) and transgenic lines (S13, S14, S15) grown on MS medium supplemented with different concentrations of NaCl for 2 months. (f) Root bending assay of wild type (WT) and transgenic plants (S13, S15) on MS medium supplemented with 0 or 150-mM NaCl. Representative pictures are shown. (g) Primary root lengths of wild type (WT) and transgenic plants (S13, S15) in the presence of different salt concentrations. Root growth under control condition (0-mM NaCl) was set to 100%. The results are shown as means and standard errors from three independent experiments (≥15 seeds of each line were sown for each experiment). *** indicates significant differences in comparison to the wild type at P < 0.001 (Student’s t-test) We also performed salt tolerance experiments with wild type and two transgenic lines grown in the greenhouse in soil. The plants were watered bi-weekly with 1/8 concentrated MS salt solution supplemented with or without 200-mM NaCl. After six weeks, distinct differences were observed between wild type and transgenic plants exposed to 200-mM NaCl (Fig. 4a). Although both sets of plants flowered under conditions of salt stress, wild type plants developed much shorter stems (Fig. 4b), produced fewer pods (Fig. 4c), and produced seeds that were not viable. We determined endogenous Na+ levels in leaves and roots of wild type and transgenic plants grown in the absence or presence of 200-mM NaCl. Both wild type and transgenic plants exposed to salt contained elevated levels of Na+ ions in leaves and roots (Fig. 4d), however, ion concentrations were not significantly different between wild type and transgenic plants (the same was true for plants grown under normal conditions). We also grew tobacco plants in hydroponic culture. In the absence of salt stress, no overt morphological difference was observed between wild type and transgenic plants. However, growth of wild type tobacco was severely inhibited in the presence of 300-mM NaCl (Fig. 4e), leading to a depression of shoot height by about one third, and of plant fresh weight by about 50% in comparison to AtIpk2β expressing tobacco plants after a two-month stress treatment (Fig. 4f). All these results indicated that AtIpk2β alleviated the negative effects imposed by salt stress on plant growth. Fig. 4Salt tolerance test to compare wild type and AtIpk2β expressing tobacco plants. For each experiment, twelve plants of each line were used. (a) Photograph of representative wild type (WT) and transgenic plants (S13, S15) watered with 200-mM NaCl for six weeks. (b), (c) Shoot height and pod numbers of wild type (WT) and transgenic (S13, S15 ) plants treated with 200-mM NaCl. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (d) Na+ content in leaves and roots of wild type (WT) and transgenic plants (S13, S15) grown in soil supplied with 0 or 200-mM NaCl solution. (e) Photograph of a representative wild type (WT) and transgenic line S15 hydroponically grown in nutrient solution supplemented with 300-mM NaCl for two months. (f) Shoot height and (g) fresh weight of wild type (WT) and transgenic lines S13 and S15 grown for two months under control (0-mM NaCl) or high salt (300-mM NaCl) conditions. Results are presented as means and standard errors from three independent experiments. ** indicates significant differences in comparison to the wild type at P < 0.01 (Student’s t-test) Transgenic plants showed increased tolerance to osmotic, drought, freezing temperature and oxidative stress In order to determine whether expression of AtIpk2β affects osmotic stress tolerance in plants, we watered wild type and transgenic tobacco plants (lines S13 and S15) with PEG-6000 (60 g/l) as described previously (Eltayeb et al. 2006). Under such experimental conditions, wild type plants were less vigorous, flowered earlier and produced smaller leaves than AtIpk2β expressing plants (Fig. 5a). We also tested the effect of water shortage on plant growth. After a 21-day drought period, wild type plants completely wilted, while transgenic plants were less severely affected (Fig. 5b). At the end of the drought tolerance test, the plants were re-watered and grown further to allow seed setting. Transgenic plants produced larger shoots than wild type plants (Fig. 5c). Fig. 5Effect of drought and osmotic stress on plant performance. For each experiment, twelve plants of wild type (WT) and each transgenic line (S13, S15) were used. (a) Wild type and transgenic plants grown in soil were watered with 60 g/l PEG-6000. Photograph was taken after 21 days of treatment. (b) Water was withheld for 21 days to impose drought stress. (c) Drought stressed plants were re-watered and grown for seed setting. (d) Proline content of wild type and transgenic plants after treatment with 100 or 200 mM NaCl for 24 h. Results are presented as means and standard errors from three independent experiments. ** indicates significant differences in comparison to the wild type at P < 0.01 (Student’s t-test) Proline accumulation in response to osmotic or salinity stress has been well documented in prokaryotic and eukaryotic organisms (Schobert 1997). Here, we compared the proline content of wild type and transgenic plants treated with 100- or 200-mM NaCl. Although proline accumulated in a dose-dependent manner in both wild type and transgenic plants, proline concentration was slightly higher in transgenic plants in the presence of 200-mM NaCl (Fig. 5d). The results presented in Fig. 4 show that AtIpk2β expression increased the salt tolerance of transgenic tobacco plants. Since salt stress exerts osmotic stress to plant cells in addition to its ionic toxicity, we further determined if AtIpk2β expression confers elevated tolerance to oxidative stress on transgenic plants. Leaf disks from wild type and transgenic tobacco plants were exposed to increasing concentrations of H2O2 for 72 h. As shown in Fig. 6, transgenic plants exhibited a strong tolerance to oxidative stress. Leaf disks from wild type plants were almost completely bleached at the end of the stress treatment, whereas those from transgenic plants showed significant resistance to H2O2-caused damage (Fig. 6a). Fig. 6Oxidative stress and short term freezing tolerance of wild type (WT) and transgenic tobacco plants (S13, S15). (a) Leaf disks were incubated in different concentration of H2O2 (1% and 2%, respectively) under continuous white light for 72 h. H2O2 induced leaf yellowing is delayed in the transgenic plants. (b) Three-week-old seedlings were stored at −20°C for 2 h, and then transferred to normal temperature for further growth. Photograph was taken seven days after the freezing treatment. (c) Quantification of the seedling survival rate (determined seven days after the freezing treatment). Results are presented as means and standard errors from three independent experiments. Fore each experiment, 40 seedlings of each line were used. *** indicates significant difference in comparison to the wild type at P < 0.001 (Student’s t-test). (d) Chlorophyll content was determined in leaf disks of wild type and T2 transgenic plants kept in H2O2 for 72 h. Disks floated in water served as control. Results are preans and standard errors from three independent experiments. ** and *** indicate significant differences in comparison to the wild type at P < 0.01 and P < 0.001, respectively (Student’s t-test) To further examine the effect of constitutive AtIpk2β expression on plant performance, the tolerance towards freezing temperature was analyzed. To this end, three-week-old seedlings were kept at −20°C for 1 or 2 h, respectively, and subsequently allowed to recover for seven days in the greenhouse. Most transgenic plants resumed normal growth after the 2 h treatment at −20°C (Fig. 6B), but only very few wild type plants did so. When treated for 1 h at −20°C, the damage was less severe, but again transgenic plants grew better than wild type plants after transfer back to the greenhouse (data not shown). Transgenic seedlings showed a superior survival rate after short-term −20°C treatment (Fig. 6c). In the presence of oxidative stress, chlorophyll loss was significantly delayed in AtIpk2β expressing plants compared to that of the wild type (Fig. 6d). Lipid hydroperoxidation is an effective indicator of cellular oxidative damage (Yoshimura et al. 2004). Changes in the rates of lipid hydroperoxide production induced by oxidative stress were measured by determining MDA content in leaf disks. At high salinity (200-mM NaCl), MDA concentration markedly increased in wild type plants, whereas only a marginal increase was observed in the two transgenic lines (Fig. 7a). Although superoxide dismutase (SOD) activity was largely unaffected in wild type and transgenic plants treated with 1% H2O2 (Fig. 7b), a slight elevation of APX activity was observed in transgenic plants (Fig. 7c). Also, catalase (CAT) activity increased in transgenic plants (Fig. 7d). These results indicate that constitutive expression of AtIpk2β enhanced tolerance to oxidative stress-induced membrane hydroperoxidation in transgenic tobacco plants. Fig. 7MDA level, anti-oxidative enzyme activity, and expression level of stress regulated genes in transgenic plants. (a) MDA levels in wild type and transgenic lines S13, S15 after treatment with 200-mM NaCl. (b–d) One-month-old seedlings were incubated for 12 h in 1% H2O2 or water (control) under continuous white light. SOD, APX and CAT activities were determined. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to the wild type at P < 0.05 and P < 0.01, respectively (Student’s t-test). (e) Two-week-old seedlings of wild type and transgenic lines (S13, S15) were used for RNA extraction. For the salt stress experiments, seedlings were treated with 300-mM NaCl for 5 h before RNA isolation. The transcriptional levels of five stress genes were determined by RT-PCR analyses. The stress genes used for the tests are as follows: lipid transfer protein (AY562132); fructose-bisphosphate aldolase (AY554169); raffinose synthase family protein/seed imbibition protein (AY554170); group 2 LEA proteins NtERD 10B (AB049336) and NtERD 10C (AB049337) Increased stress responsive gene expression in transgenic tobacco plants Many genes responding to abiotic stress were cloned in recent years and used as molecular markers to monitor the activity of stress-signaling pathways in plants (Kasuga et al. 2004). The stress experiments described above suggested that AtIpk2β plays an important role in regulating stress tolerance in plants. To address whether or not AtIpk2β affects the expression of stress responsive genes, we investigated the transcriptional levels of five marker genes in wild type and transgenic tobacco plants by RT-PCR. The marker genes chosen are identical or homologous to those encoding lipid transfer protein, fructose-bisphosphate aldolase, raffinose synthase, chloroplast ATPase, and group 2 LEA proteins of tobacco. As shown in Fig. 7, transcript levels of these genes were higher in transgenic plants than in the wild type under both, normal and salt stress conditions (Fig. 7). Discussion A number of genes encoding Ins(1,4,5)P3 3-kinase or Ins(1,4,5)P3 dual specificity 6-/3-kinases were cloned and biochemically characterized in the past years. Examples include genes from yeast (Odom et al. 2000), animals (Bertsch et al. 1999), human (Dewaste et al. 2000), and higher plants (Stevenson-Paulik et al. 2002; Xia et al. 2003; Xu et al. 2005). It has recently been reported that in yeast (S. cerevisiae) inositol polyphosphate kinase (Kcs1p) is required for resistance to salt stress, cell wall integrity, and vacuolar morphogenesis (Dubois et al. 2002), and that overexpression of D-IP3K1, a Drosophila IP3-kinase gene, confers resistance of flies to H2O2-, but not to paraquat-induced oxidative stress (Monnier et al. 2002). Further genetic studies indicated that the protective effect conferred by elevated D-IP3K1 expression is mainly due to a lowered IP3 level and thus reduced Ca2+ ion release from internal stores, rather than an increased IP4 level. Based on these observations, we were interested to know whether constitutive expression of AtIpk2β confers improved resistance to abiotic stresses on higher plants. Since the Arabidopsis AtIpk2β protein shares high amino acid sequence identity with the yeast IPK2 enzyme, which functions as a transcriptional regulator (also called Arg82 or ArgRIII) and displays dual-specificity InsP3-InsP4 kinase activity (Odom et al. 2000), we first produced transgenic Arabidopsis (ecotype Columbia-0) plants overexpressing AtIpk2β. Surprisingly, these plants did not exhibit a significant improvement to salt or osmotic stress in our preliminary studies (data not shown). This may be due to the possible overlapping/redundant functions of the two Arabidopsis inositol polyphosphate kinases (AtIpk2α and AtIpk2β) in vivo, as suggested earlier by the York group (Stevenson-Paulik et al. 2002). To examine this possibility, we further compared the growth of Arabidopsis wild type as well as transgenic AtIpk2β over-expresser (35S::AtIpk2β) and knock-out mutant (atipk2β, SALK_104995, ABRC) plants. All three types of plants had similar growth phenotypes when subjected to salt or osmotic stress (data not shown). Here we introduced AtIpk2β into tobacco plants and found that transgenic lines developed superior tolerance to various types of abiotic stresses. Previously, AtNHX1, a vacuolar Na+/H+ antiporter gene from Arabidopsis, was over-expressed in tomato and canola to improve the salt tolerance of transgenic plants (Zhang and Blumwald 2001). However, a comparative analysis of Na+ and K+ accumulation in leaves and roots of transgenic AtNHX1 over-expressers and wild type plants grown under high-salt conditions was not performed because the high (200 mM) NaCl concentration used in these experiments was lethal to the wild type plants. In the study reported here, the relatively high salt tolerance of wild type tobacco plants made it possible to perform such an analysis. In the presence of 200-mM NaCl, despite a similar increase of Na+ concentration in wild type and transgenic plants, slightly more Na+ accumulated in leaves after long-term salt stress (Fig. 4d). This phenomenon is different from what has been seen in transgenic wheat overexpressing AtNHX1 (Xue et al. 2004). Salinity causes two major damages to plant cells, water deficit resulting from the relatively high solute concentrations in the soil, and ion toxicity due to altered K+/Na+ ratios as well as excessive Na+ and Cl− contents (Apse and Blumwald 2002). The Na+/H+ exchange activity of AtNHX1 is regulated in a Ca2+- and pH-dependent manner as reported earlier (Yamaguchi et al. 2005). The increased accumulation of salt in both leaves and roots suggests that unlike AtNHX1, which mainly works through sodium compartmentation, AtIpk2β may function through both sodium extrusion and compartmentation controlled by Ca2+-dependent signal transduction, although the precise mechanism of this still remains to be elucidated. Proline contributes to osmotic adjustment (LeRudulier et al. 1989) and the protection of macromolecules during dehydration (Yancey et al. 1982), and as a hydroxyl radical scavenger (Hong et al. 2000). After treatment with 200-mM NaCl, but not 100-mM NaCl, proline accumulated to slightly higher levels in transgenic than in wild type tobacco plants (Fig. 5d). An increase in proline content was also observed in salt tolerant Arabidopsis with suppressed levels of proline degradation (Nanjo et al. 1999), transgenic salt tolerant tobacco plants with enhanced levels of proline biosynthesis (Kishor et al. 1995), and transgenic salt tolerant tomato plants overexpressing AtNHX1 (Zhang and Blumwald 2001). So, it is possible that the elevated concentration of proline in transgenic plants helps to protect anti-oxidative enzyme thus alleviating the negative effects imposed by salt on transgenic AtIpk2β plants. Salt-stress-induced generation of reactive oxygen species (ROS) has been thought as one of the major causes of adverse effects of high salinity. ROS are products of chloroplast and mitochondrial metabolism produced during stress; they cause membrane damage resulting in electrolyte leakage (Apse and Blumwald 2002). At 200-mM NaCl, a relatively strong increase of MDA production was observed in wild type plants, but only a slight increase was seen in the two transgenic lines (Fig. 7a). Furthermore, catalase (CAT) activity increased significantly in transgenic plants after treatment with 1% H2O2 (Fig. 7d). These results led us to hypothesize that constitutive expression of AtIpk2β improves cell membrane integrity. Evidence supporting this hypothesis was obtained from stress tolerance experiments with transgenic Arabidopsis overexpressing aldehyde dehydrogenase, or transgenic tobacco overexpressing Chlamydomonas glutathione peroxidase (Yoshimura et al. 2004). The induction of stress-related genes has been taken as a hallmark of stress adaptation in plants (Thomashow 1999). Earlier studies have shown that lipid transfer proteins are induced by osmotic stress (Smart et al. 2000). Genes encoding plastidic aldolase are induced by salt (Yamada et al. 2000). Transgenic plants overexpressing such enzymes exhibit improved tolerance to the respective stresses. Here, we observed that constitutive expression of AtIpk2β under the control of the CaMV 35S promoter triggered an increased expression of various stress responsive genes in transgenic tobacco. Already in the absence of salinity stress, transcript levels of genes encoding raffinose synthase family protein/seed imbibition protein (AY554170) and group 2 LEA protein NtERD 10C (AB049337) were found to be slightly elevated in the transgenic plants. At high salinity, the expression levels of genes encoding lipid transfer protein (AY562132) and raffinose synthase family protein/seed imbibition protein (AY554170) increased in the transgenic lines. These results suggest that AtIpk2β acts in one or more signal transduction pathway(s) thereby affecting the activity of stress-related genes and stress tolerance. Many stresses evoke an elevation of the cytosolic calcium ion concentration, including salinity (Knight et al. 1997), drought (Knight et al. 1997), cold (Knight et al. 1996), and oxidative stress (Sanders et al. 2002). Previously, it has been shown that salinity and osmotic stress induce a rapid and transient increase of IP3 levels (DeWald et al. 2001;Takahashi et al. 2001), and the reported accumulation of IP3 occurs during a time frame similar to that observed for stress induced Ca2+ mobilization. Therefore, the possibility exists that enhanced abiotic stress tolerance of transgenic AtIpk2β plants results from changes of Ca2+ dependent signaling. Whether this is indeed the case has to be analyzed in future studies Plant growth and productivity are severely affected by abiotic stress. Augmented resistance towards various environmental stresses has been a desirable goal for genetic improvement of plants. Stress tolerance can for example be improved by altering the expression of stress-responsive genes in transgenic plants. Representatives include genes encoding chitinase and glucanase (Hong and Hwuang 2006), betaine aldehyde dehydrogenase (Yang et al. 2005), tonoplast and plasma membrane Na+/H+ antiporters (Zhang and Blumwald 2001; Apse and Blumwald 2002), protein kinases (Xiong and Yang 2003), transcription factors (Waller et al. 2006), peroxidases (Yoshimura et al. 2004), DNA helicase (Sanan-Mishra et al. 2005), and DNA binding protein (polypeptide; Kalifa et al. 2004). Although the precise mode of action of AtIpk2β in plant responses to stress remains elusive, the results in this work provide direct evidence that alteration of AtIpk2β expression can significantly modify plant tolerance to various abiotic stresses, indicating a great potential of this gene for engineering stress tolerant crops. Electronic supplementary material Below is the link to the electronic supplementary material. Quantitative real-time RT-PCR and Western blot analyses of AtIpk2b in wild type Arabidopsis seedlings in response to different stresses. Fourteen-day old seedlings grown on MS agar plates were treated with 300-mM NaCl or 500-mM mannitol, or kept at 4°C for 24 h. (A) Quantitative real-time RT-PCR analysis of AtIpk2b transcript level. The experiments were repeated three times independently and the average calculated. Error bars represent standard error of the mean. (B) Western blot analysis of AtIpk2b expression level. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal protein loading. (TIF 457 kb)

Biochim_Biophys_Acta-2-1-2258317

Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells

Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity. 1 Introduction Calcineurin (CN) [1] is a serine/threonine specific protein phosphatase present in all eukaryotes that is responsible for the modulation of numerous cell signalling pathways. Calcineurin interacts with the ubiquitous EF-hand containing calcium (Ca2+) sensor calmodulin (CaM) [2] and this obligate association drives calcium dependent phosphatase activity, one of the primary cellular targets of which are the Nuclear Factor of Activated T-cell (NFAT) family of transcription factors. This family of gene regulatory proteins is composed of five members (NFAT1-5) [3,4], that were first identified as fundamental components of the recombinatorial immune system of vertebrates [5], but which have now been implicated in a far broader range of diverse and important physiological processes. Expression of NFAT proteins is not restricted to cells of the immune system, demonstrated by their presence and effect on key functions in a selection of other cell and tissue types including brain and muscle. In a neuronal setting, NFATs have been implicated in axonal remodelling, synaptic plasticity and memory function [6-9]. Transcriptional events mediated by activated NFATs have been linked to aspects of cardiovascular development [10,11] and they have also been suggested to drive proliferative and apoptotic pathways in epithelial cells, fibroblasts, pre-adipocytes, pancreatic β-cells and osteoblasts [12-16]. The discovery that NFAT proteins can influence cell cycle regulation has led to the proposal that they may also be involved in certain aspects of tumorigenesis [17]. NFAT proteins in unstimulated cells are heavily phosphorylated on multiple serine residues by of a number of constitutively active kinases [18]. This modification acts to mask a nuclear localisation signal and under such circumstances NFATs remain predominantly cytosolic and transcriptionally inactive. Stimulation of cells with agonists coupled to the phospholipase-C/IP3 generating pathway, depletion of endoplasmic reticulum Ca2+ stores and subsequent sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) due to opening of plasma membrane Ca2+ release activated Ca2+ channels elicits the rapid dephosphorylation of NFAT proteins, their nuclear import and upregulation of target gene expression [3]. Under such circumstances, the Ca2+ signal is integrated upstream of NFAT by CN in conjunction with CaM and it is this protein complex that represents the critical regulatory mechanism for NFAT dephosphorylation and hence activation of NFAT dependent gene expression. The relationship between CN/CaM and NFATs is so deeply intertwined that nuclear translocation of NFAT proteins has been employed as a robust assay of CN activity in intact mammalian cells [19-21] and has even found application in large scale screens for proteins involved in calcium induced calcium release [22]. CaM is the primordial member of a superfamily of small EF-hand containing Ca2+ binding proteins that function to transduce spatial and temporal cellular Ca2+ signals. Other members of the CaM family include the neuronal calcium sensor (NCS) subgroup of neuronal/neuroendocrine specific proteins [23,24]. Although only distantly related (21% sequence identity between CaM and NCS-1) published in vitro studies have identified potential overlap in target protein interactions between CaM and NCS-1 including an apparent positive interaction of both proteins with CN [25]. Since CaM exhibits high and ubiquitous expression these observations led to the speculation that NCS proteins may simply represent redundant or alternative modulators of classical CaM targets. Evidence from our laboratory and others has gone some way to disproving this idea with the identification of NCS specific binding proteins that, at present, have no documented interaction with CaM [26]. Further evidence for distinct functionality of the NCS protein family is apparent from their higher Ca2+ affinity compared to CaM, their differential cellular/tissue distributions and their non-redundancy revealed in genetic studies [23,27]. Together these data indicate that NCS proteins possess the requisite biochemical properties to allow them respond to unique Ca2+ signals and to bind to distinct effector proteins. The possibility exists however that overlap between CaM and NCS target specificities may be of physiological significance and in the case of CN, identification of a functionally relevant interaction with NCS proteins would be of great interest in view of recent findings implicating CN activity in aspects of neuronal function. In order to resolve some of the outstanding questions concerning potentially redundant functions of NCS proteins we have examined whether or not they are able to elicit CN activation in the physiologically relevant setting of intact mammalian cells using NFAT activation as a coupled reporter. We present in vitro biochemical data indicating that various recombinantly expressed NCS family members are able to directly bind CN although to lower levels compared with CaM. Consistent with these data we have also determined that expression of NCS-1, mutants of this protein that have previously been demonstrated to disrupt NCS-1 function in vivo, along with other NCS family members have no effect on NFAT dephosphorylation in live HeLa cells in response to elevation of [Ca2+]i. These data are supported by studies indicating that the nuclear translocation of NFAT on elevation of [Ca2+]i to cell nuclei is unaffected in cells expressing NCS-1 or NCS-1 mutants. We therefore suggest that NCS proteins, although exhibiting a detectable in vitro affinity for CN, may not be likely to act as physiologically relevant modulators of CN activity or NFAT mediated gene transcription in intact cells. 2 Materials and methods 2.1 Recombinant proteins All recombinant GST fusion proteins used in this study were expressed and purified as previously described [26]. Purified recombinant calcineurin was obtained from Sigma (Poole, UK) or Biomol (Exeter, UK). Unless otherwise stated all chemicals were of analytical grade and obtained from Sigma. 2.2 Small Scale GST protein binding assays In all binding assays, recombinant GST-fusions proteins (1 μM) were immobilised by incubation with 40 μl of glutathione cellulose (GST cellulose) resin (50% bead slurry, Bioline, London, UK) that had been pre-washed in binding buffer (KCl 50 mM, HEPES 20 mM (pH 7.4), EGTA 5 mM, NTA 5 mM, CaCl2 4.6 mM (giving a free [Ca2+] of 1 μM)) (+Ca2+ conditions) or binding buffer with no added CaCl2 (- Ca2+ conditions) by incubation with constant agitation for 30 min/4 °C. Recombinant calcineurin (1 μM) or bovine brain cytosol (∼ 1 mg, dialysed against the appropriate ± Ca2+ binding buffer) were then added to immobilised GST proteins in a 100 μl total binding reaction volume and samples incubated with constant agitation for 2 hrs at 4 °C. GST cellulose beads were pelleted by centrifugation (5,000 rpm/1 min/4 °C) and washed with 1 ml of the appropriate binding buffer. This wash step was repeated three times and final bead pellets extracted into 50 μl SDS dissociation buffer (125 mM HEPES pH (6.8), 10% (v/v) sucrose, 10% (v/v) glycerol, 4% (w/v) SDS, 1% β-mercaptoethanol, 2 mM EDTA) and boiling for 5 min. Samples were resolved on SDS-PAGE (12.5% gel) and transferred to nitrocellulose filters for western blotting by transverse electrophoresis. Bound calcineurin was detected using a monoclonal antibody (1:1000, Sigma) followed by incubation with anti-mouse-HRP (1:400, Sigma) and visualisation with ECL reagents. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software. 2.3 Large scale GST protein binding assays This binding assay is essentially as described in [26]. Briefly, each recombinant GST-fusion protein (5–10 mg), including free GST as control, was immobilised onto 3 ml glutathione-Sepharose 4B resin that had been pre-washed extensively with binding buffer by incubation for 2 h at 4 °C with constant agitation. Clarified bovine brain cytosolic extract was applied to the GST affinity columns and binding allowed to proceed for 16 h at 4 °C with constant agitation. Each column was washed with a minimum 50 volumes of binding buffer and specific Ca2+-dependent binding proteins eluted using binding buffer containing no added Ca2+(calcium-free or CF buffer). An additional high-salt elution step with binding buffer supplemented with 1 M NaCl and no added Ca2+ (High Salt or HS buffer) was used to isolate potential Ca2+-independent binding interactions. Eluted protein fractions were concentrated by methanol precipitation and pellets extracted into SDS dissociation buffer by boiling for 5 min. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software. 2.4 Sulfo-SBED cross-linking experiments The manufacturers protocol provided by Pierce (Rockford, IL, USA) was followed with minor modifications. All steps, except those indicated, were carried out in the dark. Bait recombinant protein of interest (∼5 mg) was dialysed against binding buffer overnight at 4 °C. Immediately before use, the contents of one tube of No-Weigh Sulfo-SBED (Pierce) was dissolved in 22 μl of DMSO, this was then added to 1 ml of dialysed protein. Sulfo-SBED/protein mixes were incubated at room temperature for 30 minutes, centrifuged briefly to remove any precipitated, hydrolysed, Sulfo-SBED, and dialysed overnight against binding buffer. Bovine brain cytosol (500 μl, ∼4 mg protein) dialysed against binding buffer was added to 500μl of the Sulfo-SBED/protein mix. This mixture was incubated on a rotator at room temperature for 1 hour. After this step, samples were exposed to long-wave UV illumination (365 nm) at a distance of ∼5 cm from source for 15 minutes. During this step an appropriate volume of Neutravidin beads (Pierce) were washed extensively in binding buffer, 100 μl added to the bait/cytosol mixture and samples incubated on a rotator for 1 hour at room temperature. Bait/cytosol mixes were then centrifuged at 13,000 rpm for 1 minute, supernatants removed and beads washed with 1 ml of binding buffer. This step was repeated 6 times. Disulfide bond reduction was then achieved by incubating with 1 ml of 50 mM DTT for 1 hour at room temperature. Beads were subsequently centrifuged and washed extensively with binding buffer. After the final wash, 100 μl of SDS dissociation buffer was added to the beads and samples boiled for 10 minutes. Samples were separated on SDS PAGE (12.5% gel) and western blotted with a monoclonal anti-calcineurin antibody (1:1000, Sigma) followed by detection with anti-mouse-HRP (1:400, Sigma) and ECL reagents. 2.5 HeLa cell cultures and transfections HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) containing 5% foetal bovine serum (Invitrogen), 1% non-essential amino acids (Invitrogen), and 1% penicillin/streptomycin (Invitrogen) at 37 °C in an atmosphere of 5% CO2 and maintained at ∼ 1,000,000 cells per 75 cm2 flask. 24 hrs prior to transfection, cells were seeded onto glass cover slips on a 24-well plate at ∼ 50,000 cells per well. Transfection mixtures comprised 3 μl Fugene™ (Roche, UK) per 1 μg plasmid DNA in a 100 μl total volume of DMEM and were incubated at room temperature for 30 minutes prior to being added drop-wise to cells. Cells were assayed 24-48 hrs post-transfection. The NFAT-GFP reporter construct was a kind gift from Professor Anjana Rao (Harvard Medical School, Boston, MA, USA). 2.6 Ionomycin induced NFAT translocation Transfected HeLa cells were washed twice in Krebs buffer (NaCl 145 mM; HEPES 20 mM (pH 7.4); D-(+)-Glucose 10 mM; KCl 5 mM; CaCl2 3 mM; MgCl2 1.3 mM; NaH2PO4 1.2 mM). Cells were subsequently incubated in 500 μl Krebs containing 3 μM ionomycin or 100 μM histamine for required times before either solubilisation in SDS dissociation buffer, to investigate the phosphorylation state of NFAT via Western blotting, or fixing in 4% formaldehyde to observe the resulting sub-cellular localisation of NFAT post-treatment via confocal microscopy. For western blotting all samples were resolved on SDS-PAGE (12.5% gel) and proteins transferred to nitrocellulose filters by transverse electrophoresis. Filters were probed with monoclonal anti-GFP (1:1000, JL-8 clone, Invitrogen) followed by incubation with anti-mouse-HRP (1:400, Sigma) and protein visualised with ECL reagents. 2.7 Confocal microscopy For confocal laser scanning microscopy, transfected cells were examined with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS microscope (Leica Microsystems, Heidelberg, Germany) using a 22 μm pinhole and a 63× water immersion objective with a 1.2 numerical aperture. EGFP was imaged using excitation at 488 nm and light collection at 500-550 nm. 3 Results 3.1 Direct binding of calcineurin to NCS proteins A previous in vitro study demonstrated that NCS-1 is capable of stimulating the phosphatase activity of CN but did not examine whether the two proteins directly associate. To confirm a direct interaction we first performed a series of in vitro binding assays utilising various recombinantly expressed and purified NCS proteins to act as baits and where either purified recombinant CN or bovine brain cytosol were employed as a source of prey protein. Initially, purified CN (1 μM) was incubated in the presence of various GST-tagged NCS proteins (also at 1 μM) in binding buffer containing 1 μM free Ca2+ (Fig. 1a, upper panel). CaM was included in this assay as a positive control for CN binding (Fig. 1a, upper panel, Calmodulin Sepharose). CN was observed to bind directly to GST-NCS-1, GST-Hippocalcin, GST-Neurocalcin-δ and GST-CaBP1 but not GST-KChIP1, despite equal loading of the proteins (Fig. 1a, lower panel), indicating some degree of interaction specificity. It was also apparent from this analysis that, under identical experimental conditions, seven-fold less CN was bound by GST-tagged NCS proteins than by CaM. We next examined if GST-tagged NCS proteins could efficiently interact with CN present as a component of a complex protein mixture. GST control protein, GST-NCS-1 and GST-CaBP1 (Calcium Binding Protein-1, a calcium sensor more closely related to CaM than the NCS family proteins [28]) were incubated with bovine brain cytosolic protein extract in a large scale pull-down experiment [26] in the presence of 1 μM free Ca2+ and Ca2+ dependent binding partners eluted by application of Ca2+ free buffer containing EGTA and NTA (Fig. 1b, Calcium Free (CF) lanes). An additional elution step using 1 M NaCl supplemented buffer was also applied to the samples to elute potential Ca2+ independent binding proteins (Fig. 1b, High Salt (HS) lanes). Western blotting for the presence of CN in eluates confirmed a strictly Ca2+ dependent interaction with both GST-NCS-1 and GST-CaBP1. No binding was detectable to GST control protein. The signal for CN in the bovine brain cytosolic extract was compared in the same experiment (Fig. 1b, Bovine brain cytosol) and it was clear that CN was not enriched over the brain extract in the Ca2+ free eluates. To provide an independent method to examine the interaction between NCS proteins and CN, we exploited a recently developed bi-functional cross-linking reagent [29] which permits the transfer of a biotin tag from a bait protein of interest to any specifically interacting prey proteins captured following a binding reaction. Samples from a GST-Neurocalcin-δ binding assay prepared via this method were probed with an anti-CN antibody and the presence of the phosphatase confirmed only in samples treated with the cross-linker (Fig. 1c, (+) Sulfo) and no CN was detectable in samples devoid of the reagent (Fig. 1c, (-) Sulfo). Despite use of cross-linking to maintain any low affinity interactions, CN was not enriched in the eluate over brain cytosol input into each condition (Fig. 1c (+) Sulfo vs. Bovine brain cytosol). The data presented in Fig. 1a suggests that the affinity of CN for CaM may be substantially greater than for the NCS proteins tested. In order to independently verify these observations we further examined the binding of CN from bovine brain cytosol to GST control protein and GST-NCS-1 in direct comparison to CaM (Fig. 1d). We observed Ca2+ dependent binding of CN to CaM (Fig. 1d, Calmodulin Sepharose (+)) that was significantly enriched in the eluate compared to bovine brain cytosolic extract, in contrast, at this exposure of the western blot, binding to GST-NCS-1 was barely detectable. On longer exposure to film, NCS-1 bound CN was detectable although it should be noted that results obtained from this small scale binding assay are not directly comparable to data presented in Fig. 1b from a large scale binding assay which employed a greater amount of bovine brain cytosolic prey protein. These data are consistent with a high affinity Ca2+ dependent interaction between CaM and CN in contrast to a comparatively weak interaction between NCS-1 and CN. 3.2 Overexpression of NCS proteins has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells Although our in vitro studies pointed to CaM as most likely the predominant interacting/activating partner for CN, we speculated that the situation in the complex physiological setting of mammalian cells might not reflect the results obtained from such a comparatively simple analysis. We therefore attempted to resolve this issue through an examination of a possible interaction between CN and NCS proteins in intact HeLa cells. For these experiments we obtained a GFP tagged NFAT reporter construct [30], encoding the phosphorylated regulatory domain of the phosphatase, that undergoes a detectable dephosphorylation and nuclear translocation in response to sustained increases in [Ca2+]i. Our rationale was that should there be a functionally relevant NCS protein/CN interaction in cells then there might be discernable alterations in dephosphorylation kinetics/nuclear translocation of the NFAT reporter in cells [30,31] over-expressing NCS proteins as observed for other NCS-1 specific functions [32-35]. In initial studies, we saw no effect on NFAT dephosphorylation or nuclear translocation of over-expression of wild-type NCS-1, hippocalcin or neurocalcin-δ. Since NCS-1 had been identified in a previous report [25] as an in vitro activator of CN enzymatic activity we decided, however, to focus on this NCS family member for this part of the study. A second reason for examining NCS-1 in further detail also related to the fact that we had at our disposal several mutant NCS-1 constructs one of which has been previously characterised as a dominant inhibitor of NCS-1 function [32]. Such mutants, we reasoned, might assist in unmasking potential functional interactions between NCS-1, CN and NFAT. The first of these mutants, NCS-1E120Q, has been employed in a number of studies due to its ability to interact normally with NCS-1 effectors however, as a consequence of impaired Ca2+ binding and conformational change, it can then exert a dominant inhibitory phenotype [32-36]. The second NCS-1 mutant we employed in this study, NCS-1G2A, is myristoylation deficient and, unlike wild type NCS-1 which is constitutively membrane associated through this lipid modification, is entirely cytosolic [37,38]. This protein was included in these studies due to the fact that the majority of cellular CN is similarly cytosolic and hence we hypothesised that the G2A mutation may exaggerate any effects of a potential NCS-1/CN interaction. In an initial analysis, HeLa cells were co-transfected with NFAT-GFP along with control empty vector, NCS-1 or NCS-1 mutant constructs (Fig. 2a). Cells were treated for 7 minutes with ionomycin/Ca2+ to elevate [Ca2+]i, lysed and NFAT-GFP detected by western blotting. A band corresponding to the fully phosphorylated form of NFAT was apparent in 0 time point samples from all transfections (Fig. 2a, P, upper arrow). Dephosphorylation of NFAT to two more rapidly migrating species after sustained [Ca2+]i for 7 minutes was observable in all transfected samples and there was no detectable difference in the dephosphorylation pattern obtained between control transfected cells and NCS-1 or NCS-1 mutant transfected conditions (Fig. 2a, DeP, lower arrows). These data are fully consistent with results obtained from parallel confocal imaging analyses of NFAT-GFP nuclear translocation in HeLa cells transfected and treated in an identical manner but which were then fixed for microscopy (Fig. 2b). For all transfection conditions at time 0 of Ca2+/ionomycin treatment NFAT-GFP was excluded from cell nuclei and was diffusely cytosolic (Fig. 2b, control, left hand panels), compatible with the presence of the phosphorylated form of the protein (Fig. 2a). After 7 minutes of Ca2+/ionomycin treatment NFAT-GFP had redistributed to cell nuclei for all transfection conditions (Fig. 2b, ionomycin, right hand panels) again consistent with the rapid dephosphorylation of the protein (Fig. 2a). We additionally verified that all NCS-1 expression constructs used in these studies over-expressed in HeLa cells to similar extents when co-expressed with NFAT-GFP (Fig. 2c). Western blotting with an anti-NCS-1 antiserum [39] of total cell lystates from HeLa cells transfected identically to those used in functional assays (Figs. 2a and b) highlighted essentially equivalent levels of over-expression of NCS-1, NCS-1G2A and NCS-1E120Q proteins. There was no detectable NCS-1 protein expression in control, pcDNA transfected, cells (Fig. 2c, pcDNA). 3.3 Overexpression of NCS-1, NCS-1E120Q and NCS-1G2A has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells over an acute time course Our analyses suggested that, over a 7 minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to [Ca2+]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an acute time course of [Ca2+]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course. For all constructs tested NFAT dephosphorylation was clearly apparent by the 5 minute time point (a 25 minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP) as determined by gel shift of the NFAT-GFP. Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells. In these experiments the calcium induced elevation in cytosolic free Ca2+ was artificially generated by high concentrations of ionomycin and external Ca2+. Since NCS-1 has a high affinity for Ca2+ (300 nM) one possibility is that it could activate CN under conditions of limited Ca2+ elevation thus increasing the overall cellular sensitivity to a Ca2+ signal. We completed, therefore, our analyses with an examination as to whether over-expression of NCS-1 or its mutants could influence NFAT dephosphorylation in response to a physiological stimulus. Histamine is a well characterised HeLa cell agonist that generates oscillatory intracellular Ca2+ signals [40] and activation of protein kinase C with phorbol esters has been shown to potentiate NFAT driven gene expression elicited by such Ca2+ oscillations [41-43]. We stimulated HeLa cells transfected identically to those in Figs. 2 and 3 with 100 μM histamine in both the presence and absence of 50 nM PMA over a three hour time course and assessed the phosphorylation state of NFAT-GFP by western blotting. No dephosphorylation of NFAT-GFP was detectable in these experiments in control vector expressing cells indicating that the stimulus was below the threshold for activation of CN/NFAT-GFP dephosphorylation. Significantly, cells expressing NCS-1, NCS-1E120Q or NCS-1G2A also failed to exhibit dephosphorylation of NFAT-GFP indicating that over-expression of NCS-1 did not enhance the sensitivity of the CN/NFAT signalling pathway to Ca2+ signals under these conditions (data not shown). For NCS-1E120Q to be able to exert a dominant negative effect it would have to bind to CN. In order to validate our use of the NCS-1E120Q mutant as a dominant negative construct in this study we therefore generated a recombinantly purified GST-tagged fusion construct encoding this protein which was used to confirm its ability to bind to purified CN (Fig. 3b). GST-NCS-1E120Q retained almost wild-type CN binding activity in this assay although with less strict Ca2+-dependency validating its use in functional assays of NFAT dephosphorylation and nuclear translocation. For GST-NCS-1 we observed a 13.7-fold increase in binding of CN in the presence of Ca2+ compared to conditions where Ca2+ was absent. In contrast, there was only a 2.2-fold increase in CN binding to GST-NCS-1E120Q in the presence of Ca2+. 4 Discussion Diverse Ca2+ signals are transduced and decoded within cells by a variety of Ca2+-sensing proteins. Of these Ca2+ responsive proteins the most intensively studied and best understood is calmodulin (CaM) [2]. CaM is the prototypical member of a super-family of evolutionarily diverse EF-hand containing Ca2+ sensors to which the NCS sub-family belongs [23]. Whilst the NCS proteins are only distantly related to CaM, comparative studies have identified significant in vitro overlap in their effector specificities [25]. It was proposed that NCS proteins might represent a set of redundant or alternative modulators of bone fide CaM target interactions [25] but the ubiquitous and high level expression of CaM would make this doubtful. Calcineurin (CN) is a cellular serine/threonine phosphatase critical to the activation of NFAT transcription factors and hence in the control of a variety of fundamental physiological processes [3]. CaM has been identified as the essential co-activator for CN function, however CN has also been identified as a potential target for NCS-1 [25] and as such represents a candidate dual CaM/NCS effector. A series of possibilities exist as a direct consequence of these observations. Firstly, NCS proteins could indeed simply represent redundant modulators for CaM targets and as such might be expected to elicit responses and to exhibit biochemical properties closely approximating those observed with CaM. Secondly, NCS proteins could feasibly interact with CaM targets in biochemically distinct ways to modulate their activities in specific, CaM independent, manners leading to the overall generation of unique physiological endpoints. The latter has been suggested for dual regulation of voltage-gated Ca2+-channels by both CaBP1 and CaM [44]. With specific regard to possible CN/NFAT activation by NCS-1, this possibility is intriguing in light of data identifying NFAT dependent gene expression as important to aspects of neuronal cell function. Thirdly, the possibility also exists that interactions observed between NCS proteins and CaM targets are physiologically irrelevant in cells. In this study we have attempted to resolve these issues through an investigation of firstly the biochemical characteristics of NCS protein interactions with CN, and secondly by examining the potential consequences of NCS protein function on activation of the classical CaM-CN-NFAT pathway in mammalian cells. In a series of in vitro biochemical assays we were able to show that there exists a selective and Ca2+ dependent interaction between various NCS proteins and both purified recombinant CN, and CN derived from bovine brain cytosolic protein extract. We noted during these analyses however that significantly more CN bound to immobilized CaM. These data are consistent with the findings of Schaad et al. indicating that NCS-1 is able to stimulate the phosphatase activity of CN in vitro but to a lesser extent than observed for CaM activation. In experiments utilising bovine brain cytosol as a source of prey protein the difference in binding of the NCS proteins for CN in comparison to CaM was more greatly magnified, and whilst considerable quantities of CN could be affinity purified by a calmodulin sepharose column, the amount of CN associated with NCS family members under identical experimental conditions was considerably lower. Previous work using a biotinylated protein overlay assay also found much less binding of CN to NCS-1 compared to CaM [45]. These data led us to conclude that the CaM/CN interaction was likely to be of a higher affinity than for interactions between CN and NCS proteins. Indeed, published biochemical parameters suggest that the in vitro Kd for CaM/CN interaction may be ≤ 0.1 nM [46] with that reported for NCS-1/CN being 350 nM [25]. The in vitro affinity of CaM for CN is therefore three orders of magnitude greater than that determined for NCS-1 which would agree with the binding data presented in this study. Importantly, CaM is expressed at 5-200 fold higher concentrations in cells than NCS-1 [25] and so CN would be predicted to be bound to CaM rather than NCS-1 at elevated [Ca2+]i levels. To assess whether NCS proteins were capable of modulating CN activity in a cellular environment we employed the dephosphorylation and nuclear translocation of a characterised NFAT-GFP reporter construct as a probe of CN phosphatase activity. Co-expression of NCS-1 did not alter the basal phosphorylation state of NFAT-GFP compared with control transfected cells nor did it exert a detectable effect on NFAT-GFP dephosphorylation/nuclear translocation after sustained elevation of [Ca2+]i during single endpoint and acute time course analyses. Co-expression of the NCS proteins Hippocalcin, Neurocalcin-δ [26], and the more closely related CaBP1 (which shares 56% similarity with CaM [47]) were similarly without effect in analyses of NFAT-GFP dephosphorylation (data not shown). From these analyses we cannot formally exclude the possibility that NCS family proteins may activate a pool of cellular CN that does not signal via NFAT dephosphorylation. This seems unlikely however taking into account our biochemical data and the fact that in numerous other studies assessing diverse triggers of CN activity, concomitant activation of the NFAT pathway is always detected. In an extension to these studies we also examined the effects of two NCS-1 mutant proteins, NCS-1E120Q and NCS-1G2A, on NFAT-GFP dephosphorylation in HeLa cells. We were able to demonstrate that the E120Q mutant of NCS-1 [32] retained approximately wild type binding properties with respect to interaction with purified CN. However, this protein, which has been routinely observed to exert a dominant negative phenotype in various other studies of NCS-1 function [32-35], was unable to generate detectable alterations in NFAT-GFP dephosphorylation. In an effort to try and understand if NCS-1 was unable to mediate an in vivo modulation of CN activity due to its restricted membrane localisation we also examined NFAT-GFP phosphorylation in the presence of co-expressed NCS-1G2A, a protein that fails to become myristoylated and as a result remains cytosolic. Since the bulk of cellular CN is also cytosolic we reasoned that expression of the G2A mutant might bias any interaction with CN to generate an observable alteration in dephosphorylation of NFAT-GFP. Our analysis of this mutant indicated that it elicited no significant change in the kinetics of NFAT-GFP dephosphorylation compared to control transfected cells. In related experiments we tested the ability of NCS-1 and NCS-1 dominant inhibitory mutants to influence NFAT dephosphorylation in response to physiological stimuli. We observed no detectable changes in NFAT dephosphorylation in the presence of over-expressed NCS-1 proteins suggesting that in addition to sustained elevation of cytosolic free Ca2+, NCS-1 proteins are similarly unable to stimulate NFAT dephosphorylation in response to agonist derived Ca2+ signals that were below the threshold for significant activation of CN and NFAT dephosphorylation. Collectively our findings would argue that, although NCS proteins are capable of interacting with CN in vitro, they may be unable to act as functionally relevant activators of the enzyme in a cellular context. The possibility remains that there may be some level of interplay between NCS proteins and CaM within protein complexes, exemplified by observations that both CaM and CaBP1 interact with IP3-receptors [40,48,49], that both NCS-1 and CaM are able to interact with Ca2+ channels [50] and G-protein coupled receptor kinases [34,51,52] and that CaM, NCS-1 and CaBP1 have all been shown to modulate the activity of the transient receptor potential channel, TRPC5 [35]. It is impossible to formally rule out direct modulation of CN by NCS proteins under all physiological conditions based on the data presented in this study however we have provided data to challenge the idea that CN is a cellular target of the NCS protein family and instead that CaM appears the more likely regulator of this key signalling phosphatase.

Anal_Bioanal_Chem-3-1-1805043

Analysis of pharmaceuticals in wastewater and removal using a membrane bioreactor

Much attention has recently been devoted to the life and behaviour of pharmaceuticals in the water cycle. In this study the behaviour of several pharmaceutical products in different therapeutic categories (analgesics and anti-inflammatory drugs, lipid regulators, antibiotics, etc.) was monitored during treatment of wastewater in a laboratory-scale membrane bioreactor (MBR). The results were compared with removal in a conventional activated-sludge (CAS) process in a wastewater-treatment facility. The performance of an MBR was monitored for approximately two months to investigate the long-term operational stability of the system and possible effects of solids retention time on the efficiency of removal of target compounds. Pharmaceuticals were, in general, removed to a greater extent by the MBR integrated system than during the CAS process. For most of the compounds investigated the performance of MBR treatment was better (removal rates >80%) and effluent concentrations of, e.g., diclofenac, ketoprofen, ranitidine, gemfibrozil, bezafibrate, pravastatin, and ofloxacin were steadier than for the conventional system. Occasionally removal efficiency was very similar, and high, for both treatments (e.g. for ibuprofen, naproxen, acetaminophen, paroxetine, and hydrochlorothiazide). The antiepileptic drug carbamazepine was the most persistent pharmaceutical and it passed through both the MBR and CAS systems untransformed. Because there was no washout of biomass from the reactor, high-quality effluent in terms of chemical oxygen demand (COD), ammonium content (N-NH4), total suspended solids (TSS), and total organic carbon (TOC) was obtained. Introduction Most pharmaceutical substances are, by nature, biologically active and hydrophilic, in order that the human body can take them up easily, and persistent, to avoid degradation before they have a curing effect. Depending on the pharmacology of a medical substance it will be excreted as a mixture of metabolites, as unchanged substance, or conjugated with an inactivating compound attached to the molecule [1]. When they enter a wastewater-treatment plant, xenobiotics are not usually completely mineralized. They are either partially retained in the sludge, or metabolized to a more hydrophilic but still persistent form and, therefore, pass through the wastewater-treatment plant (WWTP) and end up in the receiving waters. Their removal in WWTPs is variable and depends on the properties of the substance and process conditions (e.g. sludge retention time (SRT), hydraulic retention time (HRT), temperature) [2, 3]. Levels of many pharmaceutically active compounds (PhACs) are barely reduced and they are, therefore, detected in WWTP effluents. The presence of PhACs in surface, drinking, and wastewaters is well documented in the literature [1, 4–12]. Although present at low concentrations in the environment, drugs can have adverse effects on aquatic organisms. These effects are chronic rather than acutely toxic, and depend on exposure (bioavailability), susceptibility to the compound in question, and the degradability of the compound [13]. To ensure compliance with future discharge requirements, upgrading of existing wastewater-treatment facilities and implementation of new technologies is envisaged as the next step in improvement of wastewater treatment. In the last ten to fifteen years the use of membranes in wastewater reclamation has attracted much interest. Membrane technology has become a technically and economically feasible alternative for water and wastewater treatment, especially because of high SRTs achieved within compact reactor volumes. In the MBR the concentration of microorganisms can be increased to up to 20 mg L−1. Because of this high biomass concentration the rate of degradation is higher and specialists are grown for problematic compounds. Another advantage of membrane treatment is separation of suspended solids by membranes, so they are not limited by the settling characteristics of the sludge. The performance, in terms of effluent quality, is believed to be better, but there is a striking lack of knowledge about the behaviour of trace-pollutants. Literature data on this subject is still very limited and contradictory [2, 11, 14–16]. Ultrafiltration membranes do not enable greater retention of the drugs investigated in this study as a result of filtration effects—the molecular sizes of the compounds selected are at least a factor of 100 smaller than the pore size of the membranes. Additional removal of hydrophobic compounds by membranes can, nevertheless, be expected, because they are adsorbed by particles deposited as a layer on the membrane surface; this effect for hydrophilic compounds is not yet very well defined, however [11, 17]. The objectives of this work were detection of target pharmaceuticals in wastewater influents and effluents, observation of their elimination in the CAS process, and comparison with results obtained for a laboratory-scale MBR provided with a plate-and-frame submerged membrane module. The pharmaceutical products investigated were analgesics and anti-inflammatory drugs (ibuprofen, ketoprofen, naproxen, diclofenac, indomethacin, acetaminophen, mefenamic acid, and propyphenazone), lipid regulators and cholesterol-lowering statin drugs (clofibric acid, gemfibrozil, bezafibrate, pravastatin, and mevastatin), antibiotics (erythromycin, azithromycin, sulfamethoxazole, trimethoprim, and ofloxacin), psychiatric drugs (fluoxetine and paroxetine), an antiepileptic drug (carbamazepine), β-blockers (atenolol, sotalol, metoprolol, and propranolol), anti-histaminics (famotidine and loratidine), anti-ulcer agents (lansoprazole and ranitidine), an anti-diabetic (glibenclamide), and a diuretic (hydrochlorothiazide). These compounds had different physicochemical properties (i.e. neutral, ionic, hydrophilic, hydrophobic). Their chemical structures and CAS numbers are listed in the Appendix. If their behaviour during wastewater treatment could be more reliably related to process design and operating conditions, process performance could possibly be improved by alteration of these conditions in accordance with the types of molecule that are difficult to eliminate. Experimental Materials and standards Chemical standards of carbamazepine, lansoprazole, loratidine, famotidine, trimethoprim, ofloxacin, atenolol, metoprolol, azithromycin dihydrate, erythromycin hydrate, fluoxetine hydrochloride, ranitidine hydrochloride, sulfamethoxazole, propranolol hydrochloride, indomethacin, acetaminophen, mefenamic acid, clofibric acid, bezafibrate, mevastatin, and sotalol hydrochloride were purchased from Sigma–Aldrich (Steinheim, Germany), propyphenazone, pravastatin, and paroxetine hydrochloride from LGC Promochem (London, UK), ketoprofen, diclofenac, gemfibrozil, ibuprofen, and naproxen from Jescuder (Rubí, Spain), glibenclamide from SIFA Chemicals (Liestal, Switzerland), and hydrochlorothiazide from Pliva (Zagreb, Croatia). All pharmaceutical standards were of high-purity grade (>90%). Isotopically labelled compounds used as internal standards were 13C-Phenacetin, from Sigma–Aldrich, mecoprop-d3, from Dr Ehrenstorfer (Augsburg, Germany), and ibuprofen-d3, atenolol-d7, and carbamazepine-d10 from CDN Isotopes (Quebec, Canada). All solvents (methanol, acetonitrile, and water) were HPLC-grade and were purchased from Merck (Darmstadt, Germany), as also was hydrochloric acid (HCl, 37%), ammonium acetate (NH4Ac), and acetic acid (HAc). Nitrogen for drying, purity 99.995%, was from Air Liquide (Spain). Stock solutions of individual standards (1 g L−1) and internal standards were prepared in methanol and stored at −20 °C. Stock solutions of ofloxacin, pravastatin, and sulfamethoxazole were renewed monthly because of their limited stability. A standard mixture in which the compounds were at a concentration of approx. 20 mg L−1 was prepared from the stock solutions. Further dilutions of this mixture were prepared in 25:75 (v/v) methanol–water and were used as working standard solutions. A mixture of internal standards prepared by dilution of individual stock solutions in methanol was used for internal standard calibration. Membrane bioreactor (MBR) A submerged MBR of approximately 21 L active volume equipped with two flat sheet membranes (A4 size, area 0.106 m2, pore size 0.4 μm), purchased from Kubota (Osaka, Japan), was installed in a municipal WWTP in Rubí (Barcelona, Spain). Although the nominal porosity of the membranes was 0.4 μm (microfiltration) a fouling layer of proteins and microorganisms formed on the surface of the membranes reduced the effective porosity to 0.01 μm, which brought the type of filtration into the ultrafiltration range [17]. The MBR was operated in parallel with the CAS process (aeration tank and secondary settling tank). The biocenosis of the MBR was grown from inoculated sludge from the municipal WWTP (aeration basin) and cultivated over a period of approximately 1 month to reach steady-state conditions. The hydraulic retention time was set to 14 h by regulating the effluent flow and the SRT was infinite, because no sludge was discharged from the reactor. The laboratory-scale MBR was operated dynamically in intermittent permeation mode—cycles of permeation for 8 min interrupted with 2 min of halt. Influent and permeate flows were controlled by use of flow meters and computer-controlled pumps. Continuous aeration was provided by means of a sparger pipe situated at the bottom of the reaction vessel; the oxygen concentration was kept between 1 and 2 mg L−1. The temperature inside the reactor was 20 ± 2 °C throughout sampling. Wastewater-treatment plant (WWTP) Rubí WWTP was designed for 125,550 inhabitant equivalents. During the sampling programme the WWTP was operating with an average daily flow of 22,000 m3 day−1. A mixture of municipal, hospital, and industrial wastewater is treated. Treatment consists of pretreatment, preliminary treatment, primary sedimentation, and secondary (biological) treatment. Pretreated wastewater goes through a physical process of settling in a primary clarifier. Secondary treatment occurs in pre-denitrification (anaerobic) and nitrification (aerobic) tanks, and two secondary clarifiers. Secondary sludge is recirculated to a primary clarifier which improves the settling characteristics of the primary sludge and increases sludge age. A mixture of primary and secondary (activated) sludge is processed (thickening, dewatering) and anaerobically digested, and biogas produced is used to heat a digester. The hydraulic retention time of CAS treatment in WWTP Rubí, calculated for the average daily flow, is approximately 12 h. During the sampling programme the plant was operating with an SRT of approximately 3 days. WWTP effluent is discharged into the river Riera de Rubí, which flows into the Mediterranean sea. Sampling and sample preparation Sampling was conducted during May and June, 2005. Twenty-eight samples were analyzed. All wastewater samples were taken as time-proportional grab-samples, bearing in mind the HRT of the MBR and CAS processes. The sampling points were: primary sedimentation tank effluent, as the inflow to the conventional treatment plant and membrane bioreactor,CAS effluent, andMBR effluent. Wastewater samples were collected, in amber glass bottles pre-rinsed with ultra-pure water, as 24-h composite samples; the volume depended on the type of sample (100 mL for influent wastewater and 200 mL for effluent). Immediately on arrival at the laboratory they were filtered through 1-μm glass fibre filters and then through 0.45 μm Nylon membrane filters from Whatman (UK). The target compounds were extracted in one step, by a method described elsewhere [18], using a Baker vacuum system (J.T. Baker, The Netherlands) and Waters (Milford, MA, USA) Oasis HLB cartridges (60 mg, 3 mL) previously conditioned at neutral pH with 5 mL methanol then 5 mL deionised water (HPLC grade). Elution was performed twice with 4 mL methanol at a flow of 1 mL min−1. The extracts were then evaporated under a nitrogen stream and reconstituted with 1 mL 25:75 (v/v) methanol–water. Chemical analysis LC analysis was performed with a Waters (Milford, MA, USA) 2690 HPLC system coupled to a Micromass Quattro (Manchester, UK) triple quadrupole mass spectrometer equipped with a Z-spray electrospray interface. Chromatographic separation was achieved on a Purospher Star RP-18 endcapped column (125 mm × 2.0 mm, particle size 5 μm) and a C18 guard column, both from Merck. A specific multi-residue analytical method was set up for measurement of the concentrations of the pharmaceutical compounds in wastewaters [18]. Analysis was performed in multiple-reaction-monitoring (MRM) mode, in both positive and negative electrospray-ionization mode. This method was refined for analysis of hydrochlorothiazide and glibenclamide. MRM transitions selected for each compound are summarized in Table 1. In accordance with the performance characteristics defined in EU Commission Decision 2002/657/EC for confirmation and identification of pharmaceuticals when using LC–tandem MS as the instrumental technique, a minimum of three identification points are required. When using LC–MS–MS (QqQ) analysis two MRM transitions are sufficient to confirm the identity of a compound. The MRM ratio, calculated as the relationship between the abundances of both transitions and the LC retention time are also criteria used to confirm the presence of an analyte in the samples. In this study, therefore, transitions between a precursor ion and the two most abundant fragment ions were chosen for each analyte when working in MRM mode, resulting in four identification points, enough to conform with the aforementioned EU directive. When poor fragmentation was observed for the compounds, only one transition could be monitored. Confirmation of the identities of these was achieved by matching their LC retention times with those of standards. Shifts in retention times were less than 3%, so the confirmation was regarded as sufficiently reliable. For internal standards only one transition was selected, because they were isotopically labelled compounds unlikely to be found in environmental samples. Table 1MRM transitions of the compounds analyzedCompoundMRM 1MRM 2MRM 3Ibuprofen205→161Ketoprofen253→209253→197Naproxen229→170229→185Diclofenac294→250294→214Indomethacin356→297356→255Acetaminophen152→110152→93Mefenamic acid240→196240→180Propyphenazone231→201231→189Clofibric acid213→127213→85Gemfibrozil249→121Bezafibrate360→274360→154Pravastatin447→327Mevastatin391→185391→159Carbamazepine237→194237→192Fluoxetine310→44310→148Paroxetine330→192330→123Lansoprazole370→252370→205Famotidine338→189338→259Ranitidine315→176315→130Loratidine383→337383→267383→259Erythromycin734.5→158734.5→576.4Azithromycin749.5→591.4749.5→158Sulfamethoxazole254→92254→156Trimethoprim291→230291→261Ofloxacin362→316Atenolol267→190267→145Sotalol273→255273→213Metoprolol268→133268→159Propranolol260→166260→183Hydrochlorothiazide296→269296→205Glibenclamide494→369 To compensate for matrix effects from sample matrices internal standard calibration and adequate dilution of sample extracts were used, on the basis of the previously published method [18]. Recoveries of the method were determined by spiking. Influent samples and CAS and MBR effluents were spiked in triplicate with a standard mixture of selected compounds to a final concentration of 1 μg L−1. Spiked samples and a blank sample were analysed by the above mentioned method. Recoveries of the target pharmaceuticals are listed in Table 2, with method detection limits (MDL) for influent and effluent samples. MDLs and method quantification limits (MQL) were calculated on the basis of signal-to-noise ratios (S/N) of 3 and 10, respectively. Table 2Mean recoveries of the selected compounds and method detection limits (MDL) in ng L−1CompoundRecovery (%)MDL (ng L−1)InfluentMBR effluentCAS effluentInfluentMBR and CAS effluentIbuprofen131 (18.1)a68.8 (11.0)90.4 (11.0)98.020.0Ketoprofen62.8 (2.94)71.3 (3.11)59.1 (0.897)19074.0Naproxen49.2 (20.0)59.4 (1.28)53.4 (2.31)79.020.0Diclofenac83.3 (1.17)94.9 (10.0)95.0 (12.6)16040.0Indomethacin113 (2.95)120 (5.63)110 (3.78)15031.0Acetaminophen123 (17.0)108 (10.5)56.0 (7.61)20.95.35Mefenamic acid93.3 (1.95)92.1 (1.02)91.5 (5.29)5.701.85Propyphenazone60.0 (8.00)71.0 (4.00)71.0 (4.00)4.801.45Clofibric acid86.0 (10.8)104 (6.87)74.5 (1.40)16.33.75Gemfibrozil91.0 (8.47)87.5 (1.36)108 (17.2)8.702.20Bezafibrate106 (3.43)94.4 (9.30)89.4 (4.62)18.54.35Pravastatin85.6 (2.56)78.0 (12.2)96.0 (19.5)12030.9Mevastatin103 (8.61)134 (15.6)123 (9.86)9.301.30Carbamazepine84.0 (7.84)89.5 (5.20)88.0 (9.24)2.200.600Fluoxetine46.7 (2.34)93.7 (17.6)59.0 (1.60)19.81.70Paroxetine62.2 (2.15)109 (5.73)71.4 (1.49)3.500.650Lansoprazole70.0 (10.0)87.0 (5.00)86.0 (4.00)10.94.20Famotidine58.2 (7.76)55.4 (6.30)66.6 (5.39)3.100.40Ranitidine41.5 (9.85)75.8 (14.8)125 (11.7)1.400.300Loratidine72.6 (1.81)78.0 (6.97)64.5 (4.98)8.002.75Erythromycin67.7 (3.15)50.0 (13.0)66.6 (12.0)12.42.00Azithromycin30.0 (7.00)73.0 (2.00)63.0 (3.00)1.000.300Sulfamethoxazole33.7 (2.76)95.5 (9.24)78.3 (1.08)16.13.10Trimethoprim58.8 (3.29)128 (6.58)60.8 (3.87)1.300.350Ofloxacin142 (19.0)135 (5.45)138 (4.47)29.37.85Atenolol83.5 (33.8)60.8 (10.8)131 (15.5)1.700.750Sotalol47.1 (2.91)31.9 (3.05)52.0 (3.63)4.800.700Metoprolol36.7 (1.44)120 (2.64)76.7 (1.43)6.301.60Propranolol60.2 (0.506)90.8 (4.02)70.5 (5.27)2.600.300Hydrochlorothiazide39.8 (7.43)58.9 (1.62)73.4 (22.9)4.500.900Glibenclamide100 (11.7)107 (10.3)98.5 (11.7)19.22.30aThe relative standard deviation (%) of the recoveries is given in parentheses (n = 3) Results and discussion It is well documented that WWTPs are major contributors of pharmaceuticals in the environment. WWTP Rubí mainly receives domestic, hospital, and industrial wastewater and effluent concentrations of several monitored compounds exceed μg L−1 levels. Ranges of output loads for WWTP Rubi for each pharmaceutical and mean values (g day−1) are reported in Table 3. The quantities of pharmaceuticals discharged into the environment are calculated by multiplying the detected effluent concentrations by an average daily flow rate of 22,000 m3 day−1. The total amount of pharmaceutical compounds discharged by WWTP Rubi into the environment exceeded 300 g day−1 (an average value). The most abundant compounds, with average individual loads of 21–56 g day−1, were the anti-inflammatory drugs ibuprofen, naproxen, and diclofenac, the lipid regulators gemfibrozil and bezafibrate, the diuretic hydrochlorothiazide, and the β-blocker atenolol. Table 3Average daily output loads of the investigated pharmaceuticals for Rubí WWTPPharmaceuticalEffluent load (g day−1)MeanRangeAnalgesics and anti-inflammatory drugsNaproxen37.010.8–76.9Ketoprofen17.111.4–36.3Ibuprofen56.37.39–137.9Diclofenac27.317.3–43.8Indomethacin1.93nd–2.73Acetaminophen4.551.06–9.2Mefenamic acid0.440.27–0.85Propyphenazone0.680.43–0.96Anti-ulcer agentRanitidine2.770.55-5.30Psychiatric drugParoxetine0.08nda–0.16Antiepileptic drugCarbamazepine5.211.44-6.71AntibioticsOfloxacin6.932.40–11.2Sulfamethoxazole3.061.42–5.81Erythromycin2.290.95–4.51β-blockersAtenolol21.07.70–33.2Metoprolol3.321.14–5.43DiureticHydrochlorothiazide33.721.2–46.0Hypoglycaemic agentGlibenclamide0.74nd–0.98Lipid regulator and cholesterol lowering statin drugsGemfibrozil54.330.1–73.9Bezafibrate21.610.9–50.8Clofibric acid1.750.40–3.43PravastatinndndaNot detectable (below the LOQ) To assess the efficiency of elimination by the MBR, substance-specific analysis must be performed and the bulk properties DOC and COD of wastewater influents and effluents must also be determined. The performance of the MBR system is summarized in Table 4. The data are presented for the sampling period. Removal efficiencies of 98.7% for TSS and 90.4% for total COD were achieved during the membrane process. Average effluent ammonia concentration was 1.01 μg L−1 in the MBR effluent, compared with 48.41 μg L−1 in the CAS effluent. It is known that membrane processes are quite efficient at removing COD and TOC from wastewater [19, 20]. Improved COD removal is attributed to the combination of complete retention of particulate material by the membrane, including suspended COD and high molecular weight organisms, and to avoidance of biomass washout problems common in activated sludge systems. Consequently, stable conditions are provided for growth of specialized microorganisms which are the able to remove poorly biodegradable components. Table 4Summary of the performance of the MBR systemPropertyInfluentMBR effluentCAS effluentTSS (mg L−1)119.2 (17.37)a1.600 (1.770)26.72 (15.69)CODtotal (mg L−1)508.2 (124.3)48.58 (22.47)111.6 (53.35)TOC (mg L−1)67.67 (24.29)10.89 (3.470)27.33 (13.75)N-NH4 (mg L−1)49.13 (15.79)1.010 (0.4200)48.41 (12.87)pH7.52 (0.300)7.08 (0.270)7.63 (0.160)aValues are averages from n = 16 samples, with standard deviations in parentheses Of 31 pharmaceutical products included in the analytical method, 22 were detected in the wastewater entering WWTP Rubí. Box plots of measured concentrations of each pharmaceutical are showed in Figs. 1, 2 and 3. Ten measured values are given for influent and MBR effluent concentrations and eight for CAS effluent (data are missing for two sampling programmes). For each variable the box has lines at the lower quartile (25%), median (50%), and upper quartile (75%) values. The whiskers are the lines extending from each end of the box to show the extent of the data up to 1.5 times the interquartile range (IQR). Outliers are marked with + symbols. Fig. 1Removal, during MBR and CAS treatment, of the analgesics and anti-inflammatory drugs naproxen (a), ketoprofen (b), ibuprofen (c), mefenamic acid (d), diclofenac (e), indomethacin (f), acetaminophen (g), and propyphenazone (h)Fig. 2Removal during MBR and CAS treatment of the antibiotics ofloxacin (a), sulfamethoxazole (b), and erythromycin (c), the β-blockers atenolol (d) and metoprolol (e), the anti-ulcer agent ranitidine (f), the antiepileptic drug carbamazepine (g), and the psychiatric drug paroxetine (h)Fig. 3Removal during MBR and CAS treatment of the lipid regulator and cholesterol-lowering statin drugs gemfibrozil (a), bezafibrate (b), clofibric acid (c), and pravastatin (d), the diuretic hydrochlorothiazide (e), and the hypoglycaemic agent glibenclamide (f) The highest influent concentrations (μg L−1) were measured for the analgesics and anti-inflammatory drugs naproxen, ibuprofen, ketoprofen, diclofenac, and acetaminophen, the antihyperlipoproteinaemic drugs gemfibrozil and bezafibrate, the β-blocker atenolol, and the diuretic hydrochlorothiazide. For other compounds input concentrations were usually in the range 10–100 ng L−1. Because the low concentrations measured were sometimes close to the limits of quantification, any hypothesis about the efficiency of their elimination is questionable. Mean removal was, nevertheless, calculated for each of the pharmaceutical compounds; the results are presented in Table 5. The most important removal pathways of organic compounds during wastewater treatment are: biotransformation/biodegradation,adsorption by the sludge (excess sludge removal), andstripping by aeration (volatilization).Table 5Mean removal of selected pharmaceuticals by the MBR and CAS processesCompoundElimination (%) in:MBRaCASbAnalgesics and anti-inflammatory drugsNaproxen99.3 (1.52)85.1 (11.4)Ketoprofen91.9 (6.55)51.5 (22.9)Ibuprofen99.8 (0.386)82.5 (15.8)Diclofenac87.4 (14.1)50.1 (20.1)Indomethacin46.6 (23.2)23.4 (22.3)Acetaminophen99.6 (0.299)98.4 (1.72)Mefenamic acid74.8 (20.1)29.4 (32.3)Propyphenazone64.6 (13.3)42.7 (19.0)Anti-ulcer agentsRanitidine95.0 (3.74)42.2 (47.0)Psychiatric drugsParoxetine89.7 (6.69)90.6 (4.74)Antiepileptic drugsCarbamazepineNo eliminationcNo eliminationAntibioticsOfloxacin94.0 (6.51)23.8 (23.5)Sulfamethoxazole60.5 (33.9)55.6 (35.4)Erythromycin67.3 (16.1)23.8 (29.2)B-blockersAtenolol65.5 (36.2)No eliminationMetoprolol58.7 (72.8)No eliminationDiureticsHydrochlorothiazide66.3 (7.79)76.3 (6.85)Hypoglycaemic agentsGlibenclamide47.3 (20.1)44.5 (19.1)Lipid regulator and cholesterol lowering statin drugsGemfibrozil89.6 (23.3)38.8 (16.9)Bezafibrate95.8 (8.66)48.4 (33.8)Clofibric acid71.8 (30.9)27.7 (46.9)Pravastatin90.8 (13.2)61.8 (23.6)a,bValues are averages, with relative standard deviations (%) in parentheses, for n = 10a or n = 8b samplescCompounds were classified as “no elimination” if elimination was less than 10% Because of the low values of the Henry coefficients (KH) of the compounds investigated [21], the fraction removed by volatilization can be neglected [16]. The two processes abiotic (adsorption) and biotic degradation (transformation by microorganisms) could not be distinguished, because no method was developed for analysis of the target compounds in sludge. The term “removal” is therefore used here for conversion of a micropollutant to compounds other than the parent compound. Elimination efficiency of the laboratory-scale MBR and the full-scale CAS process was comparable for naproxen, ibuprofen, acetaminophen, hydrochlorothiazide, and paroxetine. All were removed to a large extent by both systems (removal was greater than 80% except for hydrochlorothiazide, for which it was between 56 and 85%). Hydrochlorothiazide and paroxetine were eliminated slightly better by conventional treatment. Similar results for the behaviour of these drugs during conventional treatment have been reported by several authors [2, 3, 9, 11]. For ketoprofen, diclofenac, bezafibrate, and gemfibrozil removal by the MBR system was very high and uniform (>90%), with the exception of two sampling programme. It is assumed this variation could have been a result of reduced microbial activity or altered sorption and flocculation conditions. No plausible explanation can be given for the drastically reduced efficiency of removal of clofibric and mefenamic acid by MBR in two sampling programmes; otherwise these were eliminated with efficiencies between 65 and 90%. High and steady removal (>80%) in the MBR was also observed for ranitidine and ofloxacin. In conventional treatment all these pharmaceuticals were eliminated with a wide range of efficiencies, always lower than those obtained by the MBR. Better removal of readily biodegradable micropollutants by the MBR could be because of the smaller flock size of the sludge, which enhances mass transfer by diffusion and therefore increases elimination. Taking into consideration the composition of sludge originating from a membrane bioreactor (specialized microorganisms, large amount of active biomass in suspended solids) improved removal is to be expected; this was confirmed by our experiments. A possible explanation of substantially greater attenuation of diclofenac by the MBR (average removal efficiency 87% compared with 50% in CAS) could be the greater age of the MBR sludge. Improved removal is observed with increasing solids retention time [14]. Another explanation could be greater adsorption potential of the MBR sludge, because the organic matter content is greater than for CAS sludge. According to results from the EU project Poseidon [22], adsorption processes affect elimination of diclofenac. Literature data on this matter is still very contradictory. Clara et al. reported poor removal of diclofenac in laboratory-scale WWTPs whereas in full-scale plants removal varied from less than 20% to between 60 and 80% for some of the facilities investigated [2]. Heberer et al. [7] reported low removal efficiencies in a WWTP whereas Ternes et al. documented significant (69%) elimination of diclofenac [8]. Removal of carbamazepine was, in contrast, very poor (<20%), and effluent concentrations for both MBR and CAS were frequently greater than influent levels. Poor elimination of this neutral drug has been reported by many authors [9, 11, 23, 24]. Glucuronide conjugates of carbamazepine can, presumably, be cleaved in sewage, thus increasing environmental concentrations [8]. Rates of removal of the antibiotic sulfamethoxazole were very variable in both treatments investigated. According to Drillia et al. its microbial degradation will depend on the presence of readily biodegradable organic matter in wastewater; this varies during both MBR and CAS treatment [25]. Also, a substantial amount of sulfamethoxazole enters WWTPs as its human metabolite N4-acetylsulfamethoxazole, which can possibly be converted back to the original compound during treatment [26]. Efficiency of removal of atenolol, metoprolol, pravastatin, erythromycin, and indomethacin varied in both MBR and CAS treatment. This could not be explained. Fluctuation of elimination efficiency was also observed for propyphenazone (44.8–82.9% for MBR and 6.82–62.6% for CAS) and glibenclamide (14.8–73.7% for MBR and 11.9–79.7% for CAS). Effluent concentrations greater than those recorded for the influent could be explained by the presence of input conjugate compounds that are transformed into the original compounds during treatment. Because these conjugates were not included in the analysis, no firm conclusion can be made about their biotransformation, especially because sampling inaccuracy can also lead to errors. Conclusion Several pharmaceutical products (e.g. ibuprofen, naproxen, acetaminophen, ketoprofen, diclofenac, bezafibrate, gemfibrozil, ranitidine, ofloxacin, hydrochlorothiazide, and paroxetine) with high rates of attenuation can be expected to be completely removed from wastewater by adsorption or degradation, or a combination of both, during membrane treatment. For most of the compounds investigated MBR effluent concentrations were significantly lower than in the effluent from conventional treatment. Elimination of hydrochlorothiazide and paroxetine was slightly better in CAS treatment. Some substances (e.g. carbamazepine) were not removed by either MBR or CAS treatment. No relationship was found between the structures of target compounds and their removal during wastewater treatment, however. The range of variation of the efficiency of removal by the MBR system was small for most of the compounds; in conventional treatment greater fluctuations were observed and removal efficiency was found to be much more sensitive to changes in operating conditions (temperature, flow rate, etc). Although membrane technology seems a promising means of removal of pharmaceutical compounds, the MBR process investigated would not completely halt discharge of micropollutants. Membrane treatment processes should be optimized by modification of the membranes (variation of the materials and reduction of molecular mass cut-off limits) and/or by modification of the treatment process (inoculation of special microorganisms). The efficiencies of diverse microbial populations in elimination of selected pharmaceuticals, and optimization of design and operating conditions of a laboratory-scale MBR will be the main objectives of our future investigations. That would provide guidelines for scale-up of a biological pilot plant and its evaluation by integration into an industrial process water-recycling system. Because of the current lack of information on the behaviour of pharmaceuticals in surface and wastewaters, however, further studies are required on the occurrence, fate, and effects of these substances in the environment.

J_Biol_Inorg_Chem-4-1-2359831

DNA cleavage and binding selectivity of a heterodinuclear Pt–Cu(3-Clip-Phen) complex

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum–copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen is 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts. Introduction DNA is a target for numerous antitumor drugs [1, 2]. Reversible or irreversible modifications of the nucleic acids can lead to disruption of the transcription and/or replication, initiating ultimately the death of cancer cells. Cisplatin [3] and bleomycin are among these efficient anticancer drugs [4]. The mechanism of action is different for both drug substances; cisplatin primarily induces distortions upon binding to DNA, whereas bleomycin is able to generate DNA strand scissions. Since the discovery of bleomycin [5], numerous metal complexes have been synthesized that are able to produce DNA cleavage. Typical examples are iron(II)–edta complexes, manganese(III)–porphyrin and Cu, Co, Ru and Rh complexes with phenanthroline [6–9]. For instance, Cu(phen)2+ in the presence of dihydrogen peroxide efficiently cleaves double-stranded DNA through the oxidative attack on deoxyribose units from the minor groove [10, 11]. The consequent DNA-cleavage products include 5′-monophosphate and 3′-monophosphate ester termini, free bases, 5-methylene furanone, and a small amount of 3′-phosphoglycolate [12–14]. Dihydrogen peroxide can be generated by Cu(phen)22+ in close proximity to the DNA strands, in the presence of a reductant and molecular oxygen [15]. The nuclease activity of Cu(phen)2 has been enhanced with the synthesis of 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine (3-Clip-Phen, 4) based on the covalent linkage of two phenanthroline units through their 3-position, leading to an increase of 60 times compared with the nuclease activity of Cu(phen)2 itself [15]. However, similarly to Cu(phen)2, the copper complexes of 3-Clip-Phen have no sequence selectivity, and cleave the DNA in a single-stranded fashion. The amine group of 3-Clip-Phen has been functionalized with different groups, such as a distamycin analog or various DNA intercalators [16, 17]. Thus, the resulting complexes show enhanced cleaving activities, and the complexes with the distamycin analog exhibit excellent targeting properties toward A·T boxes [18]. Recently, it has been shown that Cu(3-Clip-Phen) (3) attached to a cisplatin motif is able to perform direct double-strand cuts, thanks to the DNA-anchoring platinum moiety [19]. Platinum complexes have received considerable attention since the discovery of the antiproliferate activity of cisplatin in 1969 [20]. It is generally accepted that the distortion of DNA generated upon binding of cisplatin is largely responsible for its antitumor properties [3]. Subsequent drug activation via intracellular aquation reactions results in a variety of stable bifunctional DNA–platinum(II) adducts. 1,2-Intrastrand cross-links between two adjacent guanine bases d(GG) or between an adenine and a guanine residue d(AG) are primarily formed. The platinum center of complexes with a cis motif preferentially coordinates to the N7 position of both adenine and guanine in the major groove of DNA [21–24]. In the present paper, the preparation of a bifunctional complex [CuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4, 2] containing both a cis-bis(amine)Pt(II) moiety and a nuclease active 3-Clip-Phen group is reported (Fig. 1). The bridge connecting the platinum and copper moieties is very short and therefore rigid in contrast to that in the previous report, where the complexes had flexible linkers in order to have a major–minor groove interaction [19]. The platinum and the 3-Clip-Phen adjacent parts are now separated by a short linker. Consequently, in this case either the platinum species will not interact with its preferential site (the major groove) or the 3-Clip-Phen moiety will not interact with its preferential site (the minor groove). The platinum component plays two roles: (1) it acts as a DNA anchor, thus allowing the Cu(3-Clip-Phen) moiety to perform cleavages in the close proximity of the Pt–DNA adducts, and (2) it induces a sequence-selective binding of the heterodinuclear complex. Accordingly, the achievement of double-strand DNA breaks is potentially increased, because the single-strand cuts are in the close proximity of the platinum adduct and sequence-selective cleavage may be expected. Fig. 1Complexes 1–3 In the present study, the binding property of the platinum moiety and the cleavage selectivity and activity of this novel heterodinuclear complex 2 were investigated by agarose gel electrophoresis and high-resolution analysis with a 36-bp DNA fragment (Fig. 2). The results obtained are compared with those achieved with cisplatin and the Cu(3-Clip-Phen) complex. Fig. 2Nucleobase sequences of oligonucleotide I (ODN I), oligonucleotide II (ODN II) and the primer used for the binding experiments. The preferential binding sites of cisplatin are indicated by arrowheads Materials and methods Preparation of Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 and 2 All reagents and solvents were commercially available and used without further purification. Cu(3-Clip-Phen) (3) was prepared as previously described [15]. The 1H NMR chemical shifts of standard solvent molecules have been omitted in all redrawn spectra for clarity. 2-(2-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione 3-Clip-Phen (4) (100 mg, 0.22 mmol) was dissolved in 5 mL of dimethylformamide (DMF). One equivalent of N-(2-bromoethyl)phthalimide (5) (56.7 mg, 0.22 mmol) and 1 equiv of N-ethyldiisopropylamine (38 μL, 0.22 mmol) were added to 4. The reaction mixture was heated to 100 °C for 2 days. After the mixture had cooled to room temperature, 10 mL of dichloromethane (DCM) was added, and the organic phase was washed three times with 10 mL of distilled water. After being dried over Na2SO4 and evaporation under reduced pressure, the resulting crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 95:5:0.5), to yield 2-(2-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione (6) as an off-white powder (yield 34%). 1H NMR (DCCl3, 300 MHz) δ 9.05 (dd, 2H, J = 4.32, 1.67 Hz), 8.81 (d, 2H, J = 2.83 Hz), 8.10 (dd, 2H, J = 8.07, 1.62 Hz), 7.72 (dd, 2H, J = 5.43, 3.05 Hz), 7.67 (d, 2H, J = 8.88 Hz), 7.62 (d, 2H J = 8.86 Hz), 7.54 (dd, 2H, J = 6.09, 4.74 Hz), 7.48 (m, 4H), 4.27 (d, 4H, J = 5.18 Hz), 3.84 (t, 2H, J = 6.06 Hz), 3.54 (m, 1H), 3.13 (t, 2H, J = 6.12 Hz) ppm. 13C NMR (DCCl3, 75 MHz) δ 168.4, 153.7, 150.1, 146.0, 142.4, 140.3, 135.73, 133.8, 132.0, 129.3, 127.1, 125.9, 123.0, 122.0, 114.9, 67.3, 55.6, 45.4, 37.8 ppm. Low-resolution mass spectrometry (MS) (electrospray ionization, ESI, more than 0) m/z 621.00 [(M + H)+; calcd for C37H29N6O4+: 621.66], 642.94 [(M + Na)+; calcd for C37H28N6O4Na+: 643.65] Anal. calcd for C37H28N6O4·1.6 H2O: C, 68.42; H, 4.84; N, 12.94. Found: C, 68.34; H, 4.86; N, 13.21. N1-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine Two equivalents of hydrazine (15.6 μL, 0.32 mmol) were added to a solution of 6 (100 mg, 0.16 mmol) in 5 mL of pure ethanol. The mixture was refluxed overnight. The ethanol was evaporated under reduced pressure, and the crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 90:10:1) to give N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine (7) as a light-brown powder (yield 73%). 1H NMR (MeOD-d3, 300 MHz) δ 9.00 (dd, 2H, J = 4.41, 1.63 Hz), 8.84 (d, 2H, J = 2.84 Hz), 8.34 (dd, 2H, J = 8.10, 1.67 Hz), 7.90 (d, 2H, J = 2.86 Hz), 7.83 (m, 4H), 7.65 (dd, 2H, J = 8.10, 3.26 Hz), 4.51 (d, 4H, J = 4.47 Hz), 3.58 (m, 1H), 3.07 (t, 2H, J = 5.64 Hz), 2.97 (t, 2H, J = 5.23) ppm. 13C NMR (MeOD-d3, 75 MHz) δ 155.6, 150.6, 146.3, 143.0, 140.5, 137.6, 131.0, 128.7, 128.2, 127.4, 123.5, 116.7, 69.1, 57.5, 41.9 ppm. Low-resolution MS (ESI > 0) m/z 490.98 [(M + H)+; calcd for C29H27N6O2+: 491.56] Anal. calcd for C29H26N6O2·1.9 CH2Cl2: C, 56.93; H, 4.61; N, 12.89. Found: C, 56.79; H, 5.13; N, 13.23. Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 To a solution of 7 (70.4 mg, 0.14 mmol) in 6.5 mL of DMF was added a solution of 0.8 equiv of K2PtCl4 (47.69 mg, 0.11 mmol) in 3.25 mL of de-ionized H2O. The reaction mixture was stirred for 6 h at room temperature. The off-white precipitate was filtered off and washed with 30 mL of deionized water, 30 mL of MeOH and 20 mL of diethyl ether to give Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 (1) as an off-white powder (yield 42%). 1H NMR (DMSO-d6, where DMSO is dimethyl sulfoxide, 300 MHz) δ 9.04 (d, 2H, J = 2.77 Hz), 8.88 (d, 2H, J = 2.45 Hz), 8.45 (d, 2H, J = 8.00 Hz), 8.05 (br, 2H), 7.95 (m, 4H), 7.70 (dd, 2H, J = 7.99, 4.35 Hz), 4.49 (br, 4H), 4.07 (br, 5H) ppm. 195Pt NMR (DMSO-d6) δ −2,317 (complex), −3,292 (complex with one DMSO coordinated) ppm. IR (neat): ν = 3,049 (br); 1,591 (s); 1,506 (s); 1,424 (s); 1,236 (s); 1,198 (s); 1,102 (s); 1,018 (s); 876 (s), 834 (s), 728 (s), 332 (s), 325 (s) cm−1. Anal. calcd for C29H26Cl2N6O2Pt·DMF: C, 46.33; H, 4.01; N, 11.82 Found: C, 47.54; H, 4.43; N; 11.70; the deviation in calculated percentage C is ascribed to traces of solvent other than DMF. CuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4 CuCl2 (17.0 mg, 0.1 mmol) was added as a solid to a suspension of 1 (75.7 mg, 0.1 mmol) in DMF (25 mL). The reaction was stirred overnight at 50 °C. DMF was partially evaporated under reduced pressure, and the crude compound was precipitated in 100 mL of diethyl ether. The solid material was filtered off and washed with 3 × 20 mL of diethyl ether, and dried overnight at 50 °C under reduced pressure to give 2 as a green powder (yield 93%). X-band electron paramagnetic resonance (solid state): g = 2.1191. UV–vis (H2O) λmax 282 (40,100), 321 (12,500), 333 (10,100), 347 (3,280)  nm. High-resolution MS (m/z): [2M–2Cl] calcd for (C58H52Cl6N12O4Pt2Cu2)2+ 853.00852, 853.5096, 854.0097, 854.5087, 855.0088, 855.5072, 856.0073, 856.5083, 857.0100, 857.5067; found 853.0117, 853.5117, 854.0114, 854.5110, 855.0107, 855.5105, 856.0103, 856.5101, 857.0098, 857.5097. Cytotoxicity The cytotoxicity of complexes 1 and 2 has been determined for breast (MCF7), two glioblastoma (Hs683 and U373), two colorectal (HCT-15 and LoVo) and lung (A549) cancer cell lines (see supplementary material for experimental details and results). Solutions of complexes for experiments with DNA One millimolar DMSO solutions of the complexes investigated were prepared, and were subsequently diluted with Milli-Q water. Nuclease activity on supercoiled DNA One millimolar DMSO solutions of the complexes investigated were diluted to, respectively, 200, 400 and 1,000 nM with Milli-Q water. Five microliters of the complex solution was added to 10 μL of supercoiled ΦX174 DNA (Invitrogen, 7 nM, 40 μM base pairs) in 6 mM NaCl, 20 mM sodium phosphate buffer (pH 7.2), and incubated for 20 h at 37 °C. To initiate the cleavage, 5 μL of a 20 mM mercaptopropionic acid solution in water was added, and the resulting reaction mixture was incubated at 37 °C for 1 h. The reaction was quenched at 4 °C, followed by the addition of 4 μL of loading buffer (glycerol with bromophenol blue) prior to the loading on a 0.8% agarose gel containing 1 μg mL−1 ethidium bromide. The gels were run at a constant voltage of 70 V for 90 min in 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) borate edta (TBE) buffer containing 1 μg mL−1 ethidium bromide. The gels were visualized under a UV transilluminator, and the bands were quantified using a Bio-Rad Gel Doc 1000 apparatus interfaced with a computer. Time-course experiments of DNA cleavage Fifty microliters of the complex solution was added to 100 μL of supercoiled ΦX174 DNA (Invitrogen, 7 nM, 40 μM base pairs) in 6 mM NaCl, 20 mM sodium phosphate buffer (pH 7.2), and the resulting reaction mixture was incubated for 20 h at 37 °C. To initiate the cleavage, 50 μL of 20 mM mercaptopropionic acid was added, and a sample was taken out every 10 min. Four microliters of loading buffer (glycerol with bromophenol blue) was added, and the sample was directly frozen in liquid nitrogen. When all samples had been collected, they were loaded on a 0.8% agarose gel containing 1 μg mL−1 ethidium bromide. Analyses with 5′-32P-end-labeled DNA Oligonucleotides (ODNs) I and II and the primer (Fig. 2) were purchased from Eurogentec, and purified on a 15% polyacrylamide (acrylamide to bisacrylamide ratio 19:1) gel. The denaturating gel contained 7 M urea. The migrations was performed in the presence of TBE buffer at 2,500 V for 3 h. The concentrations of single-stranded ODNs were determined by UV titration at 260 nm [25]. The ODNs were end-labeled with 32P using standard procedures with T4 polynucleotide kinase (New England BioLabs) and [γ-32P]ATP for the 5′-end, before being purified on a MicroSpin G25 column (Pharmacia) [26]. Comparison of the platinum–ODN adducts formed with the different complexes 5′-end-labeled ODN I (2 μM) was annealed to 1 equiv of its complementary strand ODN II in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. Then, 60 μL of this solution was incubated with 60 μL of the complex solution (6 or 20 μM) for 20 h at 37 °C, followed by precipitation with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C before being loaded on the gel. Platinum–DNA adducts were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then by phosphorimagery. Comparison of the sequence-selective binding by primer extension experiments with Taq polymerase ODN I (2 μM) was annealed to ODN II (2 μM) in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. Sixty microliters of this solution was then incubated with 60 μL of the complex solution (6 or 20 μM) for 20 h at 37 °C, followed by precipitation with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. For primer extension, an aliquot of the redissolved samples (0.25 μM) was annealed with 5′-end-labeled primer (0.25 μM) and 1 equiv of ODN I (0.25 μM) in the enzyme buffer (the buffer contains a small amount of reductant, but not enough to induce DNA cleavage by complex 2) before the addition of 250 μM dGTP, dCTP, dATP and dTTP and 2.5 U of Taq polymerase (final concentrations are given, the total volume was 10 μL). One equivalent of unmodified ODN I was added in order to displace the ODN II from the duplex and replace it with the labeled primer. The samples were reacted at 37 °C for 30 min, and 1 μL of edta (0.2 M) was subsequently added. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C, before being loaded on the gel. Five microliters of sample was then analyzed by denaturing 20% polyacrylamide gel electrophoresis and phosphorimagery. The Maxam and Gilbert [27] sequencing scale, including a final scale of T4 polynucleotide kinase digestion to remove 3′-end phosphates, was used to analyze the DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal. Comparison of the cleavage patterns of ODN I–ODN II induced by the copper complexes The 5′-end-labeled 36mer target (2 μM) was annealed to 1 equiv of its complementary strand in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. To 60 μL of this solution was added 60 μL of complex 1, 2 or 3 solutions (20 μM). Some of the samples involving complex 2 were incubated for 19 h, followed by the addition of 1 equiv of CuCl2 per complex and subsequent incubation (1 h). The other samples were incubated for 20 h at 37 °C. Next, all samples were precipitated with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized, then dissolved to 1.33 μM in Tris–HCl buffer (13.3 mM, pH 7.4). For the cleavage experiments, to 15 μL of this solution was added 5 μL of a 0.8 mM ascorbate solution (5 μL of water was added to the controls). The samples were incubated at 37 °C for 1 h, followed by precipitation in 20 μL of sodium acetate buffer (3 M, pH 5.2) containing 1 μg of salmon testes DNA and 180 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. In order to study the DNA cleavage mechanism, additional treatments were performed on some samples: (1) heating at 90 °C in 50 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH buffer (0.1 M, pH 8.0) for 30 min, followed by ethanol precipitation; (2) heating at 90 °C in 50 μL of piperidine (0.2 M in water) for 30 min, followed by lyophilization. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C, before being loaded on the gel. Samples were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then phosphorimagery. The Maxam and Gilbert [27] sequencing scale was used to analyze DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal. Results and discussion Design and synthesis of a heterodinuclear platinum–copper complex A ditopic ligand 7 was designed to favor the simultaneous coordination of a platinum and a copper entity; hence, the resulting bifunctional complex would combine the ability to form a kinetically inert coordination bond with DNA, thanks to its cis-Pt moiety, with the cleavage properties of Cu(3-Clip-Phen). Two coordination compounds, namely, the platinum complex 1 and the heterodinuclear platinum–copper complex 2 (Fig. 1) were prepared with the ligand 7. The binding ability of the platinum unit and the cleavage selectivity of the Cu(3-Clip-Phen) moiety were investigated. The results were compared with those for the known Cu(3-Clip-Phen) complex (3). The short separation between the Pt and the Cu centers in complex 2 most likely affects the preference for platinum to bind in the major groove, and in the minor groove for copper. Accordingly, the coordination of the platinum moiety in the major groove will force the Cu(3-Clip-Phen) component to bind in the major groove, and vice versa. The general synthetic pathway to prepare complexes 1 and 2 is depicted in Scheme 1. The selective and complete platination of the ethylenediamine unit using 1 equiv of K2PtCl4 was monitored by 195Pt NMR and 1H NMR. The in situ reaction of the resulting platinum derivative 1 with 1 equiv of CuCl2 produces the heterobimetallic complex 2. Scheme 1Preparation of complexes 1 and 2. Reagents and conditions as follows: i dimethylformamide (DMF), diisopropylethylamine, 100 °C, 2 days; ii ethanol, H2N–NH2, reflux, overnight; iii K2PtCl4, MeOH/water, room temperature, 6 h; iv CuCl2, DMF, 50 °C, overnight Cleavage of supercoiled DNA The relaxation of supercoiled circular ΦX174 DNA (form I) into its relaxed (form II) and linear (form III) conformations was monitored to compare the aerobic cleavage abilities of complexes 2 and 3, in the presence of a reducing agent (Fig. 3). First, the complexes were incubated for 20 h, to allow the formation of platinum–DNA adducts. The nuclease activity was subsequently initiated by the addition of 5 mM mercaptopropionic acid. Complex 2 exhibits a markedly higher nuclease activity than Cu(3-Clip-Phen) (3) (compare lanes 5 and 9 in Fig. 3). Indeed, at complex concentrations of 100 nM, most supercoiled DNA has reacted to form circular and linear DNA in the case of 2, while Cu(3-Clip-Phen) only generates a small amount of form II (Fig. 3, lanes 4 and 8). Moreover, almost all the DNA has reacted to form smaller DNA fragments (migrating as a smear) at a complex concentration of 250 nM for 2 (Fig. 3, lane 5), whereas no smear is observed for Cu(3-Clip-Phen) (Fig. 3, lane 9). Interestingly, at a concentration of 100 nM for complex 2, form III is detected before the total disappearance of form I (Fig. 3, lane 4) [19]. This result indicates that the heterodinuclear platinum–copper complex is able to perform direct double-strand cuts since form I is still detected in the reaction. Such double-strand breaks are highly cytotoxic, since the cells have difficulties to repair such damage [28, 29]. Cu(3-Clip-Phen) is only able to perform repetitive single-strand cuts [15]. Interestingly, the cleavage activity of complex 2 is also more efficient compared with that of the platinum–copper complexes previously reported by our group [19]. Fig. 3Comparative experiments of the oxidative cleavage of ΦX174 plasmid DNA performed by 2 and 3. Lane 1 control DNA. Lane 2 250 nM solution of 2. Lane 3 50 nM 2, in the presence of 5 mM mercaptopropionic acid (MPA). Lane 4 100 nM 2, in the presence of 5 mM MPA. Lane 5 250 nM 2, in the presence of 5 mM MPA. Lane 6 250 nM 3. Lane 7 50 nM 3, in the presence of 5 mM MPA. Lane 8 100 nM 3, in the presence of 5 mM MPA. Lane 9 250 nM 3, in the presence of 5 mM MPA Time-course studies of DNA cleavage by complexes 2 and 3 were carried out to further investigate the direct double-strand cleavage event (Fig. 4). Complex 3 at a concentration of 250 nM generates a maximum of 80% of circular DNA (form II) after a reaction time of about 1 h [19]. Around 20% of linear DNA (form III) is produced via the action of 3 after 70 min. Remarkably, the formation of form III is only observed after a reaction time of 30 min when already 60% of form II has been produced. For a 150 nM solution of complex 2, linear DNA (form III) is generated from the initial stages of the cleavage reaction, with supercoiled DNA (form I) still being present (Fig. 4). Also, only a maximum of 60% of form II is generated. These features reflect the ability of 2 to perform direct double-strand breaks, most likely as a result of the binding of the platinum moiety to DNA, allowing the copper part to achieve more than one oxidative cleavage in the close proximity of the platinum coordination site. Fig. 4Time-course experiments of DNA cleavage (20 μM in base pairs) over a period of 70 min. Complex 2 (150 nM) was incubated for 24 h, followed by activation with 5 mM MPA and air Analysis of the platinum adducts on ODN I of the ODN I–ODN II duplex target High-resolution analyses with a 36-bp DNA duplex (ODN I–ODN II) were performed to investigate the coordination of the platinum component of the bifunctional complexes to DNA, (Fig. 5a). The sequence of this duplex was chosen to include GG and AG sites (which are the two major binding sites of cis-Pt(II) complexes) on one strand (ODN I). The results obtained after incubation of the complexes with the duplex labeled on the 5′-end of ODN I for 24 h were analyzed by polyacrylamide gel electrophoresis under denaturing conditions and are shown in Fig. 5a. Denaturing conditions allowed the analysis of the Pt–DNA adducts on ODN I of the duplex. Fig. 5a Polyacrylamide gel electrophoresis (PAGE) analysis of the Pt–ODN I adducts of the ODN I–ODN II duplex target (1 μM). ODN I was 5′-end-labeled with 32P-phosphate. The complexes were incubated with the DNA for 24 h before analyses. Lane 1 ODN I. Lane 2 3 μM cisplatin. Lane 3 10 μM cisplatin. Lane 4 3 μM 1. Lane 5 10 μM 1. Lane 6 3 μM 2. Lane 7 10 μM 2. b Phosphorimager data of a DNA sequencing gel comparing the sequence specificity of cisplatin, 1 and 2. All the samples were extended using Taq polymerase, starting from the 5′-end-labeled primer. Lane 1 blank experiment; Lane 2 3 μM cisplatin; Lane 3 10 μM 1. Lane 4 10 μM 2. It is noteworthy that the GTA and GGAC sites give the sequence of the opposite strand that induced the stopping of the primer extension (Figs. S1, S2). c Nucleobase sequence of ODN I, and indication of the damage sites induced by cisplatin, 1 and 2. Large arrows and small arrows represent, respectively, major and minor stop sites Molecules able to irreversibly bind to DNA will retard the rate of migration of the modified ODN, compared with that of the free ODN; therefore, ODN–Pt adducts appear as retarded bands on gels. The incubation with 3 equiv of cisplatin clearly reveals an impeded mobility of the ensuing cisplatin–ODN I adduct (Fig. 5a, lane 2). Only traces of free ODN I were detected; 89% of the ODN I had been modified. The incubation of the DNA duplex with 10 equiv of cisplatin results in a total conversion of ODN I (Fig. 5a, lane 3). Complex 1 shows the formation of Pt–ODN I adducts (Fig. 5a, lanes 4 and 5). Two distinct bands are clearly observed when 10 equiv of complex 1 is incubated with the DNA target, indicating the formation of Pt–ODN I adducts. The quantification of free ODN I reveals that as much as 84% of this DNA fragment has reacted. The use of complex 2 leads to comparable results, with the conversion of 88% of the initial ODN I (Fig. 5a, lane 7). However, the reaction between ODN I and complex 2 produces a smear (i.e., a range of products) on the gel. Our previously reported compounds showed reduced formation of Pt–ODN adducts under similar experimental conditions compared with complexes 1 and 2 [19]. Sequence selective binding of complexes 1 and 2 compared with cisplatin Primer extension experiments were performed to investigate the sequence-selective binding of the platinum units to the ODN I fragment of the ODN I–ODN II DNA duplex (Figs. 5b, c, S1, S2). The platinum complexes that did not react with DNA were removed prior to the start of the primer extension experiments. Nevertheless, it is possible to have more than one platinum moiety coordinating the ODN strand, since the concentrations of cisplatin, complex 1 and complex 2 were, respectively 3 and 10 times higher than the concentration of the ODN I–ODN II DNA duplex. Taq polymerase has proven to be a valuable tool for the determination of the sequence selectivity of various platinum complexes [23, 30–39]. Cisplatin inhibits the enzymatic polymerization at the anticipated GG and AG sites (Fig. 5b, c), but the majority of cisplatin is detected at the GG site. It should be noted that once the ODN I strand contains two Pt adducts on both the GG and AG site, the enzyme stops only at the GG site. Therefore, it is possible that the amount of modified AG sites is underestimated. Nevertheless, not all of the ODN I has been modified by the complexes (Fig. 5a). It is therefore reasonable to say that the majority of the ODN I contains only one Pt adduct and that the AG site is indeed the minor site of interaction. The stop sites observed for complex 1 are also located for a major part at the GG base pairs and for a minor part at the AG base pairs. Complex 2 shows only stops at the GG binding site. These results indicate that the platinum moiety interacts with its preferential binding site. The difference between cisplatin and complexes 1 and 2 is the precise point at which the peak intensity occurs at the damaged site. The stops of the Taq polymerase are mainly located at the A base before the GG site for cisplatin, complex 1 and complex 2. However, the enzyme stops induced at the GG site are more equally distributed among the GAC site in the cases of complexes 1 and 2 (Fig. 5b, c; the size of the arrows is an indication of the damage intensity). The binding of complexes 1 and 2 apparently produces bulky adducts, thus allowing a partial stop at the second nucleotide before the classic position, at the GG adduct, since an increase of the reaction time with the enzyme (from 30 to 120 min, Fig. S3; complex 1) induces a bypass of the stop at the C base (associated with a decrease of the peak intensity when compared with that for G). The difference between cisplatin and complex 1 is more pronounced at the AG site. Although it is the minor binding site, the damage induced by cisplatin almost exclusively takes place at the G base, at the 5′-end of ODN I. The Taq polymerase stops for complex 1 are mainly observed at the A base. Cleavage of the ODN I strand of the ODN I–ODN II duplex The cleavage of the ODN I–ODN II duplex with complexes 1–3 was investigated by polyacrylamide gel electrophoresis, the target being labeled on the 5′-end of ODN I (Fig. 6). To the best of our knowledge, studies of such bifunctional complexes have been reported only twice [40, 41]. Fig. 6a PAGE analysis of cleavage of ODN I of the ODN I–ODN II duplex (1 μM) by compounds 1–3 (10 μM when unspecified). The cleavage reactions were initiated with ascorbate (200 μM) in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine. The Maxam–Gilbert sequencing reactions A + G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On the top of the gel are indicated the conditions used during the experiments (details are given in “Materials and methods”). Lanes 3–6 are for controls without complexes. Lanes 17–22 are for experiments that were performed in the presence of 1 μM 3. b High-contrast picture of lanes 8, 10, 11, 13, 15 and 16 in a allowing easier observation of the the cleavage pattern. ΔpH 8 was a heating step of 30 min at 90 °C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH buffer (0.1 M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage Complexes 1 and 2 were preincubated for 20 h to allow the coordination of the platinum moiety to the DNA target. For complex 1, the preincubation was subsequently followed by the coordination of 1 equiv of copper, since the copper-free complex does not show any nuclease activity. The noncoordinated complexes were removed by precipitation before the cleavage was initiated by the addition of ascorbic acid (0.2 mM) under aerobic conditions. Additional treatments (HEPES pH 8.0 and piperidine) were carried out. The noncovalent interaction of Cu(3-Clip-Phen) (3) with DNA permits its abstraction from the ODNs by a simple precipitation step. A tenfold excess of 3 (compared with the ODNs) does not generate cleavage products after the precipitation step (Fig. 6, lane 24), while 1 equiv of 3 without precipitation exhibits significant cleavage (Fig. 6, lane 20). The platinum-containing complexes 1 and 2 form kinetically inert bonds with DNA, and therefore cannot be removed from the ODN during the precipitation step. In contrast, the unreacted complexes are eliminated. As expected, the cleavage pattern of 3 is nonspecific, [18] as about 20% of the target is oxidized at all nucleotides (Fig. 6, lane 20). The cleavage achieved by complex 2 in the presence of ascorbic acid results in the full and partial disappearance of, respectively, the Pt–ODN I adducts and the ODN I band (Fig. 6, lane 10). The total cleavage amounts to nearly 80%, and its pattern is different from the features resulting from the action of 3, but no clear sequence selectivity is observed. Accordingly, a smear of products is noted in contrast to 3, suggesting that the mechanism of the cleavage is different. This observation may be explained by either of the following possibilities: (1) some cuts are due to platinum adducts of complex 2 positioned on the other strand of the duplex (ODN II); (2) the extensive cleavage observed most likely indicates that the ODN I fragment experiences more than one cut; therefore, complex 2 should have been released after repetitive cuts from ODN I and should be able to cut the free ODN. To verify this hypothesis, the experimental conditions were adjusted to introduce fewer platinum adducts onto the target (20% against the 80% of modification of ODN I shown in Fig. 6), and less ascorbate (100 μM) was used to limit the recleavage events. However, the resulting cleavage pattern appears to be similar to that observed with the former conditions (Fig. 8). One equivalent of CuCl2 was added to complex 1, after the formation of Pt–DNA adducts, to analyze the cleavage selectivity of the resulting compound. A comparatively less efficient cleavage was achieved using complex 1 with extra added CuCl2, when compared with complex 2 (compare lanes 10 and 15 in Fig. 6), possibly as a result of the partial coordination of the copper ions to 1. Similarly to 2, the cleavage pattern obtained with 1 is found to be significantly different from that of 3 (Fig. 6, lane 20). Interestingly, the free ODN I is not affected during the cleavage process, as only the ODN fragments containing platinum adducts are cleaved. Furthermore, a sequence-selective cleavage (associated with the formation of fragments of ODN I including probably the 3′-phosphate end, since they comigrated as Maxam and Gilbert sequencing fragments) is observed in the close vicinity of the GG and AG sites (indicated as black bars in Fig. 6, left). The intensity of the bands is much stronger at the GG site, reflecting the results of the primer extension experiments previously obtained. The four base pairs neighboring the GG site in the 5′-direction are also affected by the cleavage of complex 1. In the 3′-direction, no clear bands are observed, but a smear is observed, which can be explained as follows: the cleavage products remain coordinated to the complex and have therefore a totally different mobility, compared with the Maxam–Gilbert sequencing fragments. The apparent smear observed in the 3′-direction supports this assumption, since such behavior is observed for Pt–ODN I adducts including the copper complex (Fig. 5a, lane 6), and can therefore be expected for cleavage fragments that coordinate to the complex. A heating step in HEPES buffer (90 °C; pH 8.0) is often used to cleave the metastable products resulting from the oxidation of deoxyribose. This treatment of the cleavage products obtained with complexes 1–3 did not show a strong increase of the DNA cuts (Fig. 6, respective comparisons between lanes 15, 16 and 10 and 11, 20 and 22). Therefore, products of strand cleavage are essentially observed during this analysis. However, the smears observed in lanes 10 and 15 are partially converted into bands, indicating that some cleavage products, arising from the oxidation of deoxyribose, are sensitive to alkaline conditions. More drastic alkaline heating (as with piperidine) allows one to detect the oxidations of the nucleobases that do not induce direct DNA cleavage [6]. Surprisingly when this treatment is applied to complexes 1 (with addition of 1 equiv of copper) and 2, clear cleavages are observed, although the systems had not been incubated with ascorbate (Fig. 6, lanes 13 and 8, respectively). This phenomenon is dependent of the presence of copper ion, since it is not observed for Pt–ODN I adducts with complex 1 without the addition of CuCl2 (results not shown). Therefore, these alkaline conditions are sufficient to induce a redox activity of the copper complex part of the hybrid molecule [this phenomenon has not been observed with Cu(3-Clip-Phen), which can be removed during a precipitation step preceding the heating in the presence of piperidine]. Interestingly, only the Pt–ODN I adducts are cleaved; none of the free ODN I is degraded and selective cleavages are observed around the position of adducts detected during primer extension experiments. Unfortunately, this activity of the copper complexes covalently linked to the target does not allow us to analyze whether or not the dual Pt–Cu complexes perform the oxidation of nucleobases. Cleavage of the ODN II strand of the ODN I–ODN II duplex The same experiments were performed with the ODNI–ODNII duplex labeled on the 5′-end of ODN II (Fig. 7). Similar amounts of Pt–ODN II adducts are formed with complexes 1 (Fig. 7, lane 8) and 2 (Fig. 7, lane 3). Most likely, only parts of the platinum components of complexes 1 and 2 are bound on ODN II at the preferential AG site, and the cleavages do not seem to be restricted to one selective site. Other adducts are probably also present. The occurrence of platinum–guanine monoadducts on ODN II (which is particularly rich in guanine bases) may explain the results observed. Further investigations are required to confirm these proposals. Fig. 7PAGE analysis of cleavage of ODN II of the ODN I–ODN II duplex (1 μM) by compounds 1–3. The cleavage reactions were initiated with ascorbate (200 μM) in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine. The Maxam–Gilbert sequencing reactions A + G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On top of the gel are indicated the conditions used during the experiments (details are given in “Materials and methods”). ΔpH 8 was a heating step of 30 min at 90 °C in HEPES–NaOH buffer (0.1 M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage Both complex 1 with added copper and complex 2 show extensive cleavage activities in the presence of ascorbic acid in air (Fig. 7, lanes 11 and 6, respectively). In the experiments with complex 2, the Pt–ODN II adducts have fully reacted, while the free ODN II is only partly altered. The cleavage fragments are poorly resolved on the gel (a smear is essentially observed), and treatment with HEPES or piperidine fails to improve their analysis. These cleavage fragments are composed of modified DNA. It can be reasonably proposed that the fragments include Pt–ODN II adducts and that these adducts produce a smear during the migration in polyacrylamide gel electrophoresis. All these results indicate that the platinum moieties of complexes 1 and 2 bind, as expected, to the preferential platinum site on ODN II, but also and mainly to single A and G nucleobases. Comparison between the cleavage of duplex and single-stranded DNA A comparison between the cleavage patterns resulting from the action of complex 2 on single-stranded ODN I and on the duplex ODN I–ODN II was made to appraise the influence of the formation of Pt–ODN II adducts on the resulting cleavage (Fig. 8). Pt–ODN I adducts are formed when the single strand is used, and these adducts are essentially positioned at the GG site of ODN I (the results of the primer extension are not shown). For this study, only 20% of the platinum adducts on ODN I of the duplex and 40% of the platinum adducts on the single strand were created in order to have a maximum of one adduct per DNA target, and thus to simplify the analysis. The cleavage was induced by the addition of a small quantity of ascorbate (100 μM) to disfavor recleavage events. Fig. 8Phosphorimager scanning of the PAGE-cleavage patterns of ODN I by complex 2. The cleavage was performed on either the ODN I–ODN II duplex (5′-end-labeled on ODN I) or on single-stranded ODN I. Cleavage was induced by the addition of ascorbate (100 μM) after the removal of the unlinked complex during a precipitation step with ethanol. The clear band area on the 3′-side of the GG site of the duplex is probably due to a covalent adduct of complex 2 on ODN II (the complementary strand of the duplex). 3′-Phosphoglycolate cleavage fragments are labeled with an asterisk. The other clear bands were attributed to fragments of ODN I including 3′-phosphate ends The cleavage of the ODN I fragment of the duplex results in a cleavage pattern exhibiting various moderate peaks in the 3′-region of the GG base pair of ODN I. The cleavage of single-stranded ODN I by complex 2 gives rise to a smear in the same region. The smears observed characterize cleaved ODN I products that are still coordinated to complex 2. Accordingly, the weak peaks in this region of the DNA sequence are most likely due to the cleavage of complex 2–ODN II adducts on both strands. Since the conditions used favor a maximum of one Pt–ODN adduct per duplex, these cleavages on nonmodified ODN I probably originate from complex 2–ODN II adducts. The results are summarized in Fig. 9. Importantly, the cleavage of ODN I by Pt–ODN II adducts appears to be partially responsible of the visible nonselective cleavage of the duplex by complex 2. Fig. 9Summary of cleavage events due to platinum–copper dual complex 2 on double-stranded DNA. The labeled strand of the DNA duplex that can be observed from denaturing PAGE experiment is labeled with an asterisk Interestingly, the weak peaks observed on the 5′-side of the cleavage fragments (and shown with an asterisk in Fig. 8) probably correspond to DNA fragments with 3′-phosphoglycolate extremities. Indeed, these species are observed at the position of the cleavage products of the duplex produced by the action of complex 3, and which have been previously characterized [15]. Such fragments were earlier identified as the result of the oxidation of the C4′ position of 2-deoxyribose. Further investigations are required to confirm these proposals. Concluding remarks The novel heterodinuclear platinum–copper complex 2 exhibits improved nuclease activity, compared with its parent compound Cu(3-Clip-Phen) (3), and is able to perform double-strand breaks. The platinum moiety acts as an anchor to DNA, forcing the Cu(3-Clip-Phen) group to generate cuts in close proximity to the Pt–DNA adducts, hence even allowing direct double-strand breaks. Mechanistic investigations on a 36-bp DNA fragment (ODN I–ODN II) revealed that platinum adducts are indeed formed with both complexes 1 and 2. The platinum moiety of complexes 1 and 2 binds to GG (primarily) and AG sites, like cisplatin. Nevertheless, the Taq polymerase enzyme stops at different base pairs for cisplatin and complexes 1 and 2. This feature suggests that complexes 1 and 2 induce different distortions (compared with cisplatin) upon their binding to DNA, most likely owing to the bulkiness of the 3-Clip-Phen moiety. Complex 1 with added copper (1 equiv) shows a sequence-selective cleavage, in the close proximity of the platinum adducts. A sequence-selective cleavage is also observed for complex 2, but to a lesser extent compared with complex 1. The opposite strand also contains Pt–DNA adducts, but no clear sequence selectivity is observed, most likely owing to the lack of preferential platinum binding sites. These atypical platinum adducts lead to the partial damage of the other DNA strand, as is clearly evidenced by comparison of the cleavage products on a single-stranded or duplex DNA. Interestingly, it has been found that piperidine is also able to activate the Cu(3-Clip-Phen) component of the bifunctional platinum–copper complexes, a feature so far not reported. Thus, a good sequence-selective cleavage is observed upon treatment of complex 1 with piperidine. Reasonable sequence selectivity is achieved with complex 2. The investigation of the mechanism of cleavage by complex 2 is currently in progress. Electronic supplementary material Below is the link to the electronic supplementary material. ESM1 (PDF 89.7 kb)

Int_J_Colorectal_Dis-4-1-2386752

Impact of transanal endoscopic microsurgery on functional outcome and quality of life

Introduction Transanal endoscopic microsurgery (TEM) is a minimal invasive technique for local excision of rectal tumours. The procedure is performed via a rectoscope with a diametre of 4 cm. The aim of this prospective study was to assess both functional outcome and quality of life after TEM. Introduction Local excision of benign rectal tumours is safer compared to radical surgery and considered treatment of choice [1–3]. Several techniques have been developed for local excision, with the transanal technique according to Parks as the most frequently used [1, 4]. Other techniques used are the dorsal transcoccygeal approach (Kraske) and the dorsal transsphincteric approach (York–Mason) [5–9]. Each procedure has its own (dis)advantages, and none of the procedures mentioned is able to achieve local excision of tumours throughout the entire rectum. Transanal endoscopic microsurgery (TEM) demonstrated to be a safe procedure capable of overcoming this shortcoming. In early publications, even distal sigmoid tumours could be locally excised with excellent results. Moreover, recurrence rates are minimal compared to other local techniques. As a result, the indication for local excision of rectal tumours has expanded dramatically [10–13]. Few studies have addressed functional outcome following TEM, and with the operation rectoscope with a length of 12 or 20 cm and a diametre of 40 mm, scepticism towards post-operative faecal continence remains. In manometric studies after TEM, there seems to be a detrimental, temporary impact on internal sphincter functioning, although without clinical significance [14–16]. Cataldo et al. [17] recently performed a prospective study on faecal continence and incontinence-specific quality of life after TEM using standardised surveys. They stated TEM does not result in significant alterations. These results are promising, especially with a relative short duration of follow-up of 6 weeks in this study. As known from other types of rectal surgery, incidence of faecal incontinence diminishes with time [18]. This could imply results after TEM may even improve with longer follow-up. Quality of life is increasingly recognised as the ultimate endpoint when assessing clinical outcomes after different surgical interventions because it measures the patient’s perspective. The precise impact of the TEM procedure on quality of life has not been well studied. This prospective study was set out to provide a comprehensive insight into the impact of TEM on functional outcome and quality of life. Patients and methods Between January 2004 and January 2006, a consecutive series of 50 patients were referred for a TEM procedure. All patients were evaluated pre-operatively according to a standard protocol including rigid rectoscopy, tumour biopsy and endorectal ultrasound. If TEM was considered feasible, patients were eligible for this study. Informed consent had to be given before inclusion. Local medical ethical committees approved this study. A full-thickness excision was always performed. The portion of the tumour located within the sphincter musculature was excised in partial thickness. Before and at least 6 months after the TEM procedure, patients were asked to fill out a questionnaire to assess anorectal functioning and quality of life. All data were collected by an independent research coordinator not previously involved in the patients’ care. We recorded the demographics, operative details, post-operative length of stay, post-operative complications and functional outcome for each participant. We evaluated functional outcome by means of a detailed questionnaire based on the Faecal Incontinence Severity Index (FISI) [19]. This system, developed by Rockwood, uses two basic components: the type of incontinence and its frequency. FISI scores range from 0 (total continence) to 61 (complete incontinence to solid stool on a daily basis). We used the validated weighting scores that are based on patients’ input. Quality of life was evaluated using both the EuroQol EQ-5D and the Faecal Incontinence Quality of Life (FIQL) score. The EuroQol EQ-5D consists of a so-called index score representing the societal value of the health state and has a scale ranging from 0 (no quality of life) to 100 (optimal quality of life). The EuroQol EQ-5D also uses a visual analogue scale, the EQ-VAS, representing the patient perspective. This scale ranges from 0 (no quality of life) to 100 (optimal quality of life). The EuroQoL EQ-5D scores were compared with a sex- and age-matched, community-based sample of healthy persons without co-morbidity [20]. The FIQL score as described by Rockwood et al. [21] measures specific quality of life issues expected to affect patients with faecal incontinence. This instrument is composed of 29 questions within four domains: lifestyle issues, coping–behaviour, depression and self-perception and embarrassment. The scores in the FIQL range from a minimum score of 1 to a maximum of 4, for all of the scales (1 = quality of life alteration present most of the time, 4 = none of the time). Data are presented as medians and standard deviations. Changes within groups were evaluated using the nonparametric one-sample Wilcoxon’s signed-rank test. Comparison of these changes between groups was conducted using the Mann–Witney U test. The Spearman’s correlation coefficient was used for correlation between the different findings. A P-value of ≤0.05 was considered statistically significant. Results Fifty patients were eligible and informed consent was obtained. Three patients were excluded. In two patients, TEM could not be performed because of bulky tumour or technical problems. An additional patient underwent low-anterior resection for locally recurrent disease within 6 months of the TEM. The remaining 47 patients completed both questionnaires and were included for analysis. All of these patients were alive without evidence of recurrent disease. The group consisted of 22 males and 25 females. Median age was 67 years (range 40–84). Pre-operative diagnosis was villous adenoma in all patients. Median distance from the distal tumour margin to the dentate line was 7 cm (range 0–15 cm) and median tumour size was 20 cm2 (range 4–53 cm2). The median rate of captured circumference of the rectal wall was 40% (range 5–80; Table 1). Median operative time, defined as beginning when the rectoscope was inserted and ending when it was removed, was 55 min (range 10–140). Complications developed in 4 of 47 (8.5%) patients. Two patients had urinary retention, one patient a urinary tract infection and one suffered from a low hemoglobin rate requiring blood transfusion. No reoperations were necessary and mortality rate was zero. Median length of stay was 4 days (range 3–9 days; Table 2). Definite histopathological examination of the resected specimens revealed an adenoma in 44 patients and an invasive carcinoma in three patients (pT1 in two patients and pT2 in one patient). These three patients were reluctant to undergo major surgery and were observed with rectoscopy and endorectal ultrasound every 3 months without signs of recurrence at 6 months after TEM. In three adenomas, excisional margin was considered microscopic irradical, resulting in 94% of tumours being radically excised. Table 1Patient and tumour characteristicsCharacteristicsNumberMale/female (N = 47)22/25Median age in years (range)67 (40–84)Median distance from dentate line in centimetres (range)7 (0–15)Median tumour size in square centimetres (range)20 (4–53)Median capture of circumference of rectal wall in per cent (range)40 (5–80)Table 2Procedure-related characteristicsCharacteristicsNumberMedian duration of operation in minutes (range)55 (10–140)Complications4/47 (8.5%)Urinary retention2Urinary tract infection1Blood transfusion1ReoperationsNoneLength of hospital stay in days (range)4 (3–9) Six months after surgery, mean FISI scores were found to be decreased (pre-operative, 10; post-operative, 7 (p  < 0.01)), depicting an improvement in faecal continence (Fig. 1). Overall, when pre-operative and post-operative FISI scores were compared, 24 patients improved, 16 patients were unchanged and seven deteriorated. Operation time or tumour size did not influence the change in FISI score. There was a significant correlation between the decrease in FISI score and tumour height (p = 0.02). Reduction of FISI was significantly greater in patients with a tumour location within 7 cm from the dentate line (p = 0.01; Table 3). Mean scores and ranges of the EuroQol EQ-5D are presented in Table 4. Mean general quality of life score from the patients’ perspective (EQ-VAS) was found to be significantly higher 6 months after TEM (p < 0.02). The observed changes in EQ-VAS showed no correlation with the post-operative alterations in FISI scores or tumour characteristics. Mean pre-operative EQ-VAS score in our group was lower compared to the mean EQ-VAS score of the sex- and age-matched general population (p = 0.02). Post-operative EQ-VAS score was comparable with the general population. Mean index score (social perspective) remained the same (p = 0.09). Both pre- and post-operative EQ-5D index scores were similar to those of the sex–age-matched general population. Comparing the change from baseline in FIQL scores, a statistically significant improvement was observed in two of the four domains (embarrassment; p = 0.03 and lifestyle; p = 0.05). The domains of lifestyle, coping and behaviour and embarrassment were correlated with the FISI (all p < 0.05; Table 5). Overall, EQ-5D and FIQL scores were not affected by age and gender of the patients. Surgical aspects and tumour characteristics did not influence the outcome. Fig. 1Mean Faecal Incontinence Severity Index (FISI) scores (±SEM) before and after Transanal Endoscopic Microsurgery (TEM)Table 3Mean FISI scores; numbers in parentheses are standard deviationsFISI scorePre-operativePost-operativeStatistical significanceOverall10 (2)6 (2)p < 0.01Duration of operation <55 min (N = 24)9 (4)7 (3)p = 0.24Duration of operation >55 min (N = 23)12 (3)4 (2)p = 0.17Tumours <7 cm from dentate line (N = 21)16 (5)5 (2)p = 0.01Tumours >7 cm from dentate line (N = 26)6 (2)7 (3)p = 0.43Median tumour size <20 cm2 (N = 27)12 (4)6 (3)p = 0.12Median tumour size >20 cm2 (N = 20)8 (3)6 (3)p = 0.32Lower values indicate better anorectal functioningTable 4Mean EuroQoL EQ-5D scores; numbers in parentheses are standard deviations Control groupPre-operativePost-operativeStatistical significanceEQ-VAS82 (7)77 (14)82 (11)p = 0.02Index score86 (6)84 (11)89 (9)p = 0.09EQ-VAS represents the patients’ perspective on quality of life; index score represents the societal value on quality of life. Higher scores indicate higher quality of life. Both scores are compared with a healthy sex- and age-matched control group.Table 5Mean FIQL scores; numbers in parentheses are standard deviationsFIQLSPre-operativePost-operativeStatistical significanceLifestyle3.7 (0.3)3.9 (0.3)p = 0.05Coping3.6 (0.5)3.8 (0.4)p = 0.10Depression3.7 (0.3)3.9 (0.4)p = 0.08Embarrassment3.1 (0.3)3.7 (0.4)p = 0.03Higher scores indicate higher quality of life. Discussion In rectal adenomas, TEM has emerged as the procedure of choice because of its safety and low local recurrence rates. Especially compared to radical surgery, TEM has proven its safety [22, 23]. However, possible adverse effects of TEM have to be addressed. The use of a rectoscope with a 4-cm diametre, introduced transanally, has lead to substantial scepticism regarding impact on anorectal functioning. In earlier studies, we already showed TEM to be superior to total mesorectal excision regarding post-operative defecation disorders, although this did not result in improved quality of life [24]. In the present study, TEM resulted in improved faecal continence as measured by the FISI. This apparent paradox may be attributed to pre-operative tumour symptoms such as mucinous or bloody discharge, prolapse, tenesmi and/or urge, giving rise to incontinence-like symptoms. Post-operative improvement of continence was most significant in tumours within 7 cm from the dentate line but disappeared in our study in tumours above 7 cm from the dentate line. Kreis et al. [25] performed manometric studies after TEM and found a significant reduction in anal resting pressure 1 year post-operative and a temporary reduction in anal squeezing pressure, resulting in a temporary rise in urge–incontinence. Kennedy et al. [26] found a significant reduction in anal resting pressure 6 weeks after TEM. This reduction was significantly correlated with duration of the procedure, but mean continence score was not changed after TEM. Both of the above studies however did not use validated questionnaires on faecal continence, and therefore comparison with our study is difficult. Cataldo et al. [17] reported on the impact of TEM on functional outcome and incontinence-specific quality of life using the same questionnaires. No significant alteration was found in faecal continence after TEM. The discrepancy between both studies may be explained by the relative short interval between the TEM procedure and post-operative questioning of 6 weeks in the Cataldo series. Also, in his study, indications for TEM were heterogeneous which may have influenced results. The positive effect of TEM on faecal continence in our series may be explained by the differences in pre-operative FISI score between both studies (10 versus 2.4), depicting more continence problems among the patients in our series. Another explanation may be the differences in tumour distance from the dentate line (present series median 7 cm, Cataldo series 11 cm). Also, in our series, tumours were larger (median 20 cm2 versus 8.75 cm2). Because tumours were larger in our series, more extensive resections were performed, often in tumours located within the sphincter apparatus. These latter resections were already shown to influence rectoinhibitory reflex, reflex sphincter contraction, rectal sensitivity and compliance [16]. Further analysis within our series upon this issue showed only tumour distance from the dentate line of less than 7 cm to be a significant contributing factor. These results however are based upon low number of patients and therefore solid conclusions cannot be drawn. Although in our study TEM resulted in a significant improvement in continence, the post-operative FISI was still worse compared to the Cataldo series (7 versus 2.4). Regarding quality of life, Cataldo found TEM was of no significant influence. In our series, mean general quality of life score from the patients’ perspective, EQ-VAS, was significantly higher after TEM. This improvement could not be explained by improved FISI scores but probably by lower pre-operative EQ-VAS scores as compared to healthy controls. Another explanation may be the rejoice phenomena, that is, patients are relieved the tumour has been excised, and in most cases an adenoma was found [27]. However, because of the low number of invasive carcinomas in our series this is purely theoretical. The societal value of general quality of life, EQ-5D, remained unchanged. Measuring quality of life using the FIQL questionnaires resulted in a significant improvement in two of the four FIQL domains (embarrassment and lifestyle). Moreover, the domains of lifestyle, coping and behaviour and embarrassment were all significantly correlated with the FISI. In conclusion, how are these results to be interpreted? This study supports the hypothesis that rectal tumours give rise to incontinence-like symptoms, especially in low-lying rectal tumours. After the tumour is excised using the TEM technique, faecal continence improves. TEM itself does not improve continence but also does not deteriorate faecal continence. Mean quality of life from the patients’ perspective following TEM is improved. Based on, as we know, the only two studies addressing anorectal functioning and quality of life after TEM in one study, it can be concluded that TEM does not impair faecal continence. Also, quality of life is not negatively influenced by the TEM procedure itself, and therefore TEM is the procedure of choice in all rectal adenomas.

J_Headache_Pain-4-1-2245994

Treatment of migraine attacks based on the interaction with the trigemino-cerebrovascular system

Primary headaches such as migraine are among the most prevalent neurological disorders, affecting up to one-fifth of the adult population. The scientific work in the last decade has unraveled much of the pathophysiological background of migraine, which is now considered to be a neurovascular disorder. It has been discovered that the trigemino-cerebrovascular system plays a key role in migraine headache pathophysiology by releasing the potent vasodilator calcitonin gene-related peptide (CGRP). This neuropeptide is released in parallel with the pain and its concentration correlates well with the intensity of the headache. The development of drugs of the triptan class has provided relief for the acute attacks but at the cost of, mainly cardiovascular, side effects. Thus, the intention to improve treatment led to the development of small CGRP receptor antagonists such as olcegepant (BIBN4096BS) and MK-0974 that alleviate the acute migraine attack without acute side events. The purpose of this review is to give a short overview of the pathological background of migraine headache and to illustrate the mechanisms behind the actions of triptans and the promising CGRP receptor blockers. The trigemino-cerebrovascular system Pain-sensitive structures such as the intracranial vessels and the meninges, especially the dura mater, are supplied with sensory nerve fibers. In fact they are innervated by the ophthalmic ramus of the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion. Those neurons project onto second order sensory neurons in the trigmenial nucleus caudalis (TNC) in the brain stem and its related extensions down to the C2-level called the trigeminocervical complex [1]. From this region a signal is transmitted to the ventroposterior thalamus leading to activation in cortical areas, including frontal cortex, insulae and cingulate cortex. This results in the experience of pain (Fig. 1). In addition, a migraine active region has been pointed out in the brainstem by using positron emission tomography (PET) [2–4]. Thus, an involvement of the trigemino-cerebrovascular system in the transmission of nociceptive information to the central nervous system is a viable position. Fig. 1In migraine attacks, sensory nerves, which innervate the cerebral blood vessels, are activated to antidromically release calcitonin gene-related peptide. They originate from the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion (TG). Those neurons project onto second order sensory neurons in the trigeminal nucleus caudalis (TNC) from where a signal is transmitted to the cortex The trigemino-cerebrovascular reflex There is a dense supply of CGRP-containing nerve fibers originating in the first division of the trigeminal ganglion that innervate intracranial vessels. Experimental studies have shown that induction of vasoconstriction provoked rapid normalization of the vascular tone due to––in part––reflex activation of the trigeminal nerve. Vasoconstriction triggers an antidromic release of sensory trigeminal neuropeptides, mainly CGRP, being a strong intracranial dilatator [5, 6]. After denervation there is no alteration in the contractile response of the vessel but the time to attain initial basal tone is markedly prolonged. This is of importance as headache may be initiated by a spreading wave of depression of cortical neurons resulting in contraction of some parts of the cerebral circulation. This phenomenon has now been shown in man [7] and could activate the trigeminal vascular system to release CGRP to reestablish the vascular tone. Thus, the trigemino-cerebrovascular reflex is probably part of a counter-balancing system, which is able to mediate dilatation of brain vessels and sustain cerebral blood flow [8]. This may assure the maintenance of local brain blood flow within normal limits as the cerebral circulation requires high and constant flow. Activation of the neurons in the trigeminal ganglion leads on the other hand to an activation of neurons in the trigeminocervical complex that project to the central nervous system (CNS) and mediate the central aspects of pain [1, 9]. Calcitonin gene-related peptide CGRP is a neuropeptide consisting of 37 amino acids and is predominantly expressed in the nervous system. It originates from the calcitonin gene encoding calcitonin and CGRP depending upon alternative splicing. Histochemical studies of cerebral perivascular nerve fibers and human trigeminal ganglia revealed that almost half of the cell bodies contain CGRP [10, 11]. Although many other signal molecules are located in the trigeminal ganglion, CGRP is the most abundant in man [12] and can be seen as a marker for trigeminal activity. Furthermore it is the only transmitter reliably released in acute migraine attacks at present. There was a marked increase in CGRP levels during migraine attacks, but no changes could be observed for other neuropeptides such as vasoactive intestinal peptide (a marker for parasympathetic activity), neuropeptide Y (sympathetic activity) and substance P (sensory activity) when their levels were analyzed in the cranial venous outflow during migraine attacks [8, 13]. In migraine attacks elicited within clinical studies by administration of nitroglycerin a good correlation has been described between increased levels of CGRP and the intensity of the headache [14, 15]. But it is noteworthy to mention that nitroglycerin elicits head pain, but not always genuine migraine attacks. There may be a need for the subject to be in a “prone phase”, where the trigemino-cerebrovascular system might be hyper-reactive as it was demonstrated in cluster headache [16]. Triptans The increased cranial venous levels of CGRP observed in conjunction with migraine attacks thus indicate activation of the trigeminal system. Administration of sumatriptan––belonging to the highly effective anti-migraine drugs called triptans or 5-HT1B/1D agonists-results in alleviation of the headache and normalization of the cranial venous CGRP levels [8]. They are thought to act mainly as powerful vasoconstrictors of cerebral vessels via 5-HT1B receptors, which have been shown to be expressed in the medial smooth muscle cell layer of the human middle meningeal [17] and cerebral arteries [18]. In addition, triptans may bind to presynaptic 5-HT1B/1D receptors on trigeminal afferents to inhibit nerve activity and hence reduce CGRP release. 5-HT1B/1D receptors were found on trigeminal sensory fibers [19], trigeminal ganglion cells [20] and in the trigemino-cerebrovascular system in man [21]. Furthermore, the 5-HT1B/1D receptors were co-localized with CGRP in the human trigeminal ganglion [20]. Autoradiographic studies using labeled zolmitriptan confirmed the presence of 5-HT1B/1D receptors in certain brain stem nuclei that are involved in cranial nociceptive processing [22] and are likely to be activated during migraine. Thus, it can be presumed that a potential relaxation of intracranial blood vessels during migraine attacks can be normalized by triptans, which activate the cerebrovascular 5-HT1B receptors and thereby cause vasoconstriction. By stimulating 5-HT1B/1D receptors, triptans inhibit the release of CGRP from the perivascular nerve terminals and the trigeminal ganglion cells. Activation of 5-HT1B/1D receptors at the level of the CNS (trigeminal nucleus caudalis) may interrupt central aspects of the headache process. Recently the relationship between pharmacokinetic and pharmacodynamic aspects of triptans in migraine was investigated. It has been observed that subcutaneous administration of sumatriptan is more effective than oral application. The difference in efficacy is most likely due to the rapid initial rise in plasma level after subcutaneous administration [23]. Furthermore migraine attacks may influence gastric stasis leading to delayed absorption of drugs [24, 25]. Thus, to increase efficacy of treatment, attention should be paid to the development of migraine drugs, which can be quickly absorbed. Triptans cause sometimes cardiovascular adverse effects as they also act as constrictors in coronary arteries. But triptans are considered as more powerful vasoconstrictors in human isolated cerebral arteries than in coronary arteries. They are at least threefold more potent [26]. The reason for the higher potency and efficiency of triptans in intracranial arteries could be due to at least three differences. First there is a higher number of 5-HT1B receptors in those vessels compared to peripheral arteries [18, 27]. Secondly there are few, if any, 5-HT2A receptors in intracranial vessels whereas the 5-HT2A receptor population dominates in coronary arteries [27]. Thirdly, there might be potential differences in coupling of the 5-HT1B receptors between cerebral and coronary arteries. In consideration of the fact that triptans are weak constrictors in coronary arteries [28, 29] and have little impact on coronary blood flow, the clinical consequences in healthy subjects are minor. However, small contractions may anyhow have proportionally greater impact in diseased artery with luminal narrowing. Triptans are hence contra-indicated in patients with known cardiovascular disease. Sensitization in migraine Treatment with triptans achieves good results in patients suffering from sensitization. Sensitization is defined as an increased afferent activity in response to an unchanged stimulus [30] and both peripheral and central sensitization may play a role in migraine attacks. Peripheral sensitization of meningeal perivascular nociceptors arising from trigeminal neurons can clinically explain phenomena like intracranial hypersensitivity (worsening pain during physical activity) and the throbbing element in migraine pain. This may be due to normally innocuous meningeal nociceptors becoming hyper-responsive to rhythmic fluctuation in intracranial pressure that is produced by normal arterial pulsations [31, 32]. Central sensitization of nociceptive second order neurons in the trigeminocervical complex can be the reason for extracranial tenderness and cutaneous allodynia. The latter is an abnormal sensory state in which otherwise innocuous stimuli are sensed as painful [31]. Two-thirds of patients develop allodynia during or even after a migraine attack [33]. Based on clinical studies new guidelines for triptan treatment in migraine has been established. They include that allodynia-free patients can receive good results from triptan treatment at any time during the migraine attack. Furthermore, patients suffering from allodynia can expect excellent results from triptans before allodynia gets established. Therefore they should take triptans as early as possible during the attack. After the onset of allodynia, migraineurs can still benefit from triptans, but pain relief may be incomplete [34–36]. However, a slightly different view has been expressed in a recent study [37]. After subcutaneous administration of sumatriptan, there was no difference in alleviation of migraine pain irrespective if the drug was given early or late. Therefore, it was suggested that the sensitization is of minor importance and that the relatively poor effect of sumatriptan late in migraine attacks is primarily due to gastric stasis [37]. CGRP receptors Local vasodilatation and release of CGRP from the vascular nerve fiber endings––caused by electrical field stimulation or capsaicin treatment [38, 39]––are attenuated by administration of triptans, but also by application of CGRP receptor blockers [40]. The proof that CGRP plays a key role in migraine was provided by demonstrating that the potent CGRP receptor antagonist olcegepant (BIBN4096) could abort acute migraine attacks to a comparable degree as sumatriptan [41, 42]. In addition, CGRP receptors are located at several sites, which play an important role during migraine, including the cerebrovasculature [43–45], the trigeminocervical complex within the brainstem [46] and the trigeminal ganglion [47]. Functional CGRP receptors consist of three components, namely a seven-transmembrane domain G-protein coupled receptor called calcitonin receptor-like receptor (CLR), a receptor activity-modifying protein (RAMP) and a receptor component protein (RCP). CLR shares 55% sequence identity with the calcitonin receptor. Co-expression of CLR with RAMP1 composes the CGRP receptor, while co-expression of CLR with either RAMP2 or RAMP3 represents the adrenomedullin (AM) receptor or possibly a combined receptor (CGRP and AM). Apart from contributing to the receptor specificity, RAMP1 is required for glycosylation and transport of CLR to the plasma membrane [48–50]. In addition, RAMP1 seems to be functionally rate limiting for CGRP receptor activity in the trigeminovascular system. It was proposed that elevated RAMP1 expression could sensitize the trigeminal ganglion of individuals to CGRP actions such as CGRP release and neurogenic inflammation [47]. The third component of the CGRP receptor, the accessory protein RCP, is required for proper biological function as it is involved in coupling the receptor to downstream signaling pathways like the protein kinase A (PKA) pathway [51]. Activation of CGRP receptors in cultured trigeminal ganglion cells increased the cAMP levels, which in turn led to elevated CGRP promoter activity and increased CGRP mRNA levels. These results point to an autoactivation of CGRP expression and indicate that elevated release of CGRP in the trigeminal system may create a self-sustaining feedback loop [47]. CGRP receptor antagonists olcegepant and MK-0974 The excellent correlation between CGRP release and migraine headache has long pointed towards the potential usefulness of a specific CGRP receptor antagonist in the treatment of primary headaches [9, 52]. Thus, a series of small molecule CGRP receptor blockers were developed. Among those is olcegepant, which demonstrates extremely high affinity for human CGRP receptors, even higher affinity than the endogenous ligand [53]. Therefore it was selected for further pharmacological investigations. Olcegepant was shown to be a pure antagonist for the human CGRP receptor and to be selective against a broad panel of enzymes and receptors such as calcitonin, amylin or adrenomedullin receptors [54]. In isolated human temporal, middle meningeal and cerebral arteries olcegepant effectively inhibits CGRP induced dilatation [44, 55]. That may be due to the block of the CGRP receptor as mRNA for CLR and RAMP1–3 has been found in human middle meningeal arteries, cerebral arteries as well as in microvessels [56] and CGRP receptor components could be detected in the human cerebral vasculature [43]. In addition, olcegepant dose-dependently inhibited vasodilatation induced by electrical stimulation of the trigeminal ganglion in primates [54]. One major advantage of a CGRP receptor blocker is the lack of vasoconstrictor ability. But blockade of the receptor of a strong vasodilatation involves a theoretical risk of causing both peripheral and cerebral vasoconstriction, especially as high affinity binding sites for CGRP have been reported in the heart, liver, spleen, intestine, urogenital system, skeletal muscles, and the skin [57–59]. But olcegepant in doses up to 10 mg did not cause any clinical relevant changes of vital signs like blood pressure and pulse rate, in the ECG or in forearm blood flow in healthy volunteers [60]. Detailed studies of peripheral arteries such as mesenteric and coronary arteries [44, 61] revealed that those arteries show weaker responses to CGRP and lower degree of antagonism by olcegepant. This is possibly related to a lower number of CGRP receptors. In addition, a study with seven healthy volunteers disclosed that olcegepant had no influence on cerebral blood flow or on the blood flow velocity in the middle cerebral artery [62]. It was concluded that CGRP receptor blockade has no effect on the cerebral circulation in man. However, it is still not clear where olcegepant acts as a CGRP antagonist. It has been reported to inhibit (1) dilatation of dural arteries initiated by CGRP [63], (2) CGRP-induced hypotension [40], and (3) increase in facial blood flow after trigeminal ganglion stimulation [54]. In contrast, olcegepant did not significantly inhibit changes in tone of cerebral arterioles or of local cortical cerebral blood flow [40]. Those results indicate that olcegepant poorly penetrates the blood-brain barrier, but is very effective in preventing vasodilatation of vessels without this feature [64]. Thus, this CGRP receptor blocker was suggested to exert its antagonistic effects mainly extracerebral, which correlates with results from a study of healthy volunteers [65]. In contrast, it was recently shown that CGRP-induced relaxation of the rat middle cerebral artery can only be inhibited by abluminally administered olcegepant as olcegepant was prevented from reaching the CGRP receptors by the arterial endothelium [66]. These results suggest that the anti-migraine effect of this CGRP receptor blocker is on the abluminal side of the blood-brain barrier. This is supported by the fact that olcegepant only has an antimigraine effect in high doses. Anyhow, olcegepant was effective in a dose of 2.5 mg in the treatment of acute migraine attacks [41]. The overall treatment effect revealed a responder rate (a moderate to severe headache going to mild or no headache) of 60% at 2 h after the start of the infusion compared with 27% in the placebo group (Fig. 2). Thus, intravenous olcegepant was found to be as efficacious as oral sumatriptan in the relief of acute attacks of migraine [42]. Fig. 2The anti-migraine effect of the CGRP receptor antagonists MK-0974 and olcegepant and the triptans rizatriptan and sumatriptan compared to placebo at 2-h pain relief. The overall treatment effect of MK-0974 (p = 0.015) and rizatriptan (p = 0.010) showed significance versus placebo (left part) [69]. A dose of 2.5 mg of olcegepant also showed significant superiority over placebo (p = 0.001) [41]. Data of sumatriptan from another study [42] were added to demonstrate the similar efficacy of olcegepant compared to triptans Significant superiority over placebo was seen regarding the pain-free rate at 2 h, the sustained pain relief at 24 h and the improvement of nausea, photophobia and phonophobia (Fig. 3). The response was still rising at 4 h pointing at a long duration of action. At 24 h the pain-free rate was better than with triptans, suggesting a lower grade of rebound. Fig. 3Comparison of the sustained pain free-rate at 24 h of the CGRP receptor antagonists MK-0974 and olcegepant, the triptans rizatriptan and sumatriptan, and placebo. MK-0974 displayed superior efficacy versus placebo (p < 0.001) [69]. In order to compare the efficacy of olcegepant to a triptan, data of sumatriptan from another study [42] were added The rate of recurrence was 19% as compared to a placebo rate of 46%. This is clearly lower than the recurrence rate of triptans which usually is around 28% [42]. Safety and tolerability studies also showed that olcegepant is well tolerated. Adverse events were, in general, minor and dose-dependent. Transient mild paresthesia was the single most frequent adverse event [60]. One major disadvantage of olcegepant remains in its structure. It is a hydrophilic molecule, has poor penetration across the blood-brain barrier and is not suitable as an oral drug. Considering the crucial advantages of an oral formulation of a drug, a small-molecule, non-peptide CGRP antagonist called MK-0974 was recently developed for the acute treatment of migraine attacks [67]. MK-0974 was, among others, tested in a noninvasive pharmacodynamic model in rhesus monkeys, in which endogenous CGRP release and a vasodilatatory response are achieved by topical application of capsaicin to the forearm of the rhesus monkey [68]. Infusion of different CGRP receptor antagonists inhibited the increase in local dermal blood flow and showed effectiveness with MK-0974 being the most potent among them (>3.5-fold) [67]. In addition, MK-0974 displayed a low clearance (7.0 mL min−1 kg−1) and a good i.v. half-life (2.8 h). The compound was finally chosen as a clinical candidate, when it showed >10,000-fold selectivity in assays including more than 160 receptors, transporters, and enzymes [67]. Recently, MK-0974 has been tested in a randomized, double-blind, placebo-and active-controlled, outpatient study with a two-stage, dose-ranging design to test safety and effectiveness [69]. Three hundred and thirty patients suffering from migraine with and without aura were treated with MK-0974 (in doses of 25–600 mg), rizatriptan (10 mg) or placebo. As doses below 300 mg were insufficient, they were discontinued. Thus, the overall treatment effect was defined as the average of the 300, 400, and 600 mg doses and showed significance vs. placebo on the primary endpoint (p = 0.015), which was defined as pain relief at 2 h after treatment (Fig. 2). MK-0974 displayed also superior efficacy vs. placebo for secondary endpoints such as pain freedom at 2 h (overall treatment effect p < 0.001), sustained pain relief at 24 h (overall treatment effect p < 0.001) or sustained pain freedom at 24 h (overall treatment effect p < 0.001) (Fig. 3). In addition, MK-0974 provided relief of migraine-associated symptoms such as photophobia, phonophobia and nausea. Rizatriptan (10 mg) was used as an active control and demonstrated effectiveness vs. placebo (p = 0.010) on the primary endpoint defined as pain relief at 2 h. By comparison, 300 and 600 mg doses of MK-0974 appeared to work as well as rizatriptan or showed even numerical superiority regarding the sustained pain freedom at 24 h and the sustained pain relief at 24 h. Concerning tolerability and safety, MK-0974 was generally well-tolerated. The reported side effects were mainly nausea, dizziness and somnolence, but the incidence of adverse experiences seemed to be comparable to the placebo group. In addition, there was no increase in adverse events with increasing dose. Thus, MK-0974 showed only mild side effects, but significant relief of migraine pain 2 h after treatment compared to placebo and the relief was sustained through 24 h [69]. Conclusion The novel CGRP receptor antagonists appear to be effective in the treatment of moderate and severe migraine attacks. They do not only provide pain relief at 2 h, but also show efficacy regarding the secondary endpoints such as pain freedom, improvement of associated symptoms and functional disability [41, 69]. In addition, they show a prolonged effect of action compared to the triptans. That may suggest not only less rebound headache but perhaps also a prophylactic possibility. Furthermore, only minor adverse effects were reported and no cardiovascular side effects could be seen so far. This is probably due to the absence of vasoconstrictor properties and it also suggests that vasoconstriction is not necessary to abort acute migraine attacks. This lack of direct vasoconstrictor activity may represent an important advantage over the triptans. Further development of drugs against the CGRP receptor will offer a new promising way of treatment.

Plant_Cell_Rep-4-1-2413081

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. Introduction Poinsettia, Euphorbia pulcherrima Willd. Ex Klotzsch, is a contemporary symbol of Christmas in most parts of the world. Since it was introduced to the United States in 1825 from Mexico, poinsettia has become the primary potted flower produced and sold in North America, Europe, Asia and Australia (Ecke et al. 2004; Williams 2005). Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the Australian region as poinsettia becomes more popular each year (Williams 2005). Global production of poinsettia has exceeded hundreds of millions and is still expanding, indicating its economic and market potential for the floral industry. Genetic engineering is an important tool for breeding ornamental plants with addition of desirable traits such as novel colour, better quality and resistance to pathogens and insects (Mol et al. 1995; Deroles et al. 2002; Hammond 2006; Hammond et al. 2006). This technology has been successfully utilized in the production of a number of important ornamental crops e.g. blue roses (Yoshikazu 2004), novel carnations (http://www.florigene.com), transgenic gladiolus (Kamo et al. 1997) and improvement of chrysanthemums (Teixeira da Silva 2004). To date, transgenic ornamentals of over 30 genera have been produced by different transformation approaches (Hammond 2006; Hammond et al. 2006). However, there are only a few reports describing genetic transformation of poinsettia: one was the US patent 7119262 (Smith et al. 1997) using the biolistic transformation approach, while the other two were electrophoresis-based transformation attempts (Vik et al. 2001; Clarke et al. 2006). Biolistic transformation requires the use of a gene gun device (Sanford et al. 1987) and tends to generate transformants with a high transgene copy number, complex transgene loci and unpredictable silencing of the transgene (Herrera-Estrella et al. 2004). Electrophoresis of DNA into meristems on a living plant was described as a simple method to generate transformants by avoiding tedious tissue culture work and was utilized in producing transgenic orchid (Griesbach 1994). However, no stable transgenic poinsettia was ever produced using electrophoresis, regardless of the strong transient expressions that were detected in both studies (Vik et al. 2001; Clarke et al. 2006). Thus, Agrobacterium-mediated transformation for poinsettia was developed in the present study. Poinsettia mosaic virus (PnMV) is a single-stranded, positive-sense RNA virus (Bradel et al. 2000) that belongs to the family Tymoviridae (Dreher et al. 2005). Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season (Fulton and Fulton 1980), which in turn decreases the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. An additional problem is that PnMV-free poinsettia tends to be rapidly reinfected, although no vector is known (Blystad and Fløistad 2002; Siepen et al. 2005). There is therefore a need for a new and effective alternative approach, like Agrobacterium-mediated transformation, which can overcome these difficulties. RNA silencing is a mechanism by which transcription or translation of a gene is suppressed. It is known to occur in plants, fungi and animals (Fire et al. 1998; Waterhouse et al. 1998; Baulcombe 2005). It is triggered by double stranded RNA (dsRNA) molecules (Meister and Tuschl 2004), which are subsequently recognized and cleaved by the host-encoded endoribonuclease dicer (Bernstein et al. 2001) into small interfering RNA (siRNA) molecules of 21–26 nucleotides in length (Hamilton and Baulcombe 1999). These siRNAs, in conjunction with the RNA-induced silencing complex (RISC), target RNA molecules with homologous sequences for sequence-specific degradation (Hammond et al. 2000; Bernstein et al. 2001; Tabara et al. 2002). In plants, RNA silencing can be achieved by genetic transformation with gene constructs that express highly transcribed sense, anti-sense or self complementary hairpin RNA (hpRNA) containing sequences homologous to the target gene (Smith et al. 2000; Wesley et al. 2001; Helliwell and Waterhouse 2003). RNA silencing has been efficiently used to generate resistance against plant viruses in many plant species including ornamentals (Metzlaff et al. 1997; Tenllado et al. 2004; Bucher et al. 2006; Hammond et al. 2006). In this study, we report the development of an A. tumefaciens-mediated transformation method for poinsettia, which has never previously been described for this plant species. This method could facilitate the improvement of poinsettia by introducing new traits into existing commercial poinsettia cultivars in order to meet market demands. Using this approach, in combination with RNA silencing technology, transgenic PnMV resistant poinsettia plants carrying PnMV-derived hpRNA constructs were produced for the first time. Materials and methods Plant materials Euphorbia pulcherrima, poinsettia cv. Millenium, plants were kindly supplied by the J. Kristiansen nursery, Grimstad, Norway, in the year 2000. The original stock plants were subjected to heat therapy to eliminate PnMV (Fløistad and Blystad, unpublished). PnMV-free cv. Millenium cuttings were grown in the greenhouse under a photoperiod of 16 h light and 8 h dark with a temperature of 22°C. Internode stem explants from 8 to 10-week-old cv. Millenium plants were used for somatic embryogenesis and A. tumefaciens-mediated transformation. Somatic embryogenesis of poinsettia cv. Millenium Internode stem explants 5–15 mm long from cv. Millenium plants were excised and used in the establishment of somatic embryogenesis for poinsettia prior to transformation. In Experiments 1 and 2, the explants were surface sterilized for 10 min in 3% NaOCl and then rinsed three times with sterilized deionized and autoclaved H2O for 5, 10 and 15 min according to Preil (1994). Because of a large number of infections encountered in Experiments 1 and 2, the stem explants were sterilized in Experiment 3 using 70% ethanol for 1 min followed by 5 min with 1% NaOCl and 3 rinsings with sterile deionized and autoclaved water for 3, 10 and 20 min, a modification of the protocol of Preil (1994). The modified protocol was repeated twice before being utilized in Experiment 3 and also in subsequent transformation experiments. After sterilization, stem segments of ca. 1–1.5 mm thickness were placed on callus induction (CI) medium [MS medium (Murashige and Skoog 1962) supplemented with 0.2 mg l−1 BAP and 0.2 mg l−1 CPA and 30 g l−1 sucrose, Table 1] for 8–10 days. Embryogenic calli were then transferred to somatic embryo induction (SEI) medium (MS medium contains 0.3 mg l−1 NAA and 0.15 mg l−1 2iP and 30 g l−1 sucrose, Table 1) for somatic embryogenesis. The somatic embryos emerged after an average of 12 weeks were then transferred to somatic embryo maturation (SEM) medium (MS basal medium containing 0.05 mg l−1 BAP and 30 g l−1 sucrose, Table 1). Shoots and plantlets derived from somatic embryos were subsequently cultured on root induction (RI) medium consisting of 1/2 strength MS, 2 mg l−1 IAA and 20 g l−1 sucrose or on hormone free 1/2 strength MS (HFMS) medium supplemented with 20 g l−1 sucrose for root induction (Table 1). Plants with well-developed roots were transferred to soil and grown in the greenhouse at 22°C. The above culture conditions and media compositions for somatic embryogenesis of cv. Millenium were developed according to Preil (1994) with a number of modifications described above. Detailed information regarding media compositions is summarized in Table 1. Table 1Media names and compositions modified according to Preil (1994)MediumMSSucrose (%)CPA (mg l−1)BAP (mg l−1)NAA (mg l−1)2iP (mg l−1)IAA (mg l−1)CIFull strength30.20.2–––SEIFull strength3––0.30.15–SEMFull strength3–0.05–––RI1/2 strength2––––2HFMS1/2 strength2–––––CI Callus induction medium, SEI somatic embryo induction medium, SEM somatic embryo maturation medium, RI root induction medium, HFMS hormone free MS medium for root induction Light conditions were 23 μE m−2 s−1 for callus and somatic embryos and 30 μE m−2 s−1 for plantlets in RI and HFMS media with a 16 h photoperiod. Temperature was constant at 24°C. A light microscope connected to a digital camera was used to follow the development of the somatic embryos. Agrobacterium strain and hairpin (hp) RNA constructs The disarmed A. tumefaciens strain LBA4404 (Hoekema et al. 1983; Invitrogen, California, USA) was utilized throughout the study. Three hpRNA constructs, named as pCP, pR2 and pR3, were generated. Construct pCP targeted the viral coat protein (CP), whereas constructs pR2 and pR3 targeted two distinct regions within the viral RNA-dependant RNA-polymerase (RdRp) (Fig. 1a). Briefly, constructs pCP, pR2 and pR3 were generated by amplifying the corresponding fragments from the viral genome and introducing the appropriate restriction sites. The following restriction sites were introduced: XhoI·KpnI (for sense orientation) and ClaI·XbaI (for antisense orientation) for R2 and R3 fragments, and XhoI·EcoRI (sense) and ClaI·XbaI (antisense) for CP fragment. The primers used are presented in Table 2. Each amplified fragment was 500 bp long. Subsequently, the fragments were inserted into pHANNIBAL vector (kindly provided by CSIRO Plant Industry, Canberra, Australia) in sense and antisense orientations, interrupted by an intron pyruvate orthophosphate dikinase (pdk) gene (Fig. 1b) as described by Helliwell and Waterhouse (2003). The three expression cassettes were cloned into pART27 (Gleave 1992, kindly provided by CSIRO Plant Industry, Canberra, Australia), a binary plasmid vector, at Not1 site under the control of CaMV 35S promoter and with ocs terminator (Helliwell and Waterhouse 2003), generating hpRNA constructs pCP, pR2 and pR3 (Fig. 1b). For selection, the neomycin phosphotransferase II (nptII) gene conferring kanamycin resistance was used under the control of nopaline synthase promoter (Nos-P) and with Nos terminator (NosT, Fig. 1b) in the binary plasmid expression vector pART27. Sequence analysis was utilized to verify the junctions and orientations in the expression vectors pCP, pR2 and pR3. All three plasmid vectors were introduced into A. tumefaciens strain LBA 4404 by electroporation according to the manufacturer’s instructions (Invitrogen, California, USA). Fig. 1Schematic representation of PnMV genome (a) and hairpin (hp) RNA constructs used in the current study (b). The locations of the primers used to generate the constructs as well as for screening purposes are presented over the PnMV genome. Sequences of the primers are presented in Table 2. The location of the probe used to detect siRNA is presented below the PnMV genome (see “Materials and methods”). CP coat protein region, R2 and R3 RNA-dependent RNA-polymerase (RdRp) regions of the PnMV genomeTable 2Primers used in this studyPrimer nameRestriction sitesSequenceaLocationR2F-XhKXhoI, KpnIctcgagggtaccTTTAGCAAAACGCAGCACAAAATCA4,371–4,395R2R-CXbClaI, XbaIcatcgattctagaTCTCCAGACACCATGATTGGGTG4,848–4,870R3F-XhKXhoI, KpnIctcgagcggtaccTTCGCTTTAAAACAGAAAGCACCA4,939–4,962R3R-CXbClaI, XbaIcatcgatgtctagaGCCTCGTAGCTTGGTTGGGTT5,418–5,438CPF-XhEXhoI, EcoRIctcgaggaattcAACCACGTCGACTCCACTCCAT5,478–5,499CPR-CXbClaI, XbaIatcgattctagaAGCTTGCCGCTCACCAGCAC5,958–5,977aPrimer sequences are presented in the 5′–3′ orientation. Sequences complementary to the viral genome are presented in uppercase letters whereas non-complementary restriction sites included in each primer are presented in lowercase letters. The primers used for screening purposes are identical to the ones presented but do not include the non-complementary restriction sites Transformation of poinsettia stem explants A. tumefaciens strain LBA4404, harbouring the plasmid pCP, pR2 or pR3, was grown overnight in 15 ml liquid LB medium supplemented with 50 mg l−1 kanamycin (Sigma-Aldrich, St Louis, USA) at 28°C with shaking at 200 rpm until an OD600 of 0.6–0.8 was reached. The bacterium suspension was collected at 2,700 rpm for 10 min, washed twice with MS basal medium supplemented with 2% sucrose (MS-2), and resuspended in 10 ml MS-2. The internode stem explants taken from 8 to 10-week-old poinsettia plants derived from cuttings were disinfected as described above, excised into stem segments with 1–1.5 mm thickness and inoculated with Agrobacterium suspension for 5 min with gentle shaking. After the infection, stem segments were blotted briefly with sterile filter paper and placed on CI medium at 24°C in the dark for 72 h without selection. After co-cultivation, the explants were blotted gently on sterile filter paper and transferred to the CI medium with selection and light conditions as described above. Selection and regeneration of transgenic plants After 8–10 days on CI medium supplemented with 500 mg l−1 claforan (Aventis Pharma Ltd, Norway) and 10 mg l−1 kanamycin, the explants were transferred to SEI medium supplemented with 400 mg l−1 claforan and 25 mg l−1 kanamycin for somatic embryogenesis. The protocol for somatic embryogenesis is detailed above. The somatic embryos obtained after about 12 weeks were then transferred to the SEM medium supplemented with 400 mg l−1 claforan and 10 mg l−1 kanamycin. Shoots and plantlets derived from somatic embryos were subsequently cultured on RI medium or on hormone free 1/2 strength MS (HFMS) medium (Table 1) for root induction and were subsequently transferred to soil and grown in the greenhouse at 22°C. Screening of transgenic poinsettia plants Screening of transgenic poinsettia plants was carried out by PCR. The primer pairs used to amplify the CP, R2 and R3 fragments were the same as those for vector constructions, but did not include any additional restriction site (Table 2). HotStarTaq PCR kit purchased from Qiagen (Valencia, California) was used in PCR screening of transformants. Twenty micro litres of reaction mixture containing 2× HotStarTaq Mastermix, 0.4 μM of each primer, 0.1 μg template DNA and H2O were subjected to PCR amplification under the following conditions: 15 min at 95°C (1 cycle), 30 s at 95°C, 30 s at 55°C, 1 min at 72°C (35 cycles) and a final extension 10 min at 72°C (1 cycle) according to the manufacturer’s instructions. All the PCR amplification was performed using Applied Biosystems 96 Thermal Cycler (Applied Biosystems). PCR products were analysed by electrophoresis on 0.8% (W/V) agarose gels. Southern blot analysis To confirm the stable integration of transgenes into the poinsettia genome and to evaluate transgene copy number, Southern blot analysis was performed, basically as described by Sambrook et al. (1989). Total genomic DNA was isolated from young leaves of control plant and the putative transformants using a modified CTAB protocol as described by Rogers and Bendich (1988). Ten micrograms of genomic DNA was digested with the restriction enzyme HindIII for 4 h and separated on a 1% (W/V) TBE agarose gel overnight at 37 V followed by transfer onto Gene Screen Transfer membrane (NEN™ Life Science Products Inc., Boston, MA, USA). The HindIII site is not present in the pCP, pR2 and pR3 vectors. The HindIII site from the pHANNIBAL vector was removed during the vector construction. Membranes were hybridized overnight with 32P-labelled probe of 1.5 kb in size targeting the CP, R2 and R3 regions (Fig. 1a). The probe was generated by amplifying a 1.5 kb fragment that included the R2, R3 and CP regions used to generate the hairpin RNA constructs pR2, pR3 and pCP. This allowed us to use one probe instead of three probes for southern blot analyses of pR2, pR3 and pCP transformants. Northern blot hybridization of low molecular weight RNA Total RNA was extracted using the Trizol reagent (Invitrogen, USA) as recommended by the manufacturer. Low molecular weight (LMW) RNA was separated from high molecular weight RNA by precipitation with 1 volume of 4 M LiCl4 at 4°C overnight. After centrifugation, the LMW RNA-containing supernatant was transferred to a new tube and precipitated with 1 volume of isopropanol. LMW RNA concentrations were measured using a GeneQuant II spectrophotometer (Amersham Biosciences, UK) and RNA quality was verified by agarose gel electrophoresis. LMW RNA (approximately 30 μg) was mixed with 1 volume of Tris-borate–EDTA–urea sample buffer (Biorad, USA) and heated at 100°C for 5 min. Subsequently, LMW RNA was separated on a 15% polyacrylamid tris-borate–EDTA–urea gel and transferred to a Hybond-N membrane (Amersham Biosciences, UK) overnight. The membranes were hybridized with a digoxigenin (DIG)-labelled (Roche Diagnostics, Germany) RNA probe (Fig. 1a), previously cleaved by alkaline hydrolysis to generated fragments of approximately 50 bp as described by Hamilton and Baulcombe (1999). After hybridization the membranes were washed at room temperature, 2 × 15 min in 5× SSC + 0.5% SDS and 2 × 15 min 1× SSC + 0.5% SDS and then exposed to films. Virus inoculation and detection Sap from poinsettia and N. benthamiana plants systemically infected with PnMV was used as an inoculum. Approximately 0.5 g of infected leaf tissue was ground with 4 ml of inoculation buffer [0.03 M sodium phosphate buffer (pH 8)] and mechanically inoculated to leaves of three to four-leaves stage plants lightly dusted with carborundum. Plants were grown in the greenhouse at 22–24°C. Detection of PnMV infection by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out at 6 and 10 weeks post-inoculation basically as described by Clark and Adams (1977). The uppermost fully expanded leaves of each plant were collected in polyvinyl bags (Bioreba, USA). Extraction buffer [100 mM phosphate buffer (pH 7.4), 20 mM Tris, 137 mM NaCl, 3 mM KCl, 2% PVP 24 kD, 0.05% Tween 20, and 0.02% NaN3] was added to the bags (1/10 w/v) and the samples were macerated. A measure of 100 μl of the homogenates were transferred to wells of microtitre plates previously coated with 100 μl of PnMV IgG (Agdia, USA) diluted 1/1,000 in coating buffer [50 mM carbonate–bicarbonate buffer (pH 9.6) and 0.02% NaN3], followed by incubation at +4°C overnight. Microtitre plates were washed with washing buffer [10 mM phosphate buffer (pH 7.4), 140 mM NaCl, 3 mM KCl, and 0.05% Tween 20] and 100 μl of alkaline phosphatase conjugated PnMV IgG (Agdia, USA) diluted 1/1,000 in extraction buffer was added to each well. Microtitre plates were incubated at +4°C overnight. Subsequently, they were washed with washing buffer and 100 μl of substrate buffer [1 M diethanolamine (pH 9.8) and 0.02% NaN3] containing p-nitrophenyl phosphate (0.5 mg ml−1) was added. Absorbance values were recorded at 405 nm with an Expert plus microtitre plate reader using the Kim software (ASYS HITECH, Austria). Values were considered positive when the A450 value was twofold higher than that of the negative control. Results Establishment of somatic embryogenesis for poinsettia cv. Millenium An efficient protocol for somatic embryogenesis of poinsettia cv. Millenium was developed. Stem explants of cv. Millenium responded well to the induction media and a large number of somatic embryos were effectively induced (Table 3; Fig. 2a–d). In Experiments 1 and 2, somatic embryos could only be induced from 21 to 37% of the explants due to infection, whereas more than 75% of the explants produced somatic embryos in Experiment 3 after the modification of the disinfection protocol. The average numbers of somatic embryos on each embryogenic callus varied from 5.3 ± 2.4 and 5.8 ± 3.1 for Experiments 1 and 2, respectively, to 7.2 ± 4.1 for Experiment 3 (Table 3). The highest induction of somatic embryos was found in Experiment 3 with 18 somatic embryos on a single callus. When globular stage somatic embryos (Fig. 2a, arrowed) reached the mature stage (Fig. 2b), they became loosely attached to the calli. Shoots derived from somatic embryos were subsequently cultured on RI medium or HFMS medium (Fig. 2c) for root induction and were transferred into the greenhouse when the roots were well established (Fig. 2d). The three experiments conducted and the efficiency of somatic embryogenesis are summarized in Table 3. Table 3Experiments and efficiency of somatic embryogenesisExperimentNo. of explants% of embryogenic explantsRange of SE/embryogenic explantMean SE/embryogenic explantaNo. of regenerated plantletsb1200212–105.3 ± 2.4702200372–145.8 ± 3.11353350753–187.2 ± 4.1768SE somatic embryosaValues are presented with their corresponding standard deviationbRegenerated plantlets after 14 weeksFig. 2Somatic embryogenesis in poinsettia cv. Millenium: a embryogenic structure and globular stage somatic embryos (arrows) that appeared on the callus (bar 1 mm), b cotyledonary stage of somatic embryos (bar 1 mm), c plantlets deriving from somatic embryos on RIM medium; and d regenerated plants established in the greenhouse The RI and HFMS media used for root induction did not show any clear differences in the percentage (85 and 87% for RI and HFMS, respectively) of plants that developed roots 3 weeks after being transferred to the rooting media. Development of the A. tumefaciens-mediated transformation method Prior to the production of transgenic poinsettia plants, a number of optimizations were carried out to reveal the most suitable conditions for inoculation and co-cultivation, concentration of antibiotic for eliminating A. tumefaciens after co-cultivation, and kanamycin selection without inhibiting somatic embryogenesis and regeneration of plants. Results showed that the optimal inoculation time was 5 min with gentle shaking, while the most suitable co-cultivation time was 72 h (Table 4). Using this combination, 26 out of the 80 (33%) explants tested PCR positive after inoculation, co-cultivation and 2 weeks on claforan (Table 4). Those explants were followed and showed normal embryogenesis and regeneration as compared to the control (data not shown). Therefore, these conditions were utilized throughout all the transformation experiments. When inoculation time exceeded 20 min followed by a co-cultivation period of 6 days, only 4% of the explants survived due to the overgrowth of Agrobacterium on the surfaces of the explants (Table 4). Table 4Number of PCR positive explants that survived 2 weeks after Agrobacterium inoculation and co-cultivation treatments. A total of 80 explants were used in each treatmentCo-cultivation time (days)Inoculation time 5 min10 min20 min29 (11%)13 (16%)21 (26%)326 (33%)21 (26%)20 (25%)610 (13%)8 (10%)3 (4%) For elimination of Agrobacterium growth after co-cultivation, the optimized concentration of claforan was 500 mg l−1 for the CI medium, whereas 400 mg l−1 claforan was an adequate concentration for the SEI and SEM media. For selection of transformed cells, 25 mg l−1 kanamycin was found optimal for the SEI medium, while 10 mg l−1 kanamycin was optimal for the CI and SEM media. Production of transgenic poinsettia plants, molecular analyses and resistance assays To produce transgenic poinsettia plants, 5 transformation experiments including 868 stem segment explants were carried out using the established inoculation, co-cultivation and selection conditions described above. Of those explants, 228 (26.3%) somatic embryo-derived plants were obtained. The results are summarised in Table 5. Table 5Summary of five Agrobacterium-mediated transformation experiments on poinsettia cv. Millenium with pCP, pR2 and pR3 constructsExperimentaNo. of explantsNo. of regenerated plantsNo. of transformantsbTransformation efficiency (%) c1 (CP)1855431.62 (R3)1326932.33 (R3)1726663.54 (R2)2542131.25 (R2)1251832.4aCP, R3, and R2 represent transformation experiments with constructs pCP, pR2 and pR3, respectivelybTransformants verified by PCR and Southern blot analysiscNumber of transformants/total number of explants transformed PCR analysis was conducted for screening the putative transformants. All 228 regenerated plants were analysed by PCR and 18 transformants were revealed. Of these, three possessed pCP constructs, while six and nine contained pR2 and pR3 constructs, respectively (Fig. 3a–c). Transformation frequency varied from 1.2 to 3.5% with an average transformation frequency of 2.1% (Table 5). Fig. 3PCR analysis. PCR positive transformants detected with primer pairs for (a) CP, (b) R2 and (c) R3 fragments respectively. Lane A1 1 kb marker; lane A2 non-transformed plant; lanesA3–5 are independent CP-transgenic lines 11-1, 11-2 and 3-1; laneA6 plasmid control. Lane B1 1 kb marker; lane B2 non-transformed plant; lanes B3-8 are R2-transgenic lines 72-1, 72-2, 75-2, 79-1, 84-2, and 84-4; lane B9 plasmid control. Lane C1 1 kb marker; lane C2 non-transformed plant, lanes C3-11 are R3 transgenic lines 18-1, 30A, 38-1, 40A, 40B, 41-2, 41-5, 56-2 and 62-1; lane C12, plasmid control. The 500 bp band of the 100 bp marker is depicted with an arrow Southern blot analysis was performed on selected PCR positive plants with satisfactory characteristics for studying transgene integration and estimating the transgene copy number. Results of Southern blot analysis confirmed the stable integration of transgene and both single- and multiple-copy transgene integration into the poinsettia genome were detected among the transformants (Fig. 4). Of the eight transformants analysed, six showed single copy integration of transgene (lanes 2–6 and 9 in Fig. 4) with fragment size greater than the length of intact T-DNA (ca 7.5 kb). However, two pR3 transformants (lanes 7–8 in Fig. 4) showed multiple-copy transgene integration with both large (intact T-DNA) and small fragments (partial, non intact T-DNA). Fig. 4Southern blot analysis of selected PCR positive transformants carrying pCP, pR2 and pR3 constructs. The HindIII-digested total genomic DNA was probed with a 1.5 kb probe homologous to the region of CP, R2 and R3 fragments (Fig. 1) allowing us to analyse all the three different types of transformants (CP, R2 and R3 transformants) at the same time. Lane1 plasmid control; lanes2-4 CP transformants 11-1, 11-2 and 3-1; lanes5-6 R2 transformants 72-2, 79-1; lanes 7-9 R3 transformants 38-1, 40B, 18-1; lane10 negative control Furthermore, Northern blot analysis of low molecular weight RNA was carried out on the transgenic plants harbouring constructs pCP, pR2 and pR3, prior to inoculation. Transgene-derived siRNA molecules of 21–26 nucleotides length were detected in transgenic lines pCP 11-1, pCP 3-1, pR2 72-1, pR2 72-2 and pR3 18-1 (Fig. 5). This data indicated that the transgene had been transcribed and converted into siRNAs (Fig. 5). Due to limitations in the amount of LMW RNA from transgenic pR3 lines 40B, 41-2, 62-1 and 41-5, these lines were not included in the experiment. Fig. 5Northern blot analysis of low molecular weight (LMW) RNA to detect small interfering RNAs (siRNA) in non-inoculated transgenic plants and non-inoculated control. aLane 1 control; lanes 2–3 CP transgenic lines 11-1, 3-1; lanes 4–5 R2 transgenic lines 72-1,72-2; lane 6 R3 transformant, 18-1. Ribosomal RNA is presented in (b) to indicate presence of LMW RNA in the gel. Lane 1 control; lanes 2 and 3 PCP transformed plants; lanes 4 and 5 PR2 transformed plants; lane 6 PR3 transformed plant Transgenic plants and non-transformed controls were mechanically inoculated with PnMV at a three to four leaves stage. The upper non-inoculated leaves were tested by DAS-ELISA at 6 and 10 weeks post-inoculation. Results showed that the control plants were systemically infected with PnMV, whereas the tested transgenic lines carrying pCP and pR2 constructs remained virus free (Table 6). However, of the five pR3-containing transgenic lines studied, two lines remained virus free, while the remaining three lines were systemically infected (Table 6). Table 6Immunological detection of PnMV on non-transformed and transformed poinsettia plants inoculated with PnMVConstructTransgenic plantaELISAb6 wpi10 wpiR272-10.10.172-20.10.2R318-10.10.240B0.82.441-20.52.662-10.10.241-50.72.8CP11-10.10.23-10.10.1Control +10.82.820.82.7Control −10.10.220.20.230.20.240.10.1wpi Weeks post inoculationaOne plant per transgenic line was challenged in this assaybELISA absorbance values (405 nm) after 60 min of incubation at room temperature are presented. Samples with twice the value of the highest negative control sample were deemed positive Discussion Significance of genetic engineering in control of virus diseases in ornamentals The application of genetic engineering in the floriculture industry has clearly become instrumental and rewarding in meeting the demand for novel desirable traits. A number of genetic engineering approaches have been developed for ornamental plants (Griesbach 1994; Kamo et al. 1995; Deroles et al. 1997; Zuker et al. 1998; Kamo et al. 2000; Kishimoto et al. 2002; Kim et al. 2004; Teixeira da Silva 2004; Hammond 2006 and the current study) to facilitate the improvement of ornamental crops with better quality and enhanced resistance against diseases and pests. Effective resistance against viruses conferred by transgenes has shown significant potential because no chemical can be applied for control of virus diseases. Regardless of the importance, only a few reports have been published demonstrating the virus resistance obtained in ornamentals through genetic engineering (Kamo et al. 1997; review by Hammond et al. 2006). Furthermore, many of the ornamentals were transformed only with marker genes like gus or selectable antibiotic or herbicide resistant markers (Deroles et al. 2002). Using the Agrobacterium-mediated transformation protocol developed in the current study, we have obtained PnMV resistant transgenic poinsettia lines expressing viral sequences targeting the CP and RdRp regions of the PnMV genome. To our knowledge, no PnMV resistance has previously been obtained using classical or molecular breeding approaches, demonstrating the potential of the transgenic lines produced in the present study for the poinsettia industry. Somatic embryogenesis and Agrobacterium-mediated transformation in poinsettia cv. Millenium One of the reasons that transformation of ornamental plants has lagged somewhat behind that of major crops is the lack of efficient regeneration systems, as these are key components leading to the successful development of transformation protocols. Moreover, both transformation and regeneration can be cultivar- or even genotype-dependent. Therefore, we developed an efficient somatic embryogenesis system for poinsettia cv. Millenium prior to transformation, although a number of somatic embryogenesis systems were reported previously for old cultivars such as Angelika, Franzi and Diamond (Preil and Beck 1991; Preil 1994; Osternack et al. 1999). A recent study on somatic embryogenesis of cv. Freedom Red and Freedom White demonstrated the influence of cultivar and even plant genotype on somatic embryogenesis in poinsettia (Castellanos et al. 2006), indicating the necessity of developing an effective regeneration system for the individual cultivar prior to the development of transformation systems. Poinsettia, like other Euphorbia species, produces latex, a milky solution exuded from cut surfaces. When explants were excised from greenhouse grown poinsettia plants, the latex was released from the cutting points, making surface sterilization difficult. In Experiments 1 and 2, a very low percentage of explants produced somatic embryos due to a severe infection found in the callus induction culture (Table 3). This is likely to have been the result of a combination of latex and insufficient sterilization based on the sterilization procedure described for cv. Angelika, Franzi and Diamond (Preil 1994), which might not be optimal for cv. Millenium (Table 3). A modified protocol was thus used for surface sterilization in Experiment 3 as well as in the five transformation experiments, and the efficiency of somatic embryogenesis was greatly improved (see Table 3), demonstrating the significance of optimization of sterilization and regeneration protocols for the individual cultivar. In addition to the availability of an efficient regeneration system, successful A. tumefaciens-mediated transformation depends on several factors, e.g. inoculation and co-cultivation times with A. tumefaciens, antibiotic selection and regeneration. The optimized infection conditions and co-cultivation times in our study (Table 4) are consistent with the range of published reports on ornamental plants, where 5–30 min infection time and 1–6 days co-cultivation have been reported (Aida et al. 1999; Kishimoto et al. 2002; Cui and Ezura 2003; Kim et al. 2004; Teixeira da Silva 2004). However, in our study, 6 days co-cultivation led to A. tumefaciens overgrowth and explant death, thus differing from the results for Begonia, in which 6 days co-cultivation is required (Kishimoto et al. 2002). These data indicate that co-cultivation times vary among different plant species. Antibiotic selection is another key factor influencing the establishment of a transformation protocol. To eliminate Agrobacterium after co-cultivation, several antibiotics such as cefotaxim (claforan), carbenecillin, and timentin have been used in previous studies. The influence of the type of antibiotic and/or concentration on each transformation system varies greatly (Ishida et al. 1996; Nauerbey et al. 1997; Sunikumar and Rathore 2001). In the present study, optimised concentrations of claforan in the CI, SEI and SEM media effectively eliminated Agrobacterium. For selecting transformed cells, the nptII gene was used. Although selection media containing 50 and 100 mg l−1 kanamycin have been documented in the production of transgenic plants (Kishimoto et al. 2002; Kim et al. 2004; Wu et al. 2005), those concentrations severely inhibited the development of embryogenic calli and somatic embryos in poinsettia. In our study the optimal concentrations of kanamycin were 10 and 25 mg l−1. In the case of Agrobacterium-mediated transformation of Platanus acerifolia, kanamycin at 20 mg l−1 was found effective for the inhibition of regeneration in non-transformed shoots (Li et al. 2007), suggesting the variability of antibiotic selection in each transformation system and the necessity of optimization of antibiotic selection for each plant species. Although transgenic PnMV resistant poinsettias were produced using the Agrobacterium-mediated transformation protocol developed in the current study, the transformation frequency was fairly low (Table 5), suggesting that further improvement is required. This will be carried out in the near future. Southern blot analysis confirmed the integration of transgenes into the poinsettia genome and detected both single- and multiple-copy integration of the transgenes among the transformants (Fig. 4). The majority (six out of eight) of the transformants showed single copy transgene integration with fragment sizes greater than that of the intact T-DNA (ca 7.5 kb), indicating that the intact T-DNA was integrated into the poinsettia genome. However, the bands with sizes around 4–6 kb, smaller than that of intact T-DNA, were also found in the transgenic lines with multiple insertions (lanes 7 and 8 in Fig. 4). According to Afolabi et al. (2004) and Zhu et al. (2006), insertion of non-intact T-DNAs were found in more than 70% of the transgenic rice lines causing 14–21% of the loci to contain only part of the T-DNA, due to T-DNA truncation. Moreover, T-DNA rearrangement is well documented in many plant species including both dicots and monocots (Deroles and Gardner 1988; Puchta et al. 1992; Azhakanandam et al. 2000; Yin and Wang 2000; Rai et al. 2007). Almost 50% of the 27 transgenic rice lines studied showed rearrangement of T-DNA inserts according to Rai et al. (2007). Consequently, such T-DNA truncations and/or rearrangements could also possibly occur in our study causing partial T-DNA insertion. Since Agrobacetrium-mediated transformation of poinsettia has never been described before, there is no available information regarding T-DNA transfer and integration into the poinsettia genome. The hp RNA constructs and PnMV resistance in transgenic poinsettia cv. Millenium A number of effective approaches have been utilized to control viral diseases (Goldbach et al. 2003). Among these, coat protein-mediated and RNA silencing-based resistance have been shown to be efficient transgenic approaches to engineer virus resistance in plants (Powell-Abel et al. 1986; Barker et al. 1998; Waterhouse et al. 1998; Smith et al. 2000; Domínguez et al. 2002; Lu et al. 2003). Furthermore, according to previous studies, hpRNA-induced RNA silencing is more efficient than sense- or antisense-mediated silencing (Chuang and Meyerowitz 2000). In the current study, the establishment of Agrobacterium-mediated transformation enabled us to transform poinsettia plants with intron-containing hpRNA constructs homologous to the PnMV genome. PnMV resistant transgenic poinsettia lines showing no systemic infection were identified after mechanical inoculation as determined using the DAS-ELISA assay (Table 6). The presence of transgene-derived siRNA molecules in non-inoculated transgenic poinsettia plants carrying pCP, pR2 and pR3 constructs, and the absence of these in the non-transformed control plants, indicates that the transgenes were expressed and subsequently cleaved. This data indicates that RNA silencing underlies the PnMV resistance observed in our study (Fig. 5). As mentioned previously, LMW Northern analysis included only one pR3 line (i.e.18-1) due to the limitations in the amount of RNA available for the remaining lines. Therefore, the underlying reason for lack of resistance in some of the pR3-containing transformants (Table 6) needs to be clarified by further studies. Poinsettia is a non-food, non-feed and vegetatively propagated ornamental plant. Pollen mediated transgene outflow is restricted, a significant advantage over sexually propagated plants. The combination of genetic engineering and RNA silencing technologies for the generation of virus-resistant transgenic ornamentals is therefore appealing, especially in an industry in which quality is so important. Transgenic vegetatively propagated ornamentals like PnMV resistant transgenic poinsettia are likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. In summary, we have developed an A. tumefaciens-mediated transformation method for poinsettia. Using this approach, transgenic PnMV resistant transgenic lines expressing PnMV-derived hpRNA constructs were produced. Phytoplasma, the poinsettia branch-inducing factor for good branching (“free-branching”) and for a compact growth habit (Lee et al. 1997), is currently being reintroduced into the transgenic poinsettias. Candidate transgenic lines with PnMV resistance and desired ornamental values will be selected for further study with the future goal of possible commercialization. The methodology developed here could facilitate the future engineering of poinsettia to meet customers’ expectations for new colours and other desirable traits.

Eur_Arch_Otorhinolaryngol-3-1-1914238

Quality of life and functional status in patients with cancer of the oral cavity and oropharynx: pretreatment values of a prospective study

We assessed the pretreatment health-related quality of life (HRQOL) and functional status of patients with advanced oral and oropharyngeal cancer. Eighty patients were investigated. HRQOL was assessed by EORTC QLQ-C30/QLQ-H&N35 questionnaires. Functional status assessment comprised speech and oral function tests. The results revealed a wide range of HRQOL and functional deficits before treatment. HRQOL appeared to be related to some extent to tumor site (patients with oral tumors reported more pain compared to patients with oropharyngeal tumors) and tumor classification (patients with T3–T4 tumors reported more trouble opening the mouth and felt more ill compared to patients with T2 tumors). Comorbidity appeared to have a major impact. Patients with comorbidity had significantly worse scores on several scales/items on both the EORTC questionnaires. Functional deficits were related to tumor site, classification and comorbidity. Patients with oral cavity tumors (versus oropharyngeal tumors), patients with T3–T4 tumors (versus T2 tumors), and patients with comorbidity (versus without comorbidity) scored significantly worse on several speech and oral function tests. Impaired speech and oral function appeared to be clearly related to global quality of life (QLQ-C30) and self-reported speech (QLQ-H&N35). Many patients with advanced oral and oropharyngeal cancer have compromised HRQOL and functional status before the start of treatment. In addition to tumor site and tumor classification, comorbidity appears to have a major impact on HRQOL and functional status. Knowledge of pretreatment HRQOL and functional status levels is useful for better understanding the impact of treatment on these outcomes over time. Introduction The most important outcome for cancer patients is overall survival. However, the disease and its treatment often have a major impact on health-related quality of life (HRQOL) and functional status in patients with head and neck cancer [1]. Therefore, HRQOL and functional status are important aspects to consider in treatment evaluation. To interpret outcome following treatment, it is necessary to assess HRQOL and functional status following diagnosis, but before the start of treatment [2]. Studies on pretreatment HRQOL [2–5] and functional status [6, 7] often include heterogeneous groups of head and neck cancer patients. However, there are substantial differences between patient groups that are related to tumor site and stage [7]. Patients with advanced oral or pharyngeal cancer, for example, often have the poorest HRQOL and functional status [5, 8]. Pretreatment HRQOL among patients with oral or oropharyngeal cancer is often compromised, although it tends to be better compared to HRQOL following treatment [9–11]. There is also evidence that functional status, including oral function, speech and swallowing abilities is significantly deteriorated before treatment [12–15]. Comorbidity is another important factor that varies substantially among subpopulations of patients, and can have a significant influence on the choice of initial treatment, the care that patients receive, and on treatment outcomes [16]. Therefore, comorbidity should be taken into consideration evaluating the HRQOL of patients, both at time of diagnosis, and over the course of treatment [17–19]. The primary objective of the present study is to assess pretreatment HRQOL and functional status in relation to tumor site, tumor classification and comorbidity, in a well-defined group of patients with advanced oral and oropharyngeal cancer. Patients and methods Patients Between January 1998 and December 2001, 92 consecutive patients diagnosed with stage II-IV oral or oropharyngeal squamous cell carcinomas were asked to participate in the study. The planned treatment was composite resection with microvascular soft tissue transfer (i.e., radial forearm free flap) for the reconstruction of surgical defects, and radiotherapy on indication. Exclusion criteria were age greater than 75 years, serious cognitive impairment and lack of basic fluency in the Dutch language. Twelve patients declined to participate, resulting in a final sample of 80 patients (response rate = 87%). All patients were treated at the Department of Otolaryngology/Head and Neck Surgery of the VU University Medical Center, Amsterdam, The Netherlands. Data collection and study measures All data were collected prior to the start of treatment, including sociodemographics (age and gender), disease stage, comorbidity, HRQOL, and functional status. Comorbidity was assessed dichotomously (yes or no) by review of medical records and on the basis of self-report, and was noted if one or more relevant medical ailments accompanied the primary medical illness. The comorbid conditions were cardiovascular, respiratory, gastro-intestinal, renal, endocrine, neurological, and immunological disorders, previous malignancy and considerable weight loss or alcohol abuse. For example, cardiovascular problems such as a myocardial infarct or hypertension, respiratory problems such as restrictive lung disease or COPD, or endocrine disorders such as diabetes mellitus with insulin usage were defined as relevant comorbid conditions. HRQOL was assessed by means of the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (version 2.0) [20] and the EORTC head and neck cancer module QLQ-H&N35 [21]. The QLQ-C30 is composed of multi-item scales and single items assessing five areas of functioning (physical, role, emotional, cognitive, and social), fatigue, pain, emesis, dyspnea, insomnia, appetite loss, constipation, and diarrhea. Overall quality of life and the perceived financial impact of the disease and treatment are also assessed. The head and neck cancer-specific QLQ-H&N35 module comprises seven symptom scales: pain, swallowing, senses, speech, social eating, social contact, and sexuality. There are 11 additional, single items covering problems with teeth, opening the mouth wide, dry mouth, sticky saliva, cough, feeling ill, weight loss, weight gain, use of nutritional supplements, feeding tubes, and painkillers. The scores of both the QLQ-C30 and of the QLQ-H&N35 are linearly transformed to a scale of 0–100, with a higher score indicating a higher (i.e., more positive) level of functioning or global HRQOL, or a higher (i.e., more negative) level of symptoms or problems. The QLQ-C30 data of the patients were compared with published normative data from the general population of Norway [22]. Comparisons were made with the normative subsample that corresponded closely to the age and gender distribution of the patient sample (i.e., age range of 50–59 years and 48% female). The QLQ-H&N35 results were compared with reference values derived from a randomly selected sample of the Swedish general population [8]. Speech and oral functional status Speech analyses were performed according to a standardized speech assessment protocol. Speech recordings of a read aloud text were performed in a sound-treated room and digitized using Cool Edit PRO 1.2 (Adobe Systems Incorporated, San Jose, CA, USA) with 22 kHz sample frequency and 16 bit resolution. Recording level was adjusted for each speaker to optimize signal-to-noise ratio. All recordings were made with a mouth-to-microphone distance of 30 cm. A computer program was developed to perform blinded randomized speech evaluation and to score overall intelligibility, and quality of articulation and nasal resonance. Overall intelligibility was assessed on a 10-point scale ranging from poor to excellent by two trained speech therapists. Scores below six were defined as insufficient intelligibility (according to the Dutch educational system). To obtain more insight into the cause of decreased intelligibility, evaluation of the quality of articulation and nasal resonance was performed by the same two speech therapists on a 4-point scale ranging from deviant (score 1–3) to normal (score 4). Speech rate was measured by calculating words per minute on a read aloud standardized text. Oral function was evaluated by a trained investigator (blinded for the clinical data regarding tumor site and stage) according to a protocol described by Teichgraeber et al. [23]. All assessments were based on 5-point scales ranging from poor to excellent (transformed scores ranging from 0–100). The oral function evaluation included three tests; (1) tongue mobility (mean score of tongue straight out, elevation of tongue tip, elevation of the base of the tongue, tongue deviation left and right, symmetry left and right, and tongue withdrawal), (2) lip mobility (mean score of general movement, spreading and rounding, symmetry left/right, lip closure without speech, and lip closure during speaking), and (3) diadochokinesis (mean score of repetitive motion ability concerning tongue movement left/right, up/down, and in/out, and repeating the syllables /ta/ /cha/, and /ka/). Tongue and lip strength were quantified by a calibrated digital voltameter. To measure tongue strength the patient was asked to push the tongue against a metallic disc with the lips positioned around a cylinder, and to resist the force. To measure lip strength, the patient was asked to keep his lips around a button with a string attached to it on which the examiner pulled. Scores ranged from 0 (no strength) to 0.5 mV (normal strength). Statistical analyses Chi-square tests were used to assess associations between the independent variables tumor site, classification and comorbid condition. Student’s t-tests were performed to test for differences between the patient sample and the normative samples. These tests were based on the mean scores of the study and normative samples and the standard deviations of the study sample only, because no standard deviations of normative samples were available. Student’s t-tests (HRQOL) and Mann–Whitney tests (functional status) were performed to determine the impact of tumor site (oral versus oropharyngeal), tumor classification (T2 versus T3–T4) and comorbidity (yes or no). Spearman correlation coefficients (r) were calculated to investigate the association between self-reported HRQOL (i.e., the QLQ-C30 global quality of life scale and the QLQ-H&N35 speech scale) and speech and oral functional status. Statistical significance was defined as a P-value less than or equal to 0.05. Results Sample description Patient characteristics are shown in Table 1. The patients’ age ranged from 23 to 74 years (mean 58 years). Forty-one percent of the sample was female. One patient was operated on for a recurrent tumor after prior transoral excision (1 year earlier) in which the rTNM stage was used, and one patient had undergone previous radiotherapy (3 years earlier) for a neck node of an unknown primary. Four patients had a synchronous second primary tumor, and in these cases the stage of the largest tumor was used. An equal percentage (50%) of patients had a tumor originating on the left or right side, and in the majority of patients (73%) the tumor did not extend over the median line. All 80 patients completed the EORTC questionnaires, speech rate tests (word count) and oral function tests (mobility and strength). In one patient speech recording could not be done due to logistical problems, and in three patients speech recordings were inadequate for interpretation due to technical problems. Speech quality analyses could thus be performed on 76 patients. Presence of comorbid conditions and tumor classification were significantly associated. Significantly more patients with comorbidity were diagnosed with a larger tumor (χ2 = 10.37, P < 0.01). No other significant associations were observed between tumor site, tumor classification, age, gender and comorbidity. Table 1Characteristics of 80 patients included in this studyAge (years) Range23–74  Mean58n (%)Gender Male47 (59) Female33 (41)General condition Comorbidity48 (60) No comorbidity32 (40)Tumour site Oral cavity38 (47) Oropharynx42 (53)T 2 35 (44) 3 42 (52) 4 3 (4)N 024 (30) 116 (19) 2a2 (3) 2b30 (38) 2c6 (7) 3 2 (3) Health-related quality of life Compared to the general population, the patients with oral or oropharyngeal cancer scored significantly worse on 5 (out of 15) scales or single items regarding the QLQ-C30: role functioning (P = 0.000), emotional functioning (P = 0.000), pain (P = 0.026), insomnia (P = 0.016) and appetite loss (P = 0.003). Conversely, patients scored significantly better regarding social functioning (P = 0.009), fatigue (P = 0.030), emesis (P = 0.001), and diarrhea (P = 0.001). Regarding the QLQ-H&N35, the patients scored significantly worse on 7 (out of 10) scales or single items compared to the general population: pain (P = 0.000), swallowing (P = 0.000), senses (P = 0.050), social eating (P = 0.000), teeth (P = 0.009), opening the mouth wide (P = 0.000) and sticky saliva (P = 0.005). Conversely, the patients scored significantly better on two items: coughing (P = 0.002) and feeling ill (P = 0.025). Regarding tumor site, the QLQ-C30 results were comparable between oral cavity and oropharynx cancer patients. Only the pain score was significantly worse (P = 0.027) for patients with oral cavity tumors as compared to patients with oropharynx tumors. The QLQ-H&N35 also revealed that patients with oral cavity tumors had significantly more pain (P = 0.002); furthermore, problems with their teeth appeared to be different (P = 0.001). No statistically significant differences were observed for any of the QLQ-C30 scores as a function of tumor classification (T2 versus T3–T4). Regarding the QLQ-H&N35, patients with T3–T4 tumors scored significantly worse on opening the mouth (P = 0.009) and reported feeling more ill (P = 0.035) compared to patients with T2 tumors. Regarding comorbidity (Table 2), patients with one or more comorbid conditions scored significantly worse on QLQ-C30 physical functioning (P = 0.002), global quality of life (P = 0.016), fatigue (P = 0.014), pain (P = 0.024), constipation (P = 0.042) and diarrhea (P = 0.017) compared to patients without comorbidity. Additionally, based on the QLQ-H&N35 data, patients with comorbidity reported significantly more pain (P = 0.007), trouble with social eating (P = 0.036), teeth problems (P = 0.034), problems with opening the mouth (P = 0.013) and feeling ill (P = 0.003). The use of pain medication was significantly higher among patients with than those without comorbidity (75 versus 50%; P = 0.025). Table 2EORTC QLQ-C30 and QLQ-H&N35 scores for comorbidityComorbidityNoYesMean (SD) n = 32Mean (SD) n = 48PEORTC QLQ-C30 Physical functioning95.0 (12.4)81.7 (21.4)0.002 Role functioning85.4 (18.3)79.2 (24.9)0.228 Cognitive functioning89.6 (18.3)87.2 (19.2)0.574 Emotional functioning69.5 (23.4)72.2 (24.6)0.626 Social functioning93.2 (13.3)88.5 (21.5)0.275 Global quality of life82.6 (19.6)71.5 (19.7)0.016 Fatigue13.9 (20.0)26.9 (24.1)0.014 Emesis1.6 (4.9)1.4 (4.7)0.874 Pain22.4 (25.3)37.8 (31.8)0.024 Dyspnea6.3 (19.7)10.4 (18.4)0.338 Insomnia36.5 (36.3)31.3 (39.1)0.550 Appetite loss7.3 (20.3)18.7 (31.4)0.072 Constipation5.2 (20.9)18.7 (32.9)0.042 Diarrhea1.0 (5.9)8.3 (16.1)0.017 Financial impact2.1 (8.2)7.6 (20.9)0.157EORTC QLQ-H&N35 Pain27.1 (17.6)41.5 (25.5)0.007 Swallowing14.6 (22.6)24.1 (22.6)0.068 Senses 6.3 (14.5)8.7 (21.5)0.577 Speech 9.4 (14.1)13.0 (17.5)0.336 Social eating14.6 (25.3)27.4 (27.1)0.036 Social contact3.1 (7.0)3.7 (6.1)0.674 Sexuality16.1 (24.9)16.3 (27.6)0.981 Teeth13.5 (26.6)30.6 (38.8)0.034 Opening mouth7.3 (20.3)25.7 (37.2)0.013 Dry mouth15.6 (26.8)22.2 (28.6)0.303 Sticky saliva12.5 (29.0)21.5 (31.1)0.196 Coughing6.3 (13.2)14.6 (21.6)0.055 Feeling ill0 (0)9.7 (18.1)0.003% Yes% YesPain medication50.075.00.022 Speech and oral functional status Abnormal scores were observed in 17% of the patients regarding overall intelligibility, in 25% regarding nasality and in 37% regarding articulation. Functional results in relation to tumor site, classification and comorbidity are shown in Table 3. Regarding tumor site, patients with oral cavity tumors scored significantly worse on intelligibility (P = 0.015), articulation (P = 0.039), nasality (P = 0.040), tongue and lip mobility (P = 0.000, 0.009), diadochokinesis (P = 0.004), and tongue strength (P = 0.001) compared to patients with oropharyngeal tumors. Regarding tumor stage, patients with T3–T4 tumors scored significantly worse on intelligibility (P = 0.011), articulation (P = 0.003), tongue mobility (P = 0.001), diadochokinesis (P = 0.012), and tongue and lip strength (P = 0.021, 0.018) than patients with T2 tumors. Patients with comorbidity scored significantly worse on articulation (P = 0.043), diadochokinesis (P = 0.026), and tongue strength (P = 0.045) as compared to patients without comorbidity. Table 3Functional status tests for tumour site, tumour stage and comorbidityTumour siteTumour stageComorbidityOral cavityOropharynxT2T3–T4NoYesMean (SD) n = 37Mean (SD) n = 39PMean (SD) n = 32Mean (SD) n = 44PMean (SD) n = 29Mean (SD) n = 47PSpeech Intelligibility  (0–10)6.0 (1.3)6.7 (1.0)0.0156.8 (1.0)6.0 (1.2)0.0116.6 (1.0)6.2 (1.3)0.132 Articulation  (0–4)3.4 (0.8)3.7 (0.6)0.0393.8 (0.4)3.3 (0.8)0.0033.8 (0.4)3.4 (0.8)0.043 Nasality  (0–4)3.6 (0.6)3.8 (0.4)0.0403.8 (0.4)3.7 (0.6)0.9113.7 (0.4)3.7 (0.5)0.771n = 38n = 42n = 35n = 45n = 32n = 48 Rate  Words per minute183 (31.6)182 (38.5)0.988189 (33.8)178 (35.8)0.201189 (31.4)179 (38.1)0.233n = 38n = 42n = 35n = 45n = 32n = 48Oral functions Mobility  Tongue (0–100)82.3 (20.0)96.4 (5.4)0.00096.0 (5.8)84.8 (19.3)0.00193.9 (9.9)89.9 (14.4)0.209  Lip (0–100)87.4 (5.7)99.7 (0.9)0.00999.4 (2.0)98.0 (5.1)0.14099.3 (2.2)98.5 (3.9)0.239  Diadoch (0–100)87.4 (20.7)97.2 (6.1)0.00497.5 (5.0)88.7 (19.6)0.01297.4 (5.1)91.5 (15.4)0.026 Strength  Tongue (0–0.5)0.2 (0.2)0.4 (0.2)0.0010.4 (0.2)0.3 (0.2)0.0210.4 (0.2)0.2 (0.2)0.045  Lip (0–0.5)0.3 (0.1)0.4 (0.2)0.3280.4 (0.2)0.3 (0.1)0.0180.4 (0.2)0.3 (0.1)0.370 Correlations between self-report and observer rated data Significant correlations (P < 0.01) were observed between self-reported global quality of life scale (QLQ-C30) and observer ratings of intelligibility (r = 0.41) and articulation (r = 0.36) (Table 4). Additionally, statistically significant (P < 0.05) but relatively low correlations were found between self-reported speech (QLQ-H&N35) and observer ratings of intelligibility (r = −0.28), articulation (r = −0.24), nasality (r = −0.27) and diadochokinesis (r = −0.28). Table 4Correlations between functional status tests and QLQ-C30 global quality of life scale/QLQ-H&N35 speech problemsScalesQLQ-C30 global quality of lifeQLQ-H&N35 speech problemsCorrelation coefficient (r) n = 76PCorrelation coefficient (r) n = 76PSpeech Intelligibility0.4110.000−0.2850.013 Articulation0.3550.002−0.2420.035 Nasality0.1520.190−0.2700.019n = 80n = 80 Rate0.1290.267−0.1420.221n = 80n = 80Oral functions Mobility  Tongue0.0780.490−0.1880.095  Lip0.1570.164−0.1760.119  Diadochokinesis0.1090.337−0.2800.012 Strength  Tongue0.1880.095−0.1750.121  Lip0.0560.624−0.0490.665 Discussion Pretreatment HRQOL and functional status were investigated in a well-defined sample of patients with advanced oral and oropharyngeal cancer. Deteriorated HRQOL and functional status before treatment have been reported in earlier studies, albeit for less well defined patient groups [9–15]. The results of the present study indicate a wide range of HRQOL and functional deficits in patients with advanced oral and oropharyngeal cancer before treatment. Impaired speech and oral function, as assessed objectively, was associated significantly with self-reported global HRQOL and speech problems, which is in accordance with the conclusions of Rogers et al. and Karnell et al. [14, 24]. Compared to the general population, patients scored significantly worse on 5 of 15 scales or items of the EORTC QLQ-C30 but, conversely, they scored significantly better on four others. Regarding the QLQ-H&N35, patients scored significantly worse on most scales or items but significantly better on coughing and feeling ill. The significantly better scores for patients compared to the reference groups on some scales or items may be explained by the fact that people from the general population may have (other) chronic conditions as well. Alonso et al. [25] reported that, 55% of the general population has one chronic health condition, and 30% has more than one chronic condition. They also found that comorbidity can have a substantial impact on HRQOL, and stated that the presence of comorbidity limits the ability to attribute HRQOL deficits to one specific disease (e.g., to head and neck cancer). In head and neck cancer patients, comorbidity has proven to be an important factor associated with complications and mortality rates [26–29]. Studies on the impact of comorbidity on functional status in head and neck cancer patients are lacking. Studies that have examined the impact of comorbid status on HRQOL are scarce, have yielded conflicting results, and have examined the post-treatment period only. Pourel et al. [17] found no significant association between comorbidity and HRQOL in 113 long-term survivors 2 years after treatment for oropharyngeal carcinomas. Similarly, Taylor et al. [30] found no impact of comorbidity on work-related disability among 384 patients after treatment for head and neck cancer. However, Terrel et al. [19] in a study on HRQOL of 570 head and neck cancer patients after treatment, reported a clear effect of comorbidity in patients with two or more comorbid conditions. In our study on patients with oral or oropharyngeal carcinomas before treatment, comorbidity was present in 60% of the patients, and proved to have a major impact on HRQOL and functional status. Patients with comorbidity had significantly worse scores on several general and head and neck specific quality of life aspects and on speech and oral function tests compared to patients without comorbidity. Unfortunately, the cause of comorbidity was not systematically noted in the present study; future studies may provide more insight into the relation between the cause of comorbidity and HRQOL. The impact of tumor site and classification on HRQOL appeared to be limited, with patients with oral cavity tumors reporting more pain, and patients with T3–T4 tumors reporting more trouble opening the mouth and feeling more ill. With regard to tumor site, comparisons with earlier studies are difficult because most studies included patients with oral cavity tumors only or reported on heterogeneous samples not stratified by (sub)sites [4, 9–11]. Regarding tumor classification, other studies have reported worse HRQOL for patients with higher tumor classifications and stages before treatment [2, 14]. Tumor site and classification were found to have a clear impact on functional status. Patients with oral cavity tumors (versus oropharyngeal tumors) and patients with T3–T4 tumors (versus T2 tumors) had worse speech and oral function scores, which is in accordance with the results of earlier studies [13, 14]. In conclusion, we observed compromised HRQOL and functional deficits among patients with advanced oral and oropharyngeal cancer before the start of treatment. In addition to the impact of tumor site and classification, comorbidity proved to have a major impact on HRQOL and functional status. Prospective studies are needed to obtain insight into the relation between pretreatment HRQOL and functional status and outcome after treatment, and the relationship between changes in HRQOL and functioning over time and tumor site, tumor classification, and comorbid conditions.

Crit_Care-6-5-130146

Antithrombin III in patients admitted to intensive care units: a multicenter observational study

Introduction The administration of antithrombin III (ATIII) is useful in patients with congenital deficiency, but evidence for the other therapeutic indications of this drug is still uncertain. In Italy, the use of ATIII is very common in intensive care units (ICUs). For this reason we undertook an observational study to determine the pattern of use of ATIII in ICUs and to assess the outcome of patients given this treatment. Introduction Antithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency [1,2,3,4,5] (see also the approval of this indication by the Food and Drug Administration); in contrast, the evidence supporting its use for other clinical indications is uncertain [6,7,8,9,10]. In Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation (DIC). The approval of ATIII by the Italian Ministry of Health was granted nearly 10 years ago (before the profound reform of the Drug Regulatory Agency made by the Italian Ministry of Health in 1993) and has remained unchanged since then. This approval of ATIII was rather generic and included 'congenital deficiency of ATIII and all clinical conditions that can cause an acquired deficiency of ATIII'. Three small randomized studies [7,8,9] and one large international trial [10] assessed the effectiveness of ATIII in sepsis, but none of these trials found a significant benefit in terms of reduced morbidity or mortality. As regards congenital deficiency, the effectiveness of ATIII is fairly well documented [1,2,3,4,5], but these patients are rare. The other clinical indications (such as acute thrombosis or thromboembolism, prevention of DIC in hepatic coma, and treatment of bleeding episodes in cirrhosis) are supported by a small series of very preliminary studies (see, for example, the Drugdex databank, CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA). To achieve a better definition of the current use of ATIII in Italian hospitals and to generate naturalistic data (based on routine practice) about the outcome of this treatment, we undertook a multicenter observational study. Methods Design of the study and aims The study was based on a multicenter observational design. From 20 May to 20 July 2001 all consecutive patients admitted to ICUs in 20 Italian hospitals and treated with ATIII were enrolled in the study. The study had the following aims: (1) surveying the use of ATIII in patients admitted to ICUs; (2) determining the outcome of patients treated with ATIII; and (3) comparing the results obtained from our observational study with those previously found in the randomized controlled trials (RCTs). A meta-analysis was also conducted to summarize the information deriving from four RCTs [7,8,9,10] that studied the effectiveness of ATIII in sepsis. Data collection The following information was recorded from each patient enrolled in the study: (1) demographic characteristics (age, sex, weight); (2) congenital deficiency (y/n); (3) baseline ATIII level; (4) ward of first admission in the hospital; (5) clinical indication for using ATIII (sepsis or DIC or any other clinical condition); (6) daily dose and duration of treatment with ATIII; (7) outcome of hospitalization (alive or dead); and (8) concurrent administration of antibiotics and/or heparin. Analysis The information collected from each patient was analyzed by standard descriptive statistics. In the subgroup of patients with sepsis, the in-hospital mortality rate observed in our study was compared with that previously reported by the four RCTs. All rates were presented together with their 95% confidence interval (CI), which was calculated by using Equations 1.26 and 1.27 of Fleiss [11]. Results The overall number of patients who were admitted to ICUs during the study period was 1648. Of these patients, 216 (13%) were enrolled in our study. The characteristics of these 216 patients are presented in Table 1. The clinical indication for using ATIII was sepsis (n = 56), DIC (n = 50), or other (n = 101). Table 1 also reports separate information for the subgroup of 56 patients treated for sepsis. The duration of ATIII therapy did not differ at levels of statistical significance between patients treated for different clinical indications (P = 0.57 according to an analysis of variance). The daily dose of ATIII showed a difference between sepsis and other indications (Table 1). Table 2 reports the outcome of hospitalization according to clinical indication. With regard to the use of ATIII in patients with sepsis, Figure 1 shows the percentage mortality rate (with 95% CI) observed in our study, together with the rates found in four previous studies [7,8,9,10]. Subgroup analyses within the patient cohort of our study did not identify any relationship between mortality and patient characteristics. The administration of heparin, which Warren et al. [10] found to have some implications for outcome, did not influence mortality in our patient series: mortality was 19.6% in the 107 patients who received heparin, compared with 30.5% in the 95 patients who did not receive this drug (P = 0.10) by Fisher's exact test; mortality was 28.6% in the 28 patients with sepsis who received heparin, compared with 42.3% in the 26 patients with sepsis who did not receive this drug (P = 0.39). Discussion The main scientific value of our observational and prospective study lies in its naturalistic design; the population of patients that we studied was in fact drawn from the everyday practice of more than 20 hospitals and was intentionally free from specific exclusion criteria. In interpreting our outcome data, one disadvantage is that the group treated with ATIII was not compared with any reference group observed prospectively within our research; neither did we include any retrospective control group not treated with the drug. However, historical retrospective controls would have raised profound problems of matching the retrospective data with the prospective ones. A prospective enrollment of controls not treated with ATIII was not feasible because the therapeutic policy of the ICUs involved in our study was to administer ATIII to virtually all patients with a diagnosis of sepsis or DIC. Regardless of our statistical indexes, a 'first-look' comparison between the data on sepsis produced by the previous RCTs (including four treatment groups and four control groups) and those observed in our naturalistic study indicates a complete overlap of the various survival rates and of their respective 95% CIs. This qualitative impression (Figure 1) is in agreement with the meta-analysis shown in Figure 2 (see Appendix2 for details of its methodology). This meta-analysis gave the following results: summary odds ratio 0.98; 95% CI 0.83–1.15, P = 0.80; χ2 for heterogeneity 1.86; 3 degrees of freedom; P = 0.60. In this meta-analysis, the large-scale trial by Warren et al. [10] outweighed the other three small RCTs in that Warren's trial included 93% of the overall cohort of the four RCTs. In the light of the above data, there seems to be no clinical benefit in administering ATIII to critical patients with sepsis; in this context, one crucial point is that the most recent large-scale trial gave very clear results and was negative. The other clinical indications reported in our patients' series were more difficult to interpret because of the nearly complete lack of previous controlled studies exploring these therapeutic issues. There has been a lively debate in the literature on the relative merits of observational studies and RCTs in providing useful evidence of clinical effectiveness [12,13,14]. Although the great majority of researchers stick to the concept that RCTs are the gold standard, common sense suggests that having information both from RCTs and from observational studies is better than having information from RCTs only. In this framework, our study advances knowledge about the use of ATIII in critical patients. In conclusion, our findings based on an observational prospective study and on an updated meta-analysis of the previous RCTs do not support the use of this drug in ICU patients with sepsis. Key messages Antithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency; in contrast, the evidence supporting its use for other clinical indications is uncertain. In Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation. Three small randomized studies and one large international trial have assessed the effectiveness of ATIII in sepsis, but none of these trials has found a significant benefit in terms of reduced morbidity or mortality. Our findings, based on an observational prospective study and on an updated meta-analysis of the previous randomized controlled trials, do not support the use of this drug in ICU patients without congenital deficiency. Competing interests In 2001 our research group received a grant from Eli-Lilly (Italy) to conduct an original study on factors influencing length of stay in critical patients with sepsis. In Italy, anti-thrombin III is marketed by Aventis-Behring and by Baxter. Appendix 1: Gruppo di Studio sull'antitrombina III (The Antithrombin Study Group) The Antithrombin Study Group includes the study coordinators (A Messori, F Vacca, M Vaiani, S Trippoli, Laboratorio di Farmacoeconomia, c/o Azienda Ospedaliera Careggi, Firenze) and a total of 51 participants. The names and addresses of the participants involved in the project were the following (all located in Italy): R Banfi, M Cecchi, E Cini, D Dupuis, T Falai, R Fornaini, A Ipponi, ML Migliaccio, F Pelagotti, L Rabatti, I Ruffino, R Silvano, E Tendi (Firenze, four hospitals); P Becagli, M Monciatti (Empoli); B Bozzone, R Casullo, F Cattel, S Pardossi, R Passera, S Stecca, U Tagliaferro (Torino, two hospitals); P Di Bartolomeo, T Faggiano, M Lattarulo (Bari); N Caboni, A Cannas (Cagliari); A Plescia, M Sorci (Rimini); L Bonistalli, M Puliti (Prato); B Ciammitti, M Costantini, F Mammini (Terni); L De Cicco, G Mazzaferro (Napoli); P Marrone, R Tetamo (Palermo); P Beneduce, MG Celeste, P Fiorani, S Galeassi, G Guaglianone, A Pecere, L Ragni (Roma, two hospitals); SM Germinario (Andria); O Basadonna, L Todesco (Camposampiero, Padova); R Calle-gari, M Pegoraro (Asolo); E Lamura (Ancona). Appendix 2: Methodology of the meta-analysis A MedLine search (PubMed, ) was performed to cover the period from January 1980 to November 2001. The search was limited to the studies published in English and was based on four index terms combined with the following Boolean syntax: "antithrombin III" AND (sepsis OR septic shock OR "disseminated intravascular coagulation"). This search was supplemented by examining the Drugdex databank (CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA). Eligible studies were included if they met the following criteria: patients were admitted to an ICU; randomized design; diagnosis of sepsis, septic shock or DIC; assessment of survival. The odds ratio was used as the main index to assess the treatment effect within each trial and to generate the overall results of the meta-analysis. The calculation of the summary odds ratios was based on a random-effect model [15,16]. Heterogeneity was assessed as described previously [17]. Abbreviations ATIII = antithrombin III; CI = confidence interval; DIC = disseminated intravascular coagulation; ICU, intensive care unit; RCT = randomized controlled trial.

Int_J_Colorectal_Dis-3-1-2134973

Colonic irrigation for defecation disorders after dynamic graciloplasty

Background and aims Dynamic graciloplasty (DGP) improves anal continence and quality of life for most patients. However, in some patients, DGP fails and fecal incontinence is unsolved or only partially improved. Constipation is also a significant problem after DGP, occurring in 13–90%. Colonic irrigation can be considered as an additional or salvage treatment for defecation disorders after unsuccessful or partially successful DGP. In this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and/or constipation after DGP is investigated. Introduction Dynamic graciloplasty (DGP) is a proven effective treatment for fecal incontinence. Success rates vary from 42–92% [1–5]. Comparison of results is difficult because the outcome of this method seems to be influenced by the etiology of fecal incontinence, pre-existing stoma, length of follow-up, method of stimulation, and the surgeon’s experience [1, 6]. DGP significantly improves quality of life and anal continence for most patients [2]. Quality of life and patient satisfaction scores correlate significantly with continence scores [7, 8]. However, in some patients, fecal incontinence is unsolved or only partially improved after DGP. Morbidity rates are high after DGP: complications associated with the technique can often be prevented or treated, but other complications like reduced sensitivity of the rectum by destruction of sensory nerves can be very hard to treat and often result in DGP failure [4, 8, 9]. Morbidity rates are lower and success rates higher in the hands of surgeons experienced in the technique [3, 10]. Constipation is a significant problem after graciloplasty, occurring in 13–90% of the patients after DGP [6–8, 11, 12]. Constipation due to technical failure, like a gracilis wrap that is too tight, can be treated by revisional surgery. Other causes of constipation can first be treated with dietary measures and medication. In case of pelvic floor dysfunction, biofeedback training can be started [9]. When this first line treatment is unsuccessful, retrograde colonic irrigation can be considered. Colonic irrigation can also be used as an additional therapy or salvage therapy for persistent fecal incontinence after failed or partially failed surgery [13]. Not much is known about the success rate of retrograde colonic irrigation, as there is only a limited number of published studies [13, 14]. Antegrade irrigation through a colostomy or appendico-cecostomy is another technique for colonic irrigation, with a reasonable success rate of 64–85% [15–17]. In this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and/or constipation after DGP was investigated. Materials and methods Patients with DGP and postoperative defecation disorders were selected for colonic irrigation between January 1999 and June 2003. The patients were offered colonic irrigation as additional or salvage therapy, as a colostomy was the final option for these patients. The inclusion criteria were invalidating fecal incontinence and/or constipation after DGP and a signed informed consent; exclusion criteria were patients not willing to perform the irrigation because of either embarrassment or wish for colostomy or patients physically and/or mentally not capable of performing irrigation. All patients visited the outpatient clinic of the University Hospital Maastricht. Relevant physical and medical history were collected. The patients could be divided in six groups according to the etiology of fecal incontinence before DGP: congenital, trauma (rupture/anal surgery), pudendopathy (PNTML > 2.6 ms), spinal cord lesion, cancer (abdominal perineal resection in rectumcarcinoma), and prolapse. The patients were asked to fill out a questionnaire. This questionnaire contained questions concerning the method of irrigation, the time needed for irrigation, the amount of water used, the frequency of irrigation, added substances to the irrigation water, and side effects of colonic irrigation. The endpoint was defined that irrigation had to be successful and satisfying for the patients. Successful irrigation was defined as reaching continence for feces (pseudo-continence) and/or complete resolution of straining, feeling of incomplete evacuation, bloating abdomen, and abdominal pain during irrigation. The patient was considered satisfied when he or she indicated that the colonic irrigation rendered a major improvement of the quality of life. The improvement of quality of life was measured by a visual analog scale/specific questions within the questionnaire. The irrigation was performed in most patients as retrograde irrigation; only four patients performed antegrade irrigation through an appendico-cecostomy or a colostomy. Patients with fecal incontinence can gain continence as a result of colonic irrigation. This is called pseudo-continence because these patients are only continent, as there is no fecal filling of the rectum and distal colon. The definition of resolved constipation was no straining and no feeling of incomplete evacuation after defecation. Defecation occurred during irrigation in most of these patients. The Biotrol® Irrimatic pump (Braun®; Fig. 1) or the irrigation bag (Braun®; Fig. 2) were used for colonic irrigation. The Irrimatic pump is an irrigation pump using a flexible tube with a cone-shaped end. The tube is introduced either in the anal canal in case of retrograde irrigation or placed in an appendico-cecostomy or colostomy in case of antegrade irrigation. The pump can hold a maximum of 2 l. The patients were instructed to start the irrigation daily with 500 ml of water. Thereafter, the frequency and the amount of water were adjusted until a satisfactory result was achieved. Water used for irrigation was at body temperature; too cold water can cause abdominal cramps or collapse and too hot water can cause burns. Soap or laxatives could be added if necessary. The pump works on a storage battery, and the speed of water ejection can be regulated. The working mechanism of the irrigation bag is similar, except for the water ejection, which is induced by gravity. Fig. 1Biotrol® Irrimatic pump (Braun®)Fig. 2Irrigation bag (Braun®) Data analysis was performed by using SPSS 14.0 (SPSS release 14.0, SPSS, Chicago, USA), with the Mann–Whitney U test. P ≤ 0.05 was considered statistically significant. Data are given as the mean values with the standard deviation. Results The questionnaire was sent to 74 patients. Fourteen patients did not wish to participate in the study for various reasons, and 14 patients did not return the questionnaire. Forty-six patients (62%) could be included for analysis of which 37 were female (80%), and these patients had an overall mean age of 59.3 ± 12.4 years. Complications of the DGP occurred in 23 (50%) patients of which 10 patients had two or more complications. Twenty-two patients suffered from constipation, 35 patients were still incontinent for feces, and seven patients had pain in the IPG pocket, leg, or anus. The medical history besides the DGP is mentioned in Table 1. On average, the patients started the irrigation 21.39 ± 38.77 months after the DGP. Eight patients already used irrigation before DGP. Table 1Medical historyMedical historyNumber of patients (percentage of total)Anal rupture during delivery13 (28%)Anal repair14 (30%)Prolapse operation19 (41%)Hysterectomy17 (37%)Abdomino-perineal resection for rectum carcinoma8 (17%)Sigmoid resection for sigmoiditis2 (9%)Cholecystectomy8 (17%)Anus atresia1 (2%) Twenty-four (52%) patients used irrigation as an additional therapy for fecal incontinence in partially failing DGP. Eleven (24%) patients used irrigation for constipation and 11 (24%) patients for both. Most patients (91%) used the irrigation pump. Three patients added soap and one patient an enema to the water to achieve a better result. These were patients with constipation or a combination of fecal incontinence and constipation. Irrigation was usually performed in the morning (70%) and sometimes in the evening (16%). The frequency of irrigation was 0.90 ± 0.40 times per day. The amount of water used for irrigation was 2.27 ± 1.75 l with duration of 39 ± 23 min. Seventy-four percent of the patients with fecal incontinence irrigated in the morning, 17% in the evening, and 9% twice a day. For the patients with constipation, these numbers are, respectively, 45, 18, and 36%. Ninety percent of the patients with a combination of defecation disorders used the colonic irrigation in the morning. There was no significant difference in the frequency of irrigation between patients with fecal incontinence and patients with constipation (0.92 vs 0.98/day; P = 0.108). The amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation, but this difference was not significant (2.31 vs 1.91 l; P = 0.484). There was no significant difference in the time needed for irrigation between patients with fecal incontinence and those with constipation (40.8 vs 32.9 min; P = 0.154). Overall, 81% of the patients were satisfied with the irrigation, 80% of the patients with fecal incontinence, 90% of the patients with constipation, and 72% of the patients with a combined defecation disorder. Thirty-seven percent of the patients with fecal incontinence reached (pseudo-) continence, and in 30% of the patients, the constipation completely resolved (Fig. 3). In 29% of the patients with a combined defecation disorder, the constipation resolved and they were (pseudo-) continent. Overall, satisfaction was related with symptomatic improvement. Only one patient with good symptomatic improvement was not satisfied because he felt that the irrigation was time-consuming and not practical. Fig. 3Success of rectal irrigation for defecation disorders Four patients, two with fecal incontinence, one with constipation, and one with both, performed antegrade irrigation through a (3) colostomy or an (1) appendico-cecostomy with good results, and all indicated that the irrigation improved their quality of life. The patients could be divided in six groups according to the underlying cause of fecal incontinence before DGP (Table 2). When the different etiologies before DGP were compared with the result during irrigation, the patients with a rupture or anal surgery had the worst results of irrigation. The patients with a spinal cord lesion seemed to have the best results. Table 2Success of rectal irrigation in different etiologies for fecal incontinence before DGPEtiologyNumber of casesSuccess (%) during irrigationCongenital11 (100%)Trauma143 (21%)Pudendopathy155 (33%)Spinal cord lesion53 (60%)Abdomino-perineal resection for rectum carcinoma83 (37%)Prolapse20 (0%)Total4515 (33%) Side effects of the irrigation were reported in 61% of the patients: leakage of water after irrigation (43%), abdominal cramps (17%), and distended abdomen (17%). Twenty-four (53%) patients changed the irrigation method during their course to improve the results. Seven (16%) patients stopped the rectal irrigation: in five patients, the result was unsatisfactory; two other patients did not need the irrigation anymore because the complaints resolved completely. Finally, two patients received a permanent stoma. Discussion Colonic irrigation can be used effectively to treat defecation disorders when other conservative treatments fail or in addition to unsuccessful or partially successful surgical treatment [18]. Colonic irrigation is usually forgotten as an alternative conservative treatment for defecation disorders, although many health care takers are familiar with colonic irrigation, particularly, for its use as perioperative colonic cleansing [19, 20]. Retrograde colonic irrigation is performed through the anorectum or via a colostomy. Only a few publications addressed to retrograde colonic irrigation are found in literature. Briel et al. [13] found a success rate of 38% of retrograde colonic irrigation for fecal incontinence and a significant improvement in quality of life. Recently, another study reported a success rate of 41% in patients with fecal incontinence and 65% in patients with constipation [18]. The success rate is based on patient satisfaction. Although patient satisfaction is the primary goal of treatment, it is a subjective measure. In future research, this should be combined with objective measures such as validated quality of life questionnaires in a prospective study design. Antegrade colonic irrigation is especially known for the treatment of evacuation disorders in small children and can be performed through an appendico-cecostomy (MACE) or a cecostomy button [16, 21–23]. Alternative enteral access is a sigmoid tube or transverse colonic conduit for patients with a left colonic evacuation disorder [24–26]. The most common problems of MACE are stoma stenosis and leakage [16, 17]. The results in our hospital of antegrade irrigation by an appendico-cecostomy were described earlier [23]. In this study, four patients performed antegrade irrigation via a colostomy or a MACE with good results. O’Bichere et al. performed an experimental study investigating the effect of retrograde vs antegrade colonic irrigation in pigs. That study demonstrates that colonic emptying is more efficient with antegrade irrigation compared to retrograde irrigation [27]. Although a reasonable success rate can be achieved with antegrade irrigation (64–85%) [15–17], retrograde colonic irrigation is preferred above antegrade irrigation as initial treatment because of its non-invasive nature and benign complications. The amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation. This can be explained by the difficulty of retaining water in the rectum in case of fecal incontinence. Theoretically, the amount of water retained in the rectum is higher in patients with constipation. The grade of colonic emptying by irrigation in constipated patients by means of scintigraphy was investigated by Christensen et al. [14]. The effect of retrograde colonic washout was significantly better in idiopathic fecal incontinence compared to idiopathic constipation, and its effect correlated with the extent to which the irrigation fluid had entered the colorectum. Surgical treatment of fecal incontinence gives an overall success of 50–84%, depending on the etiology of fecal incontinence and type of surgical procedure [28–32]. Success percentages in DGP vary from 42–92% [1–5]. The success rate of 80% of colonic irrigation is comparable to the success rate of surgical treatment for fecal incontinence. Sixty-one percent of the patients that received the questionnaire completed it. Even when we assume that the patients who did not complete the questionnaire were dissatisfied with the treatment (bad case scenario), the success rate in the present study would still be 50%. It is important to realize that this is a selected group of patients with persistent incontinence and/or constipation after DGP. Regarding the fecal incontinence, this group of patients represents the 8–58% (assuming 42–92% success after DGP) with insufficient results of DGP [1–5]. Regarding the different underlying causes for fecal incontinence before DGP, the patients with a trauma to the anal sphincter have the lowest success rate and the patients with a spinal cord lesion have the highest success rate. It is not possible to draw conclusions about the statistical significance of these results because of the limited group size. Previous studies already reported an increased incidence of constipation after DGP [3, 6, 8, 11, 12]. In this study, we included 22 patients that were constipated after DGP, of which 11 patients had constipation in combination with persistent fecal incontinence. It is important to realize that many patients are incontinent for feces after periods of severe evacuation problems. These evacuation problems are present again after treatment of the fecal incontinence. Its etiology is not well understood, but is likely to be multifactorial [33, 34]. This is known after all surgical procedures for fecal incontinence [35]. Constipation can be due to technical problems such as a tight graciloplasty, which can be solved by revision of the DGP. It is also possible that nerve tissue around the anus sustains damage during the operation, causing pudendopathy which cannot be treated by revisional surgery. Previous surgery in the pelvis, like an abdomino-perineal resection (eight patients in this study), is known to have a very high rate of obstructed defecation, probably due to reduced rectal sensitivity and pelvic floor dysfunction [36]. Constipation related to physical impairment, like a disturbed sensation and/or motility because of a congenital cause or degeneration, is difficult to treat [9]. These patients are usually served well with colonic irrigation. Surgical treatment of constipation is troublesome and only indicated in a selected group of patients with intractable constipation. The bowel frequency usually increases, but abdominal pain and bloating persists in most patients [37]. In this study, 90% of the patients were satisfied with the irrigation, and in 30%, the constipation resolved with colonic irrigation. The final step of treatment after the colonic irrigation in the patients of this study would be a colostomy or ileostomy. In the study of Norton et al., the majority of the patients with fecal incontinence managed by a colostomy reported to be positive about the stoma. However, a few could not adapt to the stoma and disliked it intensely [38]. Harris et al. investigated patients with fecal incontinence or constipation managed with a stoma. Many of these patients reported that lifestyle restrictions were imposed by the stoma, and almost half of the patients felt stigmatized. Up to a third of these patients had significant depression, especially the younger women [39]. Cultural issues also have an important impact on the acceptance of a stoma [40]. Keeping these disadvantages of a definitive stoma in mind, colonic irrigation offers an attractive alternative, which is worthwhile trying before proceeding to a definitive stoma. Conclusion Colonic irrigation is an effective alternative for the treatment of persistent fecal incontinence after DGP and/or recurrent or onset constipation additional to unsuccessful or (partially) successful DGP. Colonic irrigation is an undervalued and often forgotten treatment option, which deserves its rightful place among the other treatment modalities.

Calcif_Tissue_Int-3-1-1914224

The -1997 G/T and Sp1 Polymorphisms in the Collagen Type I alpha1 (COLIA1) Gene in Relation to Changes in Femoral Neck Bone Mineral Density and the Risk of Fracture in the Elderly: The Rotterdam Study

The COLIA1 Sp1 polymorphism has been associated with bone mineral density (BMD) and fracture. A promoter polymorphism, -1997 G/T, also has been associated with BMD. In this study, we examined whether these polymorphisms alone and in the form of haplotypes influence bone parameters and fracture risk in a large population-based cohort of elderly Caucasians. We determined the COLIA1 -1997 G/T (promoter) and Sp1 G/T (intron) polymorphisms in 6,280 individuals and inferred haplotypes. Femoral neck BMD and BMD change were compared across COLIA1 genotypes at baseline and follow-up (mean 6.5 years). We also investigated the relationship between the COLIA1 polymorphisms and incident nonvertebral fractures, which were recorded during a mean follow-up period of 7.4 years. Vertebral fractures were assessed by radiographs on 3,456 genotyped individuals. Femoral neck BMD measured at baseline was 3.8% lower in women carrying two copies of the T-Sp1 allele (P for trend = 0.03). No genotype dependent differences in BMD loss were observed. In women homozygous for the T allele of the Sp1 polymorphism, the risk of fragility fracture increased 2.3 times (95% confidence interval 1.4–3.9, P = 0.001). No such association was observed with the promoter polymorphism. In men, no association with either the Sp1 or the -1997 G/T promoter polymorphism was seen with BMD or fracture. High linkage disequilibrium (LD; D′ = 0.99, r2 = 0.03) exists between the two studied polymorphisms. We observed three haplotypes in our population: haplotype 1 (Gpromoter–Gintron) frequency (f) = 69%, haplotype 2 (Gpromoter–Tintron) f = 17.6%, and haplotype 3 (Tpromoter–Gintron) f = 13.4%. Haplotype 2 was associated with a 2.1-fold increased risk of fragility fracture in women (95% confidence interval 1.2–3.7, P = 0.001). We confirm that the COLIA1 Sp1 polymorphism influences BMD and the risk of fracture in postmenopausal Caucasian women. In contrast, we found no independent effect of the -1997 G/T promoter polymorphism on BMD or fracture. Introduction Osteoporosis is a multifactorial disease with both genetic and environmental determinants. It is characterized by a reduction in bone mineral density (BMD) and microarchitectural deterioration of bone tissue, which leads to an increased risk of fracture in later life [1, 2]. Being a predictor of bone fragility and susceptibility to fracture, BMD is used for the diagnosis of osteoporosis [3, 4]. The risk of fracture is dependent not only on BMD but also on geometry, architecture, material properties, and mass distribution [5]. The skeletal determinants of osteoporotic fracture risk such as BMD, bone loss, and bone geometry are all subject to strong genetic influences [2, 6, 7]. It has been estimated from twin studies that 60-80% of the variance in BMD is attributable to genetic factors [8, 9]. Several genes are thought to be involved in the pathogenesis of osteoporosis. Collagen type I alpha 1 (COLIA1) is one of the most prominent candidate genes, which has been consistently associated with osteoporosis in different populations [10–12]. COLIA1 encodes the alpha 1 chain of collagen type I, which is the most abundant structural protein in the bone matrix; rare mutations in this gene cause osteogenesis imperfecta, a mendelian disorder presenting with moderate to severe bone fragility [13, 14]. Previously, Grant et al. [15] identified a relatively common guanine to thymidine (G→T) polymorphism in the first intron of COLIA1. This polymorphism affects one of the binding sites of the transcription factor Sp1 and results in increased expression of collagen type I alpha 1 in bone matrix in T carriers [11] .We and others have shown that the T allele is associated with osteoporosis, lower BMD [10, 15], and increased fracture risk [10, 12, 16, 17]. Moreover, in a very large prospective meta-analysis of individual data, we observed that the Sp1 polymorphism in the COLIA1 gene is associated with reduced BMD and incident vertebral fractures independent of BMD [18]. In addition to the Sp1 polymorphism, Garcia-Giralt et al. [19] described two polymorphisms within the COLIA1 promoter region: -1997 G/T and -1663 indelT. The study showed in a small cohort of postmenopausal Spanish women that the T allele of the -1997 G/T polymorphism was significantly associated with a 7.5% decreased lumbar spine BMD and a 12% decreased femoral neck BMD. Furthermore, they analyzed compound genotypes including three polymorphic sites. However, this small study of the promoter polymorphisms in women investigated the relation in form of compound genotypes, was not extended to haplotypes of promoter and Sp1 polymorphisms, and most importantly, did not analyze fractures, the clinically most relevant end point of osteoporosis. Therefore, we investigated the influence of both COLIA1 polymorphisms independently and in the form of haplotypes in relation to baseline femoral neck BMD, change in BMD with follow-up, and risk of vertebral and nonvertebral fractures in a large population-based cohort of elderly Caucasian men and women. Materials and Methods Study Population This study was embedded in the Rotterdam Study, a population–based cohort study in which all residents of the Rotterdam suburb Ommoord aged 55 years and older were invited to take part. The design of the study has been described elsewhere [20]. Written informed consent was obtained from all participants, and the Medical Ethics Committee of the Erasmus Medical Center approved the study. Baseline data collection was conducted in January 1990 and June 1993, while two follow-up assessments were performed between July 1993 and January 1996 and from July 1996 until December 1999. A total of 7,983 subjects participated in the study (response rate 78%), and for the present study, we examined 6,280 individuals who were genotyped. Study Design This study was performed in three steps. In the first step, we performed a cross-sectional analysis where we examined the relation between the genotype and baseline BMD (n = 5,737). In the second step, we performed a longitudinal analysis to study change in BMD between baseline and the second follow-up (mean 6.5 ± standard deviation [SD] 0.6 years, n = 2,670). In the third step, we looked at the relation between COLIA1 polymorphisms and the risk of incident fracture. We studied incidence of nonvertebral fracture (mean follow-up 7.4 ± 3.3 years, n = 6,280) and vertebral fractures assessed by radiographs both at baseline (1990–1993) and at follow-up visit’ between 1997 and 1999, thoracolumbar radiographs of the spine were available for 3,469 individuals in a mean follow-up of 6.4 years. Measurements BMD (g/cm2) of the hip and L2-L4 of the lumbar spine were measured by dual-energy X-ray absorptiometry (DXA) using a Lunar DPX densitometry (DPX-L) (Lunar Radiation, Madison, WI) and reanalyzed with DPX-IQ software, under standard protocols. Methods, quality assurance, accuracy, and precision issues of the DXA measurements have been described previously [21]. The relative change of BMD from baseline was estimated as the difference in BMD between assessment periods divided by the BMD at baseline. Height (cm) and weight (kg) were measured with a stadiometer at the initial examination, in standing position wearing indoor clothes without shoes. Body mass index (BMI) was computed as weight divided by height (kg/cm2). Fracture Follow-up Information on incident nonvertebral fractures was collected from baseline (1990–1993) until January 1, 2002 (mean follow-up ± SD 7.4 ± 3.3 years, n = 6,280). Nonvertebral fracture events were retrieved from computerized records of the general practitioners (GPs) in the research area. Research physicians regularly followed participant information in GP records outside the research area and made an independent review, encoding all reported events. Subsequently, a medical expert in the field reviewed all coded events for final classification. We excluded fractures that were considered nonosteoporotic, i.e., caused by cancer and high trauma, including fractures of the hand, foot, skull, and face. Subsequently, we analyzed separately “fragility” fractures, which were defined as any fracture of the hip, pelvis, or proximal humerus that had occurred with minimal trauma at older age (mean >75 years). Vertebral Fracture Assessment Both at baseline and at first follow-up, between 1997 and 1999, thoracolumbar radiographs of the spine were obtained. The follow-up radiographs were available for 3,456 individuals, who survived an average of 6.4 (SD = 0.4) years after the baseline visit and who were still able to come to our research center. All follow-up radiographs were scored for the presence of vertebral fracture by the McCloskey/Kanis method, as described previously [22]. Genotyping Genomic DNA was extracted from samples of peripheral venous blood according to standard procedures. Genomic DNA (1–2 ng) was dispensed into 384-well plates using a Sciclone ALH3000 pipetting robot (Caliper, Mountain View, CA). Genotypes were determined using the Taqman allelic discrimination assay. The Assay-by-Design service (http://www.appliedbio-systems.com) was used to set up a Taqman allelic discrimination assay for the COL1PR-1997 polymorphism (primers: Fw, GCCTCCGGAGGGTGTCA, Rv, AAGGAGAGCAATTCTTACAGGTGTCT; probes: FAM-CCTGAGGGATGGAA-MGB, VIC-CCTGAAGGATGGAAG-MGB. The polymerase chain reaction (PCR) mixture included 1–2 ng of genomic DNA in a 2 μL volume and the following reagents: FAM and VIC probes (200 nM), primers (0.9 μM), and 2× Taqman PCR master mix (ABgene, Epsom, UK). Reagents were dispensed in a 384-well plate using the Deerac Equator NS808 (Deerac Fluidics, Dublin, Ireland). PCR cycling was performed in 384-well PCR plates in an ABI 9700 system (Applied Biosystems, Foster City, CA) and consisted of initial denaturation for 15 minutes at 95°C, 40 cycles with denaturation of 15 seconds at 95°C, and annealing and extension for 60 seconds at 60°C. Results were analyzed by the ABI Taqman 7900HT using the sequence detection system 2.22 software (Applied Biosystems). To confirm the accuracy of genotyping results, 332 (5%) randomly selected samples were regenotyped with the same method. No inconsistencies were observed. For Sp1 (intron1) an assay was set up using Primer Express Software version 2.0 (Applied Biosystems, Foster city, CA). Forward and reverse primer sequences were 5′-GTTGTCTAGGTGCTGGAGGTT-3′ and 5′-GGCGAGGGAGGAGAGAAGG-3′. The PCR mixture included 5 ng of genomic DNA in a 4 μL volume and the following reagents: FAM-CCCGCCCACATTCCCTGG-MGB probes (250 nM), TET-CCCGCCCCCATTCCCTGG-MGB probe (500 nM), primers (300 nM), 2× Taqman PCR master mix (Applied Biosystems). PCR cycling was performed in 384-well PCR plates in the ABI 9700 PCR system and consisted of initial denaturation for 15 minutes at 95°C, 40 cycles with denaturation of 15 seconds at 95°C, and annealing and extension for 60 seconds at 60°C. Results were analyzed by the ABI Taqman 7900HT using the sequence detection system 2.1 software (Applied Biosystems). To confirm the accuracy of the genotyping results, 332 randomly selected samples were genotyped for a second time with the same method. All polymorphisms had an error rate lower than 1%. Statistical Analysis Hardy-Weinberg equilibrium of the COLIA1 polymorphism genotypes was tested using the GENEPOP package [23]. Linkage disequilibrium (LD) between each pair of alleles at both polymorphic loci was calculated as D′ and r2 [24]. We stratified all analyses by gender, considering peak bone mass, changes in BMD, and fractures, following age- and sex-specific patterns. Baseline BMD and BMD rate of change were compared across COLIA1 polymorphisms using univariate analysis of variance (ANOVA). Corrections were made for age and BMI. Trend analysis assuming an underlying additive genetic model was done for the presence of zero, one, or two copies of the associated allele, incorporating the genotype variable as a continuous term in a general linear regression model. For the analysis of nonvertebral fracture follow-up data, we computed the incidence rates of fracture among genotypes and used Cox’s proportional hazards model, adjusting for age and BMI to estimate risk of fracture. For vertebral fractures, odd ratios with 95% confidence intervals (95% CIs) were calculated using logistic regression models since no data on the exact time of occurrence could be determined. We used HaploStats (available at http://www.cran.r-project.org/) to estimate the frequency of inferred haplotypes and investigate the association of haplotypes with BMD and the risk of fractures. We restricted the analysis to haplotypes with an inferred frequency of more than 0.02. The first haplotype, which was most frequent, was used as reference. Significant P values were 0.05 or lower. Finally, model assumptions were verified and model residuals checked for goodness-of-fit. SPSS 11.0 (SPSS, Chicago, IL) was used for the analyses. Results Allele and genotype frequencies of the -1997 G/T and Sp1 polymorphisms were in Hardy-Weinberg equilibrium (P = 0.61 and P = 0.10, respectively). General characteristics of the study population at baseline and follow-up are shown in Table 1. Table 1General characteristics of the study population at baseline and second follow-upWomenMenBaselineSecond follow-upBaselineSecond follow-upNumbern = 3,374n = 1,724n = 2,452n = 1,287Age (years)68.3 ± 8.272.7 ± 6.867.6 ± 7.772.2 ± 6.5Height (cm)161.1 ± 6.8160.6 ± 6.4174.6 ± 6.8174.0 ± 6.7Weight (kg)69.3 ± 11.470.3 ± 12.278.2 ± 10.879.5 ± 11.3BMI (kg/m2)26.7 ± 4.127.2 ± 4.425.6 ± 3.026.3 ± 3.2FN-BMD (g/cm2)a0.83 ± 0.140.80 ± 0.130.92 ± 0.140.90 ± 0.14Lumbar spine BMD (g/cm2)1.03 ± 0.18-1.16 ± 0.20-FN-BMD changen = 1,527n = 1,143FN-BMD change (relative % of baseline year)b−0.84 ± 1.09-Values are means ± SD. Anthropometric measurement based on 5,826 individuals at baseline and 3,011individuals at follow-upaBMD measurements based on 5,737 individual at baseline and 2,670 individual at second follow-upbFemoral neck (FN) BMD change was measured between second follow-up and baseline. Second follow-up measurements were performed on average 6.5 (SD = 0.6) years after baseline Baseline BMD by COLIA1 Genotypes In both genders, age, height, weight, and BMI did not differ significantly between genotypes for the -1997 G/T and Sp1 polymorphisms (data not shown). Table 2 shows the BMD values according to COLIA1 genotypes in men and women. Femoral neck BMD was 3.8% lower (mean difference = 0.03 g/cm2, P = 0.09) in women who were homozygous carriers of the Sp1 T allele compared to noncarriers, with evidence for an allele dose effect (P for trend = 0.03) (Table 2). No association was found between the -1997 promoter polymorphism and lumbar spine BMD or femoral neck BMD in men or women (Table 2). We did not observe any significant association between the Sp1 and -1997 promoter polymorphisms with changes in BMD during follow-up in men or women (Table 2). Table 2BMD measurements by COLIA1 genotypes at baseline and follow-upPromoter -1997 G/TIntron 1 Sp1 G/TGGGTTTP*GGGTTTP*Menn = 1,663n = 494n = 37n = 1,484n = 655n = 55Femoral neck (g/cm2)0.92 ± 0.130.92 ± 0.140.90 ± 0.141.000.92 ± 0.140.92 ± 0.140.90 ± 0.120.45Lumbar spine (g/cm2)1.16 ± 0.191.17 ± 0.201.15 ± 0.220.961.17 ± 0.191.16 ± 0.201.16 ± 0.200.91Numbern = 668n = 233n = 16n = 606n = 293n = 18FN-BMD change (relative % of baseline year)−0.46 ± 0.91−0.36 ± 0.93−0.38 ± 0.600.62−0.39 ± 0.93−0.56 ± 0.87−0.30 ± 0.820.01Womenn = 2,157n = 710n = 53n = 1,971n = 858n = 91Femoral neck (g/cm2)0.83 ± 0.140.84 ± 0.130.85 ± 0.130. 220.83 ± 0.130.82 ± 0.140.80 ± 0.140.09**Lumbar spine (g/cm2)1.03 ± 0.181.04 ± 0.181.01 ± 0.180.691.04 ± 0.181.04 ± 0.191.01 ± 0.200.52Numbern = 820n = 298n = 24n = 773n = 340n = 29FN-BMD change (relative % of baseline year)−0.84 ± 1.11−0.83 ± 1.01−0.78 ± 1.030.85−0.85 ± 1.08−0.80 ± 1.04−0.74 ± 1.490.55Values are expressed as mean ± SD. Adjustments for age and BMI. Femoral neck (FN) BMD change was measured between second follow-up and baseline*P for ANOVA. **For trend linear regression: P = 0.03 Risk of Fracture by COLIA1 Genotypes The relation between risk of fracture and COLIA1 Sp1 polymorphism is shown in Table 3. Women with two copies of the T allele of the Sp1 polymorphism had a 2.3 times higher risk of fragility fracture (95% CI 1.4-3.9, P = 0.001). Adjustment for femoral neck BMD did not essentially modify the association. A similar association was observed in men who were homozygous carriers of the Sp1 T allele, which was borderline significant (risk ratio = 2.3, 95% CI 0.9–5.8, P = 0.07). For the -1997 promoter polymorphism no association was found with any type of fracture in either men or women (Table 3). Table 3Risk of fractures by COLIA1 genotypesCOLIA1 genotypesTypes of fractureEvent (%)Risk ratio (95% CI)GGGTTTGG (reference)GT vs. referenceTT vs. referenceMenPromoter -1997 G/TNonvertebral147/1952 (7.5)49/592 (8.3)1/46 (2.2)1.0 (reference)3.18 (0.45–22.74)3.44 (0.47–29.94)Fragility72/1952 (3.7)14/592 (2.4)1/46 (1.8)1.0 (reference)1.46 (0.20–10.55)0.94 (0.12.7.14)Vertebral100/1056 (9.5)35/340 (10.3)3/28 (10.7)1.0 (reference)1.07 (0.71–1.61)1.23 (0.36–4.18)Intron 1 Sp1 G/TNonvertebral91/1254 (7.3)39/574 (6.8)4/44 (9.1)1.0 (reference)0.97 (0.71–1.33)1.40 (0.65–3.00)Fragility36/1254 (2.9)14/574 (2.4)2/44 (4.5)1.0 (reference)0.94 (0.58–1.51)2.34 (0.94–5.85)Vertebral68/719 (9.5)31/350 (8.9)2/22 (9.1)1.0 (reference)0.83 (0.55–1.24)1.59 (0.60–4.22)WomenPromoter -1997 G/TNonvertebral528/2721 (19.4)182/879 (20.7)8/63 (12.7)1.0 (reference)1.53 (0.76–3.09)1.66 (0.82–3.36)Fragility216/2721 (7.9)73/879 (8.3)4/63 (6.3)1.0 (reference)1.13 (0.42–3.05)1.19 (0.44–3.26)Vertebral159/1320 (12.0)52/445 (11.7)5/35 (14.3)1.0 (reference)0.98 (0.70–1.37)1.31 (0.49–3.45)Intron 1 Sp1 G/TNonvertebral279/1626 (17.2)121/710 (17.0)17/76 (22.4)1.0 (reference)1.05 (0.90–1.23)1.34 (0.92–1.23)Fragility98/1626 (6.0)42/710 (5.9)10/76 (13.2)1.0 (reference)1.01 (0.78–1.31)2.33 (1.39–3.87) Vertebral98/891 (11.0)51/401 (12.7)3/34 (8.8)1.0 (reference)1.21 (0.88–1.65)1.37 (0.56–3.35)Adjustments for age and BMI Haplotype Analysis High LD exists between the -1997 G/T and Sp1 polymorphisms, as assessed by D′ measure (D′ = 0.99, r2 = 0.03). We observed in our population three -1997 G/T, Sp1 G/T haplotype alleles (Fig. 1): haplotype 1 (Gpromoter–GIntron), 69.0%; haplotype 2 (Gpromoter–TIntron), 17.6%; and haplotype 3 (Tpromoter–GIntron), 13.4%. Haplotype 4 (Tpromoter–TIntron) was not present (Fig. 1). The strong LD between the -1997 G/T and Sp1 G/T polymorphism and the virtual nonexistence of haplotype 4 suggests there is absence of ancestral recombination in the region. We observed a borderline significant association (P = 0.06) between haplotype 2 (Gpromoter–TIntron) and femoral neck BMD in women. Women who carried haplotype 2 (Gpromoter–TIntron) had lower (−0.01 mg/cm2) femoral neck BMD compared with those who carried haplotype 1 (Gpromoter–GIntron). Fig. 1Schematic representation of the COLIA1 gene with the structural-1997 G/T polymorphism in the promoter region and G/T Sp1 polymorphism at binding site, with observed haplotype frequencies in the Rotterdam Study The relation between risk of fracture and COLIA1 haplotypes is shown in Table 4. Women with haplotype 2 (Gpromoter–TIntron) had a 2.1 times higher relative risk of fragility fracture (P = 0.03); in men the increase in risk was 2.0 (P = 0.31). These results were essentially unchanged after adjustment for femoral neck BMD. For haplotype 3 (Tpromoter–GIntron) we found no association with any type of fracture in either men or women. Table 4Risk of fracture by COLIA1 haplotypesTypes of fractureMen, OR (95% CI)Women, OR (95% CI)Haplotype 1Haplotype 2Haplotype 3Haplotype 1Haplotype 2Haplotype 3Nonvertebral1 (reference)1.29 (0.55–3.02)0.00 (0.00-∞)1 (reference)1.31 (0.83–2.07)0.61 (0.29–1.29)Fragility1 (reference)2.01 (0.71–5.67)0.00 (0.00-∞)1 (reference)2.12 (1.23–3.66)0.80 (0.29–2.23)Vertebral1 (reference)1.64 (0.62–4.31)1.19 (0.35–4.00)1 (reference)1.23 (0.51–2.95)1.31 (0.50–3.42)Adjustments for age and BMI. Haplotype 1, (Gpromoter–GIntron); haplotype 2, (Gpromoter–TIntron); haplotype 3, (Tpromoter–GIntron) Discussion In this large population-based study, we found that the Sp1 polymorphism influences the risk of fragility fracture in elderly women, with a similar yet not significant effect in men. Similarly, women homozygous for the T allele had 3.8% lower BMD at baseline. The -1997 G/T polymorphism showed no independent effect on fracture risk or BMD levels in both genders. The haplotype analysis showed an association with BMD and fracture in women, which appeared to be driven by the effect of the Sp1 polymorphism. A study in Spain [19] showed that the -1997 G/T polymorphism located in the promoter region of the COLIA1 gene was associated with BMD in postmenopausal women of Spanish origin. In addition, analysis of compound genotypes of the three studied polymorphisms (-1997 G/T, Sp1, and -1663 indelT) suggested that the lowest value for BMD corresponded to GG homozygous at -1997 and heterozygous at the other two loci. Furthermore, in another report, the same group observed a possible functional mechanism for the -1997 G/T polymorphism [25]. Our population-based study suggests there is no independent effect of the -1997 polymorphism on BMD and the risk of fractures. Recently, Stewart et al. [26] examined the three polymorphisms of the COLIA1 gene in forms of haplotypes in postmenopausal women. In contrast with our study, they observed an association between reduced BMD values and the promoter –1997G/T polymorphism. Since the promoter -1997G/T polymorphism is in strong LD with the Sp1 polymorphism, the observed association of haplotype 2 is driven by the SP1 polymorphism. We showed that the association between fragility fractures and Sp1 polymorphism is significant only in women. We also found that the association between fragility fracture and the Sp1 polymorphism was independent of femoral neck BMD. A possible explanation for an increased risk of fracture is the different number of fractures between men and women. There are a higher number of fractures in women compared to men (13.2% in women and 4.5% in men). This suggests that other underlying biological mechanisms beyond BMD levels, such as the role of microarchitecture and composition of mineral crystals in bone tissue, might explain the increased fracture risk [11, 27, 28]. Biomechanical testing of bone samples from heterozygous individuals with the GT genotype showed reduced bone strength compared to the homozygous GG genotype and a slight reduction in mineralization of bone [11]. Presence of the T allele in the COLIA1 Sp1 binding site leads to an abnormal relative level of COLIA1/COLIA2, which may reduce bone quality and quantity [11]. Accordingly, we assume that a weaker network of abnormal collagen cross-linking may generate a three-dimensional unstable condition that may be responsible for its relatively greater risk of fragility fracture in elderly women homozygous for the Sp1 T allele. Similarly, it is likely that the Sp1 polymorphism drives these associations since evidence of functionality of this polymorphism has been reported previously. In the Genetic Markers for Osteoporosis (GENOMOS) Study, which is the largest study examining the Sp1 polymorphism (n = 20,786) in relation to osteoporosis, an association between the Sp1 polymorphism and a 1.3 times incident risk of vertebral fractures was also observed. An effect of the -1997 promoter polymorphism due to power limitations cannot be fully excluded and should be subject to study in a larger population like that of GENOMOS. Our present study has some limitations. Survival bias may play a role if individuals who were lost to follow-up were associated to genotype. Considering this selection bias, a possible relationship of the COLIA1 polymorphisms with changes in BMD cannot be fully excluded. In conclusion, we observed an increased risk of fragility fractures in women carriers of the COLIA1 Sp1 T allele. In contrast, the -1997 G/T polymorphism by itself appears to have no influence on fracture or BMD in postmenopausal women, though the role of power limitations cannot be excluded.

BMC_Struct_Biol-4-_-331416

Consensus structural models for the amino terminal domain of the retrovirus restriction gene Fv1 and the Murine Leukaemia Virus capsid proteins

Background The mouse Fv1 (friend virus) susceptibility gene inhibits the development of the murine leukaemia virus (MLV) by interacting with its capsid (CA) protein. As no structures are available for these proteins we have constructed molecular models based on distant sequence similarity to other retroviral capsid proteins. Background The Fv1 gene is one of a series of mouse genes that control the susceptibility of mice to murine leukaemia virus (MLV) [1-3]. The gene acts in the cell to restrict virus replication [4] through a mechanism that is still uncertain. Genetic studies suggest that the target for the Fv1 gene product is the capsid protein (CA) of MLV [5,6] and it is thought to interact with CA after entry of the virus into the cell but before integration and formation of the provirus. When cloned and sequenced [7], the Fv1 gene was found to have sequence similarity to endogenous retroviruses of the HERV-L and MuERV-L families [7,8]. Based on its position within the Gag gene of these endogenous elements, it appears that Fv1 encodes a capsid-like protein. This structural assignment of the Fv1 gene is consistent with its function as it can be postulated that the gene product might act as a dominant negative mutation and interfere with the MLV capsid function [9]. Sequence alignments have been made between Fv1 and other retroviral capsid proteins [8] but besides one region of clear similarity, called the Major Homology Region (MHR), there is otherwise little that is conserved across the full family of retroviral (and related lenti-virus) CA sequences. There are now several known structures for retroviral capsid proteins in the Protein Databank (PDB). While some of these are only fragmentary, a selection can be extracted that gives a reasonable phylogenetic spread across the retroviruses, with examples from three out of six genera of orthoretroviruses. In all the known structures, the CA protein has an all-α type structure consisting of two domains: a larger N-terminal and smaller C-terminal domain with a short extended linker-region between them. As this linker enters the C-terminal domain it incorporates the MHR. There is considerable variation in the orientation of the domains and in the conformation of the loop-regions between α-helices, particularly in the N-terminal domain. In this work, we have exploited these multiple structures to construct consensus molecular models using threading methods both for the Fv1 gene product (FV1) and its target protein, the MVL CA. As threading takes known and predicted structure into account, it should provide better alignments for the regions that lie outside the MHR. However, as these methods are still far from perfect, we have constructed a model based on each known structure and the degree to which these agree has been used to assess the confidence of different parts of the model. As the threading method we have used has some 'free' parameters (such as the gap-penalty) we have introduced a novel modelling strategy in which the parameters are varied to give maximum agreement among the resulting models. Results and Discussion The sequence alignments compiled on the proteins of known structure using the Ψ-BLAST/QUEST search strategy (Methods Sectn.) were used in the modelling of both the MLV CA and FV1 sequences. These varied from 4 to 7 sequences (Table 1). Not unexpectedly, the alignments are to some extent similar, in particular each contains the sequence of the HIV-1 CA structure [1e6jP]. (QUEST is biased to retain sequences of known structure). Greatest overlap exists between the sequence sets of the two human viruses, with the HTLV-I sequences being a subset of the HIV-1 sequences (Table 0(d) and Table 0(c)). While it would be possible to extend and realign these sub-families based on structure comparisons, they were left unaltered so as to be equivalent to the MLV and FV1 alignments described below. This allows control modelling tests to be directly comparable to those performed for sequences of unknown structure. Table 1 Template sequences selected for alignment. The Ψ-BLAST/QUEST search strategy (Methods Sectn.) when started with the probe sequence of the PDB structure indicated by "SEED" selected the sequences indicated in each subtable: (a) EIAV [1eia], (b) RSV [1d1dA], (c) HIV-1 [1e6jP] and (d) HTLV-I [1qrjA]. The sequences are identified by their gene identification (gi) number (first column) and their local source databank identifier. The sequence fragment (automatically extracted by QUEST) is given as a range of residue numbers. (a) EIAV (b) RSV SEED 1eia 12084543 pdb-1E6J 0–210 6358699 gb-AAF07324 131–342 8072301 gb-AAF71968 0–155 6815746 gb-AAF28696 0–173 6649692 gb-AAF21520 5–224 12084543 pdb-1E6J 3–210 120850 sp-P18041 97–362 27803398 gb-AAO21890 120–281 294961 gb-AAA74706 116–381 5106563 gb-AAD39752 81–346 SEED 1d1dA (c) HIV-1 (d) HTLV-I 6358699 gb-AAF07324 129–340 SEED 1qrjA 22037894 gb-AAM90230 148–359 12084543 pdb-1E6J 0–210 SEED 1e6jP 22037894 gb-AAM90230 144–370 532325 gb-AAA99545 50–224 9886907 gb-AAG01643 0–222 9886907 gb-AAG01643 0–211 MLV Modelling The databank search using the MLV sequence as a probe provided a useful collection of six sequences (Table 1(a)) which, with the MLV probe sequence itself, were passed through the PsiPred secondary structure prediction protocol (Methods Sectn.). When the predicted secondary structures were viewed on the aligned sequences, several distinct α-helices were apparent, consistent with the protein having an all-α type structure similar to the other capsid proteins of known structure (Figure 1). The MLV alignment with predicted secondary structure was matched in the MST program with the four capsid proteins of known structure along with their associated aligned sequences. This was done for each parameter combination specified in Methods Sectn. and the quality of the agreement among the four resulting models quantified using both the Fu and Fw measures defined in Methods Sectn. There was reasonable overall agreement between the two measures of the parameter combinations (Figure 2) and with otherwise no basis on which to prefer one measure over the other, the combined measure (Fv) was used to select the parameters on which the final models were calculated. These were S = 7, G = 30. Figure 1 MLV sequence alignment with the sequence of 1qrjA and related sequences. The alignment is displayed twice in different colouring schemes. In the top two panels, (a) the amino half is coloured firstly by predicted secondary structure (red = α, green = β) and then below using a different colour for each amino acid type (Taylor, 1997; black = gap). The known helices (N1–N5) marked as red lines (minor helices are orange) between the two blocks. In the lower two panels (b) the carboxy half of the alignment is shown in a similar way. The sequence identifiers are given below the alignment in the same order as they are aligned. The mid-line divides the 1qrjA sub-family from the MLV sub-family. Figure 2 MLV model agreement. The degree of similarity among the four MLV CA models is plotted for different parameter combinations of MST. Combinations resulting in better agreement have colours towards the red end of the spectrum. (a) Using the unweighted RMSd measure (Fu) and (b) Using the weighted RMSd measure (Fw). (See Methods Sectn. for details.) (c) Combined score (Fu·Fu). The MST parameters varied were X-axis: S = 0→9 (in steps ofone) and Y-axis: G = 10→90 (in steps of 10). (See Methods Sectn. for details.) The best combined score is at: S = 7, G = 30. N-terminal domain The multiple structure alignment of the four models showed good agreement. In the N-terminal domain there were two extensive regions over which all models were aligned in register, covering helices N2-N3 and N5 (including the preceding short helix in the loop region). Two alternate registers were observed for helices N1 and N4, with relative shifts of 3 and 4 residues, respectively. The models based on 1qrjA (HTLV-I) and 1d1dA (RSV) were in complete agreement and the summed Fw score indicated that 1d1dA provided the best consensus model. Given that the alignment of the capsid protein sequences is ambiguous, the superposition of the models on the structures from which they were derived provides a better way to assess whether there is any significant sequence similarity that could be used as a basis on which any one model might be preferred over the others. The PSId values were: 5.6, 18.5, 10.5, 13.0 for 1d1dA, 1qrjA, 1eia and 1e6jP, respectively. (No differences were observed whether using the standard version or the sequence-biased version of SAP). Although the 1d1dA model was the best consensus representative, it has only 5.6% sequence similarity with MLV and the higher sequence similarity of the 1qrjA based model with its template (18.5%) was considered sufficient to justify its adoption as the preferred model over the 1d1dA model. Structurally, both were very similar (1.4 Å wRMSd) with the only significant structural difference being a slight reorientation of the helical region in the loop preceeding the final helix. This structure for the 1qrjA based model is shown along with its comparison with the 1d1dA model in Figure 3. Figure 3 Consensus model for the MLV N-domain. (a) The model for the MLV CA N-domain based on 1qrjA shown as a α-carbon trace with predicted α-helices coloured red (some fragments of predicted β-structure are coloured green). The molecule is in approximately the same orientation as in Figure 8(a) and residues identified by Stevens et al. (2003) are marked as small spheres. (b) The models based on 1qrjA and 1d1dA are shown superposed and coloured from blue (amino) to red (carboxy). Feint dashed lines connect identical residues. The wRMSd = 1.4 (uRMSd = 5.8) over 123 residues. It can be seen from Figure 3(a) that there is good location of the predicted and model helices with deviations occurring only at the ends of some helices and into the loop regions at the 'top' of the molecule. The ends of helices N4 and N5 and the loops at this end of the molecule also encompass the mutations identified as affecting the sensitivity of the virus to Fv1 [29]. C-terminal domain With its relatively unambiguous MHR, all the models of the C-terminal domain were in complete agreement over the first half of the domain. The more C-terminal half, however, was less consistent due to a combination of its generally less conserved nature combined with uncertainty in the location of the terminus in some of the sequences. As the C-terminal domain has been shown to be less important in determining the property of virus susceptibility, further effort was not expended to try and refine the alignment at the carboxy terminus of the molecule, especially in the more difficult alignment of the FV1 sequence described below. FV1 Modelling The databank search using the FV1 sequence as a probe provided a less useful collection of only two distinct sequences (Table 1(b)). Although other sequences were found these were rejected by QUEST as being too similar to those retained. The FV1 alignment with predicted secondary structure was matched to the four capsid proteins of known structure as described above for each MST parameter combination and agreement among the four models monitored using the F scores (Methods Sectn.). These were 50.3 and 131.874 for Fu and Fw while the corresponding score obtained for the MLV models were 78.7 and 188.0, respectively. The greater variation among the FV1 models was undoubtedly due to uncertainty in the placement of the large 'unstructured' region. Nevertheless, there was reasonable overall agreement between the two measures and both found maximal model agreement when S = 9, G = 20 (Figure 4). Figure 4 FV1 model agreement. The degree of similarity among the four FV1 CA models is plotted for different parameter combinations of MST. (a) Using the unweighted RMSd measure (Fu) and (b) Using the weighted RMSd measure (Fw). (See Methods Sectn. for details.) (c) Combined score (Fu·Fu). The MST parameters varied were X-axis: S = 0→9 (in steps of one) and Y-axis: G = 10→90 (in steps of 10). (See Methods Sectn. for details.) The best combined score is at: S = 9, G = 20. When the predicted secondary structures were viewed on the aligned sequences, although several distinct α-helices were apparent, these had a less direct correspondence with those expected for a capsid protein (Figure 5). In particular, there is little predicted α-structure for helix N4 and the alignment around N5 is ambiguous. If the secondary structure prediction were to be believed, this might indicate the introduction of a large insertion, or given that there were less sequences on which to base the predictions, it is possible that the predictions are not as accurate as those obtained for the MLV sequences. Figure 5 FV1 sequence alignment with the sequence of 1e6jP and related sequences. The alignment is displayed twice in different colouring schemes. In the top two panels, the amino half is coloured by predicted secondary structure (red = α, green = β) and amino acid type (Taylor, 1997). See the legend to Figure 2 for further details. with the known helices (N1–N5) marked as red lines between (minor helices are orange). The carboxy half of the alignment is displayed in a similar way in the lower panels. The sequence identifiers are given below the alignment in the same order as they are aligned. The mid-line divides the 1e6jP sub-family from the FV1 sub-family. N-terminal domain The multiple structure alignment of the four models showed unexpectedly good agreement. As in the MLV models, two regions were aligned in register over all the models. These included most of helix N4 and the following loop region then, after only one shift, the full register was regained for helix N5. The sum-of-F scores for each model indicated that 1eia provided the best consensus, followed by 1e6jP (to which it was most similar). The PSId values were: 9.3, 7.8, 6.6, 15.9 for 1d1dA, 1qrjA, 1eia and 1e6jP, respectively. As the 1e6jP model aligns better on its target by almost 10 PSId, this was considered sufficient to justify its adoption as the preferred model over the 1eia based model. Both models are similar in the core region (1.3 Å wRMSd) but with the significant differences in the extensive loop regions at the 'top' of the molecules. This structure for the 1e6jA based model is shown along with its comparison with the 1eia model in Figure 6. Figure 6 Consensus model for the FV1 N-terminal domain. (a) The model for the FV1 N-domain based on 1e6jP shown as a α-carbon trace with predicted α-helices coloured red (some fragments of predicted β-structure are coloured green). The molecule is in approximately the same orientation as in Figure 8(a). (b) The models based on 1e6jP and 1eia are shown superposed and coloured from blue (amino) to red (carboxy). Feint dashed lines connect identical residues. The wRMSd = 1.3 (uRMSd = 5.4) over 124 residues. Note: the modelling program rescales the secondary structure prediction values so that there is the same proportion of predicted structure as measured secondary structure on the model. It can be seen from Figure 5 and Figure 6(a) that there is reasonable location of the predicted and model helices except for helix N4 which is almost completely unpredicted. Otherwise deviations occurred at the ends of helices and in the loop regions at the 'top' of the molecule. Table 2 Target sequences selected for alignment. The Ψ-BLAST/QUEST search strategy (Methods Sectn.) when started with the two target sequences indicated by "SEED", selected the sequences indicated in each subtable: (a) MVL and (b) FV1. The sequences are identified as in Table 1. (* the C-terminal domain of this sequence appears to be replaced by an oncogene.) (a) MLV SEED AAD55051 215–432 gi-120892 sp-P03330 207–423 gi-2393894 gb-AAC58239 206–434 gi-419481 pir-A46312 199–423 gi-323873 gb-AAA43041 203–418* gi-7548235 gb-AAA4306 206–422 gi-5726238 gb-AAD48375 156–352 (b) FV1 gi-7521942 T29096 gi-23485357 gb-EAA20381.1 gi-3913713 sp-P70213 FV1 MOUSE Control Models To help assess the accuracy of the models constructed above, the protocols described in the Methods sections were applied to each of the sequences of known structure. For each protein, this results in four models based on the other structures (and its own structure). Each of these models, was then compared to the known structure using the modified SAP program and the RMSd/PSId measures reported in Table 3. Table 3 Control model similarity. The similarity of the capsid protein domains are tabulated as: uRMSd/PSId, both as calculated by the SAP program. The proteins are identified as in Table 4. The column "params" gives the MST parameter values S, G at which the four models in each row had maximum agreement as measured by the combined agreement score Fv (Methods Sect n.). str \ seq D1D QRJ E6J EIA params D1D 0.60/78.9 4.14/90.0 2.05/56.7 2.41/38.3 9,10 QRJ 5.58/100. 0.32/100. 3.58/88.4 5.51/100. 3,10 E6J 1.88/95.2 1.70/84.7 0.31/100. 1.39/100. 7,10 EIA 2.00/93.3 2.00/100. 1.28/100. 0.35/100. 8,20 With few exceptions, the wRMSd/PSId values indicated generally good models and by visual inspection, all had the correct fold. The models based on their own structures were almost identical to their templates with the exception of 1d1dA. When the uWRMSd values of the models against the known structure are plotted against number of residues ranked by local score (Figure 7) it can be seen that for 1d1dA the error rises sharply after 85 positions. Visual inspection revealed that this was due to a misaligned segment in the large loop region. The majority of models exhibited a gradual increase in wRMSd with increased error mounting as the loop regions were introduced. Two models that ran markedly below this trend were those based on 1eia and 1e6jP (22% identity), the two most similar proteins. A few models were also markedly worse. Two of these were derived from the 1qrj alignment based models which had a distinctly lower weight on the structure component (S weight) in the MST alignment. Figure 7 Control model uRMSd values. Each model was compared with its known structure and the local residue matches calculated by SAP were ranked. The uRMSd was calculated for increasingly larger sets of ranked residues and plotted against set size. This means that the best fitted residues in each comparison are found to the left of the plot with increasing divergence towards the right. Four models are plotted for each of four CA proteins of known structure: 1d1dA = red, 1qrjA = green, 1eia = purple, 1e6jP = blue. The best models are those of sequences built on their own structure. Three of these remain low throughout their length while one has poor loops and rises towards the right. Above these are two models for the most similar sequences of 1e6jP and 1eia built on each others structure (22% identity). Most models lie in the mid range with a few accumulating early errors due to shifts in the core regions. Conclusions Based on relative degrees of sequence similarity among the control models (Table 3(b)) and the MLV and FV1 sequences (MLV:1qrj = 18.5%, FV1:1e6jP = 15.9%), it would be expected that the models constructed for the MLV sequence would fall in the mid-range of the spread in quality observed in Figure 7 – typically, a good core model (3 Å RMS over 80 residues) with increasing divergence in the more variable loop regions. This is where the majority of the control models lie which were all constructed from sequence similarities that are generally lower than either of the above relationships used to model the MLV or FV1 sequences. While a similar confidence might be hoped for the FV1 model, given its overall lower sequence similarity to the proteins of known structure and the less consistent nature of its secondary structure prediction, it is more likely that it will be of lesser quality – corresponding more to the poorer models constructed among the control proteins (Figure 7). Typically, this would include shifts in the core helices (giving the characteristic immediate rise in the traces in Figure 7) with further shifts in the loop regions. Despite this, as with all of the control models, it is likely that the core fold of the protein should remain unaltered. This study has shown that reasonable models can be constructed for both the FV1 and its target MLV protein based on other retroviral capsid proteins. Although this result was suggested by the existence of the MHR in both sequences, the fluid nature of retroviral genomes does not necessarily constrain the preceding domain to remain constant in structure or even remain at all. Despite only weak sequence similarities in this region, the addition of multiple sequences with predicted secondary structure has allowed plausible models to be constructed. These models can now be used in the interpretation of experimental studies on the mode of action of retroviral susceptibility. As will be reported in more detail elsewhere [29], a series of amino acids in CA affecting the CA – FV1 interaction have been identified in the loops at the 'top' of the N-terminal domain (Figure 3). Based on the model, they suggest a potential FV1 binding domain in the MLV CA. Experiments are currently under way to test this prediction by crystallographic studies. For many years, the Fv1 gene has been the only known intracellular non-immune natural defense against a retroviral infection. Recently, two additional genes, Ref1 and Lv1, with antiviral activity have been described in human cells [30,31]. Phenotypically, they resemble Fv1 [32] but the genes themselves remain to be characterised. Understanding the mechanism of Fv1 action will provide insights into how natural defences to retroviral infection might be deployed against HIV. Methods and Data Data Sequence Data All sequences were extracted from (and searches were made over) the non-redundant protein sequence databank (NRDB) at the National Centre for Biotechnology Information (NCBI) as it was found on 28th of January 2003. The sequence of the MLV used was the gag protein AAD55051 (GI:5881091) [10] and a region was extracted from residues 215–432, corresponding to the CA protein. The sequence of the Fv1 gene [7] was taken from FV1_MOUSE (GI:3913713). The region corresponding to the CA was identified as residues 100/120–340/360 where the inner numbers represent the probable core of the protein. This range corresponds to the region of highest similarity to the MuERV-L sequence [8]. The leading 100 residues of the polyprotein may correspond with a relic matrix protein. Perhaps because of this, there is no obvious protease cleavage motif [11] to give any indication of the true terminus. However, in other situations this has not always been an accurate guide [12]. Structural Data The structures of capsid proteins were extracted from the PDB [13] with the aid of the FSSP structure comparison database [14]. Of the six structures in the FSSP alignment, only four extended over the full length of the two structural domains. There were as follows, with their PDB code (and chain delimiter, if any) shown in brackets: Rous Sarcoma Virus (RSV) [1d1dA] [15], Human T-cell Leukemia Virus (HTLV-I) [1qrjA] [16,17], Equine Infectious Anemia Virus (EIAV) p26 [1eia] [18] and Human Immunodeficiency Virus (HIV-I) p24 [1e6jP] [19]. The common core of the N-terminal domains of these proteins (in the numbering of the PDB structure) was defined as: 1d1dA 15–148, 1qrjA 16–129, 1eia 16–145 and 1e6jP 16–146. These fragments will be distinguished below as: 1d1dAn, 1qrjAn, 1eia-n and 1e6jPn and each terminates 8 or 9 residues before the conserved glutamine of the MHR. The N-terminal domain can be described as having five α-helices (N1...N5) with a long 'disordered', partly helical, loop between helices N4 and N5. For ease of reference below, this region will be called the 'top' of the molecule and its representation in the Figures will preserve this orientation. The C-terminal domains were defined as: 1d1dA 152–224, 1qrjA 132–204, 1eia 149–220, 1e6jP 149–220 and were distinguished by the suffix "c". The common core of this domain consists of an extended strand leading into the MHR region followed by four helices designated C1...C4. Despite their different sizes, both the N and the C domains have the same fold, perhaps suggesting an ancient gene duplication. This is most obvious in the HIV structure [1e6jP] where the domains can be superposed with 4.6 (2.0) unweighted (weighted) RMSd over 68 residues. The similarity of sequence and structure over these domains was calculated using the SAP structure comparison program [20] for each pair of like domains (Table 4). The N-terminal domains have clearly related structures (3–6 Å RMSd) but have no significant sequence similarity (over 20%) except for the EIAV and HIV-1 structures. The smaller C-terminal domain (which contains the MHR) has greater overall sequence and structural similarity compared to the N-terminal domain (mostly over 20% with 2–3 Å RMSd) and over 40% for the EIAV/HIV-1 pair. Table 4 Capsid protein similarity. The similarity of the capsid protein domains are tabulated as: (a) RMSd (unweighted) and (b) PSId, both as calculated by the SAP program. Upper-right triangle: over the common core of the C-terminal domain and Lower-left triangle: over the common core of the N-terminal domain. The proteins are identified as: D1D = RSV [1d1dA], QRJ = HTLV-I [1qrjA], E6J = HIV-I [1e6jP], EIA = EIAV [1eia ]. (a) structure (b) sequence N \ C D1D QRJ E6J EIA N \ C D1D QRJ E6J EIA D1D 2.88 2.64 2.46 D1D 21.9 22.2 14.7 QRJ 3.83 2.11 2.39 QRJ 12.5 30.6 26.4 E6J 6.21 4.76 1.40 E6J 8.6 10.8 41.7 EIA 5.75 4.31 3.22 EIA 11.6 12.5 22.0 Sequence Databank Searches Initially, each probe sequence was compared against the sequence databank using the Ψ-BLAST program [21] with a significance level set at 0.001 and 5 cycles of iteration. When the probe is a retroviral sequence, the number of hits found by Ψ-BLAST can be large (typically over 1000). These were reduced to manageable numbers by the use of the search program QUEST which is similar to Ψ-BLAST but incorporates a multiple sequence alignment stage in its iterations to exclude redundant sequences as well as excluding poorly related or incomplete sequences [22]. The alignments produced by QUEST typically contain between 6–12 sequences (including the probe sequences), none of which have more than 60% sequence identity (PSId) with each other. The sequences retained by QUEST are selected on the basis of associated biological information, with those including useful annotation and structural data being given preference over those with no annotation or keywords such as "hypothetical". The filters are part of the MULTAL sequence alignment program [23] which are fully described in Ref. [24]. Secondary Structure Prediction The multiple alignments resulting from the Ψ-BLAST/QUEST search protocol were passed to the program PsiPred , Version 2.3) [25]. This program normally performs its own databank searches using Ψ-BLAST to build-up an alignment. Given the problems described above that arise when searching with retroviral sequences, the PsiPred program was used locally to search only a database consisting of the sequences that had already been selected by QUEST. Each sequence in the alignment was taken in turn and used as a probe against this small local database. As the Ψ-BLAST parameters used by PsiPred were more restrictive than those used in the full search (only 3 cycles) and there are fewer sequences in the databank, each sequence may only find those to which it is more closely related. This introduces some variation into the predictions which provides a useful indication of the confidence of each predicted secondary structure element (SSE). Multiple Sequence Threading The alignment gathered on the probe sequence was then aligned with a protein structure using the multiple sequence threading MST program [26]. This program uses multiple sequence and structural information to automatically construct an α-carbon molecular model for the probe sequence with some limited remodelling in regions of insertion and deletion. Template Sequence Alignments The MST program can incorporate multiple aligned sequences along with both the probe sequence and the template structure. The latter were gathered in an identical manner to the probe sequence using the Ψ-BLAST/QUEST search protocol described above. Each search against the NRDB was started with the sequence of the protein of known structure and the resulting multiple alignments examined 'by-eye' in the light of the known secondary structures. If any large insert had been made in a secondary structure element (SSE) then it was assessed whether the gap could be shifted outside the SSE without significant loss of residue matches. Similarly, if a large insert (more than 6 residues) was made by any sequence other than the probe sequence (of known structure) then the insert was reduced to six residues by removing the positions with most gaps. Parameter Choice The MST program has parameters that allow different weights to be attached to the matching contribution of the sequences, their secondary structures, residue exposure and the residue packing in the resulting model. There is also a gap-penalty. The best values for these weights depends on the number and degree of relatedness among both the probe and the template sequences [26]. Rather than vary all these parameters individually, the weights on the structural components (secondary structure, exposure and packing) were 'ganged' together into a single parameter reflecting the contribution of structural terms relative to the sequence matching component. This gave two parameters: S (for structure) and G (the gap-penalty). Previously, the structural parameters had all been scaled into the same range so a value of S = 3 corresponds to a value of 3 for each individual weight. Although the gap-penalty is correlated with S, it cannot be linked in the same way without the risk of missing good alignments. In the current application, there was more than one available template structure and advantage was taken of this by constructing models based on all available templates and choosing the MST parameters such that the agreement among the models was greatest. The parameters were varied over the ranges: S = 0→9 (in steps of one) and G = 10→90 (in steps of 10). Measuring Model Agreement Whatever the parameters for MST, all the models constructed from the same probe have an identical sequence. These might therefore be compared using the α-carbon RMSd based on a one-to-one (100 PSId) sequence equivalence. However, using this simple measure, a 'trivial' shift in space in which, say, an α-helix shifts by one turn relative to another α-helix might result in a large RMSd between what are, topologically, similar models. It is better to allow a local relative shift in sequence of four residues to restore the spatial equivalence at the expense of residue identity. To implement this trade-off between RMSd and PSId, the models constructed for each parameter combination were compared against each other using the program SAP [20]. This program calculates both a weighted (Rw) and unweghted (Ru) RMSd for the two structures being compared and reports the percentage sequence identity of the alignment. The weighted RMSd down-weights regions of weak similarity which are mainly loop regions that can have large relative displacements. Despite its origins [27], in its current implementation the SAP program does not include a sequence matching component and this was restored (for sequence identity only) by doubling the local residue pair score for identical residue types and otherwise halving all other residue match scores in the initial score matrix. A score reflecting match quality (f) was calculated as: f = M/(1+R), where M is the PSId measured over the positions aligned by SAP and R is one of the RMSd measures. Identical structures would score 100. For a set of N models, a sum was calculated over the (N2 - N)/2 pair combinations giving an overall measure of agreement (F) among the set. For a set of four models that align perfectly (100 PSId) with 2 Å RMSd, the overall score obtained would be 200. This score was calculated for both the wRMSd and uRMSd values (giving Fw and Fu, respectively) and a combined score (Fv) as the product of Fw and Fu. While this procedure provides a general method for choosing parameter values, in the current application to a multi-domain protein it was not meaningful to calculate the RMSd over the full atomic model (because of relative domain movements). Instead, the agreement was calculated over the more distantly related N-terminal domain. Selecting a consensus model Although any model in the set could be taken as a representative, it is best to try and select one that, by some criteria, can be considered to be the most representative. To do this, we compared each pair of models usuing the structure comparison protocol described in the previous section. This provides a pairwise alignment based on structure, and even though each model has an identical sequence, the structural alignment may not match-up identical residues. The pairwise alignment were then combined into a multiple structure alignment [28] and as the models all have an identical sequence, their relative shifts can be seen easily. Rather than use a pure structure or sequence based measure of similarity between the proteins, the score F was devised in the previous section (Methods Sectn.) that combines both a sequence and a structural component. This was used to find the model with the greatest sum-of-scores to the others. An alternative selection test was also considered of selecting the model that had greatest sequence similarity when superposed with the template structure from which it was derived. As most of the sequence similarities considered below lie in the 'twilight-zone', the latter option was only used when one model was clearly better than the others. For this, we choose the criterion that it had to be 10 PSId points clear of its 'rivals'. Abbreviations Fv1/FV1, gene/gene-product of Friend Virus susceptibility locus-1; MLV, Murine Leukaemia Virus; CA, CApsid protein; HERV-L, Human Endogenous RetroVirus (L family); MuERV-L, Murine Endogenous RetroVirus (L family); MHR, Major Homology Region; NCBI, National Centre for Biotechnology Information; NRDB, Non-Redundant DataBank; MST, Multiple Sequence Threading (program); SSE, Secondary Structure Element; PSId, Percent Sequence Identity; PDB, Protein DataBank; RMSd, Root-Mean Square deviation; wRMSd, weighted Root-Mean Square deviation; uRMSd, unweighted Root-Mean Square deviation; Figure 8 Capsid protein domains. The structures of the common core of the capsid proteins are shown using the RSV protein [1d1dA] as a representative. (a) The N-terminal domain, coloured blue→red from amino→carboxy termini with the five major helices represented by thickened lines. (b) The C-terminal domain, represented as in part (a) but with the MHR region marked in white.

Cardiovasc_Intervent_Radiol-3-1-2062488

Embolization with the Amplatzer Vascular Plug in TIPS Patients

Vessel embolization can be a valuable adjunct procedure in transjugular intrahepatic portosystemic shunt (TIPS). During the creation of a TIPS, embolization of portal vein collaterals supplying esophageal varices may lower the risk of secondary rebleeding. And after creation of a TIPS, closure of the TIPS itself may be indicated if the resulting hepatic encephalopathy severely impairs mental functioning. The Amplatzer Vascular Plug (AVP; AGA Medical, Golden Valley, MN) is well suited for embolization of large-diameter vessels and has been employed in a variety of vascular lesions including congenital arteriovenous shunts. Here we describe the use of the AVP in the context of TIPS to embolize portal vein collaterals (n = 8) or to occlude the TIPS (n = 2). Portal vein collaterals supplying esophageal varices may give high risk for secondary rebleeding. Those collaterals might require embolization to decrease this risk, despite transjugular intrahepatic portosystemic shunt (TIPS) placement. Embolization of the collaterals may be particularly difficult when these vessels are large. Moreover, after creation of a TIPS, closure of the TIPS itself may be indicated when hepatic encephalopathy severely impairs mental functioning. The Amplatzer Vascular Plug (AVP; AGA Medical, Golden Valley, MN) is a device well suited to embolize large vessels and has been employed in a variety of vascular lesions such as pulmonary arteriovenous malformations and congenital shunts [1, 2]. This report describes the use of the AVP in the context of TIPS to embolize portal vein collaterals (n = 8) or to occlude the TIPS (n = 2). Description of Procedures Embolization of Portal Vein Collaterals Eight patients were referred for TIPS, seven because of multiple recurrent bleeding episodes from esophageal varices (n = 5), gastric fundus varices (n = 1), or both (n = 1) that could not be managed by endoscopic therapy. One other patient (n = 1) was in hypovolemic shock because of recurrent massive bleeding from rectal hemorrhoids. Three TIPS procedures were elective and five were done as emergency procedures for uncontrolled bleeding after initial hemodynamic stabilization had been achieved using blood transfusions, endoscopic treatment, intravenous octreotide infusion, and/or placement of a Sengstaken double balloon tube, alone or in combination. TIPS procedures were carried out under general anesthesia. As an adjunct procedure, we obliterated the portal vein collaterals supplying the bleeding varices using AVP embolization (Figs. 1 and 2). This was done either before or after creation of the TIPS based on operator preference and clinical circumstances. Embolization early in the procedure with early control of the potential bleeding site was considered relevant in emergency procedures in hemodynamically unstable patients. Also, at least theoretically, AVP embolization may be done more easily prior to TIPS whenever the collateral target vein is still widened because of portal hypertension and easily opacified because of preferential flow. After the creation of a TIPS both these phenomena may have been altered. Fig. 1Portal venous angiogram in a 53-year-old patient referred for TIPS creation because of recurrent variceal bleeding not controlled by medication plus multiple treatments with endoscopic band ligation and sclerotherapy. The varices are supplied by a single coronary veinFig. 2The same patient as in Fig. 1 immediately following TIPS creation and AVP embolization of the coronary vein (a; thin arrow). The unsubtracted image (b) shows the radiopaque sclerosing agent from prior endoscopic sclerotherapy (fat arrow) in the esophageal varices Whenever AVP embolization preceded TIPS placement, the guiding catheter for AVP delivery was introduced transhepatically into the right portal vein. At our institution the portal vein is routinely catheterized prior to TIPS with a 3- or 4-Fr catheter to visualize the portal vein as a fluoroscopic target for transjugular transhepatic puncture. For transhepatic AVP placement this catheter was exchanged for a 6- to 8-Fr hepatic introducer sheath. After AVP embolization and initial creation of the hepatic-to-portal vein shunt tract for TIPS, the hepatic introducer sheath was withdrawn in multiple small successive steps over the course of the remainder of the procedure, so that a solid sealing clot could form in the hepatic parenchymal tract. No additional embolization of the tract was performed. Whenever vein embolization was performed after the creation of a TIPS, AVP placement was done via the transjugular route. In all patients, the large-diameter coronary/gastric vein supplying extensive esophageal/gastric varices was embolized using a single AVP plug 10, 12, or 14 mm in diameter. For placement, a 6- to 8-Fr guiding catheter was first negotiated into the proximal part of the coronary vein. Then the AVP device was advanced through the guiding catheter and initially deployed by retracting the guiding catheter but without detaching the device. A control injection was made, and if needed, the AVP was retrieved into the guiding catheter and repositioned. When in a satisfactory position, the AVP was detached by anticlockwise rotation of the AVP guide wire to unscrew it from the AVP device proper. Within minutes of deployment, the collateral flow through the coronary vein had completely stopped. TIPS creation was done by placement of e-PTFE-covered stent-grafts (Viatorr; Gore, Flagstaff, AZ) 8–10 mm in diameter and 6 ± 2 cm long, resulting in a postprocedural portosystemic pressure difference of 5–10 mm Hg. All patients were hemodynamically stable immediately after the procedure. During follow-up there were no recorded episodes of rebleeding with a follow-up of 1 year maximum. The patient who was treated for bleeding rectal hemorrhoids died after 3 days because of combined liver and respiratory failure. Another patient who received emergency TIPS while in hypovolemic shock due to massive unstoppable bleeding from esophageal varices died 46 days after the procedure because of liver and kidney failure complicated by ileus. TIPS Occlusion A 47-year-old male was referred for closure of a TIPS that had been created 3 years earlier for alcoholic Child A liver cirrhosis with recurrent episodes of esophagus variceal bleeding. He now presented with problems of amnesia, difficulties concentrating, and a general, overwhelming feeling of tiredness. Spectral EEG findings were consistent with grade 2 hepatic encephalopathy. The symptoms had not improved using conservative treatment. At angiography, a patent TIPS was seen, with a portosystemic pressure difference of 10 mm Hg. Closure of the TIPS with a 14-mm AVP device (Fig. 3) resulted in a dramatic improvement in cognitive functioning and normalization of the spectral EEG. Two years after closure of the TIPS, the patient is still free of variceal bleeding. Recently, however, he developed esophageal varices grades I–II, which are currently treated with band ligation. This will be repeated every 2 weeks until complete obliteration. Fig. 3AVP device for TIPS occlusion in a patient with life-incapacitating hepatic encephalopathy. The 14-mm-diameter AVP is positioned through a guiding catheter in the central portion of the 10-mm-diameter e-PTFE stent-graft (a). By retraction of the guide wire, the unconstrained AVP sets in the intended place (b). At this point, the AVP can still be retracted into the guiding catheter if in an unsatisfactory position. The AVP is fully deployed by unscrewing the guide wire from the AVP device proper (c) Another patient, a 65-year-old male, had been referred for TIPS placement for refractory ascites following liver transplantation, which required weekly paracentesis. In the days following placement of a 10-mm-diameter e-PTFE-covered stent-graft TIPS, liver failure developed, necessitating its closure. Closure of the TIPS was technically successfully achieved using a 16-mm-diameter AVP but did not substantially improve the clinical status. Because of liver failure and refractory ascites, the patient underwent a second liver transplantation 2 days after TIPS occlusion. Discussion In this report we have described the use of AVP embolization in the context of TIPS. For closure of collateral veins supplying bleeding varices, we consider the AVP a useful device in a defined subset of patients. The AVP’s relative advantages are its efficiency to occlude large-diameter vessels in a single-step procedure and its reliability of placement. Unlike standard embolization coils, the AVP can be positioned precisely, its position can be checked with contrast injections before actual detachment, and it can be repositioned if required. Migration of the AVP during or after deployment is unlikely. Disadvantages of the AVP include the higher cost of the device itself and of the additionally required guiding catheter. Furthermore, the 6- to 8-Fr guiding catheter markedly increases the bulk of the introducer system relative to the 4- to 5-Fr catheter alternatively used for coil embolization. In our practice we are inclined to use the AVP whenever there is a single, large-diameter collateral vein which would otherwise require the insertions of multiple embolization coils and/or whenever accurate placement of the embolic device close to the portal vein may be critical. Embolization of portal vein collaterals to esophageal varices may be indicated even after successful portal venous decompression by creation of a TIPS. Especially in the life-threatening emergency situation of acute esophageal bleeding in hemodynamically unstable patients, we feel more confident providing additional control of the bleeding site by hemodynamically separating the weak-walled varices from the high-flow and potentially high-pressure portal venous bed. TIPS has proven efficient in the secondary prevention of variceal bleeding but this protection is not absolute. The rebleeding rate of TIPS alone (without embolization) has been reported as between 9% and 40.6%, with a median of 18.4% [3]. These values apply to bare-stented TIPS and initial experience suggests that the rebleeding rate is lower with e-PTFE-covered stented TIPS [4]. Whether or not performing embolization of the portal vein collaterals as an adjunct procedure provides additional protection against rebleeding is unknown. Under the assumption that it does, we routinely combine TIPS with embolization of all venous collaterals supplying the esophageal varices when creating a TIPS in the context of variceal bleeding. Another potential use for the AVP device is for closure of the TIPS itself, in circumstances where hepatic encephalopathy and/or liver failure because of the shunt may prove to be life-incapacitating. Here, the effectiveness of the AVP in occluding large-diameter high-flow vessels, its resistance to migration, and its ease of placement are particularly helpful. A potential concern when occluding a TIPS is the acute increase in portal venous pressure, which can lead to recurrence of variceal bleeding and ascites and, also, to hemodynamic changes with increased cardiac loading [5]. As mentioned, we routinely place a transhepatic catheter into the portal vein as a target for puncturing the portal vein during TIPS creation. In our experience, this increases the control over the exact entrance point into the portal vein and also reduces the number of puncture attempts while creating the TIPS tract. We have found that the reduced procedure time for the TIPS procedure proper more than makes up for the extra time it takes to insert this initial transhepatic catheter into the portal vein. We have encountered no complications because of this approach in more than 100 TIPS procedures. To our knowledge, this is the second reported use of the AVP in the portal venous system. In a recent case report, Kessler and Trerotola described a patient with gastric varices and TIPS in whom they used the AVP device for transhepatic obliteration of the portal vein collateral supplying the varices [6], a patient similar to one of our patients with flow-mediated gastric varices. We would like to emphasize that the AVP may be considered also for embolization of the more common collateral veins to esophageal varices and, if need be, the TIPS stent itself.

Cult_Med_Psychiatry-2-2-1705533

Making muslim babies: Ivf and gamete donation in sunni versus shi’a islam

Medical anthropological research on science, biotechnology, and religion has focused on the “local moral worlds” of men and women as they make difficult decisions regarding their health and the beginnings and endings of human life. This paper focuses on the local moral worlds of infertile Muslims as they attempt to make, in the religiously correct fashion, Muslim babies at in vitro fertilization (IVF) clinics in Egypt and Lebanon. As early as 1980, authoritative fatwas issued from Egypt’s famed Al-Azhar University suggested that IVF and similar technologies are permissible as long as they do not involve any form of third-party donation (of sperm, eggs, embryos, or uteruses). Since the late 1990s, however, divergences in opinion over third-party gamete donation have occurred between Sunni and Shi’ite Muslims, with Iran’s leading ayatollah permitting gamete donation under certain conditions. This Iranian fatwa has had profound implications for the country of Lebanon, where a Shi’ite majority also seeks IVF services. Based on three periods of ethnographic research in Egyptian and Lebanese IVF clinics, this paper explores official and unofficial religious discourses surrounding the practice of IVF and third-party donation in the Muslim world, as well as the gender implications of gamete donation for Muslim marriages. Introduction: Medical anthropology and science and technology studies In recent years, medical anthropologists have been turning their ethnographic attention to issues of science and technology, including how science is produced, where it is produced, by whom, and when. Anthropologists have also highlighted how scientific and technological advancements spread around the world, gradually being incorporated into the lives of ordinary people in many global sites. In order to document the production and reproduction of new scientific and technological discoveries, medical anthropologists have begun to take their ethnographic research projects into previously unexplored terrains, including hospitals, scientific research laboratories, clinical consultation rooms, and other “behind the scenes” places where the culture of science and technology is perhaps best revealed. Furthermore, most of the medical anthropologists working in this field have documented the ways in which the scientific and the divine seem to have intermingled, not only among the patients who turn to religious theodicies to make sense of their suffering, but also among the scientists and clinicians who deliver medical services and attempt to offer patients hope. These recent medical anthropological efforts to document the intersections of science, technology, and religion are clearly reflected in the theme of the 2004 annual meeting of the American Anthropological Association: “Magic, Science, and Religion.” Furthermore, this special issue of Culture, Medicine and Psychiatry reflects the ongoing importance of religion to science, medicine, and biotechnology, including in the Muslim world. Medical anthropology has much to offer to discussions of science, medicine, and religion, as demonstrated in a number of award-winning books on these subjects. To take but a few recent examples, in Testing Women, Testing the Fetus: The Social Impact of Amniocentesis in America, medical anthropologist Rayna Rapp (1999) reveals the efforts of more than ten years’ worth of extensive ethnographic research conducted at New York City hospitals, genetic counseling and testing centers, and genetics laboratories themselves. Her book focuses on the difficult decision-making of pregnant women from multiple ethnic, religious, and economic backgrounds, who are asked by clinicians and advised by genetic counselors to undergo amniocentesis in order to detect genetic anomalies in their fetuses. Although the scientific field of genetics is burgeoning with excitement, Rapp’s intent in Testing Women, Testing the Fetus is to show how women who are expected to use new genetic tests during pregnancy are put in the difficult position of being “moral pioneers:” namely, they are forced to make often heart-wrenching moral decisions about what constitutes an acceptable human life. Whereas Rapp’s book focuses on decisions about bringing life into the world, a book by medical anthropologist Margaret Lock asks us to consider decisions about ending lives considered “brain dead.” Twice Dead: Organ Transplants and the Reinvention of Death, Lock’s (2002) ethnography, examines the history of organ transplantation in North America over the past 20 years. Scientific advancements in transplant technology have made it possible for North American surgeons to perform thousands of transplants each year. In the majority of these cases, individuals diagnosed as “brain dead” are the source of the organs, without which transplants could not take place. Lock asks her readers to consider what is meant by “brain death,” and how it might be viewed differently in another culture, namely, Japan. Despite the availability of the necessary technology and expertise, Japanese society has not accepted the notion of brain death as a sufficient criterion of death, following a well-informed but heated public debate on the topic. Although brain death was legally recognized in Japan in 1997, it is authorized only in limited circumstances, meaning that organ transplantation in Japan is not widespread. Drawing on extensive interviews conducted over ten years with Intensive Care Unit (ICU) physicians, transplant surgeons, organ recipients, donor families, Japanese political activists opposed to the recognition of brain death, and members of the general public in both Japan and North America, Lock shows us that death itself is not a self-evident, biological event. Rather, it is surrounded by morally troubling cultural, medical, legal, and political dimensions. Lock’s book clearly demonstrates the degree to which medical anthropology is concerned with comparative cultural perspectives, particularly in an era of rapid globalization. Indeed, medical anthropology as a discipline can be said to focus on the “local in the global.” In other words, local considerations, be they cultural, social, economic, or political, shape and sometimes curtail the way that Western-generated scientific technologies are both offered to and received by non-Western subjects. New forms of science and technology are not transferred into cultural voids when they reach places like Japan. Thus, the assumption on the part of global producer nations that new biotechnologies—as value-free, inherently beneficial medical technologies—are “immune” to culture and can thus be appropriately transferred and implemented anywhere and everywhere is subject to challenge once local formulations, perceptions, and actual consumption of these technologies are taken into consideration (Inhorn 2003a). Indeed, the global spread of biotechnologies provides a particularly salient but little discussed example of what anthropologist Arjun Appadurai (1996) has termed a “technoscape,” or the “global configuration, also ever fluid, of technology, and the fact that technology, both high and low, both mechanical and informational, now moves at high speeds across various kinds of previously impervious boundaries.” Appadurai reminds us that this movement of technologies around the globe is both a deeply historical and an inherently localizing process. In other words, globalization is not enacted in a uniform manner around the world, nor is it simply culturally homogenizing—necessarily “Westernizing” or even “Americanizing” in its effects. The global is always imbued with local meaning, such that local actors, living their everyday lives at particular historical moments in particular places, mold the very form that global processes take (Freeman 2000). This acknowledgment of the importance of locality in the global dispersion of modern biotechnologies has been a theme of much recent work in medical anthropology. Similarly, much of this anthropological concern with locality has to do with local moral systems, or what medical anthropologist/psychiatrist Arthur Kleinman (1995) has called “local moral worlds” of pain and suffering. According to Kleinman (45), local moral worlds are “moral accounts, [which] are the commitments of social participants in a local world about what is at stake in everyday experience.” Through an “ethnography of experience,” Kleinman urges medical anthropologists to pay close attention to the moral issues that may accompany the arrival of new biotechnologies around the globe. In his recent powerful and path-breaking book, What Really Matters: Living a Moral Life amidst Uncertainty and Danger, Kleinman (2006) asks us to carefully consider what it really means to live a “moral life,” particularly in the midst of life-or-death decision-making. This focus on the “local moral” is found in another award-winning book on the topic of IVF. Titled Reproducing Jews: A Cultural Account of Assisted Conception in Israel, this book by medical anthropologist Susan Martha Kahn (2000; see also her article in this special issue) takes us into the often arcane world of Jewish Halakhic law, where male rabbis legislate on the appropriate uses of IVF for their followers. Kahn carefully describes how these rabbinical debates and decisions affect the actual practice of Israeli IVF. For example, third-party donation of gametes, including sperm donation, is allowed, since Jewishness is seen to be conferred through the mother’s side, particularly through the act of gestating and birthing the baby. However, most conservative rabbis prefer that non-Jewish donor sperm be used, to prevent adultery between a Jewish man and a Jewish woman and to prevent future genetic incest among the offspring of anonymous donors in this small, intermarrying country. Furthermore, debates have revolved around whether surrogacy should be allowed for infertile couples, using single or married surrogates. Generally speaking, single Jewish women are preferred as surrogates, both to avoid the implications of adultery for married surrogate women and to confer Jewishness through a Jewish woman’s gestation of the fetus. Finally, because the Jewish state is pronatalist—with the state subsidizing up to six cycles of IVF or up to the birth of two IVF children for any given Jewish patient—rabbis have generally been permissive when it comes to single career women, as well as lesbian Jewish mothers, conceiving children through assisted conceptive means. Kahn’s fascinating and frankly funny book details the sometimes dizzying rabbinical arguments regarding morally appropriate and inappropriate reproduction. In so doing, the book bespeaks the importance of local religious moralities in the contemporary world of Israeli assisted conception. There, doctors in many clinics serving orthodox Jewish patient populations attempt to practice IVF according to the moral dictates set forth by religiously conservative rabbis. The IVF laboratories in these clinics are full of orthodox women called maschigots, who literally peer over the shoulders of laboratory technicians to make sure that the correct sperm and correct eggs are being united—so as not to produce a mamzer, or an illegitimate child. In her book, Kahn is explicit in stating that the American consumer model of free-market reproductive medicine has yet to take hold in Israel, with its concern over religious guidelines. Nonetheless, Israel’s relative permissiveness over the use of donor gametes, surrogacy, and single and lesbian motherhood stands in stark contrast to the Muslim Middle East, including both neighboring Egypt and Lebanon, where I have conducted my own ethnographic research on IVF. Ivf in the muslim middle east With this background in mind, it is my goal in this article to take readers into the heart of the Muslim Middle East, thereby showing how the practice of IVF in Israel, the country located between Egypt and Lebanon, differs significantly from that in both of its Muslim neighbors. During more than two years of medical anthropological fieldwork in Egypt (1988–89, 1996) and Lebanon (2003), I conducted in-depth, ethnographic interviews with infertile Muslim IVF patients, both husbands and wives, now totaling nearly 500 patient couples. Furthermore, since the mid-1990s, I have focused my research on the rapid expansion of IVF technologies into this region of the Muslim world. Indeed, it is fair to assert that since the birth in 1978 of Louise Brown, the world’s first test-tube baby, IVF has spread around the globe, reaching countries far from the technology-producing nations of the West. Perhaps nowhere is this globalization process more evident than in the 22 nations of the Muslim Middle East, where a private IVF industry is flourishing. As of 2003, Egypt alone (population 70 million) boasted 50 IVF centers, and the tiny country of Lebanon (population 4 million) boasted more than 15 IVF centers, one of the highest per capita concentrations in the world. These global metrics are impressive compared to the IVF technology-producing nation of Israel, where 24 IVF centers cater to a population of 6 million (Kahn 2000). In both Egypt and Lebanon, as in other Muslim countries, nonbinding but authoritative Islamic religious proclamations called fatwas have profoundly affected the practice of IVF in ways that are not commonly seen in the West. Indeed, in the Muslim world, infertile couples are usually extremely concerned about making their test-tube babies in the religiously correct fashion. To that end, they seek out the “official” Islamic opinion on the practice of IVF in the form of a fatwa. In recent years, many such fatwas on a wide variety of reproductive health issues have been issued in Egypt and other Muslim countries (Lane and Rubinstein 1991; Zuhur 1992). But as we shall see in this paper, major divergences have occurred between Sunni and Shi’ite religious authorities regarding the permissibility of third-party gamete donation, with new fatwas emerging from the Shi’ite world in recent years. It is the differences between Sunni and Shi’a Islam with regard to third-party gamete donation that constitute the focus of this article. Sunni Islam and IVF IVF was first practiced in the Sunni Muslim world, with clinics opening in the mid-1980s in Egypt, Saudi Arabia, and Jordan, all Sunni-majority countries. The Grand Shaikh of Egypt’s famed religious university, Al Azhar, issued the first widely authoritative fatwa on medically assisted reproduction on March 23, 1980. This fatwa—issued only two years after the birth of the first IVF baby in England but a full six years before the opening of Egypt’s first IVF center—has proved to be truly enduring in all its main points (Inhorn 2006a). In fact, the basic tenets of the original Al-Azhar fatwa on IVF have been upheld by other fatwas issued since 1980 and have achieved wide acceptance throughout the Sunni Muslim world. Sunni Islam, it must be emphasized, is the dominant form of Islam found in the Middle Eastern region and throughout the Muslim world. Between 80 and 90 percent of the world’s Muslims are Sunni, and more than 90 percent of Egypt’s citizens are Sunni Muslims, the rest being predominantly Coptic Christian. The degree to which these official Sunni Islamic fatwas on IVF have affected the actual practices of the Middle Eastern medical profession is also quite striking. For physicians, the dominant Sunni religious opinion on IVF has been made known to the Middle Eastern medical community through the writings of Gamal I. Serour, one of three founding members of the first Egyptian IVF center and the director of Al-Azhar’s International Islamic Center for Population Studies and Research. In article after article (Serour 1992, 1994, 1996; Serour and Omran 1992; Serour, El Ghar, and Mansour 1990, 1991; Serour, Aboulghar, and Mansour 1995), Serour has spelled out the main points of the Sunni Islamic position on medically assisted conception, as follows: Artificial insemination with the husband’ssemen is allowed, and the resulting child is the legal offspring of the couple.In vitro fertilization of an egg from the wife with the sperm of her husband followed by the transfer of the fertilized embryo(s) back to the uterus of the wife is allowed, provided that the procedure is indicated for a medical reason and is carried out by an expert physician.No third party should intrude into the marital functions of sex and procreation, because marriage is a contract between the wife and husband during the span of their marriage. This means that a third party donor is not allowed, whether he or she is providing sperm, eggs, embryos, or a uterus. The use of a third party is tantamount to zina, or adultery.Adoption of a donor child from an illegitimate form of medically assisted conception is not allowed. The child who results from a forbidden method belongs to the mother who delivered him/her. He or she is considered to be a laqit, or an illegitimate child.If the marriage contract has come to an end because of divorce or death of the husband, medically assisted conception cannot be performed on the ex-wife even if the sperm comes from the former husband.An excess number of fertilized embryos can be preserved by cryopreservation. The frozen embryos are the property of the couple alone and may be transferred to the same wife in a successive cycle, but only during the duration of the marriage contract.Multifetal pregnancy reduction (or so-called selective abortion) is only allowed if the prospect of carrying a high-order pregnancy (i.e., twins, triplets, or more) to viability is very small. It is also allowed if the health or life of the mother is in jeopardy.All forms of surrogacy are forbidden.Establishment of sperm banks is strictly forbidden, for such a practice threatens the existence of the family and the “race” and should be prevented.The physician is the only qualified person to practice medically assisted conception in all its permitted varieties. If he performs any of the forbidden techniques, he is guilty, his earnings are forbidden, and he must be stopped from his morally illicit practice. But to what degree are these fatwa declarations—particularly the explicit prohibition on any form of third-party donation of reproductive materials—actually followed by physicians in the Muslim world? A global survey of sperm donation among assisted reproductive technology centers in 62 countries provides some indication of the degree of convergence between official discourse and actual practice (Meirow and Schenker 1997). In all of the Muslim countries surveyed in the mid-1990s—including the Middle Eastern countries of Egypt, Iran, Kuwait, Jordan, Lebanon, Morocco, Qatar, and Turkey, as well as a number of non-Middle Eastern Muslim countries, including Indonesia, Malaysia, and Pakistan—sperm donation in IVF and all other forms of gamete donation were strictly prohibited. As the authors of this global survey, Meirow and Schenker (1997), state, “In many Islamic countries, where the laws of Islam are the laws of the state, donation of sperm was not practiced. AID [Artificial Insemination, Donor] is considered adultery and leads to confusion regarding the lines of genealogy, whose purity is of prime importance in Islam” (134). In summary, in the Sunni-majority countries of the Middle East and the rest of the Sunni Muslim world, prohibitions against gamete donation have been enacted either in law or in professional medical codes of ethics. As a result, gamete donation is not legally practiced in the Sunni Muslim world, with clinics turning away (or referring to other European countries) couples who require these services. In Sunni Muslim countries such as Egypt, the thought of using donor gametes is reprehensible to most infertile Muslim (and even Christian) couples, who agree completely with the bans on third-party donation dictated by the religion and upheld through medical codes of ethics (Inhorn 2003b). As shown in the next section, however, the situation is changing for Shi’ite Muslims, whose leading cleric has taken a bold step in a new direction. Shi’a Islam and IVF For Shi’ite Muslims, attitudes toward gamete donation have changed considerably since the late 1990s. Shi’a is the minority branch of Islam found in Iran, parts of Iraq, Lebanon, Bahrain, Syria, and Saudi Arabia, as well as Afghanistan, Pakistan, and India. It has been much in the news lately because of the United States–led war in Iraq, the conflict between Lebanon and Israel, and the current tensions between the United States and Iran. Until recently, most Shi’ite religious authorities have supported the majority Sunni view: namely, they have agreed with Sunni clerics who say that third-party donation should be strictly prohibited. In the late 1990s, however, the Supreme Jurisprudent of the Shi’a branch of Islam, Ayatollah Ali Hussein Khamanei, the handpicked successor to Iran’s Ayatollah Khomeini, issued a fatwa effectively permitting donor technologies to be used. This fatwa has proved to be very significant for those Shi’a who follow the lead of Ayatollah Khamanei in Iran. This would include Lebanon’s Hizbullah leaders, who consider Ayatollah Khamanei to be their marja’ taqlid, or spiritual reference (literally, source of emulation). With regard to egg donation, Ayatollah Khamanei stated in his initial fatwa that egg donation “is not in and of itself legally forbidden.” But he stated that both the egg donor and the infertile mother must abide by the religious codes regarding parenting. Thus, the child of the egg donor has the right to inherit from her, as the infertile woman who received the eggs is considered to be like an adoptive mother. With regard to sperm donation, Ayatollah Khamanei said in his original fatwa that the baby born of sperm donation will follow the name of the infertile father rather than the sperm donor. However, as with egg donation, the donor child can only inherit from his biological father, the sperm donor, since the infertile father is considered to be like an adoptive father. The situation for Shi’ite Muslims is actually much more complicated than this, however, given two Shi’ite religious practices called ijtihad and mutca. Unlike Sunni Muslim scholars who are scripturally based in their thinking, Shi’ite religious authorities give precedence to a form of individual religious reasoning known as ijtihad. Through the use of caql, or intellectual reasoning, various Shi’ite ulama have come to their own conclusions regarding the rightness or wrongness of gamete donation. Some Shi’ite clerics continue to prohibit gamete donation for their followers, while others have allowed it under certain conditions. As many scholars of Shi’a have noted (Cole 2002; Tober 2004), the practice of ijtihad has allowed a certain flexibility and pragmatism toward new technological developments, including IVF and a number of other new medical technologies (e.g., contraception, organ transplants, transgender surgery). Furthermore, ijtihad has ultimately led to great heterogeneity of opinion and practice within the Shi’ite community. Additionally, Shi’a Islam allows a form of temporary marriage called mutca (also called sigheh in Iran), which is not recognized by Sunni religious authorities (Zuhur 1992). In Shi’a Islam, mutca is a union between an unmarried Muslim woman and a married or unmarried Muslim man, which is contracted for a fixed time period in return for a set amount of money. It is practiced in Iran (Haeri 1989), as well as in other parts of the Shi’ite world. In the past, middle-aged and older women who were divorced or widowed often engaged in mutca marriages for financial support. In Iran, following the loss of men during the devastating, eight-year Iran–Iraq war, former Iranian President Rafsanjani recommended mutca as a means of protecting the large numbers of single or widowed women who had no other source of income. For Shi’ite men, mutca marriages could be contracted while traveling, or as a way of achieving marital variety and sexual pleasure (Haeri 1989). Since the arrival of donor technologies, however, mutca has also been invoked to make egg donation legal within the parameters of marriage. Within this context of ijtihad and mutca, Shi’a religious authorities who now accept the idea of donation, but are strict in their interpretation of how donation should be practiced, argue that when a couple needs a donor, they should go to a Shi’ite religious court, where a decision can be made on a case-by-case basis;there should be a determination about which religious “reference” (i.e., source of spiritual emulation) the infertile couple follows;the decision should be made in the presence of witnesses, the IVF doctor, and with the agreement of both parties (the infertile couple and the donor);the husband should do a mutca marriage with the egg donor for the period of time in which the whole procedure (egg retrieval to embryo transfer) is taking place, because polygyny is legal in Islam and avoids the implications of zina, or adultery;but because a married Shi’ite Muslim woman cannot marry another man other than her husband (since polyandry is illegal in Islam), she cannot do a mutca marriage with a sperm donor. Technically, the child born of a sperm donor would be a laqit, or out-of-wedlock child, without a family name and without a father. Thus, in theory, only widowed or otherwise single women should be able to accept donor sperm, in order to avoid the implications of zina. However, in the Muslim countries, single motherhood of a donor child is unlikely to be socially acceptable (Inhorn 1996; Zuhur 1992). Be that as it may, divergent gamete donation practices are beginning to emerge in the Shi’ite Muslim world, as religious authorities come to their own conclusions about third-party donation. Among Shi’ite religious scholars, the major disagreements, or religious “sticking points,” revolve around the following set of issues: whether sperm donation should be allowed at all;whether the child should follow the name of the infertile father or the sperm donor;whether the child should inherit from the infertile father or the sperm donor;whether donor children and their “social” parents are related at all, and, if not, whether they could potentially marry each other, which has implications for proper comportment in domestic life (e.g., bathing, veiling, etc.);1whether donation is permissible at all if the donors are anonymous;whether a financial transaction should be allowed between gamete donors and recipients;whether the husband of an infertile woman needs to do a temporary mutca marriage with the egg donor, then divorce her after the embryo transfer (48 to 72 hours later), in order to avoid zina. For his part, Khamanei clearly stipulates that mutca marriage is not required, for he believes that zina requires the physical act of intercourse (Clarke 2006); andwhether the wife of an infertile husband can temporarily divorce her infertile husband, remarrying him after accepting sperm from a donor (Tremayne, personal communication, 5/5/06). These disagreements of opinion have played out in interesting ways. As shown in anthropologist Morgan Clarke’s (2006) recent research on the Shi’ite religious discourses surrounding gamete donation, many Shi’ite ulama do not agree with Khamanei’s permissive fatwa on donor technologies, because they do not regard him as a brilliant legal thinker. For example, Ayatollah Muhammad Husayn Fadlallah, Lebanon’s most prominent Shi’ite religious figure, does not agree with Ayatollah Khamanei’s permission of sperm donation, although he agrees with the permission of egg donation. Neither of them requires the use of mutca marriages to solve the zina issue (Clarke 2006). Ayatollah Fadlallah’s positions opposing sperm donation but supporting egg donation square with the dominant religious discourse in Iran. There, the religious rulings regarding gamete donation have evolved quickly, with sperm donation now effectively banned (Soraya Tremayne, personal communication, 23 July 2004, 31 July 2004).2 Namely, a law on gamete donation passed in 2003 in the Iranian parliament (majlis) and approved by the Guardian Council (i.e., a religious “watchdog” body that endorses every bill before it becomes law) has restricted gamete donation to married persons. Even though the law is brief (less than one page), it states clearly and succinctly who can and cannot donate and receive gametes. Egg donation is allowed, as long as the husband marries the egg donor temporarily—thereby ensuring that all three parties are married. Sperm donation, on the other hand, is legally forbidden, because a sperm donor cannot temporarily marry an already married woman whose husband is infertile. Quite interestingly, however, embryo donation—which involves both sperm and egg from another couple—is allowed in order to overcome both male and female infertility. Because an embryo comes from a married couple and is given to another married couple, it is considered hallal, or religiously permissible. The social and biological implications of embryo donation are quite interesting. For Iranian couples unable to produce a child because of male infertility, embryo donation allows them to bypass the problem of the husband’s weak (or absent) sperm. However, embryo donation does not allow a presumably fertile wife of an infertile husband to contribute her own ova, in effect severing her biological ties to the donor child. Furthermore, and most strikingly, embryos donated from another married couple involve both egg and sperm donation. Even though direct sperm donation is bypassed via the injection of another man’s sperm into the wife’s womb/ova, embryo donation still disrupts male paternity and involves the acceptance by an already married woman of another man’s (and woman’s) gametes. Moreover, a woman’s acceptance of another woman’s egg is effectively like gestational surrogacy, which is strictly prohibited in Sunni Islam. Indeed, in recent months, cases of surrogate motherhood have occurred in Iran, despite the lack of firm legislation regarding this practice. Whether these problematic complications of embryo donation have been carefully thought through by the religious and legal authorities in Iran is unclear. Based on her path-breaking research carried out at Iranian IVF clinics, anthropologist Soraya Tremayne (2005, 2006) notes, “My first and possibly superficial interpretation at this stage can only be that the moral, ethical and legal aspects of the whole matter have not yet been thought through and the consequences dawned on either the doctors and legislators, or on people themselves. It seems to me that ethics will follow, rather than lead the issue. These operations have been in place only for the past ten years ... and the cases are too new to cause problems as yet” (personal communication, 23 July 2004). It is interesting to note that many Shi’ite religious leaders both inside and outside of Iran do not agree with the relative Iranian “permissiveness” vis-à-vis donor technologies; instead, they abide by the dominant Sunni Muslim ban on all forms of third-party donation. For example, I recently attended a two-day conference in Tehran, Iran, on “Gamete and Embryo Donation,” sponsored by the Avesina Research Institute in association with the Law and Political Science Faculty of the University of Tehran.3 The conference provided a fascinating example of “ijtihad in action,” with some ulama, dressed in their stately robes and turbans (black for the sayyids, or descendants of the Prophet Muhammad), arguing against the moral permissibility of embryo and gamete donation. The disagreements generated in public between “pro” and “con” ulama were also debated in the more private recesses of the conference. For example, a Shi’ite shari’a judge from Bahrain, who was staying at our guest residence, took great pains to describe to me his opposition to all forms of gamete donation. To prove this point, he provided me with a copy of his book on Islamic personal status law, which had been translated into English and which supported his anti–gamete donation position based on evidence from the traditional Islamic scriptures. According to him, Iranian clergy, who speak Farsi rather than Arabic, are not as familiar with the original Islamic scriptures (in Arabic) that demonstrate the immorality of third-party donation. Thus, in his view, some Iranian clergy are “innovating” in ways that are religiously unacceptable, and that are at odds with the rest of the Muslim world. A case in point: ayatollahs Ali al-Sistani and Muhammad Sa’id al-Tabataba’i al-Hakim, both Shi’ite religious authorities in Iraq, advise caution against third-party donation practices, viewing them as largely unacceptable (Clarke 2006). Indeed, Ayatollah al-Sistani’s son, Muhammad Rida Al-Sistani, has devoted an entire volume of richly documented legal analysis to this debate, providing “an invaluable resource for other scholars” (26). According to Clarke, “Sistani’s work, while perhaps posing more questions than clear answers, opens up for other scholars a fascinating window into this area of Shi’ite jurisprudential debate, at a time when the Western media are just waking up to the vibrant engagement Shi’ite scholars have had with other such new technologies” (26). Indeed, the degree to which some Shi’ite clergy are “pushing the envelope” in the realm of reproductive science and technology is quite remarkable. At the recent gamete donation conference in Iran, some Iranian clergy and physicians present advocated for future laws permitting all forms of donation as well as surrogacy. Once passed into law, gamete donation of all kinds will be difficult to stop. Meanwhile, in the absence of formal legislation, some IVF physicians in Tehran—as well as in Shi’ite-dominant Lebanon, which is closely following the Iranian lead—are using the legal vacuum and the original “permissive” fatwa of Ayatollah Khamanei to practice all forms of gamete donation among their desperate infertile patients. As noted by Clarke (2006) for Lebanon, “Doctors keep Khamanei’s fatwa collection on the shelves of their surgeries to demonstrate the permissibility of such procedures to skeptical Muslim patients; and many such patients have profited from it to undertake donor sperm and egg procedures, even surrogacy arrangements, with a clear conscience” (26). Muslim patient opposition to donation Although donor law and practice are headed in interesting new directions in both Iran and Lebanon, it must be reiterated that the vast majority of Muslims, both Shi’ite and Sunni, do not accept the idea of third-party gamete donation. Why are they opposed to donation? In the hundreds of interviews that I have conducted since 1996 in Egypt (with Sunni Muslims) and Lebanon (with both Sunni and Shi’ite Muslims), the majority of infertile couples were clear that donation is haram, or forbidden by the religion (Inhorn 2006b). Patient concerns revolve around three sets of related issues: (1) the moral implications of third-party donation for marriage, (2) the potential for incest, and (3) the moral implications of donation for kinship and family life. With regard to marriage, Islam is a religion that can be said to privilege—even mandate—heterosexual marital relations. As is made clear in the original Al-Azhar fatwa, reproduction outside of marriage is considered zina, or adultery, which is strictly forbidden in Islam. Although third-party donation does not involve the sexual “body contact” of adulterous relations, or presumably the desire to engage in an extramarital affair, it is nonetheless considered by most Islamic religious scholars to be a form of adultery, by virtue of introducing a third party into the sacred dyad of husband and wife. It is the very fact that another man’s sperm or another woman’s eggs enter a place where they do not belong that makes donation of any kind inherently wrong and threatening to the marital bond. The other aspect of third-party donation that troubles marriage is the potential for incest among the offspring of unknown donors. Moral concerns have been raised about the potential for a single anonymous donor’s offspring to meet and marry each other, thereby undertaking an incestuous union of half-siblings. The final moral concern voiced by Muslim IVF patients is that third-party donation confuses issues of kinship, descent, and inheritance. As with marriage, Islam is a religion that can be said to privilege—even mandate—biological inheritance. Preserving the “origins” of each child—meaning its relationship to a known biological mother and father—is considered not only an ideal in Islam, but a moral imperative. The problem with third-party donation, therefore, is that it destroys a child’s lineage, which is immoral in addition to being psychologically devastating. Muslim IVF patients use the term “mixture of relations” to describe this untoward outcome. Such a mixture of relations, or the literal confusion of lines of descent introduced by third-party donation, is described as being very “dangerous,” “forbidden,” “against nature,” “against God”—in a word, haram, or morally unacceptable. It is argued that donation, by allowing a “stranger to enter the family,” confuses lines of descent in patrilineal Islamic societies. For men in particular, ensuring paternity and the “purity” of lineage through “known fathers” is of paramount concern (Inhorn 2006b). As one Sunni Muslim man, a high school biology teacher, summarized the problem: The most important thing is that we are Muslims. If there is faith in carrying out this operation using sperm from the husband and ova from the wife, then this is okay. We cannot accept what happens in the West. We heard some women “hire the womb” of another woman, or take sperm. According to our religion, this is called ikhtilat in-nasab, “mixing relations.” We consider it some kind of zina, prostitution. Because there are many hadiths from the Prophet Muhammad that confirm this. If you put your sperm in another woman besides your wife, you go to hell. This is adultery. There is a hadith on adultery. “If you put your sperm in another woman other than your wife, you are going to commit a sin.” People asked the Prophet, “How?” He said, “If you put it in your wife, you are going to be rewarded from Allah.” They said, “Yes.” He told them, “But this is also the case if you put it in the wrong womb. You are going to have punishment.” In addition to the consequences of mixed bloodlines and adultery, bringing such donor children into the world is considered unfair to the children themselves, who would never be treated with the love and concern parents feel for their “real” children. Such a child could only be viewed as a bastard—an ibn haram, literally “son of sin.” Thus, a child of third-party donation starts life off as an “illegal” child. The child is deemed illegitimate and stigmatized even in the eyes of his or her own parents, who will therefore lack the appropriate parental sentiments (Inhorn 2006b). As one Sunni Muslim IVF patient stated: My baby must be mine, and from my husband. This is logical. A mother will never feel this is her child if it is from another [man’s] donated sperm or ova. It’s only natural. Everything must occur naturally. If the child is from the father and mother, they will feel this is actually our baby. If not, we’ll not be a family. The feeling of the baby, and our feelings. You will feel like you’re acting, making a movie, living a life that’s not true. This is our feeling. Of course, people take babies [through adoption], but this is not so common here in Egypt. We don’t even want to think about this point! We are making our trial [of IVF] and hoping God will help us. Indeed, the firm conviction that parenthood of a donor child is an impossibility is clearly linked to the legal and cultural prohibitions against adoption throughout the Muslim world. The Islamic scriptures, including the Qur’an, encourage the kind fostering of orphans but do not allow legal adoption as it is known in the West, whereby a child takes its adoptive parents’ surname and is treated as one’s own child (Inhorn 1996; Sonbol 1995; Zuhur 1992). In the Muslim Middle East, few IVF patients, either Sunni or Shi’ite, will contemplate adoption, stating with conviction that it is “against the religion” and that the adopted child “won’t be my own son” (Inhorn 2006b). Even though legal adoption is practiced in Iran—with the child receiving a birth certificate in the adoptive couple’s name after a six-month period of adjustment—the social and cultural resistances to adoption remain strong in that country, making this a “last resort” for infertile Iranian couples (Janet Heindl, personal communication, 8 July 2004; Tober 2004). Nonetheless, Iran’s acceptance of adoption has clearly paved the way for gamete donation, with Ayatollah Khamanei’s initial fatwa deeming the infertile couple to be like adoptive parents. Indeed, Iran’s clearly adventurous path regarding both adoption and donation is leading to social transformations in other parts of the Shi’ite Middle East, in ways described in the following section. Marriage and gender relations In considering infertility and IVF in the Muslim world, it is important to ask: What happens to infertile Middle Eastern Muslim couples who are not allowed to adopt and who do not accept the use of donor gametes? In the absence of adoption and gamete donation, infertile Muslim couples have no choice but to turn to IVF and other assisted reproductive technologies to solve their infertility problems using their own gametes. In the Middle Eastern Muslim world, marriage is highly valued, and nearly all adults marry if possible in most Middle Eastern countries (Population Reference Bureau 2004; Zuhur 1992). Middle Eastern societies are also pronatalist—they highly value children for numerous reasons and expect all marriages to produce them (Inhorn 1996). Thus, the notion of a married couple living happily without children is unthinkable. Children are desired from the beginning of marriage in most cases, and are usually loved and cherished once they are born. As a result, childless couples are often under tremendous social pressure to conceive. In the Muslim world, infertile women often live in fear that their marriages will “collapse,” for Islamic personal status laws consider a wife’s barrenness to be a major ground for divorce. Although Islam also allows women to divorce if male infertility can be proven, a woman’s initiation of divorce continues to be so stigmatizing that women rarely choose this option unless their marriages are truly unbearable (Inhorn 1996). Instead, they often “cover” for their infertile husbands, accepting the social responsibility for the infertility and diffusing the embarrassment of their husbands’ reproductive emasculation (Inhorn 2003b, 2004). The emergence of the revolutionary new IVF technology called intracytoplasmic sperm injection (ICSI), however, has ironically increased the potential for divorce in the Muslim Middle East. Namely, with ICSI, infertile men with very poor sperm profiles—even azoospermia, or lack of sperm in the ejaculate—are now able to produce “biological” children of their own. As long as a single viable spermatozoon can be retrieved from a man’s body, including through painful testicular aspirations and biopsies, this spermatozoon can be injected directly into the ovum under a high-powered microscope. What ICSI requires, then, is high-quality ova, despite low-quality sperm. However, the wives of many of these men, who have “stood by” their infertile husbands for years, even decades in some cases, may have grown too old to produce viable ova for the ICSI procedure. In the absence of adoption or of any kind of egg donation, infertile Muslim couples with a reproductively “elderly” wife face four difficult options: (1) to remain together permanently without children; (2) to legally foster an orphan, which is rarely viewed as an acceptable option; (3) to remain together in a polygynous marriage, which is rarely viewed as an acceptable option by women themselves; or (4) to divorce so that the husband can have children with a younger wife. In my research in Egypt and Lebanon, the first option has proven to be the most common—namely, infertile husbands and their 40-something wives often love each other deeply, and remain together in long-term marriages without producing any children. Thus, divorce is not the immediate consequence of infertility that it is stereotypically portrayed to be. Because of the Sunni Islamic restrictions on the use of donor eggs, however, as well as lack of acceptance of this option among some segments of the Shi’ite population, at least some Muslim men are choosing to divorce or take a second wife, believing that their own reproductive destinies lie with younger, more fertile women. That being said, in the Shi’ite Muslim world, including in Iran and Lebanon, at least some Shi’ite couples are beginning to receive both donor gametes and donor embryos,4 as well as donating their gametes to other infertile couples. For infertile Shi’ite couples who accept the idea of third-party donation—as well as for Ayatollah Khamanei, who originally introduced the idea of donation to the Muslim world—the introduction of donor technologies has been described as a “marriage savior,” helping to avoid the “marital and psychological disputes” that may arise if the couple’s case is otherwise untreatable. Such disputes are clearly dramatized in the popular Iranian film Laila, which documents the painful separation of an otherwise happily married but infertile couple, and which was released in the mid-1990s before the Khamanei fatwa permitted such marriages to be saved through the use of donor technologies. In Iran today, donor egg and donor embryo programs have been set up at most IVF clinics. Donor eggs come from three sources: other IVF patients, relatives,5 and unmarried women who agree to participate as egg donors in one-day mutca marriages for a fee. Such marriages only require a witness and are not officially registered; thus, they take place in confidence in the back rooms of IVF clinics. Indeed, donors who wish to remain anonymous enter these mutca marriages only by written agreement, without ever meeting the recipients of their eggs or their temporary husbands. They receive their money following egg harvesting (usually about U.S. $550), provide no personal information about themselves to the recipient couple, receive no information about the recipient couple, and “go about their business” (Soraya Tremayne, personal communication, 31 July 2004). In short, egg donation—as well as embryo donation from other couples—is largely a financial transaction in Iran, with very little regulation or control over who donates or how donation is enacted. The same is not true for the receiving of embryos. According to Tremayne, the recent law in Iran specifies clearly that couples desiring an embryo as a result of infertility must apply in writing to a court in order to receive permission for embryo transfer. The law specifies that the couple must be morally sound and suitable as parents and must be Iranian citizens, much like the law governing adoption in Iran (Janet Heindl, personal communication, 8 July 2004). Still, the donor embryo law is so new that most IVF clinics in Iran do not yet own a copy of the legislation and are not necessarily abiding by the legal requirements for donor embryo transfer at their clinics. If the husband is infertile, the couple simply receives another couple’s embryos, with most donor couples choosing to remain anonymous. As Tremayne (31 July 2004) states, I did not get the impression that people desperate to have a child thought very far about the issues of kinship and family relations. As far as I could see, the donation is considered more a financial transaction than a donation/gift, and once you have paid the couple to buy their embryo, or paid the temporary wife for her egg, they have no further claims on you and this is the end of the story. The forms filled by the donors leave it to them to decide whether they want to give their name or not. In Lebanon where I have conducted my own research, the situation is very similar, despite the lack of a national law governing any aspect of IVF or third-party donation.6 At Lebanese IVF clinics providing donor technologies, some of the donors are other IVF patients (mostly Shi’ite Muslims who accept the idea of donation), some are friends or relatives (including egg-donor sisters), and some are anonymous donors, who provide their ova for a fee. In at least one clinic catering to a largely conservative Shi’ite clientele, some of these donors are young non-Muslim, American women who travel to Lebanon for extra payment to donate their eggs anonymously to infertile Lebanese couples. Ironically, those most likely to receive these “American eggs” are conservative Shi’ite couples, who accept the idea of donation because they follow the teachings of Ayatollah Khamanei in Iran. Thus, in Lebanon, those most likely to follow the spiritual guidance of Ayatollah Khamanei—and, hence, to receive American donor eggs—are generally members of or sympathizers with Lebanon’s Hizbullah political party, which is officially described by the U.S. administration as a terrorist organization! Furthermore, quite interestingly, in multisectarian Lebanon, the recipients of these donor eggs are not necessarily only Shi’ite Muslim couples. Some Sunni Muslim patients from Lebanon and from other Middle Eastern Muslim countries such as Egypt and Syria are quietly slipping across transnational borders to “save their marriages” through the use of donor gametes, thereby secretly “going against” the dictates of Sunni Muslim orthodoxy. Such border crossing has also been noted by Clarke (2006) for Lebanon; he writes, “Indeed patients come from other countries to benefit from this relatively relaxed regime” (26). The same is true in Iran, where, according to IVF clinic staff, scores of Persian Gulf Arabs from countries such as Saudi Arabia and Kuwait are traveling to Tehran in pursuit of donor gametes. Conclusion In conclusion, it is fair to state that global reproductive “technoscapes” (Appadurai 1996) are becoming increasingly expansive as we enter this new millennium. As this article has tried to show, the Muslim world—generally positioned on the receiving end of global reproductive technology transfers—has nonetheless embraced assisted reproductive technologies with considerable enthusiasm while, at the same time, reconfiguring them in accordance with the local religious moralities so important in this region. Although generally portrayed as monolithic, Islam itself takes several different forms, as evident in this essay. In the Sunni Muslim world, which includes most Middle Eastern countries, the use of IVF and related assisted reproductive technologies has clearly led to an entrenchment of deeply held religious beliefs about the importance of biologically based kinship, family life, and parenthood. Yet the globalization of these technologies to the Shi’ite Muslim world has fundamentally altered understandings of the ways in which families can be made and the ways in which marriages can be saved through the uses of assisted reproductive technologies. For Shi’ite Muslims, in particular, the frankly adventurous attitude on the part of some Shi’ite religious leaders toward third-party donation has led to a potential transformation in gender relations among infertile Muslim couples. For example, in Lebanon, the recent Shi’ite fatwas allowing egg donation have been a great boon to marital relations. There, both fertile and infertile men with “reproductively elderly” wives are lining up at IVF clinics to accept the eggs of donor women. Furthermore, in multisectarian Lebanon, the recipients of donor gametes are not necessarily only Shi’ite Muslim couples, but include some Sunni Muslim and Christian couples as well. In short, the arrival of donor technologies in the Muslim Middle East has led to a brave new world of reproductive possibility never imagined when these technologies were first introduced there nearly 20 years ago. These technologies have engendered (1) significant medical transnationalism and reproductive tourism; (2) mixing of gametes across ethnic, racial, and religious lines; and (3) the birth of thousands of ICSI and, now, donor babies to devout infertile Muslim couples. Infertile couples have begun to reconsider traditional notions of biological kinship, even if “social parenthood” of a donor child is still not widely embraced (Inhorn 2006b). And because donor technologies are now widely available in both Iran and Lebanon, the power of the Sunni Muslim ban on third-party donation is being weakened across the region, with some infertile Sunni Muslim couples reconsidering their own antidonation moral stances. As a result, Shi’ite gametes are finding their ways into Sunni bodies, an interesting variation on the “making of Muslim babies.” In my view, these multiple transformations are powerful indicators of the profound social effects that reproductive technologies may engender in the new world order. As the assisted reproductive technologies become further entrenched in the Muslim world, and additional forms of global reproductive technology become available, it is important to examine the new local moral worlds that are likely to arise in response to this variant of globalization. The pace of change evident in the production of assisted reproductive technologies themselves—as highlighted on the recent Nova special called “18 Ways to Make a Baby”—as well as the rapid spread of these technologies into far reaches of the non-Western world is, indeed, striking. Thus, as one science and technology studies scholar, David Hess (1994), rightly observes, “Anthropology brings to these discussions a reminder that the cultural construction of science is a global phenomenon, and that the ongoing dialogue of technoculture often takes its most interesting turns in areas of the world outside the developed West” (16). Notes To overcome the difficulties of raising a biologically unrelated donor child, some Shi’ite physicians in Lebanon are arguing for a variety of novel solutions, including inheritance through gifts and bequests; institutions of rida’ (milk kinship), whereby the mother of a donor child becomes related to it through breastfeeding; and the notion of legal guardianship of foster children (Clarke 2006). I am deeply grateful to Soraya Tremayne, who, fresh from fieldwork in Iran, has engaged in a lively e-mail discussion with me and has provided invaluable information on the practices of IVF and gamete donation in Iran since 2004. I have tried to represent her findings as accurately as possible in this paper. In Iran, I want to thank Mohammad Jalal Abassi-Shavazi, Mohamad Mehdi Akhondi, and Pegah Ebadi for so generously inviting and hosting me at the Avesina Research Center and University of Tehran conference on embryo and gamete donation. In Lebanon, anonymous sperm donation—using frozen sperm from overseas sperm banks or fresh sperm samples from mostly medical and graduate students—is “quietly” practiced at IVF clinics. One of my azoospermic Lebanese male informants produced a donor child in this way, and several others, both Muslim and Christian, had also made the decision to use donor sperm. In Iran, women commonly bring their sisters as potential egg donors. But this is not allowed, as Islam is explicitly against the marriage of one man to two living sisters. Apparently, men also bring their brothers as potential sperm donors in Iran. According to Tremayne, she observed one case where the husband did so without his wife’s knowledge; the wife believed that she was receiving her husband’s sperm instead of that of her brother-in-law. Some leading members of the Lebanese medical community are pushing for a law that bans all forms of third-party donation in the country. However, this law has yet to be debated in the Lebanese parliament and is unlikely to pass, according to some sources.

Eur_Arch_Otorhinolaryngol-4-1-2279156

Surgery did not improve the subjective neuropsychological symptoms of patients with incidentally detected mild primary hyperparathyroidism

Primary hyperparathyroidism (PHPT) is known to cause diverse subjective symptoms, in addition to those related to osteitis fibrosa cystica and kidney stones. The treatment of the disease ameliorates the subjective symptoms and improves the patients’ quality of life. In this prospective study, patients undergoing surgery for incidentally detected, mild, asymptomatic PHPT were assessed to determine whether subjective neuropsychological symptoms are improved even in patients with “asymptomatic” PHPT. From October 1995 to March 2004, 25 patients who had one or more neuropsychological symptoms preoperatively and were followed up 1 year after parathyroidectomy were enrolled. The subjective symptoms were identified using questionnaires distributed to patients; eight questions were used to determine the presence or absence of psychoneurological symptoms. Compared to their preoperative status, patients responded that their general health perceptions 1 year after surgery were improved (13 cases, 52%), unchanged (11 cases, 44%), or aggravated (1 case, 4%). There were no statistically significant differences in the patients’ responses before and after surgery with respect to individual neuropsychological symptoms, such as “tiring easily, “forgetfulness,” “decreased concentration,” “depression,” “irritability,” “uneasiness,” and “sleeplessness.” Therefore, subjective neuropsychological symptoms did not improve in otherwise asymptomatic PHPT patients following parathyroidectomy. However, patients’ questionnaire responses may not reflect their actual status as accurately as laboratory examination results. Overall, 52% of patients were subjectively satisfied with surgery; this may result from patients’ expectations of treatment. Introduction Since calcium concentration is now included in the serum biochemical analyses done by multichannel autoanalyzers, it is possible to detect patients with hypercalcemia. Consequently, an increased incidence of mild cases of primary hyperparathyroidism (PHPT) has been noted. More than 80% of PHPT patients are essentially “asymptomatic” [1, 2]. Currently, resection of the pathologic parathyroid gland continues to be the sole therapeutic modality available for curing PHPT. However, the surgical procedure is not necessarily easy for surgeons with limited experience, particularly in certain patients with mild PHPT. Under these circumstances, several investigators [1, 3, 4] have reported, based on surveys, on the natural history of patients with asymptomatic PHPT who had been conservatively followed up. Some patients reported no signs or symptoms during observation periods of various durations, although some patients developed complications specific to PHPT that required surgery. PHPT has been known to produce diverse subjective symptoms, in addition to those related to osteitis fibrosa cystica and kidney stones. The treatment of the disease ameliorates these symptoms, which improves patients’ quality of life (QOL). From the clinical standpoint, based on patient self-reporting after surgery, most surgeons have had the impression that parathyroidectomy improves psychological and mental deficits. This prospective study of patients undergoing surgery for incidentally detected, mild, asymptomatic PHPT determined whether subjective neuropsychological symptoms improved postoperatively in patients with asymptomatic PHPT. Patients and methods From October 1995 to March 2004, 62 patients with PHPT were treated at the Cancer Institute Hospital. All patients underwent surgery. In 60 patients, the pathological parathyroid glands were successfully removed; the pathology was adenoma in 58 patients, hyperplasia in none, and cancer in 2. Of the 58 patients whose parathyroid adenoma was removed, 7 had kidney stones and 14 had osteitis fibrosa cystica. Thirty-seven patients without any signs and/or symptoms of classic PHPT were diagnosed as “asymptomatic”, because all were incidentally found to have hypercalcemia on laboratory testing, and their subsequent serum examinations showed inappropriately elevated intact parathyroid hormone concentrations without concomitant clinical, biochemical, or radiological evidence of osteitis fibrosa cystica or renal stone colic. Of these 37 patients, 34 reported one or more neuropsychological symptoms preoperatively; 3 patients reported no neuropsychological symptoms preoperatively, and these 3 patients were excluded from the postoperative survey. Twelve months after successful parathyroidectomy, 25 patients responded to our questionnaire (Fig. 1) distributed at the outpatient clinic or by mail. They included 2 males and 23 females, ranging in age from 48 to 76 (mean 64) years. The other 9 patients were requested to respond by mail. Of these 9 patients, 3 gave no response, and 6 responded to less than half of the questions; thus, these 9 patients were excluded from the study. Fig. 1Survey of primary hyperparathyroid patients (pts) treated surgically during an 8.5-year period (1995–2004) Preoperative bone mineral density was measured in the lumbar spine (L2–L4) using a dual-energy X-ray absorptiometer. The serum calcium level was 10.1–12.4 (mean 11.0) mg/dl, the urinary calcium excretion rate was 51–497 (mean 246) mg/24 h, the creatinine clearance rate was 40–183 (mean 90.8) ml/min, and the bone mineral density was 0.2 to −4.8 (mean −2.5) SD. The subjective symptoms were assessed by distributing questionnaires that contained eight questions dealing with the presence or absence of neuropsychological symptoms (Table 1). The eight questions were formulated based on a textbook [5] and dealt with “tiring easily, “forgetfulness,” “decreased concentration,” “depression,” “irritability,” “uneasiness,” “sleeplessness,” and “general health”. “Uneasiness” was defined as feeling worried or unhappy about a particular situation, especially because one thinks something bad or unpleasant might happen. The questionnaires were distributed before and 1 year after surgery. To ensure that the patients understood the questions, the meaning of each question was thoroughly explained prior to surgery. To ascertain patients’ perceptions of their general health, they were asked whether they felt better 1 year after surgery than before surgery. Patients were asked to select one of the following responses to each question: the symptom is totally absent or occurs rarely (score 0); the symptom is slight or occurs only occasionally (score 1); or the symptom has recently been aggravated or occurs frequently (score 2). Table 1Questionnaire contents1. Do you tire easily?2. Are you forgetful?3. Do you lack the ability to concentrate?4. Do you feel depressed?5. Do you feel irritable?6. Do you feel uneasiness?7. Do you experience sleeplessness?8. How do you perceive your general health compared with that before surgery?aaThis question was asked only once, 1 year postoperatively Statistical analysis A two-sided Wilcoxon’s signed-rank test was used to detect statistically significant differences among the responses; the significance level was set at P < 0.05. Results General health perceptions 1 year after parathyroidectomy When compared to their preoperative responses, the patients’ general health perceptions 1 year after surgery were improved in 13 cases (52%), unchanged in 11 cases (44%), and aggravated in 1 case (4%); these changes were statistically significant (P = 0.0013). Comparison of the individual subjective neuropsychological symptoms’ grade Table 2 shows the preoperative and 1-year postparathyroidectomy comparison of individual subjective neuropsychological symptoms. Preoperatively, 17 patients (68%) reported “tiring easily,” and 1 year postoperatively the symptom had resolved in 6 and was still reported by 11, although all 11 perceived the symptom as mild. However, of the 8 patients who did not report tiring easily preoperatively, 5 reported a mild degree of tiring easily 1 year postoperatively. These changes in the number of patients who reported tiring easily were not statistically significant. Table 2Comparison of pre- and postoperative scoresNeuropsychological symptomsPreoperative1 year postoperativeP valueNo. of patientsScore012Easily tiredScore083500.2411257025140No. of patients259160ForgetfulnessScore061500.811558224040No. of patients256172Decreased concentrationScore074300.311466224040No. of patients2510132DepressionScore01210110.171844025221No. of patients251672IrritabilityScore01312100.4511155121001No. of patients251762UneasinessScore010820111347222011No. of patients2512103SleeplessnessScore01173111936025014No. of patients2510105 Changes in the number of patients reporting other neuropsychological symptoms, such as “forgetfulness,” “decreased concentration,” “depression,” “irritability,” “uneasiness,” and “sleeplessness,” before and after surgery were also not statistically significant (Table 2). Discussion In normal subjects, the serum calcium concentration is always kept within a narrow normal range and plays an important role in the physiological control of neuropsychological functions. In PHPT patients, even in those with the mild form, the abnormally elevated serum calcium concentration returns to the normal level postparathyroidectomy. It has been assumed that some subjective improvement in neuropsychological function must occur following surgery. In the present prospective study of patients with incidentally detected, mild PHPT, 52% of all patients who had successful parathyroidectomy reported improved general health 1 year after surgery. This result was statistically significant. Pasieka and Parsons [6] reported that general health improved following surgery in 60% of PHPT patients. Talpos et al. [7] studied 53 patients with asymptomatic hyperparathyroidism who were randomized into a parathyroidectomy group or an observation alone group; they were evaluated every 6 months for 2 years using the SF-36 health survey. The 28 patients who had parathyroidectomy had improved social and emotional function scores compared to the observation alone group. Edwards et al. [8], who studied 100 patients with PHPT, found that parathyroidectomy for hyperparathyroidism was associated with significant lasting improvement in subjective symptoms; they stated that the potential for long-term improvement of these QOL symptoms was a valid indication for parathyroidectomy. However, contrary to reports of long-term postoperative improvement in subjective symptoms, Okamoto et al., using the GHQ-28 in 26 patients with mild PHPT, found no improvement in symptoms except for severe depression 24 months after surgery, though they found improvement in the total GHQ score, somatic symptoms, anxiety, and severe depression 3 months following parathyroid surgery [9]. Okamoto et al. hypothesized that the transient, short-term improvement in symptoms after surgery may have been a consequence of patients’ expectations of treatment. The results of the present study showed that approximately half of the patients reported improvement in general health perceptions, and there was no statistically significant improvement in any of the individual neuropsychological symptoms. The subjective satisfaction with surgery reported by 52% of our patients may also reflect their expectations of treatment. Bollerslev et al., in a randomized study using the SF-36 involving 191 patients with asymptomatic PHPT who were assigned to surgery or medical observation, found that “no benefit of operative treatment, compared with medical observation, was found” [16]. In the present study, the numbers of patients required for comparison of differences before and after surgery, using a significance level of 0.05 and a power of 0.80, were calculated as follows: “tiring easily”, n = 39; “forgetful”, n = 382; “decreased concentration”, n = 62; “depression”, n = 55; “irritability”, n = 165; “uneasiness”, n = 1492; and “sleeplessness”, n = 1878. Therefore, between 39 and 1,878 patients would be required to have adequate statistical power to determine “whether surgery improves psychoneurological symptoms”. The sample size of the present study was 25 patients, which was too low to determine “whether surgery improves psychoneurological symptoms”. However, the present findings were similar to those of Bollerslev et al. There is a recognized need to determine the surgical indications for PHPT. In 1990, the guidelines of the US National Institutes of Health (NIH) were published. The results from a questionnaire given to the members of the Society of Surgical Endocrinology of America showed that several practices were not precisely in accordance with the 1990 NIH guidelines [11]. Sywak et al. conducted a comparative study involving one group that met the criteria of the 1990 NIH guidelines and one group that did not. They reported that nonspecific symptoms, such as fatigue, depression, irritability, mood swings, and forgetfulness, improved in both groups [12]. The guidelines were revised in 2002, and a 2002 workshop panel promulgated six criteria for PHPT surgery [13]. Since there is uncertainty concerning the specificity of subjective symptoms, the NIH conference excluded them from the criteria for parathyroidectomy. Although the present study did not find that subjective neuropsychological symptoms were improved in patients with asymptomatic PHPT following parathyroidectomy, patients’ responses to questionnaires may not reflect their actual status as accurately as laboratory test results, such as the serum calcium level, the urinary calcium excretion rate, the creatinine clearance rate, and bone mineral density. Nevertheless, obtaining information on changes in subjective symptoms is clinically important. The difficulty in quantitatively evaluating nonspecific symptoms has been previously identified, and some studies have used the Visual Analogue Scale [6, 14] or the SF-36 [7, 15, 16]. We devised an original questionnaire based on a textbook [5]. This questionnaire has not been validated as a research tool and was not tested on the normal population. However, the five questions related to “tiring easily, “forgetfulness,” “depression,” “irritability,” and “general health” are identical to those used by Pasieka et al. [6, 14], and the other three questions dealing with “decreased concentration,” “uneasiness,” and “sleeplessness” are all easily understandable neuropsychological symptoms. To ensure that all patients understood the questions, the meaning of each question was thoroughly explained to the patient prior to surgery; however, 1 year postoperatively, the questionnaires were simply sent by mail to the patients with no further explanations. To allow nonspecific symptoms to be more readily evaluated, simple, quantitative methods are required. Bollerslev et al. [10] reported that asymptomatic patients with mild PHPT have more psychological symptoms than normal controls and decreased QOL. In the present study, of the 37 patients who underwent parathyroid surgery for “asymptomatic” PHPT, 34 had one or more neuropsychological symptoms preoperatively, though it was not confirmed that these symptoms were due to the PHPT itself. The results of the survey done 1 year after successful parathyroidectomy did not show any definite improvement in any of the individual neuropsychological symptoms. The main reason for this might be the mild nature of the patients’ PHPT. Two reasons may explain why six patients gave incomplete answers and three patients did not respond to the second questionnaire: the questionnaires were mailed to patients who had stopped visiting the out-patient clinic, and patients may have found the questionnaire bothersome. If the patients had been truly symptomatic, they would have visited our hospital.

Int_J_Legal_Med-4-1-2226061

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Introduction Human body fluids such as blood and saliva are the most common sources of biological trace material found at a crime scene. Reliable tissue identification in forensic casework is important as it provides crucial insights into crime scene reconstruction and can thus contribute towards solving crimes. Blood stains are routinely tested in forensic practise using various methods including the tetrabase (4,4-bis(dimethylamino)diphenylmethane) test [1], the Kastle–Meyer phenolphthalein test, the tetramethylbenzidine test [2], the orthotolidine test [3], or the luminol (3-aminophthalhydrazide) chemoluminescence test [4], with the latter especially appropriate for detecting blood stains after cleaning attempts [2, 5]. All these presumptive—thus indicative but not identifying—tests take advantage of the peroxidase-like activity of the heme unit of the hemoglobin molecule in human blood. Therefore, false-positive results can be caused by the presence of strong oxidants, such as chlorine-containing detergents or by true peroxidases (e.g., from plants) [6]. Saliva stains are usually detected in forensic practise via an enzymatic amylase test using Phadebas [7] or with a recently developed enzyme-linked immunosorbent assay-based method [8]. However, because of amylase degradation, the time window for the successful performance of such tests can be limited [9]. Furthermore, no amylase assay can distinguish between salivary amylase and amylases from other tissues (pancreatic, urinary, etc.); therefore, the tests for saliva identification are only presumptive (similar to existing blood identification tests). On the other hand, methods for identification and quantification of mRNA are already well established, although mostly outside the forensic field. These methods make massive multiplex gene expression profiling possible—among many other applications—for the discovery of tissue-specific mRNA markers. The major concern of using mRNA markers for forensic applications is their assumed high susceptibility to degradation. However, recent studies using a few selected genes demonstrated that it is possible to isolate total RNA of sufficient quality and quantity from biological stains that are several months or even years old [10–12]. It has also been suggested, although with limited evidence so far, that different types of mRNA seem to follow different rates of degradation [13]. It is assumed that the degradation process of mRNA is influenced by many external and internal factors, including structural peculiarities like the presence of AU-rich elements (ARE motifs), protein binding properties, and cellular localization [14, 15]. However, detailed knowledge on the molecular reasons for differences in RNA degradation between different types of RNAs as well as between mRNAs of different genes is currently lacking and further investigations are sorely needed. Although a small number of mRNA markers has been tested for tissue identification in forensic science [16–19], no systematic study has yet been performed. In addition, the identification of candidate markers in previous studies was based on a mixed literature and database search, apparently without strict criteria of selection, considering only a limited number of genes and tissues, and not taking into account RNA degradation levels. Furthermore, expressed sequence tags databases, which were used previously, like the Cancer Genome Anatomy Project [18], are expected to provide heavily biased information on candidate genes because of the nonrandom character of representation of clone libraries. To find stable mRNA markers for body fluid identification in forensic practice, we performed a systematic and comprehensive whole-genome gene expression analysis on time-wise degraded blood and saliva stains using the Affymetrix U133 plus2 GeneChip. This expression array contains >54,000 mRNA probe sets, which encompass most, if not all, known and predicted human genes. Tissue-specific expression patterns of the most promising candidate genes from the array analyses were further confirmed using the GNF SymAtlas expression database [20], which covers about 100 human tissues, and finally verified by quantitative real-time polymerase chain reaction (PCR) in blood and saliva as well as in other body fluids relevant for forensic casework, i.e., semen and vaginal secretion. Materials and methods Sample collection Aliquots of 5 ml of whole blood and saliva were collected from each of five healthy volunteers (four men and one woman) of western European genetic origin under informed consent before their inclusion in the study. Native blood was collected without anticoagulation treatment to avoid disturbing effects of anticoagulation reagents on gene expression. In each sample, 75 cotton swabs were immersed. Special care was taken to shorten the time between collection and swab absorption to avoid blood coagulation. After complete absorption of the fluids, swabs were left until dry on a bench top at room temperature. When dry, the swabs were stored in dust-free nonhumid conditions (but subjected to normal daylight) for different time intervals. Swabs were visually inspected and sorted out to ensure similar liquid content between individual swabs. After 0, 1, 3, 7, 14, 21, 57, and 180 days, swabs were stored at −80°C until RNA isolation. For the time interval 0 days, samples were frozen immediately after drying. Semen and vaginal secretion samples were collected from one male and one female individual absorbed with cotton swabs and dried overnight before RNA isolation. RNA isolation RNA was isolated using the Qiagen RNeasy kit (Qiagen Benelux B.V.) according to the manufacturer’s instructions with minor modifications. These included cutting up the cotton swab into 1 × 1-mm pieces and soaking them in RLT buffer for 1 h at 4°C before the extraction. Trial experiments to lengthen this incubation time up to 24 h did not reveal any improvement in respect to RNA quantity and quality (data not shown). Microarray hybridization and gene expression data analysis Before hybridization to Affymetrix U133 plus2.0 GeneChip arrays (Affymetrix, Santa Clara, CA), RNA isolated from blood and saliva stains was amplified using the Ambion MEGAscript T7 two-cycle amplification kit (Applied Biosystems, The Netherlands). Amplification, labeling, hybridization, washing, and scanning were performed by the microarray core facility of the Erasmus MC Center for Biomics according to Affymetrix specifications. Background subtraction and probe signal summarization were calculated according to the robust multiarray analysis algorithm [21] using the R Bioconductor software [22]; the resulting log2 signal values were back-transformed to linear scale. Presence/absence calls for individual probe sets were calculated with the mas5calls function of the Bioconductor mas package. Because the constant global mean assumption does not hold true for arrays hybridized to differentially degraded RNA samples, the normalization of the signal intensities between samples was performed using the nonhuman control genes present on Affymetrix arrays (spiked-in probes). Normalization factors for each array were inferred from the average signal intensities of bioB, bioC, bioD, and Cre control probe sets. Analysis of differential gene expression was performed using the significance analysis of microarrays (SAM) algorithm [23] implemented in the TM4 software [24]. In the saliva dataset, we selected only genes with signal intensities above 50 (which is below the usually applied background threshold in expression array experiments) that had a signal intensity below 50 in the blood dataset. The selection of blood-targeted genes was done in a similar manner but with different criteria, the lower intensity limit in blood was set to 1,000 to reasonably restrict the number of candidates. Real-time PCR First strand cDNAs were synthesized with SuperScript® III RTS First-Strand cDNA Synthesis Kit (Invitrogen BV, The Netherlands) using total RNA as a template. The primers were designed with Primer3 software [25] so that forward and reverse primers were complementary to different exons of the respective genes and most closely located to the 3′-end of the corresponding RefSeq cDNA (Electronic Supplementary Material Table S1). Real-time PCR reactions with the SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen BV) were performed on an ABI 7300 PCR machine (Applied Biosystems, The Netherlands) using the following parameters: initial denaturation at 94°C for 10 min, followed by 45 cycles of denaturation at 94°C for 15 s, and a final annealing/elongation at 60°C for 30 s. Melting profiling and agarose gel electrophoresis were used to confirm the specificity of the primers and the absence of DNA contamination. Quantification of the amplified cDNA yield in comparative blood and saliva PCRs was done by the standard curve method. PCR experiments with semen and vaginal secretion were quantified using delta Ct (dCt) method. In both cases, GAPDH gene was used as an endogenous control to normalize the amplification signal between the samples from different tissues and individuals. Time points were compared to each other without normalization: Assuming the temporal degradation of all RNA molecules, no internal control gene could be used, and the only proper way to normalize RT-PCR signals was to use the same amount of template in each reaction. We found that this requirement holds true for our experiments because the GAPDH expression variability between different samples from the same tissue was relatively low (CV <25%, data not shown), which is probably because of approximately the same amount of blood or saliva absorbed with cotton swabs during material collection. Results and discussion Microarray expression data As expected, hybridization signals demonstrated high variability between individuals; however, the most striking differences were observed between the different tissues. Signal intensities in blood samples were on average about five times higher than in saliva (174.2 ± 1.9 in blood samples vs 26.9 ± 0.7 in saliva; Wilcoxon test rank sum p < 0.001). In addition, at the time-point zero, the number of the probe sets called as present according to the Affymetrix algorithm was, on average, more than three times higher in blood than in saliva (30.2% ± 0.9 vs 9.3% ± 0.6; t test p < 0.001). The SAM test with stringent parameters (false discovery rate was set to 0%) showed that, both in blood and saliva experiments, no genes demonstrated significant expression differences in a time range of 0–57 days of stain storage. Only few genes (37 and 10 significantly differential genes for saliva and blood, respectively) appeared to be differentially expressed at 180 days in comparison to other time points. This suggests that in dried blood and saliva, mRNA molecules remain relatively stable for a long period. Recent studies of Heinrich et al. [26] also revealed poor correlation between RNA degradation and postmortem time intervals. Selection of tissue-specific markers The initial selection of tissue-specific genes was performed using the normalized signal intensities of microarray hybridizations averaged across the five biological replicates at the zero experimental time point. About 500 apparent saliva-specific and 1,000 apparent blood-specific candidate genes were selected. Further refinement of tissue-specific gene sets was achieved by probing the selected candidates against the GNF SymAtlas tissue database [20] after excluding all cell lines from the database retaining only tissues and organs for the analysis. Genes were selected only if they were highly and exclusively expressed in the target tissue(s) based on the GNF SymAtlas database. For blood, target tissue in the database was defined as whole blood; while for saliva, the target tissues were salivary gland, tongue, trachea, and tonsils. The selection criteria were as follows: high expression (signal intensity >1,000) in target tissue and low expression (signal intensity <200) in nontarget tissues. Using these criteria and combining data from expression array experiments as well as GNF SymAtlas database verification, we identified six saliva-targeted genes and 15 blood-targeted genes that were highly expressed only in target tissues (or respective organs) but not, or nearly not, in the nontarget tissues (Electronic Supplementary Material Figure S1a, S1b, S1c). RT-PCR confirmation of tissue-specific markers To confirm the microarray results, real-time PCR experiments were designed for the 21 best candidate markers selected from array hybridizations and database searches and performed using RNA extracted from aged blood and saliva stains, also providing a method suitable for forensic applications. In agreement with the array results, all 21 markers analyzed showed good amplification in the target tissue but no or only marginally detectable amplification in the nontarget tissue (Fig. 1a–c). Among the candidate markers, only the PPL gene that was targeted for saliva demonstrated significant expression overlap with blood and therefore was excluded from further experiments. Our results demonstrate that, irrespective of the stain storage time, sufficient RT-PCR amplification was observed in all samples, even in the samples from the longest storage time tested (180 days), indicating marker stability over long periods of sample storage time. The only exception was the CCR2 gene for which no amplification was detected in the blood stains stored for 180 days and was therefore excluded from further analyses. A plausible explanation for this peculiarity could be the location of the PCR-amplified region, which is more than 1 kb distant from the 3′ end of the mRNA because of the very long untranslated region of CCR2. Apparently, the degree of degradation of the CCR2 mRNA after 180 days of sample storage was too high to allow its efficient reverse transcription using the oligo(dT) method that targets the 3′end of the molecule. This observation highlights the necessity to design PCR primers for the most 3′-proximal part of the mRNA molecule for successful amplification of cDNA fragments in degraded samples. Fig. 1a, b RT-PCR results for blood-targeted genes in blood and saliva stains. c RT-PCR results for saliva-targeted genes in saliva and blood stains. Genes were selected based on expression microarray results and GNF SymAtlas database. Expression values for each time point were averaged across three male and three female RNA samples; no gender-specific expression differences were detected (t test p < 0.05). B indicates blood; S indicates saliva; samples were processed after complete drying of blood and saliva at 0, 21, 57, and 180 days, respectively Expression of the candidate markers in other body fluids For additional confirmation of the tissue-specificity, we tested by RT-PCR the expression patterns of our candidate RNA markers in other body fluids that might be observed in a forensic case, i.e., vaginal secretion and semen. According to the GNF SymAtlas database, all our markers targeted for blood and saliva are not expressed in testis nor in uterus tissues. In agreement, our dedicated RT-PCR experiments revealed that two of the saliva-targeted mRNA markers (SPRR3 and SPRR1A) show no detectable expression in semen (after 50 RT-PCR cycles), and the remaining three (KRT4, KRT6A, and KRT13) show vast overexpression in saliva compared to semen (ddCt > 15, Fig. 2a), keeping with the assumption of high saliva specificity of the five proposed mRNA markers. SPRR1A and SPRR3 genes both encode cornified envelope precursor proteins and are predominantly expressed in oral and esophageal epithelia, where they are strictly linked to keratinocyte terminal differentiation [27]. Keratins 4, 6A, and 13 are known as one of the major structural proteins of oral mucosa [28, 29]. Fig. 2a RT-PCR for saliva and blood-targeted genes in semen stains. b RT-PCR for saliva and blood targeted in vaginal secretion stains. Delta Ct (dCt) values were calculated as follows: dCt = Ct (candidate gene) − Ct (endogenous control, GAPDH gene). Low dCt values correspond to high expression level of the specific mRNA. Gray bars correspond to the samples from target tissues for selected genes (either blood or saliva); black bars correspond to samples from nontarget tissues (either vaginal secretion or semen). Dotted bars represent the cases were amplification was not detected after 50 cycles, in this case, the expression values were arbitrary set to Ct value of 25 (plot maximum) For the 14 blood-targeted genes, we observed no detectable amplification in semen for nine genes (CASP1, AMICA1, C1QR1, ALOX5AP, AQP9, C5R1, NCF2, MNDA, ARHGAP26), keeping with the assumption of high blood specificity of the respective mRNA markers. These genes encode the proteins with important functions in different types of blood cells. They are known to be highly or even specifically expressed in peripheral leukocytes (AQP9, NCF2, CASP1, C5R1, C1QR1, ALOX5AP [30–35]) and myelocytes or hematopoietic cells (MNDA, ARHGAP26, AMICA1 [36–38]). However, five genes demonstrated only slightly differential or even comparable expression in blood and semen (CD36, CCR1, PF4, BIN2, and ALOX5), not expected given the information provided by the GNF database, and were therefore excluded from the final list of blood-specific markers. Thus, our microarray-based genome-wide approach to find tissue-specific mRNA markers identified the genes that are functionally relevant for the target tissues. Furthermore, and not surprisingly, RT-PCR of all saliva- and blood-targeted markers in samples from vaginal secretion revealed gene expression at a level comparable to that in blood and saliva samples (Fig. 2b). The natural occurrence of blood cells in vaginal secretion most likely explains the expression of our blood-targeted markers in vaginal secretion, whereas the high biochemical and histological similarity of oral and vaginal epithelia [40] makes the similarity of gene expression patterns between both tissues plausible. It should be pointed out that mRNA markers previously claimed to be useful for the identification of vaginal secretion such as HBD-1 [18] and MUC4 [18, 19] are known to be abundant also in oral epithelial cells and the salivary transcriptome [41–43]. Furthermore, Nussbaumer et al. [19] ruled out the potential to differentiate saliva and vaginal secretion using solely MUC4. Our results, together with previous findings, suggest that establishing mRNA markers expressed exclusively in vaginal secretion could be a challenging if not impossible task. Comparison with previously suggested mRNA markers Interestingly, tissue-specific genes, as identified here, do not overlap with the ones previously suggested for blood and saliva stain identification [18, 19]. This could be explained by the experimental setup and the systematic (but not ad hoc) approach of this study, namely, the degraded biological material analysed and the Affymetrix microarray platform applied. In contrast to previous studies, we restricted our marker ascertainment to those genes, which retained structural mRNA integrity during the stain dry-out process as well as the subsequent long-term storage of 180 days. This allows future application of detection of these markers in forensic stains of unknown age, at least up to an age of 6 months, but expectedly longer. Furthermore, our saliva-specific candidate genes were derived from mouth and pharynx epithelial cells, unlike the previously suggested STATH and HTN3 genes that are expressed in the salivary gland [18]. Secreted mRNAs that are abundant in fresh saliva are more prone to fast degradation by extracellular RNAses [39]; they are therefore not expected to be present in dried stains, explaining why they were not detected by the relatively low-sensitive microarray hybridization method used in this study. The SPTB and PBGD genes, previously proposed as blood-specific markers [18], do not demonstrate any overexpression relative to other tissues in whole blood according to the GNF SymAtlas database (data not shown). Conclusions In summary, whole-genome expression analysis in time-wise degraded samples from blood and saliva stains in combination with RT-PCR verification of various forensically relevant body fluids has resulted in the identification of stable tissue-specific mRNA markers from five genes for saliva (SPRR3, SPRR1A, KRT4, KRT6A, and KRT13) and nine genes for whole blood (CASP1, AMICA1, C1QR1, ALOX5AP, AQP9, C5R1, NCF2, MNDA, and ARHGAP26). For the first time, mRNA markers were ascertained considering almost the entire human transcriptome and based on experimental data of genome-wide gene expression as well as considering the degradation stability of mRNAs. We could demonstrate that the candidate genes identified here provide informative mRNA markers for blood and saliva identification for stains up to 180 days of age. We would like to propose their application in forensic case work (with the potential practical limitation of coamplification in vaginal secret) for stains of at least 6 months of age. However, we expect that the proposed mRNA markers will successfully identify older blood and saliva stains (respective experiments are currently in progress). Finally, we would like to remark that tissue identification in forensics should be performed in a reciprocal way; so that a tissue is identified because of the presence of markers specific for the relevant tissue together with the absence of markers specific for all other tissues in question. Clearly, more research should be dedicated towards finding the most suitable markers for tissue identification in forensics. Electronic supplementary material Below is the link to the electronic supplementary material. Table S1 RT-PCR primers for the genes amplified (DOC 79 kb). Figure S1a GNF Symatlas expression profiles of blood-specific genes (GIF 15 kb) High resolution image file (TIF 82 kb) Figure S1b GNF Symatlas expression profiles of blood-specific genes (GIF 14 kb) High resolution image file (TIF 77 kb) Figure S1c GNF Symatlas expression profiles of saliva-specific genes (GIF 12 kb) High resolution image file (TIF 74 kb)

Anal_Bioanal_Chem-3-1-1802725

Non-destructive analysis of museum objects by fibre-optic Raman spectroscopy

Raman spectroscopy is a versatile technique that has frequently been applied for the investigation of art objects. By using mobile Raman instrumentation it is possible to investigate the artworks without the need for sampling. This work evaluates the use of a dedicated mobile spectrometer for the investigation of a range of museum objects in museums in Scotland, including antique Egyptian sarcophagi, a panel painting, painted surfaces on paper and textile, and the painted lid and soundboard of an early keyboard instrument. The investigations of these artefacts illustrate some analytical challenges that arise when analysing museum objects, including fluorescing varnish layers, ambient sunlight, large dimensions of artefacts and the need to handle fragile objects with care. Analysis of the musical instrument (the Mar virginals) was undertaken in the exhibition gallery, while on display, which meant that interaction with the public and health and safety issues had to be taken into account. Introduction Advances in analytical technology have led to increasing interest in the scientific examination of precious artefacts and antiquities. These examinations may be for a variety of reasons, most commonly fundamental interest in the materials and techniques used by the artist, or understanding their conservation, condition or degradation processes. Analytical data (e.g. from spectroscopic examinations) can, by material identification, assist in dating or authenticating art objects or works of art. The availability of these techniques is therefore very important and it enables us to solve specific questions concerning the conservation, restoration and history of artworks [1–3]. In conservation science, analytical techniques which are non-destructive or micro-destructive are particularly important. Raman spectroscopy is such a technique, allowing material identification from particles down to 1 μm. This method has already been used for the investigation of different artefacts, including panel paintings [4, 5], glass [6], wall paintings [7, 8], manuscripts [9–11] and historical biomaterials [12, 13]. Although this technique used to be expensive and highly specialised, it is becoming increasingly more accessible to large museums owing to instrumental improvements and lower costs [14–17]. However, regional museums often still do not have access to this kind of analytical facility. Use of mobile equipment is one solution to this problem, since several institutions may benefit from a single instrument. In addition, mobile fibre-optic-based Raman instrumentation may facilitate the analysis of artefacts that are difficult or impossible to bring to the laboratory, such as wall paintings on the vault of a chapel [8]. In this work we evaluate the use of a mobile fibre-optic-based Raman instrument to perform investigations in a museum context. Different types of artefacts were investigated in a variety of locations, testing and demonstrating the flexibility in instrumental set-up. In particular the experimental conditions during the examination of a virginals on exhibition were challenging. Instrument stability after travelling overseas (between Belgium and Edinburgh, UK) was also thoroughly evaluated. Experimental Objects were investigated in the conservation and research laboratories, stores and an exhibition gallery of the National Museums of Scotland (NMS), Edinburgh, as well as in the Burrell Collection (Glasgow) (31 October–13 November 2005). For these surveys mobile Raman equipment was used, which has been described extensively elsewhere [19]. The core of the equipment consists of a SpectraPro-150i 150-mm spectrometer and a thermoelectrically cooled charge-coupled device detector (Roper Scientific/Princeton Instruments). The system is also equipped with a 785-nm diode laser for excitation, which has a maximal output power of 300 mW at the source. In order to avoid damaging the artefacts during these investigations, and depending on the object and the colour of the area under investigation, the output power was limited to a maximum of 10 mW (measured at the surface). The accumulation time ranged from 10 s to 1 h for weak Raman scatterers. For these investigations the 600 lines/mm grating was selected. Due to non-ideal focussing conditions, the expected minimal laser spot size of ca. 25-μm diameter (when working with a ×6 infinity-corrected objective lens) could not be reached. In practice we worked with a larger spot size of ca. 50 μm. For each area at least 10 spectra were recorded and evaluated in order to tackle paint inhomogeneity at this scale. The experimental configuration was adapted to suit the artefact under study. For example, the probe head could either be mounted horizontally or vertically on the articulating arm and extension tubes could be used on the probe head to facilitate access to the artworks [8, 18]. Results and discussion When testing the versatility of a mobile spectrometer in a museum context, the technique needs to be applied to a broad range of artefacts and questions. Throughout this text we will provide several examples, illustrating different facets of this approach. Table 1 gives an overview of the investigated artefacts, together with the analytical challenges that these investigations represent. Table 1Overview of the different artefacts that were investigated in this work and the analytical challenges they representInvestigated artefactAnalytical challengeEgyptian sarcophagus (NMS) (A.1907.569A)Pigment identification through a thick varnish layerEgyptian sarcophagus (Glasgow) (1895.167.a)Pigment analysis with strong interference from sunlight. Transport to a different museum, analyse, and return in 1 dayDean panel (H.KL 72)Analysis of a varnished paintingKalighat painting (A.1912.122)Analysis of work on paperIndian textile banner (K.2005.234)Analysis of a textile artefact of large dimensions with a thin and friable painted decorationMar virginals (H.LT 122)Investigation in the exhibition gallery open to the public The examination of Egyptian sarcophagi, both in the National Museums of Scotland (Edinburgh) and in the Burrell Collection (Glasgow Museums & Art Gallery) illustrate the comparative ease of transportation of the instrument. The capability of the technique to analyse pigments on artefacts covered by a thick varnish layer as well as to undertake the investigation with interference from sunlight are shown. During the examination of a Renaissance painted panel (one of the panels from Dean house, Edinburgh), strong inteference from a fluorescing varnish layer was also encountered. The possibilities of the technique for the non-invasive investigation of fragile artefacts on paper are illustrated by the yellow pigment identification on Indian Kalighat paintings and on a painted textile banner. During the latter survey the articulating arm was used to allow analysis of a large artefact displayed horizontally. Finally, working in the galleries represented an interesting experience as it involved undertaking analytical research whilst taking into account ambient conditions and visitors’ curiosity. This approach is illustrated by the study of the Mar virginals. Pigment analysis of antique Egyptian sarcophagi An antique Egyptian sarcophagus [A.1907.569 A], dating from the twenty first/second Dynasty (1069–715B.C.), was investigated at the NMS by using mobile Raman spectroscopy (Fig. 1a). Samples from this artefact had been examined before by Fourier transform (FT) Raman spectroscopy [19]. The aim of the present study was firstly to investigate whether non-destructive mobile Raman spectroscopy confirmed the previous findings. Secondly we wished to investigate the thick yellowed varnish layer, to see how it influenced the investigation, to establish when it was most likely to have been applied and what its visual effect was on the underlying colours. Fig. 1a Experimental set-up for the investigation of an Egyptian sarcophagus at the NMS. b Stack plot of the baseline-corrected Raman spectrum obtained from the yellow areas of this sarcophagus and the reference spectrum of orpiment (As2S3). (Experimental conditions for the spectrum of the artefact: 60 accumulations of 1 s, ×6 objective, 785 nm, ca. 5 mW at the surface) As expected, the yellow varnish generated serious interference of fluorescence radiation which overwhelmed the weaker Raman signal. Unfortunately it was not possible to identify its composition and therefore determine if it was original or whether it was the result of a previous conservation treatment. Nevertheless different pigments on the artefact could be identified. Figure 1b shows that the baseline-corrected Raman spectrum of the omnipresent yellow pigment on this sarcophagus is orpiment (As2S3). Other pigments that could be identified were hematite (Fe2O3), calcite (CaCO3) and carbon black (C). This is in agreement with the findings by Eremin et al. [19]. Although positive results were obtained, it was impossible to record a satisfactory Raman spectrum of the green and blue painted areas of the sarcophagus. It is very likely that Egyptian blue was used in this artefact; however, when stimulated with a 785-nm laser this pigment does not yield a Raman spectrum but gives rise to serious fluorescence background. Eremin et al. [19] indeed identified Egyptian blue with FT Raman spectroscopy (although the sample did not come from exactly the same area), so this illustrates one of the disadvantages of using mobile equipment. In contrast to many laboratory instruments, mobile instrumentation is usually equipped with a single laser, which makes it impossible to switch to another laser wavelength to overcome fluorescence interference. A second Egyptian sarcophagus [1895.167.a] was investigated at the Burrell Collection in Glasgow, illustrating one of the advantages of using mobile equipment: several collections can share one instrument. The sarcophagus (Fig. 2a), dating from the ancient Egyptian New Kingdom (1570–1070B.C.), was in the conservation lab, with high levels of natural light. The conservator wished to know the identity of the yellow pigment on the cover of the sarcophagus, since orpiment (As2S3) may degrade when exposed to high levels of light. Figure 2b shows that the yellow pigment on the sculptured head and hands of the sarcophagus lid indeed was indeed orpiment (As2S3), whereas the yellow pigment on the other areas was limonite (FeOOH· nH2O). Fig. 2a Experimental set-up for the investigation of an Egyptian sarcophagus in the conservation lab of the Burrell Collection, Glasgow. Ambient sunlight interfered with the investigations. b Stack plot of two Raman spectra obtained from the sculptured head and hands of this sarcophagus. (Experimental conditions: ×6 objective, 785 nm, ca. 5 mW at the surface, 120 accumulations of 1 s) Pigment investigation of a painted panel from Dean House, Edinburgh Mobile Raman spectroscopy was applied to identify the pigments in one of the Renaissance painted panels from Dean House in Edinburgh. The “Deans Panels” are thought to date from around 1627 [20]. The panel representing the sense of hearing (KL 72) is exhibited on a wall, at a height of about 3 m, which would have required the construction of scaffolding in the gallery. Therefore it was considered more practical to remove the panel temporarily from the exhibition and bring it to the conservation lab for analysis. The painted surface was found to be covered with a varnish layer, which contributed to the fluorescence background, although it still was possible to identify the pigments in the investigated areas of the artefact. Vermilion (HgS) was observed in the red areas, whereas orange areas merely contained red lead (Pb3O4). Brownish red areas were painted with a hematite (Fe2O3)-containing paint. On several occasions, mixtures of two of these pigments were encountered. Black areas contained carbon black (C), whereas the white regions were painted by using lead white (2PbCO3·Pb(OH)2). Pink zones consisted of a mixture of the latter pigment with vermilion; vermilion could also be identified in the purple regions (the blue component of which could not be identified). Finally, the yellow areas examined were painted by using lead–tin yellow, type I (Pb2SnO4). All these pigments are consistent with the early modern origin of the artwork. Identification of the yellow pigment from an Indian Kalighat painting The yellow pigment from different areas from an Indian Kalighat painting was examined by using mobile Raman spectroscopy. These are nineteenth century Indian watercolour and opaque media paintings on industrial paper. Since paper artefacts are fragile, special care has to be taken not to damage them during handling and positioning of the spectrometer. Gloves were worn and positioning of the probe head was performed with great care. On all the yellow painted areas of the Kalighat paintings that were examined, the same yellow pigment was encountered. Figure 3 shows the Raman spectrum obtained, along with the reference spectrum of chrome yellow (PbCrO4). It is clear that the yellow pigment in the Kalighat paintings is chrome yellow. Fig. 3Stack plot of the Raman spectra obtained from a yellow area from an Indian Kalighat painting and the reference spectrum of chrome yellow (PbCrO4). (Experimental conditions for the spectrum of the artefact: 8 accumulations of 150 s, ×6 objective, 785 nm, ca. 3 mW at the surface) Analysis of a painted banner The National Museums of Scotland has a large (1,080×2,030 mm) textile banner of uncertain provenance in its collection. Stylistic interpretations of the polychrome design suggest an Indian origin. The banner depicts, on a green background, colourful imagery of the different stages on the way to heaven (Fig. 4b, detail). Because of its large dimensions and the absence of appropriate non-destructive analytical methods, no previous investigation had been made and little was known of the materials that have been applied. The mobile Raman spectrometer was therefore used to identify the pigments. Fig. 4a Experimental set-up for the investigation of a large textile banner. b Lower part of the investigated textile banner. c Raman spectrum of an orange area of the textile banner, together with the reference spectra of massicot (PbO) and red lead (Pb3O4). (Experimental conditions for the spectrum of the artefact: 1 accumulation of 150 s, ×6 objective, 785 nm, ca. 10 mW at the surface). d Raman spectrum of a red area of the textile banner and reference spectrum of vermilion. (Experimental conditions: 1 accumulation of 150 s, ×6 objective, 785 nm, ca. 5 mW at the surface) In order to be able to examine a range of positions on the surface of the banner, the articulating arm was used for macro-positioning of the probe head, while the artefact was placed horizontally on a table (Fig. 4a). All the paint surfaces were thin, and in several areas the paint was friable or had already been lost. Red areas gave rise to the Raman spectrum of vermilion (HgS, Fig. 4d), whereas the orange areas yielded a more complex spectrum, which consists of a combination of massicot (PbO) and red lead (Pb3O4) (Fig. 4c). Red lead is an orange pigment, whereas massicot has a more yellowish shade. It is not clear whether massicot was intentionally added to the paint or whether the latter pigment arose as a side-product of the synthesis of red lead. Other pigments that were positively identified in the banner were calcite (CaCO3), carbon black (C), lead white (2PbCO3·2 H2O), azurite (2CuCO3·Cu(OH)2) and anatase (TiO2). Under the ambient conditions of the conservation laboratory, which did not have full light exclusion, and using the 785-nm laser, it was impossible to obtain a spectrum of sufficient quality to identify the green areas on the artefact. One solution to overcome this problem would be to use a Raman instrument with a different laser, with a smaller absorption cross section for the green pigments. Investigation of an early keyboard instrument, the Mar virginals The Mar virginals is an early keyboard instrument, which, although probably made in the Low Countries, has a long association with Lady Mary Stewart, countess of Mar and could have been made in Scotland. It probably dates from between A.D. 1560 and 1660, and its lid and soundboard contain three separate painted panels, the central one depicting Orpheus playing to the animals. We wished to compare the paintings on the lid to those on the soundboard, since similar or different pigment compositions may support or disprove the idea that the lid is contemporary to the virginals. This artefact had to be analysed while on display in the museum gallery, which enabled us to evaluate the feasibility of undertaking the analytical work in a public space. It was necessary to ensure that there were no health and safety risks, especially from direct or scattered light from the laser beam, as well as keeping the electronics and power supply cables and units away from enquiring fingers. The visiting public were clearly intrigued with what was going on and to help explain this, a poster informed the visitors about the experiments. Working in the gallery also meant that the ambient conditions were far from ideal. During this study, two issues severely hampered the Raman investigations: background light and spatial limitations. It was not possible to reduce the intensity of background light to avoid interference which tended to overwhelm the spectrum, nor on this occasion to undertake the work overnight. And since the Mar virginals was not taken out of its display case, little space was available to align the probe head with the painted surface, the cramped conditions meaning that great care had to be taken not to touch the artefact. To maximise flexibility the probe head was mounted on an articulating arm and extension tubes [8] were used to approach the painted surface (Fig. 5). Fig. 5Experimental set-up for the investigation of the Mar virginals Nevertheless, despite these non-ideal conditions, the pigments could be identified. White lead (2PbCO3·Pb(OH)2) is omnipresent: almost all the recorded Raman spectra contain the intense Raman band at 1,056 cm−1, whereas the less intense bands of white lead at 260, 203 and 154 cm−1 are only observed in a few spectra. Apparently, this pigment was applied to modify the hue of the coloured areas, making them appear brighter. From the intense Raman bands at 254, 283 and 343 cm−1, it is clear that the red areas were painted with the red pigment vermilion (HgS), whereas the bright yellow areas were painted by using orpiment (As2S3). Carbon black (C) is the black pigment which was used in the painted decoration. In general, the blue areas of the painting did not yield Raman spectra of sufficient quality to allow identification. The reasons for this are multiple. In general, blue and green pigments absorb the red (785 nm) laser light which hinders the recording of a spectrum. In addition there was a large amount of background radiation, both from the gallery lights and from fluorescence, which was probably caused by the varnish layer. It is very likely that the blue pigment azurite (2CuCO3·Cu(OH)2) is used in the artwork, since this pigment was very commonly used in Renaissance period: however, unfortunately azurite happens to be a weak Raman scatterer when using a 785-nm laser, which means that small amounts of background radiation easily overwhelm the Raman spectrum. Although only a few areas on the soundboard could be examined, the pigments on the lid were in good agreement with those on the soundboard. Despite the fact that this research project should be considered as a pilot study, this tends to support the thesis that the lid and the soundboard originate from the same instrument, or at least were painted at a similar period. However other approaches, such as stylistic studies, or taking samples to matching the spore-element compositions of the paint from the lid and the soundboard [9], are needed to be more definite. Since the investigation of this rare musical instrument was performed in one day, time was confined and only a limited number of areas could be examined. In fact, except for the absence of lead–tin yellow, none of the pigments were rare for an artwork of this period. Conclusions This work reports on the application of mobile Raman spectroscopy to the non-invasive investigation of a range of object of arts in a museum context. The different cases presented here illustrate different obstacles and analytical challenges that are often encountered during direct Raman investigations of museum objects. The investigation of two antique Egyptian sarcophagi shows the possibilities of the technique to deal with artefacts covered by a thick varnish layer as well as the investigation with the interference of ambient sunlight in the conservation laboratory. The investigation of the Dean House panel illustrates the possibilities of pigment investigation although a fluorescing varnish layer is present; the suitability of the method to analyse fragile artefacts was demonstrated by the investigation of Indian Kalighat paintings on paper; the textile banner was both fragile and large. Finally, the Mar virginals has been examined for the first time directly in the exhibition gallery with normal lighting conditions and with visitors present. All these examples illustrate that non-destructive, mobile Raman spectroscopy is able to cover a broad field of research questions on artistic and historical objects, in a range of different experimental conditions, encountered in a museum context.

Ann_Biomed_Eng-2-2-1705526

The Effects of Time Varying Curvature on Species Transport in Coronary Arteries

Alterations in mass transport patterns of low-density lipoproteins (LDL) and oxygen are known to cause atherosclerosis in larger arteries. We hypothesise that the species transport processes in coronary arteries may be affected by their physiological motion, a factor which has not been considered widely in mass transfer studies. Hence, we numerically simulated the mass transport of LDL and oxygen in an idealized moving coronary artery model under both steady and pulsatile flow conditions. A physiological inlet velocity and a sinusoidal curvature waveform were specified as velocity and wall motion boundary conditions. The results predicted elevation of LDL flux, impaired oxygen flux and low wall shear stress (WSS) along the inner wall of curvature, a predilection site for atherosclerosis. The wall motion induced changes in the velocity and WSS patterns were only secondary to the pulsatile flow effects. The temporal variations in flow and WSS due to the flow pulsation and wall motion did not affect temporal changes in the species wall flux. However, the wall motion did alter the time-averaged oxygen and LDL flux in the order of 26% and 12% respectively. Taken together, these results suggest that the wall motion may play an important role in coronary arterial transport processes and emphasise the need for further investigation. Introduction The formation of an atherosclerotic plaque is dependent upon a range of factors including local variations in haemodynamics, the transport of chemical species and cells from the blood to the arterial wall and their accumulation within the arterial wall.3,21,38The local susceptibility of coronary arteries to atherosclerosis has been putatively linked to the physiological motion arising out of their anatomical attachment to the epicardial surface of the beating heart.7,8Coronary arteries exhibit highly complex wall motion patterns such as bending, stretching, twisting, vessel torsion and vessel displacement due to the cardiac and respiratory motion of the heart.7,22 Despite their seemingly important role, to date, only a handful of studies have looked at the effects of wall motion on atherogenesis. Early studies investigated the axial and lateral movement of a straight tube model of the coronary arteries and showed that the wall shear stress (WSS) patterns were significantly affected by the axial movement and not so by the lateral movement.4,23Other studies examined the effects of time dependent curvature on the flow field and demonstrated that the flow patterns were affected by the wall motion due to instantaneous changes in curvature and that the WSS patterns were strongly dependent on the frequency of curvature variation.18,24,32,33Another study considered the cyclic flexion of coronary arteries and reported that the plaque advancement was proportional to the flexion angle.37 More recently, studies have concluded that the dynamic curvature effects could play an important role in the flow and WSS patterns in curved and in bifurcating arteries.28,40,41Nevertheless, these studies reported that the temporal WSS variations produced by the wall motion was only secondary to that caused by the pulsatility of blood flow.28,41So far studies have only paid attention to the haemodynamic effects of wall motion. However, it has now come to light that the transport of atherogenic macromolecules such as LDL and solutes such as oxygen from the blood to the arterial wall play an important role in atherogenesis.38 Interestingly, with the exception of a single study, no other past study has investigated the effects of species transport to the moving walls of coronary arteries.17 Even in that study only the effects of diameter variation (<6% of mean diameter) on oxygen transport was considered. Hence, the aim of our work was to determine the effects of wall motion on mass transport patterns in coronary arteries with physiological flow. For our investigation, we chose a geometric model of the left anterior descending coronary artery (LAD) because of its high predilection to atherosclerosis among the major coronary arteries.12 Computer aided design (CAD) models of the LAD were employed in this study as opposed to realistic geometries to simplify the complexity of the problem definition, solution and analysis of results so as to gain a fundamental understanding of the haemodynamic effects of wall motion in coronary arteries. The species of interest in this study were low-density lipoprotein (LDL) and oxygen. Methods Computational Model A 3-D computational model of the LAD (Fig. 1) was constructed based on the average LAD dimensions (diameter (d) = 0.36 cm and mean radius of curvature (Rmean) = 4 cm),9,15,16using the CFD pre-processor Gambit (v2.0.4 – Fluent Inc., Lebanon, NH). The total length (l) of the LAD considered in this study was 4.68 cm which was equivalent to 13 tube diameters. In order to make the simulation as simple as possible, the model was assumed to be rigid with a circular cross section of constant radius and a constant uniplanar curvature over its entire length at any instant in time. An unstructured grid composed of hexahedral mesh elements with a thin boundary layer was generated using the Cooper meshing scheme2 with strict adherence to mesh quality metrics. Figure 1.A schematic model of the left anterior descending coronary artery (LAD). Blood Flow Boundary Conditions The flow boundary conditions controlling the model included no slip at the tube wall, velocity inlet at the inlet, symmetry condition at the plane of symmetry and a traction free outflow condition. The LAD inlet velocity profile was assumed to be parabolic. A pulsatile physiological LAD inlet velocity waveform (Fig. 2) derived from a previous publication by Marcus et al.22 was applied at the inlet boundary through a user defined function. The time-averaged mean velocity (ν), mean flow rate (qo) Reynolds number (Re) and Dean number (κ) corresponding to this flow waveform were 9.715 cm/s, 0.99 ml/s, 105 and 22.3. The Dean number is defined as below Figure 2.The LAD inlet flow and curvature waveform. The Womersley parameter (α) of the flow was 2.78 and the period of a cardiac cycle (T) was 0.8 s. Blood was considered to be an incompressible, homogeneous, Newtonian fluid with a viscosity (μ) of 0.0035 Pa s and a density (ρ) of 1050 kg/m3. Past arterial flow dynamic studies have shown that these assumptions are reasonable enough to use in haemodynamic studies.16,29 Wall Motion Boundary Conditions The wall motion boundary condition used in this study was based on the model presented by Santamarina et al.32 The main components of coronary artery motion are solid body motion and deformation. The deformation parameter (ε) was calculated as below where R is the radius of curvature of the LAD. The present study used a deformation parameter of 0.6 which falls within the physiological range of LAD deformations.15 A sinusoidal function as given below was used to specify the time dependent variation of curvature of the LAD (Fig. 2) where ω is the angular frequency of deformation. A user-defined function was written to apply this curvature waveform as a grid motion boundary condition. The tube inlet was fixed constant in the simulations at all times. This allowed the centre of curvature to vary sinusoidally in a cardiac cycle. Mass Transfer Boundary Conditions The mass transport of species in the arterial lumen is mathematically described using the three dimensional convection-diffusion equation where C is the concentration of species, u is the velocity vector and D is the diffusivity of species in the blood. In this study, the transport of LDL and oxygen from the blood to the endothelium (fluid phase), and across the endothelium (transendothelial) were modelled. The fluid phase transport of both LDL and oxygen takes place via convection and diffusion. However, the transendothelial transport of LDL and oxygen are governed by different mechanisms due to their relative size and the endothelial function. The semi permeable endothelial membrane, acts as a barrier for the entry of macromolecules such as LDL into the arterial wall, but allows blood plasma and small solutes such as oxygen to pass through it.10 This is reflected in the permeability of LDL (K = 2 × 10−8cm/s) which is five orders of magnitude smaller than that of oxygen (K = 2 × 10−03cm/s).36 This indicates the need for modelling LDL and oxygen transport differently through appropriate mass transport boundary conditions. LDL transport was modelled as a concentration polarisation effect, a phenomenon which has been demonstrated to occur in large and medium sized arteries.5,11,39Concentration polarisation is the accumulation and elevation of endothelial surface concentration of LDL due to plasma filtration through the semi permeable endothelium. LDL mass transport boundary conditions applied in this study were uniform inlet concentration, Co (=1.2 mg/ml of blood),36 a zero gradient in concentration at the outlet and mass conservation of LDL at the luminal surface of the endothelium39 described as where Vw is the plasma filtration velocity normal to the wall (=4 × 10−6cm/s),36Cw is the endothelial surface concentration of LDL, n is the unit vector normal to the vessel wall, D is the physiological LDL diffusivity (=5 × 10−8cm2/s)39 and K is the overall mass transfer coefficient of LDL which was considered equivalent to the physiological endothelial permeability of LDL. Equation (5) was rearranged as below to implement a wall reaction boundary condition in the CFD solver. In this study, oxygen transport was modelled by applying a passive transport law for oxygen flux at the wall.31 It is known from previous estimates that the diffusion velocity of oxygen is orders of magnitude greater than the filtration velocity.1 Hence, the flux of oxygen to the wall is essentially the diffusive flux. To satisfy flux continuity at the wall the diffusive flux of oxygen to the wall should be equal to the wall flux of oxygen which can be modelled with a constant permeability wall boundary condition.31 Oxygen mass transport boundary conditions employed in this study were uniform inlet oxygen mass fraction (=0.005),20 a zero gradient in outlet concentration and a constant permeability wall boundary condition31 which is given as where the oxygen diffusivity was 1 × 10−5cm2/s1. Numerical Method The commercial CFD solver Fluent (v6.1.22 – Fluent Inc., Lebanon, NH) was used to simulate species transport in the moving LAD model. Fluent uses a finite volume technique to solve the three-dimensional (3-D) unsteady equations of momentum, mass and species conservation. QUICK scheme was employed to solve the flow and species transport equations. The time discretization and pressure–velocity coupling were achieved through second order time implicit, and PISO schemes respectively. The dynamic mesh model feature of the Fluent was used to implement the deformation of the model according to Eq. (3). The dynamic mesh model uses an arbitrary Lagrangian–Eulerian approach to move the mesh nodes. At each time step, the volume mesh is updated automatically by the solver based on the new co-ordinates of the boundary zones that were calculated by the user-defined function. At the beginning of each simulation, the radius of curvature of the model was at its maximum (=6.4 cm). At t = 0.2 s and 0.6 s the radius of curvature of the model was equivalent to its mean value. Modelling Approach To separate the effects of blood flow pulsatility and wall motion on mass transport patterns in coronary arteries so as to determine their relative importance, we undertook the following four different numerical simulations:Case 1: Steady flow inlet (qo = 0.99 ml/s) in the static geometry with the mean radius of curvature (R = 4 cm).Case 2: Pulsatile flow inlet in the static geometry with the mean radius of curvature (R = 4 cm), to isolate the haemodynamics effects of flow pulsatility.Case 3: Steady flow inlet (qo = 0.99 ml/s) with dynamic geometry, to isolate the haemodynamics effects of wall motion.Case 4: Pulsatile flow inlet with dynamic geometry, to study the combined effects of pulsatility and wall motion. Model Validation The computational method was validated by simulating Santamarina et al.’s32 coronary artery motion study in a curved model of exactly similar dimensions under identical flow and wall motion conditions. The flow and wall shear stress (WSS) patterns agreed well with that of Santamarina et al’s study and the maximum difference in the wall shear rate was less than 2%. The mesh and time step independence of the solutions were investigated by successively refining the grid and the time step size by a factor of two. Computational solutions obtained with three different mesh densities (80,310, 156,861 and 310,464 nodes) and time step sizes (0.025 s, 0.0125 s and 0.00625 s) were then compared against each other (Fig. 3). Mesh and time step independence were considered to have been achieved when the maximum difference between the time-averaged normalised outer and inner wall flux of LDL (Cw/Co) between the successive simulations was less than 2% at any axial location. The outer and inner walls were defined as the wall regions farthest and shortest from the centre of curvature to represent epicardial and myocardial surface of the curved coronary arteries. This study was only carried out for LDL transport in the dynamic model with pulsatile flow because this was the computationally most challenging problem (Schmidt number, μ/ρD = 6.7 × 105 & Peclet number, dv/D = 7 × 107) encountered in this study. Based on these criteria, a mesh containing 156,861 nodes with a time step size of 0.0125 s was chosen as the optimum case for simulation. Figure 3.Comparison of the time-averaged normalised LDL wall flux predicted with three different meshes and time step sizes to demonstrate mesh (top panel) and time step (bottom panel) independence of the computational solutions. Results Numerical simulations of oxygen and LDL transport in a 3-D curved tube model of the LAD were carried out under physiological flow and mass transport conditions. The computational solutions concerned the prediction of flow, WSS and mass transport patterns. The results will be presented only for the first 10 diameters length as this length is more relevant to realistic coronary flow situation as discussed elsewhere.32 Velocity Patterns The temporal velocity magnitude (coloured contour plots) and secondary flow patterns (vector plots) in the static and dynamic models of the LAD are shown in Fig. 4. The fundamental flow patterns in a curved tube flow situation with a characteristic velocity skewing towards the outer wall of bend and the formation of Dean vortices were clearly seen in all the models (Fig. 4). Although both the pulsatility (Fig. 4b) and wall motion (Fig. 4c) produced time dependent velocity skewing, the flow pulsation had a greater effect on velocity skewing than did wall motion. The secondary flow was also affected by both the pulsatility and wall motion (Fig. 4e and f). However, the wall motion induced time dependent curvature (Fig. 4f) seemed to have a major influence on the secondary flow patterns. Adding the dynamic motion to the pulsatile flow reduced skewing over the entire cycle (Fig. 4d). Moreover, this combination led to a reduction in secondary flow when the time dependent vessel curvature was small and vice versa during the peak curvature phases (Fig. 4g). In general, both the velocity skewing and secondary flow increased with increase in curvature and the flow rate (Fig. 4). Figure 4.Temporal axial and secondary flow patterns in a plane located at 9 diameters from the inlet in the (a) steady flow static model (R = 4 cm, qo = 0.99 ml/s), (b and e) pulsatile flow static model (R = 4 cm), (c and f) steady flow dynamic model (qo = 0.99 ml/s), and (d and g) pulsatile flow dynamic model. The outer and inner walls are located at the top right corner and top left corner of individual figures respectively. WSS Patterns The effects of pulsatility and wall motion on the instantaneous WSS distribution in a cardiac cycle at two axial locations (5D & 9D) are plotted in Fig. 5. The effect of motion in steady flow at the outer wall was to increase the WSS when the curvature was maximum. Along the inner wall the opposite effects were seen with a drop in the WSS at the maximum curvature. The wall motion also produced asymmetric WSS distribution over time in the steady flow dynamic model despite the imposed symmetric wall motion curve. Figure 5.Temporal axial inner and outer WSS distribution at 5 diameters (top panel) and 9 diameters (bottom panel) from the LAD inlet. The pulsatility of blood flow caused time varying WSS distribution along the outer and inner walls. The WSS increased with the pulsatile flow rate and overall this increase was greater along the outer wall. Hence peak WSS was noted along the outer wall at maximum flow rate. Together, wall motion and flow pulsatility produced instantaneous variations in the outer and inner WSS which were somewhat similar to the WSS distribution in the pulsatile flow static model. Although the gross distribution of WSS primarily followed the flow rate curve, the dynamic curvature effects were evident in the central section where the location and magnitude of peak WSS were different from that of the pulsatile flow static model. The time-averaged axial WSS distribution at the outer and inner walls in all the models is shown in Fig. 6. In general, the spatial dependence of time averaged WSS was evident in all the models. In the steady flow static model the outer WSS increased to a peak and thereafter was found to decrease very gradually. In contrast, the inner WSS decreased to a minimum and was thereafter found to increase slowly. In comparison, the flow pulsatility caused a big increase in the outer WSS in the pulsatile flow static model. However, the wall motion only produced a little change in the outer WSS in the steady flow dynamic model. Figure 6.Time-averaged axial WSS distribution along the inner and outer walls of the LAD. The combined effects of wall motion and pulsatility caused an increase in the outer WSS up to its peak value and a subsequent decrease in the WSS in comparison with that of the pulsatile flow static model, and an overall increase in the outer WSS when compared to the steady flow models. Along the inner wall the flow pulsatility and the wall motion produced contrasting effects whereby the former decreased the WSS and the later increased the WSS. Due to this opposing effect of wall motion and flow pulsatility their combination had only caused smaller variations in the WSS distribution. Oxygen Transport The time dependent non-dimensional oxygen wall flux (KCw/KCo) distribution at 5D and 9D is presented in Fig. 7. The oxygen wall flux did not vary appreciably over time in a cardiac cycle. At any instance the outer wall oxygen flux was higher than that at the inner wall. These patterns were similar in all the models studied. When compared to the oxygen wall flux in the steady flow static model, pulsatility caused a reduction and wall motion an increase in the oxygen wall flux along the inner wall. Along the outer wall the effects of pulsatility and wall motion were opposite to this. In total, the net effect of pulsatility and wall motion was an increase in oxygen flux at the inner wall and decrease at the outer wall when compared to the steady flow static model. This effect was more prominent in the distal region (Fig. 7c and d). Figure 7.Temporal normalised oxygen flux to the inner and outer walls at 5 diameters (top panel) and 9 diameters (bottom panel) from the LAD inlet. The time-averaged non-dimensional oxygen wall flux to the outer and inner walls of all the four models are shown in Fig. 8. The overall effects of pulsatility and wall motion were similar to that predicted with the time dependent oxygen wall flux distribution. The effect of flow pulsation was to decrease the oxygen flux to the inner wall. In contrast, the wall motion contributed to an increase in oxygen flux in the distal region of the inner wall. Hence the combined effects of wall motion and flow pulsatility were to decrease oxygen flux in the proximal and medial regions of the inner wall and a very gradual increase distally. Figure 8.Time-averaged normalised oxygen wall flux distribution along the inner and outer walls of the LAD. Along the outer wall after an initial sharp drop the flow pulsation caused a gradual increase and then a gradual decrease in the oxygen flux. The wall motion produced a net reduction in the oxygen flux to the outer wall from the inlet. When both the wall motion and flow pulsatility were included the oxygen flux decreased sharply closer to the inlet followed by a gradual increase in the medial region and a gradual decrease thereafter. Nevertheless, in all the models the time-averaged outer wall oxygen flux was more than that to the inner wall. LDL Transport The time dependent non-dimensional LDL wall flux (KCw/KCo) distribution at 5D and 9D is plotted in Fig. 9. Like oxygen wall flux the LDL wall flux did not exhibit notable variations over time. The flow pulsatility caused an increase in LDL flux to the inner wall and a decrease in LDL flux to the outer wall when compared to the LDL flux distribution in the steady flow static model. In contrast, the wall motion induced LDL wall flux was lower along the inner wall. However, along the outer wall it produced a differential distribution whereby the LDL wall flux was lower than that in the steady flow static model at 5D and vice versa at 9D (Fig. 9c and d). At 9D the outer wall LDL flux was greater than that along the inner wall. Also the outer wall flux in the steady flow dynamic model was higher than that in the pulsatile flow static model. Hence the net effect of wall motion and pulsatility produced LDL wall flux distribution in the pulsatile flow dynamic model such that it was between the range of LDL wall fluxes caused by the pulsatility and wall motion separately. Figure 9.Temporal normalised LDL flux to the inner and outer walls at 5 diameters (top panel) and 9 diameters (bottom panel) from the LAD inlet. The time-averaged non-dimensional LDL wall flux to the outer and inner walls of all the four models are shown in Fig. 10. In all the models the time-averaged LDL wall flux distribution was seen to vary appreciably with respect to the axial distance from the inlet. The flow pulsatility caused an increase and decrease in the flux along the inner and outer walls respectively when compared to the steady flow static model. In contrary, the wall motion caused a reduction in the inner wall LDL flux when compared to that of the steady flow static model. Whereas the outer wall LDL flux was lower and higher than that in the steady flow static model in the medial and distal regions respectively. The combined effects of pulsatility and wall motion led to a substantial increase in the outer wall LDL flux in the distal regions. In the proximal and medial regions the outer wall LDL flux was lower than that to the inner wall. However, the inner wall LDL flux increased to a maximum and was then seen to drop off to a value lower than that along the outer wall in the distal part. Figure 10.Time-averaged normalised LDL flux distribution along the inner and outer walls of the LAD. Discussion To determine the effects of wall motion, a factor which has received less attention in the previous mass transport studies in coronary arteries, mass transport patterns were computed in an idealised moving model of the LAD. In this study, LDL and oxygen transport were simulated because of their important role in atherogenesis. The flow patterns reported in the current study (Fig. 4) qualitatively agree with the findings of past experimental and computational studies in that they all demonstrate a general trend of velocity skewing towards the outer wall of curvature and the appearance of in-plane secondary flow.16,27,29,32The centripetal acceleration of the fluid due to the arterial curvature was primarily responsible for these flow patterns (Fig. 4a). This basic flow feature was further altered by the flow pulsatility (Fig. 4b and e) and the dynamic curvature (Fig. 4c and f). Their relative role on secondary flow can be characterised using the non-dimensional Dean number (κ). From the definition of the Dean number (Eq. 1), it is clear that higher the flow rate or smaller the radius of curvature the stronger the Dean vortices will be. This was evident in the figures as the magnitude of skewing and the strength of Dean vortices increased with increasing flow rate and curvature and were maximum at maximum flow rate in the pulsatile flow static model and at maximum curvature in the steady flow dynamic model. Therefore, the combination of flow pulsatility and wall motion were responsible for the appearance of time dependent flow patterns in the pulsatile flow dynamic model (Fig. 4d and g). The flow patterns seen in this study are qualitatively similar to that of Santamarina et al’s work which considered the effects of dynamic curvature.32 The flow patterns at t = 0.2 s and 0.6 s in the steady flow dynamic model where the time dependent curvature was equal to the mean curvature appeared to be different from that in the steady flow static model (Fig. 4c and f). This suggests that a quasi-static model cannot predict the flow patterns accurately and that the dynamic wall motion could be as important as the instantaneous geometry. Similar findings were reported by Ethier et al. who compared the flow patterns in frozen geometries and in a moving model of the right coronary artery segment.41 The WSS patterns calculated in the pulsatile flow static models are similar to those reported by other studies on coronary artery flow.16,27,29Since the WSS is directly proportional to the gradient of velocity the WSS patterns followed the trends of velocity distribution. The velocity skewing towards the outer wall subjected the outer and inner walls to higher and lower WSS respectively except up to a distance of 1D where the entrance effects were dominant (Fig. 6). The instantaneous and mean WSS distribution in the pulsatile flow dynamic model closely followed that of the pulsatile flow static model except in the central section where the curvature was high. These results suggest that the effects of wall motion on temporal WSS patterns are only secondary to the pulsatile flow effects and agree well with the findings of other dynamic model studies.28,41 Concentration polarisation of LDL has been demonstrated to occur in large arteries by several past experimental and numerical studies.5,11,39The build-up of LDL concentration at the apical surface of the endothelium due to concentration polarisation could increase the influx of LDL into the arterial wall.5,35 This, in turn, could initiate a sequence of biological and biophysical events that are associated with atherogenesis.5,25 Therefore, LDL transport was modelled as a concentration polarisation effect and the results were presented as normalised LDL wall flux (KCw/KCo). The present study also considered oxygen transport because hypoxia or low oxygen tension in the arterial wall has long been considered to be a factor responsible for atherogenesis.1,17It induces endothelial cell apoptosis, causes breakdown of endothelial barrier and forms interendothelial gaps which may cause lipid infiltration into the arterial wall. Recent evidences suggest that hypoxia may also increase vascular permeability to macromolecules by up regulating vascular endothelial growth factor (VEGF) release by vascular cells.38 Hence oxygen transport was modelled with a passive transport law for oxygen flux at the wall.31 To describe oxygen transport patterns normalised oxygen wall flux (KCw/KCo) was calculated. In general, the mass transport patterns were similar in all the models. The establishment of low velocity, low WSS regions along the inner wall due to reasons discussed earlier led to an overall elevation of LDL flux and a reduction in oxygen flux to the inner wall. In contrast, high axial flow and high WSS along the outer wall due to axial velocity skewing caused a reduction in LDL flux and an increase in oxygen flux to the outer wall. The higher the WSS the higher the convection and the convective transport of oxygen. Hence the oxygen flux to the outer wall was more than that to the inner wall. The opposite effects were noted for LDL transport because the degree of concentration polarisation is reduced where high flow and high WSS prevail.39 As a result, the LDL accumulation was generally lower along the outer wall and higher along the inner wall. The diffusive transport of LDL and oxygen however, had minor influence on their transport. This was especially true because of their constant low diffusion coefficients. These results qualitatively agree with mass transport studies in curved arterial transport models.17,17,39 Despite the large time dependent variations in the blood flow and WSS patterns, the flow pulsation only had a minimal effect on oxygen (Fig. 7) and LDL (Fig. 9) transport to the arterial wall. Consequently, the time-averaged oxygen and LDL wall fluxes (Figs. 8 and 10) in the static pulsatile flow model were only slightly different from their instantaneous values. This less significant pulsatile effects on time-averaged mass transport patterns have also been reported by other studies.11,19,34 Schneiderman et al.34 simulated oxygen transfer in straight arterial segments and concluded that the pulsatile flow has negligible effect on oxygen transport to the wall. Ma et al.19 studied the effect of pulsatility on mass transfer in a sudden expansion region and reported that the time-averaged pulsatile mass transfer patterns were similar to the steady flow mass transfer patterns. Fatouraee et al.11 simulated LDL concentration polarisation in a straight tube model of a carotid artery under pulsatile flow conditions. The variation of LDL wall concentration in their study was less than 0.014%. Time dependent variations in mass transport patterns were also not present in the dynamic models (Figs. 7 and 9). As a result, the magnitude of time-averaged oxygen and LDL wall fluxes were only slightly different from their corresponding time dependent flux values (Figs. 8 and 10). Although the wall motion did not significantly affect the temporal mass transfer patterns in the dynamic models, an overall change in the mass transfer patterns was evident (Figs. 7 and 9). This change was also reflected in the time-averaged mass transfer patterns in the dynamic models (Figs. 8 and 10). The percentage change in time-averaged oxygen wall flux and LDL wall flux along the inner and outer walls between the static and dynamic models under both pulsatile and steady flow conditions was in the order of 26% and 12% respectively. The fact that the pulsatility did not alter mass transfer patterns and that there were variations in mass transport patterns in the dynamic models suggest that the wall motion may indeed be important in determining the time-averaged mass transport patterns in the dynamic models. These variations were not significant in the proximal regions of the LAD model. However, considerable changes in the mass transport patterns were present in the medial and distal regions of the LAD (Figs. 8 and 10). This regional difference occurred due to the specification of fixed inlet boundary condition. Although this is an effect of the fixed inlet boundary condition these results once again indicate that strong motion of the coronary arteries could have a significant impact on the luminal mass transport patterns. The species transport however, does not entirely depend on the WSS distribution. Karino et al drew a scatter plot to establish a relationship between the WSS and LDL surface concentration.39 The scatter in their data suggests that the species transport is also influenced by global flow patterns, path line of fluid flow, spatial location and residence time of LDL at a site. This was also found in the present study which noticed different values of LDL and oxygen wall flux for the same WSS value along the outer and inner walls (Figs. 6, 8 and 10). The inner walls of a curved artery where low WSS prevails is known to be a predilection site for atherosclerosis.14,21 LDL and oxygen transport studies have reported impaired oxygen flux and enhanced LDL accumulation in the low shear regions.5,17,17,39Similar to these findings the present study also predicted co-localisation of low WSS regions, high LDL flux regions and hypoxic regions along the inner wall of curvature (Figs. 6 and 10) in both the static and dynamic models. Hence our results suggest that even when the wall motion is considered the susceptibility of inner wall regions of the curved arteries to atherosclerosis is maintained. Back et al.1 investigated the relationship between oxygen transport and lipoprotein accumulation in various arterial configurations. They noticed a correspondence between oxygen deprivation (hypoxia) and enhanced lipoprotein accumulation in decelerated flow regions and separation locations and concluded that the oxygen transport behaves in an opposite way to lipoprotein accumulation. This contrasting behaviour of oxygen and LDL transport was also noticed in our present study. It would be interesting to study the indirect effects of hypoxia on LDL transport through pathways involving enhancement of endothelial permeability for macromolecules.38 The present study assumed a constant endothelial permeability of oxygen and LDL which, in reality, is spatially variable and is dependent on the concentration of species at the arterial wall surface and the WSS.26,36Pulsatile variations in the WSS has been shown to alter the morphology of endothelial cells, increase macromolecular permeability to LDL, promote transendothelial diffusion and intimal entrapment of atherogenic particles and enhance oxygen wall flux.13,21Therefore, it is possible that the temporal variations in WSS caused both by the pulsatility of blood flow and wall motion may have an effect on LDL and oxygen transport if shear dependent variations in physiological parameters including the endothelial permeability are considered in mass transport studies. So far their effects on species transport have only been considered in static models30,36and it remains to be investigated how these factors would influence mass transport in moving arteries. This will be the subject of our future study. The limitations of this study centre on the assumptions that were made in the construction of computational models which include idealized geometry, wall motion, and biophysical factors governing LDL and oxygen transport. An idealised, uniformly curved model without complex geometric features such as tortuosity, twisting and non-uniform cross sections was used. Inclusion of these geometric features would be expected to create local regions of flow acceleration, deceleration, separation, recirculation and secondary motion1,27 which may exacerbate or reduce the effects of wall motion on haemodynamics and species transport. While we accept that the neglect of complex geometry may introduce some simplifications in the results it should be recognised that the geometric simplification enabled us to isolate the effects of geometry and to concentrate on the role of wall motion on species transport. The results predicted in this study are specific to the flow and wall motion used in our model and therefore would be expected to change if a different flow or wall motion were imposed. For instance, if the flow and wall motion were more out of phase it may be expected that this would reduce the instantaneous Dean number and therefore one might expect the impact of the dynamic wall motion to be decreased from what was predicted. Although the wall motion effects were secondary to the pulsatile flow effects this does not mean that the wall motion is unimportant. Given the considerable variability in coronary artery motion, geometry and flow patterns in vivo there may be instances where wall motion could play a more significant role and therefore its effects should be considered on a case by case basis. Our study assumed that the non-Newtonian effects caused by the presence of red blood cells are negligible and therefore used constant diffusivities for LDL and oxygen. However, the effective diffusivity of a species changes due to the shear induced microscopic motion of red blood cells.6 In regions of high WSS, the effective diffusivities of LDL and oxygen would increase. This would in turn enhance oxygen wall flux but reduce LDL wall flux due to LDL concentration polarisation. Therefore, the magnitude of LDL and oxygen wall fluxes predicted in our study would be expected to change but the general patterns of flux will remain the same. Conclusion In this study, we have incorporated the effects of idealised motion into a simulation of oxygen and LDL transport in the LAD. The LDL and oxygen transport were modelled differently due to their differences in in-vivo transport mechanisms. We carried out simulations under both the steady and pulsatile flow conditions in the static and dynamic models to quantify the relative importance of pulsatility and wall motion on mass transport patterns. This study showed that the effects of wall motion on mass transport in the coronary arteries could be modelled in the commercial CFD code Fluent. Our results predicted elevation of LDL flux, impaired oxygen flux and low wall shear stress (WSS) along the inner wall of curvature, a region known for its predilection to atherosclerosis. The temporal variations in velocity and WSS patterns in the dynamic model did not influence time dependent mass transport and were only secondary to the pulsatile flow effects. However, wall motion may influence mass transport if shear dependent variations in species permeability and other factors are considered in the calculations. Nevertheless, the wall motion did alter the time-averaged mass transfer in the medial and distal regions in the order of 26% and 12% for oxygen and LDL transport when compared to the corresponding static models. Taken together, these results suggest that wall motion may play an important role in coronary arterial transport processes. However, future studies on more physiologic models are warranted to gain a fundamental understanding of the role of wall motion on mass transport and atherosclerosis.

Bioinformation-1-2-1891631

A database for medicinal and aromatic plants of JK (Jammu and Kashmir) in India

High throughput screening of small molecules for a given drug target is achieved using plant materials of medicinal value. Therefore, it is important to document the availability and location of such medicinal plants in the form of a database. Here, we describe a web database containing information (botanical name, common name, local name, botany, chemistry, folklore medicinal use and medicinal uses) about the medicinal and aromatic plants available in JK (Jammu and Kashmir). The database is available for free in public domain. Background Medicinal plants documented in the literature from thick vegetations of the world are routinely used for high throughput screening in small molecular drug discovery. A large number of such plants are known to be recognized in a discrete manner. Therefore, it is important to store information related to medicinal and aromatic plants of JK in a database. This will help in the use and exploitation of the plant materials for drug discovery. Some of the plants available in this region are also known to have aromatic value. Here, we describe the development and use of a database containing information on medicinal and aromatic plants from JK. Methodology Dataset Folklore medicinal usage and other associated data were manually collected from the inhabitants (tribal/gujjars/local clinicians) of JK through direct person to person survey for each plant species. The current dataset contains information for about 133 plant species. Database Interface The database interface is designed for searching the dataset using a PHP (a programming language that allows web developers to create dynamic content that interacts with databases) server enabled script for keywords such as botanical name, local name, folklore use and medicinal use. The database is accessible for free through the internet. Database Design The database is developed in MySQL on a Linux Platform and updated regularly. Search result The search output for a given query (for example, diabetes) is given in Figure 1. Utility The database finds utility in the selection of medicinal plant species for high throughput screening in drug discovery.

Intensive_Care_Med-4-1-2244699

The influence of corticosteroids on the release of novel biomarkers in human endotoxemia

Objective Sepsis intervention studies need better patient stratification methods, and one way to realize this is the introduction of stable biomarkers. A set of recently developed novel biomarkers, based upon precursor-fragments of short-lived hormones, was previously shown to be increased during sepsis. However, it is not known whether these biomarkers are influenced by sepsis intervention strategies. Therefore we investigated the markers in a model of human endotoxemia intervened by increasing doses of prednisolone. Introduction During the past decade numerous sepsis intervention strategies have been introduced; only few of them, however, have shown beneficial effects. Major problems in sepsis intervention trials were mixed results and unclear risk stratification methods. The lack of clear stratification methods was especially disturbing, since the effectiveness of intervention in these studies correlated closely with disease severity and risk of mortality [1]. Within this context, there is a need for biomarkers to predict disease severity and mortality risk. During sepsis a wide range of inflammatory proteins is released and in theory all could be used as a biomarker. The vast majority of these proteins is biologically active though and as a consequence rapidly cleared from the circulation. A promising new approach is the measurement of stable prohormone fragments of bioactive peptide-hormones [2–4]. In theory, these fragments often have no known biological function during sepsis and are therefore less rapidly cleared and less sensitive to small homeostatic changes [5–7]. The most prominent prohormone sepsis marker is procalcitonin (PCT), which represents the precursor hormone of calcitonin. Its diagnostic and prognostic properties were superior to C-reactive protein (CRP) in multiple studies [8]. In addition, PCT responded to sepsis intervention strategies and represented a promising marker for monitoring of treatment effect [9, 10]. In recent years, additional assays have been developed for measurement of stable prohormone fragments, such as mid-regional pro-adrenomedullin (MR-proADM) [5], derived from the precursor of adrenomedullin (ADM); mid-regional pro-atrial natriuretic peptide (MR-proANP), derived from the precursor of atrial natriuretic peptide (ANP) [6]; and C-terminal proAVP (CT-proAVP), derived from the precursor of arginine–vasopressin (AVP; also known as the antidiuretic hormone) [7]. Levels of these biomarkers were higher in samples from patients with sepsis [2–4]. Yet it is not known whether these levels are influenced by a potential therapeutic sepsis intervention. In order to investigate the response of the novel biomarkers to a sepsis intervention strategy we applied the model of human endotoxemia and pre-treated subjects by increasing doses of corticosteroids, which is one of the few relatively successful, and among the most cost-effective, septic shock intervention strategies to date [11]. Methods Subjects and study design The study was designed according to the requirements of the Declaration of Helsinki and approved by the institutional scientific and ethics committees, and written informed consent was obtained from all subjects. The study was performed simultaneously with an investigation of the effects of prednisolone on activation of the cytokine network, coagulation and hemodynamic variables in the same cohort [12]. Thirty-two healthy male volunteers [mean (± SE) age 23.9 ± 0.7 years] were admitted to a hospital clinical research unit. Screening tests were all normal. Prednisolone (prednisolone sodium phosphate oral solution 5 mg/ml; prepared by institutional pharmacy) was administered orally at a dose of 0, 3, 10 or 30 mg (n = 8 per group) at 2 h prior to LPS injection. All participants were challenged at t = 0 h with LPS (Escherichia coli lipopolysaccharide, lot G; US Pharmacopeia, Rockville, MD) as a bolus intravenous injection at a dose of 4 ng/kg, which is a standardized dose [13]. Assays Blood was collected at intervals from 2 h before LPS injection to 24 h thereafter. Blood was immediately centrifuged (4 °C, 10 min, 3000 rpm) and plasma was stored at –20 °C until assayed. Uptake of orally administered prednisolone was measured by HPLC-MS/MS technology (CombinatoRx, Inc., Boston, MA, USA). MR-proADM, MR-proANP, CT-proAVP and PCT were measured by chemiluminescence label-coated tube-based sandwich immunoassays (Brahms Diagnostica, Berlin, Germany) as previously described [5–7, 14]. Statistical analysis Differences between treatment groups were analyzed using a non-parametric mixed model approach (repeated-measures ANOVA). The effects reported are treatment effects adjusted for time effects. p values < 0.05 were considered statistically significant. Values presented are given as mean ± SE. Results Pharmacokinetic data As shown in Fig. 1, uptake of orally administered prednisolone was demonstrated by a dose-dependent increase in plasma levels during endotoxemia. The levels peaked from 1 h before LPS administration to 1 h after LPS administration (peak levels 302 ± 34 ng/ml, 190 ± 19 ng/ml and 71 ± 5 ng/ml in the groups receiving prednisolone 30, 10 and 3 mg respectively; t = –1 h). Fig. 1Prednisolone. Mean (± SE) values of prednisolone concentrations after LPS administration (4 ng/kg IV, t = 0 h) to healthy male volunteers, preceded by oral administration of prednisolone 3 mg (circles), 10 mg (triangles), or 30 mg (squares) at t = –2.5 h Novel prohormone biomarkers MR-proADM levels increased after LPS administration from 0.31 ± 0.02 nmol/l at baseline to a peak value of 1.05 ± 0.11 nmol/l in the control group at t = 4 h (Fig. 2). The rise was inhibited after prednisolone 30 mg and 10 mg pre-treatment (levels at t = 4 h: 0.65 ± 0.06 nmol/l and 0.65 ± 0.1 nmol/l respectively; p < 0.05), but not after the low dose of prednisolone 3 mg. MR-proANP levels rose from 42.4 ± 2.2 pmol/l at baseline to 118.4 ± 17.2 pmol/l in the control group at t = 4 h. These levels were reduced after prednisolone 30 mg and 10 mg (peak levels: 91.5 ± 9.9 pmol/l and 64.8 ± 5.6 pmol/l at t = 2 h and t = 4 h, respectively; p < 0.05). CT-proAVP levels showed a modest increase from 12.5 ± 4.0 pmol/l to 18.0 ± 4.8 pmol/l in the control group at t = 4 h. The rise was inhibited after prednisolone 30 mg (peak level: 6.1 ± 1.0 pmol/l; p < 0.05) and not significantly changed by 10 mg (peak level: 10.6 ± 3.4 pmol/l; p < 0.1). In contrast, prednisolone 3 mg pre-treatment was associated with enhanced CT-proAVP release (peak level 34.7 ± 11.5 pmol/l; p < 0.05). PCT levels increased from a baseline level of 0.04 ± 0.02 ng/ml to a peak value of 16.7 ± 2.6 ng/ml in the control group. Peak PCT levels all occurred at t = 24 h and were not significantly affected by prednisolone pre-treatment (11.9 ± 3.0 ng/ml, 13.1 ± 2.5 ng/ml and 17.6 ± 3.5 ng/ml in the 30, 10 and 3 mg group, respectively). Fig. 2Novel prohormone biomarkers. Mean (± SE) values of MR-proADM, MR-proANP, CT-proAVP and PCT after LPS administration (4 ng/kg IV, t = 0 h) to healthy male volunteers, preceded by oral administration of prednisolone 3 mg (open circles), 10 mg (triangles), 30 mg (squares) or, in the control group, 0 mg (filled circles) at t = –2.5 h. * p < 0.05 vs. control group. NS, non significant Discussion The primary objective of this study was to determine the effect of increasing doses of prednisolone on the release of novel biomarkers during human endotoxemia. We show here that prednisolone pharmacologic concentrations peaked at the time of LPS infusion and that the release of MR-proADM, MR-proANP and CT-proAVP was subsequently inhibited in a dose-dependent way. The levels of PCT were not significantly inhibited within the study period of 24 h. However, previously inhibition of PCT was shown by anti-inflammatory agents within a period of 168 h [9]. The levels of CT-proAVP were, remarkably, increased after prednisolone 3 mg, yet the reason for this remains unclear because the pattern was unique to this biomarker. The increased levels of MR-proANP, MR-proADM and CT-proAVP reflect activation of their mature counterparts. Difficulties in measurement of the mature hormones made it difficult to determine their roles during sepsis [5–7]. The currently investigated biomarkers are produced after proteolytic processing of the same prohormone precursors as the mature hormones; thus, they reflect production levels of mature hormones in stoichiometric concentrations [7, 15]. In theory, the biomarker assays may measure levels of both cut prohormone fragments and full-length prohormones. However, the meaning of this relationship remains unclear in plasma because no well-defined methods are currently available for measurement of full-length prohormone plasma levels. The biomarker levels in this study were dose-dependently influenced by corticosteroid intervention, which is important for treatment effect monitoring. The development of new biomarkers is needed for improvement of sepsis stratification methods, which influence trial outcomes. Several sepsis intervention strategies have shown promise [16]; in this study corticosteroids were selected because they aim primarily at inhibition of the immune response, and for this intervention in particular, effects are dose-dependent and improvement of patient stratification methods is urgently needed [11, 17]. Prednisolone is among the most frequently applied corticosteroids [18]; the oral route is convenient and potentially safer than intravenous administration [19]. The doses of prednisolone used in our model were chosen according to common dose regimens for prednisolone [18]. These doses were relatively low (equivalent to 12, 40 and 120 mg hydrocortisone) in comparison to doses used for sepsis intervention (hydrocortisone 200–300 mg per day), yet sufficient to reduce the release of the investigated biomarkers during endotoxemia. Of note, it has been demonstrated that the effects of orally administered doses of prednisolone do not differ from those of comparable doses of intravenously administered hydrocortisone [20]. The human endotoxemia model differs from sepsis in many aspects: it uses a single-dose drug intervention and bolus intravenous challenge of LPS, it is self-limiting, and it lacks an infectious source [13]. The study subjects are healthy young people not using any co-medication. The intervention must be administered before LPS injection in order to reach clinically relevant drug concentrations during endotoxemia because LPS is extremely quickly cleared from the circulation. Due to these differences, data cannot be directly extrapolated to the sepsis field and must always be confirmed in patient studies. Nonetheless, the model is the best available in healthy humans and has proven useful to obtain a proof of principle of the actions of drugs and/or to study mechanisms that contribute to the activation of inflammatory pathways [13]. With these limitations in mind, we demonstrate here that the levels of the novel biomarkers MR-proADM, MR-proANP, CT-proAVP and PCT are all increased after LPS injection, and that the levels of MR-proADM, MR-proANP and CT-proAVP can be inhibited by prednisolone. These results add to their potential as biomarkers in sepsis.

J_Mol_Model-3-1-2039871

Optimization of parameters for semiempirical methods V: Modification of NDDO approximations and application to 70 elements

Several modifications that have been made to the NDDO core-core interaction term and to the method of parameter optimization are described. These changes have resulted in a more complete parameter optimization, called PM6, which has, in turn, allowed 70 elements to be parameterized. The average unsigned error (AUE) between calculated and reference heats of formation for 4,492 species was 8.0 kcal mol−1. For the subset of 1,373 compounds involving only the elements H, C, N, O, F, P, S, Cl, and Br, the PM6 AUE was 4.4 kcal mol−1. The equivalent AUE for other methods were: RM1: 5.0, B3LYP 6–31G*: 5.2, PM5: 5.7, PM3: 6.3, HF 6–31G*: 7.4, and AM1: 10.0 kcal mol−1. Several long-standing faults in AM1 and PM3 have been corrected and significant improvements have been made in the prediction of geometries. Introduction Over the past 30 years, NDDO-type [1, 2] semiempirical methods have evolved steadily. The earliest of these methods was MNDO [3, 4], which itself was a major advance over even earlier non-NDDO methods such as MINDO/3 [5]. The main advantage of MNDO over earlier methods was that the values of the parameters were optimized to reproduce molecular rather than atomic properties. When it first appeared, MNDO was immediately popular because of its increased accuracy, but, with the passage of time, various limitations were found, among the most important of which was the almost total absence of a hydrogen bond. As hydrogen bonding is essential to life, this particular fault essentially precluded MNDO being used in modeling biochemistry. In 1985 an attempt, AM1 [6], was made to improve MNDO by adding a stabilizing Gaussian function to the core-core interaction to represent the hydrogen bond. Despite the fact that this was an over-simplification of a very complicated phenomenon, the overall effect was similar, and for the first time NDDO methods gave a good, albeit limited, model of hydrogen bonding. In the course of the next several years, improvements were made to the method of parameter optimization. The result of this was the PM3 method [7–10], which culminated in the parameterization of all the elements in the main group in 2004 [11]. At the same time, various changes to the original set of approximations used in MNDO were proposed, the most important of which were the addition of d-orbitals to main-group elements [12, 13] and the introduction of diatomic parameters. Work started on the transition metals, and parameters for some of these have been reported [14, 15]. More recently, parameter sets tailored to reproduce specific phenomena such as the binding energy of nucleic acid base pairs [16], iron complex catalyzed hydrogen abstraction [17], phosphatase-catalyzed reaction barriers [18], and the redox properties of iron containing proteins [19] have been developed. Because of the way advances in NDDO developments occurred, in terms of the modifications of the approximations and the extensions to specific elements or groups of elements, there has been an inevitable lack of consistency. The aim of the current work was three-fold: to investigate the incorporation of some of the reported modifications to the core-core approximations into the NDDO methodology; to carry out a systematic global parameter optimization of all the main group elements, with emphasis on compounds of interest in biochemistry; and to extend the methodology by performing a restricted optimization of parameters for the transition metals. This resulted in the development of a new method, consisting of the final set of approximations used and the optimized parameters. This method will be referred to as parametric method number 6, or PM6. The name PM6 was chosen to avoid any confusion with two other unpublished methods, PM4 and PM5. Theory Despite the apparent complexity of semiempirical methods, there are only three possible sources of error: reference data may be inaccurate or inadequate, the set of approximations may include unrealistic assumptions or be too inflexible, and the parameter optimization process may be incomplete. In order for a method to be accurate, all three potential sources of error must be carefully examined, and, where faults are found, appropriate corrective action taken. Reference data In contrast to earlier methods, in which reference data was assembled by painstakingly searching the original literature, the current work relies heavily on the large compendia of data that have been developed in recent years. The most important of these are the WebBook [20], for thermochemistry, and the Cambridge Structural Database [21] (CSD), for molecular geometries. During the early stages of the current work, consistency checks were performed to ensure that erroneous data were not used. These checks revealed many cases in which the calculated heats of formation were inconsistent with the reference heats of formation reported in the NIST database. On further checking, many of these reference data were also found [22, 23] to be inconsistent with other data in the WebBook. In those cases where there was strong evidence of error in the reference data, the offending data were deleted, and the webbook updated [24]. For molecular geometries, gas phase reference data are preferred, but in many instances such data were unavailable, and recourse was made to condensed-phase data. Provided that care was taken to exclude those species whose geometries were likely to be significantly distorted by crystal forces, or which carried a large formal charge, condensed-phase data of the type found in the CSD were regarded as being suitable as reference data. Because earlier methods used only a limited number of reference data, most of the cases where the method gave bad results were not discovered until after the method was published. In an attempt to minimize the occurrence of such unpleasant surprises, the set of reference data used was made as large as practical. To this end, where there was a dearth or even a complete absence of experimental reference data, recourse was made to high level calculations. Thus, for the Group VIII elements, there are relatively few stable compounds, and the main phenomena of interest involve rare gas atoms colliding with other atoms or molecules, so reference data representing the mechanics of rare gas atoms colliding with other atoms was generated from the results of ab-initio calculations. Additionally, there is an almost complete lack of thermochemical data for many types of complexes involving transition metals, so augmenting what little data there was with the results of ab-initio calculations was essential. Use of Ab-Initio results Ab-initio calculations provide a convenient source of reference data; for this work, extensive use has been made of results of Hartree Fock and B3LYP density functional [25, 26] methods (DFT), both with the 6–31G(d) basis set for elements in the periodic table up to argon. For systems involving heavier elements, the B88–PW91 functional [27, 28] was used with the DZVP basis set. Within the spectrum of ab-initio methods these methods are not particularly accurate; many methods with larger basis sets and with post-Hartree-Fock corrections are more accurate. However, the methods used in this work were chosen because they were regarded as robust, practical methods, allowing many systems to be modeled in a reasonable amount of time, a condition that could not be achieved with the more sophisticated ab-initio methods. Procedure used in deriving ΔHf Reference heats of formation, ΔHf, for compounds and ions of elements for which there was a paucity of data were derived from DFT total energies in two stages. In the first stage, a basic set of ∼1,400 well-behaved compounds, for which reliable reference values of experimental ΔHf were available, was assembled. Only compounds containing one or more of the elements H, C, N, O, F, P, S, Cl, Br, and I were used. For this set, a root-mean-square fit was made to the reference ΔHf using the calculated total energies, Etot and the atom counts. Thus, the error function, S, in Eq. (1) was minimized. In this expression, the Ci are constants for each atom of type i, and the ni are the number of atoms of that type. In the second stage, the contribution to the total energy of compounds containing element X arising from the elements in the first stage was removed using the coefficients from Equation (1). A second RMS fit was then performed. In this, the function minimized, S, was the RMS difference between the reference ΔHf of compound X and the values predicted from the DFT energy, Eq. (2). In this expression, the only unknown is the multiplier coefficient Cx. After solving for Cx, the ΔHf of any compound of X could then be predicted as soon as its DFT total energy was evaluated. Training set reference data The training set of reference data used was considerably larger than that used in parameterizing PM3 [7, 8], where approximately 800 discrete species were used. In optimizing the parameters for PM6, somewhat over 9,000 separate species were used, of which about 7,500 were well-behaved stable molecules. The remainder consisted of reference data that were tailored to help define the values of individual parameters or sets of parameters. Use of rules in parameter optimization Most reference data can be expressed as simple facts. Indeed, all the earlier NDDO methods were parameterized using precisely four types of reference data: ΔHf, molecular geometries, dipole moments, and ionization potentials. During the development of PM6, however, the use of other types of reference data was found to be necessary. Because of their behavior, these new data are best described as “rules.” In this context, a rule can therefore be regarded as a reference datum that is a function of one or more other data. To illustrate the use of a rule, consider the binding energy of a hydrogen bond in the water dimer. By default, the weighting factor for ΔHf for normal compounds is 1.0 kcal mol−1. With this weighting factor, average unsigned errors in the predicted ΔHf of the order of 3–5 kcal mol−1 would be acceptable, particularly as the spectrum of values of ΔHf spans several hundreds of kilocalories per mole. However, the binding energy of a hydrogen bond in a water dimer is only 5 kcal mol−1. To have an average unsigned error (AUE) of 4 kcal mol−1 in the prediction of hydrogen bond energies would render such a method almost useless for modeling such phenomena. One way to increase the importance of the hydrogen bond in water would be to increase the weight for the ΔHf of the water molecule, −57.8 kcal mol−1, and the water dimer system, ca. −120.6 kcal mol−1. While this would have the intended effect of increasing the weight of the hydrogen bond energy, it would also have the undesired effect of increasing the weight of the ΔHf of water. An alternative would be to express the ΔHf of the water dimer in terms of the ΔHf of two individual water molecules. The difference between the two ΔHf, that of water dimer and that of two isolated water molecules, would be the energy of the hydrogen bond. If the weight assigned to this quantity were then increased, it would increase the weight for the hydrogen bond energy without also increasing the weight for the ΔHf of water. Such a reference datum is referred to here as a rule. That is, rules relate the ΔHf of a moiety to that of one or more other moieties. Thus, in the above example, the simple reference datum H, representing the ΔHf of an isolated water molecule, could be expressed as: Using a rule-based reference datum to represent the strength of the hydrogen bond, and giving a weight of 10 to the hydrogen bond energy, the ΔHf of the water dimer would then be defined as In this expression, HH2O was the calculated ΔHf, in kcal mol−1, of an isolated water molecule. This rule could be interpreted as “The calculated strength of the hydrogen bond formed when two water molecules form the dimer should be 5 kcal mol−1, and the importance should be 100 times that of ordinary heats of formation.” Rules are very useful in defining the parameter hypersurface. Examples of such tailoring are as follows: Correcting qualitatively incorrect predictions During the parameterization of transition metals, some systems were predicted to have qualitatively the wrong structure. For example, [CuIICl4]2− was initially predicted to have a tetrahedral structure, instead of the D2d geometry observed. To induce the parameters to change so as to make the D2d geometry more stable than the Td geometry, a rule was added to the set of reference data for copper compounds. This rule was constructed using the results of B3LYP calculations on [CuIICl4]2−. First, the total energies of the optimized B3LYP structure and that of the structure resulting from the semiempirical calculation were evaluated. The difference between these energies was then used in constructing the rule. In this case, the rule was that “The ΔHf of the geometry predicted by the faulty semiempirical method should be n.n kcal mol−1 more than that of the B3LYP geometry.” When such a rule was included in the parameter optimization, with an appropriate large weight, any tendency of the parameters to predict the incorrect geometry resulted in a large contribution to the error function. That is, with the new rule in place, there was a strong disincentive to prediction of the incorrect structure. Usually one rule was sufficient to correct most qualitative errors, but for a few complicated structures more than one rule was needed. The commonest need for multiple rules occurred when, initially, one rule was used to correct a faulty prediction and, after re-optimizing the parameters, the geometry optimized to a new structure that was distinctly different from either the correct structure or the incorrect structure covered by the rule. When that happened, the procedure just described was repeated, and a new rule added to the set of reference data to address the new incorrect structure. In extreme cases, several such rules might be needed, each one defining a geometry that was incorrect and should therefore be avoided. Rare gas atoms at sub-equilibrium distances For some elements, specifically those of Group VIII, there is an understandable shortage of useful experimental reference data. In addition, most simulations involving these elements are likely to involve a rare-gas atom dynamically interacting with another atom or with a molecule at distances significantly less than the equilibrium distance. This makes determining the potential energy surface at sub-equilibrium distances important. As with hydrogen bond energies, the energies involved in this domain are likely to be in the order of a few kcal mol−1. The shape of the potential energy surface (PES) can readily be mapped using DFT methods. By selecting two or three representative points on this PES, reference data rules can be constructed that describe the mechanical properties of the interactions. As with hydrogen bonding, a large weight can be assigned to these rules. Use of rules to restrain parameter values In general, uncharged atoms that are separated by a distance sufficiently large so that all overlaps between orbitals on the two atoms are vanishingly small will not interact significantly, and what interaction energy exists would arise from VDW terms: of their nature, these are mildly stabilizing. Although statements of this type are obviously true, when they are expressed as rules and added to the training set of reference data they can help define the parameter values. For a pair of atoms, A and B, a simple diatomic system would be constructed in which the interatomic separation was the minimum distance at which any overlaps of the atomic orbitals would still be insignificant. The electronic state of such a system would then be the sum of the states of the two isolated atoms. Thus, if both A and B were silicon, then, since the ground state of an isolated silicon atom is a triplet, the combined state would be a quintet. Because the two atoms do not interact significantly, a rule could then be constructed that said “The energy of the diatomic system is equal to the addition of energies of the two individual systems.” By giving this rule a large weight, any tendency of the method to generate a spurious attraction or repulsion between the atoms would be prevented. Atomic energy levels In keeping with the philosophy that a large amount of reference data should be used in the parameter optimization, spin-free atomic energy levels were used for most elements. The exceptions were carbon, nitrogen, and oxygen, where there were enough conventional reference data that the addition of atomic energy levels would not significantly improve the definition of the parameter surface. NDDO approximations do not allow for spin-orbit coupling. Therefore, spin-free levels were needed. For a few elements, there were insufficient spin states to allow the spin-free energy levels to be calculated. For all the remaining elements, spin-free energy levels were calculated. In Moore’s compendia [29–31] of atomic energy levels, observed emission spectra were used in determining the energy levels of the various states of neutral and ionized atoms. Most of these energy levels were characterized by three quantum numbers: the spin and orbital angular momenta, and the “J” or spin-orbit quantum number. The starting point for determining the spin-free atomic energy levels for a given element consisted of identifying each complete manifold of atomic energy levels for that element, that is, each set of levels split by spin-orbit coupling. If all members of the set were present, i.e., all energy levels from L+S to |L−S|, then the weighted barycenter of energy could be calculated. The spin-free energy level, E, was derived from the spin-split levels E(S,L,J) using Eq. (3). In those cases where the ground state of an atom was itself a member of a spin-split manifold, the barycenter of the ground state manifold was calculated and used in re-defining the spin-free ground state. For all elements except tungsten, this change in definition was benign. There is a 7S3 level present in tungsten that is located only 8.4 kcal mol−1 above the ground state. This puts it inside the 5DJ, manifold, which has a barycenter at 12.7 kcal mol−1. The effect of this was that, on going from a spin-split to a spin-free ground state, the ground state changed from 6d25d4 or 5D to 6d15d5 or 7S, and the 5D state now became an excited state with an energy of 4.4 kcal mol−1. To allow for this, a corresponding change was made to the ground state configuration in the PM6 definition of tungsten. Where there were relatively few other reference data, the singly-ionized, and, in rare cases, the doubly-ionized, spin-free states were also evaluated and used as reference data. Each energy level contributed one reference datum to the training set. Most atoms have a large number of atomic energy levels, so in order to minimize the probability that a level might be incorrectly assigned, each level was labeled with three quantum numbers: the total spin momentum, the total angular momentum, and the principal quantum number for these two quantum numbers. These were compared with the corresponding values calculated from the state functions. Since each set of three quantum numbers is unique, the potential for miss-assignment was minimized. In rare cases, particularly during the early stages of parameter optimization, two states with the same total spin and angular quantum numbers would be interchanged, with the result that the calculated principal quantum number would also be interchanged. All such cases always involved the ground state, and were quickly identified and corrected. Approximations Most of the approximations used in PM6 are identical to those in AM1 and PM3. The differences are: Core-core interactions In the original MNDO set of approximations, two changes were made to the simple point-charge expression for the core-core repulsion term. Beyond about five Ångstroms, there should be no significant interaction of two neutral atoms. However, in MNDO, the two-electron, two-center integrals and the electron-core interactions do not converge to the exact point charge expression; instead, they are always slightly smaller. To prevent there being a small net repulsion between two uncharged atoms, the core-core expression is modified by the exact 1/RAB term being replaced by the term used in the integrals. An additional term is needed to represent the increased core-core repulsion at small distances due to the unpolarizable core. These two changes can be expressed as the MNDO core-core repulsion term as shown in Eq. (4). This approximation works well for most main-group elements, but when molybdenum was being parameterized, Voityuk [14] found that the errors in heats of formation and geometries were unacceptably large, and good results were achieved only when a diatomic term was added to the core-core approximation, as shown in Eq. (5). When PM3 parameters for elements of Groups IA were being optimized, the MNDO approximation to the core-core expression was found to be unsuitable. In these elements there is only one valence electron so the core charge is the same as that of hydrogen. A consequence of this was that the apparent size of these elements was also approximately that of a hydrogen atom, in marked contrast with observation. For these elements, diatomic core-core parameters were also found to be essential. Further examination showed that when diatomic parameters were used, there was always an increase in accuracy; therefore, in the current work, Eq. (4) was replaced systematically by Eq. (5). As the interatomic separation increased, Voityuk’s equation converged to the exact point-charge interaction, as expected. However, for rare gas interactions, an increase in accuracy was found when the rate of convergence was increased by the addition of a small perturbation. Subsequently, the perturbed function was found to be generally beneficial. Because of this, the general form of the core-core interaction used in PM6 is that given in Eq. (6). At normal chemical bonding distances, Eqs. (5) and (6) have essentially similar behavior, but at distances of greater than about 3 Å the effect of the perturbation is to make the PM6 function significantly smaller than the Voityuk approximation. d-orbitals on main-group elements Thiel and Voityuk have shown [13] that a large increase in accuracy results when d-orbitals are added to main-group elements that have the potential to be hypervalent. During preliminary stages of this work, d-orbitals were excluded from main-group elements, and the parameters were optimized. This work was then repeated but with d-orbitals on various main-group elements. The results were in accordance with Thiel’s observation: the accuracy of the method increased significantly. Because of this, d-orbitals were added to several main-group elements: the value of the increased accuracy far outweighs the extra computational cost. The effect of the addition of d-orbitals was fundamentally different between main-group elements and transition metals. For main-group elements, the effect of d-orbitals is merely a perturbation: to a large degree the chemistry of these elements is determined by the s and p atomic orbitals. This is not the case with transition metals, where the d-orbitals are of paramount importance and the s and p orbitals are of only very minor significance. In recognition of the importance of the s and p shells in main-group chemistry, specific parameters are used for the five one-center two-electron integrals. Conversely, for the transition metals, the values of these integrals are derived directly from the internal orbital exponents. Unpolarizable core As noted earlier, the NDDO core-core interaction is a function of the number of valence electrons. For elements on the left of the periodic table these numbers are small and can cause the elements to appear to be too small. This was part of the rationale behind the adoption of Voityuk’s diatomic core-core parameters. However, even the Voityuk approximation failed during parameter optimization when, in rare cases, a pair of atoms would approach each other very closely. Examination of these catastrophes indicated that the cause was the complete neglect of the unpolarizable core of the atoms involved. To allow for its presence, the core-core interaction for all element pairs was modified by the addition of a simple function, fAB, based on the first term of the Lennard-Jones potential [32]. A candidate function was constructed, Eq. (7), using the fact that, to a first approximation, the size of an atom increases as the third power of its atomic number. The value of c was set to 10−8, this being the best compromise between the requirements that the function should have a vanishingly small value at normal chemical distances. That is, under normal conditions the value of the function should be negligible, and at small interatomic separations the function should be highly repulsive, i.e., that it should represent the unpolarizable core. Individual core-core corrections For a small number of diatomic interactions, the general expression for the core-core interaction was modified in order to correct a specific fault. Because it is desirable to keep the methodology as simple as possible, modifications of the approximations were made only after determining that the existing approximations were inadequate. The diatomic specific modifications were: O–H and N–H In the original MNDO formalism, the general core-core interaction, Eq. (4), was replaced in the cases of O–H and N–H pairs with Eq. (8). An unintended effect of this change was that at distances where hydrogen-bonding interactions are important, the diatomic contribution to the ΔHf is greater than if the general approximation, Eq. (4), had been used. This contributed to a reduced hydrogen-bonding interaction in MNDO, and was a contributor to the need for modified core-core interactions in AM1 and PM3. In PM6, the MNDO core-core approximation is replaced by Voityuk’s diatomic expression, but even with that modification, the resulting hydrogen bond interaction energy was too small. In an attempt to increase it, the Voityuk approximation was replaced by Eq. (9). At normal O–H and N–H separations, approximately 1 Å, Eqs. (5) and (9) have similar values, but at hydrogen bonding distances, ∼2 Å, the contribution arising from the exponential term is significantly reduced, resulting in a corresponding increased hydrogen bond interaction energy. C–C After optimizing all parameters, it was found that compounds containing yne groups, -C≡C-, were predicted to be too stable by about 10 kcal mol−1 per yne group. This error was unique to compounds with extremely short C–C distances, and in light of the increased emphasis on accurately reproducing the properties of organic compounds, the C–C core-core term was perturbed by the addition of a repulsive term. This term was optimized to correct the error in the yne groups and to have a negligible effect on all other C–C interactions. The optimized form of the C–C core-core interaction is given in Eq. (10). Si–O During testing of PM6, neutral silicate layers of the type found in talc, H2Mg3Si4O12, were found to be slightly repulsive instead of being slightly bound. An attempt was made to correct for this error by adding a weak perturbation to the Si–O interaction, illustrated by Eq. (11). Nitrogen sp2 pyramidalization Although PM6 predicted the degree of pyramidalization of primary amines correctly, it overestimated the pyramidalization of secondary and tertiary amines. The degree of pyramidalization of these amines was decreased by adding a function to make the calculated ΔHf more negative as the nitrogen became more planar, as shown in Eq. (12). In this equation, the angle ϕ is a measure of the non-planarity of the nitrogen environment, and is given by 2π minus the sum of the three contained angles about the nitrogen atom. For planar sp2 secondary and tertiary amines, this correction amounted to 0.5 kcal mol−1 per nitrogen atom. More elements The NDDO basis sets of many of the elements parameterized in PM6 have not previously been described. For all elements except hydrogen, which has only an s orbital, the basis set consists of an s orbital, three p orbitals, and, for most elements, a set of five d orbitals. Slater atomic orbitals are used exclusively; these are of form: Where ξ is the orbital exponent, n is the principal quantum number (PQN), and the Ylm(θ, ϕ) are the normalized real spherical harmonics. The PQN are those of the valence shell, i.e., the set of atomic orbitals most important in forming chemical bonds. For PM6, the PQN used are shown in Table 1. For most main-group elements, the s and p PQN are the same, and, when d orbitals are present, all three PQN are the same: that is, the PQN are (ns, np, nd). For transition metals, the d PQN is one less than that of the s and p shells, i.e., (ns, np, (n–1)d). An exception to this generalization occurs in the elements of Group VIII. Here, the valence shell is completely filled, so in all chemical interactions that could occur between an atom of a Group VIII element and any other atom, electron density could only migrate from the Group VIII element to the other atom. That is, when a rare gas element forms any type of chemical bond it would necessarily become slightly positive. This is an unrealistic result. In order to allow rare gas atoms to have the potential of being slightly negative, the set of valence orbitals was changed from (ns, np) to (np, (n+1)s), for the elements Ne, Ar, Kr, and Xe. Helium is the only exception to this change, because it does not have a “1p” valence shell. For helium, the valence shell used was (1s, 2p), this being considered the best compromise. Table 1Principal quantum numbers for atomic orbitals spd spdH1Kr54He12Rb55Li22Sr55Be22Y554B22Zr554C22Nb554N22Mo554O22Tc554F22Ru554Ne32Rh554Na33Pd554Mg33Ag554Al333Cd55Si333In55P333Sn55S333Sb555Cl333Te555Ar43I555K44Xe65Ca44Cs66Sc443Ba66Ti443La665V443Lu665Cr443Hf665Mn443Ta665Fe443W665Co443Re665Ni443Os665Cu443Ir665Zn44Pt665Ga44Au665Ge44Hg66As444Tl66Se444Pb66Br444Bi66 Parameter optimization Background The objective of parameter optimization is to modify the values of the parameters so as to minimize the error function, S, Eq. (13), representing the square of the differences between the values of reference data, Qref(i), and the values calculated using the semiempirical method, Qcalc(i), with appropriate weighting factors, gi. This process is initiated by rendering the reference data in the training set dimensionless. The default conversion factors are given in Table 2, with weighting factors for reference data represented by rules being much larger, typically in the order of 5–20 kcal mol−1. Table 2Default weighting factors for reference dataReference dataWeightΔHf1.0 mol.kcal−1Bond length0.7 Å−1Angle0.7 degrees−1Dipole20 Debye−1I.P.10 eV−1ElementsMultiplierCore1.0Organic0.9Main group0.8Transition metals0.7 The elements were divided into four sets: core elements, (H, C, N, and O), other elements important in organic chemistry (F, Na, P, S, Cl, K, Br, I), the rest of the main group, and the transitions metals. Elements were assigned to the different sets based on their presumed degree of importance in biochemistry, and this importance was converted into a weighting factor to be used in the parameterization optimization procedure. Reference data representing species consisting only of core elements were given their default weight. When other elements were present, the weight was set to the default weight times the smallest multiplier shown in Table 2. Thus the default weight for a reference datum involving tetramethyllead, Pb(CH3)4, would be multiplied by 0.8 reflecting the fact that this species contains an element in the main group set. For a given set of parameters, P, optimization proceeds by calculating the values of all the Qcalc(i), their first derivatives with respect to each parameter, P(j), and the second derivatives with respect to every pair of parameters. Evaluating these quantities is time-consuming, and considerable effort was expended in minimizing the need for explicit evaluation of these functions. The most efficient strategy developed [7] involved assuming that, in the region of parameter space near to the current values of the parameters, the values of the first derivatives of the Qcalc(i) with respect to P were, at least to a first approximation, constant. By making this assumption the values of the parameters could then be updated using perturbation theory. Because the assumption is only valid in the region of the starting point in parameter space, periodically the focus was moved to the new point in parameter space and a complete explicit re-evaluations of all the functions performed. The parameter optimization process terminated when the scalar of the first derivatives dropped below a preset limit. This process was fully automated, and for given sets of reference data and parameters, parameter optimization could be performed rapidly, easily, and reliably. Sequence of optimization of parameters Notwithstanding the reliability of the parameter optimization procedure, a simple global optimization of all the parameters for all 70 elements involving about over 9,000 discrete species was found to be impractical because of the large number of derivatives involved. Such an optimization would involve over 2,000 parameters and over 10,000 reference data. The set of second derivatives alone would consist of 2×1010 terms. With more powerful computers, evaluating such large sets of derivatives might be practical some day, but even then, one faulty reference datum or one faulty initial parameter value would ruin an optimization run. The strategy of parameter optimization was approached with great caution, and the procedure finally adopted was as follows: Because the elements H, C, N, and O are of paramount importance in biochemistry, and because large amounts of reference data are available, the starting point for parameter optimization involved the simultaneous optimization of parameters for these four elements. For the purposes of discussion, this set of four elements will be called the “core elements”. Once stable parameters had been obtained, parameters for other elements important in organic chemistry were optimized in two stages. First, the parameters for the core elements were held constant, and parameters for the elements F, P, S, Cl, Br, and I were optimized one at a time. Then all parameters for all ten elements were simultaneously optimized. This set (the organic elements) was then used as the starting point for parameterizing the rest of the main group. The same sequence was followed for the rest of the main-group elements. That is, parameters for each element were optimized while freezing the parameters for the organic elements. Then, once all the elements had been processed, all parameters for all of the 39 main-group elements, plus zinc, cadmium, and mercury, were optimized simultaneously. When parameters for the transition metals were being optimized, all parameters for the main group elements were held constant. There were several reasons for this. Most importantly, the reference data for the transition metals, particularly the thermochemical data, was of lower quality, so one consideration was to prevent the transition metals from having a deleterious effect on the main-group elements. Another important consideration was that most compounds involving transition metals also involved only elements of the organic set. Since parameters for these elements had been optimized using a training set consisting of all the main-group elements, the values of the optimized parameters would likely be relatively insensitive to the influence of the small number of additional reference data involving transition metals. In general, all parameters for a given element were optimized simultaneously; this was both efficient and convenient. In some optimizations, specifically those involving a new element, only sub-sets of parameters were used. Three main sub-sets were used: Parameters that determine atomic electronic properties For most elements, atomic energy levels are determined by six parameters: the one-electron one-center integrals Uss, Upp, Udd, and the internal orbital exponents ζsn, ζpn and ζdn. If the heat of ionization and sufficient atomic energy level data were available, these quantities could be uniquely defined; there would be no need for the use of molecular reference data. These parameters were the first to be optimized whenever an optimization was started for an element that had not previously been parameterized Parameters that determine molecular electronic properties Two of the more important electronic molecular properties are the dipole moment, which indicates the degree of polarization within a molecule, and the ionization potential. These properties are determined primarily by 12 parameters: the six parameters that determine atomic electronic properties and six additional parameters: βs, βp, and βd and the Slater orbital exponents ζs, ζp, and ζd. In the second stage of parameter optimization, the first six parameters were held constant at the values defined using atomic data and the second set optimized. During this operation, all geometries were fixed at their reference values. Parameters that determine geometries As soon as an initial optimized set of electronic parameters was available, the diatomic and other core-core parameters could be optimized. The most efficient process was to optimize these parameters initially without allowing the electronic parameters or the molecular geometries to optimize. If geometries were allowed to optimize, optimization of the core-core parameters would be slowed considerably, because of the tight dependency of the optimized geometries on the values of the core-core parameters, and vice versa. As soon as all parameters had been optimized using fixed geometries, the geometries were allowed to relax and the parameters that determine geometry re-optimized. After that there would be three sets of incompletely optimized parameters: the six atomic electronic parameters, the six molecular electronic parameters and the core-core parameters. The only remaining operation was the simultaneous optimization of all the parameters. If the training set of reference data was insufficient to unambiguously define the values of all the parameters, then, at that stage, the potential existed for the parameters to become ill-defined. An example of this would be where there were too few atomic energy levels to allow all six parameters in the first set to be defined. To allow for this, a penalty function was added to each parameter. If the values of a parameter exceeded pre-defined limits, the error function S was incremented by a constant times the square of the excess. No penalty was applied if the value of a parameter was between the pre-defined limits; that is, no bias was applied to the numerical value of a parameter. During the early stages of simultaneous optimization of all the parameters for a given element the penalty function was used frequently. In the later stages the penalty function was invoked rarely, and then only when there was a distinct shortage of reference data. Results Parameters for PM6 PM6 atomic and diatomic parameters for the 70 elements are presented in Tables 3 and 4, respectively. Not all elements have all parameters: where monatomic parameters are missing, the associated approximations were not used. For diatomic parameters, where an atom-pair is missing, no representatives of that type of bond were used. Table 3PM6 parameters for 70 elementsTable 4Diatomic core−core parameters Accuracy Comparison with other semiempirical methods Using the program MOPAC2007 [33], an extensive comparison was made between the results obtained using PM6 and those from PM5, PM3, and AM1. This comparison was started by generating tables of reference data (that is, ΔHf, geometries, ionization potentials (I.P.s), and dipole moments) and differences between the calculated and reference values, using each of the four methods presented here. Because of their size they are provided in the supplementary material. To simplify navigating within the tables, all species are listed in the order of their empirical formula. Average unsigned errors (AUE) for ΔHf for each element parameterized at the PM6 level are shown in Table 5, together with AUE for PM5, PM3, and AM1. The number of data used in each average varies depending on the elements available in each method. AM1 boron [34] uses a different core-core interaction expression from the other elements and was not used. AUE for bond-lengths are shown in Table 6. In those cases where a calculated bond-length was very large, indicating that the bond had broken, the bond-length was not used in the analysis. If such data had been used, the resulting statistics would have been misleading. AUE for angles are shown in Table 7. Errors in angles for many elements that form very ionic, i.e., labile, bonds are of less importance than errors involving elements that form strong covalent bonds. The angles subtended by such bonds are often determined largely by the electronic structure of the atom. Information on the accuracy of prediction of molecular electronic structure can also be inferred from the AUE of dipole moments, Table 8, and ionization potentials, Table 9. Table 5Average unsigned errors in calculated heats of formation (kcal mol−1)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen7.29303913.89234017.09234021.122270Helium0.0010.0010.0010.001Lithium7.988315.318318.028318.8482Beryllium5.923429.063429.583418.5134Boron6.4412210.8112011.84120––Carbon7.31282813.03215515.06215519.422123Nitrogen8.22106716.4576120.9676124.23744Oxygen8.42175816.59124320.13124427.681229Fluorine8.4949722.3135021.2535037.40334Neon0.0010.0010.0010.001Sodium5.72408.57399.473910.7738Magnesium9.846612.076617.946618.7166Aluminum7.617517.497519.157518.9975Silicon6.51989.289612.809617.0095Phosphorus8.2011016.019817.369820.0695Sulfur8.8142715.4033018.4433026.38323Chlorine8.2867016.6939018.7139023.06383Argon0.0010.0010.0010.001Potassium6.534312.33429.364228.3841Calcium11.874328.684343.444363.2043Scandium10.3352––––––Titanium10.2085––––––Vanadium14.2959––––––Chromium14.0960––––––Manganese12.7744––––––Iron18.3176––––––Cobalt15.5142––––––Nickel15.1051––––––Copper13.0047––––––Zinc5.565417.845432.935437.0654Gallium7.514729.124737.584746.8747Germanium9.836712.206715.866719.1267Arsenic6.944915.224916.684917.3449Selenium4.402539.582539.712532.0025Bromine7.3733017.2019925.0419928.22199Krypton0.0010.0010.0010.001Rubidium10.912416.572421.472429.3323Strontium7.723852.4638103.163857.2138Yttrium13.2851––––––Zirconium11.1846––––––Niobium8.5751––––––Molybdenum13.4170––––35.7769Technetium15.1450––––––Ruthenium13.8756––––––Rhodium20.9232––––––Palladium11.6547––––––Silver4.6714––––––Cadmium3.493834.663861.9238––Indium7.335431.535429.835432.1654Tin7.147716.837717.107720.2177Antimony5.415830.985834.615835.0058Tellurium8.204535.664546.804522.9145Iodine7.2327923.7717625.9017636.55175Xenon0.0010.0010.0010.001Cesium6.894037.014035.224055.3339Barium12.123798.2037154.6537161.0937Lanthanum10.3737––––––Lutetium7.6824––––––Hafnium8.5237––––––Tantalum14.3736––––––Tungsten7.3828––––––Rhenium10.4057––––––Osmium6.4619––––––Iridium10.2125––––––Platinum11.6177––––––Gold12.8232––––––Mercury5.945116.395117.675119.7551Thallium10.424432.634473.964573.1845Lead7.924418.084414.184416.7144Bismuth7.745399.885328.9553119.2353Table 6Average unsigned errors in bond lengths (Å)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.0442380.0562190.0322170.035181Helium0.25160.45960.18240.6555Lithium0.1751110.1911100.1671100.171105Beryllium0.076420.131420.067420.08542Boron0.0271160.0431160.066122––Carbon0.05711910.0666930.0516340.063628Nitrogen0.0906630.1453090.1242590.163253Oxygen0.09511630.1226250.1035770.117571Fluorine0.0633960.0962460.0692510.101228Neon0.35350.18220.06210.0301Sodium0.229330.200330.208300.14029Magnesium0.0891060.0671060.1671050.073106Aluminium0.045770.120720.098700.13870Silicon0.039970.056940.074950.07790Phosphorus0.0391410.078920.073920.08387Sulfur0.0943590.1072160.0912070.134200Chlorine0.0696720.0982830.0952840.130285Argon0.25840.3031––––Potassium0.139460.135470.148470.28146Calcium0.133670.177690.151670.10260Scandium0.05390––––––Titanium0.078140––––––Vanadium0.090168––––––Chromium0.08089––––––Manganese0.083107––––––Iron0.102117––––––Cobalt0.107100––––––Nickel0.065133––––––Copper0.174130––––––Zinc0.076770.084770.098770.14276Gallium0.048800.105810.192810.13581Germanium0.0381310.0451310.0561330.068133Arsenic0.073720.069700.080720.09972Selenium0.056560.094550.071540.06154Bromine0.1043580.1061840.1461820.136184Krypton0.05960.41730.62330.6023Rubidium0.413360.498370.176340.23036Strontium0.087560.199550.128320.24247Yttrium0.13269––––––Zirconium0.06365––––––Niobium0.06088––––––Molybdenum0.10489––––0.09584Technetium0.07884––––––Ruthenium0.073113––––––Rhodium0.16268––––––Palladium0.080120––––––Silver0.15141––––––Cadmium0.159540.179550.12150––Indium0.039770.085770.155770.10277Tin0.073960.065960.078960.08794Antimony0.060920.169910.083910.13592Tellurium0.070800.162790.123770.12279Iodine0.1442860.1371470.1461450.175141Xenon0.62080.58440.47220.7936Cesium0.258400.335430.372250.35843Barium0.202510.228470.207480.26151Lanthanum0.25347––––––Lutetium0.05060––––––Hafnium0.07142––––––Tantalum0.07459––––––Tungsten0.14157––––––Rhenium0.068108––––––Osmium0.07250––––––Iridium0.16971––––––Platinum0.057140––––––Gold0.15884––––––Mercury0.143640.110640.135630.13964Thallium0.202590.248550.208450.26843Lead0.140530.167530.121530.12551Bismuth0.142810.616750.225820.68275Table 7Average unsigned errors in bond angles (Degrees)ElementPM6No. in setPM5No. in setPM3No. in setAM1No. in setLithium7.79286.82283.53289.4828Beryllium6.61146.44146.94145.9814Boron3.27314.41314.6131––Carbon2.501342.791342.751312.25131Nitrogen7.32378.01376.75357.9431Oxygen12.145911.125810.17539.5742Fluorine8.32316.18326.34324.672Sodium21.0042.8743.4345.324Magnesium8.44247.282414.23247.1024Aluminum4.05205.26207.21194.3319Silicon5.25353.37352.81342.8834Phosphorus3.24354.40356.01355.0735Sulfur5.23465.64455.42415.0541Chlorine3.65519.47510.31514.805Potassium17.901110.271112.931112.7511Calcium14.991611.351616.811618.0615Scandium7.9832––––––Titanium7.8639––––––Vanadium7.4644––––––Chromium3.7719––––––Manganese6.0226––––––Iron11.2130––––––Cobalt10.6829––––––Nickel10.4448––––––Copper10.7744––––––Zinc10.922714.41278.162713.3427Gallium4.431810.861814.431813.8418Germanium4.58525.37528.95525.7152Arsenic6.29366.52366.48365.0336Selenium7.272416.162412.37235.4623Bromine12.64420.03419.2133.273Rubidium9.691110.201121.03116.6811Strontium18.162532.912532.922531.0025Yttrium12.2934––––––Zirconium10.3612––––––Niobium6.5423––––––Molybdenum8.1527––––8.7327Technetium4.9622––––––Ruthenium6.9334––––––Rhodium10.6622––––––Palladium9.1946––––––Silver23.369––––––Cadmium15.231013.521020.0910––Indium4.47177.21175.30174.9417Tin3.06344.09343.743411.8134Antimony6.494112.24416.84417.4041Tellurium4.85257.00255.33257.8725Iodine8.33112.55120.6614.531Cesium15.50128.521219.381211.7512Barium28.651028.431037.041036.1710Lanthanum9.2514––––––Lutetium7.0826––––––Hafnium5.6410––––––Tantalum9.8815––––––Tungsten10.909––––––Rhenium7.3932––––––Osmium12.6710––––––Iridium7.8618––––––Platinum5.9272––––––Gold13.5916––––––Mercury20.201520.991518.471521.4915Thallium5.731010.281019.951025.3810Lead4.33205.24204.61203.5719Bismuth8.012521.74258.282533.9925Table 8Average unsigned errors in dipole moments (D)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.622660.802650.642220.50204Lithium0.78160.95160.79160.5216Beryllium1.6311.4910.2710.531Boron0.66170.66170.7317––Carbon0.512190.622180.411760.42165Nitrogen0.61480.66480.46400.5539Oxygen0.991981.271961.05740.7475Fluorine0.801241.111210.59630.6959Sodium1.3460.8061.9761.266Aluminium0.3311.5011.7610.531Silicon0.21111.09110.72110.2911Phosphorus0.83140.79140.37100.8710Sulfur0.62281.01280.74210.7021Chlorine0.991031.271000.77470.8443Potassium0.4440.3441.3040.584Calcium0.7341.1241.2340.334Scandium1.119––––––Titanium1.028––––––Vanadium0.828––––––Chromium1.989––––––Manganese1.0611––––––Iron1.6114––––––Cobalt1.046––––––Nickel1.4015––––––Copper1.1110––––––Zinc0.2140.1840.1640.164Gallium0.2011.8111.3510.641Germanium0.63230.63230.55230.5923Arsenic0.3760.9960.3560.376Selenium0.66100.94100.61100.8010Bromine0.90881.34871.01370.5039Rubidium1.8462.4361.6560.446Strontium1.6461.3162.5561.516Yttrium1.708––––––Zirconium0.948––––––Niobium0.9110––––––Molybdenum1.098––––1.488Technetium1.7413––––––Ruthenium1.1312––––––Rhodium1.096––––––Palladium0.978––––––Silver1.989––––––Cadmium0.4222.2220.672––Indium0.4730.7830.7531.363Tin0.28130.41130.88130.8113Antimony0.5550.7750.4850.615Tellurium0.4720.7520.3121.352Iodine1.03771.54771.48281.2230Cesium1.2593.4791.8990.879Barium1.77111.29111.93111.1111Lanthanum1.238––––––Hafnium0.636––––––Tantalum0.975––––––Tungsten0.9214––––––Rhenium0.7613––––––Osmium0.638––––––Iridium0.968––––––Platinum1.078––––––Gold0.7814––––––Mercury0.6390.7790.6390.679Thallium0.8931.3530.4532.433Lead0.7360.7660.4160.826Bismuth0.4283.2181.1483.408Table 9Average unsigned errors in ionization potential (eV)ElementPM6No.PM5No.PM3No.AM1No.Hydrogen0.432260.402260.602260.52217Lithium0.89120.88121.29120.5912Beryllium0.5270.2970.9370.457Boron0.31110.34111.0111––Carbon0.412300.392300.542300.54227Nitrogen0.55430.45430.53430.4842Oxygen0.62720.56720.63720.6969Fluorine0.64670.65670.74670.8565Sodium0.3450.3451.4350.514Magnesium0.9741.0541.1041.414Aluminum0.6230.2930.4030.693Silicon0.43110.81110.70110.6811Phosphorus0.49130.47130.64130.5613Sulfur0.52460.51460.48460.6246Chlorine0.48620.58620.57600.6157Potassium0.2340.5040.5440.343Calcium0.7411.2410.5210.411Scandium3.731––––––Titanium0.091––––––Zinc0.3250.3550.9950.495Gallium0.5230.7331.2831.163Germanium0.70130.49130.93131.0513Arsenic0.6950.3150.6250.795Selenium0.38100.29100.47101.2210Bromine0.28330.39331.20330.4932Rubidium0.1830.3930.9330.223Strontium0.6310.3810.1410.261Cadmium0.3350.4650.395––Indium0.6320.8622.0620.832Tin0.70140.48141.22140.4414Antimony0.4450.9051.1650.545Tellurium0.4330.2030.2530.703Iodine0.47290.46290.48290.8929Cesium0.5840.7141.3741.114Barium0.0810.9710.0810.751Mercury0.51120.43120.74120.4912Thallium0.3030.4630.8030.533Lead0.56130.47130.93130.6513Bismuth0.9851.2850.7251.665 Comparison of the accuracy of PM6 with the other NDDO methods PM5, PM3, and AM1, was made more complicated by the fact that different sets of elements were available in each method. To allow a simple comparison, therefore, average unsigned errors (AUE) for the four common properties for various subsets are presented in Tables 10, 11, 12, 13 and 14. To ensure a valid comparison the same number of data were used in each method, except for AM1 in “whole of main group”, where data for cadmium and boron were not used. Table 10Average unsigned errors in ΔHf for various sets of elements (kcal mol–1)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O11574.644.895.605.659.41H, C, N, O, F, P, S, Cl, Br, I 17745.056.576.758.0512.57Whole of main group31886.1615.2717.7622.3470 elements44928.01Table 11Average unsigned errors in bond lengths for various sets of elements (Å)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O4130.0250.0220.0330.0210.031H, C, N, O, F, P, S, Cl, Br, I7120.0310.0360.0440.0370.046Whole of main group26360.0850.1210.1040.13170 elements51540.091Table 12Average unsigned errors in angles for various sets of elements (Degrees)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O1003.13.13.32.52.7H, C, N, O, F, P, S, Cl, Br, I2443.24.04.33.83.4Whole of main group9008.08.68.58.870 elements16817.9Table 13Average unsigned errors in dipole moments for various sets of elements (D)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O550.380.220.310.260.26H, C, N, O, F, P, S, Cl, Br, I1310.370.330.500.360.38Whole of main group3130.600.860.720.6570 elements5690.85Table 14Average unsigned errors in I.P.s for various sets of elements (eV)Set of elementsNo.PM6RM1PM5PM3AM1H, C, N, O990.450.400.410.510.45H, C, N, O, F, P, S, Cl, Br, I2290.470.410.440.510.56Whole of main group3830.500.490.680.6370 elements3850.50 Comparison with AM1* Winget, et al. [15], developed AM1* parameters for P, S, and Cl, in which Voityuk’s diatomic parameters were used for all atom-pairs involving P, S, and Cl with H, C, N, O, F, P, S, Cl and Mo. In the AM1* method, all parameters for elements other than the ones being optimized are held constant at the AM1 values. As such, AM1* could be regarded as a hybrid method: parameters for a few individual elements are re-optimized, in this case with some changes in the set of approximations, while holding the parameters for the other methods constant at their AM1 values. Tables comparing individual P, S, and Cl species calculated with AM1* and PM6 are given in the supplementary material. A summary of the statistical analysis is given in Table 15. Winget et al. also reported AM1* parameters for titanium and zirconium [15]. These parameters were not used in the comparison given here because the set of approximations used was incompatible with the set used in PM6. Table 15Average unsigned errors in phosphorus, sulfur, and chlorine ΔHf (kcal mol−1)Bond length (Å)Dipole (D)I.P. (eV)Angles (Degrees)PM6AM1*No.PM6AM1*No.PM6AM1*No.PM6AM1*No.PM6AM1*No.Phosphorus8.319.1900.0220.051560.570.49100.510.81122.53.319Sulfur6.510.61990.0290.060710.360.64140.520.50453.14.134Chlorine6.118.21560.0250.106690.550.60100.520.62253.414.64 Comparison with RM1 In 2006, ten elements, H, C, N, O, F, P, S, Cl, Br, and I, that had been parameterized at the AM1 level were re-parameterized [35]; the result was a new method, RM1. No changes were made to the set of approximations used, so that, for example, P, S, Cl, Br, and I used only the s-p basis set. That is, RM1 was functionally identical to AM1. A statistical analysis showed that RM1 was more accurate than any of the other NDDO methods, and therefore was the method of choice for modeling organic compounds. An indication of the effect of the current changes to the set of approximations can be obtained by comparing the AUE for PM6 and RM1 in Tables 10, 11, 12, 13 and 14. Voityuk reported the parameterization of molybdenum [14] at the AM1* level. These parameters were added to the standard AM1 parameters and were used in the analysis. Comparison with high-level methods A comparison of PM6, HF 6–31G(d) and B3LYP 6–31G(d) errors in predicted ΔHf for 1373 compounds is given in the supplementary material. Only compounds containing the elements H, C, N, O, F, P, S, Cl, and Br were considered, these being the more important elements in biochemistry. Ab-initio ΔHf were obtained from the calculated total energies by the addition of a simple atomic correction and conversion from atomic units to kcal mol−1. No allowance was made for thermal population effects, zero point energies, etc., the assumption being made that such effects could be absorbed into the atomic corrections. A statistical analysis of errors in thermochemical predictions for the three methods is given in Table 16. A check was also done to verify that the error distribution was approximately Gaussian. The resulting histogram, shown in Fig. 1, shows that the distribution is indeed Gaussian. Table 16Statistical analysis of errors in predicted ΔHf for various methods (kcal mol−1)StatisticPM6B3LYP*HF*Median3.263.755.10AUE4.445.197.37RMS6.237.4210.68No. of molecules in set: 1373* Basis set: 6–31G*Fig. 1Histogram of errors in calculated ΔHf Hydrogen bonding One of the commonest forms of hydrogen bonding involves a hydrogen atom attached to an oxygen atom and forming a weak bond to a distant oxygen atom. The simplest, well-characterized case is that of the water dimer. In an exhaustive analysis of this system, Tschumper, et. al. [36], characterized this system using CCSD(T) and a large basis set. They identified and characterized ten stationary points on the 12-dimensional potential energy surface of the dimer and determined that the lowest energy conformer of the water dimer was 5.00 kcal mol−1 more stable than two isolated water molecules. A comparison of the relative heats of formation of these points calculated using NDDO methods is shown in Table 17. The AUE for the various methods are as follows: PM6: 1.35 kcal mol−1, PM5: 3.35, PM3: 2.16, and AM1: 1.67. Table 17Relative energies of conformers of water dimerStructureRef.Relative ΔHf (kcal mol−1)PM6PM5PM3AM1(Non-planar open Cs)*−5.00−3.96−0.24−2.79−2.811 (Non−planar open Cs)0.000.000.000.000.002 (Open Ci)+0.520.830.500.910.643 (Planar Open Cs)0.570.660.250.930.464 (Cyclic Ci)0.700.290.112.10−0.945 (Cyclic C2)0.950.770.392.63−0.516 (Cyclic C2h)0.990.590.212.71−0.677 (Triply Hydrogen Bonded1.810.93−1.851.16−0.958 (Non-planar Bifurcated3.572.67−0.831.711.269 (Non-planar Bifurcated1.790.73−1.951.15−0.8710 (Planar Bifurcated C2v2.711.42−1.771.28−0.05*: Relative to two isolated water molecules+: Structures 2 – 10 are relative to Structure 1 The energies of various different types of hydrogen bonds were estimated from the energy released when the two small molecules involved associate to form a hydrogen-bonded system. Table 18 lists the values predicted using B3LYP and the NDDO methods. Table 18Comparison of B3LYP and PM6 hydrogen bond energies (kcal mol−1)Hydrogen-bonded systemRefPM6PM5PM3AM1Ammonia - ammonia−2.94−2.34−0.77−0.67−1.41Water - methanol−4.90−5.12−2.59−0.20−4.52Water - acetone−5.51−5.25−2.43−2.22−4.09Water, dimer, linear (O–H–O = 180°)−5.00−3.69−1.57−3.49−3.16Water, dimer−5.00−4.88−2.43−1.95−5.01Benzene dimer, T-shaped−2.34−0.83−0.22−0.56−0.07Water - acetate anion−19.22−18.72−12.28−15.77−15.91Water - formaldehyde−5.17−4.22−2.17−2.73−3.40Water - ammonia−6.36−4.32−2.75−1.53−2.90Water - formamide−8.88−7.60−4.14−4.33−7.54Formic acid, dimer−13.90−10.03−4.75−8.65−6.44Water - methylammonium cation−18.76−14.90−8.94−10.48−14.36Formamide - formamide−13.55−10.83−4.46−6.08−8.14Acetic acid, dimer−14.89−10.33−4.50−8.70−6.44 Nitrogen pyramidalization A well-documented fault in PM3 nitrogen was its exaggerated degree of pyramidalization when in the sp2 configuration. This is dramatically evident in N-methylacetamide, where the H-N-C–C torsion angle should be 180 °, but is predicted by PM3 to be 136 °. That is, the nitrogen, instead of being in a planar environment, is predicted to be highly pyramidal. The results of a survey of 19 molecules that contain sp2 nitrogen are presented in Table 19. Table 19Average errors in pyramidalization of nitrogen (Torsion angle about nitrogen, in degrees)StatisticPM6PM3AM1RM1Average signed error−1.7−13.60.29.7Average unsigned error5.015.03.519.1 Transition metals Optimizing parameters for transition metals was not as straightforward as for the main group elements. As with the main group compounds, there is a wealth of structural reference data on transition metal complexes. However, unlike main group compounds, there is a distinct shortage of reliable thermochemical data. To alleviate this shortage, the thermochemical data that was available was augmented by the results of DFT calculations. It was recognized, however, that these derived reference data were likely to be of a lower accuracy than the experimental data. Many transition metal complexes are also highly labile; a consequence of this was that some moieties that are known to exist in the solid phase were predicted to be unstable in the gas phase, at least at the PM6 level of calculation. In most cases, such moieties had a high formal charge, therefore, without any countercharge, their instability in isolation is understandable. When an intrinsically unstable ion was identified, it was removed from further consideration. Most transition metal compounds also have extensive UV-visible properties, arising from d-d transitions and from charge-transfer excitations, the presence of these absorption bands being indicative of the existence of low-lying electronic excited states. The self-consistent field (SCF) equations frequently did not converge unless special techniques were used. One of these, using the direct inversion of the iterative sub-space [37], or DIIS, would frequently yield an SCF when other methods failed. However, as a result of the way it works, the DIIS converged the wavefunction to the nearest stationary point, not necessarily to the lowest energy point. Because of the potential existence of multiple low-lying excited states, special care had to be taken when the DIIS technique was used. Conversely, the tendency to converge to the nearest stationary point was an advantage when electronic states of transition metal atoms were being optimized. In several instances, the lowest energy wavefunction corresponded to a hybrid of s, p and d atomic orbitals that did not transform as any irreducible representation of the group of the sphere. In those cases, the wavefunction could be induced to converge to the correct spherical harmonic solution by using the DIIS procedure. Sets of transition metals For the purpose of discussion, the set of 30 transition metals can be partitioned into eight of the groups of the Periodic Table, with each group containing one or more triads of elements. A detailed discussion of each element is impractical because of the wide range of compounds in transition metal chemistry. The following section, therefore, will be limited to systems where PM6 does not work well, and to systems illustrative of the structural chemistry of specific elements. Group IIIA: Scandium, Yttrium, Lanthanum, and Lutetium Possibly because of its scarcity, only a few experimental thermochemical reference data for scandium compounds were available for use in the parameterization. What reference data existed were augmented by the results of DFT calculations and with a large number of atomic energy levels for the neutral and ionized atom. Only the chemistry of ScIII was studied. Most bond lengths involving scandium were reproduced with good accuracy (for example tri(η5−cyclopentadienyl)-scandium, Fig. 2), the exception being the coordination complex [Sc(H2O)9]3+ which PM6 predicts to decompose to [Sc(H2O)7]3+ plus two water molecules. Fig. 2Tri(η5−cyclopentadienyl)-scandium Reference value in parenthesis As with scandium, very few thermochemical reference data were found for yttrium or lanthanum. To compensate for this, extensive use was made of the CSD. The chemistry of lutetium is similar to that of lanthanum, with the principal difference being that whereas LaIII has an empty 4f shell, in LuIII that shell is completely filled. Since the 4f shell is, at least chemically, virtually inert, lutetium could be regarded as a conventional transition metal, and was therefore included in this work. Group IVA: Titanium, Zirconium, and Hafnium In contrast to all the elements of Group IIIA, titanium is plentiful, and an abundance of reference data on TiIII and the more common TiIV is available. These data include many tetrahedral and octahedral inorganic complexes as well as organotitanium compounds. Most bond lengths are reproduced with good accuracy, the exceptions being the Ti-H bond in TiH4, where the predicted value, 1.36 Å, is 0.37 Å shorter than the reference, and coordination complexes which involve oxygen forming a purely dative bond to titanium. In this latter case, the Ti-O bond is typically too long by 0.1 to 0.3 Å. The behavior of zirconium and hafnium is similar to that of titanium. Group VA: Vanadium, Niobium, and Tantalum Most of the structural chemistry of vanadium in its five common oxidation states, 0, II, III, IV, and V, are reproduced with good accuracy. The common VO5 structure which occurs in bis(Acetylacetonato)-oxo-vanadium(iv), where vanadium forms a double bond to one oxygen atom and single bonds to the other four, is reproduced accurately, the V=O distance being 1.58 Å (reference, 1.56), the V-O distance 2.03 Å (1.97), and the O-V=O angle: 104.5 ° (105.9). Not all systems were reproduced with such accuracy. When there are several ligands around a vanadium atom, the effects of steric crowding are over-emphasized, and PM6 incorrectly predicts that one of the metal-oxygen bonds would break. An example is bis(bis(μ2-trifluoroacetato-O,O′)-(η5-cyclopentadienyl)-vanadium), where each vanadium atom extends bonds to four oxygen atoms and one cyclopentadienyl. In this system, PM6 predicts that one of the V-O bonds would break. In the heavier elements there is an increased tendency to form highly symmetric polynuclear complexes. An example is the tantalum dication, [Ta6Cl12]2+. This is predicted to have an octahedral structure in modest agreement with the DFT result (Fig. 3). Fig. 3Calculated structure of the complex ion [Ta6Cl12]2+ Reference value in parenthesis Transition metal complexes usually have one or more unpaired electrons; such systems can only be modeled using an open shell method such as unrestricted Hartree Fock (UHF) or restricted Hartree Fock followed by a configuration interaction (RHF-CI) correction. The UHF method is faster and more reliable, and is the method of choice when only simple properties such as heats of formation or geometries are of interest. For [M6X12]2+, M = Nb or Ta, X = Cl or Br, UHF predicts an almost octahedral complex, a very slight distortion lowering the symmetry to D4h. This distortion is also reflected in the asymmetric charge distribution. When RHF-CI is used, the geometry converges on the exact Oh structure. Group VIA: Chromium, Molybdenum, and Tungsten Most Cr–O and Cr–N bonds are reproduced well, as illustrated by [CrIII(EDTA)]- in Fig. 4. The organometallic bond in chromium hexacarbonyl is 1.90 Å, which is in good agreement with the crystal structure, 1.92 Å, found in FOHCOU01[21]. Fig. 4Chromium Ethylenediaminetetraacetate anion, [Cr(III)(EDTA)]− The octacyano-molybdate(IV) moiety, [MoIV(CN)8]4−, is a stable eight-coordinate organometallic molybdenum complex ion whose geometry in the crystal is that of a slightly distorted square antiprism. Rather unexpectedly, this structure was reproduced by PM6, the expectation being that in the absence of crystal field forces the structure would have optimized to a geometry which has a higher symmetry, i.e., converged to the exact D4d geometry. The predicted Mo-C distance was 2.22 versus 2.16 Å, again in unexpectedly good agreement for an ion with such a large formal charge. Molybdenum forms the cluster anion [Mo6(η3-Cl8)Cl6]2− in which the six molybdenum atoms form a regular octahedron. PM6 successfully reproduces this structure, and predicts the following distances: Mo-Mo: 2.30 (2.63), Mo-η3Cl: 2.75 (2.56), and Mo-Cl: 2.50 (2.43 Å). The trioxide of molybdenum can form polyoxometalates, a typical example of which is the α-keggin heteropolyoxyanion [SiO4@MoVI12O36]4−. In this structure, shown in Fig. 5, each Mo forms a double bond with one oxygen, single bonds to four other oxygen atoms, and what can only be described as a third of a bond to a sixth oxygen that is part of the SiO4 unit. Despite the apparently high symmetry, Td, this system has only a center of inversion. This low symmetry is reproduced by PM6. Fig. 5α-Keggin structure of tetraconta-oxo-silicon-dodeca-molybdenum, [SiO4@Mo12O36]4− Crossed-eyes stereo; Mo=O: 1.77 Å (1.69), Mo-O: 2.00 (1.85), Si–O: 1.52 (1.64) (Ref. in parentheses) PM6 predicts the structures of all three hexacarbonyls with good accuracy, but gives qualitatively the wrong structures for the dinuclear decacarbonyls. This failure to qualitatively predict the structure of the polynuclear carbonyls occurred frequently during the survey of the transition metals. Group VIIA: Manganese, Technetium, and Rhenium Like many other transition metals, manganese can form sepulchrates, closo polyhedral complexes of general structure 3, 6, 10, 13, 16, 19-hexaaza-bicyclo(6.6.6)icosane. In contrast to the more common open hexadentate chelates of manganese, e.g. [MnII(EDTA)]2−, the metal atom in a sepulchrate is extremely tightly bound, and cannot be removed without destroying the organic framework. A simple sepulchrate is shown in Fig. 6. PM6 predicts the Mn-N distance with good accuracy but gets the twist angle incorrect. A DFT calculation reproduced the twist angle found in the crystal, which suggests that the error in the twist angle cannot be attributed to the neglect of crystal packing forces. Fig. 6[Sepulchrate-manganese(III) ]3+ (3,6,10,13,16,19-Hexaaza-bicyclo(6.6.6)icosane)-manganese(III) §: CSD entry: HAFBUL Although there is a large amount of structural information on technetium compounds, there is a distinct shortage of thermochemical data. To make up for this, almost all the reference heats of formation of representative technetium compounds were derived from DFT calculations. Only one heat of formation was used in this derivation, that of the isolated technetium atom, therefore the reference values used almost certainly include a systematic error that may amount to many kilocalories per mole. Consequently, the reference heats of formation and the errors in PM6 predicted heats of formation of technetium compounds should be taken cum granus salis. However, this should not be construed as implying that they are meaningless: because reactions are balanced, when heats of reaction are evaluated, any systematic errors in the heats of formation are cancelled out. One of the more important technetium species is the pertechnetate ion, [TcO4]−, used in nuclear medicine. In this ion, PM6 predicts the Tc-O distance to be 1.73 Å, in good agreement with the DFT value of 1.76 Å. Group VIIIA: Iron, Cobalt, Nickel, Ruthenium, Rhodium, Palladium, Osmium, Iridium, and Platinum The geometries of most compounds of this large group were reproduced with modest to good accuracy, including the iron-porphorin complex, Fig. 7, of the type found in heme. The main exception is iron pentacarbonyl, Fe(CO)5, which in its equilibrium geometry is known unambiguously to be of point-group D3h, and which PM6 predicts to be equally unambiguously C4v. When this error was discovered, attempts were made to correct the fault by adding a rule to the training set for iron. This rule stated that “The C4v geometry was 28.7 kcal mol−1 higher in energy than the D3h geometry,” 28.7 kcal mol−1 being the difference between the energies of the two structures calculated using DFT. However, even when a very large weighting factor, 20.0, was used, the C4v structure remained more stable than the D3h, albeit the error in the relative energies was decreased. During this optimization errors in all other iron compounds increased significantly. Rather than accept a general deterioration in the predicted properties of iron compounds, the rule was removed from the training set. Fig. 7trans-7,8-Dihydro-2,3,7,8,12,13,17,18-octaethylporphyrinato-iron (II) Reference value (CSD entry BUYKUB) in parenthesis The well-known red complex nickel dimethylglyoxime is normally encountered in the quantitative analysis of inorganic nickel in solution. At the center of the molecule is the planar structure NiN4 structure, which is frequently found in nickel compounds in biochemical systems. PM6 predicts this with good accuracy (Fig. 8). Fig. 8Nickel Dimethylglyoxime Reference value (CSD entry NIMGLO10) in parenthesis One of the first polyhapto organometallic complexes discovered was Zeise’s salt. In the anion, [PtCl3(η2-C2H4)]−, platinum forms a synergic bond with an ethylene molecule. The calculated and X-ray structures of this complex are shown in Fig. 9. Fig. 9Zeise’s Salt, trichloro-(η2-ethene)-platinate Reference value (CSD entry XIVSAK) in parenthesis Group IB: Copper, Silver, and Gold Copper phthalocyanine is an extremely stable blue dyestuff. As with nickel dimethylglyoxime, the planar CuN4 moiety at the center of the porphyrin ring is typical of many copper species of importance in biochemistry. PM6 reproduces it with very good accuracy (Fig. 10). Fig. 10Copper phthalocyanine Reference value (CSD entry CUPOCY16) in parenthesis Dimethyl gold cyanide tetramer provides a good example of a square-planar AuIII complex. In this system, each gold atom forms covalent single bonds of length 1.99 Å(2.01) to the carbons of the methyl groups, a weaker, longer bond of length 2.12 Å(2.23) to the carbon of the cyanide group, and a still longer bond, 2.27 Å(2.23) to the nitrogen atom. Gold also forms small planar clusters. PM6 predicts that neutral clusters of up to about nine gold atoms should be planar, an example being the D6h Au7 cluster, in which the Au-Au distance is predicted to be 2.71 Å(2.01). Clusters of up to 12 gold atoms are also predicted to be stable, provided the cluster has a single negative charge. Group IIB Zinc, Cadmium, and Mercury These elements have completely filled d shells; therefore the valence shell can be limited to the s and p orbitals. As such, they behave like main-group elements. Discussion Methodological changes During the development of PM6, only very minor changes were made to the set of approximations. The main change was in the construction of the training set used for parameter optimization. One of the most important changes was the use of rules in the training set to define chemical information that was not a function of any single molecule. In earlier methods the training set had included only standard reference data. Of their nature, such data could not allow for chemical facts that were independent of any one moiety. For example, the strength of a hydrogen bond is of great importance in biochemistry, but it could not be expressed in terms of a single species. By use of rules, the value of some chemical quantity could be related to that of another. In the case of hydrogen bonding, the heat of formation of the water dimer was made a function of the heat of formation of two separated water molecules. Rules were particularly useful when elements of the three transition metal series were being optimized. Many complexes of these elements are highly labile, and, in the early stages of parameter optimization, there was a strong tendency for the optimized geometry of such complexes to be qualitatively incorrect. Faults of this kind could not be corrected by simply increasing the weight assigned to the correct geometry, so rules were developed to indicate that the faulty geometries were indeed incorrect. Specific points on the potential energy surface were selected, and from single-point high level calculations, the relative energy of these points above the minimum was evaluated. The points selected were precisely those qualitatively incorrect geometries resulting from the use of the then-current parameters. The fact that the incorrect geometry was predicted by high level methods to be of higher energy than the correct geometry was then added to the set of rules. A good example of such a rule was the rule concerning Fe(CO)5 mentioned above, in which the only datum that was defined referred to the relative energies of the compound in two different symmetries. No reference was made to the bond lengths, or bond angles. With such a rule in place, the parameters could be re-optimized to minimize the error arising from the rule, with the effect that the energy of the incorrect symmetry increased relative to that of the correct symmetry. In the majority of cases, one rule of this type was sufficient; less frequently, two rules were used, and, in rare cases, even more rules were necessary. Another change was the use of very large reference data training sets. In earlier parameterizations, the training set used was deliberately made as small as possible. Only when the resulting method was used in a survey of species not used in the training set could the predictive power of the method be determined. The training set used in the development of PM6 was designed to be considerably larger than the survey set. The rationale for this was that, by including in the training set reference data for unconventional species, e.g., non-equilibrium and hypothetical species, a greater region of the error-function surface could be defined. This would in turn, result in a better definition of the values of the parameters. That this is useful can be evidenced by the recent work in parameterizing chlorine at the AM1* level, where the compound 1,1′,2-trichloro-1,2,2′-trifluoroethane, C2Cl3F3 has a reported ΔHf of –173.7 kcal mol−1, but the value predicted using AM1* was –273.9 kcal mol−1. That is, the AM1* value was in error by over 100 kcal mol−1. If this compound had been included in the training set, it is highly likely that the error would have been significantly reduced. Although over 10,000 reference data were used in the PM6 training set, there are several indications that even this large number is still inadequate for the definition of the values of the parameters, and that an even larger training set would be highly desirable. In light of this, work has begun on identifying species to be added to the training set. During the testing of PM6, several faults were found in the method. Some of these were quickly traced to specific core-core parameters. One of the hydrogen atoms in the complex [ScIII (H2O)7]3+ was predicted to readily move toward the central atom with the result that a Sc-H bond was formed. Such faults could easily be corrected by the addition to the training set of appropriate reference data from high-level calculations. This was done in several instances, and the specific error was corrected, but this action then also required all the testing to be re-started. Because this was a time-consuming process, when faults were found near the end of the testing phase, the decision was taken that the fault should be noted, as in the Sc-H error mentioned here, and to take no further action at that time. A different type of error, found only near the end of testing, was the unrealistically large p electron population of some transition metals. The values of the parameters that determine the p population are defined using two very different groups of reference data: atomic energy levels and conventional properties of polyatomics. If atomic energy levels were excluded from the parameter optimization, then the p population would become very small; but if atomic energy levels were excluded, then the resulting method would not be suitable for reproducing such levels. The decision to use all available atomic energy levels in the training set was a value judgement. In the next training set, it is likely that the result of this decision-making process will be different. Detecting faults in semiempirical methods is difficult, and rather than wait until all errors of this type were found and fixed, a process that could potentially take several more years, the decision was made to freeze the parameters at their current value. Obviously, PM6 still has many errors; some have already been described. Work has already started in an attempt to correct them. Elimination of computational artifacts Earlier NDDO methods, particularly PM3 and AM1, produced artifacts in potential energy surfaces as a result of unrealistic terms in the core-core approximation, specifically in the set of Gaussian functions used. In PM6, only one Gaussian-type correction to the core-core potential is allowed, and, consequently, the potential for these artifacts has been reduced. On the other hand, because PM6 uses diatomic parameters, the likelihood of readily-characterized errors involving specific pairs of atoms, e.g. Sc and H, as mentioned earlier, is increased. Errors of this type can be easily eliminated by a re-parameterization of the faulty diatomic. There are over 450 sets of diatomic interactions parameterized in PM6, covering most of the common types of chemical bonds. But the number of potential bonds is much larger: given 70 elements, there are almost 2500 diatomic sets. If a molecule contains two elements for which the diatomic interaction parameters are missing, then, provided the elements are well separated, say by more than 4 Ångstroms, the absence of the parameters will not be important. If the two elements were near to each other, then the diatomic core-core parameters would be needed. This would involve generating a small training set of reference data that included a few examples of the type of interaction involved, and optimizing the two terms in the diatomic interaction. This ability to add diatomic parameter sets to PM6 without modifying the underlying parameterization has the advantage that more and more types of interaction can be added without changing the essential nature of the method. Accuracy PM6, being the most recent member of the NDDO family of approximate semiempirical methods, is understandably the most accurate. The development of each new method has been guided by the knowledge of the documented faults found in the earlier methods. This is reflected in the steady decrease in AUE of simple organic compounds, from 12.0 kcal mol−1 for AM1 to 4.9 kcal mol−1 for PM6. Several low-energy phenomena are predicted more accurately by PM6, with the most important of these being the prediction of the energies and geometries involved in hydrogen bonding. One consequence of this increased accuracy is that the lowest energy conformer of acetylacetone is now correctly predicted to be the ene-ol structure, and not the twisted di-one configuration. Despite the improvement in hydrogen bonding, a significant error was found in the balance of energies involved in forming zwitterions of hydroxyl and amine groups. This is best illustrated by the dimer of 2-aminophenol, where PM6 predicts that the zwitterion should be 3.6 kcal mol−1 more stable than the neutral form, but higher level calculations indicate that the neutral form should be 17.7 kcal mol−1 more stable than the zwitterion. In the solid state, CSD entries AMPHOM01 – AMPHOM10 [21], 2-aminophenol exists as the neutral species. In general, however, average unsigned errors in ΔHf have steadily decreased as semiempirical methods have evolved. Earlier NDDO methods such as PM3 and AM1 had AUE significantly larger than the 6–31G* Hartree Fock method. With the advent of PM5 and RM1 errors were intermediate between HF and B3LYP. In the current work, AUE in ΔHf are lower than those of both B3LYP and HF 6–31G*. This increase in accuracy of prediction of ΔHf relative to higher level methods should not be construed as disparaging those methods: semiempirical methods in general, and PM6 in particular, were parameterized to reproduce ΔHf. The performance of these methods when applied to non-equilibrium systems, in particular transition states, is likely to be very inferior to that of B3LYP or HF 6–31G*. As a result of the current work, there is a clear strategy for further improving the accuracy of semiempirical methods. All three potential sources of error need to be addressed. Regarding reference data, considerably more data are needed than were used here. This would likely come from increased use of high-level theoretical methods: methods significantly more accurate than those used here would obviously be needed in any future work. Parameter optimization can be performed with confidence and reliability, particularly when well-behaved systems are used. In all cases examined where problems were encountered in parameter optimization, problems also occurred in the normal SCF calculation in MOPAC2007. This implies that as faults in the SCF procedure are corrected, faults in parameter optimization would also be removed. Permanent errors Notwithstanding the optimism just expressed, not all errors can be eliminated by better data and better optimizations. Despite strenuous efforts, some calculated quantities persistently failed to agree with the reference values. Many potential causes for these failures were investigated. In each case the weight for the offending quantity was increased considerably and the parameter optimization re-run. When that was done, the specific error decreased, but errors elsewhere increased disproportionately. Since the final gradient of the error function was acceptably small, it followed that the parameter optimization was not in error. The reference data were checked to ensure that they were in fact trustworthy. Because two of the three possible origins of error had been eliminated, the inescapable conclusion was that there is a fault in the set of approximations. The most serious of these faults was the qualitatively incorrect prediction of the geometry of the exceedingly simple system, iron pentacarbonyl. Conclusions The NDDO method has been modified by the adoption of Voityuk’s core-core diatomic interaction parameters. This has resulted in a significant reduction in error for compounds of main-group elements, and, together with Thiel’s d-orbital approximation, allows extension of the NDDO method to the whole of the transition metal block. The accuracy of PM6 in predicting heats of formation for compounds of interest in biochemistry is somewhat better than Hartree Fock or B3LYP DFT methods, using the 6-31G(d) basis set. For a representative set of compounds, PM6 gave an average unsigned error of 4.4 kcal mol−1; for the same set HF and B3LYP had AUE of 7.4 and 5.2 kcal mol−1, respectively. The potential exists for further large increases in accuracy. This would likely result from the increased use of accurate reference data derived from high-level methods, and from the development of better tools for detecting errors at an early stage of method development. Electronic supplementary material Tables of errors in predicted heats of formation, geometries, dipole moments, and ionization potentials obtained using PM6, AM1, PM3, PM5, RM1, AM1*, HF 6-31G*, and B3LYP 6-31G* for individual species are provided, together with references for all reference data used. These data were used in generating the statistics presented in the discussion on accuracy. ESM DOC 23.7 MB

Neuropsychologia-2-1-2394569

Cortical circuits for silent speechreading in deaf and hearing people

This fMRI study explored the functional neural organisation of seen speech in congenitally deaf native signers and hearing non-signers. Both groups showed extensive activation in perisylvian regions for speechreading words compared to viewing the model at rest. In contrast to earlier findings, activation in left middle and posterior portions of superior temporal cortex, including regions within the lateral sulcus and the superior and middle temporal gyri, was greater for deaf than hearing participants. This activation pattern survived covarying for speechreading skill, which was better in deaf than hearing participants. Furthermore, correlational analysis showed that regions of activation related to speechreading skill varied with the hearing status of the observers. Deaf participants showed a positive correlation between speechreading skill and activation in the middle/posterior superior temporal cortex. In hearing participants, however, more posterior and inferior temporal activation (including fusiform and lingual gyri) was positively correlated with speechreading skill. Together, these findings indicate that activation in the left superior temporal regions for silent speechreading can be modulated by both hearing status and speechreading skill. 1 Introduction Auditory speech processing reliably engages perisylvian regions, particularly in the left hemisphere (e.g., Scott & Johnsrude, 2003). In hearing people, perisylvian regions are also recruited for silent speechreading. In particular, silent speechreading elicits activation in superior temporal regions, including middle and posterior portions of the superior temporal gyrus, its dorsal and ventral surfaces (i.e., lateral sulcus and superior temporal sulcus or STS, respectively) and the middle temporal gyrus (Bernstein et al., 2002; Calvert et al., 1997; Calvert et al., 1999; Calvert, Campbell, & Brammer, 2000; Ludman et al., 2000; MacSweeney et al., 2000; Paulesu et al., 2003; Pekkola et al., 2005; Ruytjens, Albers, van Dijk, Wit, & Willemsen, 2006), and inferior frontal regions (Buccino et al., 2004; Campbell et al., 2001; Nishitani & Hari, 2002; Paulesu et al., 2003; Watkins, Strafella, & Paus, 2003). Generally, seen speech appears to engage similar circuits to those activated when speech is heard. This includes portions of the superior temporal cortex reliably involved in processing auditory information. Activation in this region also appears to be modulated by speechreading skill. Hall, Fussell, and Summerfield (2005) did not find marked activation in the superior temporal gyrus at the group level when hearing adults observed silently spoken sentences, as compared to viewing facial gurning. However, their participants varied greatly in their ability to speechread, and a positive correlation was found between activation in the left posterior superior temporal gyrus and speechreading skill. Deaf people can outperform hearing people in comprehending seen speech (Bernstein, Demorest, & Tucker, 2000; Mohammed, Campbell, MacSweeney, Barry, & Coleman, 2006). Nevertheless, earlier reports suggested that superior temporal activation for speechreading was less reliably observed in deaf than in hearing people (MacSweeney et al., 2001; MacSweeney et al., 2002). However, the group size for these studies was small (n = 6), and so there may not have been sufficient statistical power to detect activation in this region. Furthermore, while the speechreading task in MacSweeney et al.'s (2002) study was easy (identify spoken numbers between 1 and 9), it was compared with a relatively high-level task—counting numbers of meaningless mouth actions. In contrast, a separate study by Sadato et al. (2005), reported activation in superior temporal regions in both hearing and deaf participants viewing speech-like actions. Here, the stimulus was a cartoon avatar opening and closing its mouth to form different vowel-like patterns, which participants may have interpreted as phonological gestures. The present study is the first to examine patterns of activation in deaf people who are proficient speechreaders while they searched for a speechread target embedded in lists of unrelated words. We anticipated that both hearing status and speechreading ability, measured outside the scanner, may determine the extent of activation in perisylvian regions. This was explored in two complementary ways. First, the group comparison between deaf and hearing activation patterns was assessed with speechreading skill entered into the analysis as a covariate. Speechreading skill was assessed using the Test of Adult Speechreading (TAS, Mohammed et al., 2006). By ‘partialling out’ individual differences in speechreading ability, we hoped to establish whether activation in brain regions could be modulated as a function of hearing status, irrespective of speechreading skill. Second, we used correlational analysis to establish, for each group in turn, which regions were sensitive to variations in speechreading skill. To summarise, this study examines cortical correlates for the perception of lists of speechread words under lexical target detection conditions. We aimed to identify regions that may be activated during observation of silently spoken lexical items that are not drawn from a closed set, and when the contrast (baseline) condition was a speaker at rest. The questions posed were: (1) To what extent do prelingually deaf people who are proficient signers and speechreaders show activation in superior temporal regions, including auditory cortical processing regions? (2) Are the patterns of activation different in deaf and hearing people? (3) In which regions is speechreading ability positively correlated with activation? 2 Method 2.1 Participants Thirteen (six female; mean age: 27.4; age range: 18–49) deaf adults were tested. All were congenitally (severely or profoundly) deaf (81 dB mean loss or greater in the better ear over four octaves, spanning 500–4000 Hz). Across the group, the mean hearing loss in the better ear was 103 dB. They were all native signers, having acquired British Sign Language (BSL) from their deaf signing parents. Thirteen (six female; mean age: 29.4; age range: 18–43) hearing, monolingual speakers of English were also tested. All participants were right-handed with no known neurological or behavioural abnormalities. Non-verbal IQ was measured using the Block Design subtest of the WAIS-R. Speechreading was measured using the Test of Adult Speechreading (TAS). The TAS comprises three subtests of silent speechreading in English: word identification, sentence identification, and short story identification (Mohammed et al., 2006). Independent-samples t-tests showed that deaf and hearing participants did not differ on non-verbal IQ (p > 0.1). However, deaf participants scored significantly higher than hearing non-signers on the TAS (t (24) = 4.779, p < 0.001), confirming earlier findings (Mohammed et al., 2006) with an independent sample of participants. Participant characteristics are summarised in Table 1. Standard scores (TAS z-scores) were derived from the populations reported in Mohammed et al.'s (2006) study, together with those for the present study. These scores were calculated separately for deaf and for hearing groups. Standard scores were used in order to correct for the differences in statistical distribution of scores within the deaf and the hearing groups, and were used in the correlational analyses (see Table 1). All participants gave written informed consent to participate in the study according to the Declaration of Helsinki (BMJ 1991; 302: 1194) and the study was approved by the Institute of Psychiatry/South London and Maudsley NHS Trust Research Ethics Committee. 2.2 Stimuli Stimuli were full-colour motion video of silently mouthed English words. Stimuli were modelled by a deaf native signer of BSL, who spoke English fluently (i.e., a BSL-English bilingual). The model was viewed full-face and torso. The words to be speechread were piloted on adult hearing volunteers who were not scanned. The final stimuli comprised only those words that were speechreadable by the hearing pilots. Stimuli consisted of both content words (nouns) and descriptive terms (both adjectival and adverbial). 2.3 fMRI experimental design and task The speechreading task was one of four conditions presented to participants. The other three conditions comprised signed language (BSL) material (not reported here). The speech stimuli were presented in blocks, alternating with blocks of the other three experimental conditions (30-s blocks for each condition), and with a 15-s baseline condition. The total run duration for all four conditions and baseline was 15 min. Both deaf and hearing participants were given the same target-detection task and instructions. During the speechreading condition, participants were instructed to watch the speech patterns produced by the model and to try to understand them. They were required to make a push-button response whenever the model was seen to be saying ‘yes’. This relatively passive task was chosen in preference to a ‘deeper’ processing task (such as semantic classification) for several reasons. First, it allowed for relatively automatic processing of non-target items to occur (as confirmed in post-scan tests). Second, it ensured similar difficulty of the task across stimulus conditions. As hearing non-signers would not be able to perform a semantic task on the sign stimuli, using a sparse target detection task enabled all participants to perform the same task during all experimental conditions. Over the course of the experiment, participants viewed 96 stimulus items, 24 in each of the four experimental conditions. Items were not repeated within the same block and were pseudorandomised to ensure that repeats were not clustered at the end of the experiment. Each participant saw five blocks of the speechreading condition. The baseline condition comprised video of the model at rest. The model's face and torso were shown, as in the experimental conditions. During the baseline condition, participants were directed to press a button when a grey fixation cross, digitally superimposed on the face region of the resting model, turned red. To maintain vigilance, targets in both the experimental and baseline conditions occurred randomly at a rate of one per block. Prior to the scan, participants practiced the tasks and were shown examples of the ‘yes’ targets outside the scanner using video of a model and words that were similar but not identical to those used in the experiment. Following the experiment, a sample of the hearing participants (8 of 13) and all of the deaf participants were asked to identify the items they had seen. Stimuli in the experimental conditions appeared at a rate of 15 items per block. The rate of articulation across all experimental conditions, including the speechreading blocks, was approximately one item every 2 s. All stimuli were projected onto a screen located at the base of the scanner table via a Sanyo XU40 LCD projector and then projected to a mirror angled above the participant's head. 2.4 Imaging parameters Gradient echoplanar MRI data were acquired with a 1.5-T General Electric Signa Excite (Milwaukee, WI, USA) with TwinSpeed gradients and fitted with an 8-channel quadrature head coil. Three hundred -weighted images depicting BOLD contrast were acquired at each of the 40 near-axial 3 mm thick planes parallel to the intercommissural (AC-PC) line (0.3 mm interslice gap; TR = 3 s, TE = 40 ms, flip angle = 90°). The field of view for the fMRI runs was 240 mm, and the matrix size was 64 × 64, with a resultant in-plane voxel size of 3.75 mm. High-resolution EPI scans were acquired to facilitate registration of individual fMRI datasets to Talairach space (Talairach & Tournoux, 1988). These comprised 40 near-axial 3 mm slices (0.3 mm gap), which were acquired parallel to the AC-PC line. The field of view for these scans was matched to that of the fMRI scans, but the matrix size was increased to 128 × 128, resulting in an in-plane voxel size of 1.875 mm. Other scan parameters (TR = 3 s, TE = 40 ms, flip angle = 90°) were, where possible, matched to those of the main EPI run, resulting in similar image contrast. 2.5 Data analysis The fMRI data were first corrected for motion artefact, then smoothed using a Gaussian filter (FWHM 7.2 mm) to improve the signal to noise ratio over each voxel and its immediate neighbours prior to data analysis. In addition, low frequency trends were removed by a wavelet-based procedure in which the time series at each voxel was first transformed into the wavelet domain and the wavelet coefficients of the three levels corresponding to the lowest temporal frequencies of the data were set to zero. The wavelet transform was then inverted to give the detrended time series. The least-squares fit was computed between the observed time series at each voxel and the convolutions of two gamma variate functions (peak responses at 4 and 8 s) with the experimental design (Friston, Josephs, Rees, & Turner, 1998). The best fit between the weighted sum of these convolutions and the time series at each voxel was computed using the constrained BOLD effect model suggested by Friman, Borga, Lundberg, and Knutsson (2003) in order to constrain the range of fits to those that reflect the physiological features of the BOLD response1. Following computation of the model fit, a goodness of fit statistic was derived by calculating the ratio between the sum of squares due to the model fit and the residual sum of squares (SSQ ratio) at each voxel. Permutation testing, as well as its freedom from many of the distributional assumptions of parametric tests, also offers the possibility of testing a number of statistics that are not easily testable parametrically. The SSQ ratio is such a statistic and is a simplified substitute for the F statistic suggested by Edgington (1995) that avoids the necessity of calculating the residual degrees of freedom of the time series following model fitting. The data were permuted by the wavelet-based method described by Bullmore et al. (2001) with the exception that, prior to permutation, any wavelet coefficients exceeding the calculated threshold (as described by Donoho and Johnstone (1994)) were removed. These were replaced by the threshold value. This step reduces the likelihood of refitting large, experimentally unrelated components of the signal following permutation. Significant values of the SSQ were identified by comparing this statistic with the null distribution, determined by repeating the fitting procedure 20 times at each voxel and combining data over all intracerebral voxels. This procedure preserves the noise characteristics of the time series during the permutation process, and the global assessment of the null distribution performed in this way provides good control of Type I error rates (Bullmore et al., 2001). The voxel-wise SSQ ratios were calculated for each subject from the observed data and, following time series permutation, were transformed into standard space (Talairach & Tournoux, 1988) as described previously (Brammer et al., 1997; Bullmore et al., 1996). The Talairach transformation stage was performed in two parts. First, each participant's fMRI data was realigned with their own high resolution -weighted images using a rigid body transformation. Second, an affine transformation to the Talairach template was computed. The cost function for both transformations was the maximization of the correlation between the images. Voxel size in Talairach space was 3 mm × 3 mm × 3 mm. 2.6 Group analysis Identification of active 3-D clusters was performed by first thresholding the median voxel-level SSQ ratio maps at the false positive probability of 0.05. The activated voxels were assembled into 3-D connected clusters and the sum of the SSQ ratios (statistical cluster mass) was determined for each cluster. This procedure was repeated for the median SSQ ratio maps obtained from the wavelet-permuted data to compute the null distribution of statistical cluster masses under the null hypothesis. The cluster-wise false positive threshold was then set using this distribution to give an expected false positive rate of <1 cluster per brain (Bullmore et al., 1999). 2.7 ANOVA Differences between the groups were calculated by fitting the data at each voxel in which all participants had non-zero data using the following linear model, Y = a + bX + e, where Y is the vector of BOLD effect sizes for each individual, X is the contrast matrix for the particular inter-group contrast required, a is the mean effect across all individuals in the groups, b is the computed group difference and e is a vector of residual errors. The model is fitted by minimising the sum of absolute deviations rather than the sums of squares to reduce outlier effects. The null distribution of b is computed by permuting data between groups (assuming the null hypothesis of no effect of group) and refitting the above model. This permutation method thus gives an exact test (for this set of data) of the probability of the value of b in the unpermuted data under the null hypothesis. The permutation process permits estimation of the distribution of b under the null hypothesis of no mean difference. Identification of significantly activated clusters was performed by using the cluster-wise false positive threshold that yielded an expected false positive rate of <1 cluster per brain (Bullmore et al., 1999). 2.8 ANCOVA Analysis of covariance was used to address behavioural differences between the deaf and hearing participants in relation to the patterns of activation for the speechreading condition (see Table 1). Differences in responses (R) were inferred at each voxel using the linear model, R = a0 + a1H + a2X + e, where H codes the contrast(s) of interest between groups, X is a covariate and e is the residual error. Maps of the standardized coefficient (size of group difference) (a1), were tested for significance against the null distribution of a1 (no effect of group membership) generated by repeatedly refitting the above model at each voxel following randomization of group membership (H). 2.9 Correlational analysis In order to examine the relationship between brain activation and speechreading skill, correlational analysis was performed between the BOLD effect data for each individual and the Test of Adult Speechreading (TAS) z-score. These were calculated separately for each group. Pearson product–moment correlation coefficients were calculated between the observed behavioural and BOLD effect data. The null distribution of correlation coefficients was then computed by permuting the BOLD data 100 times per voxel and then combining the data over all voxels. Median voxel-level maps were computed at the false probability of 0.05 and cluster-level maps, where r was significant, were computed such that the expected false positive rate was <1 cluster per brain. 3 Results 3.1 Behavioural data All participants completed the behavioural (target detection) task in the scanner reasonably accurately. Deaf participants identified the speechreading targets more accurately than hearing participants (mean accuracy (max = 5), deaf = 4.69, hearing = 3.85, t(24) = 2.99, p = 0.007). Speechreading target identification was slower in deaf than hearing participants (mean RT, deaf = 1192.63 ms, hearing = 920.08 ms, t(24) = 4.15, p < 0.001). Following scanning, participants were presented with the experimental stimuli. The deaf participants identified more words than the hearing participants (mean percent correct identification, deaf = 69%, hearing = 46%), t(19) = 4.11, p = 0.001). The behavioural data suggest that deaf participants’ greater accuracy in identification of non-target items (as indicated by the post-scan test) may have interfered with their processing of the target (as indicated by the relatively slow reaction times to targets in the scanner). 3.2 fMRI data 3.2.1 Speechreading vs. baseline In both deaf and hearing groups, extensive activation was observed in fronto-temporal cortices, bilaterally (Table 2, Fig. 1). In deaf participants, activation in the left superior temporal cortex was focused at the border between the posterior superior temporal gyrus and the transverse temporal gyrus (BA 42/41) and extended to the middle (BA 21) and inferior (BAs 37, 19) temporal gyri and the supramarginal gyrus (BA 40). This cluster of activation also extended to inferior (BAs 44, 45) and middle (BAs 6, 9) frontal gyri and precentral gyrus (BA 4). In the right hemisphere, a cluster of activation focused in the superior/middle temporal gyri (BA 22/21) extended to BAs 42 and 41 and posterior inferior temporal gyrus (BAs 37, 19). Activation in the right frontal cortex was focused in the precentral gyrus (BA 6) and extended to the inferior (BAs 44, 45) and middle (BAs 46, 9) frontal gyri. Additional activation was observed at the border of the medial frontal gyrus and the anterior cingulate gyrus (BA 6/32). In hearing participants, we observed activation focused in the left middle temporo-occipital junction (BA 37) and in the right superior/middle temporal gyrus (BA 22/21). These clusters of activation extended to include the superior and transverse temporal gyri (BAs 22, 42, 41), the postcentral gyri (BA 43) and the middle and inferior temporal (BAs 21, 37, 19, 20) and cerebellar gyri. In the left hemisphere, this cluster also extended to the supramarginal gyrus (BA 40). In both hemispheres, clusters in the inferior parietal cortex were focused in the supramarginal gyrus (BA 40). These clusters extended to angular (BA 39) and middle occipital (BA 19) gyri. The cluster in the right hemisphere extended medially to the border of the dorsal posterior cingulate gyrus (BA 31). Activation in frontal cortices was focused in the precentral gyrus (BA 4/6) of the left hemisphere and in the inferior frontal gyrus (BA 44) of the right hemisphere. In both hemispheres, frontal activation included the inferior (BAs 44, 45 47) middle (BA 46) and superior (BA 9) frontal gyri and the precentral gyrus (BAs 4, 6). In the right hemisphere, the frontal cluster extended anteriorly to the border of the frontal pole (BA 10). Additional activation was observed in the right medial frontal gyrus (BA 6), extending to medial BA 8 and anterior cingulate gyrus (BAs 24 and 32).2 3.2.2 Deaf vs. hearing Deaf native signers displayed significantly greater activation in left and right superior temporal cortices than hearing non-signers. In the left hemisphere, the cluster of activation (116 voxels) was focused at the border between the posterior superior temporal gyrus (i.e., planum temporale) and the transverse temporal (i.e., Heschl's) gyrus (BA 42/41; x = −54, y = −22, z = 10). In the right hemisphere, the cluster (61 voxels) was focused at the border between the superior and middle temporal gyri (BA 22/21; x = 51 y = −7 z = −3). Hearing non-signers showed greater activation than deaf signers in the right prefrontal cortex (128 voxels, focused in BA 44; x = 40, y = 11, z = 26). When speechreading performance, as indicated by individual TAS z-score, was entered as a covariate into this analysis, deaf participants displayed greater activation than hearing participants in the left temporal cortex. The cluster of activation (120 voxels) was focused at the border between the posterior superior temporal gyrus (i.e., planum temporale) and the transverse temporal (i.e., Heschl's) gyrus (BA 42/41; x = −54, y = −22, z = 10). The focus of this cluster was verified using probabilistic maps provided by Penhune, Zatorre, MacDonald, and Evans (1996) (25–50% probability of Heschl's gyrus) and Westbury, Zatorre, and Evans (1999) (26–45% probability of planum temporale). Based on these probability maps, 15 voxels within this cluster, displayed ≥50% probability of being located in Heschl's gyrus, and five voxels showed ≥46% probability of being in planum temporale. This cluster also extended into the posterior lateral portion of the superior temporal gyrus (BA 22) and the middle and posterior portions of the superior temporal sulcus and middle temporal gyrus (BA 21; see Fig. 2). No brain regions were significantly more active in hearing than deaf participants when speechreading was a covariate in the analysis. 3.2.3 Cortical activation for speechreading: correlations with speechreading skill Speechreading skill, as measured by performance on the Test of Adult Speechreading (TAS), varied considerably across participants (Table 1). Several brain regions were significantly positively associated with TAS z-scores in both deaf and hearing groups. 3.2.4 Deaf group In the deaf group, ten clusters of activation (≥5 voxels) were positively associated with speechreading skill (see Table 3). In the temporal lobe, clusters in the superior temporal cortex were focused in the lateral portion of the transverse temporal gyrus (BA 41) in the right hemisphere, and in the superior temporal gyrus (BA 42) in the left hemisphere. However, although the Talairach and Tournoux (1988) atlas suggests that this cluster incorporates the transverse temporal gyrus, the probability map of this region provided by Penhune et al. (1996) suggests otherwise. In fact, only one voxel (in the left hemisphere cluster) displayed a ≥50% probability of being located in this region (Penhune et al., 1996). Both clusters extended to include the posterior superior temporal gyrus (BAs 42, 22). Additional areas showing significant correlation included the middle portion of the right middle temporal gyrus (BA 21). In the frontal cortex, correlations were observed in the middle frontal gyri of both hemispheres (BA 6). In the right hemisphere, correlations were also observed in the dorsolateral prefrontal cortex (BA 46), precentral gyrus (BA 6/4) and in the anterior insula. Additional correlations were observed in the anterior cingulate gyrus (BA 32/24) and the cerebellum. 3.2.5 Hearing group In the hearing group, clusters of activation that were positively correlated with TAS z-scores included the fusiform (BA 37) and lingual (BA 18) gyri of the right hemisphere and the right postcentral gyrus (BA 4). Additional positive correlations were observed in the posterior cingulate gyrus (BA 23). 4 Discussion Deaf participants were better speechreaders than hearing participants, both in terms of their TAS performance (Table 1) and, when tested post-scan at identifying the words presented in the scanner. The finding that deaf people can be better speechreaders than hearing individuals is not new (Bernstein et al., 2000; Mohammed et al., 2006). Deaf people, including deaf people who use a signed language, rely on speechreading, whether hearing-aid supported or un-aided, to communicate in the wider hearing community. In contrast, in hearing people, where the auditory channel dominates for speech identification, reliance on silent seen speech is generally unfamiliar and unpractised. In the present study most participants, whether deaf or hearing, could speechread much of the spoken material, and it can be assumed, therefore, that some of what they were shown in the scanner was lexically processed—albeit more in deaf than in hearing participants. Interpretation of the imaging data must bear these considerations in mind. Covariance and correlational analyses allow the behavioural and neuroimaging results to be aligned. The group-level analyses, conducted separately for the deaf and hearing groups, contrasted silent speechreading with a low-level target detection task. As such, these analyses cannot allow unambiguous interpretation of the specificity of such activation in relation to speechreading alone, but they do suggest a general pattern against which the group differences can be explored. In hearing people, the pattern of activation replicates that which has been observed in many previous studies, showing extensive activation across the temporal cortex. While some of this activation must relate to visual movement detection and to the perception of biological motion, especially in posterior and inferior regions (see, for example, Zeki et al., 1991), it is likely that much of the activation in superior temporal regions relates to speechreading, since several studies contrasting speechreading with a higher-level baseline, such as observing non-speech-like mouth movements, report enhanced activation in this region (e.g., Calvert et al., 1997; Paulesu et al., 2003). The present study found that, in both hearing and deaf participants, activation associated with speechreading words included the dorsal surface of the superior temporal cortex including the junction of the superior temporal gyrus and the lateral portion of the transverse temporal (Heschl's) gyrus (BA 42/41). Spatial smoothing intrinsic to transforming data into standard brain space may limit the spatial resolution in this study. Thus the finding that activation for silent speechreading included the lateral portion of Heschl's gyrus must be interpreted with caution. Nevertheless, this finding is consistent with previous neuroimaging research that delineated this region on individual brains (Pekkola et al., 2005). In addition, left inferior frontal regions were activated when observing speech silently. This has also been observed where the contrasts were with higher-level conditions such as watching non-vocal mouth actions (Buccino et al., 2004; Campbell et al., 2001; Paulesu et al., 2003; Watkins et al., 2003) and may reflect the operation of mirror neuron systems in the observation of speech actions. The finding of superior temporal activation for speechreading in deaf people extends earlier studies exploring the neural organisation of processing a variety of oral gestures in hearing people. This pattern of superior temporal activation found in the present study is consistent with the findings recently reported by Sadato et al. (2005), who presented deaf participants with simple segmental utterances including vowel-like lip shapes. At first sight, the present results do not fit with those we have previously reported using a closed stimulus set, covert articulation and a gurning control condition conducted with a small group of deaf people (MacSweeney et al., 2001; MacSweeney et al., 2002). However, we did report activation within right superior temporal regions, when analysis combining the data from two experiments allowed for an increase in power (MacSweeney et al., 2002). A further study involving a larger group of deaf participants, and manipulating task, baseline condition and stimuli, will help establish whether our previous studies simply lacked power or whether task and stimulus factors systematically affect the extent to which superior temporal regions are recruited during silent speechreading in those born profoundly deaf. 4.1 Deaf vs. hearing When hearing non-signers were compared with deaf signers, and speechreading skill (which differed between the groups) was entered as a covariate (Fig. 2) greater activation was observed for the deaf than hearing group in left middle-posterior superior temporal regions. This cluster of activation was focused at the border between the posterior and transverse temporal gyri (BA 42/41) and extended to the middle and posterior portions of the superior temporal gyrus and sulcus, and middle temporal gyrus. No regions showed greater activation in hearing than deaf participants. In hearing people, the role of the posterior superior temporal sulcus (p-STS) has been proposed as a key ‘binding site’, responsible for cross- and supra-modal processing of co-incident auditory and visual streams in audiovisual speech processing (Calvert et al., 1999; Calvert et al., 2000). However, in deaf people, p-STS cannot play this role, since the association between seen and heard speech in deaf people is variable and relatively unsystematic. In the present study, not only was activation in this region observed in the absence of audition; it was greater in deaf than hearing people. One possibility is that activation by seen speech in p-STS is sensitive to the dominant speech modality within this multimodal region. That is, activation by silent speech in this region may be greater in deaf people because the region has developed to be sensitive to visual speech, while for hearing people it has developed to be sensitive to auditory speech characteristics, with visual speech as a secondary function. Structural imaging of the connections between p-STS and visual and auditory cortices in deaf and hearing individuals could be employed to test this hypothesis. A non-mutually exclusive possibility is that greater activation in superior temporal regions for deaf than hearing individuals reflects a more general plasticity of these regions in deaf people. Several studies suggest that brain regions considered specialised for audition can be recruited for processing stimuli from other modalities in deaf people (e.g., Fine, Finney, Boynton, & Dobkins, 2005; Finney, Fine, & Dobkins, 2001; Sadato et al., 2005). While the extent and specificity of primary auditory cortex recruitment by visual events remains unclear (Bavelier, Dye, & Hauser, 2006), some studies (e.g., MacSweeney et al., 2004) suggest that perception of signed language, and even of non-linguistic biological movement, can recruit regions within superior temporal cortex to a greater extent in deaf native signers than in hearing people exposed to a signed language from birth (hearing native signers). 4.2 Correlations of activation with individual differences in speechreading skill TAS speechreading scores and post-scan speechreading of the items seen in the scanner were positively correlated (deaf: r = 0.476, p(1-tailed) = 0.05; hearing: r = 0.673, p(1-tailed) = 0.034); thus we can infer that the higher the TAS score, the more likely it is that participants would have processed the speechread material lexically. However, TAS scores were not normally distributed across the two groups. For this reason, standard scores (TAS-z) derived for each group formed the basis for exploring the relationship between speechreading skill and cortical activation. Within each group, different patterns of association were observed. In deaf participants, the correlational analyses showed that activation in the posterior portion of the superior temporal gyri (as well as middle temporal and middle frontal gyri) was positively associated with speechreading. In the hearing participants, who were less able and more varied speechreaders than the deaf participants, speechreading skill was positively associated with activation in the right lingual and posterior cingulate gyri, which is consistent with findings from Hall et al. (2005). Additional activations displaying a positive correlation with speechreading skill included the right postcentral and inferior temporal (fusiform) gyri, perhaps suggesting relatively greater involvement of articulatory skill and face processing in hearing individuals’ speechreading, respectively. Taken together, these data show that hearing status is an important determinant of activation in left superior temporal regions when words are speechread. In particular, silent speechreading elicits greater activation in the left middle and posterior portions of the superior temporal cortex, including the superior and middle temporal gyri and the lateral portion of the transverse temporal gyrus in deaf than hearing people, even when speechreading skill is held constant. However, speechreading skill can moderate this activation, showing a positive relationship in deaf but not hearing participants. The relatively small group sizes used in the correlational analysis (n = 13 in each group), however, require that this interpretation should be provisional. Hall et al. (2005) did not find reliable activation in superior temporal gyrus for silent speechreading in contrast to viewing facial gurning in a group of 33 hearing participants, who also varied widely in speechreading skill. However, they did report a reliable positive correlation between speechreading skill and activation in this region. The inference from that study together with the present one must be that, when speechread material is linguistically processed, superior temporal regions within the left hemisphere are likely to be recruited. Additionally, the present study shows that it was deaf rather than hearing people who showed this relationship most clearly, and where individual differences in speechreading skill made an additional impact, despite the range of speechreading skill being larger in the hearing than the deaf group. We have shown that, when auditory regions are not activated by acoustic stimulation, they can nevertheless be activated by silent speech in the form of speechreading. This finding may have some practical as well as theoretical significance. Current practice in relation to speech training for prelingually deaf children preparing for cochlear implantation emphasises acoustic processing. In auditory-verbal training, the speaking model is required to hide her or his lips with the aim of training the child's acoustic skills (e.g., Chan, Chan, Kwok, & Yu, 2000; Rhoades & Chisholm, 2000). Thus, a neurological hypothesis is being advanced which suggests that the deaf child should not watch spoken (or signed) language since this may adversely affect the sensitivity of auditory brain regions to acoustic activation following cochlear implantation. Such advice may not be warranted if speechreading activates auditory regions in both deaf and hearing individuals. Speechreading gives access to spoken language structure by eye. It therefore has the potential to impact positively on the development of auditory speech processing following cochlear implantation. While there are few consistent correlates of improved post-implant speech processing in prelingually deaf cochlear implantees, efficiency in speechreading is implicated. For example, pre-implant silent speechreading skills are positively associated with general speech and language outcomes (Bergeson, Pisoni, & Davis, 2005). The possibility that superior temporal regions in deaf individuals, once tuned to visible speech, may then more readily adapt to perceiving speech multimodally should be seriously considered when recommendations concerning pediatric cochlear implantation procedures are being developed.

Matern_Child_Health_J-2-2-1592158

Preconception Care and Treatment with Assisted Reproductive Technologies

Couples with fertility problems seeking treatment with assisted reproductive technologies (ART) such as in vitro fertilization should receive preconception counseling on all factors that are provided when counseling patients without fertility problems. Additional counseling should address success rates and possible risks from ART therapies. Success rates from ART are improving, with the highest live birth rates averaging about 40% per cycle among women less than 35 years old. A woman’s age lowers the chance of achieving a live birth, as do smoking, obesity, and infertility diagnoses such as hydrosalpinx, uterine leiomyoma, or male factor infertility. Singletons conceived with ART may have lower birth weights. Animal studies suggest that genetic imprinting disorders may be induced by certain embryo culture conditions. The major risk from ovarian stimulation is multiple gestation. About one-third of live-birth deliveries from ART have more than one infant, and twins represent 85% of these multiple-birth children. There are more complications in multiple gestation pregnancies, infants are more likely to be born preterm and with other health problems, and families caring for multiples experience more stress. Transferring fewer embryos per cycle reduces the multiple birth rate from ART, but the patient may have to pay for additional cycles of ART because of a lower likelihood of pregnancy. Current knowledge Fertility problems affect about 10% of couples in developed countries [1–3]. These couples should receive preconception counseling on all factors that are discussed during preconception counseling for patients without fertility problems. Additional counseling should address success rates and possible risks from the specific fertility treatments being considered because of the couple’s diagnoses and preferences. To provide an example of an approach to preconception counseling for the infertile couple, treatment with assisted reproductive technologies (ART) such as in vitro fertilization (IVF) is reviewed here. About 1% of births in the United States are the result of ART [4]. ART care represents a unique opportunity for preconception counseling because the moment of conception is easily discernable. Counseling should address expected success rates from ART based on the woman and the man’s risk profile. Older maternal age is the strongest risk factor for not achieving a successful outcome from ART. Live birth rates average about 40% per cycle among women less than 35 years old, and gradually decrease to about 6% per cycle among women over age 42 who use their own oocytes [4]. Older women who accept donor oocytes increase their chance of a live birth to approximately the rate seen in the age group of the donor woman [5]. Cumulative live birth rates account for the fact that many couples return for further treatment if the first cycle of ART is unsuccessful. For couples who complete two to three ART cycles, cumulative live birth rates are about 50–60% for women 35 years old or younger, declining to about 30% by age 40 [6]. As men’s age increases, the time required for a couple to conceive lengthens, even after controlling for the woman’s age and other risk factors for reduced fertility [7]. The chance of having a live birth from ART therapy is influenced by the health habits and the infertility diagnoses of the couple. Current tobacco smoking by women decreases ovarian function and is manifested by increased basal levels of follicle stimulating hormone. Such women produce fewer oocytes during ART and have lower pregnancy rates [8, 9]. Current smoking by the male partner also decreases pregnancy rates through direct effects on sperm and by exposing the woman partner to side-stream smoke. Increasing body mass index (BMI) is associated with decreasing ovarian response to follicle stimulating hormone, resulting in fewer oocytes collected in a given IVF cycle, as well as increasing rates of early pregnancy loss [10]. Although no strong evidence associates underweight with adverse ART outcomes, maternal underweight (BMI < 18.5 kg/m2) is risk factor for low birth weight in the general population. As the number of specific infertility diagnoses increases for a couple, pregnancy and live birth rates from ART decrease. Moreover, certain infertility diagnoses are somewhat more difficult to overcome than others during treatment. Live birth rates after ART tend to be slightly higher if the only diagnosis is endometriosis, and slightly lower if the only diagnosis is uterine leiomyoma or hydrosalpinx [4, 6, 11]. Leiomyoma may decrease uterine receptivity, mainly when an intramural or submucosal fibroid distorts the endometrial cavity. The mechanism by which hydrosalpinx worsens the prognosis in ART is not understood, but tubal fluid in hydrosalpinx has embryotoxic properties and salpingectomy improves live birth rates [12]. Male factor infertility is present in about 40% of couples presenting for ART, and may be a barrier to achieving pregnancy unless intracytoplasmic sperm injection (ICSI) is used [4, 6, 11]. The most common adverse outcome from infertility treatment is multiple gestation. ART produced 11,544 multiple birth deliveries in the United States during 2002 [4]. Of all the ART infants born that year, 45.7% were twins and 7.6% were triplets or higher order multiples. Although health outcomes are good to excellent in many twin pregnancies, mothers of twins experience more pregnancy complications, lasting infant neurologic impairment may occur from very preterm birth, and the neonatal death rate of twins is 5–10 times greater than of singletons. For triplets, 90% of births are preterm and the relative risk of death in the first month of life is increased 20-fold [13]. To place the risk of multiple gestation from ART in perspective, estimates are available on the proportion of multiple births from unassisted conception and from treatment with ovulation medications without ART. These estimates have been made by subtracting the rate of multiple births during the era before ovulation-inducing drugs (unassisted conception) and the rate of multiple births resulting from ART from the total rate of multiple births, adjusted for trends in maternal age [4, 14]. Such an analysis shows that about 65% of twins and 15–18% of triplets are from natural conception each year. About 20% of twins and 35–40% of triplets are probably from ovulation induction without ART, and about 15% of twins and 40–45% of triplet births result from ART [14]. In general, women with the best prognosis for achieving a live birth from ART are younger than 35 years, use freshly fertilized embryos from their own eggs, and have good quality embryos—a marker of the latter is having more embryos available than are transferred. For this group, recent surveillance statistics showed that transferring one, two, three or four embryos resulted in live birth rates of 47.4, 51.8, 49.6 and 45.5%, respectively; the singleton rates among these births were 100.0, 60.4, 52.8 and 40.7%, respectively [4]. By age 38–40 years the prognosis is poorer among all women who use freshly fertilized embryos from their own eggs. Transferring one, two, three or four embryos in this group resulted in live birth rates of 10.4, 23.3, 28.9 and 29.9% respectively; the singleton rates among these births were 97.4, 81.2, 79.2 and 70.4%, respectively. Thus, for women with the best prognosis, transferring more embryos provides little improvement in live birth rates with an increasing risk of multiples. For women with a poorer prognosis, additional embryos may improve live birth rates up to a point, and the risk for multiples may increase less dramatically [4]. During ART, ovarian hyperstimulation syndrome is associated with production of a large number of follicles. Usually the only symptoms are modest abdominal pain. More severely affected women, however, experience increased capillary permeability that leads to ascites and in rare cases, death. Symptoms requiring hospitalization occur in 0–3% of IVF cycles [15]. Ovarian cancer risk from medications used to stimulate folliculogenesis has been studied because of concerns that this treatment may trigger growth of ovarian cortical cells through the process of healing at the sites of ovulation. Although studies show that women treated with ART have about a 50% increase in ovarian cancer risk compared to the general population, when compared to control women with infertility who did not receive ART, ovarian cancer risk is not elevated [16]. Thus it appears that infertility itself may be the risk factor for ovarian cancer, rather than ART. Singletons conceived with ART may be at greater risk of being born prematurely, or to be small for gestational age [17]. Available studies have been limited by lack of data on key confounders. When matching is used to control for the most important confounders, the risk difference between spontaneously conceived singletons and singletons conceived with ART remains, but appears to be relatively small. The rates of fetal growth restriction differ by about 3.7%, with an overall rate of growth restriction of 8.6% among spontaneous singletons compared to a rate of 12.3% among singletons conceived with ART. The rates of very preterm birth differ by about 1.3%, with an overall rate of very preterm birth of 0.7% for spontaneous singletons compared to 2.0% for singletons conceived with ART [17]. In some studies, an increased risk of birth defects has been noted among IVF children compared to the general population, but in other studies the difference tends to disappear when risk factors for both infertility and birth defects such as older parental age and multiple gestation are controlled [18]. Infertility is sometimes caused by sex chromosome aberrations, such as Y chromosome deletions that lead to reduced sperm counts in men [18, 19]. Sperm of men with reduced fertility has more aneuploidy [20]. Whether treatment of male factor infertility by ICSI will cause fertility problems in male offspring cannot be known until long-term follow-up is available. Adverse embryo culture conditions can cause imprinting disorders in mice during a procedure similar to ART. This lends biologic plausibility to imprinting disorders as a possible adverse effect of IVF [18, 19, 21]. Imprinting is the mechanism by which congenital disorders such as Beckwith-Wiedemann syndrome, Angelman syndrome and Prader-Willi syndrome occur. These syndromes have been reported in children conceived with ART, although the rarity of the syndromes and problems generalizing from animal experiments to humans makes a causal relationship difficult to establish. In an Australian study that used data from a national genetics testing laboratory, 37 cases of Beckwith-Wiedemann syndrome occurred in 1.3 million live births, a rate of one in every 35,580 births [22]. Four of the cases were associated with IVF compared to a matched control group in which one mother had IVF (P < 0.05). In a cohort study from the Danish national registries for IVF and for all births, however, no increase in imprinting diseases was linked to ART [23]. Recommendations for action Reproductive endocrinologists use a combination of clinical factors to advise couples of the likelihood of success from infertility treatment, and they use clinical tests such basal follicle stimulating hormone levels to further characterize prognosis. Professional practice guidelines are available that recommend transferring fewer embryos among the couples with greatest risk for multiple gestation [24]. Professional guidelines note the elements to be considered when educating patients during the informed consent process for ART [25]. Reproductive endocrinologists routinely counsel patients about the risks from ART, including multiple gestation, ovarian hyperstimulation syndrome, physical injury during procedures, and success rates [26]. It may be prudent to counsel patients about emerging scientific issues as new research is published, such as concerns about embryo culture and ICSI. This would be similar to the approach used for other potential concerns. That is, even though a causal relationship between ART treatment methods and development of ovarian cancer has not been substantiated, the majority of reproductive endocrinologists inform patients of the issue [26]. Effectiveness of interventions Little research exists on comprehensive preconception counseling for infertility patients. Studies that have examined counseling about the most common adverse outcome, multiple births, have found that many infertility patients prefer to have twins and sometimes triplets rather than a single infant [27–29]. A randomized trial of a new educational intervention was ineffective at reducing this preference, although other research has found that raising awareness of the risks of multiple births reduces patient preference for multiples [27, 28]. Integration with other preconception services Routine preconception counseling for women and men should include information on the many well-documented risk factors for experiencing infertility later in life. When patients who have trouble conceiving present for ART, reinforcing these concepts and combining preconception care at the key decision-making points should help maximize health outcomes for parents and their future children.

Biogerontology-4-1-2367389

Caloric restriction counteracts age-dependent changes in prolyl-4-hydroxylase domain (PHD) 3 expression

Caloric restriction remains the most reproducible measure known to extend life span or diminish age-associated changes. Previously, we have described an elevated expression of the prolyl-4-hydroxylase domain (PHD) 3 with increasing age in mouse and human heart. PHDs modulate the cellular response towards hypoxia by regulating the stability of the α-subunit of the transcriptional activator hypoxia inducible factor (HIF). In the present study we demonstrate that elevated PHD3, but not PHD1 or PHD2, expression is not restricted to the heart but does also occur in rat skeletal muscle and liver. Elevated expression of PHD3 is counteracted by a decrease in caloric intake (40% caloric restriction applied for 6 months) in all three tissues. Age-associated changes in PHD3 expression inversely correlated with the expression of the HIF-target gene macrophage migration inhibitory factor (MIF), which has been previously described to be involved in cellular HIF-mediated anti-ageing effects. These data give insight into the molecular consequences of caloric restriction, which influences hypoxia-mediated gene expression via PHD3. Introduction One of the hallmarks of ageing is the decline of physiological functions. This is accompanied with progressive organ failure and an increased incidence of age-related diseases. Caloric restriction remains the most reproducible measure known to extend the maximum lifespan and to retard a variety of age-associated changes in several species (Speakman and Hambly 2007). Recent evidence suggests that caloric restriction, besides preventing accumulation of energy-related metabolites like reactive oxygen species or advanced glycation end products, triggers a regulatory response (Koubova and Guarente 2003). As a result of the limited energy intake, a modulated expression of genes involved in various regulatory integrative functions has been discovered. In a recent study we described age-related changes in the expression of the prolyl-4-hydroxylase domain (PHD) 3 and as a consequence in the expression of the hypoxia-inducible factor (HIF)-1α transcription factor (Katschinski 2006; Rohrbach et al. 2005). PHD3 and HIF belong to the cellular oxygen sensing system and regulate therefore the adaptation of cells towards a decreased oxygen supply via hypoxia-inducible gene expression. This involves genes associated with angiogenesis, pH control, glucose metabolism, oxygen transport etc. (Wenger et al. 2005). In this regard, it should be noted that some ageing-associated diseases such as ischemic diseases, atherosclerosis and cancer are associated with an altered oxygen supply. Moreover, the tolerance to ischemia and hypoxia is reduced in elderly (Abete et al. 1999; Bosch-Marce et al. 2007; Mariani et al. 2000; Paolucci et al. 2003). HIF is a heterodimer comprising the oxygen labile α-subunit and the oxygen-independently expressed β-subunit, which is also called ARNT. The expression of HIF-1α is regulated by three described PHDs, i.e. PHD1, PHD2 and PHD3, at the protein level (Ivan et al. 2001; Jaakkola et al. 2001). PHD-dependent hydroxylation is directly depending on the oxygen availability (Bruick and McKnight 2001; Epstein et al. 2001). Hydroxylation of HIF-1α allows binding of the von Hippel Lindau tumor suppressor protein, which triggers rapid ubiquitination and degradation (Maxwell et al. 1999). An age-dependent decline of HIF-1α protein levels and HIF-target gene expression has been described in different organs like rat cerebral cortex, mouse heart, carotid body and smooth muscle cells isolated from rabbit aorta (Di Giulio et al. 2005; Rivard et al. 2000; Rohrbach et al. 2005). The age-related expression pattern of PHDs in other organs besides heart is not known. Caloric restriction has been consistently described to challenge biological ageing (Rohrbach et al. 2006b). Regarding the HIF system, it is interesting to note that caloric restriction counteracts age-related changes in the angiogenic response and can protect cardiomyocytes from hypoxic death in rodent models of ischemic diseases (Ahmet et al. 2005; Facchetti et al. 2007). To gain more insight into the impact of nutrient availability on the adaptive transcriptional response towards hypoxia, we quantified the expression of the HIF-1α regulating PHD1–3 in heart, liver and skeletal muscle in young and old rats undergoing a well-defined protocol of caloric restriction. Materials and methods Animals and diet protocol, 40% caloric restriction for 6 months Male young (4 months) and senescent (22 months) Sprague-Dawley rats were obtained from Charles River (Germany), caged individually with a light/dark cycle of 12 hrs and with tap water ad libitum. Food (AltrominR 1244; 2550 cal/g) was offered ad libitum. Prior to the application of the specific diet protocols, daily food intake of the normal standard diet was monitored for 14 days and averaged for each rat individually. Thereafter, rats at the age of 6 months (young rats) or at the age of 24 months (old animals) were randomly assigned to one of the following diets for the next six months: rats on “control diet” received their individual prediet average of AltrominR 1244 (2,550 cal/g), but not more, in order to avoid any degree of diet-induced obesity. Rats subjected to caloric restriction received also their prediet average, but of a calorically reduced, fibre-rich diet (AltrominR 1344/1500; 1,550 cal/g). Thus, in young rats on control diet (n = 6), the daily energy intake during six months was 64.8 ± 2.2 kcal and in young rats on -40% caloric restriction (n = 6) the daily intake was 37.8 ± 1.9 kcal. In old rats the daily energy intake amounted to 57.9 ± 1.8 kcal with control diet (n = 3) and to 36.6 ± 0.1 kcal with 40% caloric restriction (n = 3). All animals were fasted for 12 hours before killing. RNA extraction, RT-reaction and quantification by PCR The RNA was isolated from left ventricle, liver and skeletal muscle (M. gastrocnemicus) tissue as described before (Rohrbach et al. 2007). Integrity and quality of the RNA was confirmed by agarose gel electrophoresis and the concentration determined by measuring UV-absorption. Real time PCR Reverse transcription (RT) of RNA samples was carried out for 30 min at 42°C. Real-time PCR and data analysis were performed using the Mx3000P Multiplex Quantitative PCR System (Stratagene). DNA amplification was performed as follows: initial denaturation at 95°C for 10 min, 40 cycles of amplification (denaturation at 95°C for 30 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s), followed by a denaturation at 95°C for 60 s and a melting curve over the range from 55°C up to 95°C. Fluorescence data were collected at the end of the annealing stage of amplification. We performed Real-time PCR of PHD1, PHD2, PHD3, MIF-1 and 18S rRNA (18S rRNA Control kit, Yakima Yellow®-Eclipse® Dark Quencher, Eurogentec) in samples derived from rat left ventricles, skeletal muscle and liver. The following primers were used: PHD1 sense CGTGAGGCATGTTGACAATC, PHD1 antisense AACACCTTTCTGTCCCGATG; PHD2 sense TACAGGATAAACGGCCGAAC, PHD2 antisense GGCTTGAGTTCAACCCTCAC; PHD3 sense GGCCGCTGTATCACCTGTAT, PHD3 antisense TTCTGCCCTTTCTTCAGCAT; MIF sense CAGAACCGCAACTACAGCAA, MIF antisense GAACAGCGGTGCAGGTAAGT. Each assay was performed in duplicate and validation of PCR-runs was assessed by evaluation of the melting curve. All data of mRNA are given as relative units of 18S rRNA concentrations. Western blot Liver tissue was rapidly homogenized in a buffer containing 50 mmol/l Tris·HCl, 1% SDS, 1 mmol/l sodium-orthovanadate, 5 mmol/l EGTA, 1 mmol/l PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Proteins were quantified using the bicinchoninic acid protein assay (Pierce). Protein (50 μg) in 2× Laemmli SDS sample buffer were boiled for 5 min and after centrifugation loaded onto a SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 100 V for 90 min. The filters were blocked with 0.01% Tween, 2% nonfat milk, and then incubated with antibodies directed against MIF (Abcam) and GAPDH (Abcam). Statistical analysis For statistical comparison of age groups or the influence of caloric restriction, a non-paired t-test was used. A P-value < 0.05 was considered to represent a significant difference. Results Prevention of age-dependent increase of PHD3 in heart, liver and skeletal muscle Age-dependent changes of PHD1-3 expression were investigated in three different organs obtained from young (12 months old) and old (30 months old) rats. PHD1 and PHD2 mRNA expression did not differ significantly between the two age groups neither in heart, liver nor in skeletal muscle (Figs. 1 and 2). However, in all three different organs investigated, there was a significant increase in PHD3 mRNA expression in the tissues obtained from the old rats compared to the young animals (Fig. 3). Fig. 1Effect of age and caloric restriction on PHD1 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD1 mRNA quantitated by real time PCRFig. 2Effect of age and caloric restriction on PHD2 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD2 mRNA quantitated by real time PCRFig. 3Effect of age and caloric restriction on PHD3 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD3 mRNA quantitated by real time PCR Young and old rats were subsequently challenged by caloric restriction. Daily caloric intake was reduced to 40% in young and old animals for 6 months. Control animals received their prediet average caloric intake. Using this regimen, we previously have observed that thioredoxin reductase 2 is significantly reduced in ageing skeletal and cardiac muscle and renormalized after caloric restriction (Rohrbach et al. 2006a). Whereas no significant effect of caloric restriction was observed on the expression of PHD1 or PHD2 (Figs. 1 and 2), the reduced food intake significantly counteracted the age-dependent increase in PHD3 expression in heart, liver and skeletal muscle (Fig. 3). PHD3 levels in old rats after caloric restriction was comparable to the PHD3 expression levels in young animals. Caloric restriction affects the expression of the macrophage migration inhibitory factor Numerous HIF-target genes, including erythropoietin, vascular endothelial growth factor, carbonic anhydrase IX etc., have been identified (Wenger et al. 2005). Recently, the expression of the macrophage migration inhibitory factor (MIF) has been demonstrated to be inducible by hypoxia. In addition, functional HIF binding sites in the promoter have been identified. In a recent study evidence was provided that HIF-1 plays a critical role in delaying the onset of senescence in rodent cells via transcriptional activation of MIF and thereby inhibition of the p53-mediated pathway (Welford et al. 2006). To gain insight into the functional consequences of age and caloric restriction-mediated changes on the HIF-induced signal transduction pathways, we analyzed the mRNA expression of MIF as a function of age and caloric restriction. In line with the suggestion that HIF-dependent gene expression is hampered with increasing age, MIF RNA and protein expression was significantly lower in the heart, liver and skeletal muscle obtained from the old rats compared to the expression found in the young rats (Figs. 4, 5). Restriction of food intake reversed the age-dependent decline in MIF expression (Fig. 4). MIF levels after caloric restriction were comparable to the expression levels in young rats in all three tissues investigated. Fig. 4Effect of age and caloric restriction on MIF mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and MIF mRNA quantitated by real time PCRFig. 5Effect of age and caloric restriction on MIF protein levels. Liver tissue samples were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently protein was extracted and MIF and GAPDH protein levels were investigated by immunoblot analysis Discussion Caloric restriction has been described to extend life in a variety of different mammalian and non-mammalian species (Masoro 2000). In addition, restriction of food intake delays the progression of several age-associated diseases. Identification of age-dependently expressed genes is one of the most direct approaches in ageing research. In a previous study, we identified PHD3 as one age-dependently expressed gene in human and mouse heart (Rohrbach et al. 2005). The data presented here demonstrate that PHD3 expression is additionally increased in rat heart, liver and skeletal muscle with increasing age. These findings indicate that age-dependent changes in PHD3 expression are tissue- and species-independent. Furthermore our data presented here demonstrate that PHD3 expression is decreased by caloric restriction in old rats, whereas no change in the expression of PHD1 or PHD2 was observed. Previous findings indicate that each PHD-isoform displays its own tissue and cell-line-specific expression pattern (Appelhoff et al. 2004). In addition, the susceptibility of the PHD isoform expression by change in the oxygen availability differs greatly. Whereas PHD3 is highly inducible by hypoxia, there is just a slight hypoxia-mediated induction described in case of PHD2. In case of PHD1 no hypoxia-inducible expression has been observed (Appelhoff et al. 2004; Marxsen et al. 2004). Including the age-dependent and caloric restriction-induced changes in PHD3 expression, PHD3 seems to be the most flexible PHD isoform regarding stimuli-induced change in expression. In an unbiased screen using white adipose tissue it was found that energy restriction lowers the expression of genes linked to angiogenesis indicating that hypoxia-inducible gene expression is affected by caloric restriction (Higami et al. 2006). Similarly, the age-dependent and caloric restriction-induced expression of PHD3 described here seems to have consequences. Quantification of the HIF-target gene MIF demonstrates a decrease of MIF expression in heart, liver and skeletal muscle in the old animals, which was reversed by caloric restriction. In line with our observation, in previously published microarray experiments increased levels of MIF RNA were found in liver lysates from young adult mice treated with caloric restriction or with a methionine-deficient diet as well as in young mice of the long-lived Snell dwarf stock (Dozmorov et al. 2002; Miller et al. 2005; Miller et al. 2002). Since MIF affects cellular senescence (Welford et al. 2006), changes in PHD3 expression with increasing age seem to be one important feature of the molecular senescence phenotype. This conclusion is in line with a recent report demonstrating increased HIF-1α hydroxylation in aged versus young primary murine fibroblasts (Chang et al. 2007). Together with a previously published study demonstrating that under hypoxic conditions senescent cells failed to induce HIF-1α compared to presenescent cells our data indicate that the age-dependent changes in the oxygen sensing system may indeed have impact for the ageing process (Coppe et al. 2006). Up to now a reduced protein expression of HIF-1α with increasing age has been demonstrated in smooth muscle cells isolated from rabbit aorta, rat cerebral cortex, mouse lung, liver, kidney, heart and in the carotid body (Di Giulio et al. 2005; Frenkel-Denkberg et al. 1999; Rivard et al. 2000; Rohrbach et al. 2005). A decreased HIF-1α expression was also demonstrated in tissue lysates from ischemic limbs comparing 20 months old mice with 2 months old mice (Bosch-Marce et al. 2007). Hwang et al described a diminished HIF-1α stabilization in old rats in response to hypoxia, although in nomoxic breathing animals there was an age-related increase in basal levels of HIF binding to DNA. Finally, mouse and human fibroblasts isolated from different age groups express less HIF-1α as a matter age (Chang et al. 2007). However, it also should be noted that Kang et al demonstrated an increased HIF activity and HIF-1α expression in old rats and conflicting to the presented results a decrease in HIF activity as a result of caloric restriction in liver lysates (Kang et al. 2005). Comparing the chow protocols, these rats were fed a soybean protein diet, which may have resulted in the described increase in redox metabolites and HIF-1α protein expression. The degree and management of caloric restriction is not described (Hwang et al. 2007). Taken collectively, several papers indicate a diminished HIF-response with increasing age with some exceptions, which may be tissue or diet-specific. Whereas ageing thus is mostly associated with a decreased hypoxia-inducible gene expression, in endometrial cancer cells mutation of PHD2 has been associated with increased expression of HIF-1α and immortality (Kato et al. 2006). Downregulation of HIF-1α by overexpression of functional PHD2 altered the cancer cell phenotype and led to the occurrence of senescence markers (Kato et al. 2006). The impact of the HIF-system for cell fate decisions additionally was described by Welford et al demonstrating in a well defined nontransformed/nonimmortalized knock out cell model that HIF-1 delays the onset of senescence via transcriptional activation of MIF and inhibition of p53 dependent pathways (Welford et al. 2006). Alltogether these data demonstrate the necessity of tight regulation of the HIF system to prevent unrestricted cell proliferation or early cellular senescence.

Neurosci_Lett-1-5-1885960

p53 is upregulated in Alzheimer's disease and induces tau phosphorylation in HEK293a cells

p53 and tau are both associated with neurodegenerative disorders. Here, we show by Western blotting that p53 is upregulated approximately 2-fold in the superior temporal gyrus of Alzheimer's patients compared to healthy elderly control subjects. Moreover, p53 was found to induce phosphorylation of human 2N4R tau at the tau-1/AT8 epitope in HEK293a cells. Confocal microscopy revealed that tau and p53 were spatially separated intracellularly. Tau was found in the cytoskeletal compartment, whilst p53 was located in the nucleus, indicating that the effects of p53 on tau phosphorylation are indirect. Collectively, these findings have ramifications for neuronal death associated with Alzheimer's disease and other tauopathies. Tau is the major microtubule-associated protein (MAP) in neurons and functions in the formation and maintenance of axons by influencing microtubule organization. In adult human brain, there are six isoforms of tau generated by alternative mRNA-splicing. Tau has zero, one or two amino-terminal inserts and either three or four repeats of a microtubule-binding domain situated towards the carboxy-terminus [7]. Tau splicing and phosphorylation are developmentally regulated. Only the shortest tau isoform is expressed in foetal brain [8] and foetal tau is more extensively phosphorylated than tau from adult brain [10,15]. Phosphorylated tau is less efficient at promoting microtubule assembly [12,14] and elevated levels of phosphorylated tau correlate with increased microtubule dynamics associated with plasticity during development [1]. Increased tau phosphorylation is also a characteristic feature of Alzheimer's disease (AD) and tauopathies such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) [7]. In these disorders, normally soluble tau is present as paired-helical filaments (PHFs), which in turn aggregate to form neurofibrillary tangles (NFTs). p53 is a tumor suppressor protein, which induces cell cycle arrest or apoptosis. Normally, p53 is maintained at low levels by murine double minute-2 (MDM2) or the human homologue (HDM2), which inhibit the transcriptional activity of p53 and promote degradation of p53 via the proteasome [2]. Activation of p53 involves stabilization of the protein by post-translational modifications, which disrupts the interaction between p53 and MDM2. Several studies have reported an increase in p53 immunoreactivity in sporadic AD [11,13] especially in subpopulations of cortical neurons undergoing neurofibrillary degeneration [5]. Furthermore, p53−/− mice display a reduction in tau phosphorylation [6]. These findings prompted us to investigate the effects of p53 on tau phosphorylation in vitro. The following antibodies and plasmids were used in this study: mouse anti-β actin, which was from Sigma (UK). Rabbit anti-total tau from Dakocytomation (UK). Tau-1 monoclonal antibody, which was a gift from Professor L. Binder (Cognitive Neurology and Alzheimer's Disease Center, Northwestern University, USA). PHF-1 monoclonal antibody, which was a kind gift from Dr. P. Davies (Albert Einstein College of Medicine, NY, USA). AT270 and AT8 monoclonal antibodies, goat anti-mouse IgG and goat anti-rabbit IgG were from Santa Cruz Biotechnology (USA). Anti-p53 (clone DO7) was from Novocastra Laboratories (UK). Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG were from Invitrogen (UK). p53, which was a generous gift from Dr B. Vogelstein (John Hopkins, USA). 2N4R tau was a gift from Professor J. Woodgett (Ontario Cancer Institute, Toronto, Canada) BAX-Luc and p21waf-Luc were from Dr. T. Soussi (Universite P.M. Curie, Paris). Human embryonic kidney 293a cells (HEK293a) (Quantum Biotechnologies, Canada) were cultured in low glucose Dulbecco's modified essential medium (Invitrogen, UK) containing 10% fetal bovine serum (Autogen Bioclear, UK), 2 mM l-glutamine, 100 IU penicillin, and 100 mg/ml streptomycin. Post mortem brain tissue from the superior temporal gyrus was provided by the MRC London Brain Bank for Research on Neurodegenerative Diseases. Use of human tissue was approved by the South London and Maudsley Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki. Control subjects had no history or symptoms of neurological disorder. All AD cases were neuropathologically confirmed, using conventional histopathological techniques and diagnosis was performed using the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria. Total protein was extracted (50 μg) from 17 non-demented elderly controls and 53 AD cases and p53 levels were determined by Western blotting using a specific anti-p53 antibody (1:1000). p53 expression was normalised to β-tubulin by standard densitometric procedures. Values for control and AD were compared using an unpaired two-tailed t-test, *p < 0.05. For tau phosphorylation experiments HEK293a cells plated in a 6 well plate were transfected with OptiMEM (100 μl) containing FuGene 6 (5 μl) and cDNA constructs (500 ng of each) encoding human 2N4R tau independently or in combination with p53. Cells lysates were harvested 24 h after transfection. Western blotting was performed using the following primary antibodies; rabbit anti-total tau (1:10,000), mouse anti-phospho-Ser199/Ser202/Thr205 (tau-1, 1:1000), mouse-anti-phospho-Ser202/Thr205 (AT8, 1:500), mouse anti-phospho-Ser396/Ser404 (PHF-1) (1:1000) or mouse anti-phospho-Thr181 (AT270, 1:1000) according to standard protocols with HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Pharmacia, UK). To ensure equal loading membranes were reprobed with mouse anti-β actin. All experiments were performed in triplicate. For sub-cellular localisation experiments HEK293a cells were transfected with human 2N4R tau independently or in combination with p53 as described above. The following day the cells were fixed in ice-cold methanol and stained according to standard protocols. Cells were incubated with rabbit anti-total tau (1:500) or mouse anti-p53 (1:250) before being incubated with the appropriate fluorescent secondary antibody (1:200). Nuclei were counter-stained with Hoescht 33342. Immunofluorescence was captured using a Zeiss LSM510 meta-confocal microscope. All experiments were performed in triplicate; therefore, figures shown are representative of a single experiment. The transcriptional effects of p53 were examined by reporter gene assay. Four wells of HEK293a cells plated in a 48 well plate were transfected by adding 25 μl of a master-transfection mix to the culture medium. The master-mix contained 100 μl of OptiMEM (Invitrogen, UK), 4 μl FuGene 6 (Roche, UK), 400 ng of firefly either BAX-Luc or p21waf-Luc (luciferase-based reporter DNA), 50 ng phTK-Renilla luciferase (Promega, UK) to control for transfection efficiency and 800 ng of p53. Appropriate amounts of empty vector DNA were included where necessary to maintain constant DNA concentrations. Twenty-four hours post-transfection the firefly and Renilla luciferase activities were sequentially measured using Dual-Glo reagents (Promega, UK) in a Wallac Trilux 1450 Luminometer (Perkin-Elmer, UK). Firefly values were divided by the Renilla value from the same well to control for non-specific effects. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. Each assay was repeated three times. p53 levels were quantified by Western blotting in the superior temporal gyrus from a large cohort of patients comprising of 53 AD cases and 17 control subjects. In accordance with previous reports that had examined smaller patient numbers [5,11,13] we found that p53 immunoreactivity was indeed significantly elevated in tissue from AD patients compared to healthy elderly controls (Fig. 1A). Densitometry revealed an approximate 2-fold increase in p53 expression in AD (Fig. 1B). Next we explored the effects of p53 on tau phosphorylation. The 2N4R isoform of human tau was exogenously expressed in HEK293a cells (which do not contain endogenous tau) alone or in combination with p53. Co-expression of tau with p53 resulted in an increase in tau phosphorylation as demonstrated by a decrease in electrophoretic mobility using an anti-total tau antibody, which detects all tau isoforms independently of their phosphorylation state (Fig. 2A, panel a). Tau can be phosphorylated at a number of serine (Ser) and threonine (Thr) residues; therefore, we subsequently used a number of phosphorylation-specific anti-tau antibodies to examine the effects of p53 on tau further. There was an almost complete reduction of tau-1 immunoreactivity in HEK293a cells co-transfected with p53 and tau in comparison to cells expressing tau alone (Fig. 2A, panel b). Tau-1 recognises a number of amino acids including Ser199, Ser202 and Thr205 [4] when de-phosphorylated, therefore a decrease in immunoreactivity at this site reflects an increase in tau phosphorylation. Consistent with this, p53 induced an increase in tau phosphorylation as evidenced using the AT8 antibody, which recognizes tau phosphorylated at Ser202/Thr205 (Fig. 2A, panel c). In contrast, no changes in tau phosphorylation in the presence of p53 were observed using PHF-1 (Fig. 2A, panel d) or AT270 (Fig. 2A, panel e) monoclonal antibodies. Immunoblotting for β-actin illustrated that equal amounts of protein had been loaded across lanes (Fig. 2A, panel f). Confocal microscopy demonstrated that p53 and tau are compartmentally separated (Fig. 2B). p53 (red) exhibits a diffuse nuclear localisation, whilst tau (green) is present in the cytoskeletal compartment when expressed both independently and in combination. This suggests that the effects of p53 on tau phosphorylation are indirect and most likely attributable to the transcription of a p53 target gene. We verified the transcriptional properties of p53 in HEK293a cells using BAX-Luc and p21waf-Luc (Fig. 2C), which are luciferase reporter constructs derived from known p53 target genes. In summary, our findings demonstrate that p53 is upregulated in the superior temporal gyrus in AD and that p53 induces tau phosphorylation at the tau-1/AT8 epitope in HEK293a cells. We infer that the effects of p53 on tau phosphorylation are indirect as evidenced by the compartmental segregation of the two proteins. Therefore, pathological and sustained expression of p53 in adult brain might promote excessive and prolonged tau phosphorylation, which in turn might precipitate the formation of NFTs and neuronal death. Interestingly, we [9] have previously shown that TAp73 induces tau phosphorylation in HEK293a cells at the tau-1 and at the PHF-1 epitopes, which suggests that a similar mechanism of action might be shared by other p53 family members. Pertinent to our observations, it has recently been demonstrated that the expression of p53 is in part mediated by the transcriptionally active intracellular domain (ICD) of the β-amyloid precursor protein (APP), the generation of which is dependent upon the γ-secretase activities of presenilin-1 [3]. It is feasible then that in sporadic and familial AD, which both exhibit increased Aβ production, the concomitant increase in APP-ICD generation could lead to an increase in p53 expression and increased tau phosphorylation. Such a scheme forges a link between the two neuropathological hallmarks of AD, senile plaques and NFTs. In addition, it has also recently been demonstrated that Aβ itself, in particular the 42 amino acid form, binds the p53 promoter and enhances transcription [13]. Thus, p53 seems to play a pivotal role in AD, implying that modulation of cell death pathways might be of therapeutic benefit in AD and indeed in other age related neurological disorders.

Crit_Care-8-3-468887

Clinical review: Influence of vasoactive and other therapies on intestinal and hepatic circulations in patients with septic shock

The organs of the hepatosplanchnic system are considered to play a key role in the development of multiorgan failure during septic shock. Impaired oxygenation of the intestinal mucosa can lead to disruption of the intestinal barrier, which may promote a vicious cycle of inflammatory response, increased oxygen demand and inadequate oxygen supply. Standard septic shock therapy includes supportive treatment such as fluid resuscitation, administration of vasopressors (adrenergic and nonadrenergic drugs), and respiratory and renal support. These therapies may have beneficial or detrimental effects not only on systemic haemodynamics but also on splanchnic haemodynamics, at both the macrocirculatory and microcirculatory levels. This clinical review focuses on the splanchnic haemodynamic and metabolic effects of standard therapies used in patients with septic shock, as well as on the recently described nonconventional therapies such as vasopressin, prostacyclin and N-acetyl cysteine. Introduction Research interest has focused on the intestinal and hepatic circulations in various models of shock, and particularly in septic shock. The splanchnic area is reported to be the 'motor' of multiple organ failure [1] and the 'canary' of the body [2]. In fact, because of its peculiar vascular anatomy, the hepatosplanchnic area is jeopardized during septic shock, which may potentially lead to a vicious circle of inflammatory responses, culminating in multiple organ failure syndrome. The present clinical review briefly discusses the splanchnic vascular anatomy and focuses on the different therapeutic approaches that have been proposed to promote perfusion of the gastrointestinal tract during resuscitation of patients with septic shock. When possible and reasonable, we propose therapeutic recommendations. References were obtained from Medline database (from the earliest records to 2003). We used the following keywords: gastric mucosal pH or pHi, splanchnic, haemodynamics, microcirculation, sepsis, septic shock, vasoactive drugs, dobutamine, dopamine, norepinephrine, epinephrine, dopexamine vasopressin, terlipressin, prostacyclin, N-acetyl cysteine, dialysis and haemofiltration. We also reviewed the reference lists of all available review articles and primary studies to identify references not found in computerized searches. We placed emphasis on prospective, randomized, controlled clinical trials. Anatomy of hepatosplanchnic vascular bed The splanchnic vasculature includes both serial and parallel vascular beds (Fig. 1). The gut is perfused by the coeliac trunk and mesenteric arteries, and is drained via the portal system. The liver has a unique and special blood supply that includes both arterial (the common hepatic artery) and venous (the portal vein) inflow. The portal vein supplies 75–80% of the liver blood flow and the hepatic artery supplies 20–25%. Physiologically, there is an interdependent response with a compensatory blood flow between the portal vein and the hepatic artery called the hepatic arterial buffer response [3]. The hepatosplanchnic blood flow accounts for 25–30% of the cardiac output [4], and the regional oxygen extraction is slightly higher than the whole body oxygen extraction. During sepsis or septic shock, splanchnic oxygen extraction is increased compared with nonseptic patients (44% versus 30%), which leads to an increase in the hepatic venous/mixed venous haemoglobin oxygen saturation gradient [4]. In clinical practice it is generally not possible to determine portal venous flow in isolation, and measurements are taken from the hepatosplanchnic region as a whole. The flow is estimated at bedside by the method of primed, constant infusion of indocyanine green (ICG) with hepatic venous catheterization [5]. The intestinal villus is supplied by a single, unbranched arterial vessel that arborizes at the villus tip into a network of surface capillaries drained by a central villus vein. This anatomical arrangement allows countercurrent exchange and shunting of diffusible molecules such as oxygen, and hypoxia may occur at the tip of the villus even during moderate decreases in macrocirculatory flow [6]. In addition, intestinal villi perfusion is highly heterogeneous, as suggested by the wide range of intestinal surface oxygen saturation [7]. In patients with sepsis, splanchnic blood flow usually increases in proportion to cardiac output [8] and is associated with decreased hepatic vein oxygen haemoglobin saturation. Two different interpretations are possible: first, the increase in splanchnic blood flow is insufficient to meet the increased oxygen consumption; and second, hepatic arterial blood flow is reduced as a consequence of the hepatic arterial buffer response. The latter hypothesis is supported by the observations of De Backer and coworkers [9], who demonstrated that there is usually no net lactate production from the hepatosplanchnic area. In addition, the observation that splanchnic blood flow is increased does not rule out an impairment in microvascular blood flow [10-12] or the presence of cytopathic hypoxia [13]. In normal conditions the partial carbon dioxide tension (PCO2) gap, which is defined as the difference between mucosal PCO2 measured with a tonometer and arterial PCO2, is low. In case of inadequate mucosal blood flow, whether tissue hypoxia is present or not, the PCO2 gap increases. Levy and coworkers [14] recently reported that a PCO2 gap greater than 20 mmHg was associated with poor outcome in patients with septic shock. Unfortunately, there is no apparent correlation between PCO2 gap and global or regional haemodynamic measurements in septic patients [15] because the PCO2 gap mirrors both variations in microvascular flow [10] and in carbon dioxide metabolism [16]. For these reasons variations in PCO2 gap must be interpreted with caution. Therapeutic strategies Fluid challenge The mainstay of supportive treatment in patients with severe sepsis or septic shock is maintenance of adequate fluid balance, titration of appropriate oxygen delivery, and adequate perfusion pressure [17]. Hypovolaemia is a common clinical occurrence in intensive care medicine and results from several mechanisms such as fluid loss, haemorrhage, vasoplegia and capillary leak syndrome. This explains why fluid replacement therapy is a key component in the treatment of severe sepsis and septic shock. Although there is no consensus regarding the ideal type of fluid replacement, colloids are efficient in this indication [18]. There are few clinical studies focusing on the effects of colloids on splanchnic haemodynamics. In a randomized study conducted in patients with sepsis, Boldt and coworkers [19] assessed the effects on tonometric gastric mucosal acidosis of hydroxyethyl starch and albumin targeted to maintain pulmonary artery occlusion pressure between 12 and 18 mmHg. In hydroxyethyl starch treated patients cardiac index, oxygen delivery and consumption increased, and gastric intramucosal pH (pHi) remained stable whereas it decreased in albumin treated patients. In three other studies [20-22] conducted in patients with sepsis and septic shock, fluid challenges performed with hydroxyethyl starch neither altered the PCO2 gap nor influenced splanchnic haemodynamics. Moreover, a randomized comparison of hydroxyethyl starch and gelatin in haemodynamically stable septic patients revealed a beneficial effect of gelatin on the PCO2 gap [20]. These studies suggested no better effect of one colloid over the others on splanchnic haemodynamics, and the use of colloids must be weighed against their side effects [23]. Red blood cell transfusions are commonly used in intensive care units (ICUs) to enhance systemic oxygen delivery. However, proof of improved utilization of oxygen by peripheral tissues, especially in the splanchnic area, is lacking. Silverman and Tuma [24] reported the absence of improved gastric pHi with red blood cell transfusions in 21 septic patients. Moreover, there is an inverse association between the change in gastric pHi and the age of the transfused blood [25]. Finally, a recent report in 15 septic patients showed that red blood cell transfusion failed to improve oxygen utilization measured either using Fick's equation or by indirect calorimetry, and gastric pHi remained unaltered [26]. Adrenergic drugs The choice of vasoactive drugs in sepsis and septic shock is controversial. There is no evidence that any one vasoactive drug is more effective or safer than any other. Larger trials are needed to elucidate existing clinically significant differences in morbidity and mortality. A multicentre trial, which is currently ongoing, is comparing the effects of epinephrine with a combination of a fixed dose of dobutamine in addition to norepinephrine. Dopamine alone or versus norepinephrine (Table 1) The infusion of low-dose dopamine (defined as a dose lower than 5 μg/kg per min administered to normotensive patients) may not improve gut mucosal perfusion. In fact, Nevière and coworkers [27] showed that low-dose dopamine decreased gut mucosal blood flow in septic patients. Furthermore, other investigators [27-30] reported that either pHi or PCO2 gap were unchanged in patients with sepsis treated with low-dose dopamine. The effects on liver blood flow may also be variable; Maynard and coworkers [30] observed that dopamine did not affect ICG clearance and monoethylglycinexylidide (MEGX) formation from lidocaine. Interestingly, the effects of dopamine on splanchnic blood flow may differ according to basal splanchnic perfusion. Low-dose dopamine increased splanchnic blood flow that was low at baseline (seven patients) but not when splanchnic perfusion was preserved (four patients) [28]. The very small number of patients in each group limited these observations. Recently, Jakob and coworkers [31] reported that dopamine administration titrated to achieve a 25% increase in cardiac output induced a significant increase in splanchnic blood flow from 0.9 to 1.1 l/min per m2, which was associated with a significant reduction in splanchnic oxygen consumption. The results are even more controversial when dopamine is used at higher doses to restore blood pressure. Ruokonen and coworkers [32] observed that dopamine increased splanchnic blood flow and metabolism in some but not all patients with septic shock. In some patients, the same group of investigators [33] also observed an increase in hepatic vein oxygen saturation, suggesting an improvement in the balance between oxygen supply and demand during dopamine administration. However, in a pilot study, Marik and Mohedin [34] reported that dopamine administered at doses up to 25 μg/kg per min even decreased pHi. Given the very small number of patients included in these studies, no definite conclusions can be drawn regarding the effects of dopamine on splanchnic blood flow in septic patients. Comparison of the effects of norepinephrine and dopamine is difficult because norepinephrine is often combined with dobutamine, and study results are conflicting. Ruokonen and coworkers [32] reported unpredictable effects on splanchnic blood flow in patients with septic shock with norepinephrine, whereas dopamine induced a consistent increase in splanchnic blood flow. By contrast, in the randomized study reported by Marik and Mohedin [34], conducted in 20 septic patients with hyperdynamic septic shock, dopamine was reported to induce a decrease in pHi when compared with norepinephrine. More recently, De Backer and coworkers [35] reported the effects of dopamine, norepinephrine and epinephrine on the splanchnic circulation in moderate and in severe septic shock, and the main results are as follows. In moderate septic shock cardiac index was similar in dopamine-treated and norepinephrine-treated patients, and higher in epinephrine-treated patients, whereas splanchnic blood flow was the same with the three drugs. The gradient between mixed venous and hepatic venous oxygen saturation gradient was the lowest with dopamine, while PCO2 gaps were identical. In patients with moresevere septic shock cardiac index was greater and splanchnic blood flow lower with epinephrine than with dopamine and epinephrine; mixed venous and hepatic venous oxygen saturation gradient was greater with epinephrine, whereas PCO2 gap remained unaltered by any of the treatments. Given the available data (summarized in Table 1), no definite conclusions can be drawn regarding differences between dopamine and norepinephrine on splanchnic blood flow and metabolism in patients with septic shock. Dobutamine alone or combined with norepinephrine versus epinephrine (Tables 2 and 3) In patients with sepsis, a retrospective study conducted by Silverman and coworkers [24] identified a beneficial effect of dobutamine infusion on pHi [24]. Two years later Gutierrez and coworkers [36] reported an increase in pHi with dobutamine infusion in patients with sepsis syndrome who initially had low pHi. This beneficial effect, confirmed in other studies [37-39], was not related to an increase in splanchnic blood flow induced by dobutamine [39,40]. Creteur and colleagues [41] reported that dobutamine decreased the PCO2 gap in septic patients with a high gradient between the mixed venous and hepatic vein oxygen saturation (>20%), whereas PCO2 gap was not affected in patients when this gradient was less than 20%. This suggests that patients with the most severe alterations in hepatosplanchnic blood flow are also prone to decreased mucosal perfusion. Dobutamine usually, but not without exception, increases splanchnic perfusion [40-42]. The effects on splanchnic metabolism are more variable [39] and may depend on the adequacy of splanchnic perfusion at baseline. In patients with septic shock, De Backer and coworkers [43] reported that splanchnic oxygen consumption increased during dobutamine administration only in patients with an increased gradient between hepatic venous and mixed venous oxygen saturation. Combinations of dobutamine and other catecholamines have often been studied, in particular in association with norepinephrine for its effects on β-receptors, with the aim of modulating hepatosplanchnic haemodynamics. Indeed, in patients with sepsis, changing from norepinephrine (α-agonist and β-agonist) to phenylephrine (pure α-agonist), titrated to produce similar global haemodynamic measurements, led to a decrease in splanchnic blood flow, splanchnic oxygen delivery and gastric pHi. These changes were associated with decreased rates of liver lactate uptake and glucose production [44]. Whether dobutamine has a specific effect on the splanchnic circulation is still debated. In a cross-over study conducted in eight patients with septic shock, Meier-Hellmann and coworkers [45] showed that epinephrine caused lower splanchnic flow and oxygen uptake, lower gastric pHi, and higher hepatic vein lactate concentration than did the combination of dobutamine and norepinephrine. Duranteau and coworkers [11] compared the effects of epinephrine, norepinephrine and the combination of norepinephrine and dobutamine in patients with septic shock on gastric mucosal flow, as assessed using a laser Doppler technique. Epinephrine and dobutamine–norepinephrine led to a significant increase in gastric mucosal flow as compared with norepinephrine alone, but these findings were not corroborated by those reported by Seguin and coworkers [46]. Moreover, in patients with septic shock resistant to dopamine, the combination of norepinephrine and dobutamine, in comparison with epinephrine alone, restored gastric pHi more quickly and limited the increase in arterial lactate concentration. However, there was no difference in gastric mucosal PCO2 gradients between groups at 24 hours of treatment [38]. The preferential effect of dobutamine on splanchnic blood flow was not confirmed by Reinelt and coworkers [42], who studied the effects of dobutamine on fractional splanchnic flow and hepatic glucose production in septic patients resuscitated adequately with fluid and norepinephrine. Their results showed a parallel increase in splanchnic blood flow and cardiac index, unaltered splanchnic oxygen consumption and decreased rate of endogenous production of hepatic glucose. These findings suggest that splanchnic blood flow is increased in well resuscitated septic patients, and that a dobutamine test is able to reveal a oxygen delivery/consumption dependency [41,43] but it cannot exclude intraorgan blood flow redistribution at the microcirculatory level. The inadequacy of blood flow distribution is mirrored by the absence of correlation between splanchnic blood flow and the PCO2 gap. Reported data on the effects of dobutamine and norepinephrine on splanchnic haemodynamics are summarized in Tables 2 and 3, respectively. Recommendations regarding use of adrenergic drugs We suggest that both dopamine and norepinephrine can be given to septic shock patients as first-line catecholamine drugs but that their use must be weighed against the undesired neuroendocrine side effects of dopamine [45]. Epinephrine should be reserved for use as rescue therapy. If norepinephrine is chosen as the first agent, then the addition of dobutamine may be considered. Dopexamine Dopexamine hydrochloride is a dopamine analogue with vasodilating effects that may be useful in improving splanchnic microcirculation in septic shock. Twenty-five ventilated patients with systemic inflammatory response syndrome were randomly assigned to receive either a 2-hour infusion of dopexamine (1 mg/kg per min) or of dopamine (2.5 μg/kg per min) after baseline measurements of gastric pHi, MEGX formation from lidocaine and ICG disappearance rate. Dopexamine had no effects on systemic measurements but it significantly increased pHi and ICG plasma disappearance, suggesting a selective increase in splanchnic blood flow and improved hepatic function, as indicated by increased MEGX concentration [30]. A previous study from the same group showed that dopexamine at higher doses (4–6 μg/kg per min) raised gastric pHi together with a nonsignificant increase in ICG clearance [47]. Temmesfeld-Wollbrück and coworkers [7] employed reflectance spectrophotometry for direct assessment of the microvascular haemoglobin saturation and haemoglobin concentration in the gastric mucosa in patients with septic shock. Compared with healthy control individuals, patients with septic shock exhibited a reduced microvascular haemoglobin saturation with a wide distribution and with tailing of the histogram to severely hypoxic values in spite of high whole body oxygen delivery. This microvascular disturbance was associated with reduced microvascular haemoglobin concentration and a lower gastric pHi. Short-term infusion of 2 μg/kg per min dopexamine in 10 patients with septic shock increased both microvascular haemoglobin saturation and concentration, whereas whole body oxygen uptake and gastric pHi remained unaltered. Other investigators did not confirm these beneficial effects. Hannemann and coworkers [48] reported the effect of incremental doses (0.5–4 μg/kg per min) dopexamine on splanchnic circulation in 12 patients with severe sepsis haemodynamically controlled with fluid challenge and dobutamine. Splanchnic blood flow increased proportionally to cardiac output but dopexamine lowered gastric pHi in a dose-dependent manner in all patients [49]. Finally, in 12 septic shock patients haemodynamically controlled with norepinephrine, dopexamine titrated to increase cardiac output by 25% [50] increased median splanchnic blood flow whereas the fractional splanchnic blood flow was significantly reduced, and none of global or regional oxygen exchange or PCO2 was altered. In addition, those investigators found no influence of dopexamine on metabolic parameters either [51]. Given these discrepancies, it is reasonable to recommend further investigations into dopexamine before it may be routinely used in septic shock. Other vasoactive drugs Vasopressin and terlipressin Physiologically, vasopressin (a nonapeptide that is released from the neurohypophysis) plays a minor role in blood pressure regulation. Clinical data revealed that the initially very high plasma concentrations of vasopressin decrease during prolonged sepsis [52]. In the past few years clinical studies showed that blood pressure can be rapidly restored in septic shock using vasopressin, but this is mainly at the expense of cardiac output [53]. Nevertheless, in 2000 the American Heart Association and International Liaison Committee on Resuscitation recommended (grade IIB) continuous vasopressin infusion in refractory septic shock [54]. However, the effects of vasopressin on regional (i.e. splanchnic) blood flow are discussed controversially. In 1997, Landry and coworkers [52] reported on the continuous infusion of vasopressin (1.8–3.0 IU/hours) in five patients with septic shock. In all patients, blood pressure was rapidly restored and urine output increased in three. Patel and coworkers [55] randomly assigned 24 patients with septic shock to a double-blind 4-hour infusion of norepinephrine or vasopressin, and open-label vasopressors were titrated to maintain blood pressure. Although norepinephrine dosage could be significantly lowered in the vasopressin group, blood pressure and cardiac index were maintained in both groups. Urine output did not change in the norepinephrine group but increased substantially in the vasopressin group. Similarly, creatinine clearance did not change in the norepinephrine group but increased by 75% in the vasopressin group. Finally, gastric mucosal PCO2 gradient did not change significantly in either group. Recent results from Klinzing and coworkers [56], however, indicate that vasopressin may lead to a different blood flow distribution pattern in the splanchnic area as compared with norepinephrine. In 12 patients with septic shock, vasopressin was administered at a dose of 0.06–1.8 IU/min to replace norepinephrine completely. As a result, cardiac index and systemic oxygen uptake decreased significantly. Total splanchnic blood flow tended to decrease, while splanchnic blood flow expressed as percentage of cardiac output as well as the PCO2 gap were doubled [56]. By contrast, the increase in gastric PCO2 gap suggests that blood flow may have been redistributed away from the mucosa, and therefore it does not appear beneficial to directly replace norepinephrine with vasopressin in septic shock. Clinical data also suggest that low-dose vasopressin (0.04 IU/min) to compensate for endogenous deficiency could be a beneficial strategy [57-60], as was recently demonstrated by Dünser and coworkers [61], who randomly assigned 48 patients with catecholamine-resistant vasodilatory shock to receive a combined infusion of vasopressin and norepinephrine or norepinephrine alone. Vasopressin-treated patients had significantly lower heart rate, norepinephrine requirement and incidence of new onset tachyarrhythmias. Mean arterial pressure, cardiac index and stroke volume were significantly greater, and the PCO2 gap was significantly lower in patients treated with this combination. However, these patients also presented with a significant increase in plasma bilirubin concentration, suggesting an impaired liver blood flow and/or a depressed hepatic function mediated by vasopressin. More recently, terlipressin (glycinpressin), a long-acting vasopressin analogue, was proposed as a treatment for septic shock. O'Brien and coworkers [62] reported their clinical experience with terlipressin (1–2 mg) as rescue treatment in eight patients with refractory septic shock. Those investigators reported a rapid and 24 hour lasting stabilization in blood pressure, with a significant reduction in norepinephrine but a significant decrease in cardiac index. In that study, seven patients required renal replacement therapy and four patients died during their stay in the ICU. However, optimism regarding these findings must be tempered somewhat [63], in particular because detrimental effects on splanchnic blood flow have been described. Auzinger and coworkers [64] studied seven patients with catecholamine-refractory septic shock and subsequent infusion of terlipressin using gastric tonometry. During the 24-hour intervention period, terlipressin was administered as an intermittent bolus (1–3 mg). Although no changes occurred in lactate levels, the PCO2 gap progressively increased over 72 hours. Both vasopressin and terlipressin are potent vasoconstrictors and both are able to restore blood pressure in vasodilatory or septic shock. However, the effects on splanchnic blood flow are not yet fully elucidated. Clearly, adequacy of volume resuscitation is a major prerequisite for maintenance of microcirculatory blood flow. The currently available data suggest that both substances administered to compensate for endogenous vasopressin deficiency may be beneficial. Although the armamentarium for treatment of septic shock is enriched by such substances, it remains unclear whether administration during septic shock decreases morbidity or improves survival, and further research is warranted. Enoximone Modulation of the cytokine response by catecholamines might be a mechanism by which decreased morbidity and mortality are achieved with supranormal oxygen delivery in high-risk surgical patients [65]. Phosphodiesterase III inhibitors have positive inotropic, vasodilating and anti-inflamatory properties, and they may avoid the development of tolerance to catecholamines as a result of β-receptor desensitization. In a prospective, double-blind study [66], 44 patients with septic shock and conventional resuscitation were randomly assigned to receive dobutamine or enoximone to maximize left ventricular stroke work index. At 12 and 48 hours after baseline measurements, liver blood flow was assessed with hepatic venous catheterization, liver function was derived from appearance in plasma of MEGX, and release of tumour necrosis factor-α was determined to assess the severity of ischaemia/reperfusion injuries. There was a similar increase in cardiac index, systemic oxygen delivery and consumption, and liver blood flow in the two groups. Fractional splanchnic blood flow decreased slightly but significantly in dobutamine-treated patients, whereas it remained unchanged in enoximone-treated patients. In the latter group liver oxygen consumption and MEGX kinetics were significantly higher at 12 hours but not at 48 hours. The release of hepatic tumour necrosis factor-α after 12 hours of dobutamine treatment was twice as high (P < 0.05) as during enoximone treatment, suggesting a faster anti-inflammatory effect of enoximone. These interesting findings on hepatosplanchnic effects of phosphodiesterase III inhibitors were not confirmed by other studies, and further investigations are needed if these agents are to be recommended for routine clinical use. Prostacyclin Prostacyclin or its stable analogue iloprost are vasodilator substances with platelet aggregation inhibiting and cytoprotective properties. Administration of prostacyclin by the intravenous route was shown to increase oxygen delivery and consumption in septic patients [67] and to improve gastric pHi [68], as did aerosolized prostacyclin in patients with septic shock and pulmonary hypertension treated with epinephrine or norepinephrine [69]. Finally, Lehmann and coworkers [70] reported restored plasma ICG clearance without harmful effect on systemic haemodynamics in patients with septic shock treated with iloprost. More recently Kiefer and colleagues [71] reported the hepatosplanchnic effects of iloprost in 11 patients with septic shock requiring norepinephrine. Iloprost was incrementally infused to increase cardiac index by 15%, which significantly increased splanchnic blood flow in parallel, without a major fall in mean arterial pressure. Iloprost induced a decrease in endogenous glucose production rate without change in the hepatic clearance of the glucose precursors alanine, pyruvate and lactate. Similarly, the PCO2 gap was not altered. The authors avoided mean arterial pressure drop by careful exclusion of hypovolaemia before inclusion, but still the increment in iloprost doses was limited by the decrease in arterial partial oxygen tension, which raises many questions in patients with acute respiratory distress syndrome. These interesting findings on hepatosplanchnic effects of such vasodilators need further investigation before these agents may be recommended for routine clinical use [72]. Nitroglycerin Opening the microcirculation using a vasodilator is an alternative approach for treatment of the jeopardized microcirculation in patients with sepsis or septic shock. Data reported by Sprock and coworkers [73] suggest that the use of intravenous nitroglycerin results in improved sublingual microvascular flow, as assessed by orthogonal polarization spectral imaging. However, one cannot assume that the sublingual microcirculation necessarily behaves like the whole splanchnic microcirculation does. N-acetyl cysteine N-acetyl cysteine (NAC) administration was associated with a decrease in gastric pHi in septic patients [74,75] and prevented the decrease in pHi in septic patients under hyperoxic stress [76]. In a randomized, double-blind study conducted in septic shock patients, NAC given within the first 24 hours after admission to the ICU was shown to improve cardiac index and splanchnic blood flow and MEGX concentration, and to decrease gastric mucosal PCO2 gap, whereas it did not influence fractional splanchnic blood flow [75]. Nevertheless, these positive effects of NAC on the splanchnic circulation must be balanced against several negative studies. Indeed, NAC was reported to depress cardiac performance in septic patients [77], and it even worsened mortality rate when it was given more than 24 hours after hospital admission [78]. Is NAC a 'double edged sword'? This question should be answered before its use in daily practice can be recommended. Extracorporeal renal support Publications related to this topic are scarce. In 11 critically ill patients mechanically ventilated and treated with inotropic support, intermittent dialysis increased the PCO2 gap [79]. In contrast, in two recent studies conducted in patients with acute renal failure [80] and septic shock [81], the PCO2 gap remained unaltered whereas cardiac index and stroke volume, as well as splanchnic blood flow, transiently decreased [80]. Although improved cardiovascular stability during continuous veno-venous haemofiltration in comparison with intermittent dialysis has been demonstrated in retrospective studies [82], the superiority of continuous haemofiltration over hemodialysis on splanchnic circulation has not been proven [81]. Conclusion In this review we summarize different, and potentially opposing, approaches to management of splanchnic circulation in patients with septic shock. However, in these studies the measurements were focused on the effect of the drug on splanchnic blood flow or a surrogate such as the PCO2 gap, but none of these studies reported convincing results with respect to mortality and/or morbidity. Competing interests None declared. Abbreviations ICG = indocyanine green; ICU = intensive care unit; MEGX = monoethylglycinexylidide; NAC = N-acetyl cysteine; PCO2 = partial carbon dioxide tension; pHi = intramucosal pH.

Ann_Hematol-3-1-2040174

Rituximab therapy for chonic and refractory immune thrombocytopenic purpura: a long-term follow-up analysis

The aim of this study was to evaluate the long-term response to rituximab in patients with chronic and refractory immune thrombocytopenic purpura (ITP). Adults with ITP fail to respond to conventional therapies in almost 30% of cases, developing a refractory disease. Rituximab has been successfully used in these patients. We used rituximab at 375 mg/m2, IV, weekly for a total of four doses in 18 adult patients. Complete remission (CR) was considered if the platelet count was >100 × 109/l, partial remission (PR) if platelets were >50 × 109/l, minimal response (MR) if the platelet count was >30 × 109/l and <50 × 109/l, and no response if platelet count remained unchanged. Response was classified as sustained (SR) when it was stable for a minimum of 6 months. Median age was 43.5 years (range, 17 to 70). Median platelet count at baseline was 12.5 × 109/l (range, 3.0 to 26.3). CR was achieved in five patients (28%), PR in five (28%), MR in four (22%), and two patients were classified as therapeutic failures (11%). Two additional patients were lost to follow-up. The median time between rituximab therapy and response was 14 weeks (range, 4 to 32). SR was achieved in 12 patients (67%). There were no severe adverse events during rituximab therapy. During follow-up (median, 26 months; range, 12 to 59), no other immunosuppressive drugs were used. In conclusion, rituximab therapy is effective and safe in adult patients with chronic and refractory ITP. Overall response rate achieved is high, long term, and with no risk of adverse events. Introduction In immune thrombocytopenic purpura (ITP), platelets are coated with an IgG autoantibody that prompts its premature destruction and, as a result, different grades of peripheral thrombocytopenia and clinical bleeding become evident [5, 23]. Steroids are the first-line treatment for acute ITP. In case of failure to steroids, splenectomy induces a 70 to 80% response rate [10]. However, almost 30% of adults with ITP fail to respond to conventional therapies [steroids, IV immunoglobulin (IVIg), splenectomy, or immunosuppressive drugs] and, eventually, they develop a chronic refractory disease [5, 12, 23]. Refractory patients are defined as those who failed standard-dose steroids and splenectomy, requiring further treatment due to unsafe platelet counts (<30 × 109/l) or clinical bleeding. These patients are unlikely to be cured, although spontaneous remissions sometimes occur [17]. If a patient becomes refractory, some alternatives are available such as danazol, dapsone, azathioprine, high-dose steroids, or chemotherapy combinations like CHOP regimen. However, these options are not quite useful because if response is achieved it is but for a brief period and sustained responses are scarce [23]. Hematopoietic stem cell transplantation has been occasionally used in some cases, but this option represents a high-risk procedure [14]. At the end, these patients may respond poorly to subsequent therapy, may have significant morbidity from the disease and its treatment (bleeding and infections secondary to immunosuppressive therapy), and the mortality rate at 10 years may rise up to 20% [5, 11]. On the other hand, these patients have higher hemorrhagic risk imposed by trauma or surgery. Therefore, new treatment strategies for these patients are needed. Rituximab is an anti-CD20 chimeric murine-human monoclonal antibody. CD20 is a B-lymphocyte membrane protein absent on other normal cells. This monoclonal IgG-kappa antibody is used in non-Hodgkin lymphomas because it induces apoptosis or direct lysis of B cells [19]. In ITP, B cells are responsible of autoantibody production and the subsequent platelet opsonization that allows its destruction. Therefore, if B cell clones are eliminated, thrombocytopenia could be reverted. There are some reports informing that rituximab is useful in refractory ITP showing a high overall response rate, with complete remission (CR) and partial remission (PR) rates ranging from 48 to 54% [4, 8, 9, 18, 20–22]. We report herein our long-term experience with rituximab therapy in chronic and refractory ITP. Materials and methods In a 5-year period, we prospectively administered rituximab to all patients with chronic and refractory ITP who were unresponsive to prednisone, azathioprine, vincristine, danazol, and high-dose dexametasone, among other therapies, including those unsuccessfully splenectomized. Patients must have a platelet count of <30 × 109/l to be considered for this therapy. Rituximab was given in doses of 375 mg/m2, diluted in 500 ml 5% dextrose–water solution in a 4-h continuous IV infusion. This regimen was indicated weekly for four doses after prophylactic application of antipyretic and antihistamine premedication administered 30 min before rituximab infusion (paracetamol, 500 mg, PO; diphenhydramine, 30 mg, PO; and hydrocortisone, 100 mg, IV). Outpatient follow-up was scheduled monthly for 6 months. Long-term follow-up was performed every 2 months. Response was classified as complete (CR) if the platelet count was >100 × 109/l, partial (PR) if platelet count >50 × 109/l, minimal response (MR) if platelet count was <50 × 109/l and >30 × 109/l, and no response (NR) if there was no change from the baseline platelet count. Response was sustained (SR) when it was maintained for a minimum of 6 months. Numerical variables were expressed using the median and range number. The probability to achieve more than 50 × 109/l and 100 × 109/l platelets and the median time from response to end of follow-up was calculated using the Kaplan and Meier method. Before starting treatment, all patients signed an informed consent authorization. The procedures done in this protocol were in accordance with the Helsinki Declaration of 1975. The protocol was approved by the Ethics Committee of the participating hospitals. Results Eighteen patients were candidates for this therapy, and all of them were suitable for evaluation because they received the planned four doses of rituximab. Patient characteristics and response to rituximab are shown in Table 1. Median age for the whole group was 43.5 years (range, 17 to 70). Median platelet count at baseline was 12.5 × 109/l (range, 3.0 to 26.3). Median follow-up was 26 months (range, 12 to 59). Mean number of treatment regimens received before rituximab was 5.5 (range, 3 to 8). Fifteen patients (83%) had failed to splenectomy. Table 1Patient characteristics and outcomesPatient/age/genderEvolutiona (months)Previous treatmentsPC at baseline (×109/l)PC at 12 months (×109/l)Time to response (weeks)Response typeSR1/42/F37P, S, Az, D, IFN3.014624CRYes2/43/F84P, S, Az, D, IFN, V5.012324CRYes3/30/F48P, S, Az, D, IFN, V12.534. 516MRYes4b/27/F38P, S, Az, D, IFN24.47816PRbYesb5/70/M60P, Az, D, V7.8NRNRNRNo6/43/F96P, S, Az, D, IFN, V10.137.78MRYes7/53/F96P, S, Az, D, IFN, V12.55412PRYes8/36/F264P, S, Az, D, IFN, V, OE, IVIg5.030.64MRYes9/17/F38P, S, Az, D, IFN, V, OE6.51294CRYes10/57/F26P, Az, D8.3648PRYes11/36/F25P, S, Az, D, IFN, V7.04920MRNo12/63/F120P, Az, D, V26.3NRNRNRNo13/53/F348P, De, D, S, Az22.01128CRYes14/22/F266P, S, D, IFN, IVIg12.66712PRNo15/56/F137P, D, S, Az18.42234CRYes16/35/F38P, D, Az, S23.211.40NRNo17/52/F41P, D, S16.551.94PRYes18/24/F60O, D, S24.0NRNRNRNoPC Platelet count, P prednisone, S splenectomy, Az azathioprine, D danazol, IFN interferon alpha 2b, V vincristine, De dexamethasone, OE opsonized erythrocytesaTime from ITP diagnosis to rituximab therapybA second course of rituximab was given. CR was achieved in five patients (28%), PR was obtained in another five (28%), MR was seen in four (22%) patients, and two patients were considered treatment failures (11%). Two patients were lost to follow-up (11%). Ten patients (55%) achieved >50 × 109/l platelets. Median time from the first rituximab dose to achievement of any response was 14 weeks (range, 4 to 32). Twelve patients (67%) showed SR beyond the sixth month of follow-up. As expected from the results depicted in Table 1, we were unable to find any correlation between the ITP duration and the type of response to rituximab. Figure 1 shows the median platelet counts during follow-up in three different groups of patients classified according to their pattern of response (CR, PR, and MR). The median time to achieve >50 × 109/l platelets was 5 months (95%CI = 0.5 to 11.6 months), and the median time to achieve >100 × 109/l platelets was not reached (Fig. 2). One patient relapsed 22 months after the first course of rituximab. She received steroids for another 14 months; she did not obtain response, and a second course of rituximab was given using the regimen described previously. She achieved a new PR 6 months after stopping therapy. After 22 months of follow-up, PR still remains in this patient. Fig. 1Platelet count achieved after first dose of rituximab (time 0 first dose of rituximab). Diamonds, CR; ovals, PR; triangles, MRFig. 2Time required to obtain platelet counts >50 × 109/l (a) or >100 × 109/l (b) after first dose of rituximab in adult patients with chronic and refractory ITP Median time of response duration was 54 months (95%CI = 15–93 months) for patients with CR, 18 months (95%CI = 8–28 months) for patients with partial response, and 12 months (95%CI = 7–17 months) for those individuals with minimal response. Difference was statistically significant between patients in CR vs those achieving PR or MR (p < 0.05). No difference was observed between PR and MR (Fig. 3). Fig. 3Median duration of response for patients who achieved complete remission (CR; 54 months, 95%CI = 15–93 months), partial remission (PR; 18 months, 95%CI = 8–28 months), or minor response (MR; 12 months, 95%CI = 7–17 months) Side effects related to the first dose of rituximab such as fever, chills, and respiratory symptoms were common (8 out of 18 patients, 43%). There were no severe adverse events during drug administration. Neither hemorrhagic events nor infections were recorded. Neutropenia, an occasional long-term side effect of rituximab therapy, was never recorded in our group of patients (Fig. 4). In fact, the lower neutrophil count recorded during the study was 1.3 × 109/l in patient 7 after 6 weeks of treatment with rituximab. No patient needed additional immunosuppressive therapy other than rituximab. During the whole follow-up period, no other illnesses have been recorded in this group of patients. Fig. 4Mean (squares), maximum (diamonds), and minimum (triangles) neutrophil counts through the follow-up of patients receiving rituximab for chronic and refractory ITP Discussion Because almost 30% of individuals with ITP do not respond to the first- and second-line therapies, they live with low platelet counts, a situation that carries a high risk of hemorrhage and, eventually, a short life expectancy. It has been largely demonstrated that ITP with persistent low platelet counts carries a grave prognosis [6, 13]. Efforts to increase the platelet count to at least 30 × 109/l include IVIg, anti-D immunoglobulin, cyclophosphamide, polychemotherapy such as the CHOP regimen, thrombopoietin, interleukin-11, dapsone, alpha interferon, plasma exchange, and bone marrow transplantation [23]. Using these therapies, the response rate is low and the patient is exposed to unnecessary risks [10]. When the first ITP patient successfully treated with rituximab was informed, it drew the attention of physicians because this regimen offered high response rate and low toxicity. In subsequent reports, researchers have found similar results. The aim of this study was to evaluate the response rate achieved in 18 patients with chronic and refractory ITP after rituximab treatment as well as the evolution of these patients to learn about the possible long-term side effects associated with the use of this drug, information that is almost inexistent in the literature. We observed 12 favorable responses (67%) and at least a stable clinical evolution in two additional patients (11%), for a global response rate of 78%. Based on the characteristics of the evolution of our group of patients, we may emphasize some points of interest: (1) Response rate obtained was quite satisfactory considering the history of chronicity and refractoriness of the patients; (2) the monoclonal antibody was well tolerated and caused only mild allergic reactions that could be easily managed with antihistaminics and paracetamol; (3) most of the patients achieve SR; and (4) increase of platelet count was not always immediate (median time to response = 14 weeks), a fact that slightly disagrees with previous reports that found shorter time to response (3 to 8 weeks; Table 2) [4, 8, 20]. In fact, in a recent systematic review about the efficacy of rituximab in adult patients with ITP, it was found that median time to response was 5.5 weeks (ranges 2 to 18 weeks) [2]. We do not have an explanation for this discrepancy. After a careful analysis, we did not find significant differences between the results obtained in the systematic review and our results in terms of the variables that may affect the time to response to rituximab, namely, age, sex, previous use of corticosteroids, number of treatments before rituximab, rituximab dose and schedule, period between diagnosis and rituximab administration, duration of ITP before rituximab, and pretreatment platelet counts. Although splenectomy may not be a significant predictor of response to rituximab as previously suggested [2, 4, 8], it should be noted that 83% of the patients included in our report had had splenectomy as compared with 50.5% of the patients in the systematic review. Therefore, history of splenectomy seems to be the only factor related to a different time to response to rituximab in our series. Table 2Patterns of response in patients with chronic and refractory ITP (from [1, 6, 18])ReferenceNumber of patientsOR (%)CR (%)PR (%)Median time to response (weeks)Mayo clinic1250428?USA–Italy575331228Denmark353318152–8Peru22684027?Mexico1856282814OR Overall response, CR complete response, PR partial response Of course, we have no data about all possible variables influencing the pattern of response to rituximab. For example, we do not show information about B-cell counts and platelet autoantibodies before and after rituximab therapy, two variables that may influence the response as observed in other trials. The expected therapeutic effect of rituximab is a reduction in specific platelet-associated autoantibodies and the consequent increase in platelet counts. Although rituximab has not been always associated with a reduced load of platelet autoantibodies and most of the publications reporting this effect are small series of cases [1], the high overall response rate obtained in our study allows us to believe that rituximab effectively decreased the B-cell counts as well as the levels of platelet autoantibodies. Three patterns of response to rituximab have been proposed: early (before the fourth dose of rituximab), intermediate (7 to 11 weeks after rituximab), and delayed (>13 weeks after rituximab) [8]. In our study, the probability to achieve a platelet count >50 × 109/l occurred at a median of 5 months, so we speculate that the complete inhibition of antibody formation and restoration of platelet counts with rituximab may occur after at least 5 months (95%CI = 0.5 to 11.6 months) from the first dose of the antibody (Fig. 2). However, some patients may achieve a quite delayed response (as long as 1 year after therapy), a situation in which it is important to wait a reasonable time period before another treatment is planned. A previous report informed the results obtained in patients treated with the same regimen as we used [4]. After a median follow-up of 47 weeks, the authors observed a lower overall response rate of 44% (CR = 18%, PR = 15%, MR = 10%); most of the responses were sustained. They observed two response patterns: (1) an early-response group in which responses appeared within the first 2 weeks after the first dose of rituximab; (2) a late-response group characterized by an increase in platelets several weeks after rituximab. Finally, after a median follow-up of 72.5 weeks, the response rate was 54% with a majority of SR [8]. In a prospective trial performed in pediatric patients with chronic ITP, treatment with rituximab produced an increase of >50 × 109/l platelets in 11 of 36 children (31%) [3]. Median time to response was 1 week (range, 1 to 7 weeks); however, a 6% incidence of serum sickness was observed. More recently, one of the largest experiences in ITP patients treated with rituximab was published [15]. An increase of >50 × 109/l platelets was observed in 55% of the patients (CR = 46%, SR = 35%). The only predictive factor for SR was to achieve CR. Patients who were treated more intensively (more than three different treatments) and those with a longer ITP duration (>10 years from diagnosis) had the worse response. In this study, non-splenectomized patients had a higher early response rate than those splenectomized. Although some of these results are similar to those reported in our study, we believe that contrasting results seen in our study are partially explained by differences in the inclusion and response criteria used, in the evolution time of the disease, and in the number and type of treatments given before rituximab. As early relapses may be seen in ITP patients treated with rituximab and because re-treatment with this monoclonal antibody offers good results [16], this drug has been used as a long-term maintenance treatment. Based on long-lasting remissions achieved using one or two doses of rituximab [7] and the results of late responders, we feel that a single dose of 375 mg/m2 every 6 months could be an alternative for this purpose. On the other hand, because of the mechanism of action of rituximab and its interference with the immune system, we searched for possible long-term complications associated with its use, namely, neoplasias, chronic infections, or autoimmune diseases. Immune status is a major concern in patients treated with rituximab being the induction of low CD20+ counts and hypogammaglobulinemia (specifically low levels of immunoglobulin M), two previously reported secondary effects. Although we do not have data about these two immunological variables, the lack of severe infectious diseases during the long-term follow-up of our patients allows us to speculate that no severe immune abnormalities were developed in our series. As we previously stated, during the follow-up period, no associated illnesses or pathological phenomena were observed. To our knowledge, this seems to be the first report about the lack of long-term complications associated with rituximab. In conclusion, rituximab is a long-term, safe, and effective alternative treatment of chronic and refractory ITP patients. Prospective randomized clinical trials are needed to elucidate the efficacy of the drug in comparison to splenectomy in early stages of the disease

Exp_Brain_Res-3-1-1914230

Joint-action coordination in transferring objects

Here we report a study of joint-action coordination in transferring objects. Fourteen dyads were asked to repeatedly reposition a cylinder in a shared workspace without using dialogue. Variations in task constraints concerned the size of the two target regions in which the cylinder had to be (re)positioned and the size and weight of the transferred cylinder. Movements of the wrist, index finger and thumb of both actors were recorded by means of a 3D motion-tracking system. Data analyses focused on the interpersonal transfer of lifting-height and movement-speed variations. Whereas the analyses of variance did not reveal any interpersonal transfer effects targeted data comparisons demonstrated that the actor who fetched the cylinder from where the other actor had put it was systematically less surprised by cylinder-weight changes than the actor who was first confronted with such changes. In addition, a moderate, accuracy-constraint independent adaptation to each other’s movement speed was found. The current findings suggest that motor resonance plays only a moderate role in collaborative motor control and confirm the independency between sensorimotor and cognitive processing of action-related information. Introduction Given the strong inclination that people have towards behavioral mimicry when they are jointly involved in walking or speaking (Chartrand and Bargh 1999; Lakin et al. 2003), can we also demonstrate co-actors adopting each other’s movement patterns in the simple task of transferring objects? If an object relevant for my own actions is placed in front of me at a high speed, shall I then automatically pick it up and transfer it also with a high speed or will I stick to my own, preferred speed? Furthermore, will an observer be sensitive to the height of the movement trajectory with which an object is placed within reach as a potential cue of that object’s weight? Detailed questions surrounding the potential transfer of movement kinematics in a shared action sequence as specified above have not yet been systematically investigated (cf. Sebanz et al. 2006). The little work that has been done in this area was limited to unintentional synchronization of arm movements in pendulum swinging (Schmidt and O’Brien 1997), incidental entrainment of leg movements when dyads solve a puzzle through dialogue (Shockley et al. 2003), and the kinematic characteristics of cooperative versus competitive grasping movements (Georgiou et al. 2007). A recent model of joint action by Oztop et al. (2005) that makes relevant claims regarding joint-action coordination in sequential task performance presumes that when a person observes someone else’s actions she automatically will activate her own action system not only to understand the behavior of the actor being observed (Rizzolatti and Craighero 2004) but also to infer that actor’s intentions (cf. Wolpert et al. 2003; Iacoboni et al. 2005). The example Oztop et al. (2005) mentioned in this context concerns the implied meaning that is conveyed by the specific way in which people may handle a tool. For example, whereas holding a hammer with a usual grip around its handle is very likely to signal the intent of hammering, holding the hammer with a power grip around its face communicates the intention to handing over the tool to someone else rather than start hammering with it oneself. Of course, which semantics are primed by the way in which a tool is handled will depend on the shared knowledge of how the tool is used for the general purpose it was designed for (see also Cuijpers et al. 2006). In the present study we were neither interested in what a particular act might convey to an observer in terms of action semantics (Oztop et al. 2005) nor how cooperative or competitive contexts are differentially reflected in the prehension kinematics of dyads (Georgiou et al. 2007). Instead, we focused on the extent to which an observer in her subsequent movements is influenced by specific kinematic features of a co-actor’s movements bringing the object within reach. Since the participants in the present study were asked to perform complementary acts rather than identical ones this research question is therefore only indirectly related to topics like behavioral mimicry and imitation (Koski et al. 2003). Our reasoning about the possible transfer of kinematic movement parameters from one actor to another was that if during action observation people internally simulate the movements that they observe (Gallese and Goldman 1998; Rizzolatti and Craighero 2004), then the kinematic features of these simulated movements might as well be re-used for a related, self-generated movement that, in time, closely follows the observed one even when this future movement is complementary in nature. Economizing on parameter remapping costs formed the basis of this expectation (cf. Rosenbaum et al. 1986). As regards inference making we were interested in the extent to which people derive the weight of an object when seeing someone else picking up and transporting the object. A similar matter has been investigated in an fMRI study by Grezes et al. (2004) but their experiment was primarily designed to find the brain correlates of action observation in the context of deception. To this end these researchers used pictures of an accomplice lifting boxes as if they were heavy or when they were actually heavy. In the present study, however, deceit was not the issue of investigation. Moreover, the object-weight dependent variations in movement kinematics were more subtle than in the Grezes et al. (2004) study and related studies on box lifting by Hamilton et al. (2007) and Kingma et al. (2005) as we will explain next. To gain further insight into the extent to which task-dependent kinematic variations affect the subsequent complementary actions of a co-actor, we exploited two widely investigated motor-control paradigms, viz. the speed-accuracy trade-off (Fitts 1954; Fitts and Peterson 1964) and size–weight illusion (Charpentier 1891). Figure 1 depicts the transportation task that we used for this purpose. First, one of the two actors—the ‘putting-actor’—was asked to pick up a vertically positioned cylinder from a nearby location on the table and to put that cylinder into a circular-shaped target area in the middle of the table, i.e., within reach for the other actor. Subsequently, the other actor—the ‘fetching-actor’—was asked to fetch the cylinder from where the putting-actor had left it, and to reposition it, also in a circular-shaped, nearby target area. The fetching-actor was instructed not to pick up the cylinder before the putting-actor had released it. Fig. 1Top view of experimental setup depicting the experimental task. The dashed squares represent the starting areas for the (right) hand movements of both actors. At the filled circle a cylinder was positioned which had to be picked up by the putting-actor, repositioned in the central circular target area where the fetching-actor was to pick it up and place it in the target circle in the vicinity of her own starting area Three task variables were manipulated, viz. the size of the target areas in which the actors were to successively reposition the cylinder and the size and weight of the to-be-transported cylinder. The size of the target areas was expected to modulate the actors’ movement-speed and the size and weight properties of the cylinders were thought to affect the height with which the actors would lift the cylinders during transport. Before elaborating on our rationale it is important to mention that task conditions were kept constant within trial blocks of three repetitions to examine how quickly the dyads adapted to the between trial-block changes in the experimental conditions. All factors other than the repetition factor were randomized across the trial series and dyads, and at the start of each trial block the participants were unaware of the experimental conditions of that particular block (see “Method”). Two types of potential transfer of movement kinematics were foreseen in our experimental task. First, it was expected that an incorrectly anticipated weight of a to-be-picked up cylinder would be reflected in the height with which an actor would lift that cylinder (cf. Grezes et al. 2004). A lighter than expected cylinder was assumed to be lifted higher than a heavier one and vice versa, i.e., a heavier than expected cylinder was assumed to be lifted less high than a lighter one. Furthermore, if the fetching-actor would infer the weight of the cylinder that was transported by the putting-actor by observing her movements, then the fetching-actor was expected to have a more precise initial estimate of the weight of the to-be-transported cylinder than the putting-actor. The degree to which any surprise effect due to a wrongly anticipated object weight would transfer from the putting-actor to the fetching-actor thus formed a test as to whether or not the fetching-actor had actually inferred the cylinder weight. In repetitions two and three of each trial block both actors could rely, of course, on their sensorimotor memory (see Nowak and Hermsdorfer 2003) as regards the object weight since in these trials they already had handled that particular cylinder in the previous (two) trial(s) of the trial block. To investigate the predicted object-weight dependent interpersonal kinematic transfer effects, we factorially combined two cylinder weights (light vs. heavy) with two cylinder sizes (small vs. large) yielding four cylinders. The size–weight illusion that results from this particular combination of object features reflects the fact that people perceive a smaller of two equally-massed objects as heavier. For one of the suggested explanations of this illusion, we refer to Flanagan and Beltzner (2000). The size–weight illusion is so prominent that it also yields its typical effects when wielding a small and a large object of equal weight in the left and right hand simultaneously suggesting the distorted perception is due to sensory invariants rather than inferences (Amazeen and Turvey 1996). Irrespective of any size–weight illusion effects, we expected the cylinder-weight variations to result in larger between trial adaptation effects on the height with which the participants would lift the cylinders than the cylinder-size variations (Gordon et al. 1991a, b). We thus examined the extent to which the weight of an object carried from one position to another would be inferred by an observer. If the observer, i.e., the fetching-actor, would not infer the weight of the transferred cylinder, then the surprise-effect expressed in the lifting height of the fetching-actor should be equally large as that in the putting-actor. Conversely, should the fetching-actor correctly infer the weight of the object by observing the putting-actor lifting and repositioning the cylinder, then the fetching-actor should be able to anticipate the cylinder’s weight better and not show such large surprise effects as the putting-actor would. A second manipulation targeted the extent to which the two actors would adopt each other’s movement speed in the face of variations of movement-accuracy constraints. If merely by observing someone else’s movements, one would be inclined to adopt a similar speed in a subsequent complementary movement, then confronting two actors with different target area sizes was expected to modulate their speed differently than when the actors would be confronted with these target areas individually. The rationale here was based on Fitts’ law, which states that one’s movement speed systematically varies with the size of the target area (Fitts 1954; Fitts and Peterson 1964; Mottet et al. 2001). The smaller the target area to reach for, the lower the movement speed due to the higher accuracy demands that are associated with small target areas. In the present study we manipulated the target area sizes of the first and the second actor separately, where the size was either small (diameter of 9 cm) or large (diameter of 18 cm). If both actors had identical (either small or large) target area sizes, their movement speeds were most likely to become similar under the assumption that there would be a strong tendency to adopt each others’ speed. If this tendency of movement speed transfer was strong enough, then violations of Fitts’ law in the fetching-actor could be expected under conditions in which the two actors were confronted with dissimilar target area sizes. In sum, in the present study consisting of a sequential motor task involving two actors we tested whether lifting height—a parameter that potentially could reveal an object’s weight—would be picked up by an observing co-actor and determine her subsequent handling of that object and whether she would be inclined to adapt her speed to the observed speed, or, alternatively, whether the interpersonal transfer of kinematic parameters would be incidental in nature, i.e., unrelated to the task constraints with which the individuals of the dyad were confronted. Method Participants Fourteen right-handed adult dyads participated in the experiment. Their ages ranged from 18 to 30 years; mean age was 22 ± 2 years. The 28 participants, 22 females and 6 males, formed 8 female dyads and 6 dyads of mixed gender. They received either course credits or payment (6 euros per hour) for their participation. Each participant signed an informed consent form. The study was approved by the local ethics committee and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Materials A Microsoft Windows-2000 controlled PC was used to control the stimulus events and data collection. Two synchronized 3D infrared motion-tracking systems (Optotrak™, Northern Digital, Waterloo, Canada) were used to record the displacements of the wrist, thumb and index finger of the right hand of the two actors. Three infrared-light emitting diodes (IREDs) were used per participant. The IREDs were fixated on the wrist joint and the distal phalanxes of the index finger and thumb of the right arm of the participants. The IRED-position data were sampled at a rate of 100 Hz with accuracy better than 0.2 mm in X, Y and Z directions. Each participant wore a pair of earphones through which a repetitive sound was presented at approximately 60 dB throughout the experiment. This prevented the participants from speaking to each other and hearing any task-related noise, e.g., due to the cylinders making contact with the tabletop at completion of the transport phases. Each participant also wore a facial mask to avoid communication by means of facial cues. The participants were seated comfortably at opposite sides of a table on top of which a 1-cm thick marble plate was placed to prevent any object-weight information being conveyed from one actor to the other by means of vibrations via the table surface. Four cylinders of 25 cm height were used, two with a diameter of 2.5 cm and two with a diameter of 6.5 cm. Two cylinders had a lightweight of 230 g and two cylinders had a weight of 835 g. To avoid any potential confounding between the effects of cylinder and target-area size all cylinders were given a 4-mm thick, circular base of 8 cm in diameter. This ensured that the thin and thick cylinders had the same size support base and could be repositioned in a comparable stable way which was a factor that also could contribute to the accuracy constraints since the participants were asked to place the cylinders on small and large circular target areas that were clearly marked on the table surface. Procedure and task The experiment consisted of three sections. In the first section one of the two participants performed 48 single-actor control trials consisting of picking up and repositioning in random order, but blocked (three repetitions per block), the four cylinders without the presence of the other participant. In the second section, the other participant performed 48 single-actor control trials, also alone. In the third section the two participants of the dyad performed 96 joint-action experimental trials. The 48 control trials per participant resulted from three blocked replications (n = 3) of each of the factorial combinations of the factors size of starting area (small vs. large), size of target area (small vs. large), cylinder weight (light vs. heavy) and cylinder size (small vs. large). The number of joint-action experimental trials was twice the number of single-actor control trials. In the joint-action experimental trials each participant was given in half the trials the role of putting the cylinder in the centre of the table, and in the other half of the trials the role of fetching the cylinder from the centre of the table (see Fig. 1). The role was randomly assigned to the trial blocks of three repetitions. In their control trials the participants of dyads 1–9 (arbitrary numbered) performed the fetch-part of the experimental task, i.e., at the start of their control trials they were confronted with a cylinder that was placed in the middle of the table and their task was to pick it up and place it in the area which was closest to their own starting position. Dyads 10–14 performed in their control trials the put-part of the experimental task, i.e., at the start of each trial they found the cylinder on the area closest to themselves and they were instructed to put the cylinder in the target area at the centre of the table. The data analyses showed that there were no systematic differences between the two groups of dyads as far as the targeted research questions concerned so the comparisons with the control trial data will not be addressed further in the remainder of this paper. The experiment was conducted by two experimenters who sat behind the participants. Each experimenter had a clear view on a separate computer screen which displayed information that specified the experimental conditions of every trial. In-between trials the participants were asked to close their eyes. After both participants had closed their eyes, the experimenters put the appropriate target pads on the table as well as the appropriate cylinder in the starting area of the putting-actor. Then, the two experimenters simultaneously tapped the participants on their lower arm, which indicated that they could open their eyes to start performing the trial. At the same time one of the experimenters started the movement recording. The standard event sequence in a joint-action experimental trial was as follows. At the start of each trial one of the two actors, i.e., the putting-actor, found in a location close to the starting position of the right hand a cylinder that had to be picked up and moved to the target area in the centre of the tabletop (see Fig. 1). Only after the putting-actor had released the cylinder was the fetching-actor allowed to pick up the cylinder and to reposition it in a target area adjacent to her. The action sequence was to be completed within 8 s during which the displacements of the wrist and finger IREDs on both actors were sampled at a rate of 100 Hz. After every 48 trials, or whenever they asked, the participants were offered a 10-min break. Data analysis The raw IRED position data were smoothed with a second-order, low-pass Butterworth filter with a cut-off frequency of 8 Hz. Subsequently, the position data were segmented on the basis of the tangential velocity–time functions of the IREDs on the wrists of the two actors (see top-left graph in Fig. 2). Fig. 2Kinematics derived from position data of infrared-light emitting diode (IRED) on tip of the thumb, tip of the index finger and wrist as obtained in a prototypical trial. Green (light grey) functions are from the putting-actor, red (dark grey) functions are from the fetching-actor. The top-left graph shows the tangential wrist-velocity time functions, the top-right graph a 3D rendering of finger-thumb displacements, the bottom-left graph shows the aperture-time functions, and the bottom-right graph shows the height (in cm above the table surface) of the midpoint between the thumb and index-finger IREDs The movement sequence of each actor was segmented into three intervals, the first interval corresponding to grasping the cylinder starting with the right hand in the home-position on the edge of the table and ending with finger and thumb making contact with the object, the second interval corresponding to the transportation of the cylinder to the target area (see put and fetch legends in Fig. 2), and the third interval corresponding to the participant’s hand returning to the home-position. The intervals were detected by a semi-automatic search procedure for relevant local minima in the tangential velocity profiles of the wrist movements. The time indexes thus found could be adjusted with an interactive computer program using a cross-hair whenever the experimenter considered the segmentation inaccurate. Following segmentation, the data analyses focused on the displacements of the IREDs located on the tip of the thumb and index finger of each of the two actors. The top-right graph of Fig. 2 shows a 3D-graph of the typical finger–thumb displacements of the putting-actor (in green or light grey) and the fetching-actor (in red or dark grey). The three cylinders were added to this graph post hoc to indicate the phasing of the displayed movement patterns. The bottom-left graph of Fig. 2 shows the aperture-time functions of both actors. This figure was used to inspect the grasping behavior of the participants. Systematic aperture overshoots just prior to object contact and upon object release were visually checked by the experimenter to verify whether the task of each actor was performed as requested. The bottom-right graph of Fig. 2 shows the height in the Z-dimension (perpendicular to the table surface) of the midpoint between the 3D positions of the IREDs on the thumb and index finger of each actor. This parameter reflected the surprise effects due to unexpected between-trial cylinder-weight changes on the lifting height, which was defined as the difference in the vertical position of the midpoint between the tips of the digits at the time of grasping the cylinder and the maximum height realized during transportation. In the second interval of each of the three-segment movement patterns, i.e., the green function for the putting-actor and the red function for the fetching-actor, this lifting height reflected the height with which the participants lifted the cylinder while transporting it to their target area. The maximum lifting height (in cm) and the mean tangential wrist speed (in cm/s) realized by each actor during cylinder transport were used for further analyses. Statistical evaluation of the data consisted of repeated measures ANOVAs of which the designs are described in the relevant paragraphs in the results section or targeted t tests to evaluate specific hypotheses in more detail. Results Effects of cylinder properties Figure 3 shows the means and standard errors of the lifting heights realized by the putter and fetcher as a function of cylinder mass, cylinder size and the repetition factor. Table 1 summarizes the results of the repeated measures ANOVA that was conducted to evaluate these effects with a factorial design consisting of the within-dyad factors 2 roles (put vs. fetch) × 2 object masses (light vs. heavy) × 2 object sizes (small vs. large diameter) × 3 repetitions. As expected, the light cylinders were lifted higher than the heavy ones [F(1,13) = 86.5, P < 0.001]. This effect interacted with object size [F(1,13) = 14.4, P < 0.01] in line with the size–weight illusion. For the light cylinders it was the smaller one that was lifted higher than the larger one. For the two heavy cylinders, it was the larger one that was lifted higher than the smaller one. The interaction between object mass and object size on lifting height—reflecting the size–weight illusion effect—was most pronounced in repetition 1 as confirmed by the first-order interaction between object mass and repetition [F(2,26) = 123.8, P < 0.001] but remained present in repetitions 2 and 3 as reflected by the second-order interaction between object mass, object size and repetition [F(2,26) = 3.78, P < 0.05], confirming the robustness of the size–weight illusion. Fig. 3Means and standard errors of the lifting height (in cm) as a function of role (putting vs. fetching), object diameter (small vs. large) and object weight (light vs. heavy) for repetitions 1, 2 and 3 separatelyTable 1Results of repeated measures Anova on the lifting height according to a 2 role (putting vs. fetching) × 2 object mass (230 vs. 835 g) × 2 object sizes (2.5 vs. 6.5 cm in diameter) × 3 repetitions factorial designFactordfFPRole1,13<1NSObject mass1,1386.5<0.001Object size1,137.1<0.05Repetition2,2658.0<0.001Role × object mass1,13<1NSRole × object size1,131.3NSObject mass × object size1,1314.4<0.01Role × object mass × object size1,13<1NSRole × repetition2,26<1NSObject mass × repetition2,26123.8<0.001Role × object mass × repetition2,26<1NSObject size × repetition2,263.67<0.05Role × object size × repetition2,26<1NSObject mass × object size × repetition2,263.78<0.05Role × object mass × object size × repetition2,26<1NS No main effect or interaction of role (put vs. fetch) was found (all Fs < 1), which was not unexpected since cylinder-weight increases and decreases were expected to have counteracting effects on the lifting heights by the putter and fetcher if any interpersonal transfer would take place at all. Targeted paired t tests confirmed that there were systematic interpersonal transfer effects of lifting height between putter and fetcher as will be described next. A detailed time-series analysis of the lifting heights which the dyads realized indicated that the fetching-actor benefited from movement observation. The results of this analysis are shown in Fig. 4. In those cases in which from one trial block to the next the cylinder’s weight changed from heavy to light and the putting-actor showed a ‘surprise’ effect as reflected by an increase of her lifting height between these two trials of, on average, 4.69 cm, the fetching-actor showed a similar surprise effect but to a smaller extent. This reduced surprise effect in the fetching-actor of, on average, 3.67 cm, is shown in the left-hand graph of Fig. 4. The incidence of this effect was relatively high. It happened in 88% of those trials (i.e., N = 99) in which a heavy-to-light cylinder-weight change occurred. A two-tailed t test comparing the lifting heights on trial i − 1 and trial i (being the third trial of each trial block and the first of the subsequent block, respectively) for the putting-actor revealed that her surprise effect was statistically significant [t(98) = 15.98, P < 0.01]. For the fetching-actor, the smaller surprise-effect was also statistically significant [t(98) = 13.54, P < 0.01]. A repeated measures ANOVA confirmed that the relevant interaction between role (putter vs. fetcher) and trial (i − 1 = heavy and i = light) was statistically significant [F(1,13) = 7.24, P < 0.05]. Fig. 4Means and standard errors of the lifting height (in cm) of the putting-actor and the fetching-actor. Left-hand graph: whenever the putting-actor showed a lifting-height increase from trial i − 1 to trial i due to an unexpected mass reduction between these trials, the fetching-actor also showed this surprise effect but less strong. Trials i − 1 were the third trials of the trial blocks; trials i were the first trials of the subsequent trial blocks. Right-hand graph: similarly, whenever the putting-actor showed a lifting-height decrease from trial i − 1 to trial i due to an unexpected mass increase between these trials, the fetching-actor also showed this surprise effect, but again, not as strong as the putting-actor did We found a similar effect when focusing on those trial pairs in which the cylinder-weight changed from light to heavy and the putter showed a ‘surprise’ effect as reflected by a decrease of her lifting height between those trials. The results of this analysis are shown in the right-hand graph of Fig. 4. Whereas the putting-actor proved to lift the cylinder, on average, 2.21 cm less high when the cylinder turned heavier from trial i − 1 to trial i, the fetching-actor did so as well but, on average, for only 0.97 cm. The incidence of this phenomenon was at chance level, i.e., in 54% (N = 61) of the cases in which such a cylinder-weight change occurred in the experiment. A two-tailed t test comparing the lifting heights on trial i − 1 and trial i for the putting-actor and fetching-actor separately again revealed that the effects were statistically significant for both actors [t(60) = 12.08, P < 0.01, t(60) = 3.69, P < 0.01, respectively]. A repeated measures ANOVA confirmed that the relevant interaction between role (putter vs. fetcher) and trial (i − 1 = light and i = heavy) was statistically significant [F(1,13) = 31.08, P < 0.001]. The time-series analyses on light-to-heavy cylinder weight changes and vice versa were corroborated by the results of dyad-based linear regression analyses in which the lifting height of the putting-actor in the first trial of the trial blocks was used as predictor for that of the fetching-actor. In contrast to the repeated measures ANOVA, the regression analysis took into account lifting-height co-variations in both directions, i.e., increases as well as decreases. An example of these data is depicted in Fig. 5. The slopes of the resulting regression equations turned out to be smaller than 1.0 for all 14 dyads showing that the putting-actor was more surprised by a cylinder-weight change than the fetching-actor. For nine of the dyads the positive slope proved statistically significant (P < 0.05). On average, the slopes amounted to 0.43 (R2 = 0.25), indicating that the fetching-actors were less surprised by any cylinder-weight change than the putting-actors. Fig. 5Example of the linear regression analysis between the lifting-height data generated by the putter and fetcher of 1 of the 14 dyads participating in the study. The data concern the first trial within trial blocks of three repetitions of the experimental conditions. The regression analyses revealed the robustness of the observed reduction of the size–weight illusion due to movement observation in the dyads studied. The dashed line with slope = 1 represents the situation in which the lifting height of the fetcher would equal that of the putter Effects of target-area size variations Figure 6 shows the average effects on the mean tangential wrist velocity of the putting and fetching-actors as a function of the manipulations of the size of the target regions where the cylinders had to be placed. The second part of the sequential action under study, i.e., fetching the cylinder from the middle of the table, proved for no apparent reason overall to be performed somewhat slower than the first part, i.e., putting the cylinder in the middle of the table. The performance speeds of the participants were independently tuned to the sizes of the target areas with which the actors were confronted. In line with Fitts’ law, small target areas elicited low speeds and large ones yielded high speeds. This relationship applied to both actors whether or not they were confronted with similar or dissimilar accuracy constraints. Table 2 summarizes the repeated measures ANOVA to evaluate these effects with a 2 roles (put vs. fetch) × 2 sizes of putting target region (‘putting-size’; small vs. large) × 2 sizes of fetching target region (‘fetching-size’; small vs. large) factorial design. The absence of the first-order interaction between putting-size and fetching-size (F < 1) and the second-order interaction between the factors role, putting-size and fetching-size confirm the robustness of these findings. Fig. 6Means and standard errors of the mean wrist speed (in cm/s) as a function of role (putting vs. fetching) and target area size (S = Small, L = Large; p = put, f = fetch). The labels with subscript p reflect the size of the target area of the putting-actor and labels with subscript f reflect the size of the target area of the fetching-actorTable 2Results of repeated measures ANOVA on the mean wrist speed (in cm/s) according to a 2 role (putting vs. fetching) × 2 putting-size (small vs. large target area) × 2 fetching-size (small vs. large target area) factorial designFactordfFPRole1,1318.60<0.01Putting-size1,1375.07<0.001Fetching-size1,1333.90<0.001Role × putting-size1,1320.21<0.001Role × fetching-size1,1322.03<0.001Putting-size × fetching-size1,13<1NSRole × putting-size × fetching-size1,13<1NS A trial-by-trial inspection of the speed changes of both actors revealed, however, a moderate degree of performance-speed adaptation by the fetching-actor to that of the putting-actor. Figure 7 shows the results of this analysis. If from one trial to the next the size of the target areas remained constant for both actors but the putting-actor increased her performance speed for some reason, the fetching-actor followed this incidental speed increase of the putting-actor only partially as shown in the left-hand graph of Fig. 7. The incidence of this co-variation in performance speed between the two actors was below chance level, i.e., it happened in 34% (N = 150) of the cases in which the phenomenon could theoretically occur. Nevertheless, the changes in speed were, in both actors, statistically significant [t(149 = 7.63, P < 0.02 and t(149) = 5.95, P < 0.01, two-tailed, for the putting-actor and fetching-actor, respectively). Fig. 7Means and standard errors of the wrist speed (in cm/s) of the putting-actor and the fetching-actor. Left-hand graph: whenever the putting-actor showed a task-unrelated speed increase from trial i − 1 to trial i, the fetching-actor also showed such a speed increase. The trials involved concerned the second and third trials of the trial blocks in which no task conditions changed. Right-hand graph: similarly, whenever the putting-actor showed a task-unrelated speed decrease from trial i − 1 to trial i, the fetching-actor followed this speed decrease The reverse co-variation of performance speeds happened slightly more often. The right-hand graph of Fig. 7 shows that whenever the putting-actor reduced her movement speed from trial i - 1 to trial i during which the size of the target regions remained constant, the fetching-actor again partially followed this speed decrease. This co-variation in speed occurred in 42% (N = 186) of the cases in which this effect could occur. For the putting-actor and fetching-actor the speed decreases that were isolated here were again both statistically significant [t(185) = 5.95, P < 0.01 and t(185) = 3.32, P < 0.01, two-tailed, respectively]. Linear regression analyses in which the mean wrist speeds of the putting-actor as observed in the second and third trials of the trial blocks were used as predictor for that of the fetching-actor confirmed the just described results. The slopes of the regression equations proved positive for 12 of the 14 dyads indicating that the fetching-actor followed the speed changes of the putting-actor in direction but only partially in size. For 10 of the 14 dyads the positive correlations proved statistically significant (P < .05). R2s in these analyses ranged between 0 and 0.33 (mean R2 was 0.11) indicating the target-size independent speed relationship between the two actors was weak. Combined effects of size–weight illusion and speed-accuracy trade-off An inspection of the combined effects of the three task constraints that were manipulated revealed that even though on average the heavier cylinders were transported at a systematically lower speed than the lighter cylinders (mean = 43.78 cm/s; SD = 8.37 cm/s for the light cylinders and M = 38.14 cm/s; SD = 7.06 cm/s for the heavy cylinders), the speed variations due to the variations in the size and weight of the cylinders did not differentially affect the movement-speed variations as a result of the target-area size variations. Lifting-height variations due to the spatial accuracy-constraint manipulations were marginal (<2 mm) and also did not systematically influence the lifting-height variations due to the size-illusion effect. Discussion The present study examined the extent to which at the level of movement kinematics a transfer of performance parameters takes place between co-actors involved in transferring objects. The results of the main analyses that we conducted to assess the effect sizes of the experimental manipulations were largely negative. Object-mass variations induced lifting-height variations of equal size in both actors. Target-region size based speed variations also did not show interpersonal transfer. These results suggest that in sequential joint actions the tuning to each others movement parameters is absent and thus demonstrate the limits of automatic movement simulation by observation as suggested by mirror-neuron theories (Gallese and Goldman 1998; Gallese et al. 2004; Rizzolatti and Craighero 2004). However, it remains to be seen whether the mirror-neuron system represents observed actions with a precision that allows for direct imitation. This does not appear to be the case in macaque monkeys. One could claim that any hypothetical system in general could infer the goals/intentions of observed movements without explicitly representing, say, the speed of the movement. More detailed analyses of incidental interpersonal transfer effects indicated that the situation was more complex than that. When focusing on the effects of between-trial cylinder-weight changes, the actor who was confronted with such changes first showed a systematically larger surprise-effect than the actor who was asked to transport the cylinder after the first actor had done so. A key finding here was that through the realized trajectory height during object transportation, the object mass was picked up by movement observation and integrated into the observer’s movement plan. Our prediction was therefore confirmed, i.e., the movements of an actor are likely to affect a subsequent, complementary movement generated by an observer. Regarding the size of the effects it proved that the object property mass was, via lifting height, a more prominent factor determining task performance of the co-actors than movement speed. The linear regression analyses of the between-trial lifting-height changes due to cylinder-mass changes yielded larger proportions of explained variance when correlating the behavior of the fetching-actor with that of the putting-actor than the comparable analyses involving the incidental speed changes. With respect to goals and means of task performance, the dominance of object-mass over target-width makes sense given that the object is more likely to function as a goal in the task of handing over objects than the movements needed to perform such a task (Cuijpers et al. 2006). In a similar vein, the different sizes of the effects of object mass and movement speed could be taken to provide support for the importance of goal-inference making in collaborative joint action over and above a quasi-automatic, direct-matching based process of movement imitation or ‘motor resonance’ (Flanagan and Johansson 2003; Iacoboni et al. 1999; Koski et al. 2003) (Fig. 8). Fig. 8Example of the linear regression analysis between the wrist-speed data generated by the putter and fetcher of 1 of the 14 dyads participating in the study. The data concern the second and third trials within trial blocks of three repetitions of the experimental conditions. The regression analyzes revealed the robustness of the observed, task-constraint independent covariation of movement speed between the two actors. The dashed line with slope = 1 represents the situation in which the wrist speed of the fetcher would equal that of the putter Our interpretation of the current results does rely, of course, on the validity of both the weight-change effects and the co-variation of the actors’ movement speeds. It could be argued that the fetcher’s movement speed covaried with that of the putter due to a non-specific, visually primed increase in attention rather than the direct consequence of perceiving the putter’s object-transportation movements. However, attentional mechanisms are an unlikely explanation for our observations since the only visual cue that could have increased the attention in the fetcher was the putter’s movement speed. Whether the object-weight related adaptation in the fetcher to the observed movement of the putter was due to the inference of the weight of the object or to the observed kinematics per se, also remains a matter of discussion. If it was the former, then motor resonance (i.e., mirror-neuron system) may not be responsible for a direct transfer of the kinematics parameters. The effects of the object and the accuracy constraints differ in one fundamental aspect. The expected information transfer for the object is valid for the programming of the movement, while any hypothesized transfer of speed, by simple copying the observed movement to reduce dimensionality violates Fitts’ law. This implies that our hypothesis about the transfer of speed was either not valid or that the effect is intrinsically short lived because the duration of the effect is overruled by intrapersonal constraints. In other words, while the information about weight change is very relevant for movement programming, the examined speed changes were not. Apparently, movements that are being observed in a joint-action task can be used for multiple purposes. First, the observation might lead to internal simulation in order to understand the behavior that is being observed (Rizzolatti and Craighero 2004). Second, the internally simulated movements might provide clues as to the intentions associated with the observed behavior (Oztop et al. 2005; Wolpert et al. 2003; Iacoboni et al. 2005). Third, the observed movements might form the basis for prediction as to what an actor might do in the near future (Csibra 2005). Fourth, observed movements might provide clues about the task constraints at hand. In the present experiment, lifting height was such a parameter that indicated to the observer the weight of the cylinder that was transported. Fifth, observed movements may provide a global scaling parameter of ones own future actions, specifically if these actions are complementary to those performed by the actor that is being observed. Further research into the factors that determine the relative importance of these various roles which action observation may play in joint action is clearly needed. Our study also suggests that kinematic parameters picked up during movement observation may even affect a subsequent movement that is performed in a workspace that is totally differently oriented than the workspace of the actor being observed. This finding extends the findings of the recent study by Van Schie et al. (2004) who showed that action observation activates cortical motor areas as if the observer had performed the task herself, i.e., when an observer who is facing an actor sees that actor move with her right hand the right cortical motor areas of the observer become activated indicating that the observer would have executed the task with her left hand. These findings suggest that movement simulation is egocentric and viewpoint-dependent. Our demonstration of interpersonal action coordination in complementary joint action was restricted to the successive transportation of a cylinder by two actors. The movements preceding and succeeding the actual transportation of the cylinder could also have been scrutinized for signs of action coordination. However, the amplitudes of these movements by the two actors were hardly comparable. Moreover, the dyads involved in this study showed, not surprisingly, also the tendency to wanting to start task performance simultaneously. The preference of people to adopt either in-phase or out-of-phase timing patterns is not only prevalent in individual, inter-limb coordination tasks but also when people perform tasks together (Schmidt and O’Brien 1997; Shockley et al. 2003). These tendencies clearly co-exist alongside the casual transfer of kinematic movement parameters in dyadic action sequences.

J_Gastrointest_Surg-3-1-1852373

Impact of Solitary Involved Lymph Node on Outcome in Localized Cancer of the Esophagus and Esophagogastric Junction

Node-positive esophageal cancer is associated with a dismal prognosis. The impact of a solitary involved node, however, is unclear, and this study examined the implications of a solitary node compared with greater nodal involvement and node-negative disease. The clinical and pathologic details of 604 patients were entered prospectively into a database from1993 and 2005. Four pathologic groups were analyzed: node-negative, one lymph node positive, two or three lymph nodes positive, and greater than three lymph nodes positive. Three hundred and fifteen patients (52%) were node-positive and 289 were node-negative. The median survival was 26 months in the node-negative group. Patients (n = 84) who had one node positive had a median survival of 16 months (p = 0.03 vs node-negative). Eighty-four patients who had two or three nodes positive had a median survival of 11 months compared with a median survival of 8 months in the 146 patients who had greater than three nodes positive (p = 0.01). The survival of patients with one node positive [number of nodes (N) = 1] was also significantly greater than the survival of patients with 2–3 nodes positive (N = 2–3) (p = 0.049) and greater than three nodes positive (p < 0001). The presence of a solitary involved lymph node has a negative impact on survival compared with node-negative disease, but it is associated with significantly improved overall survival compared with all other nodal groups. Introduction Carcinoma of the esophagus carries a dismal prognosis, and for patients presenting with localized resectable disease, multivariate analysis has established that the presence or absence of involved lymph nodes confers the greatest prognostic significance.1 In surgical management, the extent and type of lymphadenectomy undertaken varies from no formal lymphadenectomy to two and three field dissection.2–5 The presence and extent of lymph node involvement is important as selective approaches may be considered depending on the nodal stage at presentation. In early tumors, for instance, the sentinel node concept initially developed in melanoma and breast cancer was explored to help identify patients who may not require lymph node dissection.6–8 The advent of minimally invasive esophagectomy may also highlight the need to subselect patients for lymphadenectomy.9 In the observations of the senior author (JVR), patients with solitary involved lymph nodes may achieve good outcomes, and this hypothesis was evaluated in this analysis of a large prospective database. We report herein that the cohort with a solitary node involved had cancer outcomes closer to node-negative disease than other node-positive subgroups, and suggest that this represents a distinct prognostic subgroup. Patients and Methods The study population consisted of all patients with tumors of the esophagus and esophagogastric junction who underwent surgical resection, either alone or preceded by neoadjuvant chemoradiation, between 1993 and 2005. Patients receiving multimodal therapy received cisplatin, 5-fluorouracil, and external beam radiotherapy (40–44 Gy, 2–2.67 Gy/fraction) as previously described.10 Data concerning the clinical and pathologic parameters for all patients was obtained from a detailed prospective database maintained by a full-time data manager. Pathologic parameters analyzed included the location of the tumor, tumor morphology, i.e., adenocarcinoma or squamous cell carcinoma, histological differentiation (grade), TNM staging, number and site of involved lymph nodes, and R classification after surgical resection. Staging of tumors was performed according to the American Joint Committee on Cancer TNM system.11 A subtotal esophagectomy was performed with a sutured anastomosis either in the right thorax (two-stage) or neck (three-stage). All cases underwent a formal abdominal lymphadenectomy and mediastinal lymph node dissection up to and including the subcarinal nodes. Thoracic nodes were submitted separately to abdominal nodes. Statistical Analysis Data are presented as frequencies, means, and percentages. ANOVA was used for comparison of the four demographic groups. Survival probability was estimated using the Kaplan–Meier method. Survival was calculated from the date of clinical diagnosis to date of death or date last seen. In the multivariate analysis, independent prognostic factors for survival were determined by using a Cox regression hazard model. Two analyses were performed, one for all patients and the other exclusive to node-positive patients. All statistical analyses were performed using Stata software (version 9.1 for Windows, Statcorp, TX). A p value <0.05 was considered statistically significant.12 Results Patients/histology Six hundred and four patients underwent surgery for localized malignancy of the esophagus or esophagogastric junction. The mean age was 62 ± 10.4 (median = 64, range 56 to 70). Four hundred and twelve (68%) patients were men. The mean number of lymph nodes examined per specimen was 12 ± 6 (median = 10, range = 6 to 55). Two hundred and eighty-nine patients (48%) had node-negative disease [number of nodes (N) = 0], 84 (14%) had one node positive (N = 1), 84 had two or three nodes positive, and 147 (24%) had greater than three nodes positive (N > 3). In patients with one involved node, in all cases the node was adjacent to the tumor, mediastinal for esophageal tumors, and periesophageal or along the left gastric artery for junctional tumors (Tables 1 and 2). Table 1Demographics of Nodal SubgroupsHistologic DataN = 0 (n = 289)N = 1 (n = 84)N = 2–3 (n = 84)N > 3 (n = 147)Tumor site (%)Lower esophagus138 (47)39 (46)37 (44)57 (39)EG junction80 (28)35 (42)33 (39)75 (51)Middle esophagus55 (19)10 (12)12 (14)11 (7)Upper esophagus16 (6)02 (3)4 (3)Morphology (%)Adenocarcinoma140 (48)51 (61)57 (68)113 (77)Squamous cell carcinoma140 (48)29 (35)25 (30)32 (22)Others9 (4)4 (5)2 (1)2 (1)Treatment (%)Multimodal therapy129 (44)28 (33)24 (29)21 (14)Surgery alone161 (56)56 (76)60 (71)125 (86)Residual tumor (%)R0: no residual tumor250 (86)71 (85)64 (76)108 (73)R1: residual tumor found39 (13)13 (15)19 (23)39 (27)Rx: unknown1 (1)–1 (1)–Pathological stage (%)Stage 053 (18)–––Stage I59 (20)1 (1)––Stage II170 (59)21 (25)25 (30)16 (11)Stage III5 (2)58 (29)53 (63)110 (76)Stage IV1 (1)4 (5)6 (7)20 (13)pT stage (%)Tx3 (1)02 (3)1 (0.5)Tis12 (4)000T040 (14)1 (1)2 (3)2 (1)T156 (19)5 (6)4 (5)3 (2)T235 (12)16 (19)18 (21)12 (8)T3138 (48)60 (71)54 (64)120 (82)T46 (2)2 (3)4 (5)8 (5)EG = esophagogastricTable 2Histology of Nodal SubgroupsHistologic DataN = 0N = 1N = 2–3N > 3AdenoSCCAdenoSCCAdenoSCCAdenoSCCn = 140N = 140n = 51n = 29n = 57n = 25n = 113n = 32No%No%No%No%No%No%No%No%Tumor siteLower Esophagus64(46)66(47)19(52)15(52)23(40)13(52)39(35)17(53)EG Junction73(52)6(4)31(10)3(10)33(58)0074(65)1(3)Middle Esophagus3(2)52(37)1(28)8(28)1(2)10(40)0010(31)Upper Esophagus0016(11)0(10)3(10)002(8)004(13)TreatmentMultimodal80(57)46(34)23(45)5(17)20(35)4(16)19(13)3(10)Surgery alone60(43)93(66)28(55)24(83)37(65)21(84)94(87)28(90)Path stageStage 029(21)18(13)000000000000Stage 142(30)15(10)1(2)0000000000Stage 266(47)102(73)15(29)4(14)19(33)5(20)15(13)1(3)Stage 32(1)4(3)32(63)24(83)35(61)17(68)82(73)27(84)Stage 4001(1)3(6)1(3)3(6)3(12)16914)3(10)Unknown1(1)0000000000001(3)pT stageTx2(1)1(1)0000002(8)001(3)Tis9(6)0000002(4)000000T019(14)17(12)1(2)003(5)002(2)00T139(29)15(11)4(8)0014(24)003(3)00T216(11)19(14)12(23)3(10)36(63)4(16)11(10)1(3)T353(38)84(60)33(65)26(90)2(4)17(68)91(80)28(88)T42(1)4(2)1(2)00002(8)6(5)2(6)Adeno = adenocarcinoma, SCC = small cell carcinoma, EG = esophagogastric Two hundred and two patients (33%) had multimodal therapy and 402 patients (67%) had surgery alone. Of the multimodal cohort, 129 (64%) were ypN0 on histopathologic assessment, 28 (14%) had one node positive, 24 (12%) had two to three positive nodes, and 21 (10%) had greater than three positive nodes. The attainment of an R0 resection was significantly greater in patients with none or one node involved compared with both other groups (p < 0.05). The majority of patients in all groups had pT3 tumors, 48% in the pN0 group compared with 71, 64, and 82% in the N = 1, N = 2–3, and N > 3 groups, respectively (p < 0.05). One hundred and forty (62%) of the squamous cell carcinoma cohort were node-negative (N = 0) compared with 140 (39%) of cases with adenocarcinoma (39%) (p < 0.05). Survival The median survival for all patients was 20 months at a median follow-up of 19 months (3–167). Patients who were node-negative (N = 0) had a median survival of 26 months (Table 3), compared with 16 months when one node was positive (p = 0.03). Patients who had two to three nodes positive had a median survival of 11 months, and 8 months in patients who had greater than three nodes positive (p = 0.01; N = 2–3 vs N > 3). The survival of patients with one node positive (N = 1) was significantly greater than the survival of patients with 2–3 nodes positive (p = 0.04) and the cohort with greater than three involved nodes (p < 0.0001). Table 3Univariate and Multivariate Analysis: All PatientsVariablesNo. of PatientsMedian Survival (moths)p Valuea (Univariate)HR95% CIap valueb (Multivariate)HR95% CITreatmentSurgery only401130.0771–––Multimodal203190.840.69–1.02Tumor siteUpper esophagus25160.3711–––Middle esophagus87140.9460.980.58–1.66Lower esophagus268140.6581.160.69–1.81EG junction224140.6241.130.69–1.84Depth of invasionT05755<0.00110.6521T168260.5371.160.73–1.830.4720.710.21–2.3T281260.4191.200.77–1.850.5731.110.31–3.94T337311<0.0012.281.60–3.260.8711.400.79–2.41T4197<0.0014.342.46–7.680.6492.591.42–4.08No. of nodes028926<0.0011<0.00110.63–1.87184160.0381.361.02–1.820.7741.080.83–2.432–38411<0.0011.911.45–2.520.2021.421.07–3.18>31478<0.0012.612.08–3.290.0271.84HistologySquamous361140.9161Adenocarcinoma224130.5961.050.87–1.28–––Other19260.4830.800.44–1.48Stage05355<0.00110.1181I63550.7470.920.56–1.510.5760.680.18–2.59II230200.0371.491.02–2.170.5081.550.42–4.69III22510<0.0012.711.86–3.950.5271.680.34–5.58IV316<0.0016.163.72–10.20.1823.141.14–7.76Residual tumorR049217<0.00110.0521R111081.701.37–2.121.250.99–1.58aχ2bCox regressionHR = hazard ratio, CI = 95% confidence intervals, EG = esophagogastric The 1-, 3-, and 5-year survival of the pN0 group was 78, 51, and 44%, respectively (Fig. 1). Where one node was involved, survival was 67, 41, and 35%, respectively. Where two to three nodes were involved, the 1-, 3-, and 5-year survival was 57, 25, and 13%, respectively, and where greater than three nodes were involved, this was 40, 14, and 8%, respectively. Figure 1Overall survival by number of nodes positive. Univariate analysis (Table 3) revealed nodal status, pT stage, pathologic stage, and R status as predictors of survival. Multivariate analysis revealed nodal status alone to significantly (p < 0.0001) impact on survival. By this analysis the hazards ratio increased from 1.08 for one involved node to 1.42 for two to three involved nodes, and 1.84 for greater than three nodes. Excluding node-negative patients, univariate analysis (Table 4) revealed pT stage, pathologic stage, R status, and number of nodes as predictive of survival. By multivariate analysis (Table 5), pathologic stage (p = 0.010) and number of nodes were significant determinants of survival. Compared with the cohort with one involved node, the hazard ratio for two to three nodes was 1.56 (p = 0.049) and 2.06 (p = 0.007) for greater than three nodes. Table 4Univariate Analysis: Node-positive AloneVariablesNo. of PatientsMedian Survival (moths)p valuea (Univariate)HR95% CITreatmentSurgery only241110.23410.63–1.11Multimodal74110.84Tumor siteUpper esophagus9180.6501Middle esophagus32100.5561.310.54–3.18Lower esophagus130100.1831.750.77–3.98OG junction144120.3501.480.65–3.36Depth of invasionT05110.0011T11280.9171.060.33–3.41T246240.1761.120.43–1.78T3235110.7571.430.74–2.14T41450.1572.230.74–6.78HistologySquamous86110.6381Adenocarcinoma221110.6381.070.81–1.40Other830.8481.070.49–2.35Stage1–II6319<0.0011III–IV251102.011.43–2.83Residual tumorR0259120.0351R16191.331.02–1.73No. of nodes18417<0.00112–384130.0211.671.06–2.29>31479<0.0012.531.50–3.62aχ2HR = hazard ratio, CI = 95% confidence intervalsTable 5Mutivariate Analysis: Node-positive OnlyVariablesp valuea (Multivariate)HR95% CIDepth of invasionT01T10.5440.820.31–1.75T20.6791.230.74–1.81T30.3131.490.99–2.21T40.2021.831.39–3.24StageI–II0.0101III–IV1.590.82–3.06No. of nodes112–30.0491.561.21–2.35>30.0072.061.51–2.82Residual tumorR00.2831R11.220.80–1.79aCox regressionHR = hazard ratio, CI = 95% confidence intervals Discussion Cancers of the esophagus and esophagogastric junction are aggressive tumors, which are typically diagnosed at an advanced stage of disease progression.13 This large retrospective review of a tertiary center’s experiences over 12 years highlights the importance of lymph node involvement in the prognosis of these tumors. The study shows that the presence of a solitary node, although a significantly negative factor compared with pN0 disease, is associated with significantly improved median and 1-, 3-, and 5-year survival compared with cohorts of patients with greater nodal involvement. The 5-year survival, for instance, was 35% compared with 13 and 8%, respectively, for cohorts with two to three positive nodes and greater than three positive nodes. There is no uniform consensus on the number of lymph nodes that must be sampled. In a study by Ito et al.,3 the median number of lymph nodes examined per specimen was 6 (range 0 to 35) and only 20% of patients had at least 15 lymph nodes examined. In this study, the median number of lymph nodes examined per specimen was 12 (range 6 to 55), and 24% of the patients had at least 15 lymph nodes examined. These results appear consistent with practice in the United States where an analysis of the National Cancer Database indicated that only 18% of patients undergoing surgery for gastric cancer have more than 15 lymph nodes analyzed.14 In this Unit, lymph node clearance involves a D2 dissection of abdominal nodes, and wide mediastinal clearance to the carina and paratracheal node dissection if they appear involved. No cervical dissection is performed, consistent with recommendations from another group.15 It is acknowledged that variation in lymph node yield may mask stage migration, particularly in a retrospective analysis, but the standardization of lymphadenectomy is likely to minimize the impact of this potential bias. The association between extent of nodal involvement and outcome is well described.16–18 No study to our knowledge has previously focused on the impact of one positive node on outcome in esophageal cancer. The observation, however, of the unique prognostic significance of a solitary involved node was recently reported.19 In a study of 187 patients with esophageal adenocarcinoma treated with neoadjuvant chemoradiotherapy, Gu et al.19 at the MD Anderson observed from their analysis that patients with a solitary involved node had better overall and relapse-free survival compared with other nodal groups. Moreover, the 5-year survival outcomes and 2-year relapse-free survival was not significantly different from the node-negative cohort. Although in our series survival figures were better for node-negative patients than patients with a solitary involved node, the overall pattern of outcome data in our series is consistent with the report from the Anderson group, with prognosis in this cohort closer to node-negative than other node-positive subgroups. The clinical implication of this finding is not clear at this time, but it should, at minimum, encourage a more optimistic view of patients who have a solitary lymph node identified after adequate lymphadenectomy, as approximately 35% of patients with this pathologic stage may be cured. In the future, it is possible that advances in endoscopic US staging, fluorodeoxyglucose PET, and sentinel node assessment may improve pre- and intraoperative assessment of nodal involvement, defining node-negative, solitary involved node and micrometastatic-involved subgroups, and selective lymphadenectomy and minimally invasive approaches may be evaluated in these situations. This demands prospective evaluation, but it may be noteworthy that all involved nodes in the solitary involved node cohort were close to the primary site and may possibly have been identified as sentinel nodes. In conclusion, this study shows that in a large cohort of patients, lymph node status and the number of lymph nodes positive at the time of surgical resection is directly linked to survival. Extensive nodal involvement is confirmed as carrying a dismal prognosis, but greater optimism is justified where a solitary involved lymph gland defines the pN stage after an adequate lymphadenectomy.

Diabetologia-4-1-2292423

Increasing overall physical activity and aerobic fitness is associated with improvements in metabolic risk: cohort analysis of the ProActive trial

Aims/hypothesis Our aim was to examine the association between change in physical activity energy expenditure (PAEE), total body movement (counts per day) and aerobic fitness (maximum oxygen consumption []) over 1 year and metabolic risk among individuals with a family history of diabetes. Introduction The metabolic syndrome has been defined as a cluster of closely related cardiovascular risk factors including visceral obesity, insulin resistance, hyperglycaemia, hypertension, hypertriacylglycerolaemia and low HDL-cholesterol [1]. The combination of these risk factors has been shown to predict type 2 diabetes, cardiovascular disease and all-cause mortality [2], with physical inactivity identified as a major underlying risk factor. Given the rising prevalence of obesity and type 2 diabetes, the metabolic syndrome represents a potentially large public health burden [3, 4]. Previous research has shown that low levels of physical activity are associated with the metabolic syndrome [5, 6], and that objectively measured physical activity energy expenditure (PAEE) predicts progression towards the metabolic syndrome independently of obesity [7], with some evidence of interaction by cardiorespiratory fitness [8, 9]. Additionally, lifestyle interventions targeting physical activity as a modifiable risk factor have shown positive effects upon various metabolic components [10]. However, physical activity remains poorly measured in most epidemiological studies, with many relying on self-reporting [11]. Furthermore, it is unclear whether metabolic risk is most effectively reduced by increases in overall energy expenditure (EE), fitness or total body movement. A better understanding of these relationships would inform the development and targeting of individual and population-based interventions to reduce risk of diabetes and related metabolic disorders. The ProActive (UK) trial is an explanatory trial of a theory-based intervention to promote physical activity among individuals reporting low levels of activity and who are at increased risk of the consequences of a sedentary lifestyle due to their family history of type 2 diabetes. The main trial results are presented elsewhere [12]. We recently observed a cross-sectional association between total body movement and clustered metabolic risk in this cohort [13] and are now extending these observations to examine the association between change in objectively measured PAEE (measured by individually calibrated heart rate [HR] monitoring), physical activity (total body movement measured by accelerometry) and aerobic fitness and clustered metabolic risk in the ProActive trial cohort over a period of 1 year. Methods The ProActive trial Full details of the study have been reported elsewhere [14]. In brief, ProActive aimed to evaluate the efficacy of a theoretical, evidence- and family-based intervention programme to increase physical activity among individuals defined as high-risk through having a parental history of type 2 diabetes. Potential participants were identified via diabetes registers and medical records of family history in 20 general practices in the East Anglia region of the UK. Three hundred and sixty-five individuals aged 30–50 years and reporting low levels of activity were randomly assigned to one of three interventions: brief written advice (control group), or a behavioural-change programme at two levels of intensity delivered either by telephone (distance) or face-to-face in the family home. The theory-based intervention programme [15] was delivered by trained facilitators and aimed to support increases in physical activity through the introduction and facilitation of a range of self-regulatory skills. Main trial results indicated no significant difference in the 1 year change in objectively measured daytime physical activity expressed as a ratio to resting EE between the three trial arms [12]. Consequently, the three trial arms were pooled and a cohort analysis conducted. Complete data on PAEE, aerobic fitness, anthropometry and biochemistry were available in 365 participants at baseline and 321 participants at follow-up, in addition to socio-demographic information. Total body movement measured by accelerometry was also assessed in a subsample of participants (n = 192) at baseline and follow-up and the data were included in these analyses. The measure of socioeconomic status (SES) was based on age at finishing full-time education (above or below 16 years). Ethics approval was obtained from the Eastern England Multi-centre Research Committee (MREC) and all participants gave written informed consent. Anthropometric and metabolic tests Participants attended the study centre after an overnight fast and a sample of venous blood was taken. Fasting plasma glucose, serum insulin levels and lipid profiles (cholesterol, triacylglycerol, HDL- and LDL-cholesterol) were measured using the hexokinase method and standard enzymatic methods, as described previously [14]. Weight was measured on standard calibrated scales and height was measured using a rigid stadiometer. BMI was calculated as weight (kg) divided by height (m) squared. Waist circumference (cm) was measured over light indoor clothing as the mid-point between the lower costal margin and the level of the anterior superior iliac crests. Body fat percentage was measured by bio-electrical impedance (Bodystat, Isle of Man, UK), and systolic and diastolic blood pressures were measured using an automated Accutorr sphygmomanometer (Accutorr, Cambridge, UK). Assessment of aerobic fitness, PAEE and total body movement Aerobic fitness (maximum oxygen consumption []) was predicted as oxygen uptake at maximal HR (220 minus age [years]) by extrapolation of the regression line established during the individual calibration for the relationship between oxygen consumption and HR during a submaximal graded treadmill exercise test. O2 uptake and CO2 production was measured continuously by indirect calorimetry throughout the test (Vista XT metabolic system; Vacumed, Ventura, CA, USA).Resting EE (REE) was calculated by the Weir formula [16] using and measurements obtained in the fasting state, after approximately 10 min of supine rest using the same indirect calorimetry system as previously described. PAEE was measured using the flex HR method [8]. Flex HR was calculated as the mean of the highest resting HR and the lowest HR while exercising. This point was used in the analysis of free-living minute-by-minute HR data to discriminate between rest and exercise. Below the flex HR point, EE was assumed to be equivalent to REE. EE above the flex point was predicted from the individual HR–EE regression line. Participants wore HR monitors (Polar Electro, Kemple, Finland) continuously during the waking hours over the following 4 days. PAEE was calculated by subtracting REE from the estimated average daily EE, and thereafter averaged over the 4 day period. PAEE is body size dependent, e.g. it requires more energy to move a heavier body. Similarly, aerobic fitness is highly correlated with body size or mass, e.g. larger individuals have a higher absolute aerobic capacity. Therefore, body size is a confounding factor that needs to be normalised for when analysing associations between EE variables and metabolic outcomes [17]. As such, both baseline and follow-up measures of aerobic fitness and PAEE were expressed relative to fat-free mass (FFM; kJ kg−1 FFM min−1) to adjust for between-individual differences in body size [17, 18].Free-living total body movement was assessed with an MTI Actigraph (formerly known as the CSA activity monitor) model WAM7164 (Manufacturing Technology, Fort Walton Beach, FL, USA). The accelerometer was worn over the same 4 day period as the HR monitor. Participants who did not manage to record at least 500 min/day of activity for at least 3 days were excluded from analyses. A customised program was used for data reduction and further analyses (MAHUffe; http://www.mrc-epid.cam.ac.uk). The outcome variable derived from accelerometry presented in this study is total body movement (counts per day), adjusted for monitored time, which is an indicator of the overall level of physical activity. Calculation of the metabolic syndrome z score A summary score of clustered metabolic risk based on WHO criteria [1] was calculated by summing standardised values for waist circumference, triacylglycerol, fasting insulin and glucose, systolic blood pressure and the inverse of HDL-cholesterol [7–9]. Variables were standardised by subtracting the sample mean from the individual mean and dividing by the SD. Baseline and follow-up z scores were computed with the same transformation, e.g. using the mean and SD of baseline values. This continuously distributed metabolic risk variable (zMS) was also calculated without the adiposity component (i.e. waist circumference, zMS-ob). This clustered metabolic risk score increases statistical power as variables were not dichotomised, and includes a continuous measure of glycaemia (unlike some other measures of vascular risk [19]). Additionally, the International Diabetes Federation criteria were used to define metabolic syndrome as a dichotomous variable [1]. This score predicts hard clinical endpoints [20]. Statistical analyses Descriptive summary statistics were calculated separately for men and women using means and SDs at baseline and follow-up. t tests were used to examine whether there were any differences in baseline characteristics between those with and without follow-up data, as well as those with missing accelerometer data. Fasting insulin and triacylglycerol values were log transformed (ln) due to their non-normal distribution. In order to characterise change in physical activity and fitness in the ProActive cohort, we described the association between exposure variables using correlation coefficients. We then examined the proportion of metabolic syndrome and the mean clustered metabolic risk score among participants who increased and decreased their PAEE, fitness and total body movement over 1 year. We used linear regression to model change in PAEE and fitness (both per unit FFM), and total body movement ([total counts per day]/1,000, adjusted for monitored time), against individual subcomponents of the clustered metabolic risk score at follow-up (waist circumference, blood pressure, fasting triacylglycerol, insulin and glucose, and the inverse of HDL-cholesterol). These models were adjusted for age, sex, waist circumference (except when waist circumference was modelled as the outcome), smoking status, SES (assessed by self-reporting) and baseline phenotype, and were presented as standardised β-coefficients. We then tested whether change in PAEE, fitness and total body movement was associated with the clustered metabolic risk score at follow-up. The first model (obesity dependent) included all metabolic subcomponents (zMS) and was adjusted for age, sex, smoking status, SES and baseline zMS, while the second model excluded waist circumference from the zMS (obesity independent zMS-ob) and was adjusted for age, sex, smoking status, SES, baseline zMS-ob and waist circumference. Finally, we examined whether there was evidence of interaction between change in PAEE, total body movement and aerobic fitness and clustered metabolic risk, as well as interaction by age and sex. All data were analysed in continuous form, although some data were dichotomised for illustrative purposes. All analyses were completed using Stata Version 8.0. (STATA, College Station, TX, USA). Results Table 1 shows the anthropometric and metabolic characteristics of participants with complete data for baseline and follow-up (n = 321) stratified by sex. More women met the inclusion criteria and agreed to take part in the study than men. Participants with missing data at follow-up were slightly shorter than those with complete data (difference = 3.1 cm, p < 0.05) but not significantly different for all other baseline variables. There were also no significant differences in baseline characteristics between participants with and without accelerometer data (data not shown). In general, men were significantly taller and heavier than women but had a lower percentage body fat at baseline. In terms of the six metabolic syndrome subcomponents, waist circumference, systolic blood pressure, triacylglycerol, fasting glucose and insulin were all significantly higher in men, while HDL-cholesterol was significantly higher in women. Statistically significant differences were also observed for PAEE (kJ kg−1 FFM min−1) and (ml kg−1 FFM min−1), with men achieving higher levels of physical activity and fitness even after adjustment for FFM. There were no significant differences in total body movement between men and women at baseline. Table 1Anthropometric and metabolic characteristics of ProActive participants with complete baseline and follow-up data (n = 321), stratified by sex MenWomenBaseline (n = 129)Follow-up (n = 129)Baseline (n = 192)Follow-up (n = 192)Age (years)40.2 (5.8)N/A40.8 (6.1)N/AHeight (cm)177.8 (6.8)177.8 (7.0)163.4 (6.1)a163.2 (5.9)cWeight (kg)89.3 (15.5)89.5 (15.6)73.7 (14.6)a74.0 (15.2)BMI (kg/m2)28.2 (4.3)28.3 (4.4)27.6 (5.2)27.8 (5.4)dBody fat (%)25.6 (5.3)25.8 (5.2)34.6 (6.9)a34.8 (7.1)Waist circumference (cm)100.3 (11.1)100.9 (11.5)87.9 (11.8)a89.2 (12.5)cSystolic blood pressure (mmHg)127.1 (11.3)124.5 (11.3)d120.6 (13.8)a116.8 (13.3)cDiastolic blood pressure (mmHg)81.4 (8.8)79.6 (9.7)d76.2 (9.3)a73.9 (9.9)cTriacylglycerol (mmol/l)1.8 (1.6)1.7 (1.3)1.2 (0.6)a1.1 (0.7)Total cholesterol (mmol/l)5.2 (1.0)5.3 (1.0)5.1 (0.9)5.2 (1.0)HDL-cholesterol (mmol/l)1.2 (0.3)1.2 (0.3)1.6 (0.4)a1.6 (0.4)Fasting plasma insulin (mmol/l)70.2 (71.6)72.2 (52.3)55.0 (35.6)b56.3 (35.4)Fasting plasma glucose (mmol/l)5.1 (0.9)5.2 (1.0)4.7 (0.5)a4.8 (0.5)dPAEE (kJ kg−1 FFM min−1)0.13 (0.07)0.14 (0.08)d0.10 (0.08)b0.11 (0.07) (ml kg−1 FFM min−1)60.7 (10.54)61.2 (11.42)57.10 (10.72)b58.06 (10.38)Daily physical activity (total count)e265,000 (79,000)275,000 (106,000)269,000 (109,00)280,000 (110,000)Data are means (SD)ap < 0.001, bp < 0.05 for women vs men at baseline; cp < 0.001, dp < 0.05 for baseline vs follow-up (separately in men and women)eThese values are based on data from n = 192 ProActive participants for whom accelerometer data were available at baseline and follow-up For men, there were no significant differences in mean height, weight, BMI, body fat, waist circumference, triacylglycerol, HDL-cholesterol, fasting insulin and glucose from baseline to follow-up. The only metabolic subcomponent that changed significantly over time was systolic blood pressure, which reduced from 127.1 to 124.5 mmHg. In terms of the activity variables, men significantly increased their physical activity (PAEE) from 0.125 to 0.139 kJ kg−1 FFM min−1, but increases in fitness and total body movement did not achieve statistical significance. For women, there were no significant differences in mean weight, body fat, triacylglycerol, total cholesterol, HDL-cholesterol and fasting insulin from baseline to follow-up. Mean height decreased very slightly, while BMI increased from 27.6 to 27.8 kg/m2 and waist circumference increased from 87.9 to 89.2 cm. Values for both diastolic and systolic pressure decreased significantly, while fasting plasma glucose increased slightly from 4.7 to 4.8 mmol/l. Increases over the year in PAEE, total body movement and fitness did not reach statistical significance. There was no significant change in the use of cardiovascular therapies during follow-up (data not shown). There was no evidence for a correlation between change in fitness and change in PAEE (r = 0.1) or total body movement (r = 0.0). There was limited evidence for a positive correlation between change in PAEE and total body movement (r = 0.3). The overall prevalence of metabolic syndrome was 20% at both baseline and follow-up, although there was some movement between groups. The proportion of metabolic syndrome at follow-up was higher among participants who failed to increase their PAEE, fitness and total body movement over 1 year (Fig. 1a). Similarly, clustered metabolic risk scores were lower among individuals who managed to increase their PAEE, fitness and total body movement during follow-up (Fig. 1b). None of the differences between these groups reached statistical significance but were in the expected direction of effect. Fig. 1a Proportion of ProActive participants with metabolic syndrome at 1 year follow-up by those who increased and decreased their PAEE, fitness and total body movement. b Mean metabolic summary score for ProActive participants at 1 year follow-up by those who increased and decreased their PAEE, fitness and total body movement (bars represent 95% CIs) Table 2 shows the crude and adjusted associations between change in PAEE (kJ kg−1 FFM min−1), (ml kg−1 FFM min−1) and total body movement ([counts per day]/1,000) with standardised subcomponents of the clustered metabolic risk score and the whole score (zMS) at follow-up. As all outcomes are expressed in the same unit (SD), it is possible to directly compare the magnitude of associations between the different components of activity with each of the outcomes, assuming they are measured with the same degree of measurement error. In adjusted analyses, fasting glucose, insulin and HDL-cholesterol at follow-up were significantly associated with a change in fitness over time; on average, individuals who increased their fitness had reduced levels of glucose and insulin, and increased levels of HDL-cholesterol at follow-up. Similarly, fasting glucose and insulin were associated with change in total body movement over time. All other subcomponents of the clustered metabolic risk score failed to show an association with change in either physical activity, fitness or total body movement. In adjusted analyses, change in fitness, but not change in PAEE, was associated with clustered metabolic risk at follow-up with (zMS; p = 0.034) and without the obesity component (zMS-ob; p = 0.04). In addition, change in total body movement from baseline to follow-up was associated with clustered metabolic risk in both the overall (zMS; p = 0.004) and obesity-independent score (zMS-ob; p = 0.01). Table 2Crude and adjusted associations (standardised β-coefficients) between change in PAEE, and total body movement with standardised subcomponents of the metabolic summary score and the whole score (zMS) at follow-up in the ProActive cohort over 1 yearOutcome (SD)Standardised β-coefficient (95% CI)Change in PAEE (n = 318)Change in (n = 318)Change in total body movement (n = 191)CrudeAdjustedaCrudeAdjustedaCrudeAdjustedaWaist circumference−0.061 (−0.172, 0.051)−0.025 (−0.065, 0.016)−0.074 (−0.185, 0.037)−0.017 (−0.058, 0.024)−0.117 (−0.261, 0.027)−0.019 (−0.071, 0.032)Systolic blood pressure0.062 (−0.047, 0.171)0.074 (−0.004, 0.152)−0.017 (−0.126, 0.093)−0.035 (−0.113, 0.044)−0.017 (−0.158, 0.125)0.012 (−0.094, 0.118)Logged triacylglycerol−0.008 (−0.127, 0.110)−0.024 (−0.100, 0.051)−0.059 (−0.178, 0.060)−0.009 (−0.084, 0.067)−0.026 (−0.180, 0.128)−0.054 (−0.149, 0.041)Logged fasting insulin−0.064 (−0.175, 0.046)−0.024 (−0.093, 0.045)−0.094 (−0.203, 0.016)−0.069 (−0.137, −0.002)b−0.097 (−0.239, 0.045)−0.101 (−0.186, −0.017)bFasting plasma glucose−0.005 (−0.133, 0.123)−0.024 (−0.114, 0.067)−0.139 (−0.266, −0.012)b−0.090 (−0.180, 0.000)b−0.163 (−0.328, 0.002)−0.147 (−0.259, −0.035)bHDL-cholesterol (inverse)−0.053 (−0.165, 0.059)−0.056 (−0.125, 0.014)−0.115 (−0.227, −0.004)b−0.097 (−0.166, −0.029)b−0.027 (−0.167, 0.113)−0.026 (−0.121, 0.069)zMS−0.031 (−0.112, 0.050)−0.017 (−0.054, 0.020)−0.081 (−0.160, −0.001)b−0.056 (−0.093, −0.020)b−0.074 (−0.176, 0.028)−0.066 (−0.111, −0.021)bzMS-ob−0.023 (−0.103, 0.058)−0.012 (−0.053, 0.029)−0.083 (−0.163, −0.004)b−0.062 (−0.102, −0.022)b−0.065 (−0.166, 0.037)−0.068 (−0.119, −0.016)baAll coefficients adjusted for age, sex, smoking status, SES, waist circumference and baseline phenotype, except for waist circumference, which was adjusted for age, sex, smoking status, SES and baseline phenotypebp < 0.05 There was no evidence of interaction between change in EE and aerobic fitness (p = 0.28), EE and total body movement (p = 0.13) and aerobic fitness and total body movement (p = 0.13) with clustered metabolic risk at follow-up. While there was no evidence of a significant interaction between age or sex and change in the activity variables, the β-coefficients for change in PAEE appeared stronger in men than in women, and also in older rather than younger individuals. Discussion Our results suggest that small increases in physical activity, measured using accelerometry and aerobic fitness, were associated with improvement in clustered metabolic risk over a 1 year period in middle-aged individuals at high risk of developing type 2 diabetes. Our results were independent of age, sex, smoking status, socioeconomic group and baseline phenotype. This confirms our earlier cross-sectional result [13] in a prospective analysis, which provides stronger inferential evidence for the association between total body movement and metabolic risk. Further, this result is supported by our finding that the proportion of metabolic syndrome at follow-up was higher, albeit non-significantly, among groups who failed to increase their PAEE, fitness and total body movement over 1 year. Although PAEE was not significantly associated with clustered metabolic risk in our analyses, we were able to demonstrate an association between overall body movement (i.e. physical activity) assessed by accelerometry and clustered metabolic risk. This is a novel finding given that previous prospective associations between objectively measured physical activity and metabolic risk were observed in a population of healthy middle-aged Europids [7], rather than the overweight, sedentary younger adults with a high risk of developing type 2 diabetes described in this cohort. We also observed that the benefits of increased fitness and total body movement acted primarily through changes in serum glucose, insulin and HDL-cholesterol, which may suggest that blood pressure is less sensitive to changes in activity and fitness. In a comparable study, Ekelund et al. [21] observed that an increase in activity was associated with fasting insulin, triacylglycerol, 2 h glucose and clustered metabolic risk but not with systolic or diastolic blood pressure. Contrary to our findings, previous literature has shown an association between PAEE and clustered metabolic risk, both cross-sectionally [8] and prospectively over a period of 5.6 years, independently of fitness [7]. This apparent discrepancy might be explained in a number of ways. First, while the trial was powered to detect a difference in physical activity-related EE between trial arms equivalent to 2 metabolic energy equivalent (MET) h/day or approximately 30 min of brisk walking, the sample size was relatively small for a cohort analysis and may not have been large enough to detect smaller but still biologically important differences. Nonetheless, all the PAEE β-coefficients were in the same direction of effect as those for the accelerometry data but they did not reach statistical significance. Second, the precision in the methods we used to assess PAEE and body movement may affect the observed associations. PAEE from individually calibrated HR monitoring is an integrated measure of EE above rest calculated from free-living HR data. In sedentary populations, much of the daytime is spent in the region around the flex HR, which is used to discriminate between rest and physical activity. The association between HR and EE is less precise in this region, which may influence the accuracy of predicted PAEE on an individual level. Furthermore, the flex HR method may be sensitive to the fitness level of the people under investigation [22]. In contrast, accelerometry measures the vertical accelerations of the body, i.e. physical work, and is likely to be less sensitive to the characteristics of the population in terms of a sedentary lifestyle and fitness level. If most of the activities performed are locomotor activities, such as walking, measurement of body movement by accelerometry may be superior to HR monitoring when examining associations with disease risk. Ideally, the two different measurement techniques should be combined into one single piece instrument [23]. Finally, by standardising the clustered metabolic risk score, we assumed each component made an equal contribution towards defining metabolic risk. It is unlikely that each component is equally strongly associated with metabolic risk and some variables will be more important that others in different populations. It would therefore be ideal to weight each component of the score, but data are currently unavailable. Our results suggest that small, feasible changes in physical activity (total body movement) and fitness in an at-risk population may prevent progression towards the metabolic syndrome. This supports previous literature, which has demonstrated an inverse relationship between fitness, physical activity and risk of developing metabolic syndrome in a number of settings [5, 24–26]. At a population level, our findings have important implications. This group of middle-aged, slightly overweight individuals, identified through primary care registers via their first degree family history of diabetes, represents an accessible population who might benefit from increased physical activity and fitness levels. Primary care or public health practitioners might target this group for preventive action. Findings can also be extrapolated to inform the characteristics of public health initiatives. Increasing total body movement can be achieved through small changes in lifestyle activity, such as taking the stairs and parking the car further away from work, which may be more palatable to sedentary and at-risk populations than targeting changes in moderate to vigorous intensity activities. Regular physical activity participation also has multiple positive effects upon other diseases in addition to metabolic syndrome, such as stroke, coronary heart disease and cancer [27, 28]. However, currently there is limited evidence regarding the effectiveness of lifestyle interventions for individuals, at-risk groups or populations in the treatment and prevention of metabolic syndrome and this is an area of research that warrants further investigation. It is important to clarify whether metabolic risk is most effectively reduced by increases in overall EE, fitness or total body movement to inform the development and nature of preventive interventions. This study had several methodological strengths. The ProActive trial allowed objective measurement of several components of physical activity and fitness over a period of 12 months in a well-defined group of individuals accessed through primary care who were at risk of diabetes. There was a high follow-up rate (88%) and standardised measures were used throughout. We used objective measures of physical activity, which reduces the error and bias commonly associated with self-report measures and our objective measures of EE, physical activity (PAEE, total body movement) and fitness have been extensively validated in the laboratory and during free-living conditions [18]. In conclusion, small increases in physical activity assessed by accelerometry and in aerobic fitness were associated with improved metabolic risk over 1 year. Further research is needed to clarify the relationship between physical activity, EE, fitness and metabolic risk to inform advice and intervention development. We may need even more precise methods of measurement to capture the true dose effect of such relationships.

Oecologia-4-1-2270366

Scaling of offspring number and mass to plant and animal size: model and meta-analysis

The scaling of reproductive parameters to body size is important for understanding ecological and evolutionary patterns. Here, we derived allometric relationships for the number and mass of seeds, eggs and neonates from an existing model on population production. In a separate meta-analysis, we collected 79 empirical regressions on offspring mass and number covering different taxa and various habitats. The literature review served as a validation of the model, whereas, vice versa, consistency of isolated regressions with each other and related ecological quantities was checked with the model. The total offspring mass delivered in a reproductive event scaled to adult size with slopes in the range of about 3/4 to 1. Exponents for individual seed, egg and neonate mass varied around 1/2 for most heterotherms and between 3/4 and 1 for most homeotherms. The scaling of the progeny number released in a sowing, clutch or litter was opposite to that of their size. The linear regressions fitted into a triangular envelope where maximum offspring mass is limited by the size of the adult. Minimum seed and egg size scaled with weight exponents of approximately 0 up to 1/4. These patterns can be explained by the influence of parents on the fate of their offspring, covering the continuum of r-strategists (pelagic–aquatic, arial, most invertebrates, heterotherms) and K-strategists (littoral–terrestrial, some invertebrates, homeotherms). Introduction Scaling of processes and patterns to body size has fascinated biologists for centuries (e.g., Peters 1983; Damuth 2007). Plant and animal characteristics have been correlated to organisms’ body mass (m) by simple scaling relationships of the form a·mb or, if log transformed, log(a) + b log(m). Properties include various reproductive parameters, such as the mass and number of the offspring, important for understanding life history strategies of species. The scaling of reproduction reflects several important trade-offs, e.g., investment of energy in somatic versus gonadic growth, in continuous or intermittent release and in many small or a few large offspring. Studies have derived empirical regressions for offspring mass and number within one taxon (see review by Peters 1983). In addition, several partly contradictive physical, biochemical and ecological constraints related to parental energy investment, have been hypothesized to be responsible for the observed patterns (May 1978; Gordon 1989; Reiss 1989; Charnov and Downhower 1995; Hendry et al. 2001; Charnov 2001, 2002; Aarssen 2005). Although the trade-offs in reproductive investment reflect universal dilemmas that every species has to face, most work so far has covered either several progeny characteristics within a single species group or a single offspring parameter within at most two taxa (Moles et al. 2005a; Grubb et al. 2005). In the present paper, we will focus on the relationship of offspring mass and number to adult weight for miscellaneous groups of species. The aim is to obtain a set of straightforward equations that are coherent with allometric theory and underpinned by empirical regressions available in literature. To this end, we will first derive body-size functions for the mass and number of offspring delivered in a reproductive event which comply with allometric macroecology (e.g., West et al. 1997; Hendriks 1999). Next, we will compare the acquired relationships to independent empirical regressions collected in a meta-analysis. Separating model development and testing allows for a comparison of theoretical and empirical evidence, reducing weaknesses of both. In this approach, model predictions of, e.g., offspring mass, are underpinned by independent measurements. Likewise, isolated empirical regressions for this reproductive parameter are checked for consistency with body-size dependence of related ecological factors, such as population production or survival. In our analysis, we will seek a balance between model transparency and accuracy, needed for underpinning assumed mechanisms and identifying exceptions. The equations are not intended to cover detailed or speculative differences, observed in empirical regressions for sufficient taxa. In our interpretation, we will follow the classical distinction between r-strategists and K-strategists (MacArthur and Wilson 1967; Pianka 1970) as well as more recent applications thereof (Winemiller and Rose 1992; McCann and Shuter 1997; Cox and Moore 2005). However, it should be emphasized that the traditional classification of many small offspring versus few large offspring as used in the present paper is no more than a convenient way to structure the discussion. Materials and methods Regression collection and treatment Allometric regressions derived from data before 1983 were collected from an extensive review (Peters 1983). Papers published afterwards were obtained by screening books and by an electronic literature search in Web of Science in 2006. All body-size correlations acquired for the meta-analysis were checked in the original papers. Collection and treatment of data by the authors was assumed to be adequate. Where multiple types of regressions were reported, we took the least-squares equations, to increase compatibility with the other studies. If regression characteristics were not given or suspected to be wrong, we derived them from the original data or digitized graphs. To allow for comparison, all values were converted to fresh biomass, using dry–wet weight fractions. A database containing dry matter fractions of 110 species, collected by the present authors for various purposes, yielded averages (with 95% confidence intervals) of 20% (18–22%) for herbs, 22% (19–25%) for invertebrates, 25% (18–32%) for cold-blooded vertebrates and 30% (21–39%) for warm-blooded vertebrates. In addition, the literature reports means of 53% for wood, 15–21% for fruit, 87% for seeds, 23 for insects, 19% for marine invertebrates, 25% for fish and 29% for birds and mammals (Peters 1983; Suzuki 1999). Although the samples may be somewhat biased towards particular taxa, average levels do not deviate substantially. In the present analysis, we therefore used dry–wet weight fractions of 20% for fruits, forbs, herbs and invertebrates, of 25% for cold-blooded vertebrates, of 30% for warm-blooded vertebrates, of 40% for shrubs and trees (wood and leaves together) and of 80% for seeds. Differences between species around these values have, on average, a negligible influence on the intercept of the regressions. Even more apparent, conversion does not affect the slope of the allometric relationships. Seeds mass was multiplied by 80/20% = 4 to arrive at the fresh weight of sprouts with a water content that is comparable to that of eggs and neonates. Since total plant weight is not used as the independent variable for vegetation regressions, we took the closest alternatives, being the above-ground or stem mass. In one study on insects, animal size was expressed on a length basis instead of a weight basis (Berrigan 1991). For these regressions, length (m) was converted to weight (kg) as mass = 1/152π × 103 length3, assuming that the total size equals 1.5 times the elythra length and twice the abdomen length. Rate constants for reproduction of plants were reported as annual standing reproductive mass delivered by all plants, including both reproducing and non-reproducing individuals. We assumed that the seed and fruit mass are renewed each year. For animals, total clutch mass reflected the total reproductive mass released in an event or present in ovaries, sometimes calculated in the original studies as a product of the total number of eggs (neonates) in a clutch (litter), the number of eggs per clutch (neonates per litter) and the egg (neonate) weight. To allow for comparison between plants, invertebrates, cold-blooded vertebrates and warm-blooded vertebrates, we calculated the arithmetic average of all slopes (b) reported for each taxonomic group. To correct for differences in the amount of information included in the regressions, we also computed an average weighted by the number of data. For instance, the weighted average of a regression with b = 0.5 and n = 10 and another with b = 0.3 and n = 20 was calculated to be 0.5 × 10/30 + 0.3 × 20/30 = 0.37. As we are interested in both the absence and presence of links to organism mass, weak and insignificant correlations were included in the analysis as well. Where used, however, they will be explicitly mentioned in the text. Model development The relationship of offspring mass and size to adult weight can also be derived theoretically from previous modeling efforts (Hendriks 2007). The starting point in this model is production because coefficients and slopes of allometric regressions for this parameter differ only slightly for various groups of species. The rate constant for population turnover (production kp), measured as the production/biomass ratio (P/B) (kg kg−1 day−1) is related to adult mass (m) as: The exponent (κ) was close to 1/4 for a wide range of cold-blooded species but tended to be somewhat higher for homeotherms (Peters 1983; Hendriks 1999, 2007). The coefficient (γp) represents the intercept at the standard temperature of 20°C and has a universal value of 7.5 × 10−4 kgκ day−1 for all species (Hendriks 2007). Following biochemical reaction kinetics, the intercept increases by a factor of qT as a function of temperature (Gillooly et al. 2001). Production kp by warm-blooded species with a body temperature of 37° is qT = 3.5 times higher than that of equally sized cold-blooded species at 20°C (Hendriks 2007). Production kp is allocated to somatic and gonadic growth. We will consider the rate constant for reproduction (reproduction kpr) to be a fraction (prp) of total production kp according to: In Spermatophyta, 7% of the total dry weight production is diverted to reproduction via seeds (Niklas and Enquist 2002). After correction to a dry–wet weight fraction of 20% as described in the methods section, the reproductive fraction (prp) equals 28% (Table 1). In animals, prp varies between 0 for juveniles and 1 for adults without somatic growth. Filling in the slope and the intercepts, yields the reproduction rate constant as a function of m (Fig. 1). Table 1Main factors used in the equationsSymbolDescriptionUnitValuea, sourceγpScaling coefficients for productionkgκ day−17.5 × 10−4 (Hendriks 2007)kp, kprRate constants for production and reproductionday−1Equation 1κScaling exponent–p,c,w1/4, w1/3 (Hendriks 2007)mAdult masskgVariablemrTotal reproductive mass in a batchkgEquation 5meSeed, egg or neonate masskgmin(me) = 10−10, i10−10, p,acv10−7, tcv10−3… mr/3prlFraction inter-reproductive period of average life time–0.2prpFraction of production directed to reproduction–p0.28 (Niklas and Enquist 2002), i,v1R0Fecundity, number of offspring released in a reproductive eventno. individual−1Equation 6qTTemperature quotient–p,c1, w3.5 (Hendriks 2007)τrInter-reproductive perioddayEquation 4aSuperscripts: p plants, c cold-blooded, w warm-blooded, i invertebrates, v vertebrates, a aquatic, t terrestrialFig. 1Reproduction rate constants (kpr) (kg kg−1 day−1) decrease with adult mass (m) (kg) to the power of about 1/4–1/3. Model estimations (thick lines with letter) as well as empirical regressions (thin lines) for vascular plants (A, green dotted), invertebrates (B, yellow dashed-dotted), cold-blooded vertebrates (C, blue dashed) and warm-blooded vertebrates (D, red solid), as listed in Table 2. d−1 Day−1 Usually seeds, eggs and neonates are not delivered continuously but released in discrete batches, i.e., sowings, clutches and litters. The total offspring mass (mr) accumulated in a period between two successive reproductive events (τr) equals: The inter-reproductive interval (τr) might be related to the organisms’ metabolism and to environmental conditions. Initially, we did not find empirical regressions that link τr to organism size. However, many other biological time parameters, including gestation and weaning time of homeotherms, scale to m with an exponent of κ (Peters 1983; Hendriks 1999). For an average mammal, the age at birth, weaning off and average death is at about 2, 3, 50% of the total period from conception to death, respectively (Peters 1983). τr, covering at least gestation and weaning (2 + 3%), thus equals at least (2 + 3%)/50% = 10% of the average lifetime τl, i.e., τr = prl·τl > 10%·τl. To allow for additional time to recover and mate we set the inter-reproductive period at a fraction (prl) = 20% of the average lifetime τl. This lemma is indirectly demonstrated by one long-term study on metabolism, reproductivity and average lifetime (τl) of earthworms (Mulder et al. 2007). In fact, although no evidence of a correlation between τr and the offspring was found, τl and m were strongly correlated. Following a more general theory, τl can indeed be defined here as the mean residence period in the population and can thus be replaced by 1/kp (Odum 1983). In this way, τr can now be assumed to equal: Filling in the parameter values for mammals yields τl = 0.3/(3.5 × 7.5 × 10−4 m−1/4) = 114 m1/4. This is a factor of 2 larger than the value of 55 m0.23 (r2 = 0.37, n = 41) derived from a compilation of data on South American mammals that was published after our estimation (Pereira and Daily 2007). Obviously, this relationships needs to be confirmed for other species groups. Substituting Eq. 4 into Eq. 3 yields the reproductive mass (mr) accumulated between batches as a linear function of adult size (m) as (Fig. 2): Fig. 2Clutch or litter (reproductive) mass (mr) (kg) increases with m (kg) to the power of about 3/4 to 1. Legend as in Fig. 1. Regressions listed in Table 3 The total reproductive mass derived from Eq. 5 can be divided into many small or a few large young. At one end of the interval, species called “r”-strategists here, maximize the number of young per female (R0) up to a limit set by the absolute minimum size [min(me)] of a seed, egg or neonate. The existence of such a threshold has been attributed to various restrictions but, to our knowledge, supporting quantitative evidence is still lacking. Since each individual starts off as a single cell, the average size of parental cells may serve as a first indication for the minimum egg or seed mass (me). The weight of a cell is reported to be in the range of 10−15–10−11 kg for plants and animals (Raven 1998; West et al. 2001; Mulder et al. 2005). Lower taxa with multi-cellular organization, such as mycorrhizal conidia and fruit bodies of basidiomycetes, actually release offspring as single cells (Mulder et al. 2003). Higher species reproduce by multi-cellular sexual spores, seeds, eggs and neonates, with sizes at or above that range. With this variability in mind, min(me) was tentatively set at the minimum observed for the taxon covered (Table 1). At the other side of the range, “K”-type organisms invest all reproductive mass into a few young. In this strategy, seed, egg and neonate mass are maximized by dividing the total reproductive mass over a few offspring. Low values are noted for, e.g., birds with an average of 4.8 eggs per clutch and for mammals with means of 2.7–2.8 neonates per litter (Blueweiss et al. 1978; Ernest 2003). Apparently, the risk of premature death is typically spread over, on average, at least three young, so that the maximum mass of individual offspring [max(me)] is calculated as max(me) = mr/3 = prp·prl·m/3. With seed, egg and neonate mass ranging between min(me) and max(me), we can now derive R0 as: The number of seeds, eggs and neonates (R0) is thus expected to scale to m with exponents in the range of 0–1, for K-strategists and r-strategists, respectively. The corresponding intercepts are prp·prl/min(me) and 3, respectively. Intermediate values indicate that both types of species are present in the sampled taxon. If strategies are uniformly distributed among species, one expects a mean slope of 1/2, with the geometric average of prp·prl/min(me) and 3 as a coefficient. Vice versa, the mass of a seed, egg or neonate (i.e. me) varies between min(me) ∝ m1 and max(me)  ∝ m0, also with an average at m1/2 (Figs. 3, 4). Fig. 3Egg or neonate mass (me) (kg) increases with m (kg) to a power varying around 1/2 for cold-blooded animals and in the range of 3/4–1 for warm-blooded animals. Legend as in Fig. 1; in addition, model estimations for organisms in general (E, dashed-dotted, me = 0.0000046 m1/2, me = 0.2 m). Regressions listed in Table 3Fig. 4Seed, egg or neonate number in ovaries, clutch or litter (R0) (young/individual) increases with m (kg) to a power varying around 1/2 for cold-blooded animals and close to 0 for warm-blooded animals. Legend as in Fig. 1; in addition, model estimations for organisms in general (E, dashed-dotted, R0 = 45,000 m1/2, R0 = 1). Regressions as listed in Table 3 Equations 4–6 apply to cases where τr is related to the metabolism of the organisms. Alternatively, τr may depend on cyclic processes in the environment. Since optimal conditions for offspring usually occur in specific seasons, synchronization of reproduction to annual cycles of temperature, light or food availability by e.g., hibernation or fetal growth retardation, is most likely. With τr = 365 days, Eq. 3 becomes: indicating that the mass released in a yearly sowing, clutch or litter (mr) scales to m with an exponent of 1−κ. Organisms with an intrinsic τr close to 1 year can exploit such synchronization most readily. Smaller species may somewhat postpone their reproduction, increasing the gonadic mass released. Larger species may do the opposite, decreasing the slope of the regression towards 3/4. Fitting Eq. 1 into 4 and writing m explicitly gives: Using the typical values for the parameters (Table 1), cold-blooded and warm-blooded organisms with a τr of 365 days are expected to have an m of about 4 and 500 kg, respectively. Organisms that are much smaller mature sufficiently fast to allow multiple generations within a year or growing season. Species which are substantially larger need more than 1 year to produce one egg or neonate. Results Reproduction rate constant We will now compare the derived equations to the empirical regressions obtained in the meta-analysis. The few correlations available for gonadic growth show that reproduction kpr indeed decrease significantly with increasing adult mass (P < 0.0001…<0.002, Table 2). Average slopes for plants and animals are in the range of −0.16 to −0.39 (Table 4). Slopes for warm-blooded species tend to be steeper than those for cold-blooded species. The empirical regressions for seed plants were derived from one study, with annual reproductive mass expressed as a function of foliage and stem mass because total weight correlations were not provided (Fig. 1; Niklas and Enquist 2003). The invertebrate regressions apply to short-term studies on different types of copepods. The outlier noted in field experiments with sac spawners was not confirmed by other correlations, including those for high food levels (Hirst and Bunker 2003, p 1,995). Low intercepts for fish and mammals apply to long-term observations, including intervals without gonadic growth (Charnov 2001; Charnov et al. 2001). High values were noted for reproductive growth in gestation periods (Payne and Wheeler 1968; Blueweiss et al. 1978). Reproduction within primates is slower, as earlier noted for production (Table 2, no. 34; Western and Ssemakula 1982; Hendriks 2007). Table 2kpr (kg female kg−1 day−1) as a function of m (kg) according to empirical regressions (y = amb) collected in the meta-analysis. For abbreviations, see Table 1NoTaxonabnr2PSource1Spermatophyta1.8 × 10−4−0.162790.75<0.0001Niklas and Enquist (2003)2Spermatophyta3.0 × 10−4−0.334180.75<0.0001Niklas and Enquist (2003)11Copepoda9.7 × 10−4−0.2530810.06<0.001Hirst and Bunker (2003)12Copepoda1.3 × 1000.174520.05<0.001Hirst and Bunker (2003)13Copepoda2.7 × 10−3−0.26350.320.002Kiørboe and Sabatini (1995) 14Copepoda1.2 × 10−3−0.26100.720.002Kiørboe and Sabatini (1995)21Osteichthyes3.0 × 10−4−0.191390.74<0.0001Charnov et al. (2001)31Mammalia4.9 × 10−4−0.331920.89<0.0001Charnov (2001)32Mammalia1.9 × 10−3−0.43920.65<0.0001Blueweiss et al. (1978)33Mammalia3.6 × 10−3−0.40300.90<0.0001Payne and Wheeler (1968)34Primates8.4 × 10−4−0.44150.93<0.0001Payne and Wheeler (1968) The differences between regressions for the reproduction kpr (kg kg−1 day−1) follow the trends expected from the model, based on average turnover kp (Eq. 2). That is to say, slopes −κ for both reproduction and production tend to be close to −1/4 for heterotherms and at or below −1/3 for homeotherms (Table 4; White and Seymour 2004; Hendriks 2007). Intercepts for seed growth in plant populations, including all life stages, follow Eq. 2, independently confirming the value of 28% for the prp. The reproduction rate regressions for animals are expressed on a female basis. With the exception of deviations noted for some regressions on copepods and primates, levels for true gonadic growth in heterotherms are within a factor of 3 of the model, based on a γp of 7.5 × 10−4 kg kg−1 day−1 previously derived for production (Fig. 1). In general, the variability of the intercepts for reproduction reflects differences in methods, conditions and species and is similar to the variability observed for, e.g., individual growth or consumption (Hendriks 2007). Keeping this variability in mind, we might approximate reproduction by 0.28 kp in plants and 1 kp in adult animals as predicted by Eq. 2 of the model. Total offspring mass in a reproductive batch With average production kp as a predictor of reproduction, we can now compare measurements and estimations for the reproductive masses and numbers (Fig. 2). Regressions indicate that total seed, egg and neonate weight increase with parent size (P < 0.0001…<0.05 in Table 3, Fig. 2). Most 95% confidence intervals for the average of the slopes encompass 3/4 or 1, but the whole animal size range can only be covered by an exponent of 1 (Table 4). The outcome for plants is dominated by a regression with a slope of 0.67 observed for fruit versus stem mass (1 in Table 3). Expressed on leaf mass, annual standing reproductive weight in plants scaled to 0.84 (Niklas and Enquist 2003). Comparing weighted averages, the mean slope for invertebrates 0.95 is significantly higher than the confidence interval calculated for plants and cold-blooded vertebrates (Table 4). The confidence intervals noted for exponents of birds and mammals cover 3/4 but not 1. All intercepts for cold-blooded and warm-blooded species are close to each other with the exception of one low value noted for fish (Fig. 2, Table 3; Stolz 2005). This outlier, however, reflects year-round observations while other regressions apply to peak gonad mass. Similar intercept differences were noted for the reproduction kpr. Table 3Reproductive mass [sowing, clutch, litter (mr); seed, egg, neonate (me)] (kg) as well as the number of offspring (seed, egg, neonate; R0) as a function of m (kg), according to empirical regressions (y = amb) collected in the meta-analysis. Values in italics approximated by us because not/wrongly reported in the original study. Offspring mass or number measured in event (clutch, litter) or organ (ovary, fruits). n.a. Not available, n.s. not significant; for other abbreviations, see Tables 1 and  2No.Taxone, omr (kg)me (kg)R0 (no./female)Sourceabnr2Pabnr2pabnr2P1Spermatophytaao1.1 × 10−10.674180.75<0.0001Niklas and Enquist (2003)2Angiospermaeao1.1 × 10−11.02150.87<0.00015.8 × 10−60.23150.080.302.1 × 1051.01150.490.004Aarssen and Jordan (2001)3Spermatophytao5.7 × 10−21.08130.94<0.00011.0 × 10−50.60130.260.076.3 × 1020.43130.200.13Greene and Johnson (1994)4Angiospermaeo5.5 × 10−20.95570.68<0.00011.4 × 1030.562200.38<0.0001Shipley and Dion (1992)5Spermatophytao8.0 × 10−50.472240.43<0.001Moles et al. (2005b)6Spermatophytao4.6 × 10−60.602260.25<0.0001Grubb et al. (2005)11Rotifera–3.6 × 10−50.60430.550.001Walz et al. (1995)12Copepoda–4.0 × 10−30.93210.87<0.001Kiørboe and Sabatini (1995)13Copepoda–1.2 × 10−50.62410.75<0.001Kiørboe and Sabatini (1995)14Crustaceae2.5 × 10−21.16230.99<0.00015.2 × 10−80.24220.82<0.00012.5 × 1040.59230.59<0.0001Blueweiss et al. (1978)15Dipteraao6.7 × 10−11.00480.75<0.052.1 × 10−60.31480.19<0.052.1 × 1050.62480.19<0.05Berrigan (1991)16Hymenopteraao1.1 × 10−10.96310.80<0.051.4 × 10−30.89310.75<0.057.9 × 1010.07310.02n.s.Berrigan (1991)17Coleopteraao6.7 × 10−20.79560.82<0.051.5 × 10−30.69560.72<0.054.6 × 1010.10560.05n.s.Berrigan (1991)18Araneomorphaee1.3 × 1001.09130.900.00012.7 × 10−50.43330.620.00014.7 × 1040.66390.700.0001Marshall and Gittleman (1994)19Aphidaee1.2 × 1030.161370.35<0.0001Llewellyn and Brown (1953)20Cephalopoda–2.2 × 10−50.26180.080.25Wood and O’dor (2000)21Osteichthyeso1.3 × 10−21.04200.85<0.001Stolz (2005)22Osteichthyes-Reptiliaoe1.1 × 10−10.92850.86<0.00011.2 × 10−50.43630.820.00071.2 × 1040.551210.56<0.0001Blueweiss et al. (1978)23Osteichthyeso1.5 × 10−11.0890.91<0.0001McCann and Shuter (1997)24Osteichthyeso4.4 × 10−20.86200.91<0.0001McCann and Shuter (1997)25Osteichthyese4.7 × 1040.791080.62<0.0001Winemiller and Rose (1992)26Osteichthyese1.4 × 1050.781080.35<0.0001Winemiller and Rose (1992)27Salamandraee2.2 × 10−20.64740.89<0.012.0 × 10−50.25250.190.027.1 × 1020.31250.250.01Kaplan and Salthe (1979)28Salamandrae–1.8 × 10−40.37250.58<0.0001Kaplan and Salthe (1979)29Salamandrae–7.2 × 10−40.49210.550.0001Kaplan and Salthe (1979)30Reptiliae1.5 × 10−10.88350.96n.a.7.5 × 10−30.42350.70<0.00012.1 × 1010.48540.78<0.0001Blueweiss et al. (1978)31Avese2.9 × 10−10.641140.69<0.0016.9 × 10−20.67760.88<0.0014.1 × 100−0.081140.10<0.001Cabana et al. (1982)32Avese2.1 × 10−10.742200.85<0.00015.3 × 10−20.771600.83<0.00014.9 × 100≈01000n.a.Blueweiss et al. (1978)33Avese1.9 × 10−10.723500.79n.a.5.4 × 10−20.772300.93n.a.Western and Ssemakula (1982)34Mammaliae1.1 × 10−10.84760.93<0.0014.0 × 10−20.941140.94<0.001Cabana et al. (1982)35Mammaliae1.6 × 10−10.821100.97<0.00015.6 × 10−20.922000.94<0.00012.7 × 100≈01000n.a.Blueweiss et al. (1978)36Mammaliae1.1 × 10−10.79920.95n.a.5.1 × 10−20.94910.94<0.00012.4 × 100−0.14910.74<0.0001Western and Ssemakula (1982), Sacher and Staffeldt (1974)37Mammaliae2.7 × 10−20.71950.88<0.013.9 × 1000.001000n.s.Millar (1977)38Mammaliae3.4 × 100−0.16290.54<0.001Millar and Zammuto (1983)39Mammaliae2.2 × 1020.9540.98<0.00012.8 × 100−0.12630.32<0.0001Purvis and Harvey (1995)40Primatese8.3 × 10−20.851000.93<0.0001Ross (1998)Table 4Allometric scaling of reproductive parameters as listed in Tables 2 and 3 plotted in Figs. 1–4. Measured arithmetic mean and data-weighted mean (in italics) for the regression exponent b with 95% confidence interval and model values. For abbreviations, see Tables 1 and  2ParameterReproduction rateBatch massOffspring massOffspring numberSymbolpap·kpmrmeR0mr/meUnitd−1kgkgNumber of individualsEmpirical averagePlants−0.33, −0.160.93 (0.65–1.22)0.48 (0.20–0.75)0.66 (−0.10 to 1.43)0.450.78 (0.65–0.91)0.53 (0.45–0.60)0.58 (0.38–0.78)0.25Invertebrates−0.15 (−0.49 to 0.19)1.00 (0.83–1.17)0.55 (0.35–0.75)0.32 (−0.04 to 0.68)0.45−0.20 (−0.33 to −0.07)0.95 (0.87–1.03)0.56 (0.51–0.62)0.27 (0.13–0.42)0.39Cold-blooded vertebrates−0.190.90 (0.74–1.06)0.39 (0.28–0.50)0.58 (0.26–0.91)0.510.85 (0.78–0.91)0.40 (0.27–0.53)0.67 (0.54–0.81)0.45Birds0.70 (0.56–0.84)0.74 (0.59–0.88)−0.04 (−0.55 to 0.47)−0.04Mammals−0.39 (−0.53 to −0.25)0.82 (0.75–0.88)0.88 (0.77–1.00)−0.08 (−0.28 to 0.11)−0.06Model valueCold-blooded−1/411/21/2Warm-blooded−1/4 to −1/3(3/4)–1(3/4)–10 For the overall range, empirical slopes for heterotherms approach the value of 1 expected from Eq. 5 of the model. In addition, the intercepts for plants and animals are at the level expected from the coefficients prl·prp of Eq. 5. Exponents collected for homeotherms and some large-sized heterotherms tend towards 3/4, suggesting synchronization to annual cycles as explained by Eq. 7. The steep lines observed for invertebrates in comparison to the flatter slopes of cold-blooded vertebrates confirm the prediction that heterotherms of ca. 4 kg are likely to experience some kind of synchronization. In addition, exponents for mammals, including large species weighting up to 500 kg, are close to 3/4. Exponents for birds are smaller and obviously their weight range does not include this value. Seed, egg and neonate mass Regressions show that seed and egg masses are significantly related to adult size for most heterothermic species groups (P < 0.05, Table 3). Exceptions are noted for some correlations that cover a small size range or a few data (P ≥ 0.07;Table 3, nos. 2 and 3 and 20). The slope varies within a small interval around the mid-point value of 1/2 (Fig. 3). Averages for invertebrates are higher than those for plants and cold-blooded vertebrates, mainly because regressions on egg-carrying copepods and wasps have slopes closer to 1 (Table 3, 12 and 16; Berrigan 1991; Kiørboe and Sabatini 1995). The egg and seed masses (me) for plants, invertebrates and (semi-)aquatic vertebrates of the same size are within 1 order of magnitude of each other (Fig. 3). Eggs of aquatic crustaceans tend to be smaller than those of terrestrial spiders and insects with equal body size (Table 3, nos. 12–14 < 14–18). Likewise, intercepts for cold-blooded vertebrates increase in the sequence of fish, water-bound salamanders, land-dwelling salamanders and reptiles (Table 3, 22 < 27 < 28 ≤ 29 < 30; Blueweiss et al. 1978; Kaplan and Salthe 1979). Warm-blooded animals distribute their reproductive effort over a small and size-invariant R0, leading to exponents for egg and neonate mass (me) between 3/4 and 1 (Fig. 3). The partitioning of the reproductive mass over seeds, eggs and neonate thus follows a combination of both strategies distinguished in the model. The empirical regressions collected are within the minimum and maximum boundaries set by Eq. 6 of the model (Fig. 3). The slopes for plants, most invertebrates and all cold-blooded vertebrates vary around ½ indicating some intermediate value between min(me)–m0 and max(me)–m1. Egg-carrying copepods, wasps, birds and mammals largely consists of K-strategists, with egg and neonate mass (me) exponents near those noted for clutch and litter mass (mr). Almost all regressions are above the intermediate intercept calculated by the model for an overall minimum offspring size min(me) of 10−10 kg (Table 1). Using typical minimum values min(me) of 10−7 kg for plants, fish and amphibians and of 10−3 kg for reptiles rather than the overall 10−10 kg gives a better fit of the model. Seed, egg and neonate number The number of seeds and eggs released by heterotherms scales to size with exponents largely between 1/2 and 1 (Table 4, Fig. 4). The difference between intercepts reflects the fraction of the lifetime covered, being near 100% for herbs and a few percent of less for trees, reflecting lifetime and batch fecundity (R0), respectively (Table 3, 2 vs. 3–4; Shipley and Dion 1992; Greene and Johnson 1994; Aarssen and Jordan 2001). The amount of eggs delivered by crustaceans, flies, spiders and aphids is significantly correlated to adult size but those for wasps and beetles are size-independent (Table 3, nos. 14–15, 18–19 vs. 16–17; Llewellyn and Brown 1953; Blueweiss et al. 1978; Berrigan 1991; Marshall and Gittleman 1994). The low intercept for vertebrate heterotherms applies to reptiles (Table 3, 30; Blueweiss et al. 1978). The clutch and litter size of warm-blooded species does not increase with adult size. In fact, four out of seven regressions have slightly negative slopes. As expected, average slopes of regressions for R0 are in the same range as those calculated for the ratio of total and individual offspring mass (mr/me) (Table 4). The largest deviation between arithmetic means is noted for plants and equals 0.66−0.45 = 0.21. However, this difference is due to one study in which sowing weight and number but not seed weight scale significantly to m (b = 1.01…1.02, P < 0.00001…0.004, vs. b = 0.23, P = 0.30). With parameters set at typical values, the model follows these trends (Table 1). Discussion Data variability The meta-analysis yielded regressions for various species groups. However, important phylogenetically lower taxa, such as bacteria, algae, fungi, mosses, ferns, Protozoa and annelids have not been included in allometric relationship on reproductive parameters. Budding or binary fission in unicellular organisms yields new cells that become independent at about 30–50% of the parent cell volume, respectively (Woldringh et al. 1993). Unicellular taxa thus maximize the mass rather than the number of their offspring. Fungi, mosses and ferns release spores that are at the lower end of the palynological range noted for seed plants (e.g., Nilsson 1983; Haig and Westoby 1991; Moore et al. 1991; Mulder and Janssen 1999). Their strategy is thus comparable to that of some seed plants, such as orchids. Additional study of the size-related reproduction of these cryptogams is required. Plant offspring is characterized by seed and fruit mass with or without ancillary tissues, while adult mass refers to leaves and/or stems. Animal studies either cover the whole annual cycle or reproductive periods only. In addition, deviations from average values of other factors, such those for length–weight conversion or life-stage may increase variability. Slopes on the trade-off between reproduction and production Despite uncertainties, clear patterns on the average trends emerge (Fig. 5). Reproduction kpr (kg kg−1 day−1) decrease with size, as expected from the model. According to Eq. 2, reproduction is considered to be a fraction (prp) of the production kp, representing the P/B of a population. Reproduction kpr by homeotherms has steeper slopes and larger intercepts in comparison with heterotherms, as earlier noted for production kp (Fig. 2, Table 2). It indicates that the trade-off between somatic and gonadic growth is rather invariant to size and metabolism. The fraction of the production directed to reproduction (prp) was found to be 28% for plants after conversion to seedling wet weight, indeed independent of the species weight (Niklas and Enquist 2002). The similarity between slopes for reproduction and overall population production indicates that such a relationship also exists for animals. However, the derivation of such a fraction is yet impeded by large variability among intercepts, by a lack of information on many heterothermic species and by the focus on adult animals instead of whole populations. Fig. 5Theoretical (lines) and empirical (dotted area) trends of seed, egg or neonate mass (mr, me) (kg) and number (R0) versus m (kg) for several species groups Slopes on the trade-off between offspring number and mass The meta-analysis in the present paper shows that the mass released in a single reproductive event (mr) (kg) is a constant fraction of the parents’ weight for a remarkable variety of plant and animal species (Table 3, Fig. 2). Over the whole range of species studied, the slope is close to 1 but exponents for specific groups, such as homeotherms and possibly plants, are smaller. Ignoring differences in slopes and intercepts, about 20% of the adult mass is released in a sowing, clutch or litter (Eq. 5). This supports, though not necessarily implies, the existence of a size-independent prp. In addition to the trade-off between somatic and gonadic growth, organisms may divert their reproductive mass over many small or a few large offspring (Fig. 5). In other words, the R0 is inverse proportional to seed, egg or neonate mass (me). As elaborated in the Model development section, one may distinguish between species with r-tactics that maximize the number of offspring with increasing adult mass (R0~m1, me~m0) and K-strategists that amplify seed, egg or neonate weight (me~m1, R0~m0). Slopes of regressions on R0 and me of the offspring generally vary around the intermediate value of 1/2, expected if species with an r- and K-strategy are equally distributed (Table 3). However, regressions on egg-carrying copepods, wasps, birds and mammals have exponents close to 1 for egg and neonate size (me) and near 0 for R0 (Table 3). These species groups thus largely consist of K-strategists. Graphically, seed, egg and neonate mass are confined to a triangular envelope that contains small species with small offspring and large species with small or large offspring (Figs. 3–5). Such a pattern has been previously recognized in original data on plants and fish. The triangle was found to be right-angled for fish, with a minimum egg size [min(me)] of 10−7 kg independent of the m (e.g., Duarte and Alearaz 1989). In plants, the envelope was reported to be obtuse-angled, where the 5%-tile and the 95%-tile of seed size scaled to total plant mass with exponents in the range of 0.21–0.32 and 0.38–0.68, respectively (Grubb et al. 2005; Moles et al. 2005b). The slopes for the lower boundary encompass the value of 1/4, known for scaling of biological time variables (Peters 1983; Hendriks 1999). Although the period to adulthood has been suggested to limit egg and seed size, such a relationship may be more complex than a 1/4 power scaling to size (see, e.g., Kiflawi 2006). In addition, some proof for the underlying mechanisms, including an explanation for its absence and presence in different species, as well as taxon-specific slope values, is yet lacking. Similar considerations apply to the upper boundary. The egg and neonate mass (me) in warm-blooded species is proportional to the weight of their parents. Each of the slopes is steeper than observed for clutch or litter mass (mr). However, confidence intervals for the averages indicate that 3/4 may be the appropriate exponent in addition to 1, at least within small size ranges such as noted for birds. Slopes of 3/4 can be understood from inter-reproductive periods that synchronize to annual cycles rather than to adult mass (Eq. 7). Yet, the difference between cold-blooded (≈1/2) and warm-blooded [(3/4)–1] species indicate that metabolism-related mechanisms may be important too. Just as size of warm-blooded adults appears to be constrained by heat exchange, neonate mass may be also be determined by thermodynamic principles. However, the exploration and underpinning of other values for the slopes is beyond the scope of the present paper. For now, we assume that the lower and upper end of the range scale to between 0 and 1. Most species groups contain both an r- and K-strategists yielding an average of slope of about 1/2, whereas some animal taxa were shown to scale to 1. Future refinement by thermodynamic principles may lead to a smaller range between, e.g., 1/4 and 3/4, still yielding average slopes around 1/2. Intercepts on the trade-off between aquatic and terrestrial habitats Having described the possible values of the slopes, we will now take a look at the intercepts. The variability of the exponents excludes detection of subtle differences between coefficients. Yet, there is an evident increase in egg size from aquatic to terrestrial habitats, both among invertebrates and vertebrates. The difference has historically been attributed to oxygen limitations as its diffusion in air is 10,000 times faster than in water (Hendry et al. 2001). However, experiments within the same species do not confirm this explanation (Einum et al. 2002). We therefore suggest that an alternative, more universal, mechanism may be more important. A parent is more willing to invest in the size of a seed or egg when it can influence its fate. Organisms have little control of transport and development in large homogeneous compartments such as water and air. Consequently, clutches of aquatic animals consist of many small eggs (Fig. 4; Blueweiss et al. 1978; Kaplan and Salthe 1979; Berrigan 1991; Winemiller and Rose 1992; McCann and Shuter 1997; Wood and O’dor 2000). Even within fish, pelagic species reproduce by smaller and more eggs than demersal counterparts of equal size (Duarte and Alearaz 1989). The intercepts for the amphibians increase in the sequence of pond breeders that attach eggs to plants followed by stream and land breeders with nests (Table 3, 27 < 28 < 29; Kaplan and Salthe 1979). Within the heterogeneous terrestrial environment, land animals can direct their offspring to sites that provide shelter or food to avoid wasting reproductive mass in unfavorable patches (Blueweiss et al. 1978; Berrigan 1991; Marshall and Gittleman 1994). The small seed size of terrestrial plants in comparison to egg masses in reptiles can be understood from the extra dispersal function of propagules in the former case. Obviously, other factors that covary with the water–land gradient may be responsible for the observed patterns. For instance, the dry–wet weight fraction of adults increases from aquatic to terrestrial habitats and a similar trend might exist in their offspring too. However, this explanation, and the related interpretation, is unlikely to cover the orders of magnitude of the noted differences. Integration of theoretical and empirical evidence Integrating these trends, we can now derive common patterns within the triangular envelope of offspring and adult mass (Fig. 5). In general, r-strategists are found amongst small adults (Fig. 5, left-hand side) with many small offspring in aquatic environments (Fig. 5, lower end). Large adults (Fig. 5, right-hand side) with a few large young live in terrestrial habitats (Fig. 5, upper end). Obviously, these patterns cannot cover all reproductive and non-reproductive characteristics associated with r−K gradients. For instance, ruderal plants typically reproduce as r-strategists, but more K-type species, especially in close canopies, may also regenerate via wind dispersal of small seeds (Grime et al. 1997; Grime 2001; Aarssen 2005). In addition, seed mass is known to be negatively correlated with the leaf size or the life span of 640 plant species (Díaz et al. 2004). Such results for plants strongly imply the existence of correlated traits in other taxa and demand further investigation.

Bioinformation-2-2-2174422

TeCK Database: A comprehensive collection of telomeric and centromeric sequences with their associated proteins

Telomeres and centromere are two essential features of all eukaryotic chromosomes. They provide function that is necessary for the stability of chromosomes. We developed a comprehensive database named TeCK, which covers a gamut of sequence and other related information about telomeric patterns, telomere repeat sequences, centromere sequences and centromeric patterns present in chromosomes. It also contains information about telomerase ribo-nucleoprotein complexes, centromere binding protein and centromere DNA-binding protein complexes. The database also includes a collection of all kinetochore-associated proteins including inner, outer and central kinetochore proteins. The database can be searched using a user-friendly web interface. Background Telomeres [1] are a series of repeated DNA sequences located at the end of chromosomes, with strand asymmetry in GC content, resulting in one G-rich strand and one C-rich strand. They serve to assure that a chromosome is replicated properly during cell division. During this process some of the telomeres are lost. Eventually little or no telomere remains, and the cell dies. [2] Telomerase is an enzyme that adds specific DNA sequence repeats, (“TTAGGG” in all vertebrates) to the 3’ end of DNA strands, in the telomere regions at the ends of chromosomes, thus preventing it from getting shorter. Centromeres [3] are DNA sequences contained in the heterochromatin, responsible for the segregation of each chromosome into daughter cells during cell division. The kinetochore [4] assembled on the centromere links the chromosome to the mitotic spindle during mitosis. As a cell tends to become cancerous [5], it divides more often, and its telomeres become very short, causing death of cells, which can be prevented by activating the enzyme telomerase. By understanding these regions of chromosomes, cancer and aging can be treated effectively and thus is a potential target of drug design approach. Hence, a database for analyzing and interpreting chromosomal information is essential. So far there had been no attempts to annotate and curate these parts of chromosomes. Methodology TECK is developed using MySQL [6] a relational database management system that serves as the backend for storing data. IIS (Internet Information Server) is used as the web server and PHP5 (Hypertext preprocessor) [7] a widely used scripting language driven by Zend engine is used as the web interface. The nucleotide information is retrieved from NCBI, [8] a national resource repository for molecular biological information. About 160 entries are obtained for both telomere and centromere. The information about the associated proteins is retrieved from SWISSPROT, [9] a protein database. About 154 entries are obtained for telomerase, 162 entries are obtained for centromere binding proteins and 170 entries are obtained for kinetochore. Hyperlinks are provided to the corresponding databases for each entry with their IDs displayed. Each entry is given an unique ID called TeCK ID consisting of three parts, the first part indicating the class and the second depicting the organism and the last denoting the position of that particular entry in the table. About 16 tables had been created all of which are linked using their unique TeCK ID. Each protein entry is described using twenty parameters such as protein name, gene name, source, lineage, function, other interacting proteins etc. The nucleotide information is described using parameters such as chromosome number, keywords, lineage, the articles along with the author details and references. BLAST [10] is a search tool for comparing biological sequences. A stand alone BLAST Program obtained from NCBI has been installed for searching similar sequences. Accessibility TeCK database can be accessed via internet and the screen shot of the homepage is displayed [Figure 1A]. There are three ways by which the user can query the database. The first one is the keyword search in home page that can be performed for five categories (Telomere, Centromere and its associated proteins along with Kinetochore proteins) by giving any text data related to organism name, protein name, function etc. to retrieve the required data. The second one is the advanced search option for specific requirements [Figure 1B], where the user can fill the form by specifying the input parameters such as SwissProt, Interpro, Pfam, BLOCKS, PDB, ModBase IDs or TeCk ID. Alternatively, the users can also browse the database [Figure 1C] by organisms and category. On selection of the required query, a result page containing the number of hits and the list of all entries ID with a short description is displayed. Detailed information can be obtained by clicking each entry ID [Figure 1D]. The sequences are displayed in FASTA format and the retrieval can be done using a separate ‘retrieve in FASTA format’ link. BLASTn can be done against TeCK database & NCBI and BLASTp against TeCK database, SwissProt & PDB. Utility As TeCK database includes name, function, sequence, subunits, and other literature information. The data for TeCK database were obtained from various curated databases and hence TeCK database helps end users to study, compare, analyze and interpret them and finds utility in control of cancer and ageing. Caveats It should be noted that the consistency depends on the source of the original data.

Oecologia-3-1-1915602

Impact of foliar herbivory on the development of a root-feeding insect and its parasitoid

The majority of studies exploring interactions between above- and below-ground biota have been focused on the effects of root-associated organisms on foliar herbivorous insects. This study examined the effects of foliar herbivory by Pieris brassicae L. (Lepidoptera: Pieridae) on the performance of the root herbivore Delia radicum L. (Diptera: Anthomyiidae) and its parasitoid Trybliographa rapae (Westwood) (Hymenoptera: Figitidae), mediated through a shared host plant Brassica nigra L. (Brassicaceae). In the presence of foliar herbivory, the survival of D. radicum and T. rapae decreased significantly by more than 50%. In addition, newly emerged adults of both root herbivores and parasitoids were significantly smaller on plants that had been exposed to foliar herbivory than on control plants. To determine what factor(s) may have accounted for the observed results, we examined the effects of foliar herbivory on root quantity and quality. No significant differences in root biomass were found between plants with and without shoot herbivore damage. Moreover, concentrations of nitrogen in root tissues were also unaffected by shoot damage by P. brassicae larvae. However, higher levels of indole glucosinolates were measured in roots of plants exposed to foliar herbivory, suggesting that the development of the root herbivore and its parasitoid may be, at least partly, negatively affected by increased levels of these allelochemicals in root tissues. Our results show that foliar herbivores can affect the development not only of root-feeding insects but also their natural enemies. We argue that such indirect interactions between above- and below-ground biota may play an important role in the structuring and functioning of communities. Introduction Over the past 20 years, studies exploring community-level processes have been based primarily on bi- and tri-trophic interactions in above-ground systems, usually involving associations among plants, insect herbivores, and their natural enemies such as parasitoids or predators (Price et al. 1980; Turlings et al. 1990; Vet and Dicke 1992; Dicke 1999). While providing valuable data on a range of selective forces shaping community structure and function, these studies have generally ignored biological processes that occur below-ground. Recently, however, it has become apparent that above-ground trophic interactions can be strongly influenced by interactions between the host plant and soil-dwelling organisms (Gange and Brown 1989; Masters and Brown 1992, 1997; Masters et al. 1993; Masters 1995; Gange 2001; Van der Putten et al. 2001; Bardgett and Wardle 2003; Bezemer et al. 2003). For instance, recent studies have shown that soil-dwelling organisms—such as root-feeding insects, arbuscular mycorrhiza fungi, and nematodes—can also influence the growth, development, and behavior of higher trophic levels below-ground, including parasitoids (Masters et al. 2001; Gange et al. 2003; Wurst and Jones 2003; Bezemer et al. 2005; Guerrieri et al. 2005) and even hyperparasitoids in the fourth trophic level (Soler et al. 2005). It is also known that above-ground herbivory can affect the development of below-ground insect herbivores (reviewed by Blossey and Hunt-Joshi 2003), when the shared host plant has previously been exposed to intense shoot damage. For example, Tindall and Stout (2001) reported that severe damage by insect foliar herbivores was correlated with a significant reduction in the body size of an insect herbivore feeding on root tissues of the same host plant, compared with conspecifics developing on roots of plants without foliar herbivores. Other studies have shown similar effects (Masters et al. 1993; Hunt-Joshi and Blossey 2005). However, the effects of foliar herbivory on the development of natural enemies in the soil, such as predators and parasitoids, remain largely unexplored. Thus far, the effects of above-ground herbivory on the performance of below-ground consumers have been explained mainly by quantitative changes in the amount of root biomass, when an extreme amount of leaf damage results in partial or complete death of the root system. However, minor levels of shoot damage, while not leading to a significant reduction in root biomass, may still affect the quality of the root tissues influencing the development of root-associated organisms (reviewed in Bezemer and Van Dam 2005). Furthermore, interactions between above- and below-ground herbivores may also be mediated by qualitative changes in the shared host plant. For example, insect herbivory frequently leads to a systemic defense response within the host plant, resulting in enhanced concentrations of secondary plant compounds in plant organs that have not been damaged by herbivory (Karban and Baldwin 1997). Secondary plant compounds commonly have a negative impact on herbivore performance (Karban and Baldwin 1997). Several studies have shown that root herbivory can result in enhanced concentrations of secondary plant compounds in the foliage, although the reverse, e.g., the effects of foliar herbivory on changes in concentrations of root secondary plant compounds, has rarely been reported (reviewed in Bezemer and Van Dam 2005). Moreover, herbivory can induce a stress response within the host plant, which can lead to a reallocation of plant compounds such as carbohydrates and soluble nitrogen between root and shoot tissues, and the concentration of these compounds is known to directly affect insect growth parameters (Masters et al. 1993; Masters and Brown 1997). It is important to stress that the mechanisms described above are not necessarily mutually exclusive. In this study, we examined the effects of above-ground herbivory on the development and survival of a below-ground insect herbivore and its endoparasitoid. Our study is based on interactions involving a naturally occurring system in Western Europe. The black mustard, Brassica nigra L. (Brassicaceae), is a widely distributed annual crucifer that is common along rivers and on disturbed sites in The Netherlands (Schaminee et al. 1998). It is attacked by several adapted specialist herbivores, including the large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae) whose larvae feed on the shoots and flowers of this and related species (Harvey et al. 2003). Mustard plants are also attacked by specialized root-feeding herbivores, including the cabbage root fly, Delia radicum L. (Diptera: Anthomyiidae) (Coaker and Finch 1971). Trybliographa rapae (Westwood) (Hymenoptera: Figitidae) is one of the main parasitoids of D. radicum in Europe (Lahmar 1982). The main aim of this investigation was to determine whether the growth, development, and survival of D. radicum and its endoparasitoid, T. rapae, are affected by foliar herbivory by P. brassicae. We also examined whether above-ground herbivory leads to qualitative and/or quantitative differences in root tissues. We compared levels of root biomass in plants with and without shoot damage, as well as levels of nitrogen in root tissues. Finally, we compared levels of allelochemicals (glucosinolates) in root tissues of plants under the two treatments. We hypothesized that above-ground herbivory by P. brassicae would result in reduced root-plant quality which would negatively affect the performance of D. radicum. Because T. rapae is a koinobiont parasitoid, D. radicum larvae continue feeding and growing during much of the parasitoid–host interaction (reviewed in Harvey 2005). As a consequence of this close association between host and parasitoid, we hypothesized that the effects of P. brassicae on D. radicum would be carried over to T. rapae. Since parasitoids are highly susceptible to very small changes in the quality of the hosts, the effect on the parasitoid should be stronger than that on the host. Materials and methods Studied species Brassica nigra, like other members of the Brassicaceae, possesses potent inducible direct defenses via the production of glucosinolates and their breakdown products; thus, the plant provides excellent potential for the study of multitrophic interactions (Van Dam et al. 2003). Pieris brassicae is a specialist chewing foliar herbivore that feeds on several species of wild and cultivated crucifers. The female butterfly typically lays broods of 7–140 eggs on the underside of a leaf. After hatching, the larvae feed gregariously until the third instar, when they disperse on the food plant. Delia radicum is a specialist chewing root herbivore that feeds on several species of crucifers. Females oviposit near the root-stem interface. The newly hatched maggots feed by boring galleries in the upper roots of the plant. Trybliographa rapae is a fairly specialized solitary koinobiont endoparasitoid that parasitizes L1–L3 instar larvae of D. radicum, as well as several closely related species. Adult parasitoid females forage via antennation, ovipositor probing, and vibrotaxis in order to find their hosts (Vet and Van Alphen 1985). The wasps readily parasitize larvae that are actively feeding in the root galleries but refuse hosts extracted from the root (Jones 1986). Females use their sharp, but flexible, ovipositor to explore infested roots to find hosts and evaluate their quality (Neveu et al. 2000). Seeds of B. nigra were collected from a single population in the Northwest of Wageningen, The Netherlands. Seeds were surface sterilized and germinated on a bed of glass pearls (pearls of 1 mm diameter). One week after germination, seedlings were transplanted into 1.2-l pots, filled with soil collected from a restoration area that was abandoned in 1996, at De Mossel in Ede, The Netherlands. The sandy loam soil was sieved (2 cm) and mixed with 10% white sand (to improve the drainage of the soil), and sterilized using gamma radiation (25 KGray) to eliminate all soil organisms. The plants were grown in a greenhouse, at 70% relative humidity, at 22 ± 1°C (day) and 16 ± 1°C (night) on a 16-h:8-h day:night cycle. Natural daylight was provided by metal halide lamps (225 μmol s−1 m−2 PAR; 1 lamp per 1.5 m2). Plants were watered daily and supplemented with nutrients (Hoagland solution) once when the plants were 3 weeks old. Pieris brassicae was obtained from the insect culture maintained at the Laboratory of Entomology of Wageningen University, The Netherlands. D. radicum and T.rapae were obtained from insect cultures maintained at the Laboratory of Ecobiology of Insect Parasitoids, of Rennes University, France. Experimental design and insect measurements To test the effect of above-ground herbivores on the performance of soil insects, we reared D. radicum and its parasitoid T. rapae on roots of plants previously exposed (treatment) and not exposed (control) to P. brassicae larvae. Twenty-five days after transplanting, three newly hatched L1 larvae of P. brassicae were carefully placed with a small brush on the youngest fully developed leaf of each plant, and enclosed in a cylindrical clip cage (5.5 cm diameter) in order to ensure that the damage was localized. After 3 days, the larvae were moved to another new leaf on the plant. The larvae were allowed to feed for a further 3 days, after which each larva and clip cage was removed. Thus, each foliar-damaged plant was exposed to feeding by three L1 P. brassicae larvae for a total of 6 days. A second set of plants was kept undamaged and served as control. To standardize the possible effects of the clip cages on plant shoots, they were also placed on control plants, following the same methods as for the foliar-damaged plants. A total of 72 plants were used in the experiment (36 plants with and 36 without foliar herbivore damage). One day after removing the P. brassicae larvae, four L1 D. radicum larvae were introduced to each plant by carefully placing the larvae with a brush onto the soil surface immediately adjacent to the stem. Using a magnifying glass, each plant was carefully checked 30 min after inoculation, to ensure that all the root fly larvae had successfully entered into the soil. For each plant, two of the larvae were healthy and two had been parasitized by T. rapae within approximately the prior 12 h. In a previous study, we reported that there was no significant difference in final root biomass in B. nigra plants with initial densities of five unparasitized D. radicum larvae per plant when compared with undamaged plants (Soler at al. 2005). Furthermore, parasitized larvae consume less plant material and attain smaller final masses than healthy hosts (Grandgirard 2003); thus, we assumed that plant root biomass would not be a limiting factor for root herbivore/parasitoid development. It is also important to stress that larvae of D. radicum are not cannibalistic (A.M. Cortesero, unpublished observations), making it possible for unparasitized and parasitized larvae to share a host plant. To obtain parasitized D. radicum larvae, approximately 300 first instar larvae were offered to 60 mated T. rapae females over the course of 4 h. To facilitate and ensure high rates of parasitism, a slice of B. napus (turnip) root of 1.5–2 cm in height (15 cm diameter) that had previously been infested with the larvae of D. radicum was offered to the parasitoid females. The larvae were collected from the root after 4 h, and 144 larvae were then transferred to the experimental B. nigra plants. Because direct observations of parasitism are almost impossible in T. rapae, 100 additional larvae from the infested turnip slice were removed and placed on a new turnip, where parasitoid emergence was monitored. From these 100 larvae, 92 T. rapae adults emerged, and none produced D. radicum adults. Following the below-ground inoculation of unparasitized and parasitized D. radicum larvae, experimental plants were placed individually into large meshed cylindrical cages (height 1 m, diameter 35 cm) to ensure that newly emerged adult flies and wasps did not escape and could be immediately captured. Plants from the two treatments were randomly distributed in the greenhouse and were moved around the room every 2–3 days to avoid any potential effects of positioning on insect development. The cages were checked twice daily (once in the morning and once in the afternoon) to record the emergence of adult flies and parasitoids. Newly emerged adults of D. radicum and T. rapae were collected with an aspirator. At emergence, the following data were recorded: (1) development time between inoculation and adult eclosion in days and (2) adult body size, based on the measurement of hind tibia length in both the herbivore and parasitoid. Hind tibia length has been commonly used as a measure of adult size in many insects, especially parasitoids (Godfray 1994; Harvey et al. 1994; Bezemer and Mills 2003). The hind tibia lengths of D. radicum and T. rapae were measured using a calibrated slide under a stereomicroscope. Adult survival (calculated as the proportion of insects that emerged relative to the number of larvae introduced in each pot) was also recorded. Plant responses Because the measurement of glucosinolate and nitrogen concentrations requires the destructive sampling of plants, root samples were taken from an additional set of plants (nine plants per treatment). The plants in the additional set were treated in exactly the same way as the foliar-damaged and control plants described above. Root material was collected 1 h before the other set of experimental plants was inoculated with parasitized and unparasitized D. radicum larvae to evaluate the quality of the control and foliar-damaged plants that larvae encountered after inoculation. After carefully washing the roots of each plant, the fine roots were separated from the main root. Roots less than 1 mm thick were considered fine (with a hair-like appearance) and those thicker than 1 mm were considered to be main roots. The roots were immediately frozen at −80°C, then freeze-dried and ground. Glucosinolate contents were determined using high performance liquid chromatography (HPLC) as described by Van Dam et al. (2004). Nitrogen content was determined using a Carlo Erba/Fisons, type EA1108, CHN analyzer. The total root biomass of the experimental plants was determined at the end of the experiment. The roots of each plant were harvested, oven-dried at 70°C and, subsequently, weighed on a microbalance. Statistical analysis The effects of foliar herbivory (with or without) and larval status (parasitized or unparasitized) on survival of D. radicum and T. rapae were tested using a generalized linear mixed model (with binomial distribution and logit link function, including plant as random term). The effects of foliar herbivory on developmental time and hind tibia length of D. radicum and T. rapae were tested using a generalized linear model (with normal distribution and identity link function). The effects of foliar herbivory and root type (primary or secondary roots) on plant quality (nitrogen and glucosinolate levels) were tested using a linear mixed model, including plant as random term to consider that the fine and main roots were sampled from the same plants. To test the effect of foliar herbivory on plant quantity (root biomass), we used analysis of variance. Normality, independence, and homogeneity of variance were checked by inspection of the residuals after model fitting. Analyses were carried out in Statistica and Genstat. Results Foliar herbivory significantly affected the survival of the root herbivore D. radicum and its parasitoid T. rapae (Wald = 12.17, P < 0.001). Relative to control plants, the survival of D. radicum and T. rapae decreased by more than 50%, when the host plants were previously exposed to foliar herbivory (Fig. 1), and the effect was qualitatively similar for root herbivore and parasitoid responses indicated by a non-significant interaction term (Wald = 0.49, P = 0.48). There was a significant effect of foliar herbivory on D. radicum and T. rapae adult size, measured as hind tibia length (Wald = 5.88, P = 0.02). Both herbivore and parasitoid adult sizes were reduced in the presence of foliar herbivores on the shared host plant (Fig. 2). Again, the negative effect of foliar herbivory did not differ between host and parasitoid (interaction term Wald = 0.02, P = 0.86). Development times of D. radicum and T. rapae were not significantly affected by foliar herbivory (Wald = 3.34, P = 0.07). Fig. 1Mean (±SE) survival of D. radicum (herbivore) and T. rapae (parasitoid) on B. nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars), and on control plants (open bars). Within herbivore and parasitoid, significant differences (P < 0.05) between treatments are indicated by different lettersFig. 2Mean (±SE) hind tibia length of D. radicum (herbivore) and T. rapae (parasitoid) on B. nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars), and on control plants (open bars). Within herbivore and parasitoid, significant differences (P < 0.05) between treatments are indicated by different letters Plants under both treatments demonstrated similar root biomass (F1,30 = 0.008, P = 0.92; Fig. 3a). Root-nitrogen levels also did not differ significantly between control plants and plants exposed to foliar herbivory (Table 1). Independent of the treatment, nitrogen levels were significantly lower in secondary than in primary roots (Table 1). Foliar herbivory significantly affected the levels of indole glucosinolates (neoglucobrassicin and glucobrassicin) in the roots (Table 1). In the presence of foliar herbivory, the levels of neoglucobrassicin (Fig. 4a) and glucobrassicin (Fig. 4b) significantly increased in both primary and secondary roots. Independent of the treatment, levels of neoglucobrassicin and glucobrassicin were significantly higher in secondary than in primary roots. Concentrations of gluconasturtin (Fig. 4c) and sinigrin (Fig. 4d) did not differ significantly between the roots of control plants and plants that had been exposed to foliar herbivory (Table 1). Independent of the treatment, gluconasturtin levels were significantly higher in primary than in secondary roots. Sinigrin levels did not differ significantly between primary and secondary roots. Fig. 3Mean (±SE) root biomass (a) and percentage of nitrogen in primary and secondary roots (b) of Brassica nigra plants that had been exposed to foliar herbivory (by P. brassicae) (shaded bars) and in those of control plants (open bars )Table 1Approximate F-test (F and P values) for the fixed effects and variance component estimates from REML (restricted maximum likelihood); analysis of the effect of foliar herbivory (by P. brassicae) and root-type on nitrogen and glucosinolate levels of B. nigra rootsFactorsNitrogenNeoglucobrassicinGlucobrassicinGluconasturtinSinigrinFPFPFPFPFPFoliar herbivory (1)1.60.215.0<0.00115.3<0.0010.50.41.70.2Root-type (2)28.4<0.00128.5<0.00189.5<0.0014.570.030.0010.9Interaction (1.2)0.40.52.90.080.20.50.70.31.40.2Fig. 4Mean (±SE) levels of glucosinolates (μmol g−1 dry mass) (a neoglucobrassicin, b glucobrassicin, c gluconasturtin, d sinigrin) in primary and secondary roots of Brassica nigra plants exposed to foliar herbivory (by P. brassicae) (shaded bars) and in those of control plants (open bars). Within each root type, significant differences (P < 0.05) between treatments are indicated by different letters Discussion This study provides evidence that shoot-feeding insects, via systemic changes in the root quality of the shared host plant, can influence the development of root-feeding insects sharing the host plant. Moreover, the effects can be carried over at least one trophic level higher, affecting the performance of a parasitoid. We found that foliar herbivory was significantly correlated with a reduction in successful development of Deliaradicum and Trybliographarapae. Survival of the herbivore and its parasitoid was two to three times higher on plants that were undamaged above-ground than on plants that were previously exposed to foliar herbivory. In addition, when compared with those on control plants, newly emerged adult D. radicum and T. rapae were significantly smaller when the insects developed on plants that had been initially damaged by foliar herbivores. Several authors have argued that body size is the main target of selection in parasitoids and other insects, because it is correlated with life-history and demographic characters such as searching and mating efficiency, longevity, and fecundity (Godfray 1994; Harvey et al. 1994; Bezemer and Mills 2003; Harvey 2005). Consequently, our results reveal not only that D. radicum and T. rapae suffer from increased mortality on plants previously exposed to foliar herbivory compared with control plants, but that fitness of the surviving adults is also compromised. In a recent study based on the same multitrophic system, we reported that root herbivory by D. radicum reduced the performance of the foliar feeder P. brassicae (Soler et al. 2005). However, several others found no significant relationship between root herbivory and the performance of above-ground herbivores (Moran and Whitham 1990; Salt et al. 1996; Hunt-Joshi and Blossey 2005), and yet others found a positive relationship (Masters and Brown 1992). These results clearly show that the effects of below-ground herbivores on their above-ground counterparts are likely to be association-specific, although this contrasts with the rather consistent negative effect thus far observed in most reciprocal studies of above-ground herbivory on below-ground insects (this study; Moran and Whitham 1990; Masters and Brown 1992; Salt et al. 1996; Tindall and Stout 2001; Hunt-Joshi and Blossey 2005). More work is clearly required to elucidate possible patterns in interactions between above- and below-ground systems, and to determine whether there are any clear patterns emerging from them. We also found that the amount of root biomass did not differ significantly between the foliar-damaged and control plants, suggesting that the negative effects of above-ground herbivory by P. brassicae on the development of D. radicum and T. rapae were based on qualitative, rather than quantitative, changes in the root system. Because only three young caterpillars were allowed to feed from the foliage for a limited period of time, the physical amount of shoot tissue removed was negligible (R. Soler, personal observation). Changes in root biomass as a result of source–sink relationships within the plant are likely to occur when above-ground herbivore damage is much more extensive (Hunt-Joshi and Blossey 2005). Root nitrogen levels were also not significantly affected by foliar herbivory, and thus it is highly unlikely that changes in this important (and often limiting) primary plant compound were responsible for the observed effects. However, levels of indole glucosinolates were significantly increased by about 30% in plants that had been exposed to foliar herbivores. Glucosinolates and their breakdown products, including indoles, have been shown to be involved in reducing plant quality for both generalist and specialist insect herbivores and nematodes (Li et al. 2000; Van Dam et al. 2005). Although indoles constituted only a small fraction of total glucosinolates in the plant (with sinigrin being by far the most prevalent), studies with other secondary compounds have shown that only trace amounts in the diet are required to exert strong effects on the development of herbivores and their parasitoids (Barbosa et al. 1986). Consequently, the negative relationship between above-ground herbivory and the development of D. radicum may be, at least partially, correlated with increased levels of indole glucosinolates in the roots of the shared host plant that were induced by feeding damage from P. brassicae larvae above ground. It has been well established that allelochemicals in the herbivore’s diet can exhibit negative effects on the morphology, development, size, and survival of their predators and parasitoids (reviewed in Harvey 2005; Ode 2006). T. rapae is a koinobiont parasitoid of D. radicum and, therefore, after being parasitized, Delia larvae continue feeding and growing until the final larval instar has been reached. At this time, the developing parasitoid larvae begin destructive feeding and exponential growth (Harvey et al. 1994). At the end of the host’s larval development, allelochemicals that may be present in the hemolymph and other tissues, such as fat body, are rapidly consumed by the koinobiont parasitoid (Harvey 2005). Due to the tight linkage between host and koinobiont parasitoid development, in our study, increased mortality and reduced adult size observed in the T. rapae adults on plants that had been exposed to foliar herbivory could probably be attributed to a corresponding increased mortality and reduced size of its host D. radicum. Parasitoids, in particular, are highly susceptible to very small changes in the quality of the host’s internal biochemical environment (Harvey 2005). Other studies have shown that parasitoids can be more affected than their hosts by the quality of the host plant (Harvey et al. 2003; Soler et al. 2005). This appears not to be the case in our studied trophic chain, since we found comparable effects of foliar herbivory on the root herbivore and its parasitoid. In summary, this study has reported that herbivore damage of plant shoots can have significant negative effects on the development of below-ground herbivores and their natural enemies. Combined with the results of a previous study based on the same system, we have demonstrated that these effects are largely reciprocal (Soler et al. 2005). Our research further highlights the importance of integrating the below-ground domain with above-ground systems in order to better understand the myriad of factors that shape the evolution, assembly, and functioning of communities and ecosystems (Wardle et al. 2005). Studies over larger spatial scales have already shown that above-ground herbivores can influence the community structure of the below-ground system and vice-versa (De Deyn and Van der Putten 2005; Wardle et al. 2004). Further studies, combining interactions between above-ground and below-ground communities in a multitrophic framework, and which seek to explore both processes and mechanisms, are required if we are to develop a better understanding of the many complex factors that influence community structure and function.

Exp_Brain_Res-3-1-2137946

Children with cerebral palsy exhibit greater and more regular postural sway than typically developing children

Following recent advances in the analysis of centre-of-pressure (COP) recordings, we examined the structure of COP trajectories in ten children (nine in the analyses) with cerebral palsy (CP) and nine typically developing (TD) children while standing quietly with eyes open (EO) and eyes closed (EC) and with concurrent visual COP feedback (FB). In particular, we quantified COP trajectories in terms of both the amount and regularity of sway. We hypothesised that: (1) compared to TD children, CP children exhibit a greater amount of sway and more regular sway and (2) concurrent visual feedback (creating an external functional context for postural control, inducing a more external focus of attention) decreases both the amount of sway and sway regularity in TD and CP children alike, while closing the eyes has opposite effects. The data were largely in agreement with both hypotheses. Compared to TD children, the amount of sway tended to be larger in CP children, while sway was more regular. Furthermore, the presence of concurrent visual feedback resulted in less regular sway compared to the EO and EC conditions. This effect was less pronounced in the CP group where posturograms were most regular in the EO condition rather than in the EC condition, as in the control group. Nonetheless, we concluded that CP children might benefit from therapies involving postural tasks with an external functional context for postural control. Introduction Poor postural control has been suggested to underlie the delays and deviations in motor skill acquisition and development observed in children with cerebral palsy (CP) (e.g., Berger et al. 1984; Liao et al. 1997; Liao and Hwang 2003). In particular, children with CP encounter problems during static upright standing in altered sensory environments (Cherng et al. 1999; Rose et al. 2002) and when rapid weight shifts during standing are required, either in gait initiation (Stackhouse et al. 2007) or in reaction to external perturbations (Nashner et al. 1983; Woollacott and Burtner 1996). Postural control is often assessed by means of posturography, that is, the quantitative analysis of centre-of-pressure (COP) trajectories as measured with a force platform. Several descriptive statistics of the COP time series, which are often derived by averaging out the assumed noisy or random character of postural sway, have been shown to change with various motor, sensory and cognitive processes involved in the control of standing (e.g., Horak 2006; Woollacott and Shumway-Cook 2002). In the past two decades, however, new concepts and methods for studying postural control have been introduced based on the assumption that the act of maintaining an erect posture may be viewed as a stochastic process (Collins and De Luca 1993; Newell et al. 1997; Riley et al. 1999; Frank et al. 2001). Based on these insights, new measures have been developed for examining the time-varying or dynamical characteristics of COP trajectories. These dynamical measures may be more informative with regard to changes in postural control with task constraints, aging or pathology than the more global summary statistics that have been typically employed for this purpose, or at least provide important additional information (see Baratto et al. 2002; Raymakers et al. 2005; Roerdink et al. 2006). Recent research has suggested that the regularity of COP trajectories is a particularly revealing and theoretically important dynamical measure. As a case in point, Roerdink et al. (2006) found that COP trajectories were more regular (as indexed by reduced sample entropy) in stroke patients than in healthy elderly while COP trajectories became progressively less regular with recovery. These findings may be viewed in line with the notion of pathological regularity versus healthy complexity (e.g., Goldberger 1996, 1997). Moreover, when performing a secondary cognitive task COP trajectories became less regular (Roerdink et al. 2006). The latter finding was replicated in young healthy adults, albeit only in the more attention-demanding eyes closed condition (Donker et al. 2007). Based on these results, a direct relation was proposed between the regularity of COP time series and the amount of attention invested in postural control (or, inversely, between COP regularity and the degree of automatism of postural control; see Donker et al. 2007; Roerdink et al. 2006). In particular, the introduction of a secondary cognitive task withdraws attention from the regulation of posture (i.e., towards the performance of the attention-demanding secondary task), resulting in less regular COP trajectories. This interpretation is in line with the claim and corresponding findings of Wulf and colleagues that adopting an external focus of attention is beneficial for the performance and learning of motor skills, whereas an internal focus of attention (e.g., directing attention to one’s own body and bodily movements) can be detrimental as it may disrupt the ‘automatisms’ with which well-learned skills are normally performed (for a review see Wulf and Prinz 2001; see also Wulf et al. 2001; McNevin and Wulf 2002; McNevin et al. 2003). When applied to CP children this insight may imply that their postural control may be improved by means of instructions or task conditions that direct their attention away from the regulation of posture itself to its consequences in the world. Providing CP children with concurrent visual feedback about postural sway may help create a more external focus of attention (i.e., the external consequences of postural sway become readily apparent via visual cursor movements). Visual feedback of postural sway provides a functional context for postural control (e.g., minimise cursor movements), which is likely to enhance the performance of the primary task (cf., Lin et al. 1998; Van der Weel et al 1991; Volman et al. 2002). For example, Volman et al. (2002) showed that the quality of reaching movements of the affected arm in children with spastic hemiparesis could be enhanced when reaching to press a light switch to turn on a light (task with functional context) compared to reaching to a marker (non-functional task). We therefore expected that postural control could benefit from postural visual feedback as it constitutes a task with a functional external context for postural control, inducing a more external focus of attention. Although postural visual feedback is sometimes used by physical therapists to train postural control and steadiness (i.e., the ability to maintain a given posture with minimal sway, Nichols 1997), evidence for this practice is limited (e.g., Rougier 2003; Ledebt et al. 2005). The recent conceptual and methodological developments in the analysis of COP trajectories alluded to in the preceding provide an alternative approach for studying the consequences of concurrent visual feedback for postural control and thus of its potential for possible therapeutic applications. Motivated by this combination of theoretical and practical considerations, the aim of the present study was (1) to compare postural sway fluctuations of children with CP with that of typically developing (TD) children and (2) to investigate the effect of visual information on postural sway in CP children and in TD children. For this purpose, the availability of visual information was manipulated relative to standing with eyes open (EO condition) by means of visual deprivation (standing with eyes closed, EC condition) and external provision of COP feedback (FB condition). Two kinds of measures were used to analyse the COP trajectories recorded during those conditions, the one scale-dependent and the other scale-independent. The conventional, scale-dependent measures pertained to the ‘amount of sway’, whereas the more recent scale-independent measures pertained to the ‘regularity of sway’ (see Methods section for details). We hypothesised that (1) compared to TD children, CP children exhibit a greater amount of sway and more regular sway and (2) concurrent visual feedback (providing a functional external context for postural control, creating a more external focus of attention) decreases both the amount of sway and sway regularity in TD and CP children alike, while closing the eyes (increasing task difficulty through deprivation of visual information, promoting a less automatic mode of postural control) has opposite effects. Methods Participants Ten children with congenital hemiplegia or cerebral palsy (CP children; six boys, four girls; mean age = 7 years, range = 5–11 years), reduced to nine in the analyses (see below) and nine typically developing children (TD children; five boys, four girls; mean age = 8 years, range = 5–11 years) without known motor impairments or movement-related disorders volunteered to participate in the experiment. All children and their parents gave their informed consent prior to participation. The experiment was carried out in compliance with the Helsinki Declaration and was endorsed by the VU University Medical Centre Amsterdam. Procedure and apparatus Children stood upright on a 1 m × 1 m custom-made strain gauge force plate1 with their arms hanging relaxed alongside the body. Their feet were placed parallel to the anterior–posterior axis of the force plate and the distance between the feet corresponded to the width of the pelvis. Children wore their own shoes, including ankle-foot ortheses and/or insoles to correct for leg-length discrepancies. Children participated in three conditions that were presented in random order: standing upright with eyes open (EO condition), standing upright with eyes closed (EC condition) and standing upright with visual COP feedback (FB condition). As explained, the FB condition was introduced to provide an external functional context for postural control, creating a more external focus of attention. To this end, a 2.5 m × 2.5 m vertical screen was placed in front of the child at a distance of 1.3 m. On this screen the force plate was depicted as a 40 cm × 40 cm square onto which the COP was projected as a red dot. The children were asked to keep this dot within the target area, which consisted of a 4 cm × 4 cm square. Deprivation of visual postural sway information was effectuated by blindfolding the children in the EC condition. Trial duration was 60 s. Participants performed each condition once, with the proviso that, as part of a separate study, five CP children performed each condition twice (the first trial was used for further analyses). Because in the latter set of recordings trial duration was 30 s, the first 3,000 samples of all trials were used in the analyses to avoid differences in the reliability of the dependent measures. One CP child did not want to be blindfolded and was therefore excluded from further analyses, which rendered the number of CP children equal to the number of control subjects. Data analysis Prior to all analyses, the mean was subtracted from mediolateral and anterioposterior COP trajectories, which transformed the original time series into x and y time series, respectively. Subsequently, x and y time series were bi-directionally filtered (second-order low-pass Butterworth filter, cut-off frequency 12.5 Hz) and the resultant distance time series r was constructed using (see Prieto et al. 1996), with i = 1, 2, 3,..., N and N indicating the total number of analysed samples in the COP time series (i.e., 3,000 samples). The ‘amount of sway’ was quantified by means of two conventional, scale-dependent measures (see Prieto et al. 1996). First, the average COP distance to the origin of the mean-centred posturogram was determined by taking the mean of the r time series (i.e., mean amplitude rmean in mm). Second, sway path length (SP in mm) was determined by taking the sum of the distances between consecutive points in the conventional posturogram, using . To examine the structure of COP trajectories in more detail, independent of its size or scale, x and y were normalised to unit variance by dividing those time series by their respective standard deviations σx and σy, resulting in a normalised posturogram. Subsequently, two scale-independent COP measures were quantified. First, the sway path of the normalised posturogram (SPn) was determined in a similar manner as described above for the conventional posturogram. Because the posturograms were normalised to its size, differences in SPn could only be the result of changes in the structure of the posturogram, with a larger SPn indicating a larger amount of ‘twisting and turning’ or ‘curviness’ in the COP trajectory (cf., Donker et al. 2007). Second, COP regularity was quantified by calculating the sample entropy (cf., Richman and Moorman 2000; Lake et al. 2002; see also Roerdink et al. 2006), which is a slightly improved version of the approximate entropy (cf., Pincus 1991). The sample entropy (SEn) is the negative natural logarithm of an estimate of the conditional probability that subseries (epochs) of length m (in our case, m = 3) that match pointwise within a specific tolerance also match at the next point (for a more formal and detailed explanation see Richman and Moorman (2000; Lake et al. 2002). In other words, smaller SEn values imply more regular COP time series, that is, a greater likelihood that sets of matching epochs in a time series will be followed by another match within a certain tolerance. On the other hand, highly irregular COP time series are characterised by the fact that sets of matching epochs tend to be followed by data samples of different values, resulting in larger SEn estimates. In the present study, SEn was quantified from the r time series, which was first normalised to unit variance. To optimise the choice of the tolerance for a given m (see also Roerdink et al. 2006) we applied the approach of Lake et al. (2002) and selected the median value of the optimal tolerance over all trials (in our case, 0.05). The code used for calculating sample entropy was obtained from PhysioNet2 (Goldberger et al. 2000). To rule out the possibility that COP trajectories exhibited regular or deterministic patterns by chance, we compared the SEn estimates obtained for the COP trajectories to those obtained for surrogate data (cf., Theiler et al. 1992). We used time-randomisation to preserve the probability distribution of the data (e.g., mean, variance) while destroying the temporal correlations in the data, resulting in very large values for SEn. In contrast, phase-randomisation randomises the phases of the data in the Fourier domain but preserves the probability distribution of the data and its spectral power distribution (cf., Kantz and Schreiber 2004). Consequently, estimates of SEn for phase-randomised COP data should be somewhat increased, although less pronounced as for the time-randomised COP data. Statistics A repeated-measures analysis of variance (ANOVA) was conducted on all dependent variables with group as between-subject factor (two levels: CP and TD children) and condition as within-subject factor (three levels: EO, EC and FB conditions). The effect of randomisation was evaluated using a one-way repeated measures ANOVA with the factor randomisation (three levels: original, phase-randomised and time-randomised time series), including each individual time series as a sample. Effect sizes for main and interaction effects are reported as partial eta squared (εp2). Besides significant effects (P < 0.05), also tendencies towards significance (P < 0.10) are reported in view of the limited sample size. Paired-samples t-tests were used for post-hoc analyses of significant condition or randomisation effects. The analyses were performed using SPSS (SPSS, Inc., Chicago IL, USA). Results The results (F, P and εp2 values) of the group by condition ANOVA for the dependent COP variables are presented in Table 1. In Fig. 1, the group by condition effects are summarised for all dependent measures. CP children’s sway amplitude was greater than that of TD children (i.e., rmean = 12.8 mm and 5.7 mm, respectively, although this difference was strictly speaking not significant, P = 0.062). Neither a significant main effect of condition nor a significant group by condition interaction effect was observed for rmean. Furthermore, no significant main or interaction effects were observed for the sway path in the conventional posturogram (SP; mean 1,367 mm, standard error 108 mm), whereas the sway path in the normalised posturogram (SPn) differed significantly between groups and conditions in the absence of a significant interaction (see Table 1). Specifically, SPn was significantly larger for the TD children (328) than for the CP children (212). Moreover, post-hoc analysis revealed that concurrent visual feedback significantly increased SPn (310) compared to both standing with eyes open (SPn = 259: t(17) = 2.41, P < 0.05) and standing with eyes closed (SPn = 243: t(17) = 2.49, P < 0.05). Table 1Main and interaction effects of group (between-subject factor, two levels: CP and TD children) and condition (within-subject factor, three levels: EO, EC and FB) on mean sway amplitude (rmean), sway path of the conventional posturogram (SP), sway path of the normalised posturogram (SPn) and sample entropy (SEn)GroupConditionInteractionF(1, 16)aP-valueεp2F(2, 32)aP-valueεp2F(2, 32)aP-valueεp2rmean4.010.0620.201.35ns0.081.11ns0.06SP1.86ns0.100.13ns0.010.13ns0.01SPn11.11<0.0050.415.12<0.020.242.04ns0.11SEn13.49<0.0050.465.53<0.010.263.230.0560.17ns not significantaIn case the assumption of sphericity was violated, the number of degrees of freedom was adjusted using the Huynh–Feldt method (corrected degrees of freedom are not listed here)Fig. 1Effects of group (CP and TD children, represented by black and grey bars, respectively) and condition (EO, EC and FB) on the ‘amount of sway’ (upper panels, mean sway amplitude rmean and sway path of the conventional posturogram SP) and on the ‘regularity of sway’ (lower panels, sway path of the normalised posturogram SPn and sample entropy SEn; note that lower SEn values imply more regular posturograms). Error bars represent the standard error As can be seen in Table 1, the main effects of group and condition were both significant for sample entropy, while the group by condition interaction tended towards significance (P = 0.056). SEn was significantly larger for the TD children (1.01) than for the CP children (0.71). Moreover, post-hoc analysis revealed that the FB condition significantly increased SEn (0.98) compared to both standing with eyes open (SEn = 0.86: t(17) = 2.37, P < 0.05) and standing with eyes closed (SEn = 0.83: t(17) = 2.42, P < 0.05). The observed tendency of the group by condition interaction towards significance (see Fig. 1, lower right panel) may be due to the fact that for CP children SEn increased gradually over EO, EC and FB conditions (0.71, 0.73 and 0.76, respectively), whereas in the TD children SEn first reduced in the EC condition (0.93) and then increased for the FB condition (1.21) as compared to standing with eyes open (1.01). The surrogate analysis revealed that SEn differed significantly with randomisation (F(2, 106) = 2915.90, P < 0.001, εp2 = 0.98). Post-hoc t-tests indicated that SEn differed significantly over all comparisons (all t(53) > 7.2, all P < 0.001), indicating that the regular or deterministic patterns observed in the original data did not occur by chance. SEn was on average (standard error) 0.89 (0.04), 1.06 (0.04) and 3.44 (0.02) for original, phase-randomised and time-randomised COP data, respectively. Discussion In the present study we compared posturograms of children with CP with those of TD children under three conditions. Across conditions, we expected the posturograms of CP children to reflect a greater amount of sway and more regular sway than those of TD children (Hypothesis 1). Furthermore, in line with the recently postulated relation between the amount of attention invested in posture and COP regularity, we expected that concurrent visual COP feedback, providing a functional external context for postural control leading to a more external focus of attention, would result in a decrease in both the amount of sway and sway regularity, whereas the EC condition, in which visual information is absent, would have opposite results (Hypothesis 2). The data were largely in agreement with both hypotheses, as will be discussed next. Furthermore, the finding that SEn estimates were significantly increased after time- and phase-randomisation of the original data ruled out the possibility that the regular or deterministic properties observed in the original data occurred by chance. CP children exhibit greater and more regular sway than TD children Posturogram characteristics of children with CP differed markedly from those of TD children (see Fig. 1). As expected (Hypothesis 1), the CP children exhibited a larger amount of sway than the TD children. In particular, a near significant (P = 0.062, accompanied by a large effect size) increment in mean sway amplitude (rmean) was observed in the CP children compared to that in the TD children (see Table 1 and Fig. 1). Although, strictly speaking, not significant due to the large interindividual variations, this effect suggests that ‘postural steadiness’ is reduced in CP children, consistent with previous results on balance control in CP children (e.g., Cherng et al. 1999; Ferdjallah et al. 2002; Liao and Hwang 2003; Rose et al. 2002). An interesting and unexpected finding was that the total COP excursion (i.e., SP) did not differ significantly between CP and TD children. In combination with the apparent increase in sway amplitude, this finding hints at the presence of marked differences in the dynamical structure of posturograms between CP and TD children. To expose those differences, we focused on the sway regularity using two scale-independent measures. Scale-independence was achieved by normalising the data to unit variance. As expected, sway path length of the normalised posturogram was significantly reduced in CP children, indicating less ‘twisting and turning’ in the posturogram (see also Donker et al. 2007). Relatedly, resultant COP trajectories of the CP children were more regular than those of the TD children, as indexed by significantly lower sample entropy values. Recently, increased regularity in COP trajectories has been reported for a variety of pathological conditions, including athletes with a sports-related cerebral concussion (Cavanaugh et al. 2005 using approximate entropy), patients with Parkinson’s disease (Schmit et al. 2006 using recurrence quantification analysis) and stroke patients (Roerdink et al. 2006 using sample entropy). These findings are in line with the more general notion of ‘dynamical diseases’ (Glass and Mackey 1988; Belair et al. 1995) and the ‘pathological regularity versus healthy complexity’ hypothesis (Goldberger 1996, 1997; Goldberger et al. 2002; Kyriazis 2003; Lipsitz 2002), according to which less ‘complex’ or more ‘regular’ physiological time series reflect less effective physiological control. In a similar vein, the observed increased COP regularity in CP children may be viewed as the dynamical signature of poor postural control. Effect of visual information We further expected that the ‘amount of sway’ would be reduced in the presence of concurrent visual COP feedback, providing a supposedly beneficial functional external context for postural control (cf., Van der Weel et al. 1991; Volman et al. 2002; Wulf and Prinz 2001), as opposed to deprivation of visual information in standing with eyes closed. However, no significant main effects of condition were found for the scale-dependent COP measures (i.e., mean amplitude and sway path, see Table 1). This unexpected result is in line with some recent studies showing that the use of visual COP feedback to facilitate quiet standing has no effect on conventional COP parameters in TD children (Lebiedowska and Syczewska 2000) and healthy elderly and stroke patients (Dault et al. 2003) alike. These findings suggest that participants may require some practice to benefit from visual COP feedback. This may explain why, in spite of the apparent lack of immediate effects, Ledebt et al. (2005) found that balance training with visual feedback improved CP children’s quiet standing ability as well as their functional limits of stability as assessed by a weight-shifting task. In contrast to the results obtained for the ‘amount of sway’ under the various conditions, the scale-independent measures pertaining to the ‘regularity of sway’ did show significant differences between the FB condition and the EO and EC conditions. This indicates that the dynamical structure of the posturogram must have been different in the FB condition compared to the EO and EC conditions, which was precisely what we anticipated when formulating our expectation that sway would be less regular in the FB condition (i.e., longer sway path of the normalised posturogram and higher sample entropy) and more regular in the EC condition (Hypothesis 2). Sway path of the normalised posturogram was indeed significantly longer with visual feedback than for standing with eyes open or closed, indicating more twisting and turning (see also Donker et al. 2007). In parallel, sample entropy increased with concurrent visual feedback compared to standing with eyes open and eyes closed. Thus, in spite of the fact that the ‘amount of sway’ did not change significantly over conditions, the observed changes in ‘regularity of sway’ were as expected: sway was more regular in the EC condition and less regular in the FB condition. Our expectations concerning the ‘regularity of sway’ with conditions were derived from the proposed direct relation between the amount of attention invested in posture and the regularity of COP trajectories (cf., Donker et al. 2007; Roerdink et al. 2006). This interpretation is in line with the insight of Wulf and colleagues that adopting an external focus of attention, i.e., a focus on the consequences of one’s movement in the external world, is beneficial for the performance and learning of motor skills (McNevin et al. 2003; McNevin and Wulf 2002; Wulf and Prinz 2001; Wulf et al. 2001). Specifically, we expected that providing CP children with concurrent visual feedback about postural sway would help shift the focus of attention to the external consequences of postural sway (i.e., cursor movements) by constituting a functional external context for postural control (e.g., minimise cursor movements). Providing a functional context for the task at hand generally enhances its performance (e.g., Lin et al. 1998; Van der Weel et al. 1991; Volman et al. 2002). In line with these notions, we expected that standing with concurrent visual feedback, a functional task inducing a more external focus of attention, would lead to less regular sway compared to the EO condition, whereas standing with eyes closed would lead to more regular sway in the EC condition compared to the EO condition because task difficulty increased due to the absence of visual information. This predicted pattern of results was only found for the TD children and not for the CP children (see lower right panel of Fig. 1). As it turned out, COP regularity in the CP children gradually decreased from the EO condition (most regular), via the EC condition, to the FB condition (least regular). As a result of this difference in group behaviour, a strong tendency towards a significant group by condition interaction on sample entropy was found. From this latter observation one could tentatively infer that CP children’s posture actually improved when standing with eyes closed compared to standing with eyes open. In support of this somewhat counter-intuitive inference, Rose et al. (2002) showed that the percentage of CP children with abnormal sway characteristics did not differ between EO and EC conditions, while the number of sway path outliers even reduced when standing with eyes closed in that particular CP group. Following a similar line of interpretation, Newell et al. (1997) suggested that very young children may create self-induced perturbations when using visual information in controlling posture, a phenomenon which may be particularly prominent in CP children given that the development of proper neural control mechanisms is strongly delayed compared to TD children (viz., Forssberg 1999). From the opposite SEn results between CP and TD children with eyes open and eyes closed it is fair to conclude that postural control in CP children is organised differently than in TD children, especially with regard to the use of normal sway-induced visual information. It has been suggested that creating an external focus of attention would allow for a more automatic control of movements (e.g., Donker et al. 2007; McNevin and Wulf 2002; Roerdink et al. 2006), which is often found to be more efficient (e.g., Blanchard et al. 2005; McNevin et al. 2003). Although no significant main effect of condition on mean amplitude was found, the finding that during quiet standing with visual COP feedback sample entropy increased (i.e., less regularity in the posturogram) in both TD and CP children supports this idea. Moreover, it corroborates the suggestion that visual COP feedback may be advantageous when seeking to improve postural control, although it must be emphasised again that it requires training (see Ledebt et al. 2005). Conclusion The findings of the present study indicate that postural control in CP children is deteriorated compared to TD children, most likely due to a slowed development of neural control mechanisms in CP children. Moreover, visual deprivation (EC condition) influenced sway characteristics differently in CP and TD children, testifying to the altered nature of postural control in CP children. In line with the proposed relation between the amount of attention invested in posture and COP regularity, we found in CP and TD children alike that, compared to the EO and EC condition, COP trajectories were less regular in the presence of visual COP feedback, corresponding to a functional context mediated external shift in the focus of attention. Future studies should examine the merits of adopting an external focus of attention to enhance postural control in CP children in training or therapy, for example by providing visual COP feedback leading to a posture specific functional task or by introducing an attention-demanding secondary cognitive task.

Dig_Dis_Sci-3-1-1914242

Gastrointestinal Endoscopic Terminology Coding (GET-C): A WHO-Approved Extension of the ICD-10

Technological developments have greatly promoted interest in the use of computer systems for recording findings and images at endoscopy and creating databases. The aim of this study was to develop a comprehensive WHO-approved code system for gastrointestinal endoscopic terminology. The International Classification of Diseases, 10th edition (ICD-10), and the ICD-10 clinical modification (ICD-10-CM) were expanded to allow description of every possible gastrointestinal endoscopic term under conditions defined by the WHO. Classifications of specific gastrointestinal disorders and endoscopic locations were added. A new chapter was developed for frequently used terminology that could not be classified in the existing ICD-10, such as descriptions of therapeutic procedures. The new extended code system was named Gastrointestinal Endoscopic Terminology Coding (GET-C). The GET-C is a complete ICD-10-related code system that can be used within every endoscopic database program for all specific endoscopic terms. The GET-C is available for free at http://www.trans-it.org/. Introduction Technological developments and the introduction of hospital information systems have greatly promoted interest in the use of computer systems for recording findings and images at endoscopy. Several database systems have been developed for this purpose, most working only as a report generator. These systems are, however, hampered by the lack of a specific code system for endoscopic diagnoses and terminology. As a result of this shortcoming, the composition of reports and storage of data differ considerably between systems. Systems use different database structures and variable terminology and are thus not compatible or comparable. The International Classification of Diseases (ICD) is designed to promote international comparability in the collection, processing, classification, and presentation of mortality statistics. This ICD is developed, published, and maintained by the World Health Organization (WHO). The system is, in its current setting, not applicable for endoscopy, as it does not include descriptive terminology. However, endoscopic findings are classified according to their appearance. Adequate description of this appearance in uniform terminology is essential for the interpretation of endoscopy reports and allows important conclusions to be made with respect to therapy and prognosis. Thus it is important to uniformly store and code endoscopic findings. This is, however, hampered by the lack of a specific, uniformly applicable code system for endoscopic terminology and findings. In addition, only about 80% of report systems are able to use a code in their database [1]. Different coding systems for medical data are being used. The ICD, 9th edition (ICD-9), and the clinical modification of the ICD-9 (ICD-9-CM) have already been used and proven to be of value in a specific and defined gastrointestinal endoscopic setting [2]. The ICD has been revised periodically to incorporate changes in the medical field. The tenth edition (ICD-10) differs from the ninth edition in several ways, although the overall content is similar. First, the ICD-10 has alphanumeric categories rather than numeric categories. Second, some chapters have been rearranged, some titles changed, and conditions have been regrouped. Third, the ICD-10 has almost twice as many categories as the ICD-9. The ICD-10 has been available since 1992 and was translated into Dutch in 1997 [3]. Other medical specialities, such as oncology and dentistry, have produced an extended code system for their field of interest based on the ICD-10 [4]. The aim of this project is to develop a comprehensive code system for gastrointestinal endoscopic terminology, based on the widely accepted ICD-10 code system, that can be used in any gastrointestinal endoscopic information system. Methods As a basis for the new coding system the latest edition of the ICD, the ICD-10, was chosen. The reason for choosing this code system was that a predecessor has already been in use for some gastrointestinal endoscopic databases [2]. In addition, the current version of the ICD is now accepted in a majority of countries throughout the world [5]. Extensions of the ICD-10 are made according to the recommendations of the WHO, without changing the structure of the ICD-10. The first four characters are never changed, but one or more characters are added behind these first four descriptors. In this way it is always possible to delete the extension to come back to the original ICD-10. We used the Dutch translation of the ICD-10. Also, the draft version of the ICD-10-CM of the National Center for Health Statistics (NCHS) was studied, which is available on the Internet [6]. Conflicts with this ICD-10 CM system were avoided, to rule out coding conflicts. A working group, the TRANS.IT working group, was founded, comprised of representatives from two academic hospitals (Utrecht, Amsterdam) and four general hospitals. This working group used the Endobase III system (Olympus). In this system, the endoscopist can choose among three methods of writing reports. Besides the Minimal Standard Terminology (MST) [7], text-blocks and standard reports are also available. A standard report is a complete report based on a diagnosis or a combination of diagnoses. With text-blocks, different portions of the text are combined to compose a complete report. These two variants enable more rapid generation of reports and are well accepted and widely used in the Netherlands [8]. In the TRANS.IT project group, which was founded as a peer reference group, standard reports, text-blocks, and linkage to the new code system were discussed. A list of every endoscopic finding, intervention, or complication described using one of the three methods of report writing available for TRANS.IT users was generated. All items on this list were linked to a specific code. Results We first generated a list of the different fields necessary for evaluation of endoscopic data (Table 1). The program automatically generated the data during the composition of endoscopic reports. All these fields are necessary to generate a comprehensive anonymous database with endoscopic information for extensive research. In addition, demographic data such as gender and age of patients and specialty of referring doctor are recorded.Table 1Different fields that need a specific code in endoscopic information systemsReason for endoscopyMedication useSedation and medication during endoscopyPreparationProcedure for investigationEndoscopic diagnosis/findingsTherapeutic and diagnostic interventionsHistology resultsTherapy startedAdvice to referring doctorComplications We included all available standard reports, text-blocks, and diagnoses of Minimal Standard Terminology (MST) accepted by the TRANS.IT group. The endoscopic findings, interventions, and complications were extracted and linked to a specific code. In this way, a total of 316 standard reports and 1571 text-blocks were coded, to assure coding of every item within this project. In total, 2593 different items were extracted and received a specific code. Of these items, 630 (24%) could be coded with the existing ICD-10. Thus, 1963 new codes were required and added to the ICD-10 system, in order to be able to specifically code every endoscopic term. The ICD-10 used was originally designed for mortality and discharge statistics. The problem with that design is that the structure used in the ICD-10 is not very detailed. This is true especially for gastrointestinal endoscopic terms, which do not always include a mortality risk but, most of the time, descriptive items essential for prognosis and therapy. For example, specific descriptions of peptic ulcers are relevant for determination of outcome risks, therapy, and prognosis. We therefore introduced the Forrest classification for description of gastric and duodenal ulcers [9]. We thus, for instance, changed the diagnosis K25.2 (acute gastric ulcer with both hemorrhage and perforation) to K25.21 (acute gastric ulcer with spurting bleeding and perforation; Forrest Ia) in the Gastrointestinal Endoscopic Terminology Coding (GET-C; Table 2).Table 2Comparison of ICD-10 and GET-C codes for gastric ulcerICD-10DescriptionGET-CDescriptionK25.0Gastric ulcer, acute with hemorrhageK25.0Gastric ulcer, acute with hemorrhageK25.01Gastric ulcer, acute with spurting bleeding (Forrest Ia)K25.02Gastric ulcer, acute with nonspurting active bleeding (Forrest Ib)K25.1Gastric ulcer, acute with perforationK25.1Gastric ulcer, acute with perforationK25.2Gastric ulcer, acute with both hemorrhage and perforationK25.2Gastric ulcer, acute with both hemorrhage and perforationK25.21Gastric ulcer, acute with spurting bleeding and perforation (Forrest Ia)K25.22Gastric ulcer, acute with nonspurting active bleeding and perforation (Forrest Ib)K25.3Gastric ulcer, acute without hemorrhage or perforationK25.3Gastric ulcer, acute without hemorrhage or perforationK25.31Gastric ulcer, acute with visible vessel (Forrest IIa)K25.32Gastric ulcer, acute nonbleeding with overlying clot (Forrest IIb)K25.33Gastric ulcer, acute with hematin-covered basis (Forrest IIc)K25.34Gastric ulcer, acute with clean ulcer ground (Forrest III)K25.5Gastric ulcer, chronic or unspecified with perforationK25.5Gastric ulcer, chronic or unspecified with perforationK25.6Gastric ulcer, chronic or unspecified with both hemorrhage and perforationK25.6Gastric ulcer, chronic or unspecified with both hemorrhage and perforationK25.61Gastric ulcer, chronic or unspecified with spurting bleeding and perforation (Forrest Ia)K25.62Gastric ulcer, chronic or unspecified with non- spurting active bleeding and perforation (Forrest Ib)K25.7Gastric ulcer, chronic without hemorrhage or perforationK25.7Gastric ulcer, chronic without hemorrhage or perforationK25.71Gastric ulcer, chronic with visible vessel (Forrest IIa)K25.72Gastric ulcer, chronic non-bleeding with overlying clot (Forrest IIb)K25.73Gastric ulcer, chronic with hematin-covered basis (Forrest IIc)K25.74Gastric ulcer, chronic with clean ulcer ground (Forrest III)K25.9Gastric ulcer, unspecified, without hemorrhage or perforationK25.9Gastric ulcer, unspecified, without hemorrhage or perforationK25.91Gastric ulcer, unspecified, with visible vessel (Forrest IIa)K25.92Gastric ulcer, unspecified, nonbleeding with overlying clot (Forrest IIb)K25.93Gastric ulcer, unspecified, with hematin-covered basis (Forrest IIc)K25.94Gastric ulcer, unspecified, with clean ulcer ground (Forrest III) Besides such an adaptation of the ICD-10 system, endoscopy is still developing and new terms and endoscopic techniques are continuously being created. For example, new disease classifications are being introduced from time to time, such as the semirecent Los Angeles classification for reflux esophagitis [9, 10]. For several fields, there was no ICD-10 code available, and the item thus could not be categorized under an existing ICD-10 code. For example, the procedure for investigation and interventions could not be covered within the ICD-10 or even the ICD-10-CM system. Because these items are essential for good analysis of endoscopic data, a new chapter with the same structure as the ICD-10 was developed. The items in this new chapter start with the letters OG. We chose to categorize therapeutic interventions in this new chapter also because new therapies are developed frequently and can now be adapted. Adaptations were made in different chapters of the ICD-10. In Chapter 1, Certain Infectious and Parasitic Diseases (A00–B99), the exact locations in the gastrointestinal tract of some specific infections were added. In Chapter 2, Neoplasms (C00–D48), besides the exact locations in the gastrointestinal tract, the endoscopic characteristics of polyps were described and coded in more detail. It is important to register whether a polyp is pedunculated, sessile, or flat or has a villous endoscopic aspect. Also, the number of polyps found in a specific part of the intestine must be coded, because of important diagnostic and therapeutic options for patients and the follow-up of these patients. In Chapter 9, Diseases of the Circulatory System (I00–I99), hemorrhoids and varices were described according to a classification. Different gradings of protrusion were used for hemorrhoids. Paquet’s endoscopic classification for esophageal varices was coded [11]. Most changes were, however, made in Chapter 11, Diseases of the Digestive System (K00–K93). The description of esophagitis was divided into different origins and severity. Gastric and duodenal ulcers were coded according to the Forrest classification and their specific location in the stomach or duodenum. Gastritis and duodenitis were coded according to their specific location and the endoscopically suspected cause. Inflammatory bowel diseases were coded according to their endoscopic severity and location. Some complications related to gastrointestinal procedures were coded in more detail. In Chapter 18 symptoms, signs, and abnormal clinical and laboratory findings that are an indication for endoscopic investigations were coded in more detail. In Chapter 21 postoperative situations important for gastrointestinal endoscopic investigations such as gastric and colon operations were extensively coded. In the new, thirteenth chapter of the GET-C, about 446 new codes were generated. These codes start with the letters OG. The first part of this chapter includes coding of various indications for endoscopy that could not be categorized in the ICD-10. The second part consists of specific codes for detailed locations in the gastrointestinal tract. The third part includes procedures, which are divided into diagnostic and therapeutic procedures. Diagnostic procedures include, for example, sampling of histology and culture specimens. Examples of therapeutic procedures are different kinds of polypectomies, endoscopic mucosal resections, placement of endoprotheses, and dilatation of stenoses. Another aspect of these therapeutic interventions was used for the management of gastrointestinal bleeding such as injection therapy with or without coagulation, band ligations, and clipping. Preparation and procedure for the endoscopic examination are also coded in this chapter. The extensions of the GET-C were checked by the Dutch translation board of the ICD-10 to preclude any conflicts between the two code systems. Discussion The increased use of computer systems in health care and the need for communication between these systems necessitate the availability of generally accepted code systems. Good coding provides epidemiological information for research purposes. Coding is of importance for hospitals and professionals because most of the financial systems used in health care are based on different codes. In addition, good coding makes statistical analysis of different diagnoses and collection of rare diagnoses easier. With an internationally accepted code, it is possible to compare and share information in one field of interest. Most currently used coding systems are regional or national. Only some coding systems, such as the ICD-10, are translated into different languages and used in different countries. For endoscopic report writing and endoscopic databases, different systems have been developed. These systems are now being used at many hospitals. We used the Endobase III system from Olympus Europe in our project to compose reports and record different codes. The system runs as a network version with different workstations as well as a stand-alone unit. The Endobase III system enables the generation of reports via three pathways; in addition to standard reports and text-blocks, the latest translated version of the MST is also used to compose an endoscopic report. Thus it is essential that all differently generated data are coded in the same way to enable analysis of the data anonymously. In this system, all standard reports and text-blocks are linked to one or more specific GET-C codes. The choice of a standard report or text-block leads directly to the recording of the correct GET-C code in the Endobase III database. Due to the structure of the MST, the automatic link for it is more complex to realize. We chose to link the diagnoses with the GET-C code, which is separately selected within the MST by the endoscopist at the end of the report. Automatic linking is essential to ensure correct and complete selection of codes and to make the system workable for endoscopists. In the TRANS.IT project, an anonymous central database of endoscopic investigations was built. In this central database, only coded data instead of free text can be collected, for privacy reasons. After 3 years of use, a database of about 120,000 investigations using the same coding system has been built by this working group. The GET-C system allows the encoding of any data collected during endoscopic investigations and recorded in any endoscopic database. By extending the ICD-10 with respect to the structure developed by the WHO, it is always possible to extract the original ICD-10 code from the GET-C. Thus, it is possible to link the endoscopic database with other health-care systems throughout the world. Because endoscopy is an evolving medical specialty, new techniques are continuously becoming available. These require new codes, which will be discussed within the TRANS.IT project. The Dutch Association of Gastroenterologists has accepted the GET-C. The GET-C will be available for use in other systems and was discussed at the WHO Family of International Classifications Network meeting in October 2004 [12]. The GET-C is available for free at http://www.trans-it.org. We hope that this coding system will help to make endoscopic databases and endoscopic report writing programs more meaningful.

Int_J_Med_Sci-4-2-1796947

Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis

Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA), excision repair cross complementing group 1 (ERCC1), ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.14 - 1.49). We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95). Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples. 1. Introduction Sporadic cancer is a multifactorial disease that results from complex interactions between many genetic and environmental factors 1. This means that there will not be a single gene or single environmental factor that has large effects on cancer susceptibility. Environmental factors (e.g. tobacco smoke, dietary factors, infectious agents and radiation) add to the carcinogenic load to which humans are exposed, but exact numbers for added risk are generally less well established. Cancer is the result of a series of DNA alternations in a single cell or clone of that cell, which leads to a loss of normal function, aberrant or uncontrolled cell growth and often metastasis. Several of the genes that are frequently lost or mutated have been identified, including genes that function to induce cell proliferation under specific circumstances (e.g. the ras and myc proto-oncogenes) and those which are programmed to halt proliferation in damaged cells (e.g. the TP53 and RB1 tumor suppressor genes). Other mutations in genes involved in DNA repair are also necessary. About 150 human DNA repair genes have been identified to date 2, but the real number is probably higher, since less than 50% of known and putative genes have an identified function. The association between defects in DNA repair and cancer was established by Cleaver in 1968 3, who showed that xeroderma pigmentosum (XP) is caused by deficient nucleotide excision repair (NER). For more than a quarter of a century after that it was thought that only rare syndromes, such as XP, Cockayne syndrome (CS) and ataxia telangiectasia, were associated with DNA repair defects 4. Novel, common polymorphisms in DNA repair genes are continuously being identified, and these polymorphisms may play a pivotal role in sporadic carcinogenesis. A growing body of literature, including observations of inter-individual differences in measures of DNA damage, suggests that these polymorphisms may alter the functional properties of DNA repair enzymes. At least four pathways of DNA repair operate on specific types of damaged DNA. Base excision repair (BER) operates on small lesions, while the NER pathway repairs bulk lesions. Mismatch repair corrects replication errors. Double-strand DNA break repair (DSBR) actually consists of two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). The NHEJ repair pathway involves direct ligation of the two double strand break ends, while HR is a process by which double-strand DNA breaks are repaired through the alignment of homologous sequences of DNA. The following sections review the literature on DNA repair genes in more detail, specifically those involved in the NER pathway. NER is a versatile DNA repair system that removes a wide range of DNA lesions including UV-induced lesions. There are two subpathways in NER. One is transcription-coupled DNA repair (TCR), which preferentially removes DNA damage that blocks ongoing transcription in the transcribed DNA strand of active genes. The other is global genome repair (GGR), which removes lesions throughout the genome, including those from the nontranscribed strand in the active gene 5. Three rare, autosomal recessive inherited human disorders are associated with impaired NER activity: XP, CS and trichothiodystrophy (TTD) 6. XP has been studied most extensively. XP patients develop skin tumors at an extremely high frequency (1000 fold increased incidence as compared to normal individuals) because of their inability to repair UV-induced DNA lesions. These clinical findings are associated with cellular defects, including hypersensitivity to killing and the mutagenic effects of UV and the inability of XP cells to repair UV-induced DNA damage 7. Approximately 80% of XP patients who have been classified have a defect in the NER pathway. These patients are said to have "classical" XP, in contrast to the remaining 20% of patients who are designated as XP variants (XPV) and most likely have a defect in post-replication repair. In XPV patients, DNA replication stops or is interrupted at sites of UV-damage. Furthermore, de novo DNA synthesis opposite cyclobutane pyrimidine dimer lesions is prone to errors, leading to the fixation of multiple DNA mutations and ultimately to cancer. Seven different DNA NER genes, which correct seven distinct genetic XP complementation groups (XPA, XPB (ERCC3), XPC, XPD (ERCC2), XPE, XPF (ERCC4) and XPG (ERCC5, this gene causes CS)) and XPV have been identified 6. XPA, ERCC3/XPB, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG have a defect in TCR and GGR, while XPC and XPE have a defect in GGR only. ERCC6 and ERCC8 are also known as CS type B (CSB) and CSA, respectively. Approximately 20% of patients have been assigned to the CSA complementation group Essentially CS shows some overlap with certain forms of XP. In contrast to XP and TTD, however, the NER defect in CS is limited to the TCR pathway. As with XP, TTD involves mutations in XP genes, usually XPD, which encodes a component of the transcription factor TFIIH 8. However, it has been suggested that the functions of XPD associated with TTD are distinct from those of XPD associated with XP. Approximately half of the patients with TTD display photosensitivity, correlated with the NER defect. The aim of this article is to review and evaluate associations between genes in the NER pathway and lung cancer risk, focusing on genes encoding five key enzymes in this pathway: XPA, ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. 2. Materials and methods 2-1. Identification and eligibility of relevant studies We conducted MEDLINE, Current Contents and Web of Science searches using "XPA", "ERCC1", "ERCC2/XPD", "ERCC4/XPF", "ERCC5/XPG", "lung cancer" and "polymorphism" as keywords to search for papers published (from January 1, 1966 through May 31, 2006). Additional articles were identified through the references cited in the first series of articles selected. Articles included in the meta-analysis were in any language, with human subjects, published in the primary literature and had no obvious overlap of subjects with other studies. We excluded studies with the same data or overlapping data by the same authors. Case-control studies were eligible if they had determined the distribution of the relevant genotypes in lung cancer cases and in concurrent controls using a molecular method for genotyping. Using the MEDLINE database, we identified 5 genetic epidemiological studies 9-13 that provided information on lung cancer occurrence associated with the XPA G23A polymorphism (one of the identified 6 candidate studies was excluded due to overlapping data 11). We identified 5 studies of the ERCC1 T19007C polymorphism (all of 5 candidate studies were independent 13-17). We gathered 18 articles on the ERCC2 312/751 polymorphisms found through literature searches and checked their references for additional relevant studies. Of the relevant 18 studies, 2 studies appeared to be on populations already reported 14, 18, 19, leaving 15 independent studies (11 studies for the Asp312Asn polymorphism 11, 13, 14, 17-24 and 14 studies for the Lys751Gln polymorphism 11, 13, 14, 17-19, 21-28. Less than 5 studies each have been reported on the ERCC1 C8092A, ERCC4/XPF Arg415Gln, ERCC4/XPF Ser835Ser, ERCC5/XPG His46His, ERCC5/XPG Asp1104His SNPs. 2-2. Data extraction and assessment of study quality For each study, characteristics such as authors, year of publication, ethnic group of the study population, source of control population, number of genotyped cases and controls, crude odds ratio (OR) and the method for quality control of genotyping were noted. For studies including subjects of different ethnic groups, data were extracted separately for each ethnic group whenever possible. Methods for defining study quality in genetic studies are more clearly delineated than those for observational studies. We assessed the homogeneity of the study population (Caucasian or Asian). 2-3. Meta-analysis Data were combined using both a fixed effects (the inverse variance-weighted method) and a random effects (DerSimonian and Laird method) models 29. The Cochrane Q statistics test is used for the assessment of heterogeneity. The fixed effects model is used when the effects are assumed to be homogenous, while the random effects model is used when they are heterogenous. In the absence of between-study heterogeneity, the two methods provide identical results. The presence of heterogeneity can result from differences in the selection of controls, age distribution, prevalence of lifestyle factors, histologic type of lung cancer, stage of lung cancer and so on. The random effects model incorporates an estimate of the between-study variance and tends to provide wider CIs when the results of the constituent studies differ among themselves. As the random effects model is more appropriate when heterogeneity is present 29, the summary OR and prevalence were essentially based on the random effects model. The meta-analyses were performed on crude ORs, since the adjusted ORs were not comparable because of the inclusion of different covariates in the multivariate regression models. Using individuals with the homozygous common genotype as the reference group, we calculated ORs for individuals with the heterozygous genotype and homozygous rare genotype separately whenever possible (information available in at least two studies). In some cases, we combined the heterozygous genotype with the homozygous rare genotype due to a low prevalence of the rare allele in several polymorphisms. The Q statistic was considered significant for P<0.10 30, 31. Publication bias is always a concern in meta-analysis. The presence of publication bias indicates that nonsignificant or negative findings remain unpublished. To test for publication bias, both Begg's 32 and Egger's 33 tests are commonly used to assess whether smaller studies reported greater associations than larger studies. Publication bias is considered significant for P<0.10. Publication bias may be always a possible limitation of combining data from various sources as in a meta-analysis. The idea of adjusting the results of meta-analyses for publication bias and imputing "fictional" studies into a meta-analysis is controversial at the moment 34. Sutton et al. concluded that publication or related biases did not affect the conclusions in most meta-analyses because missing studies changed the conclusions in less than 10% of meta-analyses 34. All of the calculations were performed using STATA Version 8.2 (Stata Corporation, College Station, TX) software. 3. Results 3-1. DNA repair capacity and lung cancer risk Cigarette smoke contains several thousand chemicals that are known to chemically modify DNA 35 and lead to the formation of mutations 36. Most of these compounds are procarcinogens that must be activated by Phase I enzymes, such as cytochrome P450s. All activated carcinogens can bind to DNA and form DNA adducts that are capable of inducing mutations and initiating carcinogenesis. The capacity to repair DNA damage induced by activated carcinogens appears to be one of the host factors that may influence lung cancer risk. A critical cellular response that counteracts the carcinogenic effects of DNA damage is DNA repair. As stated earlier, there are several known pathways of DNA repair, all of which act to remove DNA lesions and prevent mutations, thereby restoring genetic integrity. Several studies have investigated whether reduced DNA repair capacity (DRC) is associated with an increased risk of cancer 37. The reduced DRC of benzo(a)pyrene-7,8-diol-9,10-epoxide (an active form of benzo(a)pyrene)-DNA adducts is associated with an increased risk of lung cancer (2.1-fold, 95% confidence interval (CI) = 1.5 - 3.0) 38. The reduced DRC has been shown to be associated with a 5.7-fold (95% CI = 2.1 - 15.7) increased risk of developing lung cancer 39. Likewise, the reduced DRC of bleomycin-induced damage was found to be associated with an increased risk of lung cancer 40. These studies suggested that a low DRC of various DNA repair mechanisms predisposes individuals to lung cancer, and this realization prompted us to search for defined DNA repair activities that may be risk factors for lung cancer. Polymorphisms in DNA repair genes may be associated with differences in the DRC of DNA damage and may influence an individual's risk of lung cancer, because the variant genotype in those polymorphisms might destroy or alter repair function. 3-2. XPA G23A polymorphism and lung cancer risk The heterotrimeric replication protein A (RPA) is required for NER and may play an important role in the damage recognition process. The XPA protein is required for NER and is involved in the DNA damage recognition process. Both RPA and XPA preferentially bind damaged DNA, and because RPA and XPA directly interact in the absence of DNA, the RPA-XPA complex has been implicated as a key component in the earliest stage of damage recognition 41. There is also evidence that the XPC-hHR23B protein complex may initiate recognition of DNA damage for the global genomic repair pathway of NER 42. Recent evidence also implicates the damaged DNA binding protein heterodimer in damage recognition, because the complex binds damaged DNA with high affinity 43 and can dramatically increase the repair rate of certain DNA adducts, including cyclobutane pyrimidine dimers, in conjunction with XPA and RPA 44. The XPA maps on chromosome 9, at 9q22.3. In the XPA gene, a polymorphic site was identified that was in the 5' untranslated region (UTR) of the gene and which consisted of a G-to-A (or A-to G) substitution in the fourth nucleotide before the ATG start codon (dbSNP rs 1800975) 45. SNP alleles with higher frequencies are more likely to be ancestral than less frequently occurring alleles although there may be some exceptions. As the 23G allele was more prevalent than the 23A allele (Table 1), we regarded the 23G allele as ancestral (wild-type or major) allele for descriptive purposes (the XPA 23 polymorphism caused by the G-to-A substitution is the XPA G23A polymorphism). The polymorphism, termed the XPA G23A polymorphism (at position 23 in the transcript, four nucleotides upstream of the start codon), is in the Kozak sequence near the start codon and thus may affect the XPA protein levels in cells 46. A functional association between the XPA G23A polymorphism and DRC has been reported 10. It has been shown that healthy subjects with at least one 23G allele have significantly higher DRC. When the combined A/A and A/G genotype was used as the reference, the G/G genotype was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.56, 95% CI = 0.35 - 0.90) in Koreans 9. A significant protective effect of the combined G/A and G/G genotypes on lung cancer risk was reported in Americans (adjusted OR = 0.69, 95% CI = 0.53 - 0.90) and Mexican-Americans (adjusted OR = 0.32, 95% CI = 0.12 - 0.83) 10. Likewise, a protective and nonsignificant effect was seen among Germans 11 and Danes 12. As compared with the combined G/A and A/A genotypes, the G/G genotype was, however, associated with a significantly increased risk of lung cancer (adjusted OR = 1.59, 95% CI =1.12 - 2.27) in a Norwegian population 13. Summary frequencies of the 23A allele among all and Caucasian populations, based on the random effects model, were 0.368 (95% CI = 0.308 - 0.429) and 0.352 (95% CI = 0.277 - 0.428), respectively (Table 1). Summary ORs for the G/A genotype and G/G genotype among 5 studies in 7 populations were 0.73 (95% CI = 0.61 - 0.89) and 0.75 (95% CI = 0.59 - 0.95), respectively (Table 1). Evidence for heterogeneity was absent in both analyses. Among Caucasian studies, the summary ORs for the G/A genotype and the A/A genotype were 0.72 (95% CI = 0.58 - 0.89) and 0.82 (95% CI = 0.61 - 1.11), respectively. The Cochrane Q test for heterogeneity did not show a statistical significance. The Egger's test was statistically significant for publication bias in a subgroup analysis of Caucasians (P = 0.073, G/A genotype vs. G/G genotype). Two studies investigated associations between cigarette smoking and the G23A polymorphism in relation to lung cancer. When stratifying by smoking status, there was a significant protective effect for current smokers who possessed the G/G genotype (adjusted OR = 0.23, 95% CI = 0.07- 0.71) but not for former or never smokers 9. Ever smokers (current and former) with at least one copy of the 23G allele showed a significantly reduced risk of lung cancer (adjusted OR = 0.68, 95% CI = 0.51 - 0.91) among Caucasians 10. The presence of the 23A polymorphism, however, was associated with a statistically significant reduced risk in subjects who smoked >29 pack-years (OR = 0.53, 95% CI = 0.17 - 0.97) 13. Interactions between cigarette smoking and the polymorphism were not determined in the studies 9, 10, 13. No associations were seen between the G23A polymorphism and any histologic types of lung cancer 11, while the G/G genotype was associated with a significantly decreased risk for small cell lung cancer (OR = 0.23, 95% CI = 0.07 - 0.71) 9. The XPA G23A polymorphism may, thus, be a promising SNP for lung cancer. It is thought that cigarette smoking modifies the association between DNA repair polymorphisms, as well as metabolic polymorphisms, and lung cancer risk. Since interactions between the G23A polymorphism and smoking have not been fully elucidated, further studies are needed to better understand the associations between the XPA G23A polymorphism and lung cancer risk. 3-3. ERCC1 polymorphisms and lung cancer risk The ERCC1 coding region is 1.1 kb long and comprises 10 exons. This gene is located on 19q13.2 - q13.3. Shen et al. 47 have identified polymorphisms of three of the exons of the ERCC1 gene, all of which resulted in silent mutations. No amino acid substitutions were observed among the ERCC1 polymorphisms 48. The functional effects of the silent polymorphisms in ERCC1 have not been fully elucidated; however, some of the variant alleles of the polymorphisms in DNA repair genes may be associated with the reduced DRC. The studies have focused on polymorphisms of the 3′ UTR (C8092A, dbSNP no. rs3212986) and codon 118 (Asn118Asn, T19007C, dbSNP no. rs11615) in ERCC1. For the T19007 C (Asn118Asn) polymorphism, although the T/T genotype generates the less commonly associated triplet codon sequence encoding the amino acid and has been termed the "variant" by convention, the T/T genotype indeed has been reported to occur at higher frequencies. Hence, the T/T genotype is used as reference in this paper. The C/C genotype of the C8092A polymorphism is used as reference on the same score. The C/C genotype of the T19007C polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.32, 95% CI = 0.19 - 0.55) in a Norwegian population 13. A lack of association between the T19007C polymorphism and lung cancer risk was observed in a Danish population 14, a large American population 15, a Chinese population 16 and a nonsmoking European population 17. As shown in Table 2, summary frequencies of the 19007T allele among all and Caucasian populations, based on the random effects model, were 0.499 (95% CI = 0.387 - 0.611) and 0.575 (95% CI = 0.529 - 0.622), respectively. The summary ORs for the T/C genotype and the C/C genotype were 0.82 (95% CI = 0.62 - 1.08) and 0.72 (95% CI = 0.46 - 1.11), respectively. Even if the analysis was restricted to Caucasian studies, the ORs did not materially change. The Cochrane Q test for heterogeneity showed a statistical significance in any analysis. In comparison of the T/C genotype with the T/T genotype, the Begg's test was statistically significant in an overall analysis (P = 0.086) and a subgroup analysis of Caucasians (P = 0.089). Two studies examined an interaction between the T19007C polymorphism and cigarette smoking. When stratified by smoking status, the interaction between smoking and the polymorphism was not statistically significant 15, 16. Only one study provides information on the T19007C polymorphism and lung cancer risk in histologic types. There was no difference in risk estimates according to the histological type of lung cancer 16. As for the C8092A polymorphism, no association was found between the polymorphism and lung cancer risk in Norwegians 13 and Americans 15. The C8092A and T19007C polymorphisms have been reported to be in linkage disequilibrium 15. Although harboring at least one 19007C allele may be associated with a deceased risk of lung cancer, the protective effect of the 19007C allele needs to be confirmed in other independent studies. Furthermore, additional studies are needed to detect the function of the ERCC1 polymorphisms. 3-4. ERCC2/XPD polymorphisms and lung cancer risk The ERCC2/XPD protein plays a role in the NER pathway, which recognizes and repairs a wide range of structurally unrelated lesions such as bulky adducts and thymidine dimers. ERCC2/XPD works as an ATP-dependent (5'→3') helicase joined to the basal TFIIH complex used to separate the double helix. The ERCC2/XPD protein is necessary for normal transcription initiation and NER. ERCC2/XPD maps on chromosome 19, at 19q13.3 and covers 21.14 kb. Mutations in the ERCC2 gene can diminish the activity of TFIIH complexes, giving rise to repair defects, transcription defects and abnormal responses to apoptosis 49. A number of polymorphisms in the ERCC2/XPD gene have been reported. Whereas polymorphisms in the codons 199, 201 and 575 are rare, those in codons 156, 312, 711 and 751 are common. Two ERCC2/XPD polymorphisms, Asp312Asn (db SNP no. rs1799793) and Lys751Gln (db SNP no. rs13181), have mainly been investigated in relation to phenotypic endpoints relevant to lung carcinogenesis. With regard to the Asp312Asn polymorphism, most of the reported data indicate a higher level of DNA adducts in subjects with the Asn allele. The interpretation of this finding is a lower DRC for the Asn allele than the Asp allele. This is also true for the ERCC2/XPD Lys751Gln polymorphism. The Gln allele is associated with a higher DNA adduct level or lower DRC. The Asp/Asp genotype of the ERCC2/XPD Asp312Asn polymorphism was found to have an increased risk of lung cancer when the combined Asp/Asn and Asn/Asn genotypes served as reference (OR = 1.86, 95% CI =1.02 - 3.40) in Polish men 20. A large American lung-cancer study also reported an elevated risk (adjusted OR = 1.5, 95% CI = 1.1 - 2.0; Asn/Asn genotype vs. Asp/Asp genotype) 18. Likewise, Chinese subjects homozygous for the Asn/Asn genotype had an increased risk of lung cancer (adjusted OR = 10.33, 95% CI = 1.29 - 82.50) compared with subjects homozygous for the Asp/Asp genotype 19. No association with this polymorphism was seen in an admixed population 21, a small Swedish population 22 and among Finnish smoking men 23. Two meta-analyses have been published in 2004 50 and 2005 51, respectively. Both of them are based on the same published data from 6 individual case-control studies 18-23. The first meta-analysis showed that individuals with the Asn/Asn genotype had a 27% (95% CI = 1.04 - 1.56) increased risk of lung cancer compared with individuals with the Asp/Asp genotype. The results supported the hypothesis that individuals with the Asn/Asn genotype are at higher risk of developing lung cancer 50. The second meta-analysis was somewhat different from the first one, because unadjusted ORs were summarized in the first one. The summary OR associated with the Asn/Asn genotype was 1.18 (95% CI = 0.84 - 1.67). No significant association between the ERCC2/XPD Asp312Asn polymorphism and lung cancer was found in the second meta-analysis 51. Regardless, these meta-analyses indicate that the excess lung cancer risk from the Asn/Asn genotype may be less than 30%. Five studies have been reported since the publication of these two meta-analyses. They revealed that the Asp312Asn polymorphism was not associated with lung cancer risk in Germans 11, Norwegians 13, Danes 14, Europeans 17 and Chinese 24. As shown in Table 3, the summary frequency of the 312Asp allele among Caucasians (0.645, 95% CI = 0.572 - 0.719) was significantly lower than that among Asians (0.936, 95% CI = 0.925 - 0.946). Summary ORs associated with the ERCC2/XPD Asp312Asn polymorphism are also shown in Table 3. No significant association between lung cancer and the heterozygous Asp/Asn genotype was found for all of the studies combined or by ethnicity. The Cochrane Q test for heterogeneity did not show a statistical significance in all analyses. Although no evidence of publication bias was found in overall analyses, both Begg's (P= 0.035) and Egger's (P = 0.003) tests showed a statistical significance in a subgroup analysis of Caucasians (Asn/Asn genotype vs. Asp/Asp genotype). When stratifing by smoking dose, the risk of lung cancer was significantly higher in light-smokers with the Asp/Asp genotype than in those with the Asn/Asn genotype 20. Similar findings were not seen for never-smoker or heavy-smokers 20. A significant interaction between smoking (smoking status, pack-years and duration) and the polymorphism was observed in one study 18 but not in two other studies 16,19. Stratification analysis revealed that the increased risk was mainly confined to squamous cell carcinoma of the lung, with the ORs being 20.50 (95% CI = 2.25 - 179.05) for the 312Asn/Asn genotype 19. Table 4 shows the association between the ERCC2 Lys751Gln polymorphism and lung cancer risk. The Gln/Gln genotype was associated with an increased risk for lung cancer compared with the 751Lys/Lys genotype (adjusted OR = 2.71, 95% CI = 1.01 - 7.24) in Chinese 19. Stratification analysis revealed that the increased risk was mainly confined to lung squamous cell carcinoma, with the OR being 4.24 (95% CI = 1.34 - 13.38) for the Gln/Gln genotype 19, however. Although David-Beades et al. reported that the Gln/Gln genotype was associated with a significantly increased risk of lung cancer in Caucasians (USA), a multivariate-adjusted OR was no longer significant 25. No association with the Lys751Gln polymorphism was seen in two Caucasian populations 18, 22, an admixed population 21, a Finnish population 23, African-Americans 25, a Chinese population 26 and a Korean population 27. The meta-analysis by Hu et al. (2004) showed that the Gln/Gln genotype had a 21% (95% CI = 1.02 - 1.43) increased risk of lung cancer compared with individuals with the Lys/Lys genotype 51. The meta-analysis by Benhamou and Sarasin (2005) reported that the summary OR for the Gln/Gln genotype was 1.18 (95% CI = 0.95 - 1.47) 51. Both of the meta-analyses were based on the same published data from 8 individual case-control studies 18, 19, 21-23, 25-27. No significant association between the Lys751Gln polymorphism and lung cancer was found in the two meta-analyses 51. These meta-analyses indicate that the excess lung cancer risk from the Gln/Gln genotype may be about 20%. Six studies 11, 13, 14, 17, 24, 28 have been reported after the two meta-analysis. Danish subjects with the Gln/Gln genotype were at a 2.01-fold (95% CI = 1.20 - 3.35) higher risk of lung cancer risk than those with the Lys/Lys genotype 14. Similarly, the Gln/Gln genotype was associated with significantly increased risk of lung cancer (adjusted OR = 1.60, 95% CI = 1.10 - 2.30) in Norwegians 13. German individuals with the Gln/Gln genotype were at a borderline increased risk (adjusted OR = 1.59, 95% CI = 0.95 - 2.67) 11. However, individuals with the Gln allele had a 61% (95% CI = 14 - 83) reduction of lung cancer risk in a Chinese population 24. No association with the Lys751Gln polymorphism was seen in a European cohort 17 and in non-Hispanic Caucasians (USA) 28. The summary frequency of the 751Lys allele among Caucasians (0.634, 95% CI = 0.614 - 0.655) was significantly lower than that among Asians (0.843, 95% CI = 0.763 - 0.924). A statistically significant ethnic difference was observed between Caucasians and Asians. Summary ORs for the Gln/Gln genotype and Lys/Gln genotype were 1.06 (95% CI = 0.97 - 1.16) and 1.30 (95% CI = 1.14 - 1.49), respectively. Evidence of publication bias was absent in all of the analyses. The effect of the Gln/Gln genotype on lung cancer risk was stronger in Caucasians (OR = 2.25, 95% CI = 0.97 - 5.23) than in Asians (OR = 1.02, 95% CI = 0.20 - 5.27). This may only be due to a difference in sample sizes. Reasons for this difference in risk among different ethnic populations are as yet unknown but, if real, may be related to other genetic or environmental factors. The Cochrane Q test for heterogeneity showed a statistical significance among Asian studies (P = 0.040, Gln/Gln genotype vs. Lys/Lys genotype). There was no interaction between smoking (smoking status, pack-years and duration) and the polymorphism 14, 19, 26, 27. Although the Lys/Lys genotype was associated with a statistically significant increased risk (OR = 2.0, 95% CI = 1.15 - 3.41) among subjects who smoked>29 pack-years, an interaction between cigarette smoking and the polymorphism was not determined 13. When stratified by histological type, no statistically significant association between the polymorphism and lung cancer risk was found 26, 27. Several studies have investigated the possible association of ERCC2/XPD Asp312Asn and Lys751Gln polymorphisms with lung cancer with inconsistent results. The Lys751Gln polymorphism has been more studied than the Asp312Asn polymorphism, because the frequency of the 751Gln allele is more prevalent than the 312Asn allele. The Asp312Asn polymorphism is in linkage disequilibrium with the Lys751Gln polymorphism 19, 20, 21, however. The inconsistent associations in previous studies of the ERCC2/XPD polymorphisms could be due to differences in study populations, the small sample sizes of earlier studies and possible environmental interactions. 3-5. ERCC4/XPF polymorphisms and lung cancer risk ERCC4/XPF is an essential protein in the NER pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER enzymes, ERCC4/XPF and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC4/XPF-ERCC1 complex, which makes incisions at the 5′ end of DNA loops, may contribute to the repair of large trinucleotide repeat containing loops that are generated due to replication slippage and that are too long to be repaired by the postreplicative DNA mismatch repair system 52. Polymorphisms in enzymes involved in large loop repair could be responsible for the observed variation in the stability of similar-sized trinucleotide repeat disease alleles among different individuals. The ERCC4/XPF gene is evolutionarily conserved. Extensive homology exists between human ERCC4/XPF, Drosophila Mei-9, Saccharomyces cerevisiae RAD1, and S. pombe Rad16 53, all of which have similar functions in NER. The ERCC4/XPF gene contains 11 exons, spans 28.2 kb and is located on chromosome 16p13.2 - p13.13. Several polymorphisms exist in the coding region of ERCC4/XPF, a few of which have been associated with cancer risks. Genetic instability of simple repeated sequences might also be influenced by the ERCC4/XPF polymorphisms. The ERCC4/XPF G1244A polymorphism is a G-to-A change in exon 8 (Arg415Gln, dbSNP no. rs1800067) that results in a change from arginine to glutamine. The ERCC4/XPF polymorphism in exon 8 has been reported to be associated with an increased risk for developing breast cancer 54. The T2505C polymorphism is a T-to-C change in exon 11 (Ser835Ser, dbSNP no. rs1799801) that results in no amino acid change (serine is conserved) 55. Functionally significant SNPs in the ERCC4/XPF gene may also contribute to individual differences in the fine details of DNA repair. A lack of association was found between the G1244A (Arg415Gln) polymorphism and lung cancer risk (adjusted OR = 1.11, 95% CI = 0.59 - 2.07; Arg/Gln genotype vs. Arg/Arg genotype) in Koreans 9. The C/C genotype of the T2505C polymorphism was nonsignificantly associated with an increased risk of lung cancer (adjusted OR = 1.71, 95% CI = 0.52 - 5.58) in Chinese 24. 3-6. ERCC5/XPG polymorphisms and lung cancer risk ERCC5/XPG is responsible for a 1186 amino acid structure-specific endonuclease activity that is essential for the two incision steps in NER. The ERCC5/XPG nuclease has been suggested to act on the single-stranded region created as a result of the combined action of the XPB helicase and the ERCC2/XPD helicase at the DNA damage site. In human cells, ERCC5/XPG catalyses an incision approximately 5 nucleotides 3' to the site of damage but is also involved non-enzymatically in the subsequent 5' incision. It is further involved in the stabilization of a pre-incision complex on the damaged DNA. The ERCC5/XPG gene contains 17 exons, spans 32 kb and is located on chromosome 13q32.3 -q33.1. Several polymorphisms in the coding sequence of the EECC5/XPG gene have been identified. The association between lung cancer and two common polymorphisms, T335C (His46His, dbSNP no. rs1047768) and G3507C (Asp1104His, dbSNP no. rs17655), have been investigated. The functional effects of these two SNPs are still unknown. However, it is likely that the SNPs in the coding DNA sequences may result in a subtle structural alteration of the ERCC5/XPG activity and modulation of lung cancer susceptibility. The Asp/Asp genotype of the Asp1104His polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.60, 95% CI = 0.38 - 0.95) in a Korean population 56. Similarly, the Asp/Asp genotype was inversely associated with lung cancer (adjusted OR = 0.65, 95% CI = 0.39 - 1.1) in an admixed population (composed mostly composed of whites) 57. However, the Asp/Asp genotype was not associated with lung cancer risk in a Chinese population 24. As for T335C polymorphism, the C/C genotype was associated with a significantly increased risk of lung cancer (adjusted OR = 1.79, 95% CI = 1.19 - 2.63) in Norwegians 13 but not in Chinese 24. 4. Discussion Epidemiological studies of common polymorphisms in DNA repair genes, if large and unbiased, can provide insight into the in vivo relationships between DNA repair genes and lung cancer risk. Such studies may identify empirical associations which indicate that a polymorphism in a gene of interest has an impact on lung cancer, independent of metabolic regulatory mechanisms and other genetic and environmental variability. Findings from epidemiological studies can complement in vitro analyses of the various polymorphisms, genes, and pathways. In addition, epidemiological studies of common polymorphisms can lead to an increased understanding of the public health dimension of DNA-repair variation. We conducted a systematic literature review to evaluate the associations between sequence variants in DNA repair genes and lung cancer risk. We found an increased risk of lung cancer among subjects carrying the ERCC2/XPD 751Gln/Gln genotype (OR = 1.30, 95% CI = 1.14 - 1.49). The Gln allele of the ERCC2/XPD Lys751Gln polymorphism is associated with a higher DNA adduct level or lower DNA repair efficiency, except in research published by Duell et al. (2000) who found no correlation between the ERCC2/XPD Lys751Gln polymorphism and the level of polyphenol-DNA adducts in human blood samples 58. Matullo et al. (2003) demonstrated a higher level of DNA adducts, measured by 32P-postlabeling, in lymphocytes from nonsmokers with the ERCC2/XPD 751Gln/Gln genotype 59. Similarly, Palli et al. (2001) reported a higher level of DNA adducts in workers with at least one Gln allele who were exposed to traffic pollution in comparison with workers with the two common alleles 60. An increased number of aromatic DNA adducts was found by Hou et al. (2002) in peripheral blood lymphocytes from subjects with the ERCC2/XPD 312Asn and ERCC2/XPD 751Gln alleles 22. The combined Asn/Asn and Gln/Gln genotypes showed a higher level of DNA lesions than did other genotypes. In contrast, we found a protective effect of the XPG G23A G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95) on lung cancer risk. The G23A polymorphism itself may alter the transcription and/or translation of the gene. Because this polymorphism is located in the vicinity of the translation initiation codon, it may alter translation efficiency. The nearby proximal nucleotides to the AUG initiation codon are important for the initiation of translation because the 40S ribosomal subunit binds initially at the 5'-end of the mRNA 61. The consensus sequence around the start codon is GCCRCCAUGG, which is known as the Kozak consensus sequence 62. The R at position -3 and the G just downstream of the start codon are especially important, and the lack of these bases leads to read-through of the start codon 63. However, there has been no precise explanation of the mechanism by which the recognition of the start codon is aided by a purine at position -3 62, which is the core nucleotide of the Kozak consensus. The polymorphism XPA G23A is a G/A transversion occurring 4 nucleotides upstream of the start codon of XPA and possibly improving the Kozak sequence 9. The sequences (CCAGAGAUGG) around the predicted initiator methionine codon of the XPA gene agree with the Kozak's consensus sequence at positions -3 and +4 64. Although both the A and polymorphic variant G nucleotides at the -4 position of the XPA gene do not correspond to the original consensus Kozak sequence containing the nucleotide C at position -4, it is possible that a nucleotide substitution of A to G at position -4 preceding the AUG codon may affect ribosomal binding and thus alter the efficiency of XPA protein synthesis. To investigate whether the transition from G to A changes the translation efficiency, an in vitro transcription/translation analysis and a primer extension assay of the initiation complex will be necessary in the future. Furthermore, a functional association between the G23A polymorphism and DRC was reported 10, which showed significantly higher repair efficiency in healthy subjects with at least one G allele. An alternative explanation could be that the protective XPA 23G allele is in linkage disequilibrium with an allele from an adjacent gene which is the true susceptibility gene. Several DNA repair pathways are involved in the maintenance of genetic stability. The most versatile and important one is the NER pathway, which detects and removes bulky DNA adducts, including those induced by cigarette smoking 65. However, there are several conflicting reports on the association between this polymorphism and lung cancer risk among various populations. Although the reasons for the inconsistencies in the studies are not clear, possible explanations are: 1) low frequency of the "at-risk" genotype, which would reduce the statistical power of the studies and 2) small size of the studies. Ethnic differences in the roles of the polymorphism may be caused by gene-gene interactions, different linkages to the polymorphisms determining lung cancer risk and different lifestyles. The most important problems facing lung cancer research are identifying "at-risk" individuals and implementing clinical surveillance, prevention practices, and follow-up care. Repair pathways play an important role in lung cancer risk, and genetic variations may contribute to decreased DRC and lung cancer susceptibility. Although the increased/decreased risk associated with individual DNA repair SNPs may be small compared to that conferred by high-penetrance cancer genes, their public health implication may be large because of their high frequency in the general population. It is thus essential that epidemiological investigations of DNA repair polymorphisms are adequately designed. Unfortunately a fairly good number of studies are limited by their sample size and subsequently suffer from too low power to detect effects that may truly exist. Also, given the borderline significance of some associations and multiple comparisons that have been carried out, there is a possibility that one or more findings are false-positives 66. Large and combined analyses may be preferred to minimize the likelihood of both false-positive and false-negative results. In addition, controls should be chosen in such a way that, if they were cases, they would be included in the case group; when controls are matched to cases, it is essential to account for matching in the analysis. When appropriate, confounding factors should be controlled for, with particular consideration of race and ethnicity. An additional major concern is the grouping of genotypes for calculation of ORs. Without functional data to dictate genotype groupings, it seems prudent to present two ORs per polymorphism (one for heterozygotes vs. common-allele homozygotes and one for rare-allele homozygotes vs. common-allele homozygotes) so that dominant, codominant, or recessive patterns may be elucidated. Continued advances in SNP maps and in high-throughput genotyping methods will facilitate the analysis of multiple polymorphisms within genes and the analysis of multiple genes within pathways. The effects of polymorphisms are best represented by their haplotypes. Data from multiple polymorphisms within a gene can be combined to create haplotypes, the set of multiple alleles on a single chromosome. None of the studies reviewed here reported haplotype associations, although several studies analyzed multiple polymorphisms within a gene, sometimes with inconsistent results. The analysis of haplotypes can increase the power to detect disease associations because of higher heterozygosity and tighter linkage disequilibrium with disease-causing mutations. In addition, haplotype analysis offers the advantage of not assuming that any of the genotyped polymorphisms is functional; rather, it allows for the possibility of an ungenotyped functional variant to be in linkage disequilibrium with the genotyped polymorphisms 67. An analysis of data from multiple genes within the same DNA-repair pathway (particularly those known to form complexes) can provide more comprehensive insight into the studied associations. Such an analysis may shed light on the complexities of the many pathways involved in DNA repair and lung cancer development, providing hypotheses for future functional studies. Because of concerns over inflated type I error rates in pathway-wide or genome-wide association studies, methods of statistical analysis seeking to obviate this problem are under development 68. The ability to include haplotype information and data from multiple genes, and to model their interactions, will provide more powerful and more comprehensive assessments of the DNA repair pathways. This review, which is limited by the bias against publication of null findings, highlights the complexities inherent in epidemiological research and, particularly, in molecular epidemiological research. There is evidence that some polymorphisms in DNA repair genes play a role in carcinogenesis, most notably the ERCC2/XPD Lys751Gln and XPA G23A polymorphisms. The variant allele of each of the three polymorphisms was associated with about a 30% decrease or increase in lung cancer risk. Although the summary risk for developing lung cancer in individuals of each genotype may not be large, lung cancer is such a common malignancy that even a small increase in risk can translate to a large number of excess lung cancer cases. Therefore, polymorphisms, even those not strongly associated with lung cancer, should be considered as potentially important public health issues. In addition, it is important to keep in mind that a susceptibility factor in one population may not be a factor in another. There are differences in the prevalence of DNA repair polymorphisms across populations. In a population where the prevalence of an "at-risk" genotype in a given polymorphism is very low, the "at-risk" allele or "at-risk" genotype may be too infrequent to assess its associated risk. At a population level, the attributable risk must be small simply because it is an infrequent allele. Finally, the major burden of lung cancer in the population probably results from the complex interaction between many genetic and environmental factors over time. Most environmental carcinogens first require metabolic activation by Phase I enzymes to their ultimate forms which then bind to DNA, forming aromatic-DNA adducts that are thought to be an early step in tumorigenesis. On the other hand, these activated forms are detoxified by Phase II enzymes. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance among Phase I enzymes, Phase II enzymes and DNA repair enzymes 69. Further investigations of the combined effects of polymorphisms between DNA repair genes and drug-metabolizing genes may also help to clarify the influence of genetic variation in the carcinogenic process. Consortia and international collaborative studies, which may be a way to maximize study efficacy and overcome the limitations of individual studies, are needed to help further illuminate the complex landscape of lung cancer risk and genetic variations.

J_Chem_Ecol-4-1-2239252

Barbarea vulgaris Glucosinolate Phenotypes Differentially Affect Performance and Preference of Two Different Species of Lepidopteran Herbivores

The composition of secondary metabolites and the nutritional value of a plant both determine herbivore preference and performance. The genetically determined glucosinolate pattern of Barbarea vulgaris can be dominated by either glucobarbarin (BAR-type) or by gluconasturtiin (NAS-type). Because of the structural differences, these glucosinolates may have different effects on herbivores. We compared the two Barbarea chemotypes with regards to the preference and performance of two lepidopteran herbivores, using Mamestra brassicae as a generalist and Pieris rapae as a specialist. The generalist and specialist herbivores did not prefer either chemotype for oviposition. However, larvae of the generalist M. brassicae preferred to feed and performed best on NAS-type plants. On NAS-type plants, 100% of the M. brassicae larvae survived while growing exponentially, whereas on BAR-type plants, M. brassicae larvae showed little growth and a mortality of 37.5%. In contrast to M. brassicae, the larval preference and performance of the specialist P. rapae was unaffected by plant chemotype. Total levels of glucosinolates, water soluble sugars, and amino acids of B. vulgaris could not explain the poor preference and performance of M. brassicae on BAR-type plants. Our results suggest that difference in glucosinolate chemical structure is responsible for the differential effects of the B. vulgaris chemotypes on the generalist herbivore. Introduction Glucosinolates and their breakdown products are involved in plant defense against a wide variety of potential plant enemies. They can also serve as feeding and oviposition stimulants for specialist herbivores (Chew 1988; Louda and Mole 1991; Wittstock et al. 2003). The composition of glucosinolates is genetically variable within plant species, and influences the feeding choices of insect herbivores (Kroymann et al. 2001; Lambrix et al. 2001). A heritable glucosinolate polymorphism in Barbarea vulgaris results in two discrete chemotypes (Van Leur et al. 2006). In this study, we investigate the effect of these two chemotypes on the preference and performance of two lepidopterans. The most common and genetically dominant chemotype of B. vulgaris forms mainly (S)-2-hydroxy-2-phenylethylglucosinolate (glucobarbarin, BAR-type). Although depending on the presence of cofactors like ESP, pH, or metal ions (Burow et al. 2006), the most likely initial hydrolysis product of glucobarbarin is an isothiocyanate. Due to the 2-hydroxylation of the glucosinolate side chain, it spontaneously cyclizes to 5-phenyloxazolidine-2-thione (Kjaer and Gmelin 1957). This glucosinolate breakdown product is known to reduce infection by the soil fungus Plasmodiophora brassicae (Ludwig-Müller et al. 1999). Hardly anything is known about other ecological activities of oxazolidine-2-thiones (Wittstock et al. 2003). The less abundant and genetically recessive chemotype of B. vulgaris contains mainly 2-phenylethyl glucosinolate (gluconasturtiin, NAS-type). 2-Phenylethyl glucosinolate is also present in Arabidopsis thaliana and other Brassicaceae (Reichelt et al. 2002), predominantly in root tissues (Sang et al. 1984). Its most likely breakdown product is 2-phenylethyl isothiocyanate. Isothiocyanates are the predominant breakdown products of glucosinolates and are generally toxic to various herbivores (Wittstock et al. 2003). 2-Phenylethyl isothiocyanate negatively affects a broad range of phytophages, e.g., nematodes (Potter et al. 1999, 2000; Serra et al. 2002; Lazzeri et al. 2004), snails (Kerfoot et al. 1998), flies, aphids, mites (Lichtenstein et al. 1962), fungi (Sarwar and Kirkegaard 1998), and several generalist and specialist Lepidoptera (Wadleigh and Yu 1988; Borek et al. 1998). Despite counter-adaptations of specialists to reduce or circumvent negative effects of glucosinolates (Ratzka et al. 2002; Wittstock et al. 2003), isothiocyanates can still reduce survival and growth, and increase development time of specialists (Agrawal and Kurashige 2003). In contrast to the oxazolidine-2-thiones formed in BAR-type plants, which can increase the incidence of goiter in mammals, the 2-phenylethyl isothiocyanate formed in NAS-type plants has chemopreventive effects against tumorigenesis in mammalian organisms (Canistro et al. 2004). Based on the specific biological effects of the expected breakdown products of glucobarbarin and gluconasturtiin, we hypothesized that NAS-type and BAR-type B. vulgaris plants have differential effects on insect herbivores. Generally, it is expected that chemical plant defenses are more effective against generalist herbivores than against specialists (Cornell and Hawkins 2003). Therefore, we compared the preference and performance on the two chemotypes of a generalist (Mamestra brassicae) and a specialist (Pieris rapae) herbivore, which are both well-studied, important crucifer pests (Theunissen et al. 1985; Finch and Kienegger 1997). M. brassicae is a generalist feeding on plants in 70 species and 22 families, of which Brassicaceae are among the most preferred (Rojas et al. 2000). Even though M. brassicae can detect glucosinolates by receptor cells on the sensilla (Wieczorek 1976), and its oviposition is stimulated by damaged cabbage plants (Rojas 1999), no physiological adaptations of this species to glucosinolates have been yet described. The larvae of P. rapae, on the other hand, can detoxify glucosinolates by shifting hydrolysis products from isothiocyanates to less toxic nitriles by using the myrosinase directing nitrile-specifier protein (NSP). This enables P. rapae to consume foliage that is otherwise well defended (Wittstock et al. 2003, 2004). Female adults of P. rapae can detect intact glucosinolates in leaves of Brassicaceae with specialized receptor cells, and are stimulated to lay eggs on glucosinolate containing plants. We assessed herbivore preference on the level of adult oviposition and larval feeding. Female oviposition preference initially determines the host of the larvae (Akhtar and Isman 2003). As the isothiocyanates from NAS-type plants are expected to be more toxic than the oxazolidine-2-thiones produced by BAR-type plants, we expected M. brassicae to be repelled by NAS-type plants and to prefer BAR-type plants for oviposition. Oviposition of P. rapae is affected by glucosinolates at the leaf surface (Renwick et al. 1992; Van Loon et al. 1992) and known to be stimulated by glucobarbarin as well as by gluconasturtiin in a dose-dependent way (Chew 1988; Huang and Renwick 1994; Huang et al. 1994a, b). As larvae can eventually leave their initial host (van Dam et al. 2000), we also assessed larval preference. If the B. vulgaris chemotypes differ in toxicity or palatability, we expect to see the largest effect on larval preference and larval performance for the unadapted generalist M. brassicae and no or minor effects on the glucosinolate specialist P. rapae. Although we observed no differences in morphology, growth, or germination between the chemotypes (van Leur and van Dam, unpublished results), pleiotropic effects or close linkages could possibly cause other genes and metabolites to be consistently different between the chemotypes. As the nutritional value of plants is important for herbivore performance and preference (Simpson and Simpson 1990; Berenbaum 1995), additionally, we analyzed the sugar content and amino acid level of the tissue on which the herbivores were feeding. Materials and Methods Plant Material B. vulgaris seeds were collected from 10 individual BAR and their nearest neighbor NAS-type maternal plants, which were freely cross pollinated in a natural population of B. vulgaris. The population was located in Elderveld, The Netherlands (51.95°N; 5.87°E) and consisted of 22% NAS-type plants (Van Leur et al. 2006). We selected offspring of maternal NAS-type plants “EL44” (68% BAR-type offspring) and “EL13” (62% BAR-type offspring). Plants were grown in a glasshouse, at 21°C (day) and 16°C (night), with 60% relative humidity and natural daylight supplemented with sodium lamps to maintain the minimum PAR at 225 μmol m−2 s−1 with a photoperiod of 16:8 (L:D). One week after germination on glass beads, seedlings were transplanted to a mixture of peat soil (Potgrond 4, Lentse Potgrond BV., Lent, The Netherlands) and 20% sand. After 2 weeks, the seedlings were transplanted to 1.1-l pots, watered, and fertilized regularly with half strength Hoagland’s nutrient solution with a doubled KH2PO4 content. Chemical Analysis For quantification of glucosinolates, soluble sugars, and amino acids, one global extraction was used. In a 2-ml Eppendorf tube, 50.0 mg of lyophilized finely ground plant material were dissolved in 1.0 ml 70% MeOH in water (v/v), vortexed, and immediately boiled for 5 min to kill the remaining myrosinase activity. Tubes were placed in an ultrasonic bath for 15 min and centrifuged (10 min 10,000 rpm). The extraction was repeated for the pellet omitting the boiling step. For each sample, both supernatants were combined in a new 2-ml Eppendorf tube and supplemented individually with 70% MeOH to attain the average mass (N = 3) of a 2-ml Eppendorf tube that contained 2.0 ml 70% MeOH. This “stock” extract was stored at −20°C until further analysis.Half (1.0 ml) of the stock extract was used for glucosinolate analysis and applied to a DEAE-Sephadex A 25 column (EC 1990), desulfated with arylsulfatase (Sigma, St. Louis, MO, USA) and separated on a reversed phase C-18 column on HPLC with a CH3CN–H2O gradient as described in van Dam et al. (2004). Glucosinolate analysis was performed with a PDA detector (200–350 nm) with 229 nm as the integration wavelength. Desulfoglucosinolate peaks were identified by comparison of HPLC retention times and UV spectra with authentic standards isolated from B. vulgaris as previously described (Agerbirk et al. 2001), as well as standards kindly provided by M. Reichelt, MPI Chemical Ecology, and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R). The same response factor was used for glucobarbarin as for gluconasturtiin (Buchner 1987). To calculate glucosinolate concentrations in the plant tissue, the obtained values were multiplied by two and divided by dry mass.To analyze soluble sugar content, a 10-μl aliquot of the stock extract was diluted with 990 μl MilliQ water. Soluble sugars were analyzed by injecting 5 μl of the diluted extract on Dionex HPLC system, equipped with a Carbopac PA1 column (2 × 250 mm) and a Carbopac PA1 guard column (2 × 50 mm, Dionex, Sunnyvale, CA, USA). An isocratic gradient mixture of 10% 1 M NaOH and 90% MilliQ water was used to separate sugars at a flow rate of 0.25 ml/min. Column temperature was kept at 20°C. A “10-ppm” reference solution that contained 54.9 μM sorbitol and manitol, 29.21 μM trehalose, sucrose, and melbiose, and 55.51 μM glucose and fructose, was diluted to obtain 7.5, 5, and 2.5 ppm calibration standards to obtain a reference curve. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of sugars in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.Amino acids were analyzed on a Dionex HPLC system by integrated pulsed amperometric detection. An aliquot of 20 μl of the stock extract was diluted with 980 μl MilliQ. Of this diluted extract, 25 μl were injected, and amino acids were separated with a ternary gradient (see DIONEX application update 152, Method 1, standard AAA gradient; condition 60/2 in Hanko and Rohrer 2004) on a 2 × 250 mm AminoPac© PA10 column with a 2 × 50 mm AminoPac© PA10 Guard column (Dionex, Sunnyvale, CA, USA). Eluents, flow rates, waveform, and working electrode conditions were all as specified under Method 1 in Dionex application update 152 and in Hanko and Rohrer (2004). The Sigma AA-S-18 amino acid standard (Sigma, St Louis, MO, USA) that contained 17 amino acids was supplemented with asparagine, glutamine, and tryptophane (2.5 μmol/ml each) to obtain a reference sample that contained the 20 most common amino acids. This reference solution was diluted to obtain calibration standard ranging from 1–8 μM for each amino acid, except for cysteine, which had a range of 0.5–4 μM. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of the amino acids in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.To determine the chemotype of each plant, glucosinolates were extracted from the first full grown leaf and analyzed on HPLC as described above. When the peak area of glucobarbarin divided by the peak area of gluconasturtiin was >10, the plant was considered a BAR-type. When this ratio was <0.1, it was considered a NAS-type. Oviposition Insects were obtained from the Laboratory of Entomology of Wageningen University the Netherlands. Stock colonies of P. rapae and M. brassicae were maintained on Brassica oleracea var. gemnifera L., cultivar Cyrus, in a climatized room at 20–22°C, 50–70% relative humidity, and a photoperiod of 16:8 (L:D). We used ca. 4-month-old half-sib plants of family EL44 that were clipped 1 month before use to ensure abundant fresh leaf material. At the start of the experiment, plants were moved from the glasshouse to a climatized room at 21°C (day) 16°C (night), 60% relative humidity, illuminated to 200 PAR at plant height (Philips Master TLD 50W/840 HF and 60 W lights) and a photoperiod of 16:8 (L:D). In the same room, 1- to 3-day-old adult insects were held in a mesh cage (40 × 45 × 65 cm3) provided with sugar solution. Males and females were held together to mate for at least 24 h. Oviposition preference was assessed by introducing individual pairs into one of the 11 oviposition mesh cages (40 × 45 × 65 cm3) each containing one BAR-type and one NAS-type plant and a source of sugar. Oviposition was checked every day at 10.30 and 15.30 h. M. brassicae oviposition was recorded 24 h after the first egg up to 7 days after introduction (N = 68). P. rapae oviposition was recorded for at least 5 h after the first egg and up to a maximum of 24 h in total (N = 35). Eggs deposited on the cage, pot, or on the label were not included in the analyses. To obtain sufficient replicates, four randomly chosen plants were used twice for M. brassicae, but oviposition preference was never tested on the same combination of plants. Larval Preference For P. rapae as well as for M. brassicae, we used plants from EL44 and EL13 half sibs (one plant per chemotype per half-sib family and per herbivore). Two 0.9 cm diameter discs of each chemotype were cut from fully expanded leaves and placed in a circle (in an alternated design) in a 12 cm diameter Petri dish. We tested neonate larvae and 5-day-old larvae, which, until use, were feeding on Brassica oleracea var. gemnifera L., cultivar Cyrus leaves. The larvae were released individually at the center of the Petri dish with equal distance to all leaf discs (N = 20 per species per age). After 4 h, the amount of leaf material consumed was recorded visually and categorized as follows: 0 = no damage, 1 = only consumption of the lower leaf layers, 2 = less then 10% was consumed, 3 = between 10% and 50% of the leaf disc was consumed, and 4 = more than 50% was consumed. We were not able to assign consumption categories in the experiment with neonates because they caused so little damage, but 5-day-old larvae showed distinct feeding patterns. Larval Performance Larval performance was tested by forcing neonate larvae to stay on selected plants and measuring larval biomass and survival every other day. We selected forty 6-week-old plants (20 EL44 half sibs and 20 EL13 half sibs; 10 of each chemotype per half-sib family). To obtain a total of 80 plants, every plant was multiplied by cutting it into two halves and growing each plant in fresh pots. After 3 weeks, the plants were used to test performance of M. brassicae. Larvae were kept on the plant using 25 mm diameter meshed clip cages. After 4 days, these clip cages were replaced by 55 mm diameter meshed clip cages. After 8 days, M. brassicae larvae had consumed such large amounts of leaf tissue that the experiment had to be stopped. The remaining larvae were removed and weighed. All plants were clipped and after 3 weeks of regrowth used to asses performance of Pieris in the same way as we did for M. brassicae. However, P. rapae larvae were kept on the plants till day 18 when most larvae had pupated. Until day 4, each larva was feeding on a single leaf that we harvested to measure the consumed leaf area and sugar, glucosinolate, and amino acid content. These samples were considered to provide an estimate for the chemotype differences over the first 8 days of feeding.The long-term performance of M. brassicae larvae was studied in a second experiment, in which three neonate larvae were placed on one plant (15 BAR and 15 NAS-type plants of EL 13). The larvae could freely move on the plant, but could not move to other plants due to a plastic cylinder and a water barrier around each plant. After 8 and 13 days, we determined larval mass and the number of dead or lost larvae. After 13 days on BAR-type plants, all but five larvae had died or were lost, so that we stopped the experiment. As dead larvae had often dried out, we excluded their biomass from all calculations. Statistical Analysis In the oviposition preference experiments, total egg load may differ among individuals. Therefore, the number of eggs on each chemotype per individual female was treated as a paired sample. Per paired sample, we considered the chemotype with the highest egg load as the preferred type. Overall preference was tested with a Sign test. The number of eggs per plant and number of clusters per plant were not normally distributed, and were analyzed with the Wilcoxon matched-pair signed-ranks test. As there were no paired observations for cluster size, these data were analyzed with the Mann–Whitney U test.To analyze larval preference, we compared the average food consumption category between the two chemotypes by using the Wilcoxon matched-pair signed-ranks test.In the larval performance experiments, the larval masses were not normally distributed and therefore analyzed for differences between the chemotypes with separate Mann–Whitney U tests for each day followed by Bonferroni correction for multiple comparisons (for M. brassicae α = 0.05/4 = 0.0125; for P. rapae α = 0.05/8 = 0.0062). Data on the leaf consumption of each herbivore on the two chemotypes were also tested with the Mann–Whitney U test. The relationships between larval biomass and leaf consumption and between larval biomass and total glucosinolate concentration were tested by using Pearson product–moment correlations. The data were log-transformed to get a normal distribution. Before analysis of variance (ANOVA) total glucosinolate, sugar, and amino acid content data were log-transformed to meet the assumptions of ANOVA. Statistical analyses were performed with STATISTICA (data analysis software system), version 7.1. (StatSoft (2005); http://www.statsoft.com). Results Oviposition Preference Generalist and specialist adult herbivores did not prefer one chemotype over the other. Of the 68 M. brassicae females tested, 33.8% oviposited on the plants, 47.1% preferred to oviposit on the cage, whereas 19.1% did not oviposit at all within 1 week. The chemotypes did not receive significantly different numbers of eggs (Table 1). M. brassicae, which is a gregarious species, deposited a total of 19 clusters on BAR-type and 19 on NAS-type plants. The number of clusters per plant and the average cluster size, ranging from 6 to 465 eggs per cluster, were not significantly different between chemotypes (Table 1). Table 1Oviposition of Mamestra brassicae and Pieris rapae on BAR-type and NAS-type Barbarea vulgaris  Mamestra brassicaePieris rapaeBARNASPZBARNASPZPreferencea14110.8380.20412190.4720.720Eggsb220 ± 40.9193 ± 51.70.3990.84326.9 ± 18.226.6 ± 14.50.9220.100Clusters/plantc1.36 ± 0.21.73 ± 0.30.9510.061Cluster sized162 ± 23.4112 ± 32.90.8000.254aTimes when that chemotype received most eggs in a pairwise comparisonbAverage number of eggs per plant ± SEcAverage number of clusters per plant ± SEdAverage number of eggs per cluster ± SEOf the 35 P. rapae pairs tested, 32 females oviposited on plants, two females did not oviposit at all, and one female died. With an average of 53.5 ± 5.1 eggs per female, 61% deposited most eggs on NAS-type plants, and one deposited an equal amount of eggs on both chemotypes. The average numbers of eggs deposited on each chemotype did not significantly differ between chemotypes (Table 1). In total, BAR-type plants received 861 eggs and NAS-type plants 850 (Table 1). Larval Preference Of the 20 5-day-old M. Brassicae larvae tested, only one preferred to feed on BAR-type leaf discs, whereas 19 preferred the NAS-type (Sign test: Z = 3.67, P < 0.001). This preference for NAS-type leaf discs was confirmed when corrected for the consumed quantity (feeding categories). M. brassicae larvae consumed more from NAS-type leaf discs than from BAR-type (Fig. 1; Wilcoxon matched-pairs test on feeding categories: M. brassicae N = 20, Z = 3.743, P < 0.001). In contrast, the 20 5-day-old P. rapae larvae did not show a significant preference. Five larvae preferred NAS-type, 11 BAR-type, and four consumed from BAR-type as well as NAS-type leaf discs (Sign test: Z = 1.25, P = 0.211). Although P. rapae larvae consumed more from BAR-type leaves, the difference in feeding categories was not significant (Fig. 1; Wilcoxon matched-pairs test on feeding categories: P. rapae N = 20, Z = 1.629, P = 0.103). Consequently, we found no significant preference of P. rapae larvae for either chemotype. Fig. 1Consumption of BAR-type (grey bars) and NAS-type (white bars) leaf discs by 5-day-old larvae of Mamestra brasscicae and Pieris rapae (feeding category averaged per Petri dish ± SE) Larval Performance and Chemotypes Over the first 8 days, the larval biomass accumulation of the generalist M. brassicae was affected by chemotype (Mann–Whitney U test: P < 0.001 for each day of recording). When forced to stay on NAS-type plants, M. brassicae larvae grew exponentially (Fig. 2a). On BAR-type plants, however, M. brassicae larvae hardly increased in biomass and were moribund. This difference in performance was positively correlated to the difference in leaf consumption after 4 days of feeding (correlation analysis: r = 0.942, r2 = 0.887, P < 0.01). Consequently, M. brassicae larvae caused more damage to NAS-type leaves than to BAR-type (Fig. 3a; Mann–Whitney U test: U = 12, Z = –7.583, P < 0.001). Larval survival after 8 days on the plants was in line with these results. On NAS-type plants, all larvae were still alive, whereas on BAR-type plants 37.5% of the M. brassicae larvae had died. The results of the second experiment, in which the larvae could move freely on the plants, confirmed the results of the clip cage experiment. After 13 days, 89% ±10 of the larvae had stayed and survived on NAS-type plants and weighed 42.13 ± 2.69 mg, whereas only 20% ±10 of the larvae had stayed and survived on BAR-type plants and weighed only 4.11 ± 0.02 mg. Fig. 2Biomass accumulation of aMamestra brassicae and bPieris rapae larvae during 8 days of feeding BAR-type (grey bars) and NAS-type (white bars) Barbarea vulgaris plants ± SEFig. 3Leaf characteristics after 4 days of larval feeding of Mamestra brassicae (left graphs) or Pieris rapae (right graphs) on Barbarea vulgaris ± SEM: a and b consumed leaf area (cm2), c and d total glucosinolate content, e and f total sugar content, g and h total amino acid content. Results are depicted per chemotype (grey bars BAR-type plants; white bars NAS-type plants) and per half-sib family (no hatching EL13, hatching EL44)In contrast to M. brassicae larvae, P. rapae larvae grew and survived equally well on both chemotypes. P. rapae larval biomass accumulation over time showed no significant difference between chemotypes over the first 18 days (Fig. 2b; Mann–Whitney U test: P > 0.05 for each day of recording) and was positively correlated to leaf consumption after 4 days (correlation: r = 0.768, r2 = 0.589, P < 0.01). Leaf damage due to P. rapae feeding was not different between the chemotypes (Fig. 3b; Mann–Whitney U test: U = 673, Z = −1.217, P = 0.223). After 8 days, larval mortality was 7.5% on NAS-type and 12.5% on BAR-type plants. On day 14, the first P. rapae larvae started pupating. After 16 days on BAR-type plants, 45% had pupated, and on NAS-type plants this was 47.5%. At the end of the experiment, after 18 days, larval weights were still not significantly different on the chemotypes (on BAR-type: 161.9 ± 7.37 mg, on NAS-type: 175.24 ± 5.63 mg; Mann–Whitney U test: U = 238, Z = −1.131, P = 0.265). Larval Performance and Total Glucosinolate Content The leaves on which larvae were feeding differed in glucosinolate composition (glucobarbarin vs. gluconasturtiin) and in total glucosinolate content (Fig. 3c,d). In both chemotypes, we detected glucobarbarin, gluconasturtiin, glucosibarin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin. In both families, BAR-type leaves had on average 1.5 times higher total glucosinolate levels than NAS-type leaves (Fig. 3c; ANOVA type effect: F1,143 = 22.831, P < 0.001). Because M. brassicae fed more on NAS plants, overall a negative—but weak—correlation of total glucosinolate content with the consumed leaf area was observed (correlation: r(X,Y) = −0.26, r2 = 0.065, P < 0.05). To analyze in more detail whether total glucosinolate level determines larval consumption and performance, we also examined the correlation within each chemotype. Within chemotypes, the range of glucosinolate concentrations was substantial (factor 6–7), but there was no negative correlation between glucosinolate level and larval biomass (Fig. 4; Correlation: within BAR, r(X,Y) = 0.15, r2 = 0.024, P = 0.932; within NAS, r(X,Y) = 0.28, r2 = 0.078, P = 0.090). Moreover, larvae on BAR-type leaves with similar low levels of glucosinolates as NAS-types consumed and performed considerably worse (Fig. 4; glucosinolate levels <35 μmol g−1 dry mass). There was also no difference in total glucosinolate level from BAR-type leaves on which the larvae survived or died (with living larvae: 31.32 ± 2.95 μmol g−1, with dead larvae: 32.81 ± 6.49 μmol g−1; ANOVA: F1,29 = 0.059, P > 0.05; Fig. 4). As the performance of P. rapae larvae was similar among chemotypes and families, there was no correlation of larval biomass with total glucosinolate level. Fig. 4Correlation of total glucosinolate content and the larval biomass after 4 days feeding of Mamestra brassicae experiment on NAS-type (white dots), BAR-type (black dots), and dead larvae of on BAR-type (black crosses) Barbarea vulgaris plants with their correlations per chemotype (black line) and their 95% confidence intervals (dotted lines) Larval Performance and Nutritional Value Total sugar content of both chemotypes was composed of five consistently detected sugars, which were glucose, sucrose, sorbitol, fructose, and trehalose (from high to low average concentration). Total amino acid content of both chemotypes was composed of 11 consistently detected amino acids, which were threonine, isoleucine, arginine, serine, glutamate, aspartate, glutamine, asparagine, phenylalanine, tyrosine, and histidine (from high to low average concentration). Because the individual sugars and amino acids showed effects similar to the total levels (results not shown), we only present data on total levels.In contrast to the total glucosinolate level, sugar and amino acid content did not differ between the chemotypes (Fig. 3e–h). Although in the M. brassicae experiment the total sugar content was overall higher in BAR-type plants, there was a significant interaction between chemotype and family (Fig. 3e; ANOVA type effect: F1, 75 = 12.014, P < 0.001; type × family F1, 75 = 10.252, P < 0.05). Similar interactions between chemotype and family were shown for amino acid content of the plants in the M. brassicae experiment and for sugars and amino acid content of plants in the P. rapae experiment. In all chemotype × family interactions, the BAR-type of the EL13 half-sib family had higher levels of primary metabolites than the NAS-type of that family (Fig. 3e–h). In the EL44 half-sib family, the sugar and amino acid level differences of the chemotypes were negligible in plants during the M. brassicae experiment or even opposite to those of the EL13 during the P. rapae experiment. As sugar and amino acid content did not vary consistently with chemotype, these cannot explain the differences in larval performance and preference between the chemotypes. Discussion NAS and BAR chemotypes of B. vulgaris differentially affected preference and performance of the generalist herbivore M. brassicae but not the specialist P. rapae. Larvae of M. brassicae grew exponentially and had 100% survival on NAS-type leaves, but hardly grew and had a high mortality when feeding on BAR-type plants. As the dose-dependent effect of total glucosinolate content on herbivores is well-known (Li et al. 2000; Agrawal and Kurashige 2003; Mewis et al. 2006), the poor performance of M. brassicae could have been caused by the on average 1.5 times higher total glucosinolate content of BAR-type plants compared to NAS-type plants (Van Leur et al. 2006). However, we did not observe any dose-dependent effect within the chemotypes. Moreover, M. brassicae larvae on BAR-type plants with total glucosinolate levels similar to NAS-type plants still performed significantly worse. Hence, we exclude total glucosinolate level as explanatory factor for the differences found between chemotypes on larval biomass and leaf consumption. In addition to total glucosinolate content, sugar and amino acid contents differed between chemotypes, but the pattern was inconsistent among half-sib families. The statistical interaction between chemotype and family found for sugar and amino acid levels was not found for herbivore performance and preference (Fig. 3). Therefore, total sugar content and total amino acid content can not explain the observed effects on M. brassicae. In a different B. vulgaris polymorphism, described by Agerbirk et al. (2003b), there were P- and G-type plants that differed in resistance to Plutella xylostella. Other than our BAR/NAS polymorphism, the P-type and G-types also differed in trichome density, did not co-occur in natural populations, were hard to cross, and neither of the Danish types had a high content of gluconasturtiin in the leaves (Agerbirk, personal communication).The resistance of the G-type to P. xylostella was due to a difference in saponin content (Agerbirk et al. 2003a). Based on these findings, we compared saponin levels between BAR and NAS-type plants by using LC-TOF-MS (see supplemental data). This analysis revealed that the levels of the saponin described by Agerbirk et al. (2003a) and a saponin described by Shinoda et al. (2002) did not differ between the NAS and BAR chemotypes (supplemental data). Therefore, we exclude these saponins as an explanatory factor for the poor performance of M. brassicae on BAR-type plants. The observed effects are likely caused by difference in glucosinolate structures and their break-down products. BAR-type plants were more toxic and deterrent to M. brassicae than the NAS-type plants. This suggests that M. brassicae can deal effectively with gluconasturtiin and isothiocyanates, but not with glucobarbarin and resulting oxazolidine-2-thiones. The latter compounds have received hardly any attention in chemical–ecological literature. Generalist herbivores possess broad-spectrum detoxification enzymes such as P450 enzymes and mixed-function oxidases (MFO) that enable them to deal with a wide range of allelochemicals, including glucosinolates (Schoonhoven et al. 1998; Li et al. 2000). Which detoxification mechanism is present in M. brassicae and why it can handle the presumably more toxic isothiocyanates, but not the oxazolidine thiones is unknown. The non-different performance of P. rapae on the chemotypes may indicate that NSP enzymes are equally effective in redirecting the hydrolysis pathways of both chemotypes towards the generally less toxic nitriles (Wittstock et al. 2004). Identification of the bioactive compounds could be acquired by using bioassay-guided fractionation. Neither herbivore species significantly preferred one of the chemotypes as a host plant for oviposition. Even though isothiocyanates are known to elicit anemotaxis in herbivores at extremely low concentrations (Finch and Skinner 1982), the concentrations of volatile cues emitted by our undamaged plants may have been below the detection limit (Finch et al. 1978). Upon damage or induction by herbivores, volatile levels may rise and affect oviposition preference (Rothschild and Schoonhoven 1977; Bruinsma et al. 2007). Next to chemical suitability, the surface on which to oviposit can be an important factor (Renwick and Chew 1994). In our experiment, however, leaf surface structure was unlikely to affect oviposition preference between chemotypes because the chemotypes did not have apparent differences in leaf surface (e.g., trichomes) in contrast to the completely different B. vulgaris polymorphism reported by Agerbirk et al. (2003b). As M. brassicae larvae performed poorly on BAR-type plants, the lack of oviposition preference of adults seems to be non-adaptive. The discrepancy between larval performance and oviposition preference on B. vulgaris has also been shown for the Diamondback moth (Plutella xylostella) (Serizawa et al. 2001; Badenes-Perez et al. 2006). It may occur when insects or plants are new to an area and there has not been enough time for evolutionary adaptation (Agosta 2006). Whether this is the case for M. brassicae and the B. vulgaris chemotypes is unknown. Co-evolutionary theory suggests that the variation of plant defense compounds is maintained by sequential adaptations of specialist herbivores and plants (Agrawal et al. 1999; Cornell and Hawkins 2003). The good performance of P. rapae on both chemotypes suggests that neither chemotype is effectively defended against this specialist. On the contrary, BAR-type plants are defended against the generalist M. brassicae. Although we have no experimental evidence indicating which chemotype is the newest form, the effective defense of BAR-type plants against M. brassicae suggests an evolutionary adaptive step in which BAR-type evolved from NAS-type. This matches with the biosynthetic origin of these glucosinolates. Gluconasturtiin is presumably the precursor that is hydroxylated to produce glucobarbarin. Moreover, gluconasturtiin occurs in five times more genera than glucobarbarin (Fahey et al. 2001). This does not preclude, however, that the NAS-types found locally in the Netherlands are due to a loss-of-function mutation from the BAR-type. In natural populations of B. vulgaris, there are only 0–22% NAS-type plants (Van Leur et al. 2006). This results in a potentially limited genetic basis for NAS-type plants compared to BAR-type plants, which could contribute to linkage disequilibrium between the BAR/NAS locus and other loci (for instance closely linked loci). However, the natural population from which we selected seed batches for these experiments had a phenotype frequency close to a Hardy–Weinberg equilibrium (22% potential NAS-type fathers observed instead of 25% expected). We estimate the chance of linkage disequilibrium effects in our experiments to be relatively small. Additionally, there are no a priori indications of closely linked loci that affect herbivore performance. For the plant, the benefit of increasing its defense against M. brassicae will be larger than increasing its defense against P. rapae. Being gregarious and larger, M. brassicae larvae are more harmful to the chosen individual (many larvae feeding from one plant) and will more strongly reduce plant fitness than the solitary P. rapae (only one or few larvae feeding on a plant). Therefore, irrespective of the degree of specialization of the herbivores, it will be more important for B. vulgaris to be defended against M. brassicae than against P. rapae. Based on our results, we would expect BAR-type plants in the field to suffer less from herbivory than NAS-type plants. Besides being the chemotype that is most severely damaged by M. brassicae larvae, the NAS chemotype is also the recessive genotype. Therefore, if herbivorous insects are the only selective force, we expect that natural selection will drive natural populations towards 100% BAR-types. European populations of B. vulgaris indeed are mainly dominated by BAR-type plants, but a minority number of populations has still up to 22% NAS-type plants. This indicates that in these populations there may be other factors that play a role in maintaining this chemical polymorphism. Other factors that may differ between the two types and that determine plant fitness, e.g., belowground herbivory, higher trophic level interactions, and inter- and intraspecific competition, need to be included in future studies. Electronic supplementary material Below is the link to the electronic supplementary material. ESM (DOC 52.5 kb)

Histochem_Cell_Biol-4-1-2323033

Cell fusions in mammals

Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host cells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work together with a number of other proteins to regulate the cell fusion machinery. Cell fusions in the developing mammal The fusion between the sperm and the egg marks the beginning of a new individual and ensuing development of either man or beast depends upon a number of additional cell fusion events (reviewed by Ogle et al. 2005). Thus, mononuclear cytotrophoblasts fuse to form the syncytiotrophoblast layer of the placenta in man and certain other mammals. Syncytiotrophoblasts control the exchange of gases, nutrients and waste products between the fetus and the mother, protect the fetus against the maternal immune system and are also responsible for the production of hormones, which like chorionic gonadotropin, regulate the continuation of pregnancy. In addition, in the developing individual, myoblasts fuse to form multinucleated skeletal muscle fibers, while cells of monocytic origin fuse to form osteoclasts, which participate in bone sculpturing and remodeling as well as in the regulation of serum calcium concentrations. Cell fusions contribute to tissue repair It is well-known that skeletal muscle is regenerated through fusion of muscle fibers with satellite cells (Bischoff 1994) and that macrophages may fuse to form multinucleated giant cells with enhanced phagocytic capabilities in response to injury and antigenic challenges (Vignery 2005). Recent data indicate that additional cell fusions may contribute to tissue repair in the adult (Vassilopoulos et al. 2003; Wang et al. 2003). Bone-marrow-derived (BMD) cells have been shown to fuse with hepatic cells, nerve cells and gastrointestinal cells and the theory has been put forward that such fusions may serve to repair damaged or corrupted cells (Alvarez-Dolado et al. 2003; Rizvi et al. 2006; Vassilopoulos et al.2003; Wang et al. 2003). However, it is still debated whether such fusions are important to tissue repair and whether they engage BMD stem cells or more differentiated fusogenic cells like monocytes/macrophages (reviewed by Vignery 2005). Intriguingly, the accumulation of macrophages in injured organs may reflect not only the need to remove debris but also to repair compromised cells through heterotypic fusions (Vignery 2005). Moreover, in recent transplant experiments, irradiated mice showed evidence of fusions between crypt stem cells and donor BMD cells (Rizvi et al. 2006). In thus transplanted mice, representatives of all cell lineages derived from intestinal crypt stem cells (absorptive columnar, enteroendocrine, goblet and Paneth cells) showed expression of chromosomal markers characterizing donor BMD cells. This phenomenon appeared to depend upon tissue injury since expression of donor cell markers never was detected in non-irradiated mice (Rizvi et al. 2006). An alternative to fusions has been suggested by Holmgren et al. (1999) and de la Taille et al. (1999), who have demonstrated that cells phagocytosing apoptotic cells may acquire functional DNA. Moreover, a mechanism of cell–cell invasion (entosis) was recently described and could potentially transfer DNA between cells (Overholtzer et al. 2007). Thus, viral as well as normal sequences from apoptotic cells have been found to be active also in phagocytosing cells (Holmgren et al. 1999). This observation challenges our understanding of apoptosis as an efficient way of clearing corrupted or alien DNA. Cell fusions during viral infections Infections with enveloped viruses such as HIV-1 may also lead to cell fusions. The viral genome encodes envelope (Env) proteins, which bind to cell surface receptors and assist the virus in entering the cell. The infected cell commences to synthesize Env proteins, which, upon insertion in the plasmalemma, engages receptor proteins in neighboring cells and initiate fusions. Interestingly, modifications of the cytoplasmic tail of several Env proteins appear to modify fusions. Thus, proteolytic cleavage of the cytoplasmic tail regulates fusions and also cellular signaling events, such as tyrosine kinase activity, may be involved in regulation of virally induced cell fusions (Kubo et al. 2003). Receptor proteins may bind Env proteins from different classes of viruses. Thus, a cell infected by a specific virus becomes resistant to infections with other viruses that bind to the same receptor. This phenomenon, which probably reflects receptor saturation, has led to the grouping of retroviruses into different subtypes, which bind to the same receptor. Other types of viruses encode fusogenic proteins (like influenza hemagglutinin), which do not bind to surface receptors, but which liberate a fusion peptide in the acidic milieu of endosomes and thus initiate viral–host cell fusion following endocytic uptake of viral particles. Both these proteins and the Env proteins are referred to as class I viral fusion proteins and contain hydrophobic fusion peptides buried within their sequences. Following receptor engagement or endosomal acidification the fusion peptides are liberated by proteolytic cleavage. The cleaved peptides undergo conformational changes, resulting in hairpin-like, alpha-helical bundles, which bring the viral and cell membranes into close apposition and thereby facilitates fusion (reviewed by Chen and Olson 2005). Cancer–host cell fusions In 1911 Aichel suggested that cancer cells might spontaneously fuse with host cells to produce hybrids with supernumerary chromosomes that could evolve into cells of increased malignancy. Subsequent studies have confirmed this prediction by demonstrating that cells with mixed phenotypes spontaneously appear in co-cultures of normal and malignant cells (Barski and Cornefert 1962; Busund et al. 2002; Mortensen et al. 2004; Wakeling et al. 1994) (Figs. 1, 2, 3). Additionally, transplanted tumor cells of animal (Busund et al. 2003; Chakraborty et al. 2000; Fortuna et al. 1990; Kerbel et al. 1983; Larizza et al. 1984a; Wiener et al. 1974a, b) or human (Goldenberg et al. 1974; Goldenberg and Pavia 1982; Mortensen et al. 2004) origin may fuse with and acquire phenotypic characteristics of normal host cells (Fig. 4). In some of the in vivo studies, data suggested that the putative fusion partner was of macrophage or endothelial origin but, in most cases, definite identification was not achieved. In a few cases, it has also been demonstrated that the fused cells expressed genetic markers of both parental cell types (Goldenberg et al. 1974; Mortensen et al. 2004; Wiener et al. 1974a, b) (Figs. 2, 3). Mortensen et al. (2004) demonstrated that, initially after fusion, bi- or multinucleated cells formed and that such cells had the parental genomes seggregated in diffferent nuclei (heterokaryons) (Fig. 2a–e). Subsequently, mitotic figures appeared showing an admixture of the parental chromosomes (Fig. 2f–j). Eventually, cells with the parental genomes mixed in a single nucleus (synkaryons) were detected (Fig. 2k–o). In agreement with the notion that synkaryons appear after mitotic divison of heterokaryons, synkaryons are usually detected in pairs (Fig. 3). A few recent studies present compelling evidence that cells with genetic characteristics of hybrid cells also may appear in human tumors (Andersen et al. 2007; Chakraborty et al. 2004; Yilmaz et al. 2005; Streubel et al. 2004). Thus, in two cases of renal carcinomas, arising in bone marrow transplant recipients, some tumor cells showed genetic markers characterizing the healthy donor (Chakraborty et al. 2004; Yilmaz et al. 2005). Moreover, in patients with multiple myelomas, osteoclasts have been shown to contain an admixture of nuclei, of which some possess tumor-specific chromosomal translocations while others are devoid of translocations (Andersen et al. 2007). Finally, lymphoma-specific genetic abnormalities were described in endothelial cells in B cell lymphomas (Streubel et al. 2004). Although these data do not formally prove that tumor–host cell hybrids did form in these patients, the combined results from studies in vitro and in vivo do present compelling evidence that cell fusions do occur in tumors (reviewed by Pawelek 2005). Fig. 1Cancer–endothelial cell fusion. Co-culture of human breast cancer (MCF-7) cells and human umbilical vein endothelial cells (HUVEC). Cells were stained for the cancer cell marker cytokeratin (red, c), the endothelial cell marker vimentin (green, b) and DNA (bisbenzimide, blue, d). Note in the merged image (a) one fused, multinucleated cell reacting for both cytokeratin and vimentin (orange-yellow)Fig. 2Chromosomal markers and cell fusions. Co-cultures of human breast cancer cells and bovine endothelial cells. The cultures were subjected to fluorescent in situ hybridization (FISH) with probes recognizing all bovine chromosomes (green; b, g, l) and all human chromosomes (red; c, h, m) and counter-stained for DNA with DAPI (blue; d, i, n) and observed in differential interference contrast (DIC; e, j, o). Merged red-green images are shown in a, f, k. Note that initially, binuclear cells (heterokaryons) form, having one bovine and one human nucleus (a–e). Occasionally, tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected. At longer times after mixing, mitotic figures, containing an admixture of bovine and human chromosomes appear (f–j) and, eventually, cells with a single nucleus, containing an admixture of the two genomes in mixture (synkaryons) are detected (k–o)Fig. 3Chromosomal markers and cell fusions. Mixed culture of human breast cancer cells and bovine endothelial cells submitted to double FISH as in Fig. 2 (a double FISH; b combined DAPI and DIC). Low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei (arrows). In addition, nuclei hybridizing only for the bovine (green) or only for the human (red) genome occurFig. 4Cancer–host cell fusion in vivo. a, b Section from lung of a nude mice injected with human breast cancer (MDA-MB-231) cells in the tail vein (Mortensen et al. 2004). The section underwent FISH for the mouse genome (red) and the human genome (green) (a) and DNA was counterstained with DAPI (b; blue). Note one nucleus in which the human and mouse genome co-localize (arrow). C: Similar section, stained with an antibody detecting human (but not mouse) p53 (red; p53 is mutated and overexpressed by the breast cancer cells), an antibody to beta-catenin (to mark cell membranes) and for DNA with bisbenzimide (blue). Note a micrometastasis of human breast cancer cells having violet (red + blue) fluorescent nuclei. d Section stained for human p53 (red) and the endothelial marker von Willebrand factor (green) and DNA (blue). Note a human cancer cell with a violet (red + blue) nucleus showing membrane-staining for von Willebrand factor. Since von Willebrand factor is not normally expressed by the breast cancer cells, this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell. Similar results were obtained using double FISH for the human and mouse genome and imunofluorescent staining for von Willebrand factor (described by Mortensen et al. 2004) The essential question regarding cancer–host cell fusions is, of course, if they are relevant to the patient. In fact, there are two opposing views. The first is based on early experiments on fusions induced to occur between cancer cells and normal cells in culture. With few exceptions, such experiments revealed that malignancy was suppressed (Harris et al. 1969; Harris 1988; Stanbridge 1976; Wiener et al. 1974a, b). These studies were, in fact, seminal to the discovery of tumor suppressor genes (reviewed by Anderson and Stanbridge 1993). Since tumor suppressor genes, like p53 and Rb, frequently are inactivated in cancer cells, fusions would present cancer cells with unperturbed tumor suppressors from the normal fusion partner and consequently initiate cell cycle arrest or apoptosis. Although, this certainly applied for the cell types studied in the contributions cited above, it may not be a general rule. Thus, production of monoclonal antibodies depends upon the fact that it is possible to fuse antibody-producing spleen cells with myeloma cells to obtain hybridomas that retain the unlimited proliferative ability of the tumor cell partner and the antibody production of the normal cell (Kohler and Milstein 1975). In fact, several studies documented that some fusions may lead to cells of increased malignancy (Barski and Cornefert 1962; Busund et al. 2003; Chakraborty et al. 2001; De Baetselier et al. 1981; Kerbel et al. 1983; Larizza et al. 1984b; Pawelek 2000; Rachkovsky et al. 1998). Possibly, the genetic make-up of tumors may dictate the outcome of individual cancer–host cell fusions. Moreover, fusions lead to aneuploidy and chromosomal instability, which characterizes most cancers and may, by itself, stimulate carcinogenesis (Duelli et al. 2007). Mechanisms behind cell–cell fusions Fusions between normal cells Cell fusion events must be extremely well controlled. Due to their major importance to fertilization, placentation, muscle development, bone structure, calcium homeostasis and the immune defense system, much effort has gone into elucidating mechanisms underlying cell–cell fusions. Additionally, the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field. In spite of this, much less is known about cell–cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t-SNAREs. Interestingly, engineered flipping of the v- and t-SNARE machinery has been shown to promote cell–cell fusions (Weber et al. 1998). However, it is evident that this mechanism is not a physiological mediator of cell–cell fusions. Interestingly, v- and t-SNAREs act similar to class I viral fusion proteins in that they form bundles of alpha-helices, which result in membrane apposition and fusion (Blumenthal et al. 2003; Jahn et al. 2003). Of the many proteins, which to date have been shown (or proposed) to be involved in cell fusions in mammals, only the syncytin family appears to use a similar alpha-helical mechanism. That they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral Env sequences. The founding family member, syncytin-1, was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts (Blond et al. 2000; Mi et al. 2000). This capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence. Syncytin-1 represents the Env protein of the human endogenous retroviral (HERV) W sequence, which entered the primate genome 25–40 million years ago. In contrast, most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences. Molecular studies have shown that syncytin-1 (Env W) shares a structure similar to class I viral fusion proteins, especially in the region of the N- and C-terminal heptad repeats (NHR and CHR), and shares a common fusion mechanism with these proteins (Chang et al. 2004; Gong et al. 2005). A synthetic peptide derived from the CHR is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism (Chang et al. 2004). Syncytin-1 binds to the D-type retroviral receptor ASCT-2 (Blond et al. 2000) and may use another neutral amino acid transporter (ASCT-1) as an auxiliary receptor (Lavillette et al. 2000). Syncytin antibodies, syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 CHR peptide have been shown to inhibit fusions between trophoblast-derived cells (Blond et al. 2000; Chang et al. 2004; Mi et al. 2000). Agents increasing cellular levels of cAMP or cAMP analogues have been shown to promote cytotrophoblast fusions in vitro (Keryer et al. 1998) and such agents are also known to elevate protein and mRNA levels of syncytin-1 in isolated cytotrophoblasts (Frendo et al. 2003). Also estradiol may regulate syncytin-1 expression (Carino et al. 2003) and the placenta-specific transcription factor GCMa interacts with two upstream sites in the HERV-W 5′-long terminal repeat and stimulates syncytin-1 transcription (Yu et al. 2002). Additionally, experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity (Drewlo et al. 2006), similar to what has been observed for some virally derived Env proteins (reviewed by Kubo et al. 2003). However, if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated. A second syncytin-family member, syncytin-2, was subsequently found also to be expressed in the placenta (Blaise et al. 2003). Syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the HERV FRD sequence (Blaise et al. 2003). Immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts (Fig. 5). The exact localization is somewhat controversial (reviewed by Potgens et al. 2004), which may reflect differences in antibodies, fixation procedures and controls. A major point of concern is also the degree of cross-reactivity to the related sequences in syncytin-2, which recently was localized to a subpopulation of cytotrophoblasts (Malassiné et al. 2008). The syncytin-1 receptor protein ASCT-2 was recently localized to cytotrophoblasts (Hayward et al. 2007). More studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur. Fig. 5Human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum. Staining occurs in the syncytiotrophoblasts, which predominate at this stage. Nuclei are lightly counterstained with haematoxylin Trophoblast cell fusions are, however, not unique to the primate placenta. Thus, also in the mouse placenta, trophoblasts fuse to form syncytiotrophoblasts but mice do, of course, not express HERV sequences. Amazingly, an in-silico search of the mouse genome unraveled the existence of two murine endogenous retroviral (MERV) Env genes, labeled syncytin-A and -B (Dupresssoir et al. 2005). These genes were also fusogenic and orthologous genes were present in additional species of muridae (rats, gerbils, voles and hamsters) and appear to have entered the rodent lineage some 20 million years ago (Dupresssoir et al. 2005). They were expressed in the placenta and at least syncytin-A has been shown to be involved in the formation of syncytiotrophoblasts in mice (Gong et al. 2007). This represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions. The ability of syncytins to induce cell fusions may not be their only physiologic role. Thus, recent studies have shown that while syncytin-2 and syncytin-B also possess immunosuppressive activity, syncytin-1 and syncytin-A do not (Mangeney et al. 2007). Syncytin-1 expression was originally detected in the placenta and testis (Blond et al. 2000; Mi et al. 2000) but subsequent studies have revealed the presence of syncytin-1 also in the brain (Antony et al. 2004) and in breast, colon and endometrial cancers (Bjerregaard et al. 2006; Strick et al. 2007, Larsen, Talts, Andersen, Bjerregaard and Larsson: work in progress). Syncytin-1 may also regulate production of inflammatory mediators (Antony et al. 2004). A number of additional molecules have been shown to be important to cell fusions. In fact, in all systems studied so far, from mating yeast to man, a bewildering array of mechanisms have been identified (see Chen and Olson 2005; Chen et al. 2007; Oren-Suissa and Podbilewicz 2007 for recent reviews). Molecules potentially involved in mammalian cell fusions include ADAM (a disintegrin and metalloproteinase domain) 12 (meltrin alpha), which has been associated with myoblast and osteoclast cell fusions (Abe et al. 1999; Galliano et al. 2000; Gilpin et al. 1998; Yagami-Hiromasa et al. 1995). In addition, ADAMs 1 and 2 (fertilins) may be involved in sperm–oocyte fusions, but do not seem indispensable for this function and, in man, the fertilin alpha gene is dysfunctional (Chen and Olson 2005; Cho et al. 1997, 1998, Evans et al. 1998; Jury et al. 1997, 1998). In contrast, the tetraspanin protein CD9 is needed for sperm–oocyte fusions (Le Naour et al. 2000). CD9 is also expressed by BeWo trophoblast tumor (choriocarcinoma) cells and has been linked both to BeWo invasiveness and to invasion during mouse embryo implantation (Hirano et al. 1999; Liu et al. 2006). CD9 has, together with another tetraspanin family member, CD81, also been linked to myoblast fusion and myotube maintenance (Tachibana and Hemler 1999). Moreover, antibodies to either CD9 or CD81 have been shown to block fusions induced by Mason—Pfizer monkey virus—a D-type retrovirus (Duelli et al. 2005). Tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via EWI (Glu-Trp-Ile) and ERM (ezrin–radixin–moesin) proteins (Hemler 2003; Sala-Valdés et al. 2006). Possibly, their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions (Zivyat et al. 2006). They do not express characteristics of fusogenic proteins like the syncytins, SNAREs and class I viral envelope proteins, but are coexpressed with syncytin-1, at least in BeWo cells. A macrophage fusion receptor (MFR, SIRPalpha), resembling CD4–the cell surface receptor for HIV—has been identified in macrophages (Saginario et al. 1995, 1998; van den Berg et al. 2005). MFR, which belongs to the immunoglobulin superfamily, binds another member of this family, CD47. CD47 is also structurally related to proteins expressed by Vaccinia and Variola viruses (Chen et al. 2007). Whereas expression of MFR is restricted to myeloid cells and neurons, CD47 is ubiquitously expressed. MFR is transiently induced in macrophages at the onset of fusion while CD47 expression is constant. It has been hypothesized that CD47 initially binds to a long form of MFR to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer (5–10 nm) for fusion (Vignery 2005). Additionally, or alternatively, CD47 may promote calcium entry by forming a membrane pore (reviewed in Chen et al. 2007). Also the hyaluronan receptor, CD44, is induced transiently when macrophages start to fuse. CD44 and MFR are subsequently cleaved by proteases during fusion. As mentioned above, CD9 and CD81 may also contribute to macrophage fusion. Additionally, DC-STAMP seems to be required for macrophage fusions. Mice lacking DC-STAMP are osteopetrotic and lack multinucleated osteoclasts and giant cells (Yagi et al. 2005). DC-STAMP is a seven-transmembrane receptor, somewhat similar to the HIV co-receptor CXCR4, but a ligand has yet to be identified. These and many more molecules, including integrins, vacuolar ATPase and receptors and their ligands, as well as different signaling intermediates have been associated with cell fusions. With the exception of the syncytins, most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions (reviewed by Chen and Olson 2005; Chen et al. 2007; Oren-Suissa and Podbilewicz 2007). However, this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions. It is noteworthy that, in both man and mouse, two different syncytins are expressed and are both fusogenic. It is possible that, in the mammalian system, fusion requires a flotilla of molecules organized into membrane microdomains by proteins like CD9, encompassing receptors/ligands, signaling entities, proteases and fusogens of retroviral origin that are capable of forming alpha-helical bundles, which bring membranes closer. Syncytins and related retroviral envelope sequences may, in this connection, function both as fusogens and receptor ligands. However, it seems unlikely that as irreversible an event as a cell–cell fusion should depend upon a single receptor–ligand interaction. Thus, both facilitatory and inhibitory factors are expected to be part of the flotilla. Cancer cell fusions Studies by Mortensen et al. (2004) documented that human breast cancer cells fused with endothelial cells in culture. Stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions (Blond et al. 2000; Mi et al. 2000), we examined whether a similar mechanism could account for cancer–endothelial cell fusions. Expression of syncytin-1 was documented in the breast cancer cell lines examined (MCF-7 and MDA-MB-231 cells; Bjerregaard et al. 2006, SK-BR-3 cells: Talts, Bjerregaard and Larsson: unpublished data). Moreover, we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor ASCT-2. Use of phosphorthioate-protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative RT-PCR or Western blotting and inhibited breast cancer–endothelial cell fusions, whereas a scrambled oligonucleotide control was without effect. Additionally, the syncytin-1 CHR peptide, referred to above, also inhibited the fusions whereas a control peptide was without effect (Bjerregaard et al. 2006). However, neither the antisense nor the CHR peptide experiments effected a total inhibition of cancer-endothelial cell fusions. There may be several reasons to this. Thus, the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the CHR peptide could potentially be ascribed to proteolytic degradation. However, we also detected that the breast cancer cells produced syncytin-2 and cannot exclude that also this molecule contributed to the cancer–host cell fusions. Further experiments using shRNA-directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this. Nevertheless, these data provide strong evidence that syncytin-1 is involved in mediating cancer–endothelial cell fusions in vitro. We next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence. In addition, tumors were also screened for expression of ASCT-2 using a peptide antiserum. Preabsorption of the antisera with the corresponding peptides, but not with irrelevant peptides, abolished staining (Larsson et al. 2007b) (Fig. 6). The results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed ASCT-2 (Fig. 6). Moreover, significant expression of ASCT-2 was detected also in many tumor cells (Fig. 6). The degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease-free survival of the patients (Larsson et al. 2007b). Multivariate analysis included age dichotomized at 40 years, tumor size dichotomized at 20 mm, grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease-free survival Also when used as a continuous variable, syncytin expression emerged as a significant prognostic indicator for disease-free survival in the Cox model (P = 0.02) (Larsson et al. 2007b). Fig. 6Human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen-preabsorbed antiserum (b) and for ASCT-2 (c, e) or with antigen-preabsorbed antiserum (d). Nuclei are lightly counterstained with haematoxylin. Note presence of syncytin immunoreactivity in tumor cells and of ASCT-2 immunoreactivity in endothelial cells (arrows) and in tumor cells The involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by Strick et al. (2007), working with endometrial carcinomas. In their study, downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells. In agreement with findings on placental cells (vide supra), both cAMP elevating agents and estrogens upregulated syncytin-1 expression. However, only cAMP elevating agents stimulated cell fusions (Strick et al. 2007). This apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated TGFbeta, which interfered with syncytin-induced fusions. Thus, estrogen did, in fact, stimulate cell fusions if TGFbeta was immunoneutralized. Conversely, additions of TGFbeta 1 or 3 reduced fusions induced by cAMP-elevating agents. This effect was observed both in endometrial carcinoma cells and in trophoblast-derived cells. These results show that both cAMP and estrogens positively may regulate syncytin-1 expression in tumor cells and that the TGFbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells (Strick et al. 2007). The role of syncytin-1 and cell fusions in cancer needs further study. Thus, our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by Strick et al. (2007) show that additional regulators, such as TGFbeta isoforms may be important modulators of cell fusions. So far, our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor. This is not the same as to say that cell fusions may be universally beneficial to cancers. First, syncytins may have additional effects within the tumor environment (Larsson et al. 2007a, b). Secondly, it seems likely that several factors within the cancer and its stroma (inactivated tumor suppressor genes, activated oncogenes, expression of fusogens and of CD9 and CD81 as well as TGFbeta) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above. Interestingly, however, expression of CD9 has been analyzed in a number of tumors and seems, like syncytin expression, to predict a good prognosis (Funakoshi et al. 2003; Hashida et al. 2002; Higashiyama et al. 1997; Houle et al. 2002; Miyake et al. 1995; Uchida et al. 1999). We propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events, acting in concert with other molecules with both enhancing and inhibitory regulatory effects.

Planta-4-1-2249615

Tie-dyed1 and Sucrose export defective1 act independently to promote carbohydrate export from maize leaves

tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) are recessive maize (Zea mays) mutants with nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars. In addition, both mutants display similar growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaves. As tdy1 and sxd1 are the only variegated leaf mutants known to accumulate carbohydrates in any plant, we investigated whether Tdy1 and Sxd1 function in the same pathway. Using aniline blue staining for callose and transmission electron microscopy to inspect plasmodesmatal ultrastructure, we determined that tdy1 does not have any physical blockage or alteration along the symplastic transport pathway as found in sxd1 mutants. To test whether the two genes function in the same genetic pathway, we constructed F2 families segregating both mutations. Double mutant plants showed an additive interaction for growth related phenotypes and soluble sugar accumulation, and expressed the leaf variegation pattern of both single mutants indicating that Tdy1 and Sxd1 act in separate genetic pathways. Although sxd1 mutants lack tocopherols, we determined that tdy1 mutants have wild-type tocopherol levels, indicating that Tdy1 does not function in the same biochemical pathway as Sxd1. From these and other data we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Our genetic and cytological studies implicate Tdy1 functioning in veins, and a model discussing possible functions of TDY1 is presented. Introduction Sugars synthesized in photosynthetic leaf cells must be transported into the veins for distribution to nonphotosynthetic tissues. Maize (Zea mays) is a C4 plant which displays Kranz anatomy in its leaves (Esau 1977). Veins are surrounded by bundle sheath cells which in turn are surrounded by mesophyll cells. Mesophyll and bundle sheath cells cooperatively perform the reactions of photosynthesis and carbon assimilation. Sucrose is synthesized in the cytoplasm of mesophyll cells (Lunn and Furbank 1999), diffuses through plasmodesmata into bundle sheath cells, and then into vascular parenchyma cells (Russin et al. 1996). Maize is an apoplastic phloem loading species (Evert et al. 1978), whereby sucrose is exported from the vascular parenchyma cells to the apoplast by an unknown mechanism (see Lalonde et al. 2004 for discussion). Sucrose is then imported into phloem companion cells and/or sieve elements by sucrose transporters located in the plasma membrane (Aoki et al. 1999; Lalonde et al. 2004; Sauer 2007; Scofield et al. 2007). The energy to transport sucrose into the phloem is derived from H+-ATPases that generate the proton motive force across the plasma membrane (Bush 1993; Gaxiola et al. 2007). In addition, the abundance of the sucrose transporter that controls phloem loading is transcriptionally regulated by a sucrose-sensing signal transduction pathway (Chiou and Bush 1998; Vaughn et al. 2002; Ransom-Hodgkins et al. 2003). If carbon transport into the vein is blocked, carbohydrates accumulate in the photosynthetic cells, leading to down-regulation of photosynthetic gene expression and reduced chlorophyll levels (chlorosis) (Sheen 1990; Goldschmidt and Huber 1992; Riesmeier et al. 1994; Krapp and Stitt 1995; Burkle et al. 1998; Gottwald et al. 2000; Jeannette et al. 2000). Even though the transport pathway for assimilated carbon has been studied in maize leaves for almost 40 years (Hofstra and Nelson 1969), the mechanisms that regulate sucrose export from leaves remain poorly understood. To date, two recessive maize mutants have been described with defects in carbon export from leaves. tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) mutants develop nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars (Russin et al. 1996; Provencher et al. 2001; Braun et al. 2006). Other mutants with variegated leaves have been characterized, but none have carbohydrate accumulation within the chlorotic regions (Rhoades and Carvalho 1944; Fisher and Eschrich 1985; Madore 1990; Yu et al. 2007). tdy1 and sxd1 both display growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaf tissues. However, their phenotypes also show significant differences. sxd1 mutant leaves progressively exhibit chlorosis which initiates at the leaf tip and basipetally spreads toward the base. Interestingly, in sxd1 mutants, only the leaf minor veins are affected in phloem loading (Russin et al. 1996). The tdy1 mutant phenotype is distinct from sxd1 mutants in that large chlorotic regions develop throughout leaf blade tissue. Further, tdy1 chlorotic regions form only during a limited time as the leaf emerges from the whorl and, once formed, do not expand (Braun et al. 2006). Lastly, in tdy1 mutants, chlorotic tissues are often bounded by lateral veins, implicating this vein class in limiting the expansion of a tdy1 chlorotic sector (Baker and Braun 2007). Sxd1 encodes tocopherol cyclase, an enzyme functioning in tocopherol (vitamin E) biosynthesis, and sxd1 mutants lack tocopherols (Sattler et al. 2003). Absence of tocopherols results in callose deposition over the plasmodesmata at the bundle sheath-vascular parenchyma cell interface of leaf minor veins (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). This occludes these passageways and prevents sucrose from moving into the vein, resulting in the build-up of carbohydrates in photosynthetic cells. The function of tocopherols in preventing callose deposition in the phloem is evolutionarily conserved. RNAi-mediated suppression of tocopherol cyclase activity in potato results in callose deposition in vascular-associated cells and carbohydrate accumulation in the photosynthetic cells (Hofius et al. 2004). In addition, in Arabidopsis tocopherol cyclase mutants and other vitamin E deficient mutants, callose is deposited in phloem parenchyma transfer cells and the photosynthetic cells accumulate carbohydrates if the plants are subjected to nonfreezing low temperatures (Maeda et al. 2006). It is not known how lack of tocopherols causes callose deposition. Because both mutants show defects in carbon export from leaves, we examined whether Tdy1 and Sxd1 function in the same pathway to limit leaf carbohydrate accumulation. Using aniline blue fluorescence microscopy and transmission electron microscopy (TEM) we determined that carbohydrate accumulation in tdy1 mutant leaves does not result from a similar impediment to phloem loading as in sxd1 mutants. To genetically test whether the two genes function in the same pathway, we analyzed F2 families segregating both mutations. We observed that the double mutant plants showed an additive interaction for growth-related phenotypes and soluble sugar accumulation indicating that the two genes function in separate genetic pathways. To determine if Tdy1 functions in the same biochemical pathway as Sxd1, we measured tocopherol levels in mutant plants. We found that tdy1 mutants contain wild-type levels of tocopherols, indicating that Tdy1 does not function in the same biochemical process as Sxd1. Thus, from the combination of cellular, genetic and biochemical investigations we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Additionally, genetic and cytological studies suggest that Tdy1 functions within the veins, and a model of possible TDY1 functions is discussed. Materials and methods Plant materials Plants were grown in the summer in the Rock Springs Agronomy Farm, Pennsylvania State University, and in the winter in Juana Diaz, Puerto Rico. The tdy1-Reference (tdy1-R) (Braun et al. 2006) and sxd1-1 (Russin et al. 1996) mutations were backcrossed to the B73 inbred line five times prior to crossing together. F2 families were generated by crossing tdy1-R and sxd1-1 mutant plants and self-pollinating the F1 individuals. Morphological analyses Plant height was measured from the soil surface to the tip of the central spike of the tassel. Tassel length was measured from the node of the flag leaf to the tip of the central spike. Anthesis was recorded as the day of first pollen shed. Plants were scored as having produced ears if either (1) ears were clearly visible, (2) silks were visible emerging from the leaf sheath, or (3) ears developed such that they were identifiable by peeling back the ear leaf sheath. For wild-type and tdy1-R single mutants n = 18 for all measurements. For sxd1-1 single mutants, 12 of the 16 plants produced a tassel that shed pollen, and 13 produced an ear. For tdy1-R; sxd1-1 double mutants, 7 of the 15 plants shed pollen and 8 made an ear. Barren tassels that did not shed pollen were not included in the tassel length or anthesis measurements. Aniline blue staining Leaf strips 1 cm in width were cut from mutant and wild-type leaves. The abaxial epidermis and the mesophyll cells directly beneath it were gently scraped away with a single-edge razor to expose the inner leaf tissues. The leaf strip was placed on a slide, briefly stained with a 0.05% aniline blue and immediately viewed using a 360–370-nm excitation filter and a 420-nm long pass emission filter on a Nikon Eclipse 80i fluorescent microscope. Epifluorescent illumination was provided by a 100-W mercury lamp, and images were recorded using a DXM1200F Nikon digital camera. TEM analyses For tissue fixation, leaf samples were diced into 3 × 1 mm pieces, placed in 4% glutaraldehyde, 1% paraformaldehyde, 0.3% Tween 20, 50 mM sodium cacodylate, pH 7.4, and vacuum infiltrated on ice for 4–6 h. Samples were post-fixed in 1–2% osmium tetroxide at 4°C overnight, subsequently dehydrated through a graded acetone series and embedded in Spurr’s epoxy resin (Spurr 1969). Thin sections (90–100 nm) were cut on a Leica Ultracut E ultramicrotome with a glass knife and lifted onto 200-mesh copper grids. The grids were stained in 2% uranyl acetate, followed by Reynold’s lead citrate and observed with a JEOL JEM 1200 EXII at an accelerating voltage of 80 kV. Chlorophyll quantification Relative levels of total chlorophylls were quantified from leaves using a SPAD 502 Data Logger Chlorophyll Meter (Spectrum Technologies). For each tissue, 30 samples were measured, and the experiment was performed three times. Representative data from one replicate are shown. Sugars and starch quantification Leaf tissues were collected at the end of the photoperiod, weighed and frozen at −80°C. Carbohydrate extraction was performed according to Dinges et al. (2001). Sugars and starch were quantified using commercial assay kits according to the manufacturer’s instructions (R-Biopharm). Six samples from each tissue type were measured in triplicate. For visualizing starch in leaves, samples were collected at the end the photoperiod, decolored by boiling in 95% ethanol and stained with iodine-potassium iodide (IKI) (Ruzin 1999). Tocopherol quantification Leaf discs of 11 mm were harvested with a cork borer from leaf tissues, weighed and immediately frozen on dry ice. Total lipids were extracted from collected tissues and tocopherols measured according to (Sattler et al. 2003). Results tdy1-R and sxd1-1 single mutants both display variegated chlorotic leaves tdy1-R and sxd1-1 mutants show striking parallels in their leaf phenotypes. Both develop chlorotic sectors that hyperaccumulate starch and soluble sugars in leaf blades (Fig. 1). However, there are also distinctions. Whereas wild-type leaves have a uniform, dark green color, sxd1-1 mutant leaves develop a chlorotic phenotype that shows a continuum of expression from strongest at the tip to mildest toward the base (Fig. 1a, b). In addition, at the tip and leaf margins, the chlorotic tissues progressively accumulate anthocyanin and excess starch (Fig. 1b, e). In sxd1-1 leaves, no clear, delineated boundaries occur between green and chlorotic regions. Conversely, tdy1-R mutants display a variegated pattern of chlorotic and normal appearing green regions throughout leaf blade tissue (Fig. 1c). Additionally, the boundaries between tdy1-R chlorotic and normal appearing green tissue are sharp and distinct, and they tend to occur at lateral veins (Baker and Braun 2007). As in sxd1-1 mutants, chlorotic regions in tdy1-R mutants hyperaccumulate starch relative to green tissues (Fig. 1f). tdy1 and sxd1 are the only known leaf variegation mutants that hyperaccumulate carbohydrates in any plant; therefore, we investigated whether Tdy1 and Sxd1 function in a common pathway influencing carbon partitioning in leaves. Fig. 1tdy1-R and sxd1-1 leaves display chlorotic sectors that hyperaccumulate carbohydrates. a Wild-type leaf showing uniform dark green color. b sxd1-1 mutant leaf displaying a chlorotic gradient, strongest at the tip to mildest toward the leaf base. Anthocyanins accumulate in the chlorotic tissues at the leaf tip and margins. c tdy1-R mutant leaf containing variegated chlorotic and green sectors throughout the leaf blade. d–f Cleared, IKI stained leaf tissues showing that mutant chlorotic regions hyperaccumulate starch. d Wild type. e sxd1-1. f tdy1-R. Scale bars represent 4 cm Carbohydrate accumulation in tdy1-R mutants occurs by a different mechanism than in sxd1-1 plants To address whether the two genes function in the same pathway, we investigated if the mechanism for carbohydrate accumulation was similar in the two mutants. Carbohydrate accumulation in sxd1-1 mutants results from a blockage in the symplastic pathway of sucrose movement (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). Specifically, at the bundle sheath–vascular parenchyma cell interface of leaf minor veins, callose is deposited over the plasmodesmata in bundle sheath cells. This blockage prevents sucrose from being loaded into the phloem and results in carbohydrate accumulation in the photosynthetic cells. To determine if the tdy1-R leaf phenotype is similarly caused by callose deposits plugging the plasmodesmata, we performed aniline blue staining and fluorescence microscopy. Aniline blue binds callose and fluoresces a blue–white color under UV light. In wild-type plants, only the sieve plates in the end walls of phloem sieve elements stain positively for callose (Fig. 2b). As previously reported, sxd1-1 mutants display many punctate callose deposits at the vascular parenchyma-bundle sheath cell interface of minor veins (Fig. 2c) (Botha et al. 2000). In tdy1-R chlorotic regions, no ectopic callose deposits were detected at this interface (Fig. 2d). Further, no callose deposits were observed in any cells of minor or lateral veins in tdy1-R chlorotic tissues, nor in any cells of tdy1-R green regions (data not shown). These data suggest that tdy1-R mutants do not accumulate carbohydrates due to callose blocking symplastic sucrose movement. Fig. 2Aniline blue staining of ectopic callose deposits in leaf minor veins. a Cross section of a wild-type minor vein shown by UV autofluorescence indicating the cell types and orientation of view (arrow) for b–e. Abaxial cells were removed below the black dotted line to view the bundle sheath–vascular parenchyma cell interface. X xylem, P phloem, VP vascular parenchyma, BS bundle sheath, M mesophyll. b–eAniline blue fluorescence images of paradermal sections along minor veins. b Wild-type cells lack punctate callose deposits indicating no blockages along the symplastic pathway. The bright fluorescence observed in the phloem corresponds to sieve plates (arrowhead). c sxd1-1 minor veins contain many callose deposits at the bundle sheath–vascular parenchyma cell interface (arrow). d tdy1-R chlorotic tissue lacks callose deposition over plasmodesmata in any cells. e tdy1-R; sxd1-1 double mutants contain callose deposits (arrow) at the bundle sheath–vascular parenchyma cell interface comparable to the level seen in sxd1-1 single mutants. Scale bar in a represents 50 μm, and in b–e 10 μm Although we did not observe ectopic callose deposition in any cells in tdy1-R mutant leaves using aniline blue staining, it is possible that symplastic sucrose transport is precluded due to a different defect in plasmodesmatal structure. To investigate this possibility, we performed TEM to inspect the plasmodesmata ultrastructure along the symplastic pathway. In minor veins from wild-type leaves, the plasmodesmata between bundle sheath and vascular parenchyma cells were unobstructed, spanned the cell wall and connected the cytoplasms between the two cells (Fig. 3a). Consistent with previous reports, minor veins from leaf tips expressing the sxd1-1 mutant phenotype showed occlusions over the plasmodesmata on the bundle sheath cell side of the bundle sheath–vascular parenchyma cell interface (Fig. 3b) (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). In tdy1-R mutant leaves, we found no occlusions over the plasmodesmata or alterations in their structure between the bundle sheath and vascular parenchyma cells in minor veins (Fig. 3c). In examining plasmodesmata along the symplastic pathway at all cellular interfaces in tdy1-R leaves, we did not observe any structural perturbations or occlusions (Fig. 3e–j). These data indicate that carbohydrate retention in tdy1-R mutant leaves does not appear to result from physical blockage or plasmodesmatal structural changes. Together, the aniline blue staining and TEM studies indicate that the cellular basis for the tdy1-R and sxd1-1 phenotypes is different. Fig. 3TEM images of cellular interfaces along the symplastic pathway of minor veins. a-d Bundle sheath–vascular parenchyma cells. Arrows indicate the location of the plasma membrane in the bundle sheath cell. a Wild-type plasmodesmata span the cell wall, lack occlusions, and connect with the plasma membrane. b sxd1-1 chlorotic leaf tips contain occlusions over the plasmodesmata on the bundle sheath cell side of the cell wall. c tdy1-R chlorotic sectors contain normal appearing plasmodesmata that lack occlusions. Note the plasmodesmata span the cell wall and connect with the plasma membrane. d tdy1-R; sxd1-1double mutant chlorotic tissues contain deposits over the plasmodesmata in the bundle sheath cell similar to sxd1-1 single mutants. Additional cellular interfaces along the symplastic pathway of wild type (e, g, i) and tdy1-R chlorotic tissues (f, h, j) are shown. In e–j the plasmodesmata spanned the cell wall connecting the cytoplasms of adjacent cells and lacked occlusions. e, f BS–BS cells, g, h BS–M cells, i, j M–M cells. BS bundle sheath, M mesophyll. Scale bars represent 200 nm Double mutant plants show more severe growth defects than either single mutant Both tdy1-R and sxd1-1 single mutant plants have growth-related defects due to the retention of carbohydrates in leaves. To determine whether the two genes function in the same genetic pathway promoting carbon export from leaves, we created F2 families segregating both mutations. Both mutations are recessive and the F2 segregation ratio fits the expected 9:3:3:1 prediction, indicating that neither mutant shows epistasis (Table 1). sxd1-1 is a molecular null allele lacking transcripts (Provencher et al. 2001), and based on dosage analysis, tdy1-R is a genetic null allele (Braun et al. 2006). If Tdy1 and Sxd1 act in a linear genetic pathway, we would expect that the phenotype of the double mutant would be similar to one or the other mutant. If the two genes function in separate pathways, then we predict that the double mutant would be more severe than either single mutant. Table 1Genetic segregation in F2 familiesFamily #TotalWild typetdy1-Rsxd1-1tdy1-R; sxd1-1χ2DB 55039348221672.0P > 0.05DB 55129552231552.1P > 0.05DB 55239355181550.6P > 0.05Total2811556346172.9P > 0.05χ2 analyses of tdy1-R and sxd1-1 segregation in three F2 families conform to a 9:3:3:1 expectation. Observed numbers of individuals are shown To examine the interactions between tdy1-R and sxd1-1, we quantified the growth parameters of plant height, tassel size, time to anthesis and ear production (Fig. 4, Table 2). At the whole plant level, the double mutants express a more severe phenotype and show a stronger reduction in growth characteristics than either single mutant (Fig. 4a). Compared to wild-type, tdy1-R single mutants have an 11% decrease, sxd1-1 mutants have a 24% reduction, and the tdy1-R; sxd1-1 double mutant plants show the greatest reduction in plant height of 35.5%. Similar to plant height, tassel size is reduced 13% in tdy1-R plants, 29% in sxd1-1 mutants, and 50% in tdy1-R; sxd1-1 double mutants (Fig. 4c, Table 2). In addition, approximately half of the tdy1-R; sxd1-1 double mutant plants produced very reduced tassel branches lacking spikelets (Fig. 4d). This phenotype was observed in 25% of the sxd1-1 single mutants. Correlated with the tassel height defects, both mutations retarded time to flowering. Both tdy1-R and sxd1-1 single mutants first shed pollen eight days later than wild-type siblings (Table 2). Double mutant plants were delayed even further by approximately another 5 days. Similarly, wild-type and tdy1-R mutant plants invariably made ears, and sxd1-1 mutants produced ears 81% of the time. In contrast, only 53% of the double mutant plants produced an ear (Table 2). All of the growth defects observed in the single and double mutants are consistent with a restriction in assimilates transported to the growing portions of the plants. The double mutant plants showed the most severe phenotypes suggesting they have a greater reduction in carbohydrates exported out of leaves than in either single mutant. In support of this, the leaves of tdy1-R; sxd1-1 double mutant plants are more strongly chlorotic than either single mutant, with a corresponding reduction in the amount of green tissue (compare Figs. 4b with 1b, c). In double mutant leaves, the tip to base gradient of chlorosis and anthocyanin pigmentation characteristic of the sxd1-1 phenotype is superimposed on the tdy1-R chlorotic and green sectored pattern. Double mutant leaves still contain some green tissues located at the leaf base as in sxd1-1 single mutants; however, the boundaries between the chlorotic and green regions are sharp and distinct as in tdy1-R mutants (Fig. 4b). Thus, the double mutant leaves express the phenotypes of both single mutants, and double mutant plants display stronger growth retardation than either single mutant. Fig. 4Phenotypic comparisons among plants from a F2 family segregating tdy1-R and sxd1-1 mutants. In a and c the order from left to right corresponds to wild type, tdy1-R, sxd1-1, and tdy1-R; sxd1-1 double mutant. a Double mutants show the greatest reduction in plant height compared with either single mutants or wild-type siblings. b Double mutant leaves are severely chlorotic with lighter green tissues restricted to the leaf base. Distinct borders between the chlorotic and green tissues characteristic of tdy1-R are visible at the leaf base. The strong tip to base chlorosis gradient of sxd1-1 expression is evident. c Tassel height is most strongly reduced in tdy1-R; sxd1-1 double mutants compared with sxd1-1 and tdy1-R single mutants or wild-type siblings. d Approximately half of the tdy1-R; sxd1-1 double mutant plants produced barren tassels lacking spikelets. Scale bars represent 4 cmTable 2Morphometric analyses in F2 familiesPhenotypePlant heightwt%Tassel lengthwt% AnthesisEars (%)Wild type220.4a ± 0.3910055.2a ± 0.1410071.9a ± 0.06 100tdy1-R196.8b ± 0.9289.247.9b ± 0.28 86.880.3b ± 0.13 100sxd1-1168.9c ± 1.2476.639.3c ± 0.1871.280.3b ± 0.0981tdy1-R; sxd1-1142.2d ± 0.7964.526.8d ± 0.34 48.684.9c ± 0.1353Values are the means ± SE. Plant height and tassel length are measured in cm. Anthesis is measured as the number of days after planting until first pollen shed. Percent ears indicates the frequency each phenotypic class produced an ear. Different letters in superscript within a column denote statistical significance determined using Student’s t test Since double mutants have more extensive chlorosis in their leaves, we ascertained whether the callose deposits at the vascular parenchyma-bundle sheath cell interface observed in sxd1-1 single mutants were more severe in the double mutants. Using aniline blue fluorescence, the double mutants showed no enhancement of the callose deposition phenotype and appeared similar to sxd1-1 single mutants (Fig. 2c, e). Similarly using TEM, we found blockages over the plasmodesmata on the bundle sheath cell side of the bundle sheath–vascular parenchyma cell interface of minor veins in the double mutants comparable to what we observed in sxd1-1 single mutant leaves (Fig. 3b, d). In neither case was there any increase in the severity of the phenotype. Hence, tdy1-R and sxd1-1 do not display a synergistic interaction as the double mutant leaves express both single mutant phenotypes. Rather, these data suggest that the phenotypes observed in the double mutants are additive, and that the genes act in independent pathways. Leaf tips of double mutant plants have the greatest reduction in chlorophyll levels tdy1-R and sxd1-1 single mutants both show variegated chlorotic regions in their leaves (Fig. 1b, c). We quantified total chlorophyll levels to determine whether the double mutants were more strongly affected than either single mutant (Table 3). We observed a decreasing series in chlorophyll content with the strongest reduction in the tips of double mutant leaves < the chlorotic regions of either tdy1-R or sxd1-1 single mutant leaves < the green base of sxd1-1 or double mutant leaves < green regions of tdy1-R mutants or wild-type leaves. The leaf bases of sxd1-1 mutants and the double mutants displayed a mild chlorosis phenotype with approximately 70% as much chlorophyll as wild-type leaves indicating that these tissues are affected by the sxd1-1 mutation. As the tips of double mutant leaves showed a more severe chlorosis phenotype than either single mutant, it suggests that Tdy1 and Sxd1 function in separate pathways. Table 3Chlorophyll quantification in F2 plantsPhenotypeChlorophyllWild type55.7a ± 0.55tdy1-R green54.8a ± 0.57sxd1-1 green41.0b ± 0.75tdy1-R; sxd1-1 green42.2b ± 0.67tdy1-R chlorotic20.9c ± 0.62sxd1-1 chlorotic21.5c ± 0.66tdy1-R; sxd1-1 chlorotic11.4d ± 0.64Chlorophyll levels were measured in different phenotypic (green or chlorotic) tissues from different genotypes. Values represent the means ± SE in relative units (n = 30). Statistical significance was determined using Student’s t test and indicated by different letters in superscript Sugar accumulation in double mutant leaves is greater than in either single mutant Both single mutants accumulate excess starch in the chlorotic regions of their leaves (Fig. 1e, f). To investigate whether Tdy1 and Sxd1 act in the same or separate genetic pathways limiting carbohydrate accumulation in leaves, we determined the starch and soluble sugars content in different phenotypic regions of F2 plants (Fig. 5; Table S1 in electronic supplementary material). For both the green regions and the chlorotic regions, we found that the double mutants contained similar amounts of starch as the single mutants. In contrast, we determined that the levels of soluble sugars differed with respect to genotype and phenotype. As previously reported, we found green regions of tdy1-R leaves had sucrose, glucose and fructose levels indistinguishable from wild type (Braun et al. 2006). However, green leaf bases of the double mutants and sxd1-1 single mutants contained approximately fourfold higher levels of glucose and fructose as wild type or tdy1-R green tissues. Among the chlorotic leaf tissues, the double mutant leaf tips had the highest concentrations of sugars, with approximately 33% greater amounts of glucose, and a significant increase in sucrose content compared with either single mutant. Double mutant leaf tips also contained ∼30% more fructose than sxd1-1 leaf tips and approximately two-fold greater levels than in tdy1-R chlorotic regions. Overall, sugar concentrations in green tissues of double mutant leaves are more similar to sxd1-1 leaf bases than to tdy1-R green regions, whereas in chlorotic tissues, the double mutants contain more soluble sugars than either single mutant. Because the double mutants show greater individual sugar accumulation than either single mutant, these data support the hypothesis that Tdy1 and Sxd1 function in independent pathways promoting carbon export from leaves. Fig. 5Carbohydrate quantification in leaves of wild-type, tdy1-R, sxd1-1 and double mutant plants. Units for all panels are mg carbohydrate/g fresh weight. Values are the means of 18 samples ± SE. Abbreviations are wt, wild-type, dm, tdy1-R; sxd1-1 double mutant. Different letters indicate statistically significant differences between samples determined using Student’s t test Tdy1 does not function in the same biochemical pathway as Sxd1 Sxd1 encodes tocopherol cyclase, the penultimate enzyme in the vitamin E biosynthesis pathway, and sxd1 mutants lack tocopherols (Sattler et al. 2003). To determine whether Tdy1 functions in the same biochemical pathway as Sxd1, we quantified tocopherol levels in tdy1-R plants (Table 4). We found no statistically significant changes in tocopherol quantities in tdy1-R chlorotic or green regions in comparison to wild-type tissue. sxd1 mutants had undetectable levels of tocopherols as previously reported (Sattler et al. 2003). Hence, tdy1-R does not have a lesion in tocopherol synthesis or accumulation, and Tdy1 does not function in the same biochemical pathway as Sxd1. Table 4Tocopherol quantificationPhenotypeTocopherolWild type118.6a ± 3.59tdy1-R green101.0a ± 3.17tdy1-R chlorotic112.3a ± 6.17sxd1-1ndValues are means of six samples ± SE measured in μg/g fresh weight. nd indicates none detected. Values indicated by letter “a” in superscript were not statistically different as determined using Student’s t test Discussion Tdy1 and Sxd1 independently promote carbon export in maize leaves tdy1 and sxd1 are the only known mutations in plants that confer a variegated leaf phenotype with sectors that hyperaccumulate carbohydrates. Using a combination of biochemical, cytological and genetic analyses we investigated whether Tdy1 and Sxd1 act in a common pathway. Whereas sxd1 mutants lack tocopherol, we determined that tdy1-R mutants accumulate wild-type levels of tocopherols. Therefore, Tdy1 does not function in the same biochemical process as Sxd1. sxd1 mutants accumulate carbohydrates in leaf tissues due to callose being deposited over the interface of bundle sheath-vascular parenchyma cells in leaf minor veins which blocks sucrose symplastic movement. Using aniline blue fluorescence microscopy we did not identify any ectopic callose deposition in tdy1-R mutant leaf tissues. Moreover, using TEM we did not observe any structural alterations to the plasmodesmata, indicating that the symplastic loading pathway is not physically impeded. This suggests that tdy1-R mutants do not accumulate carbohydrates due to an occlusion, but rather due to a different cellular mechanism. This was confirmed by double mutant analyses which showed that the two mutations had an additive interaction. We found that the double mutants expressed both single mutant leaf phenotypes, that the reductions in plant height, tassel size and chlorophyll levels were additive, and that the accumulation of sugars in chlorotic leaf tissues of the double mutants was greater than in either single mutant. In addition, half of the double mutants produced ears whereas the tdy1-R mutant frequency was indistinguishable from wild type and 81% of the sxd1-1 single mutants made ears. The increased frequencies in the failure to make ears and production of barren tassels observed in the double mutants are most likely explained by an additive retention of carbohydrates in leaf tissues and a concomitant failure to export sucrose to the developing inflorescences. Collectively, these data lead us to conclude that Tdy1 and Sxd1 function independently, and that tdy1 defines a distinct genetic pathway affecting carbon partitioning in maize leaves. Chlorotic leaf tissues do not retain sink identity We previously suggested that one possibility to explain the excess carbon accumulation in tdy1-R chlorotic leaf sectors was a block in the developmental transition from sink to source identity, such that the chlorotic sectors continued to import photoassimilates from neighboring green tissues (Braun et al. 2006). If the chlorotic tissues were to remain sinks, the large amounts of green source tissues could potentially provide the carbohydrates accumulating in the chlorotic regions. However, based on our genetic analyses, our data do not support this hypothesis. In the tdy1-R; sxd1-1 double mutants, the amount of green leaf tissue is greatly reduced and is restricted to a narrow region at the very base of the leaves. Additionally, this green tissue displays mild chlorosis and an increase in carbohydrate accumulation indicating that it is perturbed in carbon export. As this tissue is proximal to and up to a meter distant from the chlorotic tip of adult leaves, it is highly unlikely to be providing the carbohydrates that accumulate in the cells near the tip. Furthermore, half of the double mutant plants produced a tassel and an ear. These are sink tissues dependent on the photosynthetic source leaves for their carbohydrates. Because the great majority of the area of double mutant leaves is severely chlorotic, yet still must be capable of exporting photoassimilates to produce the developing reproductive tissues, our data argues against the chlorotic tissues remaining sinks and does not support the hypothesis that Tdy1 functions to regulate the sink to source transition. Rather, the new data lead us to favor the hypothesis that the cells in the tdy1 chlorotic region are source tissues that are partly blocked in carbon export capacity. A model for TDY1 function In wild-type maize leaves, sucrose is synthesized in mesophyll cells, diffuses through plasmodesmata into bundle sheath cells and then into the vascular parenchyma cells (Fig. 6a). Sucrose is exported from the vascular parenchyma cell to the apoplast, and then loaded into the phloem by sucrose transporters in the plasma membrane of the companion cell and/or sieve element (Lalonde et al. 2004; Sauer 2007). sxd1 mutant leaf tips are blocked in this pathway at the bundle sheath–vascular parenchyma cell interface (Fig. 6b) (Russin et al. 1996; Botha et al. 2000; Provencher et al. 2001). Fig. 6Model for TDY1 functions in phloem loading of sucrose. Diagrams represent the pathway of sucrose movement from photosynthetic cells into the phloem. M mesophyll, BS bundle sheath, VP vascular parenchyma, CC companion cell, SE sieve element. Green and yellow ovals in M and BS cells represent chloroplasts. White circles within chloroplasts represent starch grains. Magenta box on VP plasma membrane represents sucrose efflux transporter, and blue boxes on CC and SE plasma membranes depict sucrose transporters. Arrows indicate the direction of sucrose movement. a Wild-type tissue with normal, unimpeded sucrose movement into the phloem. b sxd1-1 mutant chlorotic tissue has callose deposits (red box) over the plasmodesmata at the BS–VP cell interface blocking sucrose movement and resulting in carbohydrate accumulation in M and BS cells. c tdy1-R chlorotic tissue has normal appearing plasmodesmata and lacks callose deposits. We hypothesize that either the sucrose efflux transport step is inhibited (red X) or the CC–SE sucrose transporters (orange X) fail to load sucrose into the phloem. This would result in the carbon hyperaccumulation observed in the photosynthetic cells tdy1 chlorotic sectors accumulate excess carbohydrates indicating that they have a defect in phloem loading. In agreement with this, we previously localized the site of TDY1 function to the innermost tissue layer of leaves comprised of the veins, bundle sheath cells, and interveinal mesophyll cells (Baker and Braun 2007). Due to the limits of the experiment it was not possible to further delineate where within this tissue layer TDY1 acts. However, the cytological investigations presented resolve this ambiguity. As we observed no blockages or alterations to plasmodesmatal structure along the sucrose symplastic pathway in tdy1-R leaves, the carbon accumulation points to a defect at a later step in phloem loading. Nonetheless, even though the plasmodesmata appear unobstructed, we cannot be certain that they are functional. However, we propose that a molecular blockage of plasmodesmata precluding sucrose movement is an unlikely scenario for the following reason. In sxd1 mutants, specific plasmodesmata are occluded and unable to transit sucrose, causing the vascular parenchyma cells to appear plasmolysed due to their low osmotic concentration (Russin et al. 1996). Accordingly, we carefully inspected for, but never observed, plasmolysis of any vascular parenchyma, mesophyll or bundle sheath cells in tdy1 mutants in our microscopic analyses. Therefore, these data suggest that sucrose is able to pass freely between cells along the symplastic pathway, and hence that TDY1 likely functions in the veins (Fig. 6c). From the variegated phenotype of tdy1 mutants, we hypothesize that TDY1 acts as a regulator of sucrose transport rather than as a transporter itself, as loss of transporter function leads to uniform carbohydrate accumulation and chlorosis phenotypes (Riesmeier et al. 1994; Burkle et al. 1998; Gottwald et al. 2000; see Braun et al. 2006; Baker and Braun 2007 for discussion of mechanisms for tdy1 variegation). Several possibilities are envisioned for Tdy1 function. The first possibility is that Tdy1 may regulate sucrose efflux out of vascular parenchyma cells. Failure to export sucrose to the apoplast would create a bottleneck restriction leading to the build-up of sucrose in the vascular parenchyma cells and, in turn, to carbohydrate accumulation in the bundle sheath and mesophyll cells. The second possibility is that Tdy1 may function to regulate the activity of the H+-ATPases or sucrose transporters within the companion cells and sieve elements. Lack of function of either the H+-ATPases or sucrose transporters results in failure to load sucrose into the phloem and thereby to excess accumulation in the apoplast. Sucrose accumulation in the apoplast would shift the equilibrium for sucrose export from vascular parenchyma cells leading to increased concentration within these cells, and ultimately to carbon accumulation in the photosynthetic cells. In support of this, antisense expression or mutation of a phloem H+-ATPase or sucrose transporters causes an accumulation of carbohydrates in photosynthetic cells and chlorosis (Riesmeier et al. 1994; Burkle et al. 1998; Gottwald et al. 2000; Zhao et al. 2000). A third possibility is that TDY1 may function in the sucrose signal transduction pathway that regulates the abundance of the sucrose transporter controlling phloem loading (Chiou and Bush 1998; Vaughn et al. 2002; Ransom-Hodgkins et al. 2003). Our analyses of the tdy1-R mutant phenotype and genetic interactions between tdy1-R and sxd1-1 have shown that Tdy1 acts independently of Sxd1. Thus, the tdy1 mutation identifies a separate genetic pathway limiting carbon accumulation in maize leaves. Future work characterizing the molecular function of Tdy1 will determine the mechanism by which Tdy1 influences carbon partitioning. Understanding the control of carbon export from leaves will lead to novel strategies to manipulate carbon allocation and biomass deposition which may have applications in the production of biofuels. Electronic supplementary material Below is the link to the electronic supplementary material. (DOC 31 kb)

Eur_J_Nutr-2-2-1705523

Effect of sesamin on serum cholesterol and triglycerides levels in LDL receptor-deficient mice

Background Sesamin, a major lignan from sesame seeds has been associated with cholesterol reduction in previous reports, but recent studies suggested differences in the response to sesamin intake depending on the model studied as well as the nature of the sesamin preparation used. Introduction The research on natural substances affecting cholesterol metabolism for prevention of hypercholesterolemic atherosclerosis has particular therapeutic importance. In particular, the identification of dietary components that can be added to foods to lower or regulate cholesterol levels has gained special interest. Sesame seeds in general and its major lignan sesamin in particular, have been associated with various biochemical actions, mainly related to lipid metabolism, including a hypocholesterolemic effect in both human and animal-based trials (see [1, 2] for review). Although the general hypolipidemic effect of dietary sesamin seems to be clear in studies with rats, recent studies pointed out discrepancies in the response to sesamin between animal species [3] raising doubts about a possible extrapolation of these results to humans. Additionally, the heterogeneous nature of the sesamin preparations used in the different studies seems to be another point to consider, since different biological actions and mechanisms have been proposed for the two epimers of dietary sesamin [4]. In this study, we aimed to further investigate the hypocholesterolemic action of sesamin minimizing the above described variables and using only pure sesamin epimer and selecting low density lipoprotein (LDL) receptor-deficient mice as experimental animals. This model aims to reproduce homozygous familial hypercholesterolemia and it is characterized by decreased removal of cholesterol from the circulation due the lack of functional LDL receptors [5] that leads to increased serum cholesterol levels. Materials and methods Animal model Female ldlr−/− mice (strain number, B6.129S7-Ldlrtm1Her) quality SPF were obtained from the Jackson Laboratory (Bar Harbor, Maine, US), and maintained at the Central Animal Laboratory of Raisio Benecol Ltd. (Raisio, Finland) in accordance with European guidelines (European Treaty Series No. 123, EU No 609/86, Official Journal of the European Communities No. L 358) approved by the Animal Care and Use Committee of the University of Turku (Finland) approval number 1263/02. Animals were 17–18 weeks at the beginning of the study/15–20 g and after 4 weeks of acclimatization, mice were housed (Polycarbonate Macrolon III, Scanbur AS, Denmark) in groups of 1–2/cage and randomly given free access to I–IV diets for 4 weeks. Ambient temperature was 21 ± 3°C, humidity ranged 55 ± 15% and illumination consisted in 12-h dark/light cycle. Free access to community tap water was allowed, except during the experiments. Blood samples were collected for the determination of the concentrations of serum cholesterol and triglycerides at the beginning and at the end of the 4-week dietary intervention. Mice were sedated with CO2/O2-mixture and the blood samples were collected from the orbital sinus (at the beginning) or through a cardiac puncture (at the end). At the beginning of the study the blood was collected into 500 µl gel tubes and allowed to clot at least for 1 h. Samples were then centrifuged for 15 min at 4000 rpm. Serum was separated and stored at −20°C until analyses. At the end of the intervention, blood samples were collected in heparin tubes and centrifuged within 1 h. The plasma samples were frozen at −20°C and stored until analyses. About 50 µl was used for cholesterol and triglyceride analyses and the rest was stored at −20°C for further analyses. During the intervention, the animals were weighed twice a week and observed daily (morning and afternoon) for general well being. Test compounds and diets Plant-derived stanol ester was produced by Raisio Benecol Ltd. (Raisio, Finland). Sesamin epimer (CAS No. 607–80-7, >95% purity, Fig. 1A) was synthesized by Industrial Research Ltd. (Wellingtown, New Zealand). During acclimatization/isolation period the diet was RM1 (E) SQC, (Special Diet Service, Witham Essex, UK). Experimental atherogenic diets containing 0.25% of cholesterol were prepared at Raisio Benecol Ltd. based on Clinton-Cybulsky diet premix (D12106pxc, Research Diets Inc., New Brunswick, NJ) in which the test compounds were administered, as follows: control (I) diet (n = 8), stanol ester (II) diet (n = 8, 7 mg/kg), sesamin (III) diet (n = 8, 1 mg/kg) and combined (IV) diet (n = 7, 7 mg/kg stanol ester and 1 mg/kg sesamin). Diets were stored at +2 to +8°C and allowed to warm to room temperature before delivery to the animals (twice a week) in 300-ml glass cups attached on the cage floor. Fig. 1Chemical structure of sesamin epimers (A) sesamin and (B) episesamin (or asarinin) Rationale for dose selection Fat and cholesterol levels in the atherogenic diet were based on the literature [6]. The use of stanol ester as control test model has been proven several times to induce cholesterol reduction (Raisio Benecol Ltd., unpublished results) and the concentration in this study was selected to exert sufficient but not the maximal response, enabling the possible combination effect with sesamin. Sesamin concentration, that was adopted from previous reports [7–9] allowing comparison of the results was also considered as a rationale level to be used in clinical trials. Analysis Serum cholesterol and triglycerides were determined by Ecoline CHOD-PAP and Ecoline 25 GPO-PAP assay kits (1.14856.0001, Merck KGaA, Darmstadt, Germany), respectively. Control serum was Qualitrol HS N (Merck KGaA, Darmstadt, Germany). Serum metabolic profile of lignans was carried out by HPLC with coulometric electrode array detection [10] and the absence of plant lignans other than sesamin epimer in the diets was confirmed by isotope-dilution GC-MS [11]. Statistics Graph Pad Prism software version 3.02 (GraphPad Software Inc., CA) was used for statistical data treatment. The differences in changes from the basal concentrations during the intervention between the groups were analyzed by ANOVA. Pair-wise comparisons between the groups were performed by Tukey’s Multiple Comparison Test. Two-way ANOVA was used to analyze differences in the body weights between the groups. A P-value less than 0.05 was considered as statistically significant. All analyses were performed as two-sided tests. Results Body weight gain The body weights of the animals increased in all groups during the 4-week intervention period (Fig. 2). The increment tendency in group I was less than those in the other groups but analysis did not reveal a significant group × week interaction (P = 1.00). However, ANOVA showed a significant group effect (P = 0.0002), which indicates that the body weights differed between the groups during the intervention. Fig. 2Body weights of the animals in the different treatment groups during the study (n = 7–8) Effects of the atherogenic diet and test compounds on serum cholesterol and serum triglyceride concentrations The atherogenic diet (group I) induced an almost 3-fold increase in the s-cholesterol levels but no effect was seen in the triglyceride levels. Stanol ester alone (group II) or together with sesamin (group IV) significantly attenuated the elevation of the cholesterol levels. Sesamin alone (group III) either normalize the elevation of cholesterol levels nor did it enhance the effect of stanol ester (group II versus group IV). Diet-induced changes in the triglyceride levels did not differ between groups (P = 0.89). Results are presented in Fig. 3. Fig. 3S-cholesterol and -triglyceride concentrations before (Basal) and after (Intervention) the 4-week intervention with experimental diets and the difference between basal and Intervention levels (Difference). **P < 0.001 in comparison with Group 1 (Tukey’s Multiple Comparison Test) Effects of the atherogenic diet and test compounds on plasma lignan profile after intervention Presence of sesamin epimer in the diets resulted in the appearance of enterolignans in plasma, although concentrations varied greatly between animals (Table 1). Significantly increased levels of enterodiol (P = 0.00), but not enterolactone were found. Table 1Enterolignan (enterolactone and enterodiol) values after 4-week intervention period nmol/l (Mean ± SEM, n = 8)GroupnENLaENDENL + ENDI, Control84.61 ± 0.406.11 ± 2.109.18 ± 2.14II, Stanol ester83.01 ± 0.3510.0 ± 4.6913.0 ± 4.85III, Sesamin88.01 ± 0.98569 ± 54.2**577 ± 55.0**IV, Stanol ester + sesamin76.54 ± 1.99388 ± 90.7**394 ± 91.8****P < 0.001 in comparison with Group 1 (Tukey’s Multiple Comparison Test)aENL, Enterolactone; END, enterodiol Discussion As one of the major components of sesame seeds, the lignan sesamin has received a great deal of interest regarding its potential as a hypocholesterolemic agent, especially after the positive results reported by Hirata et al. [7] in humans. Thereafter, different approaches have been used to confirm this observation in which different sources of dietary sesamin have been used. Studies using sesame seed-based diets provided results that are difficult to discuss since the hypocholesterolemic effect could not be primarily attributed to sesamin. As for the studies using pure sesamin supplementation, the preparations used so far contained a mixture of sesamin epimers (sesamin and episesamin in a 1:1 ratio) that they are known now to possess a different behavior both in their metabolic fate [12] and biological action [4] supporting the need to differentiate between different sesamin epimers in future studies. A clear hypocholesterolemic effect elicited by sesamin (alone or in combination with vitamin E) was reported in studies conducted in rats [9, 13–15] or in cultured rat cells [16] and similarly, other biological effects proposed for sesamin [17–26], have also been studied using rats as experimental animal. Very few studies though, have used other animals than rats to test the effects of sesamin [27, 28]. In the recent study of Kushiro et al. [3] where sesamin was tested in different experimental animals, results for rats were confirmed, but no effect was observed in hamsters or mice. Only one study has investigated the hypocholesterolemic effect of sesamin in humans [7] finding a significant effect of dietary sesamin in the reduction of total cholesterol and LDL-cholesterol. In this study however, dietary sesamin was administered together with vitamin E and therefore the observed effect might be due to the reported synergism between this two dietary components [14] and since no separated sesamin-group was included in the study, the hypothetical action of sesamin could not be confirmed. In order to minimize the commented variables, in the present experiment only pure sesamin epimer was used and additionally, transgenic mice were selected for they offer the most convenient model to investigate the two proposed mechanisms for the hypocholesterolemic effect of sesamin i.e. reduction of endogenous cholesterol synthesis and reduction of dietary absorption. Due to the lack of functional LDL-receptors, the animals used in this trial have a spontaneously elevated serum cholesterol concentration, and in addition, they are prone to diet-induced modulation of the serum cholesterol concentration [5, 6]. Hepatic HMG-CoA reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway and its inhibitors are very effective in lowering plasma cholesterol. Sesamin has been reported to inhibit HMG-CoA reductase in rats [13] and through this mechanism it has been proposed to reduce plasma cholesterol. However, in this study no reduction of serum cholesterol or triglyceride levels was observed in the group supplemented with sesamin alone (group III) and therefore it is concluded that sesamin does not affect HMG-CoA reductase in this animal model. Similarly, cholesterol supplementation (0.25%, w/w) for 4 weeks increased plasma total cholesterol levels approx. 3-fold in the absence of a functional LDL receptor. However, the introduction of stanol ester in the atherogenic diet significantly reduced the total plasma cholesterol level, independently of the presence of sesamin. It is known that plant stanols inhibit the absorption of dietary cholesterol and the reabsorption (enterohepatic circulation) of endogenous cholesterol from the gastrointestinal tract [29]. It seems though that sesamin does not affect dietary cholesterol absorption in this animal model. The metabolism of sesamin seems to play an essential role in its further biological action. It has been recently suggested that in humans, sesamin is absorbed through the portal vein reaching the liver where extensive metabolism takes places generating demethylenated (catechol) derivatives secreted as glucuronidates via the bile [26]. The same metabolites were found after in vitro fermentation of pure sesamin standard with human fecal inoculum and furthermore extensive in vivo conversion to enterolactone was reported after sesame seed supplementation in humans [11]. We hypothesized then that the absorption of sesamin could certainly take place via the portal vein and that transformation to catechol derivatives could be possible, as well as further absorption to the general circulation where they may exert the biological actions proposed for sesamin. Nevertheless, in this study as in our previous trial [11], extensive conversion of sesamin to enterolignans was observed suggesting that non-absorbed sesamin and possibly biliary catecholic sesamin derivatives undergo metabolism by gut microflora leading to the production and further absorption of enterolignans. Postprandial levels of sesamin in plasma differed considerably between rats [12] and humans [11], suggesting a different metabolic pathway depending on species. So far, our data suggest that a limited absorption or a too rapid enterohepatic circulation of sesamin could be the explanation for the lack of antihypercholesterolemic effect in mice/hamsters and possibly in humans but this hypothesis needs to be confirmed. Furthermore, the characteristics of our animal model did not allow to study a third mechanism for cholesterol reduction that sesamin could use; the increment of LDL-receptor activity. This hypothetical mechanism has been suggested for other dietary components [30], and it is a likely explanation of the negative results reported in this paper. In conclusion, possible confounding factors have been minimized with the use of a specific animal model and the selection a pure sesamin epimer. It can be therefore stated that sesamin does not seem to affect cholesterol biosynthesis or absorption in mice. The so far contradictory results hinder the extrapolation to humans, and only a clinical trial with separate epimeric forms and the elucidation of the complete metabolic pathway of sesamin in humans will contribute to clarify the possible utilization of sesamin as a hyocholesterolemic agent.

Oecologia-4-1-2373415

Swan foraging shapes spatial distribution of two submerged plants, favouring the preferred prey species

Compared to terrestrial environments, grazing intensity on belowground plant parts may be particularly strong in aquatic environments, which may have great effects on plant-community structure. We observed that the submerged macrophyte, Potamogeton pectinatus, which mainly reproduces with tubers, often grows at intermediate water depth and that P. perfoliatus, which mainly reproduces with rhizomes and turions, grows in either shallow or deep water. One mechanism behind this distributional pattern may be that swans prefer to feed on P. pectinatus tubers at intermediate water depths. We hypothesised that when swans feed on tubers in the sediment, P. perfoliatus rhizomes and turions may be damaged by the uprooting, whereas the small round tubers of P. pectinatus that escaped herbivory may be more tolerant to this bioturbation. In spring 2000, we transplanted P. perfoliatus rhizomes into a P. pectinatus stand and followed growth in plots protected and unprotected, respectively, from bird foraging. Although swan foraging reduced tuber biomass in unprotected plots, leading to lower P. pectinatus density in spring 2001, this species grew well both in protected and unprotected plots later that summer. In contrast, swan grazing had a dramatic negative effect on P. perfoliatus that persisted throughout the summer of 2001, with close to no plants in the unprotected plots and high densities in the protected plots. Our results demonstrate that herbivorous waterbirds may play a crucial role in the distribution and prevalence of specific plant species. Furthermore, since their grazing benefitted their preferred food source, the interaction between swans and P. pectinatus may be classified as ecologically mutualistic. Introduction Herbivores may have considerable impact on the distribution and population dynamics of plants (e.g. Maron and Crone 2006) as well as vegetation community structure (e.g. Augustine and McNaughton 1998), but non-grassland examples of such impacts are few. Similarities between aquatic and terrestrial systems and their dependence on top–down regulation are increasingly being appreciated by ecologists (Cyr and Pace 1993; Chase 2000). However, in these broad-scale inter-system comparisons, the predation of zooplankton on algae still plays a front-stage role in the representation of aquatic plant–animal interactions. The importance of vertebrate grazing on submerged macrophytes community structure remains disputed. Among the studies reported to date on interactions between aquatic herbivores and plants, few have considered the possibility of herbivory mediating competition between plants (but see Lauridsen et al. 1993; Van Donk and Otte 1996; Santamaría 2002; LaMontagne et al. 2003), and none have experimentally shown that this can lead to a specific herbivore-induced vegetation distribution and composition. Considering that the impact of herbivory on community structure in terrestrial systems increases with grazing intensity (Augustine and McNaughton 1998), the study of some aquatic plant–animal interactions with sometimes extremely high grazing pressures on both above- and belowground parts may prove rewarding. Potamogeton pectinatus and P. perfoliatus are two species of aquatic macrophytes typical of eutrophic lakes, often co-occurring, with a more or less cosmopolitan distribution. They hibernate mainly vegetatively with rhizomes and turions (P. perfoliatus) or specialised tubers (P. pectinatus) in the sediment (Fig. 1). Many species of waterbirds rely on the leaves and seeds of these two species of water plants (Cramp and Simmons 1986). Among waterbird foragers, swans form a special group of consumers that are also capable of uprooting and consuming the belowground parts of P. pectinatus and P. perfoliatus. In particular, Bewick’s (Cygnus columbianus bewickii) and whooper swans (C. cygnus) are highly dependent on the nutrient-rich tubers of P. pectinatus (Nolet and Drent 1998; Nolet et al. 2001, 2002; Beekman et al. 2002). Fig. 1Vegetative reproductive organs of Potamogeton perfoliatus and P. pectinatus. The tubers of P. pectinatus may considerably vary in size between approximately 2 and 16 mm. The depicted tuber is 15 mm long The spatial distributions and segregation of P. pectinatus and P. perfoliatus have been studied extensively. Typically, P. pectinatus is found in shallower waters than P. perfoliatus. Lehmann et al. (1997) argued that the higher tolerance of P. pectinatus to wave exposure in shallow water and the higher tolerance of P. perfoliatus to light attenuation in deep water could be a major causal factor of this distribution of the two species. Scheffer et al. (1992) was able to explain about 50% of the variation in the distribution of both species by considering water depth, transparency, spring water temperature and exposure to waves for P. pectinatus and water depth and transparency for P. perfoliatus. During extensive studies on the distribution of aquatic vegetations and swan foraging in two of our major research sites where P. pectinatus and P. perfoliatus co-occur (Pechora Delta, in Arctic Russia, and Lake Ringsjön, in southern Sweden), we noticed that P. perfoliatus occurred not only in deep water but also in very shallow water seldom visited by tuber-feeding swans. We thus hypothesised that swan foraging, which is limited to areas of intermediate depth (Beekman et al. 1991; Nolet et al. 2001; Sandsten 2002), at least partly determines the distribution of P. perfoliatus. In order to investigate how swan feeding on tubers of P. pectinatus affects the growth of P. perfoliatus, we monitored vegetation and swan foraging and performed a transplant experiment in Eastern Lake Ringsjön, Sweden. Potamogeton perfoliatus was planted in a P. pectinatus stand after which the development of both species was followed in the presence and absence of swan grazing. Methods Eastern Lake Ringsjön (55°52′N, 13°32′E) is a eutrophic, 20.5-km2 large lake with a maximum and mean depth of 16.4 and 6.1 m, respectively. The experiment was set up in Fulltofta Bay (0.7 km2, <1 m deep; Fig. 2), which is a wind exposed, shallow and sandy part of Eastern Lake Ringsjön, with an aquatic vegetation that nearly exclusively consists of P. pectinatus and P. perfoliatus. Every year from September to November, Fulltofta Bay hosts large numbers of migrating waterbirds (up to 1600 individuals, Sandsten 2002). Many of these migrating waterbird species, notably swans (whooper swans C. cygnus, Bewick’s swans C. columbianus bewickii, and mute swans C. olor), feed on tubers of P. pectinatus and rhizomes and turions of P. perfoliatus by digging in the sandy sediment (e.g. Cramp and Simmons 1986; Nolet and Drent 1998; Sandsten 2002). Coot (Fulica atra) and various species of ducks (mallard Anas platyrhynchos, pintail Anas acuta, wigeon Anas penelope, goldeneye Bucephala clangula, tufted duck Aythya fuligula, pochard Aythya ferina) may also forage on tubers, but nearly always in association with swans since they are unable to dig up the tubers from the sediment by themselves (e.g. Cramp and Simmons 1986; Sandsten 2002). Geese (mainly Anser anser) roost frequently in the area, but they very seldom feed on tubers (Cramp and Simmons 1986; personal observations, H. Sandsten). Fig. 2Map of Lake Ringsjön with an enlargement of Fulltofta Bay and an overview of the bird-monitored and plant-monitored areas as well as the plant-experimental site. Enlargement of the plant-experimental site shows the six replicates of three treatments, with gray squares representing plots where no P. perfoliatus was planted and no protection from waterbirds was provided until 18 December 2000 at which time all treatments were covered with metal nets on the bottom (treatment p). Black squares represent plots with planted P. perfoliatus that were continuously protected by nets (treatment ). White squares represent plots with planted P. perfoliatus that were unprotected from 8 August to 18 December 2000 (treatment P) To quantify our impression that P. perfoliatus occurs only in very shallow and relatively deep parts of Fulltofta Bay, we mapped the occurrence of P. pectinatus and P. perfoliatus in relation to water depth in Fulltofta Bay on 10 August 2000 (Fig. 2). Vegetation and water depths were examined on 173 points regularly spaced (at 20–30 m distance from point to point) in an area (0.12 km2) running from the shore to a depth of 1.2 m. The geographical position of each sampling point was measured with a GPS (Garmin Personal Navigator 12 XL). On each of the points we investigated plant occurrence three times by throwing a 50 × 50-cm metal frame into the water and diving down to look for plants. For P. pectinatus, a cover index was used, where 0 was no ramets m−2, 1 was 1–20 ramets m−2, 2 was 21–80 ramets m−2 and 3 was >80 ramets m−2. We counted all shoots emerging from the sediment surface as ramets. Potamogeton perfoliatus shoots were less frequent than P. pectinatus shoots and seldom found in the frames—possibly because of less favourable growing conditions but at least in part because of its morphology and growth strategy. We therefore used data on absence/presence in the proximity (within a radius of 10 m) of the sampling point. The data were entered into a geographic information system, GIS (Surfer 6.04) and interpolated over the entire monitored surface using intermediate distance interpolation. Swans were counted over an area of 0.54 km2 from an observation point (Fig. 2) south of Fulltofta Bay on 5, 17 and 30 August, 10 and 23 September, 5, 18 and 28 October and 9 November 1999. Swans foraging on tubers tread water to create a water current that flushes the sediment and uncovers tubers and rhizomes, resulting in the appearance of pits and piles of sediment on the bottom at the sites where foraging activity has taken place. Wave action will ultimately level the sediment again (Sandsten 2002). Depth heterogeneity was used as an indirect measure of waterbird foraging on tubers in the sediment. In order to investigate the timing of swan foraging in relation to water depth we measured depth heterogeneity along five fixed transects running parallel to the shore within the plant-monitored area. The transects were aimed to be at equidistant intervals parallel to the shore, but in fact they were located at a distance of 110, 180, 260, 340 and 400 m from the shore (mean depths were 37, 45, 60, 75 and 90 cm on 5 August 1999). Depth heterogeneity was obtained by calculating the SD from 5 × 10 measurements of water depth along each transect. Within every second metre along the 11-m-long transect ten water depths were measured at a distance of 10 cm, resulting in five SD from each transect. These five SD were entered as replicates from each transect in the statistical analysis and were also the basis for the graphical presentation of “mean standard deviation”. Measurements were conducted prior to and during the main swan grazing period on 5, 17 and 30 August, 10 and 23 September, 5 and 18 October and 1 and 12 November 1999. Depth heterogeneity in relation to time of measurement and mean water depth was statistically analysed using repeated measures ANOVA (Statistica ver. 5.1; StatSoft, Tulsa, OK). Normal distribution of residuals was checked with a probability plot, and heteroscedasticity of variances was checked for with Levene’s test. If the variances were not homogenous or the plots of residuals were not linear, the data were transformed to meet the assumptions of ANOVA. The transplant experiment consisted of three separate treatments with six replicate plots (1.4 × 1.4 m), all situated in an established (at least for 5 years) stand of P. pectinatus in Fulltofta Bay (Fig. 2). The experiment ran from 31 May 2000 until 15 August 2001. One treatment, consisted of continuously protected plots where rhizomes with ramets of P. perfoliatus had been planted among the P. pectinatus plants. The second treatment, P, consisted of plots where ramets of P. perfoliatus had also been planted among the P. pectinatus plants, but where swans were allowed to graze during the autumn migratory period (8 August until 18 December 2000). The last treatment, p, consisted of P. pectinatus plots without P. perfoliatus where swans were allowed to graze permanently. This last treatment was designed as a control treatment to check for any effects of the planting of P. perfoliatus on P. pectinatus. Ramets of P. perfoliatus were collected in the eastern part of the nearby Lake Krankesjön (55°42′N, 13°28′E) by scuba diving on 30 May 2000 just after onset of sprouting. The collected ramets with roots and intact meristems and varying in size from 30 to 70 cm were stored in plastic bags at 5°C overnight. On the following day, 22 ramets (density 11 m−2) with roots and intact meristems were planted in each of the and P plots. The randomly selected ramets were distributed evenly over the plots and planted at a depth of 10 cm. The plots were subsequently protected with a 1.6-m high plastic net (mesh size 25 × 32 mm). The p plots were not protected since summer grazing on above- and belowground biomass of P. pectinatus is considered negligible (Santamaría 2002). In all 18 plots, the above- and belowground biomass of the vegetation was surveyed by scuba diving and core sampling prior to the migratory and main grazing period of the swans, on 8 August 2000. After the main swan-grazing period (verified by counts on 18 July, 8 and 18 August, 8 September, 4 and 24 October and 2 November 2000, yielding 22, 18, 34, 10, 10, 10 and 10 swans km−2, respectively), the belowground biomass was again surveyed by core sampling on 18 December 2000. Finally, the effect of swan grazing on the aboveground plant biomass during the autumn of 2000 was again assessed by scuba diving on 6 June and 15 August 2001 the following growth season. Aboveground biomass was evaluated by counting all ramets emerging from the sediment in two perpendicular transects (5 cm wide, 2.0 m long) running diagonally across each plot. Belowground biomass was sampled using four sediment core samples (diameter 11 cm, length 30 cm) in each plot. Roots, rhizomes and tubers of P. pectinatus and roots and rhizomes of P. perfoliatus were dried at 105°C for 24 h before dry weight (DW, g) measurements were taken. In December, we wanted to retain most of the P. pectinatus tubers for other purposes. Consequently, we first measured the fresh weight (FW, g) of each collected tuber and then assessed the DW on a subsample of randomly selected tubers. The regression of these DW estimates on FW was used to estimate total tuber DW of the whole sample (DW = 0.2953 FW, P < 0.0001, R2 = 0.958, n = 33). Lake Ringsjön is usually covered with ice during the winter, which poses a potential threat to the fences around the plots. Therefore, on 18 December 2000, when the majority of birds had left the bay, the fences of treatment were removed. Subsequently all plots, of treatments P and p, were covered with metal nets (mesh width 10 × 10 cm) to protect them from belowground winter grazing by swans during occasional periods when the lake was not ice-covered. These bottom-covering nets remained in place throughout the experiment, and counting of ramets in 2001 was carried out without removing the nets. The experimental data were analysed with analysis of variance, ANOVA, in Statistica ver. 5.1. Heteroscedasticity and residuals were examined, and transformations were carried out using the same methods as for the depth heterogeneity data. Tukey’s honestly significant difference (HSD) was used to test all pairwise comparisons among means when the overall treatment effect in the ANOVA was (near) significance. Results Vegetation and water depth mapping The shallowest (<20 cm) and deepest (>95 cm) parts of Fulltofta Bay were void of submerged macrophytes (Fig. 3). Potamogeton pectinatus grew at depths varying from 20 to 95 cm, with the highest ramet densities at intermediate water depths. In contrast, P. perfoliatus was found at relatively few points and mainly in deep water (>70 cm). Some P. perfoliatus was also found in shallow water (20–30 cm) but except for one finding at a depth of 46 cm, no P. perfoliatus was found at intermediate water depths (30–70 cm).Fig. 3Maps on vegetation and water depth in Fulltofta Bay, Lake Ringsjön. The vertical line shows the same point in the three maps. The upper map shows the distribution of P. perfoliatus on deep and shallow water. Grey represents areas where the probability of encountering P. perfoliatus on the sampling points was larger than 50%. The middle map shows the distribution and abundance of P. pectinatus. The lower map is a three-dimensional presentation of water depths in the monitored area Swans Figure 4 (upper panel) shows swan densities in Fulltofta Bay during the autumn of 1999; swan grazing followed a similar temporal pattern in 2000 (Sandsten 2002). Depth heterogeneity largely coincided with swan densities and increased with time, indicating that swan grazing on P. pectinatus tubers increased from August to early November (Fig. 4, lower panel). Depth heterogeneity was significantly affected by time and the interaction between water depth and time [F8,160 = 14.38 P < 0.001, F32,160 = 3.65 P < 0.001, F4,20 = 1.80 P = 0.168; respectively; data was ln (Y + 0.1) transformed]. The interaction between time and water depth indicates that over time the swans’ main foraging depth changed. As shown in Fig. 4, birds started foraging on macrophytes at shallow depths (37 and 45 cm) in August, shifting to deeper areas (60, 75 and 90 cm) in October and November. During the last days of October, stormy weather events evened out the sediment, resulting in little depth heterogeneity being recorded on 1 November.Fig. 4The upper panel shows swan densities in Fulltofta Bay (area 0.54 km2) over the period August–November 1999. Swans include the whooper swan (Cygnus cygnus), Bewick’s swan (C. columbianus bewickii) and mute swan (C. olor) The lower panel shows water depth heterogeneity measured as 5 SD of water depth at five locations with different water depths. The columns show mean + standard error of these SD. Depth heterogeneity is used as an indirect measure of swan foraging on tubers in the sediment Transplant experiment During the summer following planting, P. perfoliatus established well in the and P plots, as indicated by the August 2000 sampling (Fig. 5) prior to the peak swan-grazing period (compare Fig. 4). A comparison of the and P treatments revealed that neither belowground biomass nor ramet density of P. perfoliatus differed significantly at the start of the experiment (Table 1; Fig. 5). As expected, no P. perfoliatus was found in the p plots where no P. perfoliatus had been planted.Fig. 5Above- and belowground biomass (means and standard errors) of P. pectinatus and P. perfoliatus in the transplant experiment during various times of the year in 2000 and 2001. six plots where P. perfoliatus was planted in May 2000 and where above- and belowground biomass was protected against waterbird herbivory until December 2000, P six plots where P. perfoliatus was planted in May 2000 and above- and belowground biomass was protected against waterbird herbivory until August 2000, p six “unplanted” plots without protection against herbivores until December 2000. All 18 plots were protected from waterbirds after December 2000 with metal nets covering the bottomTable 1ANOVA table for above- and belowground data of Potamogeton perfoliatus and P. pectinatus sampled in the three treatments on 8 August 2000 before the peak of waterbird herbivorySource of variationPotamogeton sp.FdfPRamets (number m−2)P. pectinatusa0.621,10NSP. perfoliatus a1.61,10NSP. pectinatus b1.02,15NSBelow-ground DW (g m−2)P. pectinatusa0.861,10NSP. perfoliatusa2.21,10NSP. pectinatusb3.32,15NS (0.065)*Tubers (number m−2)P. pectinatusa3.01,10NSP. pectinatusb1.72,15NS*Tukey’s HSD multiple comparison between treatments resulted in a near significant effect (P = 0.058) for the comparison between and p; NS, not significantaPlots without planted P. perfoliatus excludedbAll plots included The August sampling also showed a healthy stand of P. pectinatus in all plots before the peak swan-grazing period. Although there seemed to be a tendency for P. pectinatus to perform worse in the presence of P. perfoliatus, no significant differences in belowground DW, ramet density or P. pectinatus tuber density were detected between the treatments (Table 1; Fig. 5). In December, when the majority of swans had left the lake, belowground DW was again estimated (Fig. 5). Potamogeton perfoliatus was found in three of the and none of the other plots. Although this result suggested a negative impact of swan grazing on P. perfoliatus, it did not allow for any meaningful statistical analysis. In December, P. pectinatus belowground biomass and tuber density were higher in the plots than in the P plots [F1,10 = 7.7, P < 0.05 after ln(Y + 1) transformation, and F1,10 = 12, P < 0.001 after square root (Y + 1) transformation, respectively], revealing the effect of tuber consumption by swans. Ramet densities in the plots were counted in June 2001. There were significant treatment effects both on P. pectinatus and P. perfoliatus, and both species were present at higher densities in the plots than in the P plots (Fig. 5; Table 2). In August 2001 there was still a difference in P. perfoliatus ramet density but not in P. pectinatus (Fig. 5; Table 2).Table 2ANOVA table on P. pectinatus and P. perfoliatus ramet density in P and plots on 6 June and 15 August 2001Source of variationPotamogeton sp.FdfPRamets (n m−2) in JuneP. pectinatus431,10***Ramets (n m−2) in JuneP. perfoliatus5651,10***Ramets (n m−2) in AugustP. pectinatus1.11,10NSRamets (n m−2) in AugustP. perfoliatus451,10******P < 0.001; NS, not significantAll data is ln(Y + 1) transformed Discussion Our vegetation mapping study supports our earlier impression that the distribution of P. perfoliatus is limited to those areas where the pressure of swan grazing is low. It also shows that P. pectinatus grows at intermediate water depths, while P. perfoliatus is found mainly in deep water and occasionally at shallow depths. This is consistent with reports in the literature that P. perfoliatus mainly grows deeper than P. pectinatus (Scheffer et al. 1992; Lehmann et al. 1997), although we found no other report on P. perfoliatus growing in very shallow water. Depth heterogeneity substantiated our impressions of the spatial variation in foraging activity of the swans in Fulltofta Bay. Swans showed a tendency to forage at intermediate depths that are largely void of P. perfoliatus. They started to forage in relatively shallow water and moved to deeper parts, where foraging is more expensive (Nolet et al. 2001), later in the season. A similar phenomenon was observed in Bewick’s Swans foraging on P. pectinatus in the Russian White Sea (Nolet and Drent 1998). This shift into deeper water over time may not only have been a consequence of depletion—it may also have been related to a decrease in the water table by 0.2 m from August to early November (Swedish Meteorological and Hydrological Institute, unpublished data). When the spatial variation in foraging activity was considered over the whole season, there was a tendency for intermediate water levels to be used more frequently than the shallow and deep parts of the bay. This result is in accordance with those from an experiment on tuber depletion at different water depths in Fulltofta Bay (Sandsten 2002) and with previous observations on P. pectinatus foraging swans elsewhere (Beekman et al. 1991; Nolet et al. 2001). The observed tendency of swans to preferentially forage at intermediate depths becomes even more striking when we study the depth heterogeneity in Fulltofta Bay during the month of October 1999, when swan numbers were peaking. The results of the transplant experiment showed that subterranean swan grazing had a dramatic negative effect on P. perfoliatus and, consequently, on the spatial depth-related distribution of P. pectinatus and P. perfoliatus in Fulltofta Bay and possibly elsewhere. When faced with increased subterranean herbivory, P. pectinatus seems to increase the depth at which its tubers are buried—likely as a defence mechanism to reduce tuber loss (Klaassen and Nolet 2007; Santamaría and Rodríguez-Gironés 2002). However, during the August–December 2000 period, P. pectinatus winter biomass was considerably reduced as a result of swan foraging. Both belowground tuber biomass and tuber numbers were negatively affected. At the same time, swan foraging also had a marked negative effect on the occurrence of P. perfoliatus in the following year. Potamogeton perfoliatus had virtually disappeared from the unprotected sites where waterbirds had free access: in December, directly after herbivory, no belowground remnants of P. perfoliatus were detected; in June and August of the following year, the aboveground presence of P. perfoliatus was marginal in the unprotected plots, which contrasted sharply with the situation in the plots that had been continuously protected from subterranean herbivory by swans. Although not as dramatic as in P. perfoliatus, tuber herbivory by swans also had an effect on the aboveground biomass of P. pectinatus in the spring, but differences in aboveground biomass between protected and unprotected plots were no longer detectable by August 2001. The small number of tubers remaining in P was probably sufficient to produce a large number of tubers during the following autumn. This result is consistent with observations by LaMontagne et al. (2003) and the model of Jonzén et al. (2002), which suggest that tuber herbivory in winter leads to a lower density of ramets the following spring, but that compensation (or even overcompensation; compare Nolet 2004) of tuber production may occur the following autumn. In Sweden, the investments of P. perfoliatus and P. pectinatus in vegetative reproduction are large. It is highly likely that the difference in their wintering strategy determines their susceptibility to subterranean foraging by swans and, consequently, their prevalence in sites regularly frequented by digging swans. The small, round and relatively robust tubers of P. pectinatus are a highly preferred food source by foraging swans (e.g. Cramp and Simmons 1986; Sandsten 2002; Nolet and Drent 1998). However, if missed and not eaten, they are very likely to survive the digging and uprooting activity of the swans. In contrast, the rhizomes and turions of P. perfoliatus are long, interconnected and relatively fragile. Compared to the solitary tubers of P. pectinatus, they are easily detected and, if not eaten, fragmented by the digging and uprooting of swans. The experiment reported here was mainly designed to study competition between P. pectinatus and P. perfoliatus under autumnal swan grazing. However, during the early experimental phase, which was without grazing until August 2000, no effect of P. perfoliatus on P. pectinatus growth was observed. Nevertheless, it is rather conceivable that both species compete for nutrients and light and that the removal of P. perfoliatus by swans will ultimately be of benefit to P. pectinatus. Therefore, the swans ultimately increase the profitability of the vegetation to themselves by promoting the nutrient-rich tuber-producing P. pectinatus. Although subterranean feeding in P. pectinatus stands may also favour the establishment of new macrophyte species (LaMontagne et al. 2003), the current findings suggest that tuber-feeding herbivores may actually stimulate the development of P. pectinatus by mediating competition with other macrophyte species, notably P. perfoliatus. This outcome implies a potentially mutualistic relationship, at least in an ecological sense (sensu Järemo et al. 1999), between a predator (swan) and its prey (P. pectinatus). Following Järemo et al.'s (1999) classification of mutualistic plant–herbivore interactions, the current relationship may represent a “plant–herbivore antagonism”, where P. pectinatus has adapted itself better than competing plants to subterranean herbivory (by producing specialised clonal propagules and hiding; Klaassen and Nolet 2007) thus obtaining a relative fitness advantage over less well-adapted species such as P. perfoliatus. However, since overcompensation to subterranean grazing may occur (Santamaría and Rodríguez-Gironés 2002; Nolet 2004), the relationship between P. pectinatus and swans may also be considered as an “evolution of overcompensation relationship”, in which P. pectinatus has an absolute fitness advantage to grazing, whereas its competitors have not. Our data support the view that waterbirds may play a crucial role in the prevalence of certain plant species with potential consequences for the functioning of the system as a whole (e.g. Engelhardt and Ritchie 2001). Reviewing the (terrestrially-skewed) literature, Augustine and McNaughton (1998) concluded that the impact on vegetation communities tends to increase with grazing intensity. In aquatic systems, belowground grazing may be facilitated by the generally softer and easily penetrable sediments, notably if grazers make use of water jets to uncover the vegetative plant parts. Grazing intensity may thus reach very high levels in these systems, resulting in very rapid alterations in vegetation composition that are uncommon in terrestrial systems.

Eur_Spine_J-2-2-1602193

Osteoma in the upper cervical spine with spinal cord compression

Osteoma is a common benign tumor. It occurs dominantly at the skull bone. Outside skull osteoma is rare, and primary intra-canal osteoma is extremely rare. To the author’s knowledge, only 14 cases of osteomas of the spine had been reported, in which only seven cases were in English literature. The authors reported two rare cases of intra-canal osteoma of the upper cervical spine with cord compression. Included are pertinent history, physical examination, rontgenographic evaluation before and after operation, surgical interventions, pathological study, and outcome. The available literature is also reviewed. On systemic examination and rontgenographic study, these two cases were found to have bone tumor in the upper cervical canal. Surgical interventions were performed, one with an en bloc excision, the other with a subtotal excision. The pathological study demonstrated a diagnosis of osteoma. After a follow-up with 20 and 15 months, the clinical symptoms of both cases significantly improved. Introduction Osteomas are rare, slow growing lesions composed of compact bone that occur almost exclusively in the skull [2, 7], most often in the inner and outer table of the calvarium or in the bones of the face or mandible [1, 12, 13]. The prevalence of this disease amounting those who have had sinus radiographs has been estimated to be 0.42% [7]. Although these lesions may cause sinusitis or exophthalmos when they arise from the wall of the nasal sinuses or the orbit, cranial osteomas often cause no symptoms and are discovered incidentally. Primary intra-canal osteomas are extremely rare. To the authors’ knowledge, at the time of writing, only 14 cases of osteomas of the spine had been reported, in which only seven cases were in the English literature [4–6, 8, 9, 11]. The purpose of this article is to report on two patients who presented with cervical intra-canal osteoma. Case report Case One A 56-year-old man had presented with paresis and weakness of his extremities after a hyperextension injury of the cervical spine in August 2002. The man had been asymptomatic prior to the accident. Physical examination demonstrated limitation in neck rotation, and motor weakness of the extremities with strength 3/5 in upper extremities and 4/5 in lower extremities. He could eat with chopsticks to a limited degree. He could walk. However, he needed cane or aid on stairs. Hypoesthesia was present at the upper extremities, bilaterally. The biceps tendon reflex, patellar tendon reflex, and Achilles tendon reflex were hyperactive, and the Hoffman and Babinski reflex were positive bilaterally. Sustained ankle clonus was present, bilaterally. Neurological examination was consistent with upper neuron cell injury. The Japanese orthopedic association (JOA) score for cervical myelopathy was 11. Preoperative imaging study demonstrated a cortical like solid lesion. The results were shown in Fig. 1a–e. Fig. 1a Cervical A–P view X-ray showed densification at left side at C2–3 level. b Cervical lateral view X-ray showed high density at C2–3 level, the facet joints between C2\C3 were obscure. c CT scan through the intervertebral foramen between C2 and C3 showed the tumor occupied more than 60% canal and the vertebral shape changed, caused by the chronic pressure. d Three dimensions CT reconstruction showed the three-dimension relationship between the tumor and the cervical spinal canal. e Middle sagittal plane T1WI MRI showed the spinal cord was pressed by the tumor. The signal of the tumor was low. f The same plane as c after operation, showed the whole decompression of the spinal canal, part of the tumor, near the vertebral artery was left. g The same plane as e after operation, showed the whole decompression of the cord and spinal canal. h, i The pathological study showed that the lesion was composed of uniformly dense, compact, cortical-like mature lamellar bone. H: ×100, I: ×400 We performed a leminectomy of C2 and C3. In the operation, we found that the tumor was adhesive with the left side vertebral artery. To avoid the injury of the artery, part of the tumor, near the vertebral artery, was left. However the decompression of the spinal cord was enough (Fig. 1f–g). The pathological study showed that the lesion was composed of uniformly dense, compact, cortical-like, mature lamellar bone (Fig. 1h–i), and a diagnosis of osteoma was made. Two weeks after the operation, this patient’s clinical symptoms improved significantly. Then, with hard cervical brace protection, the patient was permitted to ambulate. He noted gradual improvement of strength in his upper extremity. Three months later, the brace was removed. Twenty months later, the muscle strength was 5/5 in all extremities. He could eat with chopsticks, but awkwardly. He could walk with no disability. No hypoesthesia was present. The patient’s JOA score increased from 11 to 16 (Table 1). Table 1JOA score of the two cases before and after therapyBefore therapyFollow-upCase 1Case 2Case 1Case 2Motor Upper extremity2133 Lower extremity2243Feeling Upper extremity1122 Trunk2222 Lower extremity2222Sphincter2233Total11101615 Case two A 49-year-old man had experienced paresis and weakness of his extremities after a weight-hit injury of the head in November 2002. The man had been asymptomatic before the accident. Physical examination showed moderate motor weakness and hypoesthesia of the upper extremity, with strength 3/5 in upper extremities and 4/5 in lower extremities. He could eat with spoon but could not with chopsticks. He could walk. However, he needed a cane or aid on stairs. The feel of trunk and lower extremity is fair. The biceps, patellar, and Achilles tendon reflexes were hyperactive, and the Hoffman and Babinski reflexes were positive bilaterally. Ankle clonus was noted continuously on both sides. Preoperative imaging study demonstrated lamina fracture of C3, traumatic instability of C3–5, and a cortical like solid lesion at the left side of C2 lamina. The results were shown in Fig. 2a–c. Fig. 2a Cervical lateral view X-ray showed high density at C2–3 level, the facet joints between C2\C3 were obscure, traumatic instability of C3–5. b CT scan through C2 showed the tumor at the left side of the lamina, occupied about 20% canal, and the left side lamina of the C2 became thicker. c Sagittal plane T2WI MRI showed the spinal cord was pressed by the tumor. The signal of the tumor was low. d Three-dimension CT reconstructive image showed complete decompression We performed a laminectomy of C3–C4 and part of C2, as well as internal fixation of C3–5, using pedicle screw system. The tumor was removed completely and the spinal cord was decompressed. The pathological study showed that the lesion was also composed of uniformly dense, compact, cortical-like, mature lamellar bone without nidus (not shown). Lateral view X-ray after operation showed the reconstruction of the stability by the pedicle screw system and lamina decompression of C3–4 and part of C2. Three dimension CT reconstructive image showed complete decompression of the spinal cord (Fig. 2d). A month after the operation, this patient’s clinical symptoms improved significantly. Then, with hard cervical brace protection, the patient was permitted to ambulate, and he noted gradual improvement of strength in his upper extremity also. Three months later, the brace was removed. After 15 months, the muscle strength was 4/5 in upper extremity and 5/5 in lower extremity. He could eat with chopsticks, but awkwardly. He could walk without cane or aid, but slowly. No hypoesthesia was present. The JOA score increased from 10 to 15 (Table 1). Discussion Congenital disease, rheumatoid arthritis, fracture and dislocation, and other systemic diseases can cause the compression of the upper cervical spinal cord [14]. However, osteoma is one of the rare reasons. Osteoma is defined by WHO as a benign lesion consisting of well differentiated mature bone tissue with a predominantly laminar structure, and showing very slow growth. Osteomas are bosselated, round to oval sessile tumors that project from the subperiosteal or endosteal surfaces of the cortex [3]. They are composed of a composite of woven and lamellar bone that is frequently deposited in a cortical pattern with haversian-like systems. Some variants contain a component of trabecular bone in which the intertrabecular spaces are filled with hematopoietic marrow [10]. Osteoma usually occurs in the skull. Only occasionally have these lesions been reported in extracranial locations (Table 2). In 1980, Pecker et al. reported the first osteoma in cervical inter-vertebral foramen. They used a Lateral interscalenic approach to excise the lesion [8]. In 1986, Lantsman reported six cases of osteoma in spine, but the article was in Russian, no more details could be reviewed [4]. In 1993 [5] and 1996 [6] Laus reported the same case in Italian and English. To excise the lesion, they performed an anterior prevascular extra oral approach. In 1996 [9], Peyser reported 11 cases of extracranial osteoma, in which five were in spine, one in the cervical spine, two in the lumbar spine and two in the scrum spine. In 1998 [11], Rengachary reported that another osteoma affected the cervical spine. Table 2Cases reported in the literatureReferencesArticle languageCase numberSite of the lesionRecurredRengachary [11]English1CervicalNoPeyser [9]English5Cervical (1), lumbar (2), sacrum (2)One pain recurredLaus [6]English1CervicalNoLaus [5]Italy1CervicalNoLantsman [4]Russian6SpineNoPecker [8]French1CervicalNoTotal14aaLaus M. reported the same case in English and Italy The diagnosis of intra-canal osteoma is still confusing. In Peyser’s series [9], none of the cases had correct diagnosis before the operation. In our cases, the rontgenographic study before operation could give some clues, but the final diagnosis still depended on the pathological findings. Histologically, the reactive bone induced by infection and trauma may be similar with a true osteoma [10]. However the differential diagnosis can be made by the tumor’s site and pertinent history. Traumatic reactive bones are most frequently observed in relation to the femur, beneath the quadriceps, in relation to the adductor magnus (the so-called rider’s bone) or in relation to the medial collateral ligament of the knee joint. The condition is also not infrequent at the elbow, the exostosis forming in the intermuscular planes of the brachialis following dislocation. Injury leads to the formation of a subperiosteal hemorrhage, the periosteum being detached by muscular traction. If the periosteum remains intact, the hematoma may become absorbed; but if the periosteal reapposition and the absorption of the blood clot are prevented, traumatic bone overgrowth may occur. However, this condition could not occur at the endosteum. In the case of infective reactive bones, an infection history of relevant site should be noted. The clinical features of osteoma in the spine may be confusing. Cranial osteomas often cause no symptoms and are discovered incidentally, although these lesions may cause sinusitis or exophthalmus when they arise from the wall of the nasal sinuses or the orbit. It would be expected that the symptoms of spinal osteoma would depend on the location of the lesion and its relationship with the peripheral tissue. As Rengachary [11] and Laus [5, 6] reported, the symptom of the spinal osteoma was caused from spinal cord or nerve root pressure. However in Peyser’s series all but one of the patients was symptomatic and had dull, aching pain at the time of the initial presentation. In our series, the two cases had no symptom before the injuries. It may be because osteomas are benign and very slow-growing lesions. And the spinal cord and never roots may adapt to the chronic pressure. Since the lesions cause spinal canal stenosis, they can cause the spinal cord to be more easily damaged by minor injury. The growth potential of osteoma remains unclear, as we know the cranial osteomas grow very slowly. Peyser’s excellent work showed us the growth potential feature of the spinal osteoma [9]. In their series, one patient who had a juxtacortical osteoma of the cervical spine, showed no major change on serial CT scans performed twelve years apart. However, another patient’s sacral osteoma showed significant growth on serial radiographs performed 18 years apart. The method of surgical management of an osteoma depends on the growth feature of the tumor. Cranial osteoma does not need surgical excision if there are no symptoms. The aim of surgical management of spinal osteoma is decompression and stabilization if instability exists. Rengachary and Laus performed total excision of the lesion, with no tumor recurrences. Peyser performed three total and two subtotal excisions, in the case series of osteomas, none recurred. However in one of the patients, partial resection of an osteoma from the body of the fifth lumbar vertebra resulted in no relief of pain and necessitated an additional procedure to remove the remainder of the lesion. In our series, it was relatively easy to perform an en bloc excision in the second case. In the first case, in view of a patient’s age and risk of vertebral artery injuries, a subtotal excision was performed, also with satisfied short time outcome. However, the follow-up was not long enough to observe the recurrence. Conclusion The incidence of intra-canal osteomas is rare. It can cause upper cervical myeolopathy. The rontgenographic study before operation can give some clues, but the final diagnosis still depends on the pathological findings. Surgical treatment, either en bloc or subtotal excision of the lesion, may be effective.

J_Abnorm_Child_Psychol-4-1-1915634

Homotypic Versus Heterotypic Continuity of Anxiety Symptoms in Young Adolescents: Evidence for Distinctions Between DSM-IV Subtypes

Objective: to investigate homotypic and heterotypic longitudinal patterns of symptoms of separation anxiety disorder (SAD), generalized anxiety disorder (GAD), social phobia (SoPh), panic disorder (PD), and obsessive compulsive disorder (OCD) in young adolescents from the Dutch general population. Introduction Anxiety disorders are among the most prevalent psychiatric disorders in children and adolescents (Costello et al., 1996; Essau et al., 2000; Verhulst et al., 1997), are persistent, and are associated with impaired functioning (Canino et al., 2004; Ferdinand & Verhulst, 1995; Ferdinand et al., 1995; McGee & Stanton, 1990; Pine et al., 1998; Verhulst et al., 1997). The high degree of comorbidity amongst anxiety disorders in children and adolescents seems to point in the direction of one taxonomic construct, instead of a number of separate disorders. High comorbidity rates have been reported by many authors (Essau et al., 2000; Masi et al., 1999; Newman et al., 1996; Verduin & Kendall, 2003). Evidence for a higher order factor that explains the presence of different types of anxiety has been found in children (Nauta et al., 2004) and adults (Hettema et al., 2005; Krueger, 1999; Vollebergh et al., 2001). Negative affectivity (NA) (Chorpita, 2002; Clark, 2005; Clark & Watson, 1991; Lonigan et al., 1999, 2003) may be one of the higher order factors that may explain the finding of heterotypic continuity. NA represents displeasurable engagement with the environment and a sense of high subjective distress (Lonigan et al., 2003), and is often considered as a temperament trait that is associated not only with anxiety, but with depression as well (Clark, 2005). Using data collected at the first assessment wave of a study that was also used to conduct the research that is being described in the present manuscript, support was found for the presence of one single anxiety dimension, instead of a number of separate anxiety concepts (Ferdinand et al., 2006). The sample of this previous study consisted of 10- to 12-year-olds from the Dutch general population, who completed a self-report questionnaire for anxiety symptoms. Based on item scores on this questionnaire, latent class analysis did not detect classes of individuals with, for instance, high scores on items tapping separation anxiety, and simultaneously low scores on items tapping panic or social anxiety. Instead, high scores on one anxiety dimension implicated high scores on the other anxiety dimensions as well. However, other studies found evidence for separate anxiety dimensions. By performing factor-analyses, several authors found that DSM-IV anxiety disorders, such as generalized anxiety disorder, separation anxiety disorder, social phobia, and panic disorder represent different problem dimensions in children and adolescents (Chorpita & Daleiden, 2000; Muris et al., 2002; Spence, 1997). Longitudinal studies can provide valuable information regarding taxonomic constructs. For instance, it was found that symptoms of social phobia in adolescents predicted similar symptoms in adulthood (Pine et al., 1998). The prediction of a disorder by the same disorder is called homotypic continuity. However, social phobia symptoms also predicted simple phobia in adulthood. The prediction of a disorder by another disorder is called heterotypic continuity (Costello et al., 2003). Several mechanisms may explain heterotypic continuity. Heterotypic continuity may occur by chance. In other words, disease A may disappear, and disease B may occur subsequently, as a coincidence. However, in that case, continuity would not be reflected in statistical significance. More likely reasons for heterotypic continuity would be that disease A would be the cause of disease B, or that disease A and B share a common vulnerability factor. The aim of the present study was to investigate homotypic and heterotypic longitudinal patterns of symptoms of separation anxiety disorder, generalized anxiety disorder, social phobia, panic disorder, and obsessive compulsive disorder in young adolescents from the Dutch general population. For this purpose, individuals from a community sample, who were assessed for the first time when they were aged 10 to 12 years, were followed up across a period of two years. At both assessments, anxiety symptoms were assessed with a self-report questionnaire. Given mixed results of previous studies, we did not formulate specific hypotheses regarding the level of homotypic or heterotypic continuity of different types of anxiety. Methods Sample and procedure The TRacking Adolescents’ Individual Lives Survey (TRAILS) is a prospective cohort study of Dutch early adolescents aged 10–12 years, who are followed biennially. The present study used data from the first (2001–2002) and second (2003–2004) assessment wave. The TRAILS target sample consisted of young adolescents from five municipalities in the North of the Netherlands, including both urban and rural areas. More details about the sample selection have been published elsewhere (de Winter et al., 2005). Of all subjects who were approached at wave 1 (N = 3,145), 6.7% were excluded. The exclusion criteria were (1) adolescent incapable to participate because of mental retardation or a serious physical illness or handicap and (2) Dutch-speaking parent or parent surrogate not available, and not feasible to administer a part of the measurements in parent’s own language. Of the remaining 2,935 young adolescents, 24% did not want to cooperate, and 76.0% cooperated with the study at wave 1 (N = 2,230, mean age 11.09 years, SD .55, with 50.8% girls). Most frequent reasons for non-response were ‘not interested’ (33.8%), participation in other research or unfavorable experiences with research (15.4%), too much of a burden on the child (12.2%), lack of time (10.3%), concerns about privacy and confidentiality (8.0%), and the child’s refusal to participate because friend(s) did not participate (4.0%). In 34 cases (1.2%) we failed to contact anyone of the household (de Winter et al., 2005). Responders and non-responders did not differ with respect to the proportion of single parent families, or the prevalence of teacher-rated problem behavior. Furthermore, no differences between responders and non-responders were found regarding associations between socio-demographic variables and mental health outcomes (de Winter et al., 2005). To assess anxiety symptoms, the Revised Child Anxiety and Depression Scale (RCADS) (Chorpita & Daleiden, 2000) was used at wave 1, and also at wave 2. For 20 cases, RCADS data were not obtained at wave 1 because respondents were not present during the measurements that were conducted in the classrooms, and could not be reached afterwards. Hence, RCADS data of 2,210 pre-adolescents were available at wave 1. At the second assessment wave, following similar procedures as at wave 1, RCADS information was obtained from 2,067 individuals. This was 95.5% of those for whom wave 1 RCADS information had been collected (51.4% girls). To examine possible selective attrition, a stepwise logistic regression analysis was performed with ‘wave 2 RCADS information available’ as a dependent variable, and wave 1 age, sex, and the wave 1 RCADS Total Anxiety score (that was constituted by summing scores on the five anxiety dimensions that were assessed with the RCADS in the present study, see below) as possible predictors. The RCADS Total Anxiety score and sex did not predict attrition. However, younger age predicted attrition significantly (odds ratio = .17, Wald = 93.1, p < .001; Model chi-square = 109,551, df = 1, p < .001). Cox and Snell R-square of the regression model was .048, which indicated that the effect of age was small. Further, most importantly, the level of anxiety at the initial assessment did not influence cooperation at wave 2.Table 1RCADS itemsSADSoPhFears being alone at homeWorried when does poorly at thingsScared to sleep aloneWorried when somebody angryScared to sleep away from homeWorried will do badly at schoolFears being away from parentsWorried about mistakesWorried in bed at nightWorried what others thinkTrouble going to schoolScared to take a testAfraid of being in crowded placesWorried might look foolishAfraid to talk in front of classAfraid to look foolish in front of peopleGADPDWorried something awful will happen to familySuddenly trouble breathing without reasonWorried bad things will happen to selfWhen has a problem, feels shakyWorried something bad will happen to selfSuddenly trembling, shaking without reasonThinks about deathSuddenly dizzy, faint without reasonWorried about thingsWhen has a problem, stomach feels funnyWorried about what will happenWhen has a problem, heart beats really fastSuddenly feeling scared without reasonSuddenly heart beats too fast without reasonWorried suddenly get scared without reasonOCDCan’t get silly/bad thoughts out of headKeeps checkingHas to think thoughts to stop bad eventsHas to do things over and over againHas to do things just right to stop bad eventsBothered by bad/silly thoughts or images Measures The Revised Child Anxiety and Depression Scale (RCADS) (Chorpita & Daleiden, 2000) is a revision of the Spence Children’s Anxiety Scale (SCAS) (Spence, 1997). It is a self-report questionnaire with 47 items, that are scored on a 4-point scale (0 = never, 1 = sometimes, 2 = often, 3 = always). The questionnaire covers six scales, corresponding with DSM-IV dimensions of anxiety disorders and depressive disorder. The following five scales were used for the present study: separation anxiety disorder (SAD), generalized anxiety disorder (GAD), social phobia (SoPh), panic disorder (PD), and obsessive compulsive disorder (OCD) (see Table 1). The scale major depressive disorder (MDD) was not used. The internal consistencies of the scales that were used were (respectively at wave 1/wave 2) .66/.59 for SAD, .80/.72 for GAD, .78/.88 for SoPh, .75/.72 for PD, and .68/.66 for OCD. The factor structure—for all six scales together—that was originally based on data from 1,641 children and adolescents from a community sample from Hawaii (Chorpita & Daleiden, 2000), was confirmed by confirmatory factor analysis in the TRAILS sample at wave 1 (fit indices of NNFI = .96, RMSEA = .05, and SRMR = .05, indicating an adequate fit to the sample data) (Ferdinand et al., 2006). The association of RCADS dimensions of anxiety with corresponding DSM-IV anxiety disorders was supported by previous research (Nauta et al., 2004). Statistical analyses First, to obtain information regarding comorbidity between different types of anxiety problems in the study sample, correlations among wave 1 RCADS scale scores were computed for each sex. Then, Pearson correlations were computed between wave 1 and wave 2 RCADS scale scores, separately for each sex group. Correlations provide insight in the associations between measures. However, by just computing correlations, it can not be judged if continuity is homotypic or heterotypic. For instance, the magnitude of a correlation between wave 1 SAD and wave 2 SoPh scores depends on the correlation between wave 1 SAD and wave 1 SoPh scores. The higher correlations between wave 1 SAD and wave 1 SoPh are, the higher the correlation between wave 1 SAD and wave 2 SoPh will be. In other words, if assessment of continuity would solely be based on correlations, comorbidity at wave 1 would artificially inflate estimations of the extent of heterotypic continuity between wave 1 and wave 2. To correct for the effects of wave 1 comorbidity rates, regression analyses were conducted. First, it was assessed which part of continuity in anxiety problems was typically homotypic. For this purpose, for scores on each of the five RCADS scales at wave 2, a set of regression analyses was conducted, with wave 2 RCADS SAD, GAD, SoPh, PD, and OCD scores as dependent variables. These analyses were conducted to investigate how much of the variance in a specific RCADS scale score at wave 2 was not accounted for by an overall elevation in different types of anxiety at wave 1, but instead, specifically by its own counterpart at wave 1. We will now describe the regression analyses that were conducted for wave 2 SAD. Those for GAD, SoPh, PD, and OCD were similar. In the first block of the analyses, wave 1 scores on GAD, SoPh, PD, and OCD were entered simultaneously as predictors. Then, in a second block, wave 1 scores on the SAD scales were added, to see how much of the variance in wave 2 scores was predicted specifically by wave 1 SAD scores, and not by scores on the other RCADS scales at wave 1. This variance reflects specific homotypic continuity. In the third block, sex was added. In the fourth block, an interaction between sex and SAD was added. If this interaction was significant, analyses were conducted for girls and boys separately. For each next block, the variance that was accounted for by the variable in this block was computed (R2). Second, it was assessed which part of continuity in anxiety problems was specifically heterotypic. For this purpose, for scores on each of the five RCADS scales at wave 2, a set of regression analyses was conducted, with wave 2 RCADS SAD, GAD, SoPh, PD, and OCD scores as dependent variables. These analyses were conducted to investigate how much of the variance in a specific RCADS scale score at wave 2 was not accounted for by its own counterpart at wave 1, but instead, by the other wave 1 anxiety scale scores. We will now describe the regression analyses that were conducted for wave 2 SAD. Those for GAD, SoPh, PD, and OCD were similar. In the first block of the analyses, wave 1 SAD scores were entered as predictor. Then, in the second block, scores on wave 1 GAD, SoPh, PD, and OCD scales were added, to see how much of the variance in wave 2 scores was specifically predicted by other RCADS scales at wave 1. This variance reflects specific heterotypic continuity. In the third block, sex was added. To judge the magnitude of effects, Cohen’s rules for effects sizes can be used (Cohen, 1988). According to Cohen, R2 between 1.0% and 5.9% is small, between 5.9% to 13.8% medium, and above 13.8% large. Results Means and standard deviations at wave 1 were calculated for SAD (mean=.375, SD=.356), GAD (mean=.666, SD=.454), SoPh (mean=.779, SD=.427), PD (mean=.428, SD=.363), and OCD (mean=.597, SD=.445). Means and standard deviations were also calculated for wave 2 SAD (mean=.236, SD=.291), GAD (mean=.485, SD=.427), SoPh (mean=.684, SD=.465), PD (mean=.301, SD=.321), and OCD (mean=.339, SD=.348). Means reflect mean item scores for each RCADS scale. Correlations among wave 1 RCADS scale scores for each sex separately are presented in Table 2. It is shown that all correlations were in a close range, almost similar across sexes, and generally above .50 which can be regarded as high (Cohen, 1988). For both sexes the highest correlations were found between PD and OCD (r=.61 in boys and girls), and the lowest between SAD and OCD (boys r=.47; girls r=.51).Table 2Correlations among wave 1 RCADS scale scoresWave 1 RCADS scaleWave 1 RCADS scaleGAD b/gSoPh b/gPD b/gOCD b/gSAD.52/.58.52/.52.51/.52.47/.51GAD—.59/.54.55/.54.58/.58SoPh—.56/.55.54/.53PD—.61/.61OCD—Note. Correlations are presented for boys (b), girls (g) separately.Table 3Correlations between wave 1 and wave 2 RCADS scale scoresWave 1 RCADS scaleWave 2 RCADS scaleSAD b/gGAD b/gSoPh b/gPD b/gOCD b/gSAD.30/.38.23/.32.35/.29.28/.27.21/.25GAD.27/.27.34/.38.29/.28.22/.25.23/.27SoPh.29/.29.35/.29.42/.41.29/.28.25/.25PD.22/.29.25/.31.24/.27.32/.42.25/.29OCD.23/.30.25/.32.28/.27.27/.32.31/.37Note. Correlations are presented for boys (b), girls (g) separately. Correlations between wave 1 and wave 2 RCADS scale scores can be found in Table 3. For instance, in boys, the correlation between wave 1 and wave 2 SAD scores was .30, whereas the correlations between wave 1 GAD, SoPh, and PD scores and wave 2 SAD scores were .27, .29, and .22 respectively. Hence, heterotypic correlations were almost as high as the homotypic correlation. In girls, a similar result was found for wave 2 SAD. Heterotypic correlations ranged between .27 and .29, whereas the homotypic correlation was .38. Similar relatively small discrepancies between homotypic and heterotypic correlations were found for wave 2 GAD, SoPh, and PD scores.Table 4Specific homotypic continuity (prediction by target scale). Prediction of wave 2 RCADS scale scores by wave 1 scale scores and sexWave 2 RCADS scaleSADGADSoPhPDOCDWave 1 predictorsR2/F change/p1R2/F change/p1R2/F change/p1R2/F change/p1R2/F change/p1Non-target scales (block 1).119/69.68/.000.150/91.19/.000.132/78.35/.000.126/74.19/.000.106/61.22/.000Target scale (block 2).050/123.57/.000.023/58.06/.000.069/178.54/.000.037/90.39/.000.025/59.20/.000Sex (block 3).023/58.92/.000.036/93.32/.000.037/100.65/.000.032/81.21/.000.009/21.34/.000Target-scale*sex (block 4).001/2.06/ns.002/5.12/.024.000/.81/ns.009/23.81/.000.002/5.38/.020Models for girls/boys separately in case of interaction target-scale*sex Girls  Non-target scales (block 1)—.145/44.70/.000—.128/38.75/.000.109/32.82/.000  Target scale (block 2)—.024/31.06/.000—.060/77.54/.000.038/47.13/.000 Boys  Non-target scales (block 1)—.136/39.32/.000—.107/29.72/.000.089/24.41/.000  Target scale (block 2)—.018/20.85/.000—.020/22.82/.000.021/23.05/.000Note. Target-scale---Wave 1 RCADS scale identical to the wave 2 RCADS outcome scale in the regression model (prediction indicating specific homotypic continuity). Non-target scales---all other wave 1 RCADS scales, not identical to the wave 2 RCADS outcome scale. R2---explained variance. The results of the regression analyses are presented in Tables 4 and 5. Analyses that were aimed at assessing specific homotypic continuity (Table 4) indicated that variances reflecting homotypic continuity were 2.3% for SAD, 3.6% for GAD, 3.7% for SoPh, 3.2% for PD, and .9% for OCD. Analyses aimed as specifically investigating heterotypic continuity revealed variances of 2.0% for SAD, 4.2% for GAD, 1.3% for SoPh, 2.3% for PD, and 2.7% for OCD (Table 5). In analyses aimed at homotypic continuity, some anxiety*sex interactions were significant. A marked sex difference was found for PD. Table 4 shows that homotypic continuity was much stronger for girls (homotypic R2=6.0%) than for boys (homotypic R2=2.0%). Discussion The present study assessed homotypic and heterotypic continuity of symptoms of separation anxiety disorder, generalized anxiety disorder, social phobia, panic disorder, and obsessive compulsive disorder in individuals from a community sample, who were assessed for the first time when they were aged 10 to 12 years, and for the second time two years later. Variances reflecting homotypic continuity were roughly equal to those reflecting heterotypic continuity for SAD (2.3% versus 2.0%) and GAD (3.6% versus 4.2%). Variances reflecting homotypic continuity were larger than for heterotypic continuity for SoPh (3.7% versus 1.3%) and PD (3.2% versus 2.3%), and smaller for homotypic than for heterotypic continuity for OCD (.9% versus 2.7%) (Tables 4 and 5). Applying cross-sectional research designs, previous studies found high comorbidity rates among different types of anxiety problems (Essau et al., 2000; Masi et al., 1999; Newman et al., 1996; Verduin & Kendall, 2003). The present study extended the knowledge about the taxonomy of anxiety problems in young adolescents with longitudinal data. In accordance with previous cross-sectional work, SAD, GAD, SoPh, PD, and OCD symptoms appeared to be intertwined in a longitudinal fashion as well. However, considerable homotypic continuity was found as well. Separation anxiety For SAD, homotypic and heterotypic continuity were roughly equal. Previous studies indicated considerable comorbidity between SAD and the other anxiety problems, especially with SoPh (Compton et al., 2000). In the present study comorbidity rates at wave 1 were high as well, not only with SoPh, but also with other types of anxiety. This suggests that SAD and the other anxiety problems may represent two sides of the same coin. However, homotypic continuity was as strong as heterotypic continuity, which supports the usefulness of SAD as a separate diagnostic construct. The distinction between SAD and other types of anxiety was further supported by another study, in which we conducted latent class analysis to assess the boundaries between SAD and SoPh in referred 8- to 11-year-olds (Ferdinand et al., 2006). Four different classes of individuals were detected; those with (1) low SAD and SoPh item scores on a self-report questionnaire, (2) high SAD and high SoPh item scores, (3) high SAD and low SoPh scores, and (4) low SAD and high SoPh scores. This also supported the idea that, despite high comorbidity rates, SAD may at least partially represent a separate phenomenon, that, for instance, may be subject to specific etiological influences that differ from influences that affect the course of other anxiety problems.Table 5Specific heterotypic continuity (prediction by non-target scales). Prediction of wave 2 RCADS scale scores by wave 1 scale scores and sexWave 2 RCADS scaleSADGADSoPhPDOCDWave 1 predictorsR2/F change/p1R2/F change/p1R2/F change/p1R2/F change/p1R2/F change/p1Target scales (block 1).391/373.39/.000.375/337.53/.000.436/483.57/.000.380/347.98/.000.337/263.45/.000Non-target scale (block 2).020/83.77/.000.042/86.59/.000.013/103.79/.000.023/80.00/.000.027/82.20/.000Sex (block 3).027/81.60/.000.041/90.95/.000.040/107.45/.000.028/82.80/.000.012/55.90/.000Note. Target-scale---Wave 1 RCADS scale identical to the wave 2 RCADS outcome scale in the regression model. Non-target scales---all other wave 1 RCADS scales, not identical to the wave 2 RCADS outcome scale (prediction indicating specific heterotypic continuity). R2---explained variance. Generalized anxiety For GAD, homotypic continuity was almost as strong as heterotypic continuity. This is—at least partly—consistent with a previous study (Pine et al., 2001) in adolescents from the general population indicating that separation anxiety disorder and social phobia in adolescents did not predict future generalized anxiety disorder. This suggested rather strong homotypic continuity of GAD. However, continuity of GAD was more heterotypic than in the Pine et al. study, because in our study, heterotypic continuity was also considerable. This contrast may be due to methodological issues such as differences in sample characteristics (Pine et al. investigated older adolescents), assessment procedures (Pine et al. applied standardized interviews instead of self-report questionnaires), or statistical approach (Pine et al. used categorical diagnostic samples whereas the present study used dimensional scale scores). Further, Pine et al. did not use a block design for their regression analyses, but included all predictors in a forward stepwise logistic regression analysis. So, in essence, they did not test if one predictor predicted future GAD, over and above the effect of other predictors. Social phobia Compared to heterotypic continuity, homotypic continuity of SoPh symptoms was relatively strong. Pine et al. (2001) found that SoPh, but also GAD, in adolescence predicted future SoPh, independently of other types of anxiety, whereas SAD did not. Remarkably, in their study, GAD was a better predictor of future SoPh than SoPh itself. The aforementioned methodological differences between the Pine et al. study versus the present study may explain differences between findings. Panic Homotypic and heterotypic continuity of PD did not differ much. Analyses for boys and girls separately (Table 4) indicated that homotypic continuity was higher in girls than in boys. Previous studies already indicated that panic disorder tends to have a chronic course, in children as well as in adults (Biederman et al., 1997; Bruce et al., 2005). The prevalence of full blown panic disorder, and even of panic attacks, in adolescents is very low (Essau et al., 1999). The present study nevertheless suggests that in girls, the disorder may already begin with a chronic homotypic course at a very young age which is in accordance with the higher prevalence in females versus males (Goodwin et al., 2005), and with studies that retrospectively investigated age at onset, and that often point to childhood or adolescence as a starting point (Sheehan et al., 1981). Obsessive compulsive disorder Comorbidity rates of OCD with other anxiety disorders are generally high (Heyman et al., 2001; Tukel et al., 2002), which was supported by the correlations among the wave 1 RCADS scale scores. The present study showed that heterotypic continuity of OCD symptoms was higher than homotypic continuity. To our knwoledge, previous studies that assessed homotypic continuity of OCD symptoms versus heterotypic continuity with other types of anxiety in young adolescents are not available, so we cannot compare our findings with previous work. Homotypic continuity was somewhat stronger in girls than in boys. This seems to suggest that, longitudinally, OCD symptoms in boys correlate differently with comorbid conditions than OCD symptoms in girls. Again, we were not able to find previous work on this topic. Future studies are needed to investigate if the differences between boys and girls we found can also be found in other samples, countries, and cultures. Practical implications In many studies, anxiety disorders are treated as one group of disorders (Barrett et al., 2001; Lipman et al., 2001; MacMillan et al., 2001; Roza et al., 2003; Shortt et al., 2001), and, even, some widely used assessment instruments do not contain scales that tap different anxiety dimensions (Achenbach, 1991a, b). Previous studies found considerable associations between different types of anxiety symptoms, which suggested the presence of one higher order factor (Nauta et al., 2004). Several studies with adults also found evidence for a higher order factor that explained the presence of different types of anxiety (Hettema et al., 2005; Krueger, 1999; Vollebergh et al., 2001). Given the magnitude of heterotypic continuity in the present study, a higher order factor is likely to be present. However, the present study also showed that considerable homotypic continuity is present as well, occurring separately from a general propensity for high anxiety levels. This indicates that each type of anxiety problem may, at least partly, represent a distinct taxonomic construct. Homotypic continuity was found specifically for SAD, GAD, and SoPh, and for PD in girls. This may indicate that SAD, GAD, SoPh, and PD represent diagnostic constructs that are at least partially distinct. Hence, in clinical practice, instruments are needed that measure different anxiety dimensions separately. Intruments that just assess on single anxiety dimension may not be sufficient. Further, the distinctions between different anxiety constructs indicate that, despite the evidence that similar treatment methods are generally efficacious for different types of anxiety problems, each type of anxiety might require a slightly different treatment approach, and development of specific treatment modules. Limitations The sample consisted of young adolescents only. For older adolescents, different homotypic and heterotypic continuities could apply. Furthermore, questionnaires were used instead of clinical interviews; information about the presence or absence of DSM-IV (American Psychiatric Association, 1994) clinical diagnoses was not obtained. Even though RCADS symptom dimensions have proved to reflect corresponding DSM-IV anxiety disorders (Nauta et al., 2004), still, it may be the case that different results would have been obtained if DSM-IV diagnoses, that take account of the level of functional impairment, would have been used instead of RCADS scale scores. Since different informants may provide different information, this study would have been more valuable if information regarding symptoms of different types of anxiety would also have been gathered from parents or teachers. Unfortunately, such information was not available. Conclusion In the present study’s sample of young adolescents from the Dutch general population, evidence for homotypic continuity was found, especially for symptoms of separation, social, and generalized anxiety, and for symptoms of panic disorder in girls.

Mol_Immunol-1-5-1994210

Modulation of Aire regulates the expression of tissue-restricted antigens

Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity. 1 Introduction Thymus has an essential role in establishing immune tolerance. Previous studies have demonstrated that tissue-restricted antigens (TRAs) are expressed in thymus and that this expression is needed for the deletion of self-reactive T cells (Kyewski and Klein, 2006). A central feature in this process is the promiscuous expression of TRAs by epithelial cells in thymic medullary region, where the TRAs are presented and encountered by the thymocytes, leading to the induction of tolerance either by clonal deletion or functional inactivation (Derbinski et al., 2001). In this context, medullary thymic epithelial cells (mTEC) highly express MHC molecules with costimulatory signals and act as professional antigen presenting cells (APCs) in thymus. A detailed study of the gene expression pattern in mTEC revealed that many of the TRAs and, in particular, almost all putative autoantigen targets of experimental animal models and human diseases are expressed by mTEC (Derbinski et al., 2001; Gotter and Kyewski, 2004). Altogether, the pool of promiscuously expressed genes in thymus appears to be highly diverse including tissue and sex-specific genes and genes specifically involved in development (Kyewski and Klein, 2006). An important molecule in regulation of TRA expression in mTEC is autoimmune regulator (Aire) (Nagamine et al., 1997). The Aire protein has several features such as SAND and PHD finger domains that are characteristic to proteins involved in transcriptional control and has been reported to bind directly to DNA (Kumar et al., 2001) and to a common transcriptional regulator and histone acetyltransferase, CREB binding protein (CBP) (Pitkanen et al., 2000). In the thymus and cell lines, the Aire protein is subcellularly located to the nuclear bodies (Bjorses et al., 1999; Heino et al., 1999), which have been associated with several functions, including modulation of chromatin structure, transcriptional control, DNA repair and antiviral response (Everett and Chelbi-Alix, 2007). Initial studies have shown the Aire protein to be predominantly expressed in mTECs and suggest it has a role in regulation of immune tolerance (Blechschmidt et al., 1999; Heino et al., 1999). In humans, mutations in AIRE cause autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED), a syndrome characterized by the presence of autoantibodies to multiple self antigens and lymphocytic infiltration of endocrine glands, leading to autoimmune endocrine disorders (Perheentupa, 2006; Peterson and Peltonen, 2005). In agreement with the human disease, the Aire deficient mice have autoantibodies and tissue infiltration, although the full development of autoimmune disease appears to depend on the genetic background of the mouse (Anderson et al., 2002; Kuroda et al., 2005; Ramsey et al., 2002). The Aire deficiency affects negative selection since there is a complete failure to delete the organ-specific thymocytes in this mouse model (Liston et al., 2003). More importantly, the microarray analysis of mTEC population shows a decreased or abolished expression of multiple tissue specific genes in the Aire deficient mouse suggesting thus that Aire plays a role in modulating TRAs in the mTEC (Anderson et al., 2002; Derbinski et al., 2005; Jiang et al., 2005). This study aims to further clarify whether Aire can directly regulate the TRA expression by analyzing the expression of four antigens in several experimental settings where Aire's expression has been modulated. The study aims to establish whether there is a dose-dependent correlation between the number of Aire allele copies and TRA expression level in thymic epithelial cells, and whether TRAs are co-expressed with Aire during thymic development and involution. We also studied whether the over-expression of Aire as a sole factor is sufficient to induce TRA expression and whether thymic microenvironment plays a role in the expression of Aire and TRAs. 2 Material and methods 2.1 Mice and cell cultures Aire deficient mice (C57BL/6J and Balb/c background) were generated at The Walter and Eliza Hall Institute (Melbourne, Australia). The inserted targeting construct containing LacZ gene replaced mouse Aire exon 8. For genotyping, the genomic DNA was extracted using JetQuick Tissue DNA Spin Kit (Genomed), and wild-type (WT) and knockout (KO) alleles were amplified using primers: 1042 5′-cagaagaacgaggat-3′, 1045 5′-cagactgccttggga-3′ or 1043 5′-ctgtcttctgtgaaggcttctagg-3′. As shown in Fig. 1A, primers pair 1042/1043 and 1043/1045 detect WT and KO alleles, respectively. Thymuses from 4- to 6-week-old WT, Aire HET (heterozygote) and Aire KO mice were used. Embryonic (E13.5, E15.5 and E17.5), newborn, neonatal D11 and adult (6 weeks, 6 months and 12 months) mouse tissues were used in developmental dynamics analysis. Mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. TEC 1C6 cell line (Mizuochi et al., 1992) was kindly provided by G. Holländer (University of Basel, Switzerland). Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Gibco BRL). 2.2 EGFP and Aire adenovirus construction and infection The pAdTrack-CMV (Stratagene) vector expressing enhanced green fluorescence protein (EGFP) gene was used as pAd-GFP plasmid. The mouse Aire gene was amplified from pcAire vector (Heino et al., 2000) using the primers: mAire-5-SalI 5′-tttgtcgac agatggcaggtggggatggaatg-3′ and mAire-3-NotI_stop 5′-tttgcggccgctcaggaagagaagggtggtgtc-3′ and cloned into SalI and NotI sites of pAdTrack-CMV resulting in AdAire-GFP. HEK293 cells (Invitrogen), which constitutively express AdEasy deleted E1 genes in-trans, were used for expression analysis of adenoviral vectors and for virus growth. To make recombinant adenoviruses, pAd-GFP and pAdAire-GFP plasmids were recombined with pAdEasy-1. The amplification and harvest process was repeated to generate higher titer viral stocks. Subsequently, the lyzates from several amplification steps were purified by CsCl gradient centrifugation (He et al., 1998). Virus bands was collected and mixed with 2× preservation buffer (10 mM Tris pH 8.0, 100 mM NaCl, 0.1% BSA, 50% glycerol). We quantified the Ad-GFP and AdAire-GFP virus particles by absorbance measurement at 260 nm to be equivalent to 1012 particles with virus titers >2 × 108 pfu/ml. The adenoviruses were next verified for their expression by infection of HEK293 cells for 48 h and analyzed by immunoblotting with rabbit polyclonal anti-mouseAire and mouse monoclonal anti-G3PDH (Ambion). The signal was detected by Supersignal® West Pico Chemoluminescent Substrate (Pierce Biotechnology) according to the manufacturer's instructions. For adenoviral infection of thymic primary culture and TEC 1C6, the cells were infected with Ad-GFP and AdAire-GFP at approximately 70% confluence. Cells were incubated in 500 μl serum-free OptiMEM (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B) and infected with equal amounts of virus for 1 h. The infection rate was further assessed by quantitative real-time PCR analyzing intake of viral genomic DNA. 2.3 Thymic stromal cell isolation Small cuts were made into the capsules of thymi dissected from eight to twelve 4-week-old mice and thymocytes were released by repetitive pipeting. The remaining thymic fragments were incubated in 0.5 mg/ml dispase/collagenase (Roche) and 1.5 μg/ml DNase I (AppliChem) in PBS at 37 °C for 20 min, with gentle agitation using a Pasteur pipette every 5 min. Released cells were collected to separate fractions and fresh enzyme solution was added four times. The cells were resuspended in 5 mM EDTA in 10 ml of RPMI-1460. Isolated cells were used either for stromal cell isolation or for growing thymic primary culture. For thymic primary culture, the isolated cells were cultured for 4 days in DMEM supplemented with 10% FCS. For stromal cell isolation cells were counted in each fraction. To obtain 2 × 108 cells required for isolation, the final fraction was used first and then collected backwards through the collected fractions. The fractions were pooled and passed through a 100 μm mesh to remove clumps. 2.4 Cell sorting For CD45 depletion CD45 MicroBeads (Miltenyi Biotec) were used according to manufacturers instructions. For cortical epithelial cell (cTEC) isolation, the CD45− cells were stained with H213-HB Ab (anti-CDR1) followed by goat anti-rat IgG Microbeads (Miltenyi Biotech) and AutoMACS separation (isolation mode: Possel-S). The positive fraction (CDR1+) contained magnetically bound cTEC-s. For mTEC isolation CDR1− cells were stained with G8.8 (anti-EpCAM, generated from a G8.8 hybridoma cell line) followed by goat anti-rat IgG Microbeads (Miltenyi Biotec) and separation as previously described. The purity of mTEC and cTEC was >80% as assessed by staining with anti-CD45 FITC (30F11, Miltenyi Biotech) and anti-I-Ab PE (AF6-120.1, BD Biosciences) using FACSCalibur flow cytometer (BD Biosciences). 2.5 Immunofluorescence and microscopy Cryostat sections (5μm) of fresh-frozen 10-day-old mouse thymus were thaw-mounted onto SuperFrost Plus microscope slides (Menzel–Gläzer) and fixed in cold acetone (−20 °C) for 5 min. Sections were permeabilized in PBS/0.5% Triton X-100/1% normal goat serum (DAKO) for 15 min. Slides were blocked with 1% normal goat sera for 20 min at room temperature and incubated with rat monoclonal G8.8 (1:100) or rabbit polyclonal anti-mAIRE (1:2000) and then incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (H + L) or Alexa Fluor 488-conjugated goat anti-rabbit (Fab)2 (both from Molecular Probes, Eugene), followed by washing six times in PBS. The slides were incubated with 15 μg/ml DAPI (Roche) and mounted with fluorescent mounting medium (DAKO). The images were acquired by fluorescence microscopy (Eclipse TE2000-4; Nikon, Melville, NY). 2.6 Thymic reaggregate organ culture Reaggregated thymic organ-cultures were done as described previously (Jenkinson et al., 1992). Briefly, thymic stromal cells from E17.5 C57BL/6 mice were prepared by disaggregating fetal thymic lobes, which had been previously cultured for 7 days in 1.45 mM deoxyguanosine (Sigma, St. Louis, MO) using 1× trypsin (Life Technologies, Grand Island, NY). Reaggregates were formed by mixing together stromal cells and thymocytes at a 3:1 cell ratio and cultured for 3 days at 37 °C. 2.7 Real-time PCR RNA was isolated using TRIzol (Invitrogen, Life Technologies) and reverse-transcribed to cDNA using the SuperScript™ III Reverse Transcriptase (Invitrogen, Life Technologies). Real-time PCR was performed with the ABI Prism 7900HT Fast Real-Time PCR System instrument (Applied Biosystems) using qPCR SYBR Green Core Kit (Eurogentec) according to the manufacturer's instructions except that 2 mM MgCl2 concentration was used. The amplification program included an initial denaturation step at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min, for 45 cycles. SYBR Green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by melting curve analysis. Primers used to amplify specific gene products from murine cDNA were K2-8 sense, 5′-aggagctcattccgtagctg-3′; K2-8 antisense, 5′-tctgggatgcagaacatgag-3′; Aire 11/12, 5′-ccccgccggggaccaatctc-3′; Aire 12/13, 5′-agtcgtcccctaccttggcaagc-3′; Tff3 sense, 5′-tacgttggcctgtctccaag-3′; Tff3 antisense, 5′-cagggcacatttgggatact-3′; Ins2 sense, 5′-gacccacaagtggcacaac-3′; Ins2 antisense, 5′-tctacaatgccacgcttctg-3’, Mup1 sense, 5′-tctgtgacgtatgatggattcaa-3’; Mup1 antisense, 5′-tctggttctcggccatagag-3′; Spt1 sense, 5′-aacttctggaactgctgattctg-3′; Spt1 antisense, 5′-gaggcctcattagcagtgttg-3′. The relative gene expression levels were calculated using the comparative Ct (ΔΔCt) method (according to Applied Biosystems), where the relative expression is calculated as , and where Ct represents the threshold cycle. Every sample was run in three parallel reactions. 3 Results 3.1 Decrease in Aire expression down-regulates the TRA expression in a dose-dependent manner We initially set out to confirm by real-time PCR the previously published array results (Derbinski et al., 2005) demonstrating decreased TRA mRNA levels in the Aire deficient mouse. In order to study whether the self antigen expression is dependent on Aire we chose four TRAs; Tff3, Ins2, Mup1 and Spt1, which were downregulated in the Aire deficient mouse according to publicly available microarray data (Derbinski et al., 2005). Throughout the study, we normalized our data to the expression level of keratin 8 (K8) mRNA, which in thymus is specifically expressed in epithelial cell fraction and is not influenced by Aire gene expression (Anderson et al., 2002; Derbinski et al., 2005). The real-time PCR analysis showed almost complete absence of TRA mRNA signal in C57BL/6 Aire KO thymus samples, which was uniformly seen with all four antigens studied (Fig. 1B). Furthermore, Aire allele dose-dependency was observed, as heterozygous mouse thymus consistently showed lower expression levels compared to the WT thymus levels. The expression level of all four TRAs in heterozygous mice thymus was approximately 10–20% of the expression level in WT mice. In order to determine whether Aire's effect on TRA expression depends on the genetic background, we also measured expression levels of the four TRAs in Aire KO and Aire HET mice backcrossed to Balb/c WT mice (Fig. 1C). Again, we observed a clear allele dose-dependency for all TRAs studied and almost no expression of the TRAs in the Aire KO mouse. We next determined whether Aire has a similar effect on TRA expression in the lymph nodes and quantified the expression of TRAs in the lymph nodes from C57BL/6 mice. Although we could clearly detect Aire mRNA in the lymph nodes at level that was even higher than the one of the whole thymus (Fig. 2A), most of the analyzed TRAs were undetectable or close to the detection limit. The higher expression of Aire in lymph nodes was relative to the epithelial cell marker K8, limiting the detection of Aire mRNA signal to the epithelial cell fraction. However, the mRNA signal for the Ins2 was clearly present in lymph node samples but, interestingly, did not depend on the presence of Aire (Fig. 2B). In order to establish whether Aire co-localizes in the thymus with TRAs, we purified the thymic mTEC based on the cell-surface marker EpCAM (Fig. 3A) and analyzed the expression of the TRA genes. As seen in Fig. 3B, the expression of the Tff3, Ins2, Mup1 and Spt1 antigens was limited to the mTEC population, i.e. the cell population of Aire expression. The cTEC population showed a very low expression for all four TRAs both in the WT as well as Aire KO mouse. Collectively these data show that Aire dose-dependently regulates TRA expression in thymus but not in the lymph nodes, and confirms by real-time PCR the previously published microarray data, suggesting that both Aire and TRAs are predominantly expressed in thymus medullary epithelium. 3.2 TRAs follow the expression of Aire during normal development and involution Thus far, the expression of Aire and TRAs has been studied in fetal or neonatal mice using WT versus Aire KO mice. If the expression of self antigens is directly dependent on Aire, this should be evident throughout the development of thymic tissue. However, the thymic cell content and volume changes significantly during development. To limit our analysis to the epithelial cell subsets only, we normalized our data again to the K8 gene. Thymuses from different embryonic, neonatal, young or adult developmental stages were analyzed for the Aire and TRA expression (Fig. 4). Very low Aire expression was detectable already at day E13.5 but showed a significant increase at E15.5. Thus, the start of Aire expression coincides with the influx of the first wave of the hematopoietic cells to the thymus. We observed the highest expression level at postnatal D11 and a gradual decrease thereafter until the very last time-point studied. Aire expression was, however, clearly present even in 12-month-old mice. The expression of TRAs closely followed the pattern of Aire reaching their peak at D11 followed by an obvious decrease, suggesting a role for Aire in their regulation. 3.3 Over-expression of Aire results in an increase in TRA expression Although the lack of Aire has been shown to have a negative effect on TRA expression, the ability of Aire as a single factor to up-regulate TRA expression has not been demonstrated. We used an adenoviral expression system (AdAire-GFP versus Ad-GFP) to determine whether the specific over-expression of Aire has any effect on TRAs. Infection with the AdAire-GFP resulted in an increased production of Aire protein (Fig. 5A). The AdAire-GFP infection also resulted in an increase of expression of all four TRAs studied, which was detected in primary thymic stromal cells (Fig. 5B) as well as in thymic medullary epithelial cell line TEC 1C6 (Fig. 5C). The results demonstrate that Aire can indeed act as a single inducer of TRAs in thymic epithelial cells even in the absence of signals from other cell-types normally present in thymus. 3.4 Thymic microenvironment is needed for the expression of Aire and TRAs Although the precise mechanism is not fully understood, it has been demonstrated that the complex 3D structure of thymus is essential for mTECs to function properly (Anderson et al., 2006). To study whether the microenvironment plays a role in Aire and TRA expression, we used ex vivo culture of thymocyte-depleted dissaggregated and reaggregated thymic stromal cells. As shown in Fig. 6, the disaggregation of thymus to 2D culture resulted in a dramatic decrease in expression of Aire as well as most of the TRAs. This effect was, at least partly, reversed by reaggregation suggesting a critical role for the thymic microenvironment in Aire as well as TRA expression. 4 Discussion In this paper, we report Aire dependent expression of four TRAs in mouse thymus. TRA expression was previously reported to be substantially decreased in Aire deficient mTEC subpopulations by microarray analysis (Anderson et al., 2002; Derbinski et al., 2005), which prompted us to follow the expression of four TRAs as marker antigens. All four genes have highly selective tissue specific expression. For example, Tff3 is restricted to mucin producing epithelial cells, with high expression in stomach and intestine (Hoffmann and Jagla, 2002; Karam et al., 2004), Spt1 is expressed in salivary and lacrimal glands (Dickinson et al., 1989) and Mup1 is expressed in liver but also in salivary, lacrimal and mammary glands (Shaw et al., 1983). Of the two insulin genes present in the mouse genome and encoded from separate loci, we selected Ins2 due to its high expression in the thymus as well as pancreatic beta cells, and previously reported Aire dependent expression pattern (Chentoufi and Polychronakos, 2002; Derbinski et al., 2005). Furthermore, it has been reported that Ins2 deficient mouse with low insulin expression in thymus has T cell reactivity to proinsulin (Chentoufi and Polychronakos, 2002). The expression signal of all four TRAs tested was readily detectable in whole thymus and sorted mTEC samples by real-time PCR analysis. Interestingly, the TRA expression in heterozygous mouse thymus, both on C57Bl/6 and Balb/c background, was repeatedly only 10–20% and not 50% of the expression level seen in WT mouse, which would be expected when one of the Aire genomic alleles remains intact. This expression at lower levels than expected in the heterozygous mouse suggests Aire haploinsufficiency in regulation of target TRA genes. In haploinsufficiency of transcriptional regulators, only one intact gene copy is not sufficient for the functional activity of the regulated target gene product. The phenomenon has been explained by a stochastic expression model where diploid cells have a higher probability than haploid cells in maintaining the abundance of an expressed gene product above a low threshold level (Cook et al., 1998; Kaern et al., 2005). Further support to Aire haploinsufficiency comes from the report by Liston et al (Liston et al., 2004) demonstrating that the loss of one copy of the Aire allele in TCR-insHEL double transgenic mice caused severe functional defects in negative selection of autoreactive T cells and resulted in pancreatic cell insulitis, with diabetes incidence comparable to the Aire KO mouse. It should be noted, however, that the activation of target genes by a transcriptional factor in vivo can be dependent on the specific gene and the physiological context, such as in the case of cardiac transcription factor Nkx2–5 (Jay et al., 2005). A recent study reported that lymph node stroma can also express Aire as well as a range of TRAs and contributes accordingly to tolerance induction (Lee et al., 2007). Here we report that, although Aire as well as Ins2 expression is clearly present in the lymph nodes of WT mice, the lack of Aire does not lead to a decrease in Ins2 expression in the Aire KO mice. The data suggests that, unlike in the thymus, other Aire-independent factors are likely to control Ins2 expression in the lymph nodes. Regarding the rest of the TRAs studied, we found very low, if any, expression by real-time PCR, which did not allow us to quantify the changes. In order to determine whether the thymic expression of Aire and TRAs follow the same pattern during development, we monitored the expression throughout mouse development from E13.5 to 12 months. We detected an increase in Aire expression at day E15.5, which is in concordance with earlier reported results (Sousa Cardoso et al., 2006). The expression was at its highest at D11 and decreased thereafter, but was still present even in 12-month-old mouse tissue. The expression of TRAs followed a similar pattern to Aire expression, indicating a correlation between the amount of Aire and TRAs. The data demonstrate that the dynamics of Aire and TRA expression closely follow the dynamics of thymic function in general, being most active during the postnatal period and followed by a gradual decline in activity (Gray et al., 2006). Aire dependent TRA expression is further illustrated by adenoviral experiments enforcing Aire expression in thymic epithelial cells. We show that over-expression of Aire as a single factor is sufficient to induce the expression of all four TRAs studied, providing evidence that modulation of Aire can directly lead to alterations in TRA levels and may thus also affect the maintenance of central tolerance. The finding that Aire, in addition to primary thymic stromal cells, can also modulate TRA levels in the thymic medullary epithelial cell line, suggests that there is no need for other cell types for the Aire-induced up-regulation of TRAs to occur. The disruption of normal thymic architecture is known to affect the expression pattern and functionality of thymus, and it has been suggested that interactions between epithelial cells and thymocytes control the development of the thymic microenvironment and T cell development (Van Ewijk et al., 1994). Although the maturation of thymic epithelial stroma during the fetal period apparently occurs independently of thymocyte-derived signals (Jenkinson et al., 2005) and is mainly regulated by thymic mesenchyme (Jenkinson et al., 2003), thymocytes deliver signaling molecules, which are needed to maintain the normal adult thymic microenvironment. For example, lymphotoxin that signals through the lymphotoxin receptor and directs the alternative NfkappaB pathway, is needed for development of the thymic medullary compartment. Consequently, lymphotoxin receptor deficient mouse thymus had subnormal levels of Aire and TRAs (Boehm et al., 2003; Chin et al., 2003). Thymic medullary atrophy and lower expression of Aire and TRAs have been reported in mouse models deficient in several genes involved in the NFkappaB pathway, such as TRAF6, NIK, RelB or p52 suggesting an important role of this pathway in development of thymic medulla (Akiyama et al., 2005; Burkly et al., 1995; Kinoshita et al., 2006; Zhang et al., 2006). A recent study suggests that Aire deficiency may also cause changes in the organization and composition of the medullary epithelial compartment (Gillard et al., 2007). Thus, it is presently unknown whether the reduced levels of TRA expression seen in Aire KO mice are predominantly the result of changes in transcriptional activity or changes in thymic epithelial cell development. In this study, we show a sustained Aire and TRA expression in 2-deoxyguonosine treated FTOC, which rapidly disappeared after the disruption of the three-dimensional thymic meshwork into two-dimensional culture. Aire as well as TRA expression was regained in RTOC, however, the presence of thymocytes did not further augment this effect. These results show that Aire and TRA expression is dependent on the three-dimensional structure of epithelial microenvironment. This expression, however, seems to be independent of the presence of thymocytes being in line with previous data demonstrating the Aire expression signal in RAG deficient and CD3etg26 transgenic mice, in which T-cell development is blocked (Jenkinson et al., 2005; Zuklys et al., 2000). In conclusion, we show that Aire has a dose-dependent effect on TRA expression in thymus but not in the lymph nodes. Both, Aire as well as TRAs localize in the thymic medulla and are co-expressed during normal development and involution. We also show that Aire can directly induce TRA expression in medullary epithelial cells although the thymic microenvironment plays a crucial role for the maximal expression to occur. Our data suggest a clear correlation between the expression of Aire and TRAs and indicate that approaches to stimulate Aire expression in thymic epithelium could be considered to modulate tolerance induction to peripheral antigens.

Eur_J_Nutr-2-2-1705483

Diet supplementation for 5 weeks with polyphenol-rich cereals improves several functions and the redox state of mouse leucocytes

Background Cereals naturally contain a great variety of polyphenols, which exert a wide range of physiological effects both in vitro and in vivo. Many of their protective effects, including an improvement of the function and redox state of immune cells in unhealthy or aged subjects come from their properties as powerful antioxidant compounds. However, whether cereal-based dietary supplementation positively affects the immune function and cellular redox state of healthy subjects remains unclear. Introduction Nowadays, consumption of natural products is a matter of major importance as regards health. Indeed, consumption of fruit and vegetables was already associated with a reduced risk of chronic diseases and age-related functional decline several years ago [1]. Nevertheless, less attention has been focused on the importance of phytochemicals from cereals in maintaining health. It is well known that the nutritional status is a central factor contributing to immune system competence [2] and that an adequate function of the immune system is related to health promotion and disease prevention [3]. Nutrients that have traditionally been shown to be required for correct immune system function include different types of vitamins, zinc, copper, and selenium. However, there is an increasing amount of data showing the implication of the so-called “non-nutrients”, i.e., phytochemicals, in the immune system responses [4]. Further, the phytochemicals found in natural products have been involved in protecting against a wide range of oxidative stress-related chronic diseases [5]. The overproduction of oxidants and the overwhelming of the antioxidant defence lead to oxidative stress, that can cause oxidative damage to large biomolecules, such as proteins, DNA, and lipids, resulting in an increased risk of disease [5, 6]. For this reason, to forestall an excessive production of reactive oxygen species (ROS), sufficient amounts of antioxidants need to be consumed. However, the generation of ROS is a key part of normal immune system function [7]. These molecules play a key role in the defence against infectious agents and seem to be involved in regulating many biological processes, serving as second messengers [8]. Thus, it is important to maintain a balance between ROS generation and antioxidant defences within immune cells to ensure a correct functioning of the immune system and help to prevent the onset of chronic diseases. Cereals naturally contain a wide variety of polyphenols such as the hydroxycinnamic acids, ferulic, vanillic, and p-coumaric acids [9], which show a strong antioxidant power and may help to protect from oxidative stress and, therefore, can decrease the risk of contracting many diseases. Flavonoids are present in small quantities, even though their numerous biological effects and their implications for inflammation and chronic diseases have been widely described [10, 11]. The mechanisms of action of polyphenols go beyond the modulation of oxidative stress-related pathways [6]. Cereal phytochemicals exert their health benefits through multifactorial physiological mechanisms, including enhancement of the immune system and hormones, facilitation of substance transit through the digestive tract, metabolism, modulation and antioxidant, antibacterial and antiviral activities [12]. It is known that unhealthy or aged subjects are more susceptible to infection, showing an impairment of function and redox state of immune cells and that such impairment can be counteracted by antioxidant administration [13, 14]. However, whether dietary antioxidant supplementation affects the functional and redox state of immune cells of healthy subjects remains unclear. Not all subjects are likely to benefit in the same way from supplementation, i.e., there are certain groups within the apparently healthy, general population that benefit from adequate intakes of certain compounds [15]. Since there is an increasing interest in consuming products naturally rich in phytochemicals able to provide health benefits and prevent a wide variety of diseases, in this context we have investigated in the present work the effects of diet supplementation for 5 weeks with different types of cereal fractions naturally rich in polyphenols on certain parameters of immune function and redox state of peritoneal leukocytes from adult healthy mice. Material and methods Animals One hundred-fifty female ICR (CD-1) mice (Mus musculus) (Harlan Ibérica, Barcelona, Spain), which were 24 weeks old on arrival to our laboratory, were used. Mice were checked periodically by animal care personnel and were specific pathogen free, as tested by Harlan according to FELASA recommendations, and did not show any sign of malignancy or other pathological processes. The animals were randomly housed in polyurethane boxes, at a constant temperature (22 ± 2°C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France), on a 12/12 h reversed light/dark cycle. All animals were fed water and standard Sander Mus (A04 diet from Panlab L.S., Barcelona, Spain) pellets ad libitum. The diet was in accordance with the recommendations of the American Institute of Nutrition for laboratory animals. Mice were treated according to the guidelines of the European Community Council Directives 86/6091 EEC. Although we have previously observed that the oestrous cycle phase of mice has no effect on this experimental assay, all females used in the present work were at the beginning of dioestrous. Experimental groups The animals were marked for their individual follow-up. Five experimental groups, one control (n = 30) and four supplemented (30 mice per group), were used. The supplementation started at 28 weeks of age. Then, the supplemented groups were fed for a period of 5 weeks, and at the age of 33 ± 2 weeks samples were obtained to evaluate certain parameters of function and redox state of peritoneal leukocytes. The experiments were carried out in January at a constant laboratory temperature of 25 ± 2°C. Food intake was measured weekly at the same time (9:00–11:00 h). Each animal was fed weekly 50 g of diet (control or supplemented) and, after 1 week, the uneaten remains were weighed. Body weights were determined once every week. No significant differences on food consumption and body weight among diet groups during the study were observed. Cereal products and preparation of extracts All cereal products (wheat germ, buckwheat flour, fine rice bran and wheat middlings, named B, C, D and E; respectively) used in this study were provided by Danone Vitapole (France). The samples were stored and kept at −20°C prior to analysis. Moisture content of the cereal samples was evaluated by drying at 130°C for 90 min. The analyses were done in duplicate. The different extracts were analyzed by Danone Vitapole (France). Briefly, the cereal products were sequentially extracted with water at 50°C followed by 80% methanol and ethyl acetate. Extraction with each solvent was carried out twice for 30 min under N2, in the dark and constant agitation (200 rpm). The ethyl acetate extracts were evaporated under vacuum and re-dissolved in 80% methanol. The final insoluble pellet was dried. The soluble fractions and the insoluble pellet were stored at 4°C (in the dark and under N2) prior to analysis of polyphenols and determination of antioxidant activity. Analysis of total polyphenols and antioxidant activity The amount of total phenolic compounds in all extracts was determined by Danone Vitapole (France), according to the Folin–Ciocalteu method [16]. The total phenolic concentration is calculated from a standard curve using a range of concentrations of gallic acid. The results are expressed as milligrams of gallic acid equivalents per gram of material. The antioxidant in vitro activity of the extracts obtained from selected cereal products was evaluated using an Oxygen Radical Absorbance Capacity (ORAC) procedure (Brunswick Laboratories, Wareham, MA, USA). Values are expressed as μmol of Trolox equivalents (TE) per g of dry matter. This assay was conducted in triplicate. There was also a positive correlation between ORAC and total polyphenol content (r2 = 0.843). Preparation of diets The control group only received a standard maintenance diet (AO4 diet from Panlab L.S. Barcelona, Spain; see Table 1) and the remaining groups received 80% of control diet plus 20% of cereal fractions naturally rich in polyphenols (B, C, D and E; see Table 1). Control and supplemented diets were prepared by mixing the standard maintenance diet or one of the cereal fractions with water, forming a biscuit-shape preparation. Both types of preparations were daily turned upside down and maintained in a dark room for 3 days to obtain a dry preparation. Then, the humidity of the different diets was measured and each of them was stored at 4°C for less than 3 weeks, avoiding exposure to light in order to prevent their oxidation. Table 1Nutritional composition of standard (control) and experimental (B, C, D and E) dietsDiet ingredientsa (mg/100g)ControlBCDEHFLFbHFLFbLFbLFbHFLFbPolyphenols  Gallic acid————0.49————  Catechin———10.80.192.043.34——  p-HB acid—0.270.99—0.78——1.04—  Vanillic acid—0.5413.191.82—1.93—0.860.46  p-coumaric acid———0.210.161.2818.910.060.68  Sinapic acid————0.454.73———  Ferulic acid—25.9755.600.420.0911.7569.1923.8363.06  Quercetin——0.17a—2.42a————  Rutin——3.94a—11.85a————  Oryzanol—————4.03a——Vitamins  Vitamin A0.45————  Vitamin D30.0038————  Vitamin E213.650.21.335.55  Thiamin0.11.870.411.811.48  Riboflavin0.4————  Niacin2————  Pantothenic acid1.5————  Pyridoxin0.40.870.20.180.68  Biotin0.01————  Folacin0.12914569103  Cobalaminec2————  Choline50————Fatty acids  Palmitic acid460————  Palmitoleic acid39————  Stearic acid130————  Oleic acid650————  Linoleic acid1,390————  Linolenic acid130————Minerals  Calcium88025107862  Phosphorum590————  Chloride310————  Sodium250————  Potasium600————  Magnesium140259174910203Proteinsd15.433.611.521.316.85Carbohidratesd101.521.167.47.428Fibred3.97.73.140.429.3Each control (non-supplemented) animal received a 100% of A04 maintenance diet (Panlab, Barcelona, Spain). Each supplemented animal received an 80% of A04 maintenance diet plus 20% of cereal fractions (B, C, D or E) naturally rich in polyphenols. B = wheat germ; C = buckwheat flour; D = fine rice bran; E = wheat middlings. ORAC (μTrolox Equivalents/g): B = 106; C = 81; D = 121; E = 49. Total polyphenols content (eq mg gallic acid/g): B = 11.15; C = 8.48; D = 15.24; E = 9.35. HF (mg/100g) = hydrosoluble fraction; LF (mg/100mg) = lyposoluble fraction; p-HB = p-hydroxybenzoic. amg/100 g. bmg/100 mg. cμg/100 g. dg/100 g Collection of peritoneal leukocytes Peritoneal suspensions were obtained between 8:00 and 10.00 h, without sacrificing the animals, following the method previously described [17]. Peritoneal leukocytes (macrophages and lymphocytes) were collected, identified, quantified by their morphology using optical microscopy (40×) and their concentration was adjusted depending on the function assayed [14, 18]. The cellular viability, determined in each experiment using the trypan-blue exclusion test, was in all cases higher than 95%. Leukocyte functions The chemotaxis assays were performed according to a technique previously described [14]. The results were expressed as chemotaxis index (C.I.) by counting the number of macrophages and lymphocytes at the lower face of the filters of the Boyden’ chambers [14]. The intracellular superoxide anion (O2·−) levels were evaluated as described elsewhere [17]. The results were expressed as nmol of O2·− per 106 cells. The lymphoproliferation assay and the determination of interleukin-2 (IL-2) levels were carried out following the methods previously described [14]. The proliferation results were expressed as percentage of stimulation by the mitogens Concanavalin A (Con A) or lipopolysaccharide (LPS) with respect to spontaneous proliferation that was considered to be 100. In the case of IL-2 the results were expressed as pg/ml. Leukocyte redox state Total glutathione and its oxidized form (GSSG) were spectrophotometrically evaluated according to [17]. The results are expressed as nmol/106cells. To calculate the GSSG/GSH ratio, the GSH (reduced form) was obtained by substracting the GSSG values from the total glutathione values. The secretion of mouse tumor necrosis factor alpha (TNFα) was measured in culture supernatants stimulated with LPS, using an ELISA kit (HyCult Biotechnology, Uden, The Netherlands). The results were expressed as pg/ml, being the minimum detectable value 8 pg/ml. Catalase activity (CAT) was carried out according to [19], slightly modified. Samples of peritoneal suspension (adjusted to 106 cells/ml) were resuspended in 50 mM phosphate buffer, sonicated and centrifuged at 3200 g (4°C) for 20 min. The reaction was initiated by the addition of H2O2 and spectrophotometrically measured at 240 nm (25°C) for 50 s. The results were expressed as U/106 cells. Malondialdehyde (MDA) levels were evaluated according to a technique previously described [20], slightly modified, and quantified by high performance liquid chromatography (HPLC). Aliquots of peritoneal suspension (adjusted to 106 cells/ml) resuspended in 50 mM KH2PO4 (Sigma) pH 6.8 were sonicated and added to a mixture reaction that contained 0.44 M ortophosphoric acid (H3PO4, Fluka, Steinheim, Germany), 2-thiobarbituric acid (0.6%), (TBA, Sigma) and 3 mM butylated hydroxytoluene (BHT, Sigma). It was heated at 95°C for 30 min and after cooling on ice, the samples were extracted adding n-butanol (Panreac), centrifugated at 13,000 rpm (4°C) for 5 min and 50 μl of supernatants (organic phase) were injected in the HPLC column (Novapack C18, 15 cm × 3.9 mm, Waters). As mobile phase 50 mM KH2PO4 pH 6.8/methanol (90/10 v/v) was used. The flow rate of the mobile phase was adjusted to 0.4 ml/min. MDA was monitored at λ = 532 nm in a WATERS 486 ultraviolet detector. Standard curves were prepared fresh daily using MDA bis (dimethyl acetal, Sigma). The results were expressed as nmol/106cells. Statistical analysis Statistical analysis was performed using the software SPSS 11.5 (Chicago, IL, USA). The data are expressed as the mean ± standard deviation (S.D.) of the eight values corresponding to the number of experiments. Each value is the mean of the data from assays performed in duplicate. The normality of the samples was confirmed by the Kolmogorov–Smirnov test and the homogeneity of variances by the Levene test. The data were analysed by the one-way analysis of variance (ANOVA) followed by the Tukey test. The Kruskall Wallis test for non-parametric data was used. The minimum level considered statistically significant was P < 0.05. Results Immune system response Figure 1 shows the chemotaxis capacity, lymphoproliferative response to the T-cell mitogen Concanavalin A (ConA) or the B-cell mitogen LPS and release of IL-2 of peritoneal lymphocytes from adult female mice supplemented for 5 weeks with cereal fractions naturally rich in polyphenols. Mice supplemented with cereals showed a strongly significant increase of lymphocyte chemotaxis compared with that of control mice (P < 0.001 with B, D and E and P = 0.031 with C) (Fig. 1a). As regards lymphoproliferative response to Con A (Fig. 1b), mice supplemented with any of the cereals B (P = 0.027), C (P = 0.002), D (P < 0.001) or E (P < 0.001) showed a significantly higher capacity compared with that of control mice. The supplementation with the cereals C (P < 0.001), D (P = 0.024) or E (P < 0.001), but not with B, was able to increase lymphoproliferation in response to LPS with respect to that of non-supplemented mice (Fig. 1c). The secretion of IL-2 by peritoneal leukocytes from mice supplemented with cereal B (P < 0.001), C (P = 0.004) or E (P = 0.006) was higher than that of control mice (Fig. 1d). The chemotaxis capacity of macrophages increased significantly in cereal E-supplemented mice in comparison with that of control mice (434 ± 39 versus 290 ± 42, P < 0.001). The intracellular superoxide anion (O2·−) levels in non-stimulated peritoneal leukocytes significantly increased after supplementation for 5 weeks with cereal B and C with respect to non-supplemented mice (Controls: 24 ± 5; B: 42 ± 7, P < 0.001; C: 41 ± 7, P < 0.001). The release of intracellular O2·− from peritoneal leukocytes stimulated with latex beads was significantly higher in mice supplemented with cereals B, C and E (Controls: 52 ± 5; B: 63 ± 4, P = 0.045; C: 67 ± 8, P = 0.002 E: 64 ± 7, P = 0.021). Fig. 1(a) Chemotaxis capacity of peritoneal lymphocytes, (b) lymphoproliferation in ConA (1 μg/ml)-stimulated 48 h-culture supernatants, (c) lymphoproliferation in LPS (1 μg/ml)-stimulated 48 h-culture supernatants and (d) release of interleukin-2 (IL-2) in supernatants of 48 h-cultures of peritoneal leukocytes stimulated with Con A (1 μg/ml) from adult female ICR (CD-1) mice supplemented for 5 weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean ± SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P < 0.05 **P < 0.01 and ***P < 0.001 versus the control group. Lymphoproliferation in response to both mitogens, Con A and LPS, was expressed as % of stimulation with respect to spontaneous proliferation, the latter considered as 100. Spontaneous lymphoproliferation (in counts per min): Control = 1245 ± 253; B = 1348 ± 239; C = 1071 ± 171; D = 1186 ± 166; E = 1052 ± 169 Cellular redox state The content of oxidized glutathione (GSSG) and the GSSG/GSH ratio, the levels of TNFα, CAT activity and MDA levels in peritoneal leukocytes are shown in Fig. 2. All cereal fractions studied decreased significantly the GSSG levels (P < 0.001) compared to those of control mice (Fig. 2a). The GSSG/GSH ratio (Fig. 2b) was significantly decreased after supplementation with any of the cereals B (P < 0.001), D (P = 0.002) or E (P < 0.001) compared with that of non-supplemented mice. Supplementation for 5 weeks with any of the cereals did not cause changes in the GSH levels of peritoneal leukocytes with respect to the values of controls (2.29 ± 0.23). The secretion of TNFα decreased in leukocytes from mice supplemented with cereal C (P = 0.005), D (P = 0.007) and E (P = 0.004) in comparison with that of non-supplemented mice (Fig. 2c). The CAT activity of peritoneal leukocytes from mice supplemented with cereal C (P < 0.001), D (P = 0.003) and E (P = 0.040) was higher than the activity of this antioxidant enzyme in control mice (Fig. 2d). Finally, the lipid oxidative damage in peritoneal leukocytes, evaluated as MDA levels (Fig. 2e), was lower (P < 0.001) in mice supplemented for 5 weeks with cereals than in the control mice. Fig. 2(a) Oxidized glutathione (GSSG) content, (b) GSSG/GSH ratio, (c) tumor necrosis factor (TNFα) levels in LPS-stimulated (1 μg/ml) culture supernatants, (d) catalase (CAT) activity and (e) malondialdehyde (MDA) levels in peritoneal leukocytes from adult female ICR (CD-1) mice supplemented for 5 weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean ± SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P < 0.05 **P < 0.01 and ***P < 0.001 versus the control group Discussion It is widely accepted that antioxidant compounds may help to protect against oxidative stress, with resulting health promotion and disease prevention [21]. Moreover, an adequate intake of these compounds can enhance certain aspects of immune function, which is related to health [4]. Thus, it is becoming clear that diet supplementation with appropriate amounts of antioxidants enhances the immune function and improves the redox state of unhealthy subjects [15]. However, whether antioxidant supplementation has these effects in healthy subjects, still remains unknown. In the present study the cereal fractions used were able to improve several aspects of immune function. Thus, important events of innate immune response, i.e., migration capacity (chemotaxis) and microbicidal activity (intracellular superoxide anion levels), appear to be effectively affected by the supplementation with cereals naturally rich in polyphenols. Akbay et al. [22] showed that the three flavonoid glycosides from Urtica dioica L., quercetin-3-O-rutinoside, kaempherol-3-O-rutinoside and isorhamnetin-3-O-glucoside increased, in vitro, the chemotaxis and the intracellular killing activity of neutrophils, using methods similar to those of the present work. In our study, the fraction E is only able to enhance macrophage chemotaxis, whereas all the cereal fractions were able to show a strong stimulatory effect on this function in lymphocytes. This fact suggests that the polyphenols present in cereal fractions could exert a cell-dependent effect on migration capacity. Since a higher NF-κB expression is linked to an increased production of migratory inhibitory factor (MIF), thus being responsible for a decreased migration capacity [13], and several polyphenols are able to inhibit NF-κB activation [23], it seems plausible that some of the polyphenols present in the cereals used in our study could enhance the chemotaxis capacity through their action on NF-κB activation. All cereal fractions used in the present work, with the exception of D, have been shown to increase the microbicidal activity of peritoneal leukocytes. Since p-hydroxybenzoic acid is not present in fraction D, this compound could be responsible for that effect. However, to date, there is no evidence of a microbicidal activity increase following p-hydroxybenzoic acid administration. Lymphoproliferation is a crucial event of the acquired immune response. Several studies have demonstrated a strong stimulatory effect on this capacity and on the cytokine well-known as enhancer of proliferative response, namely the IL-2, after antioxidant supplementation. Thus, Bub et al. [4] showed an increase in both functions in peripheral blood mononuclear cells (PBMC) from healthy subjects after diet supplementation with fruit juices rich in polyphenolic compounds. In ethanol-fed mice, Cheshier et al. [24] demonstrated an increased IL-2 production by mitogen-stimulated splenocytes after in vitro administration of Pycnogenol®, a commercial combination of phenolic compounds from pine bark. To date, no studies on the effect of the isolated polyphenolic compounds of the cereals used in the present research on these functions have been carried out. Glutathione (GSH) plays important roles in antioxidant defence, nutrient metabolism and regulation of many cellular events, and its deficiency contributes to the oxidative stress-related pathogenesis of several chronic diseases [25]. Both the oxidized glutathione (GSSG) content and the GSSG/GSH ratio, the latter being considered a sensitive marker of cellular oxidative stress [26], were significantly decreased after supplementation with all the cereal fractions, although fraction C only showed a tendency to diminish the GSSG/GSH ratio. However, the GSH content of peritoneal leukocytes was not changed after cereal-based supplementation, suggesting that the lower GSSG/GSH ratio observed is primarily due to diminutions of the GSSG content. Rosenblat et al. [27] demonstrated that isoflavonoid glabridin (25 μg/kg/day) supplementation was able to reduce the GSSG content in murine peritoneal macrophages. However, the same authors also observed increased GSH levels in these immune cells. Tumor necrosis factor is one of the most common proinflammatory cytokines produced predominantly by macrophages that has been reported to increase in many oxidative stress-related chronic diseases [28]. Our study showed that each of the cereal fractions, except B, was able to decrease the secretion of TNFα by peritoneal leukocytes. In agreement with Wang and Mazza [29], catechin at a concentration of 31 μM inhibited the TNFα production of LPS/IFNγ-activated RAW 264.7 macrophages. However, our results suggest that p-coumaric acid could also have an important anti-inflammatory role because this common phenolic acid is present in each cereal fraction, except the B. Moreover, in rats with endotoxemia, two different doses (75, 150 mg/kg, i.p.) of a water extract of propolis, a natural product rich in p-coumaric acid, were able to inhibit TNFα production in plasma [30]. Nevertheless, other authors did not observe any effect on TNFα production in whole blood cultures in the presence of phenolic compounds like p-coumaric acid at the concentrations of 10−7–10−4 M [31]. The most probable mechanism triggered by phenolic compounds for blocking LPS-induced production of TNFα by macrophages could be the inhibition of NF-kB activation [23]. Catalase plays a fundamental role in protecting cells against oxidative damage [32]. Decreased CAT activity may compromise the overall enzymatic defence system. In the present study, each of the cereal fractions increased CAT activity in peritoneal leukocytes, although fraction B only showed a tendency to increase this enzymatic activity. In other study, the renal CAT activity was significantly increased in rats fed black rice, one of the most important cereals rich in phenolic acids [33]. Khan et al. [34] showed that following oral feeding (0.2% w/v) for 30 days of polyphenols from green tea, a popular beverage rich in catechin, CAT activity significantly increased in the small bowel, liver and lungs of mice. In fact, fractions C and D (the fractions rich in catechin) showed the greatest effect. The present data suggest that p-coumaric acid, which is present in almost all the cereal fractions (C, D and E) might also have a relatively important role in modulating the CAT activity. Indeed, an oral administration of p-coumaric acid (100 mg/kg) for five days ameliorated the CAT activity in the heart from rats treated with doxorubicin, an anticancer antibiotic [35]. This potential protective action of p-coumaric acid is likely to be due to its free radical scavenging power. ROS generation is able to promote oxidative damage to fatty acids present in biological membranes via an autocatalytic process known as lipid peroxidation. Malondialdehyde is one of the end-products of this process [36], which is related to the onset of chronic diseases [37]. In the present work, all the cereal fractions strongly decreased the MDA levels in peritoneal leukocytes. It has been observed that highly reactive and destructive hydroxyl radicals are effectively scavenged by oral administration of p-coumaric acid (317 mg/day) for 30 days in rats [38], which might decrease the potential damage to biological membranes by lowering MDA production. Several authors have shown that rutin, quercetin or catechin, which are some of the flavonoids present in the cereals used in the present work, are able to decrease lipid oxidative damage [39, 40]. The same happens with ferulic acid, the most common phenolic acid in cereal cell walls, which inhibit peroxidation [41]. Red wine polyphenols, together with ascorbic acid, inhibit lipid peroxidation in human muscle tissue [42]. The same authors demonstrated that, in the presence of catechin, ascorbic acid at pH 3.0 works in a sinergystic way preventing lipid oxidative damage and β-carotene cooxidation. This fact might explain the central role that consuming natural products rich in polyphenolic antioxidants, like cereals, has in promoting health and preventing the onset of chronic diseases. The present data demonstrate the importance of polyphenol-rich cereal consumption because not only has shown to be very effective for modulating several parameters of immune function, but also is able to improve to a large extent the redox state of immune cells from mice. More importantly, we have demonstrated that cereal-based antioxidant supplementation can exert beneficial effects in healthy mice and thus we suggest that normal healthy populations may benefit from this supplementation. Indeed, a recent study has provided evidence for potential protective effects of moderate consumption of polyphenol-rich red wine in healthy subjects [43]. It has been reported that subjects susceptible to infections or suffering from chronic inflammatory processes showing an impairment of function and redox state of immune cells likely to benefit from antioxidant administration [13, 14]. However, it is difficult to know whether there is changes or not in the healthy mice from the present study may be a reliable predictor for the nutrient-immune requirements in disease-associated, unhealthy mice. To confirm this possibility we should know the optimal combination and quantities of nutrients, the potential interactions that might occur between these nutrients and the most appropiate time at which that immunonutrition should be provided. In words of Berger [15], “the efficiency of supplementation is a question of timing. Antioxidant nutrients cannot cure an installed disease, but they may prevent its promotion”. It is important to point out that the cereal fractions used by us contain other compounds, both nutrients and non-nutrients, such as vitamins, minerals or fibre and, therefore, we cannot rule out that they can result in health benefits. However, the present cereal fractions show a good correlation coefficient between total antioxidant capacity and content of phenolic compounds, thus suggesting that polyphenols are largely responsible for the antioxidant activity of the different cereal fractions, which ultimately may help to protect from oxidative stress and, consequently, can decrease the risk of many potential diseases. Nevertheless, it is important to take into account some issues. First, the doses used play a crucial role, since it is known that excessive amounts of antioxidant compounds can impair the immune function [44] or even becomes potentially prooxidants [45]. Yet, we have not used “megadoses”, but nutritional doses of polyphenols. Second, the present recommendation is not a single polyphenol-based supplementation to modulate the antioxidant status or the immune system, but it is the combination of different substances as the best way to provide protective action [46, 47]. Therefore, the cereals used by us contain a complex natural mixture of polyphenols of different size, polarity and solubility. Third, we can speculate that one of the reasons for the protective effects of cereals could be that the metabolism of polyphenols carried out by the gut microflora [48] is able to provide immune protection beyond the gut. Fourth, it should not be ruled out the possibility that synergistic and/or additive effects occur. Several studies have shown that polyphenols can work synergistically with other antioxidant compounds [42, 49]. Thus, it is of great physiological importance that many polyphenols of different solubility will interact in the body cells and compartments, with antioxidants recharging adjacent antioxidants in an integrated manner [50]. Finally, we consider the present nutritional intervention in animals as preventive, but not therapeutic, since it is able to maintain the normal functional and redox status of the body cells from a general population of mice. Therefore, we do not deliver a cereal-based antioxidant supplement to unhealthy subjects, i.e., in conditions caused by excessive free radicals generation. In conclusion, we strongly believe that polyphenols naturally present in cereals may have important implications for health preservation, by acting, al least partly, as modulators of immune function and redox state. Although we emphasize the importance of further studies on this subject, we suggest the use of this type of supplementation for the general population since “prevention is a much more effective approach that treatment of disease”.

Psychopharmacologia-3-1-2080349

Medial prefrontal serotonin in the rat is involved in goal-directed behaviour when affect guides decision making

Rationale Across species, serotonin (5-HT) depletion in the prefrontal cortex (PFC) has been shown to cause impaired performance on tests of cognitive flexibility and the processing of affective information (e.g. information with an ‘emotional’ content). While recent work has explored the specific role of the orbital PFC herein, the role of the medial PFC remains unclear. Introduction Certain aspects of cognitive flexibility (i.e. the ability to adapt behaviour, see for instance, Fuster 1980; Kolb 1984; De Bruin et al. 1994; Dalley et al. 2004) depend critically on central serotonin (5-HT). Recent experiments in humans with tryptophan depletion, a method resulting in a transient decrease in 5-HT synthesis, show impairments in the ability to learn changed stimulus–reward associations (Rogers et al. 1999) and in the performance on tests of cognitive flexibility (Rubinsztein et al. 2001). Lesion studies in non-human primates corroborate these findings, showing that it is the 5-HT innervation of the prefrontal cortex (PFC) that is critical for adapting behaviour when stimulus–reward contingencies are reversed in a two-choice discrimination (reversal learning; Clarke et al. 2004, 2005). Similar observations have been made after central 5-HT depletion in rats, indicating that 5-HT depletion leads to impaired flexibility (Harrison et al. 1999), increased perseverative responding (Beninger and Phillips 1979; Morgan et al. 1993) and impaired impulse inhibition (Winstanley et al. 2004, 2006). In addition to effects on cognitive flexibility, tryptophan depletion has also been reported to lead to impairments in affective processing (i.e. the ability to evaluate and integrate emotional content, see for instance, Hariri et al. 2006) and reward discrimination (Rubinsztein et al. 2001; Rogers et al. 2003; see also Roberts and Wallis 2000). Such changes in affective processing (see also Remijnse et al. 2005) have, however, not yet been reported in studies involving selective prefrontal 5-HT lesions in either primates or in rats. Elucidation of the neural substrates underlying this special role of 5-HT in affective processing is one aim of the current study. A second issue that is central to studies of the PFC in cognitive flexibility is that of specific contributions of the different cortical sub-areas. Evidence for a functional dissociation within the PFC regarding cognitive flexibility comes from studies across species, and it is important to note that the functional differentiation (specificity) of the anatomical subdivision of the rat brain PFC is, to a large extent, comparable to that of the primate PFC (Uylings et al. 2003). In terms of homology between the primate and rodent PFC, we believe, given the data reviewed in Uylings et al. (2003), that the rat medial PFC (mPFC) shows a functional overlap with both lateral and medial frontal regions of the primate brain. The orbital PFC has been shown to support reversal learning in both primates (Iversen and Mishkin 1970; Butters et al. 1973; Dias et al. 1996; Clarke et al. 2004; Kringelbach et al. 2003; Hornak et al. 2004) and rats (Schoenbaum et al. 2002; Chudasama and Robbins 2003; McAlonan and Brown 2003) and is implicated in the coding of reward-related information in non-human primates (Hikosaka and Watanabe 2000; Tremblay and Schultz 2000; Schultz 2006), rats (Schoenbaum et al. 2003; Bohn et al. 2003) and humans (O’Doherty et al. 2001; Kringelbach 2005). The mPFC, on the other hand, supports rule switching and attentional set shifting in rats (De Bruin et al. 1994; Joel et al. 1997; Ragozzino et al. 1999a,b; Birrell and Brown 2000). This functional dissociation between reversal learning and attentional set shifting is also observed in primates, in which the latter is dependent on the lateral PFC (Dias et al. 1996; see also Robbins 2005). However, this particular distinction between PFC functions might be rather specific to visual- and odour-based learning in rats (Chudasama and Robbins 2003; Schoenbaum et al. 2002) and primates (Clarke et al. 2005, 2007), as several groups have reported an involvement of the mPFC in reversal learning using spatial discriminations (Kolb et al. 1974; Li and Shao 1998; Salazar et al. 2004; De Bruin et al. 2000). Furthermore, the mPFC has been shown to be involved in the initial processing of reward-related information, as recording of single neuron activity indicated this area to be involved in the coding of reward information in relation to spatial cues (Hok et al. 2005). From this, we conclude that the mPFC may have a specific role in spatial reversal learning. On a more general level, these findings have been paralleled and extended by human studies. Knutson et al. (2005), for example, reported on mPFC-mediated reward coding, and evidence for a mPFC involvement in implementation of that (reward) knowledge in existing stimulus–reward contingencies comes from Ridderinkhof et al. (2004). Given the fact that 5-HT is involved in reversal learning supported by the orbital PFC (see above) and that 5-HT lesions lead to functional deficits such as perserverative responding and loss of impulse inhibition (Beninger and Phillips 1979; Soubrié 1986; Harrison et al. 1999; Dalley et al. 2002; Winstanley et al. 2004, 2006), which are also observed after lesioning of the mPFC (Morgan et al. 1993; Passetti et al. 2002; Salazar et al. 2004), we feel that investigation of the involvement of 5-HT in the mPFC in spatial reversal learning is warranted. This forms the second main aim of these experiments. Given the primate literature on the involvement of 5-HT in affective processing and of the mPFC in the implementation of reward information, we hypothesise that the medial prefrontal 5-HT is selectively involved in cognitive flexibility when affect guides decision making (i.e. when ‘emotional’ content drives decision making). Moreover, based on the ‘reversal’ literature, we hypothesise that 5-HT in the mPFC is especially important for successful behavioural adaptation when the stimulus modality is spatial but not odour-based. To test our hypotheses, we conducted an experiment in which we selectively destroyed 5-HT terminals in the mPFC of male Wistar rats by means of local infusion of the toxin 5,7-dihydroxytryptamine (5,7-DHT). Both sham controls and lesioned animals were then assessed on a series of behavioural tests in which functions thought to underlie cognitive flexibility and affective processing were addressed (De Bruin et al. 2000; Schoenbaum et al. 2006). To explore the hypothesis that mPFC 5-HT is involved in affective processing, we tested the impact of altered reward value on reversal learning. For half of the sham and control animals, we switched the reward presented after a correct response from non-preferred reward pellets to preferred pellets at the time of the first reversal. Our expectation was that the introduction of this affective shift, on top of the reversal, would cause lesioned animals to show altered task performance because of an inability to accurately process the altered reward information. For the hypothesis that stimulus modality determines specific involvement of the mPFC, we tested reversal learning across different stimulus modalities. In line with our studies on mPFC lidocaine inactivation (De Bruin et al. 2000), we subjected the animals to a test of spatial discrimination and reversal learning. In addition, we included a second, operant, odour-based go/no-go reversal task. Based on our hypothesis regarding selective involvement of the mPFC in spatial learning, we expected impaired reversal learning in the spatial but not in the odour-guided task in lesioned animals. To gain more insight in the mechanisms that might underlie impaired cognitive flexibility, we tested the effect of the lesion on extinction learning and impulse inhibition, and we assessed the animals’ behaviour during extinction of the ‘spatial’ task and responding on ‘go’ and ‘no-go’ trials in the ‘odour’ task. Materials and methods All experiments were approved of by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences and were carried out in agreement with Dutch Laws (Wet op de Dierproeven 1996) and European regulations (Guideline 86/609/EEC). Subjects Subjects were 32 male outbred Wistar rats (Harlan/CPB, Horst, The Netherlands), weighing 175–200 g upon arrival. The animals were socially housed in groups of four in standard type IV Macrolon cages, where they were kept under a reversed day/night cycle (dimmed red light from 7 a.m. until 7 p.m., white light from 7 p.m. until 7 a.m.) for the duration of the experiment. After surgery, the animals were food restricted (16 g/animal per day) to maintain their body weight at 90% of free-feeding weight. Water was available ad libitum throughout the experiment. Surgery Two weeks after arrival, the animals were randomly assigned to either the experimental lesion groups or the sham groups and subjected to surgery. The aim was to selectively destroy the serotonergic (5-HT) innervation of the mPFC. To prevent loss of dopaminergic (DA) and noradrenergic (NA) terminals in the target area because of the treatment, all animals were intraperitoneally injected with 20 mg/kg Desipramine HCl (Sigma, Zwijndrecht, The Netherlands) dissolved in MilliQ water (10 mg/ml) 30 min before surgery. The weight of the animals at the time of surgery was 247–289 g. Anaesthesia was induced with 5% isoflurane in oxygen and maintained for the duration of the surgery with 2% isoflurane. For the surgical procedure, the animals were mounted in a stereotactic frame with the toothbar set at −2.5 mm. Bilateral stainless steel cannulas (outside diameter 0.3 mm), connected to a microinfusion pump (801 syringe pump, Univentor, High Precision Engineering, Zejtun, Malta) with flexible PEEK tubing (0.51 mm outside diameter, 0.013 mm inside diameter; Aurora-Borealis, Schoonebeek, The Netherlands), were then placed in the mPFC at an angle of 12°, anterioposterior +30, lateral ±16; ventral −40 (Paxinos and Watson 1998). After cannula placement, 0.5 μl of either 5,7-DHT (5,7-DHT creatinine sulphate salt, Sigma; 32 μg/μl, dissolved in 0.1% ascorbic acid) or vehicle was infused into the target area over a period of 1 min. After infusion, the cannulas were kept in place for an additional 2 min to allow for diffusion. The wound was closed with surgical stitches after the cannulas were removed from the brain. Post-operative pain reduction was achieved with Finadyne (flunixin meglumide 50 mg/ml, Schering-Plough, Segré, France), 0.01 ml/100 g body weight subcutaneously, approximately 2 h after surgery. After surgery, the animals were returned to their home cages. Apparatus Two behavioural tasks were used. For the first (two-lever discrimination) task, locally constructed operant chambers were equipped with a set of two retractable levers positioned to the left and right of a food dispenser (distance lever–feeder, 10 cm). A house light was placed on the opposite wall, two lights were positioned above the levers, signalling their presentation, and one inside the food dispenser, signalling the delivery of a 45 mg food pellet (Noyes Formula AI or P, Research Diets, New Brunswick, NJ). Nose pokes were detected by an infrared sensor located inside the food well. Operation of the Skinner boxes was controlled by a personal computer running the Med-pc™ software (Med Associates, Sanddown Scientific, Middlesex, UK). For the second (odour-based go/no-go) task, commercially available operant chambers (Med Associates, Sanddown Scientific) were equipped with a retractable lever and an odour-sampling unit positioned on opposing walls (spaced 34 cm apart), allowing the presentation of distinct (non-aversive natural) odours. A food dispenser, from the same company, was positioned next to the odour-sampling port. Trial lights indicated odour, lever and reward presentation. Pellet rewards To manipulate the affective value of the rewards, we used two different types of pellet rewards (Noyes ™ AI and P; for additional information see www.researchdiets.com) that were shown to be differentially preferred by the animals (see “Pellet preference”). Before the start of the experiment, all animals were familiarised with both types of pellets in their home cage. During the reversal phases of both tasks, the preferred pellet reward was only available to half of the animals (for groups, see below). Pellet preference To establish whether male Wistar rats showed preference for a specific food pellet, we pre-tested three types of rewards (Noyes ™ formula AI, P, and FP; see Fig. 1) in a separate group of eight animals. The testing apparatus was a standard-size T-maze with trap doors in which both arms were baited. Five daily habituation sessions were given during which the animals were allowed to freely explore the entire T-maze and familiarise themselves to the position of the two different rewards that were tested that day. Subsequently, the animals were given the choice between the two arms, each baited with one of the three types of reward (15 trials). In these trials, the trap door was lowered after an arm entry was made to prevent the rat from entering the second arm. All three possible combinations of pellets were given over 3 consecutive days to all rats. To avoid ‘sequence’ effects, the rats were pseudo-randomly assigned to a particular pellet order. The animals were housed and food restricted as the experimental animals. Table 1Results of HPLC analysis of sham and 5-HT-lesioned animals in seven brain areas Treatment5-HTDANAmPFC—15,7-DHT63.2 ± 8.6 (9.1%)**35.5 ± 7.1 (66.9%)417.6 ± 41.1 (66.6%)**Sham691.5 ± 119.553.1 ± 8.0627.0 ± 56.8mPFC—25,7-DHT68.8 ± 14.9 (28.0%)**60.9 ± 11.1 (93.0%)446.3 ± 41.8 (104.8%)Sham245.3 ± 46.565.5 ± 5.0425.8 ± 25.4mPFC—35,7-DHT42.5 ± 6.9 (18.0%)*71.1 ± 19.1 (70.3%)272.6 ± 23.0 (74.1%)Sham236.0 ± 70.1101.1 ± 33.7367.7 ± 48.0olPFC—15,7-DHT319.1 ± 125.2 (107.2%)11.3 ± 1.6 (87.5%)239.4 ± 15.7 (93.4%)Sham297.6 ± 28.012.9 ± 1.8256.4 ± 13.0olPFC—25,7-DHT175.6 ± 40.5 (80.4%)23.7 ± 3.4 (74.2%)293.6 ± 42.9 (126.6%)Sham218.3 ± 32.332.0 ± 4.5231.8 ± 14.0olPFC—35,7-DHT278.2 ± 96.5 (107.7%)77.1 ± 14.0 (94.7%)235.5 ± 19.7 (93.5%)Sham258.2 ± 25.681.4 ± 9.8251.9 ± 20.4M-25,7-DHT138.7 ± 19.2 (72.1%)*53.4 ± 6.9 (74.6%)199.4 ± 14.7 (112.3%)Sham192.3 ± 15.171.5 ± 10.2177.5 ± 9.5Tissue concentrations of serotonin (5-HT), dopamine (DA) and noradrenaline (NA) in three adjacent (anterior–posrterior) medial prefrontal (mPFC 1–3) and orbital prefrontal (olPFC 1–3) areas, and control area (M-2) in sham (n = 16) and 5,7-DHT- (n = 14) lesioned animals. Group averages (±SEM) are presented in ng/g*Significant group differences p < 0.01**Significant group differences p < 0.05 Experimental procedure Approximately 9 days after surgery, a period that a pilot experiment had indicated to be necessary to achieve maximum 5-HT depletion (data not shown), the behavioural experiments started. On experimental days, which typically lasted from 9 a.m. to 5 p.m., the animals were transferred to the experimental room, where they remained in their home cages until testing. In both the animal facility and the experimental room, a radio played to mask background noises. Before the behavioural testing, both lesion and sham groups were further divided to create a total of four groups. Two groups, one lesion and one sham, received non-preferred pellets throughout the experiment, and two groups started receiving non-preferred pellets but only received preferred pellets from the start of the first reversal. The two behavioural tasks were applied consecutively in the same order to all animals. At the start of the second task, all animals received non-preferred pellets again. Behavioural task 1. Two-lever discrimination task. (weeks 1–4) This task was used to assess the effects of mPFC 5-HT depletion on the acquisition of an operant response, discrimination between two spatial stimuli and reversal and extinction of the acquired responses (De Bruin et al. 2000). First, the animals were trained, in a maximum of nine (64 trials) shaping sessions, to press a lever for a food reward. During this shaping phase, a single lever was presented at each trial (randomly the left or right lever). In the course of the sessions, the animals were required to increase the number of lever responses per trial from a fixed ratio (FR) of one to three responses to obtain a reward. All of the trials in the two-lever spatial discrimination and reversal task (including shaping) were discrete choice trials; that is, the lever(s) were retracted upon responding or, in case of an omission, at the end of the trial duration (60 s). The inter-trial interval was set at 25 s. Thereafter, a second lever was introduced, and the animal had to learn to press either the left or right lever to obtain a food reward (two-lever discrimination). Pressing the incorrect lever or omitting a response (i.e. not responding within 60 s) led to trial termination and an inter-trial interval timeout. After reaching criterion, i.e. obtaining 90% of the food pellets in a session, the animals were subjected to serial reversals. For two of the four groups, this coincided with the switch from non-preferred to preferred pellets. During this reversal phase, the rewarded and non-rewarded levers were reversed daily for 4 consecutive days. The final phase was an extinction phase in which both levers were presented but none were rewarded. On both the two-lever discrimination and the serial reversal tests, the animals were tested twice daily: a morning and an afternoon session. The extinction phase consisted of a single daily session for 4 consecutive days. All sessions comprised eight blocks (with eight trials each), which were divided for analysis into two sets of four blocks. Session duration ranged from 45 min (two-lever discrimination) to 2 h (extinction). Behavioural task 2. Odour go/no-go inhibition task (weeks 5–9) This task was used to assess the acquisition of a two-odour discrimination, the inhibition of lever pressing and the reversal of the acquired discrimination. In this task, the animals were first trained to sample an odour in an odour port and subsequently press a lever once for a food reward (FR1). This was achieved in four (30 trial) sessions over 2 days. Once they acquired these two operant behaviours, they were trained in six (30 trial) daily sessions to discriminate between two odours, one indicating a ‘go’ trial in which the animal was required to press the lever to obtain a food reward and another odour that signaled a ‘no-go’ trial in which the animal had to withhold pressing the lever to obtain a food reward. Failure to press the lever on a ‘go’ trial or inhibit a lever response on a ‘no-go’ trial, as well as a failure to sample the odour or respond within 60 s (omission), triggered a 30-s inter-trial timeout during which the house light was turned off and no pellet was rewarded. The final phase was a single reversal in which the stimulus–response contingencies were switched and the animals were required to reverse their previously acquired go/no-go responses. For two of the four groups, this coincided with the switch from non-preferred to preferred pellets. In this final phase, the animals were tested in 24 (45 trial) daily sessions over 12 days. The number of trials per session during this phase was increased to 45 to facilitate task acquisition. Behavioural measures Pellet preference test The mean cumulative number of arm entries/pellet choices per rat were grouped and compared over all, 15 trials, sessions. General activity To assess the possible effects of the lesion on general activity, the groups were compared regarding average number of head entries into the food dispenser and lever presses per block. These measures were taken during testing on the two-lever discrimination task. To exclude the influence of performance on these activity measures, data from the last acquisition session (both sets) were used. In this session, the performance of both groups was stable and statistically identical. Tests of cognitive flexibility The data obtained from both behavioural tasks were taken as measures of performance in the following fashion: the amount of obtained rewards as a fraction of the amount of obtainable rewards was taken as an index of overall performance on all test phases except the extinction phase of the two-lever discrimination. For this phase, the total number of lever responses was taken as the index, and, as this measure is prone to interference from pre-existing differences between groups in the total number of lever presses, the amount of lever presses per block of eight trials was expressed as a percentage of the mean number of lever presses per group in the same block on the previous day. Response accuracy as an index of the amount of response errors was scored as the number of correct lever presses relative to the total amount of lever presses. This measure, together with the number of lever omissions, was taken to explain sub-optimal performance. Performance on the two-odour go/no-go task was further divided into success and failure of responding on the ‘go’ trials and the ‘no-go’ trials as a measure of inhibition. The effects of reward type (preferred vs non-preferred pellets) on reversal learning were measured by comparing performance scores of the reversal phase of both groups. The success of task acquisition of any test phase was established by calculating the aforementioned performance. In case of the extinction phase, the success on the test was measured by the total number of lever presses. Lesion evaluation Neurochemistry After all experimental procedures were completed, the animals were numbed with a mixture of O2/CO2, after which they were decapitated. Dissection of the brain and removal of the regions of interest immediately followed decapitation. Both the medial (prelimbic, infralimbic and anterior cingulate cortex) and orbitolateral (ventral orbital, ventrolateral orbital, lateral orbital, agranular insular) PFC were completely removed in three adjacent (anterior–posterior from frontal pole to genu of the corpus callosum) slices as well as a single control area (motor cortex, M-2; Paxinos and Watson 1998; Uylings et al. 2003). After dissection, all samples were stored in −80°C until analysis. All medial and orbitolateral PFC areas were analysed separately to gain more insight into the spread of the lesion. Tissue from both hemispheres were, however, analysed together. Tissue samples were weighed and homogenised in ice-cold 0.1 mol/l perchloric acid and centrifuged for 15 min at 14,000 rounds/min. After centrifuging, the unfiltered supernatants were transferred to an autosampler and 20-μl aliquots were injected onto a column for high-performance liquid chromatography (HPLC) analysis (Waters 600E pump and Waters 717plus autosampler, Waters Chromatography b.v., The Netherlands; Decade VT-03 electrochemical detector, Antec Leyden, The Netherlands) and Shimadzu Class-vp™ software (version 5.03, Shimadzu Duisburg, Germany). The mobile phase consisted of 0.06 mol/l sodium acetate, 9 mmol/l citric acid, 0.37 mmol/l heptanesulphonic acid and 12.5% methanol. The flow rate was kept constant at 0.65 ml/min. Separation of 5-HT, DA and NA from other components was achieved with a Supelcosil column (LC-18-DB 25 cm × 4.6 mm × 5 μm), with a 2-cm guard column of the same material (Supelco Superguard ™, Supelco, USA) kept at a constant temperature of 28°C. Quantification was achieved by means of electrochemical detection (oxidation potential set at +0.65 V). The transmitter/metabolite content was calculated against a calibration curve of external standards. The lowest detectable concentration in the supernatant of our main transmitter of interest, 5-HT, was 800 pg/ml, at a signal to noise ratio of 2:1. Data analysis Pellet preference The data of the pellet-preference experiment were analysed using a Friedman rank-order test with the cumulative number of choices per pellet as rank sums. Post-hoc testing consisted of two-group Mann–Whitney U comparisons. p values (two-tailed) were set at p < 0.01. Behavioural tasks 1 and 2 Behavioural data taken during these experiments were analysed using SPSS for Windows (version 11.0; SPSS, Gorinchem, The Netherlands). Shaping data from both behavioural tasks were analysed as the number of trials needed to reach a performance level of greater than 90%. Acquisition and reversal data obtained from both behavioural tasks were arcsine transformed to reduce the interference from ceiling effects (Microsoft Excel, Seattle, WA). The data of the extinction phase of the two-lever discrimination, where no ceiling effect was present, were not transformed. Based on previous results, in which effects of local mPFC inactivation were often observed only in one half of a learning session, we divided the data of each session of the two-lever discrimination task in two sets of four blocks, each consisting of eight trials (De Bruin et al. 2000). In contrast, data from the go/no-go task execution were analysed between sessions and not between sets or blocks because the observed learning in this task was slow and group differences spread out over multiple days. Because of technical problems, some data points were lost. For the data of both tasks, a repeated-measures analysis of variance (ANOVA) was used with treatment (control/lesion) and pellet (AI/P) as ‘between-subjects’ factors and block as the ‘within-subjects’ factor. When a ‘between-subjects’ effect was found, a one-way ANOVA and Student–Newman–Keuls (SNK) test was used to test for group differences. For the shaping and acquisition phases (which preceded the introduction of the different pellets), the behavioural data of both control groups and lesion groups were combined. This was done to increase power and because both control and lesion groups received the same pellet. In case a treatment effect was found, an independent-samples t test was used as the post-hoc test. Activity measures over the last acquisition session were analysed in a similar fashion. Neurochemical data Transmitter tissue content was analysed against a calibration curve of external standards. Group comparisons were made using one-way ANOVA, and post-hoc testing was done with an independent-samples t test. Results of the 5,7-DHT mPFC lesion experiment Pellet preference Figure 1 shows the mean cumulative scores or arm entries for the three pellet types tested. Comparison of rank sums using a Friedman rank-order test revealed significant group differences (F(9,2) = 12.667, p = 0.002). Repeated post-hoc Mann–Whitney U tests revealed a food preference for pellet type ‘P’ over pellet types ‘FP’ and ‘AI,’ p < 0.001. Fig. 1Pellet preference test. Rats were tested over 3 consecutive days in a T-maze for pellet preference. Mean cumulative number of arm entries/pellet choices is displayed. Asterisk, different from ‘AI’ and ‘FP,’ p < 0.001 Neurochemical examination Postmortem tissue analysis of brain tissue revealed that 5-HT levels were reduced in lesioned animals compared to control rats in all mPFC areas. Table 1 summarises the results. Spread to neighbouring areas was limited to a small but significant reduction of 5-HT in M-2. Pre-surgery desipramine administration prevented significant loss of DA terminals in all inspected areas, although reductions in both medial and orbital areas were observed. A modest but significant reduction in NA was found in the most frontal mPFC area (med-1). Two animals were excluded from the experiment and further data analysis for showing insufficient loss of 5-HT in the mPFC (i.e. less than 30%) or loss of transmitter in neighbouring areas (i.e. more than 50% of both DA and NA). Body weight and general activity Post-surgery body weight did not differ between the lesion group and animals that underwent sham surgery (data not shown). The behavioural measures, head entries into the food dispenser and average number of lever presses, taken to assess overall activity of the treatment groups, did not differ, respectively (F(1,28) = 0.719, p = 0.404 and F(1,28) = 0.001, p = 0.977). Table 2 shows the averages for both parameters. Table 2General activity measures ShamLesionHead entries into food dispenser15.48 ± 1.4317.38 ± 1.76Lever presses26.71 ± 0.7126.73 ± 0.39The average number (±SEM) of head entries into the food dispenser and the average number of lever presses for sham control and lesioned animals. No statistical differences of the lesion on these parameters were observed. Behavioural analysis Effects of mPFC 5-HT depletion on the two-lever discrimination task Shaping During the initial shaping phase, the animals learned to press a lever for a food reward. No differences between any of the groups were observed on number of trails needed to reach criterion (i.e. >90% of pellets obtained; F(3,28) = 1.597, p = 0.215). Shaping took maximally nine sessions spread out over 5 days. Performance, expressed as a percentage of obtained rewards, exceeded 90% before initiation of the next test phase. Two-lever discrimination Shaping on the operant task was followed by a two-lever discrimination. A main effect of block, showing that learning took place, was found over all blocks of the morning session (F(7,168) = 17.019, p = 0.000) after which performance levelled off. Initially, the overall performance of the sham controls fell behind that of lesioned animals (interaction effect), which made fewer errors in the second set of the morning session (F(1,24) = 5.599, p = 0.026; see Fig. 2). An independent-samples t test over this set showed blocks 2 and 3 to differ between lesion and control groups, respectively (t = −2.509; p = 0.025 and t = −2.534; p = 0.024). No difference in the number of omissions was observed (F(1,24) = 1.045, p = 0.316). Group differences had disappeared by the second session. Fig. 2Acquisition of the two-lever discrimination. Depicted is the percentage of correct responses for both lesion and control groups on the first acquisition day. White and grey bars indicate sets of four blocks (32 trials). Asterisk indicates group differences, p < 0.05 Overall performance over the second set of the final acquisition session showed that all groups (four) had obtained at least 90% of the rewards and did not differ significantly in performance (F(1,23) = 0.980, p = 0.334; see Fig. 3a). Fig. 3Performance on the final acquisition phase and initial reversal. a Performance on the final acquisition sessions. All groups obtained at least 90% of the rewards over the last set of the afternoon session, and no group differences were found. b Performance over the first reversal sessions. Control animals that switched to preferred pellets fell behind the other groups. White and grey bars indicated sets of four blocks (32 trials). Asterisk indicates group differences, p < 0.05 Spatial reversal After reaching criterion on the two-lever discrimination, the animals were subjected to four serial reversals. As each group (lesion and control) was now divided into a non-preferred- and preferred-pellet group, the number of groups was four from now on. A main effect of block (F(7,133) = 51.715, p = 0.000; not treatment or pellet, p > 0.3) was observed over the morning session of the first reversal (F(1,19) = 51.715, p = 0.000). During the afternoon session, a block effect over both sets taken together indicated that learning continued (F(7,140) = 22.669, p = 0.000). Main effects on performance for both treatment (F(1,23) = 5.245, p = 0.032) and pellet (F(1,23) = 6.158, p = 0.021) as well as a treatment/pellet interaction (F(1,23) = 5.701, p = 0.026) were observed over the first set. A one-way ANOVA with SNK post-hoc test revealed that sham animals receiving preferred pellets obtained fewer pellets than any other group in blocks 1 (F(3,27) = 4.163, p = 0.017), 2 (F(3,27) = 5.176, p = 0.007) and 4 (F(3,27) = 5.520, p = 0.012; see Fig. 3b). No differences for treatment, pellet or interaction were found over the second set of the afternoon session (p > 0.3). A covariate analysis revealed that the effects found during the reversal could not be attributed to a pre-existing performance difference between groups. To examine if the group differences could be attributed to either an increase in errors or an increase in omissions, further analyses were performed. The data indicated that the control animals that were switched to preferred pellets exhibited a decrease in accurate lever pressing, i.e. increased errors (F(1,24) = 4.638, p = 0.042; see Fig. 4), as opposed to increased omissions (F(1,24) = 1.606, p = 0.213), in this group in the first set of the afternoon session. After the initial reversal, all groups performed in similar fashion in the three subsequent reversals. Fig. 4Omissions and response accuracy in the first reversal of the two-lever spatial discrimination task. The graph shows the percentage omissions (top) and percentage correct responses (bottom) during the initial reversal. Decreased lever press accuracy (increased errors) of control animals that receive preferred pellets is seen during the first set of the afternoon session. White and grey bars indicated sets of four blocks (32 trials). Post-hoc testing revealed the second block of trials in this set to be statistically different from all other groups. No differences were observed for the percentage of omissions Extinction After four consecutive reversals, the animals were subjected to an extinction phase during which lever pressing was no longer rewarded. When comparing the total amount of lever presses corrected for baseline lever pressing, all groups showed a steady decrease over both sets of each of the four sessions (main effect of block: F(7,175) between 29.237 and 10.048; p = 0.000; see Fig. 5). There were, however, no main effects of treatment or pellet or any interaction effect between the groups (p > 0.2). Over the course of 4 days, lever pressing was never totally extinguished. Analysis of the uncorrected extinction data yielded identical results. Fig. 5Lever responding during extinction of the two-lever spatial discrimination task. The cumulative number of lever presses per group during the extinction phase of the two-lever discrimination task, when lever pressing is no longer rewarded is shown. Although a decrease is seen over each individual session, lever pressing is never totally abolished. White and grey bars indicated sets of four blocks (32 trials). No significant group differences were found Effects of mPFC 5-HT depletion on the odour-based go/no-go task Shaping During the initial shaping phase, the animals learned to sample an odour and press a lever for a reward. Both groups acquired the responses (i.e. >90% of pellets obtained) in maximally eight sessions, spread out over 6 days, without showing any treatment effect (F(3,28) = 0.667, p = 0.590). Two-odour discrimination After shaping, the animals were trained, over six sessions, to respond to one odour with a lever press, while inhibiting a lever press after sampling a second, different, odour. Results show an overall increase in performance over these sessions, i.e. a main effect of block (F(5,140) = 16.074, p = 0.00) for all groups (data not shown). No effect of treatment was found (F(1,28) = 1.375, p = 0.251). Response preference for either the ‘go’ or ‘no-go’ response were not observed. Odour reversal After the acquisition of the odour discrimination, the stimulus–response contingencies were reversed, and the animals had to switch their response to the odours. In this treatment, the two groups were again divided into sub-groups for preferred and non-preferred pellets. In contrast to the quickly acquired spatial reversal, the odour reversal took 12 sessions to acquire (reaching ‘pre-reversal’ performance). Figure 6 shows the performance of all groups. Fig. 6Performance on the final acquisition phase and reversal of the two-odour go/no-go discrimination task. a Performance over the four final acquisition sessions. All groups obtained at least 90% of the rewards over the final four acquisition sessions, and no group differences were found. b Performance over the reversal sessions. Control animals that were rewarded non-preferred pellets fell behind the other groups. Asterisk indicates group differences, p < 0.05 Over 6 consecutive test days (12 sessions), main effects of both session (F(1,10) = 40.163, p = 0.000) and treatment (F(1,10) = 9.025, p = 0.013) as well as a treatment/pellet interaction were observed (F(1,10) = 5.764, p = 0.037). Further analysis (one-way ANOVA and SNK post-hoc test) revealed that in sessions 7 (F(3,13) = 6.851, p = 0.009), 8 (F(3,13) = 3.898, p = 0.044) and 9 (F(3,13) = 5.563, p = 0.017), control animals receiving non-preferred pellets obtained fewer rewards than all other groups. Further analysis of the data showed that unlike the initial reversal task, none of the groups differed in response accuracy (F(1,10) = 0.158, p = 0.699). However, a treatment/pellet interaction (F(1,10) = 5.898, p = 0.036) was observed for the average number of omissions (see Fig. 7). Post-hoc testing showed this measure in sessions 8 and 10 to differ significantly between sham animals on non-preferred rewards and all other groups, respectively (F(3,13) = 4.647, p = 0.028 and F(3,13) = 4.194, p = 0.037). Fig. 7Omissions and response accuracy during reversal learning of the two-odour go/no-go discrimination task. Mean percentage of omissions (top) and percentage correct responses (bottom) per group during the ‘odour-reversal’ are shown. An increase in the number of omissions is seen for control animals that received non-preferred pellets on sessions 8 and 10. No differences were observed for the percentage of correct responses Discussion With the current experiment, we found support for our hypothesis that medial prefrontal 5-HT is selectively involved in cognitive flexibility when affect guides decision making. Local depletion of mPFC 5-HT prevented the reversal learning impairments that were induced by a change in the reward value during operant behaviour. This effect was observed both in spatial two-lever discrimination reversal learning and during the reversal of the odour-based go/no-go task. In the former, control animals that were switched to preferred pellets showed increased erroneous lever pressing compared to all other groups, whereas in the latter, non-switched control animals showed increased omissions. Apparently, the lesion rendered the animals less sensitive to changed reward value. Similar effects, however, were not observed during extinction learning, when rewards were no longer available. Taken together, these data suggest that depletion of mPFC 5-HT impairs goal-directed behaviour by rendering rats less capable of responding to a change in reward value (incentive learning; Balleine and Dickinson 1998; Balleine 2005; Niv et al. 2006), without disturbing their ability to detect the presence or absence of a reinforcer and to utilise this information to adapt behaviour accordingly. The data moreover indicate that this effect occurs independent of the stimulus modality of the behavioural tasks, thereby opposing our hypothesis that reversal deficits induced by mPFC 5-HT lesioning would be limited to spatial tasks, as opposed to odour-guided ones. In the current experiment, we lesioned 5-HT terminals in the mPFC by means of a local microinjection of the toxin 5,7-DHT. HPLC analysis showed that this procedure was successful as a marked loss of serotonergic innervation (70–90%), compared to sham controls, which was observed in the target area. This reduction was observed without spread to neighbouring areas whilst sparing both DA and NA innervation. Two behavioural paradigms were employed to study cognitive flexibility and affective processing. First, we used a task previously developed in our lab with which we showed mPFC involvement in spatial reversal learning (De Bruin et al. 2000). A second, symmetrically reinforced go/no-go odour reversal task was newly designed for the current experiments. These tasks were chosen to give insight into reversal learning across different stimulus modalities and to allow the study of different aspects of reversal learning, such as inhibitory processes involving both response extinction (spatial discrimination task) and impulse inhibition (odour-based, go/no-go task). Data showed that whilst the stimulus discriminations for both tasks were relatively quickly acquired, reversal learning took significantly longer for the odour stimuli. Whereas the ‘spatial’ lever press reversal was acquired within a day, the ‘odour’ go/no-go reversal took up to 12 days. These differences are in line with existing literature on the acquisition of the spatial lever-press task (De Bruin et al. 2000; Van der Meulen et al. 2003) and a comparable odour-based go/no-go task (Schoenbaum et al. 2006). Our hypothesis that medial prefrontal 5-HT depletion would have a stronger impact on reversal learning in the ‘spatial’ task than in the ‘odour’ task could not be proven. When comparing the performance of lesioned animals to that of control animals, we were unable to find any indication for impaired reversal learning on either task. Not only did 5-HT lesioned animals make fewer errors during the initial acquisition, these animals showed to be fully capable of acquiring both types of reversals, demonstrating intact cognitive flexibility across stimulus modalities when the reward identity remains constant. These findings suggest that in rats, the acquisition of a spatial reversal task, which depends on the mPFC, does not depend on 5-HT innervation of that area. This contrasts the finding that in primates, the orbital PFC involvement in visual reversal learning does depend on 5-HT. To test our hypothesis that mPFC 5-HT is involved in affective processing, we used a pellet switch to induce a change in affective value of the reinforcer during both reversal tasks. The rationale for this was that the impact of the serotonergic lesion in the mPFC on cognitive flexibility would be greater in a situation where the affective value of the reward can be used to guide decision making. Whereas in primate literature, the term ‘affective shifts’ has been used for a switch in the value associated with the stimulus (Dias et al. 1996), we wanted to introduce a switch in affective value of the reinforcer. Similar to the experiments of Tremblay and Schultz (2000), we changed the relative reward value from non-preferred, although still salient, to preferred (see also Watanabe 1996), at the time of the reversal. To determine food preference, food-deprived rats were pre-tested on three types of food reward in a T-maze, where all rats chose Noyes ™ pellet P over AI. These were subsequently implemented in the task as ‘preferred’ and ‘non-preferred’ rewards, respectively, to examine the effect of this affective shift in both groups during reversal learning. To exclude a ‘novelty’ effect, the animals were acquainted with both types of pellets before testing. As we observed pellet switch-induced performance effects in control rats during both tasks, we conclude that the pellet rewards were sufficiently different. The possibility that the lesion induced an indifference to either reward does not seem likely. If lesioned animals were in fact indifferent to reward outcome, they would have performed as non-switched control animals throughout the experiment, but this was not the case (see below). During initial ‘spatial’ reversal learning, lesioned animals behaved as non-switched controls, showing a quick reversal, without performance deficits on reversal learning per se. We did, however, observe that switching intact animals to preferred rewards resulted in reduced performance on the reversal task compared to lesioned animals. These results suggest that in intact animals behavioural adaptation is at least partly guided by reward value and, in this particular case, further complicates acquisition of the reversal. A second instance of such a pellet-switch-induced performance deficit was observed during the ‘odour’ reversal. Whereas the initial reversal impairment in switched control animals was due to increased errors, during the ‘odour’ reversal, non-switched control animals fell behind in performance because of increased omissions. As the pellet switch did not affect performance of lesioned animals at any time, we pose that an inability to use affective information (i.e. pellet value) in lesioned animals underlies the observed group differences. These data suggest that initial behavioural adaptation guided by altered reward outcome is dependent on medial prefrontal 5-HT. In the following section, we will first discuss why, in our opinion, a number of possible mechanisms cannot explain the observed effects induced by the pellet switch. We will then propose how our observations fit and extend current literature. Among the possible causes for these effects, altered associative learning is not a likely one. Although lesioned animals did make fewer errors during the acquisition of the spatial discrimination task, an effect that has also been observed by others (e.g. Ward et al. 1999), and suggests possible improved stimulus–outcome learning, performance of neither lesioned group (switch or non-switched) exceeded performance of the control animals during the subsequent testing. Moreover, the performance impairments observed in the control group occurred only during the early stages of reversal learning, leaving the acquisition of subsequent (spatial) reversals or stimulus–outcome learning unaffected (see also Harrison et al. 1999). Despite reports on involvement of both the mPFC (Salazar et al. 2004, Winstanley et al. 2006) and 5-HT (Beninger and Phillips 1979; Soubrié 1986) in impulsive responding and extinction processes, our tests indicate that reduction in mPFC 5-HT does not lead to decreased extinction learning or increased impulsivity. No effects of the lesion were observed during extinction of lever pressing or increased lever pressing during the go/no-go inhibition task. Although these results do not seem to fit the current literature on 5-HT in impulsivity, recent findings by Chamberlain et al. (2006) in human subjects show that impulse inhibition might be mediated via the NA system, rather than the classically implicated 5-HT. Therefore, the possibility that reversal learning improved in lesioned animals through altered inhibition or extinction processes is unlikely. Furthermore, subsequent analysis showed no difference between groups on general motor activity, as assessed by the average number of lever presses and nose pokes into the food dispenser. The possibility that a motivational effect of 5-HT depletion underlies the observed deficits does not seem likely either. Although the control animals showed an increase in the number of omissions towards the end of the go/no-go task, an effect suggesting decreased motivation towards the end of the experiment for the non-preferred reward, none of the groups (lesion or sham) showed any sign of increased or decreased lever pressing during the preceding test phases, suggesting that motivational processes were intact in lesioned animals. These findings are supported by other reports where motivational changes after general 5-HT depletion in humans (Evers et al. 2005), primates (Clarke et al. 2004) and rodents (Harrison et al. 1999) were not observed either. Earlier work by others (e.g. Roberts et al. 1994) suggests that (global) 5-HT depletion may induce increased reinforcing efficacy for both psychomotor stimulants and food rewards (but also see Tran-Nguyen et al. 2001). The evidence for such an effect is, however, inconclusive, and the observed changes can also be ascribed to increased impulsivity and insensitivity to punishment (e.g. Harrison et al. 1997). The current data, moreover, does not support these findings, as lesioned animals showed ‘normal’ extinction learning, whereas decreased extinction learning would be expected in case of increased reinforcing efficacy. From these findings, we conclude that the behavioural effects are not due to a generalized decrement of overall functioning but are rather specific to an inability to effectively incorporate reward value. These results suggest that mPFC 5-HT depletion induce a specific deficit in goal-directed behaviour that is limited to the ability to use current reward value to guide behaviour (Balleine and Dickinson 1998; Niv et al. 2006). Human literature provides a framework in which we think the current findings can be placed. Miller (2000) has described the PFC as a control centre over overt behaviour that regulates ‘active maintenance of patterns of activity that represent goals and the means to achieve them.’ Later work by Miller and Cohen (2001) and Ridderinkhof et al. (2004) extends this model by incorporating different cortical sub-areas and describes how these areas connect to combine (reward-related) information and lead to goal-directed behaviour. In this model, the orbital PFC can be seen as the area where reward-related information is encoded (as discussed in “Introduction”) and the mPFC functions as an area that regulates or signals the orbital PFC to implement performance adjustments (based on reward information). A unified model for the rodent PFC, similar to that of Miller for the human PFC, has not been proposed, but the available experimental data obtained in rodent areas that show functional overlap with the human mPFC (Uylings et al. 2003) parallel and extend these findings. Balleine and Dickinson (1998), for example, showed that lesions of the prelimbic area (part of the mPFC) render the animals insensitive to variations in the contingency between a response and a specific reward. Moreover, the same authors (De Wit et al. 2006) showed that inactivation of the mPFC leads to impaired resolving of response conflict through loss of inhibitory function, without any apparent impairment in discriminative function for foods or loss of reinforcing function of food pellets and sugar. Further indications for a parallel between the human and rodent mPFC comes from Cardinal et al. (2001), who showed that lesions of the mPFC yielded what Dalley et al. (2004) later described as a flattening of the shift from large-delayed rewards to small-immediate rewards. Although the authors ascribed this to a ‘timing impairment,’ these findings fit the described, human, model of mPFC-mediated performance monitoring and adjustment of behaviour. Finally, Hok et al. (2005) reported on neurons recorded in the mPFC that encode the motivational salience of places. These results add to the experimental evidence presented earlier of orbital PFC-mediated reward encoding by showing that reward-related information is also processed in the mPFC. Together, the presented rodent data support a role for the mPFC in the monitoring, processing and implementation of reward-related information, as suggested for the human mPFC. Experiments concerning the underlying neurochemical mechanisms involved in the processing of reward-related stimuli have mainly focused on DA. Direct evidence for DA involvement comes from measurements of DA ventral tegmental area neurons in non-human primates that show reactivity to reward (Schultz 1998), probability of reward (Fiorillo et al. 2003) and adaptive neural firing in response to reward-predicting stimuli (Tobler et al. 2005). These findings are further supported by a recent study that shows midbrain activation in humans during reward anticipation (Wittmann et al. 2005). The presented data, however, show that cognitive flexibility, when affect guides decision making, might be dependent on intact serotonergic projections. Although we cannot rule out possible compensatory mechanisms, our analysis shows that DA innervation in the lesioned animals remained intact. Support for our findings can be found in human studies where global depletion of 5-HT through tryptophan depletion caused altered processing of reward cues, lesading to unfavourable choice behaviour in a gambling task (Rogers et al. 2003). The current experiments support previous notions on the mPFC as a performance monitoring structure, responsible for the implementation of adjustments in cognitive control. Moreover, they extend the current model by implicating 5-HT, in addition to DA, in cognitive control. With regards to the initial research questions whether cognitive flexibility is dependent on mPFC 5-HT, the present data suggest that in both the spatial and the odour tasks, the lesioned animals are fully capable of acquiring a reversal, show appropriate extinction learning in the absence of a reinforcer and exert normal inhibitory control. The current observations, however, also suggest that when the value of a reinforcer changes but not its presence or absence, mPFC 5-HT is necessary to adapt the behaviour accordingly.

Immunome_Res-1-_-1289288

AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data

AntiJen is a database system focused on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. Compared to its progenitor JenPep, the interface has been completely rewritten and redesigned and now offers a wider variety of search methods, including a nucleotide and a peptide BLAST search. In terms of data archived, AntiJen has a richer and more complete breadth, depth, and scope, and this has seen the database increase to over 31,000 entries. AntiJen provides the most complete and up-to-date dataset of its kind. While AntiJen v2.0 retains a focus on both T cell and B cell epitopes, its greatest novelty is the archiving of continuous quantitative data on a variety of immunological molecular interactions. This includes thermodynamic and kinetic measures of peptide binding to TAP and the Major Histocompatibility Complex (MHC), peptide-MHC complexes binding to T cell receptors, antibodies binding to protein antigens and general immunological protein-protein interactions. The database also contains quantitative specificity data from position-specific peptide libraries and biophysical data, in the form of diffusion co-efficients and cell surface copy numbers, on MHCs and other immunological molecules. The uses of AntiJen include the design of vaccines and diagnostics, such as tetramers, and other laboratory reagents, as well as helping parameterize the bioinformatic or mathematical in silico modeling of the immune system. The database is accessible from the URL: . Introduction There is a vast, and ever increasing, volume of important information that has accumulated from decades of experimental analysis within immunology. This will only become compounded as high-throughput techniques begin to impinge upon the immunological biosciences. The only efficient way for this information to be properly utilized requires the development of databases that store it and systems that use it. Although the type of data archived may alter from case to case, nonetheless the creation, use, and manipulation of databases containing biologically important information is the most crucial feature of current bioinformatics, both as it supports the genomic and post-genomic revolutions and as a discipline in its own right. There is nothing new in developing databases focusing on immunology: many spotlighting the in-depth sequence analysis of individual immunomacromolecules have existed for some time [1]. Functional or epitope-orientated databases are a more recent development. Examples include the now defunct MHCPEP database [2], FIMM [3], SYFPEITHI [4], the HIV sequence database [5], the HLA ligand database [6], the EPIMHC database [7], and the MHCBN database [8]. An epitope is any molecular structure that can be recognised by the immune, or other biological, system. Epitopes, or the antigen from which they are derived, can be composed of protein, carbohydrate, lipid, nucleotide, or a combination thereof. It is through recognition of foreign, or non-self, epitopes that the immune system can identify and, hopefully, destroy pathogens. Hitherto, peptide epitopes have been the best studied, and have, traditionally, have been categorized as either T cell or B cell epitopes. T cell epitopes are peptides presented to the cellular arm of the immune system via the MHC-peptide-TCR complex. B cell epitopes represent surface regions of an antigen that are bound by soluble or membrane-bound antibodies. If this region of a protein antigen is comprised of residues distally separated within the primary structure, and brought into local proximity by protein folding, then it is termed a discontinuous or conformational B cell epitope. Linear or continuous B cell epitope residues are sequential in both primary structure and thus as a region on the proteins' surface. Such epitopes are predominantly identified by antigen-specific antibody cross-reactivity with peptides. There is a need to create a databank for the wider disciplines of immuno-vaccinologists, which can act as a central repository and resource. Our aim is to complement other databanks [2-8] and thus we have developed AntiJen, a computational information resource for immunology and vaccinology that integrates quantitative kinetic, thermodynamic and biophysical data, with functional and cellular information. AntiJen v2.0, a development of our earlier database system JenPep [9,10], contains functional data on T cell and B cell epitopes. Moreover, the B cell archive is now sub-divided into linear and conformational epitopes. These epitopes form the basis of the humoral immune response and, unlike T cell epitopes, methods of prediction are often inaccurate [11]. A more in-depth B cell epitope archive should aid the development of prediction strategies. Antigen recognition by the Major Histocompatibility Complex (MHC) is vital to T cell activation hence, the inclusion of thermodynamic data on the binding of peptides to MHC molecules and T Cell Receptor (TCR) binding to peptide-MHC (pMHC) complexes. This data is complemented by kinetic data based on the same molecular interactions. Data on antigen processing and presentation is also included in AntiJen. Binding data derived from peptide interactions with the Transporter Associated with Antigen Processing (TAP transporter) are included in the archive. Additionally, quantitative specificity data from position-specific peptide libraries is included. AntiJen also incorporates thermodynamic data on protein-protein interactions, within an immunological context, such as co-receptor and superantigen binding, plus interactions with the MHC. All of these interactions are, potentially, key factors for the successful computational design of vaccines. AntiJen also contains biophysical data, including diffusion coefficients and cell surface copy numbers, on a variety of immunological molecules. Such data provides insight into the number of target receptors, which is an important, if under explored, component of binding between cells. Indeed, the number of molecules expressed on the membrane can alter depending on disease. The final addition to the databank focuses upon antigen binding to antibodies. One key innovation is a greatly increased compendium of experimental conditions, which, in conjunction with a greatly enhanced search capacity, consolidates our databases as a unique, value-added data source, fostering developments within both in silico immunology and the wider community of immunovaccinology. The database is available from the URL: . Database development Relative to the database system used for JenPep [9,10], the interface to AntiJen is entirely new, having been completely rewritten. AntiJen has been designed and implemented using postgreSQL, a system comprising a relational database and database server, and has thus established increased database robustness, creating an improved infrastructure for foreseeable issues of data storage and data growth. Data within AntiJen is structured into twenty-four normalised tables. Each is category specific and holds either statistical or experimental data. Additional tables accommodate the keyword data – which powers our protein-orientated antigen search and allows integration of the BLAST search – and there is also a structural data table to accommodate links to external structural databases. The user interface consists of a series of HTML forms. The search requests from these forms target PERL scripts integrated with SQL which in turn query the database. Database content Compared to its progenitor JenPep [9,10], the data archived in AntiJen v2.0 has grown considerably in depth (additional data types such as experimental conditions), breadth (addition of new data to existing databases), and scope (addition of extra sub-databases containing novel kinds of information). Additions to AntiJen have been derived from exhaustive searching of the primary literature, to give a dataset of > 31,000 entries. AntiJen v2.0 now consists of 11 sub-databases; details of the different databases are given in Table 1. The relative sizes of the databases and the growth from JenPep are summarised in Table 2. Table 1 AntiJen sub-databases and content. DATABASE CONTENT T Cell Epitopes Contains T Cell epitope peptides (known binders). B Cell Epitopes Contains B Cell linear and conformational epitope peptides. MHC-Peptide Binding data relating to antigenic peptides and MHC interactions. TCR Binding data relating to antigenic peptides – TCR – MHC interactions. TAP Binding data relating to antigenic peptides and TAP interactions. Kinetics Kinetic binding data for MHC peptide interactions. IPPI Binding data for a collection of immunological protein interactions. Diffusion Coefficient Collection of Diffusion and Friction coefficients for surface peptides. Copy Number Number/Abundance of cell surface molecules. Peptide Libraries Relative binding data for antigenic peptide amino acid substitutions. Antibody-Peptide A variety of antibodies known to bind proteins. Table 2 Size of AntiJen relative to JenPep. The number of peptides for each category in the AntiJen database is given, distinguishing between class I and class II categories, where appropriate. Growth versus JenPep 1 and 2, the progenitors of AntiJen, is included. For certain data categories, most obviously TAP binding data, re-evaluation of the quality of data within JenPep has seen it decrease rather than increase, however the expansion of the data is clearly seen. DATABASE JenPep v1.0 JenPep v2.0 AntiJen v1.0 AntiJen v2.0 Class 1 Class 2 Total Class 1 Class 2 Total Class 1 Class 2 TOTAL Class 1 Class 2 TOTAL T cell epitotes 1266 795 2061 2060 1158 3218 2247 1578 3825 2402 1585 4158 MHC peptide binding 3196 2652 5848 6411 5925 12336 6853 7772 14625 7304 8114 15454 TAP peptide binding 432 441 408 1106 B cell epitotes 816 1295 3541 TCR – peptide-MHC 49 375 124 594 527 253 782 MHC peptide kinetics 704 243 947 897 294 1150 IPPI 805 2675 Copy Number 161 243 414 Diffusion coefficients 759 Peptide Libraries 897 Antibody 395 AntiJen contains both generic and dataset-specific data. For each entry, we record the peptide sequence (eg. YTSDYFISY) of the epitope using the standard one-letter code, its length (9 in this case), and, by linking to the sequence database Swiss-Prot or NCBI , the antigen to which the peptide sequence most closely matches (in the case of YTSDYFISY: C-ests-1 (p54), SWISS-PROT code P41156). The description of the antigen is, wherever possible, obtained directly from the literature. AntiJen is also linked to PUBMED. This allows us to record the original citation associated with the data. For example, for YTSDYFISY, we cite: Journal of Immunol 1994 volume 152 pages 3913–3924, PUBMED ID 8144960. For the T cell epitope, MHC ligand, and TCR-pMHC complex categories, we also record, for each peptide, the MHC restriction in terms of the host species, class (class I vs. class II), and, where the data is available, the serotype and allele. For YTSDYFISY, these data would be human, class I, HLA-A1, and A*0101. Entries within AntiJen are, in turn, linked to external databases, which enables further in-depth cross referencing. As we have said, protein sequence identifiers, which may be the source of an antigenic peptide or immunological co-receptor, link directly to details in the Swiss-Prot database [12] or the NCBI protein database. Journal references can be viewed via a link to the PUBMED database , and thus to full literature references, where available. AntiJen also links to structural data, currently derived from the MPID database [13] and the Protein Data Bank [14]. The database aims to provide access to background data where available. Allele identifiers serve as a link to the IMGT/HLA database at the European Bioinformatics Institute [15]. Inherent variability in the way MHC alleles are named within the primary literature prevents us from unambiguously standardizing nomenclature within AntiJen. HLA nomenclature follows that of the HLA Informatics Group . An allele is named using a defined pattern. For example, for HLA-A*0101: the HLA-A refers to the HLA locus; the first 01 to the serologically recognized A1 antigen and the final 01 to the individual HLA allele protein sequence. AntiJen stores the antigen classification (i.e. HLA-A1) and, when available, the specific allele. We have often encountered problems with the nonstandard allele reporting. A 4-digit HLA name necessarily implies the two digit serological antigen, a two digit classification clearly does not imply a specific allele. During database compilation, a sequence search allows us to identify the protein from which an epitope sequence originates. However, because epitopes are generally short, their sequences may be present in several potential antigens: in orthologues, paralogues, or in totally unrelated sequences. As epitopes are processed from whole proteins via a complex proteolytic pathway, one can use the sequence context to infer preferred proteasomal or endoplasmic protease cleavage patterns, but not if its context is defined incorrectly. Moreover, assuming that AntiJen is used subsequently to assign the antigenic status of proteins, wrongly identifying particular proteins as antigens can lead to the percolation of annotation errors [16,17]. AntiJen is, where possible, a quantitative database archiving continuous measures of binding. This is a fundamental feature of several sub databases, such as the MHC ligand and pMHC-TCR databases. The binding of an immunological macromolecule to a peptide or other biomacromolecule is quantified as are other receptor-ligand interactions: R+L↔RL Here R is the receptor (an MHC or TCR), L the ligand (peptide or pMHC), and RL, the receptor-ligand complex (pMHC or pMHC-TCR complex). The rate of the forward reaction is proportional to [L] [R]. The rate of the reverse reaction is proportional to [RL] as no other species are involved in dissociation. At equilibrium, the forward and reverse rates are equal, and so using kon and koff as the respective constants: kon[R][L] = koff[RL] Rearranging: Here KD is the equilibrium dissociation constant, which represents the concentration of ligand that occupies 50% of the equilibrium receptor population, and KA is the equivalent association constant. Experimentally, the measurement of equilibrium dissociation constants is often addressed using radio-ligand binding assays. Saturation radio-ligand binding assays measure equilibrium binding, at a range of peptide concentrations, to establish affinity (Ka) and receptor number (Bmax). Competitive binding experiments determine binding at a single labelled ligand concentration in the presence of a range of concentrations of unlabelled ligand. AntiJen records a hierarchy of different binding measures in its different sub-databases. Equilibrium constants are the most dependable and sit atop this hierarchy. Next come IC50 values, which can be obtained from a competitive radio-ligand or fluorescence assay. These are the Fmost commonly reported binding measures. Values obtained from radio-ligand or fluorescence methods may be significantly different. IC50 values for a peptide may vary between experiments depending on the intrinsic affinity and concentration of the standard radiolabelled reference peptide, as well as the intrinsic affinity of the test peptide. IC50 values vary with the equilibrium dissociation constant, at least within a single experiment. In practice, the variation in IC50 is often small enough that values can be compared between experiments. For the peptide discussed above, YTSDYFISY, the radiolabelled IC50 value recorded in AntiJen is 5.3 nM. BL50 values are also obtained from a peptide binding assay and are commonly reported. They are the half maximal binding levels calculated from mean fluorescence intensities of peptides binding to MHCs bound on the surface of RMA-S or T2 cells. Cells, pre-incubated with peptides, are labelled with a fluorescent monoclonal antibody. An overview of the thermodynamic and kinetic binding data within AntiJen is given in Table 3. Table 3 AntiJen Thermodynamic and Kinetic Data. An overview of the 6 AntiJen databases that provide binding data. It must also be noted that several of the databases contain additional data not present in any of the other databanks. MHC-Peptide Kinetics IPPI TAP pMHC-TCR Antibody TOTAL IC50 8562 0 247 1000 0 4 9813 Kon 0 188 563 0 157 87 995 Koff 0 146 610 0 150 101 1007 KD 359 156 1143 16 227 70 1971 Ka 65 0 37 0 28 132 262 t1/2 0 207 72 0 148 0 427 AntiJen also now contains experimental conditions, such as temperature and pH. A summary of this data is given in Table 4. The accuracy of data depends greatly upon the experimental method used. The grouping of data with respect to specific experimental techniques allows a more thorough assessment of training sets. Figure 1 shows the distribution of data for each type of analysis with respect to each database. The MHC Kinetics and TAP databases highlight the problems outlined above. The kinetics database contains data determined from over 14 methods while the TAP database is derived from 4 methods, with radiolabelled assays accounting for 80% of the data. Table 4 Experimental conditions and associated information archived in AntiJen. Number of recorded experimental conditions stored within the AntiJen database. For each condition (temperature, pH, etc.) we show here the number of entries within a particular sub-database. [Standard] is the concentration of labelled standard peptide in an assay. Likewise, [competitor] is the concentration of competitor peptide within a competition assay. [peptide] is the concentration of peptide in a kinetic experiment. The Method category refers to a standard procedure used to perform a particular assay. Differences in the number of recorded data, relative to figures in Table 1, arise primarily from the omission of key details from particular papers. Archiving of experimental conditions is on-going. DATABASE TOTAL pH Temperature [standard] Stand. peptide seq. [competitor] Method [peptide] MHC Binding 15454 6679 9831 10893 12796 5007 1251 MHC Kinetics 1150 677 1101 1149 606 TAP Binding 1106 22 243 1092 1101 86 981 TCR-pMHC 782 426 632 668 IPPI a 2675 726 1371 2600 Copy Number 414 183 278 414 Peptide Libraries 897 897 897 Diffusion Coefficient 759 321 668 736 Antibody 395 119 115 372 Figure 1 The distribution of experimental methods applied within each database. The number of different experimental methods and the abundance of data relating to the method is shown within the figures. The 'OTHERS' category refers to methods for which there is a relatively small number of entries. The compilation process has highlighted the considerable inconsistency within the immunological literature regarding the recording of such fundamental data. AntiJen contains, however, a direct, verbatim transcription of data from the primary literature. As such, we do not attempt, as a matter of policy, the comprehensive and retrospective correction of potential errors. To undertake such correction would only compound any errors, introducing the kind of percolating inconsistencies so much a feature of other database systems [16,17]. Further inaccuracies may stem from our logistic inability to verify data independently, therefore we must trust those values reported in the literature. Subsidiary Databases in AntiJen The AntiJen database contains a number of sub-databases. Each of these contains data on different aspects of the biological function and/or biophysical properties of different classes of immunomacromolecule. We describe the nature and content of each sub database below. B Cell Epitopes Epitopes are the principal chemical moieties recognized by the immune system. Although the importance of non-peptide epitopes, such as carbohydrates and lipids, is now increasingly well understood, peptidic B cell and T cell epitopes remain the principal tools by which the intricacy of the immune response can be explored. B cell epitopes are regions of the surface of a protein, or other biomacromolecule, recognized by soluble or membrane-bound Antibody molecules. In developing AntiJen, we have discarded the contents of our previous B cell archive and constructed one de novo. It contains an entirely new data set with a substantially different data structure. There are two forms of epitopes: linear and discontinuous. A linear B cell epitope is composed of a single stretch of sequential residues. A discontinuous B cell epitope is composed of sequentially separate residues brought into close proximity in a conformationally-dependent arrangement. The data we archive is primarily focused upon linear epitopes. This is due to the far greater amount of experimental data available for linear epitopes, which reflects both the relatively facile experiments needed to identify them and an implicit belief in their utility as potential vaccine epitopes. By contrast, discontinuous epitopes are thought to be more prevalent within folded proteins, but are far more challenging to determine experimentally. The archive catalogues the sequence of binding peptides, and also gives the length and source: TTGDVIASS, a 9 amino acid peptide from Escherichia coli non-fimbrial adhesion. Residues identified as important in binding to the antibody are recorded. This may correspond to a whole peptide or a subsequence, such as TTGDVI in the above example. The peptides are also categorized in terms of their relative observed immunodominance. Antibodies host organism and isotype are recorded. The current B cell epitope archive contains 3,541 epitopes. T Cell Epitopes T cell epitopes are short peptides bound by major histocompatibility complexes (MHC) and subsequently recognized by T cells. Epitopes recognized by both CD4+ and CD8+ T cells are included in the database. Such epitopes can be identified in many different ways. However, this diversity of measurement imposes a certain need for consistency, necessitating the requirement for recording a range of different experimental methods. The archive has expanded to include 4,158 entries. The entries contain the epitopes, ranging in length from 4 to 38 amino acids, peptide information, detailing the source, with links to Swiss-Prot and the corresponding MHC restriction data such as Serotype, Allele and Class. Additionally, the peptides are categorized in to groups such as Allergens, Bacterial, Cancer, Human, Viral and Self peptides. MHC – Peptide binding AntiJen continues to archive quantitative data on the thermodynamics of peptide interactions [18,19], and it has expanded in number and content, with additions such as experimental conditions, plus specific Standard and Competitor peptide concentrations used in the assays. The current archive contains 15,454 entries. The sequence of the binding peptide, along with the source, plus relevant MHC restriction data is recorded. The restriction alleles currently include those from Human, Mouse, Rat, Rhesus Monkey, Cotton-top Tamarin, and Chimpanzee. AntiJen contains IC50 values, binding affinity measurements from competitive binding assays, for which the standard and competitor peptides and concentrations are recorded, plus BL50 values, calculated from peptide stabilizing assays. Where possible, antibodies and the concentrations used to calculate BL50 values are archived. Additionally, but on a somewhat smaller scale, equilibrium association (KA) and dissociation (KD) constants are recorded for peptide-MHC interaction. Melting temperatures (Tm) and signal wavelength are also recorded; this is the temperature and wavelength at which 50% of the MHC protein is denatured as measured by circular dichroism. AntiJen also records so-called Weak/Non-binders. This indicates that the peptide has been tested in an MHC restriction assay and has been found to exhibit a binding affinity, i.e. an IC50 value > 10,000 nM for a radio-ligand assay, so low that it can be categorized as inactive. pMHC-T Cell Receptor interaction The TCR sub-database contains 782 entries, which records thermodynamic and kinetic binding data for the interaction of peptide-MHC (pMHC) complexes with TCRs. Different MHCs exhibit a distinct selectivity for certain peptide sequences. T cell receptors, in their turn, also exhibit different affinities for peptide-MHC complexes. The entries contain epitope, peptide source and MHC restriction data, as described above, plus TCR structure information, located at the MMBD database . Furthermore, any mutations are noted and a designated name for the TCR is archived. In addition, the peptides are recorded as either agonists or antagonists. The binding data is given as equilibrium constants (KD), EC50 values, rate of association (Kon), rate of dissociation (Koff), association constant (KA) and the half-life (t1/2) of the TCR-peptide interaction. TAP Binding This dataset contains binding data for the interactions between peptides and the TAP transporter, one of the principle steps in antigen presentation. As with the peptide-MHC database, the data is established from competitor binding experiments, based on labelled assays. Therefore, standard and competitor peptide sequences and their concentrations are recorded. The binding data is given as IC50 and KD values. The database currently contains 1,106 entries, with peptides from Human, Rat and Mouse sources. Based on IC50 values > 10,000 nM, the peptides are categorized as weak/non-binders. The entries have increase in number from the level found in JenPep, although several entries were removed in an effort to increase the accuracy and consistency of the archive (Table 2). Peptide-MHC Kinetics AntiJen's kinetics sub-database, which contains 1,150 entries, mostly relates to Class I MHC data. It records measurements for forward and reverse rate constants for complexation events. This complements the thermodynamic measurements on peptide-MHC binding described above. The data currently focuses upon both the half-lives of binding interactions, as well as association and dissociation rate constant values (Kon and Koff) for the recorded epitopes. Additionally, concentrations of the peptide, MHC and TAP are archived. The half-life for radioisotope labelled β2-microglobulin dissociation from an MHC class I complex, as measured at 37°C, is also archived. This is a kinetic measurement rather than a thermodynamic one, although it is often assumed that the greater the half-life the stronger the peptide-MHC complex. The half-life (t1/2) equals: Here the t1/2 corresponds to the dissociation of the MHC-β2 microglobulin complex rather than the kinetics of the protein-ligand interaction, but is still peptide dependent, as well as kinetic in nature. Immunological Protein-Protein Interactions The immune system is built around protein interactions therefore we developed another new sub-database which deals with Immunological Protein-Protein Interactions (IPPI). This archive contains 2,675 entries based on a variety of binding data, such as Kon and Koff rates, for a range of macromolecules implicated in physiological or pathological interactions, as well as KD, KA and IC50 values. The molecules include receptors such as CD4 or CD8 molecules, superantigens and other microbial virulence factors, cytokine receptors and cell adhesion molecules. The entries detail both protein partners involved in the binding interaction, with links provided to the NCBI-Entrez database. Additional data for MHC receptors is archived, whereby the reactive epitope is recorded and the co-receptors are categorized into viral, bacterial and self peptides. MHC data outlined in the previous databases is given, where appropriate including any mutations to the MHC. Antibody – Protein Binding AntiJen also contains a further sub-database, which comprises thermodynamic data relating to antibody-antigen binding. The dataset contains 395 entries for antigen proteins and antibodies, mostly derived from viral and mammalian sources. Reported values were obtained using radiolabelled assays and BIAcore analysis. This archive should aid in the selection of antibodies and peptides for in vitro studies. The entries list the antibodies and the binding/kinetic data, consisting of KD and KA values and to a lesser extent Kon, Koff and IC50 values. Peptide Libraries This archive further complements our MHC binding databases by indicating the relative contribution of residues within peptide libraries to MHC binding. 897 entries contain quantitative specificity data derived from position specific peptide libraries [20]. This catalogues the relative effect on affinity, in the form of IC50, log relative SD50 and log SI values, of all substitutions, at all peptides positions, against a random sequence backdrop. All of the libraries relating to a known peptide binder are designated a name within AntiJen, this usually consists of the author and year of publication. The archive contains the core peptide, along with the mutation position and the substituted amino acid. The corresponding MHC data is given as mentioned above. Diffusion Co-efficients To further increase the range of data archived, AntiJen also contains 759 records of cellular biophysical data, in the form of diffusion co-efficients, recorded as cm2s-1, for a diversity of cell surface molecules, including MHCs (Mouse and Human), viral peptides and other receptors [21]. The molecules are either chemically or fluorescently labelled and then measured using one of two methods: Single Particle Tracking (SPT) or Fluorescence Photobleaching Recovery (FPR). SPT monitors the lateral motion of a labelled molecule while FPR measures the rate of subsequent infiltration from a photobleached section of the membrane. Friction co-efficient data is also given, which measures of the velocity and force applied to an antibody-coated bead. Records contain the cell or cell type where diffusion is occurring, the name of the diffusing protein along with the form of labelling applied. Furthermore, specific experimental data is given such as antibody bead size. In this case the diffusion of the beads is monitored. Increasingly, data relating to photobleaching is included, such as beam power, bleaching duration, pre- and post-bleach time, etc.. Copy Numbers The final sub-database contains 414 measures of cell surface populations of different molecules, called cell surface copy numbers hereafter. This database focuses on an array of molecules, including Class I MHCs (22) and TCRs [23]. The entries are given as number and type of MHC molecules, number of MHC-peptide complexes or abundance of peptides associated with each MHC serotype, generally defined by mass spectroscopy. The entries list the cell type, the antibody bound to the MHC and, if appropriate, the binding epitope. This only applies to the number of MHC-peptide complexes and the abundance of peptides associated with each MHC. Searching the database Search mechanisms within AntiJen are significantly improved and allow either a detailed or a broad search from a simple user interface. From our experience with JenPep, we recognize that accessibility to the data in a user friendly manner is a vital requirement, and have improved our current search mechanisms and developed new search interfaces. Two different search mechanisms are available. One is based on BLAST [24] and the other is a bespoke system, allowing several alternative searches. Within a typical search, the user-entered search criteria are carried from an HTML form to a category specific PERL/SQL script, which performs the database queries. The BLAST search allows querying of a peptide or nucleotide sequence against the proteins contained in AntiJen; all entries containing data within AntiJen which are relevant to a protein sequence are linked via the BLAST output. A local database of protein sequences is searched with BLASTP or BLASTX [24] using the BLOSUM64 matrix. All BLAST control variables are fixed. An HTML Front-End (Figure 2), where a sequence can be entered or uploaded, connects to a web server-based PERL/CGI scripts, which interacts with BLAST. An annotated version of the default BLAST output is produced and links to AntiJen entries via SWISS-PROT [12] accession codes, which act as a query within a Keyword search. This allows AntiJen entries to be viewed directly from the BLAST output. Figure 2 Overview of the different search methods within AntiJen. The example search is focused upon an MHC ligand. The MHC ligand data can be searched directly (A) from a link on the AntiJen homepage, a broad search and specific search is available. A search for the epitope AMALLRLPLV, has one hit (D), this leads to the entry (G). All of the other sub-databases can be searched in this manner from the homepage. The two other searches are more generalised. A Keyword search (B) carries out a broad search on the whole database, for the criteria – Bacteria. This search gives 139 hits (E) and all of the sub-database entries can be selected from this output. The final search method is a BLAST search (C). The peptide (or nucleotide) sequence is queried against a local protein database. The output (F) provides links to the sub-databases relative to the protein. At present, peptide string, keyword, and protein name index searches are available within the bespoke system, which allows querying of individual peptide sequences or at the level of whole protein antigens. An overview of the AntiJen search systems is given in Figure 2 and 3. Epitopes, MHC, TCR, TAP peptide binding and kinetics databases can all be searched using sequence strings. The search protocol first returns an epitope list and a count of epitope matches. Subsequently, experimental criteria can be accessed for each selected epitope. Peptides can be searched using an amino acid orientated query: a sparse peptide string, similar in form to a peptide binding motif [3] or a PROSITE pattern [25], is used to identify all matching sequences. See Figure 4. Alternately, a list of protein antigens within AntiJen can be searched using keywords; the thermodynamic binding data such as MHC-peptide, TCR-pMHC and TAP, plus the B and T cell archives, related to the search criteria can then be selected from the matches based upon the SWISS-PROT accession codes [12], displaying all corresponding entries in the database.The other search method, allows the IPPI, diffusion co-efficients, peptide libraries, antibody-protein and copy number sub-databases to be searched using an index method, within a user-friendly HTML drop-down menu. Each of these methods can also be moderated using subsidiary search filters, data size ranges, and result presentation alternatives, such as peptide length or IC50 values. Minimum and maximum values can be used to restrict results, as can selection of MHC restriction alleles. Figure 3 Searchable database types within AntiJen. The database contains 3 types of searchable sub database: a set of Antigens searchable by keyword, various databases of functional and thermodynamic data searchable by peptide sequence, and a database of immunological protein-protein interactions searchable through an index. Peptide sequence searches can be explicit or "motif" based. Searches can also be focussed by setting the value ranges for properties, such as IC50 etc, recorded in the databases. Currently there is a link between all the thermodynamic and kinetic databases and between them and the database of antigens. Only antigens with data in one of the other sub-databases are included in the antigen database. Links to external databases are also indicated. The BLAST search provides an overall search of the databases except the Protein-Protein Interactions archive. Figure 4 Sparse peptide sequence search. Examples of the two search types available in AntiJen: (Search 1) a substring query and (Searches 2 and 3) PROSITE-like sparse queries allowing sets of variable (indicated by asterisks) and alternative (encased in square brackets) amino acids in the search. In our example, each query is an extension of the previous search. The initial search type returns a single hit, which is a weak binder. By introducing variable amino acid positions within the query string, the second query permits access to a larger data set with 39 hits being returned. The third search utilises both variable and alternative amino acid request functionality and returns 1996 hits. The number of entries returned can be reduced by specifying the epitope length, limiting IC50 values and restricting by one MHC allele. This search is constrained to peptides of amino acid length 9, which returns 280 hits. Constraining further by MHC allele HLA-A*0201, reduces this to 120. An additional constraint using the IC50 data filter, and requesting values below 500 only (the epitope range), reduces this again to 60. Discussion The name given to our new database, AntiJen, reflects a shift from a peptide orientated database structure, which was inherent within our earlier JenPep database, to one which can properly balance its focus on both protein antigens and isolated peptides. As such, it represents an important, integrated, immunological data resource. AntiJen now provides broad insight into both T cell and B cell mediated antigen recognition. In addition, through the auspices of the IPPI sub-database, the database also throws light on co-stimulation by co-receptors and gives important insights into the innate immune response. Our approach to protein-protein interactions, focusing on measured affinities, complements other methods, such as the Yeast-2-Hybrid system, which, while giving greater volumes of data, has problems of accuracy [26]. This, while not devoid of experimental artifact, gives a usefully different perspective on cataloguing protein-protein interactions. The further addition of weak binding notation to the MHC-peptide and TAP provides a greater overview of the nature of antigenic epitopes. This is further improved by the addition of the peptide libraries database, whereby key peptide residues can be highlighted. New databases have expanded the breadth of AntiJen to include biophysical data such as diffusion co-efficients and cellular data such as abundance of molecules. The antibody-antigenic protein sub-database will also provide a key resource for in vivo and in vitro studies, aiding in the selection of antibodies and peptide/protein targets. AntiJen distinguishes itself from the other specific binding databases [2-8] in several ways. Firstly, more data is recorded; our MHC-peptide database contains over 2,000 more entries than MHCPEP [2] and 10,000 more entries than EPIMHC [7]. Additionally, we have not restricted our archive to only high binders or to a specific category, as seen in EPIMHC and the HIV sequence databases [5]. Furthermore, AntiJen is currently a curated database, which is constantly expanding. Most obviously, AntiJen is useful in the design of epitope and subunit vaccines. Additionally, AntiJen is helpful in the design of clinical diagnostics and other laboratory reagents, such as the selection of peptides for tetramer design. AntiJen is also useful in the parameterization of mathematical models in theoretical immunology [27]. The redevelopment of the database has focused not only on content, but also on infrastructure. The current system, based on epitope string, keyword and index searches, along with an overall BLAST search, plus the redesigned HTML interface, leads to much greater accessibility and usability. Finally, the database acts as a repository of quantitative, continuous data, for the development of data-driven in silico predictive models, such as prediction of epitopes and MHC binding [18,19,28,29] through QSAR modeling. Future work Future tasks in the development of AntiJen, fall into two principle categories: eliminating deficiencies, errors, and inconsistencies within the database and simultaneously reinforcing it by expanding its depth, breadth, and scope. We also need to monitor updates within external databases, so that any alterations are mirrored within the archive. Like all other such repositories, AntiJen is prone to both systematic and random errors within the data accumulation process. User feedback and our interactions with immunologists will hopefully address persisting errors. Deficiencies in our database include our current inability to encode chemically or post-translationally modified peptides, non-natural MHC mutants and non-amino acid peptidomimetic MHC ligands. Additionally, it would also be interesting to complement our existing data on TAP binding with information on antigen presentation pathways, such as proteasomal and cathepsin cleavage patterns. Moreover, the compilation of B cell or antibody epitope data is an area ripe for robust development. Linear and conformational B cell epitopes are very much larger in number than our current compilation, leaving us scope to greatly increase recorded epitopes. Conclusion The development of a database is always a work in progress. Not simply because the easily accessible literature is typically always increasing, but also because of the desire to capture as much of the existing, but hidden, literature, as possible. In the post-genomic era, the database has formed the bedrock and language of bioinformatics; increasingly databases are coming to underpin our modern understanding of biology as a whole. Traditionally, databases have arisen as a response to need, answering the individual and idiosyncratic questions posed by biologists. However, the history of bioinformatics databases has shown the extraordinarily diverse ways in which archived data can be used. In creating AntiJen, we were motivated partly by our desire, and the desire of collaborators, to use the data within it to build predictive in silico models [16,17,28,29], and partly by a more altruistic desire to generate a useful, integrated database system with a quantitative focus. AntiJen has many potential uses throughout the immunological discipline, from immunoinformaticans to experimental immunologists and vaccinologists. By increasing the degree to which data is machine readable and web accessible, we open up new, and previously unthought-of, avenues for the bioinformatic exploration of immunological data. AntiJen is a primary data resource, amongst the most complete of its kind, yet, like SWISSPROT [12] or GenBank [30] decades ago, it is still relatively small and offers much scope for improved annotation. We see the database as a foundation from which to consolidate, through time, thus achieving a comprehensive resource of immunological data.

Pflugers_Arch-4-1-2270919

Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis. Introduction The role of biological cell plasma membrane endocytic pits, e.g. clathrin-coated pits [19] and caveolae [15], has been extensively studied over the last decades and still remains a hot topic. They not only provide cells with a means of transport across the membrane but also work as signalosomes aggregating receptors and ion channels [4, 17]. More recently, another membrane-microdomain-associated protein flotillin (1 and 2), traditionally thought to be associated with various receptors such as insulin receptor [1], has been shown to be responsible for clathrin- and caveolins-independent endocytosis [6], providing an alternative route of trafficking through the cell membrane. Whilst it is well known that clathrin and caveolin form basket-shaped pits in cell membranes [17, 25], the surface structures created by flotillin remain unknown. Current methods for endocytic pit function and dynamics studies include confocal microscopy, total internal reflection (TIR) microscopy [6, 20, 26], scanning and transmission electron microscopy [25] and atomic force microscopy [5]. Although fluorescence confocal microscopy is the one of the most commonly used microscopy techniques in cell biology, it provides limited information about single endocytic pit due to its lack of resolution. TIR has better vertical resolution and signal-to-noise ratio and has been used for single endocytic event following [2]. However, TIR is limited to observation of events that occur on the basal cell membrane only since it relies on imaging to a depth of about 100 nm from the glass surface and is not applicable to studying apical cell surfaces. In addition, using fluorescence-based microscopy techniques, it is impossible to determine the precise location of the endocytic pit relative to the cell membrane since the membrane position is hard to define. On the other hand, electron microscopy techniques have more than sufficient resolution and provide much more detailed information about the surface topography than optical images since the cell membrane can be imaged. However, these require fixation and specialised treatments of the cells, which potentially can alter the structure of interest and are thus incompatible with live studies. Scanning probe microscopy techniques may address a number of these issues by providing topographical data of surfaces. Atomic force microscopy has been used to study cell surfaces but, due to the direct interaction of its probe with the sample, imaging soft and mobile cell surfaces is difficult, limiting most studies to fixed cells or isolated membrane leaflets which are more rigid [5]. Scanning ion conductance microscopy (SICM), which uses a glass micropipette as an imaging probe [9], has no such disadvantage and allows non-contact visualisation of the topography of living cells [13, 14]. This has recently been combined with simultaneous confocal fluorescence imaging, resulting in a new technique—scanning surface confocal microscopy (SSCM) [7]. Also, since its inception, the resolution of the technique has been significantly improved, making it possible to visualise structures as small as protein complexes on surfaces of live cells [23]. This novel technique allows us to identify fluorescence located on the surface of the cell and localise it with topographically observed nanometre-scale structures (unpublished results). Materials and methods SSCM SSCM is based on a combination of SCM and SICM. SICM is a scanning probe microscopy technique[14, 23] in which the ion current flowing into a nanopipette is used to control the vertical (z axis) position of the cell relative to the pipette tip. As shown diagrammatically in Fig. 1a (not to scale), in SSCM, the cell is moved up and down in the z direction while scanning in the x and y directions, so its surface is always the same distance from the nanopipette. A laser is passed up a high numerical aperture objective so that it is focused just at the tip of the nanopipette, and a pinhole is positioned at the image plane so that the confocal volume is just below the pipette, as described [22]. Thus, a fluorescence image of the cell surface is obtained in a single scan, as well as a simultaneously captured image of the cell topography. Fig. 1Topographical imaging of endocytic pits in living cells by SICM. a Schematic diagram of the scanning ion conductance microscope. b SICM topographical image of live Cos-7 cell. c High resolution topographical SICM image of live Cos-7 cell membrane revealing numerous pits. d High resolution topographical SICM image of a fixed Cos-7 cell membrane revealing numerous pits. e Zoomed image showing a single pit (top). Topographical profile of a pit in a live cell (bottom). f Zoomed image showing two pits (top). Topographical profile of pits in fixed cell (bottom) The SCIM scanning head was developed in collaboration with Ionscope Limited, UK and mounted on a Nikon TE2000-U Inverted Microscope (Nikon Co., Japan). The sample holder was attached to a 100-μm HERA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany) used for lateral scanning. Vertical measurement and modulation was provided by 12-μm LISA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany). Both piezo stages were mounted on 25-mm translation stage DC motors (Physik Instrumente (PI) GmbH & Co., Germany) to provide coarse lateral and vertical approach. The setups were controlled via a computer with a SBC6711 DSP board equipped with A4D4 ADC/DAC modules (Innovative Integration, USA) using SICM software v. 1.2.00 (Ionscope Limited). The time to acquire a 512 × 512 pixel image was approximately 10 min. Two types of nanopipettes were used for the experiments. For low resolution images, nanopipettes with internal diameters ~150 nm were pulled from borosilicate glass capillaries. High resolution imaging was made using quartz nanopipettes with internal diameters ~70 nm. The nanopipettes were made from 1.00-mm outer diameter by 0.5-mm inner diameter capillaries with inner filament (Sutter Instrument, USA) using a laser-based Brown–Flaming puller (model P-2000, Sutter Instrument, San Rafael, CA, USA). The nanopipettes, backfilled with phosphate-buffered saline (PBS) and lowered in PBS, produced a resistance of approximately 300 MΩ for quartz and 100 MΩ for borosilicate pipettes. The maximum ion current measured using an Axopatch 200B (Axon Instruments, USA) was ~0.7 nA for quartz and ~1.5 nA for borosilicate pipettes. The set-point for imaging was 1% of the maximum of modulated ion current. The excitation light source was provided by a GPNT-02 laser diode (532-nm wavelength, IQ1A 635-nm laser; Power Technology Inc., USA). The optical recording system consisted of a Nikon TE2000-U Inverted Microscope equipped with a ×100 1.3-N.A. oil-immersion objective. The excitation light was fed through an epi-fluorescent filter block and emitted light was collected by a photomultiplier with a pinhole (model D-104-814; Photon Technology International, UK). Image processing and data analysis Matching VLP topographical structure to its corresponding fluorescent signal was done as follows: fluorescent confocal images were threshold to subtract the background, and the positions of individual fluorescent spots were marked by arrowheads and the multiple spots (where individual signals could not be resolved) circled. All positional markers were then grouped into one template and placed over the simultaneously recorded topographical image. As the result of this procedure, those topographical features having corresponding fluorescent signals were marked. Image contrast enhancement included slope correction and high-pass filtration performed similarly as previously described [10, 18]. The slope was calculated and subtracted from images by least squares algorithm [21] resulting in image flattening. A high-pass filter is a filter that passes high frequencies efficiently but reduces frequencies lower than the certain specified (cutoff) frequency. When applied to a 3D image, high-pass filtering results in finer, i.e. high frequency, details to stand out because larger features are eliminated. Cell culture and plasmids Monkey Cos-7 cells were routinely maintained at 37°C in 5% CO2, using DMEM (GIBCO/BRL) containing 5% (vol./vol.) FCS. The plasmid DNA used in the experiments were in pCi (Promega) containing either caveolin-green fluorescent protein (GFP), clathrin-GFP, flotillin-1-GFP or flotillin-2-GFP. Transfection procedure Cos-7 cells (1 × 106 cells per flask) were plated into a T25 flask and incubated overnight at 37°C in DMEM containing 5% FCS. Cells were washed prior to transfection with PBS, and complexes of Lipofectamine (Invitrogen) and plasmid DNA at a ratio of 1 μl to 1 μg were added in Optimem (Invitrogen) without FCS to the cells. For flotillin transfections, a mixture of flotillin-1- and flotillin-2-GFP plasmids were complexed together and added to the cells. After 2 h, the media was replaced with DMEM containing 5% FCS. After 24 h, the cells were trypsinised and plated onto coverslips (5 × 104 per well) and incubated at 37°C overnight. Cells where then either used for live imaging or fixed for 20 min with 3% formaldehyde containing 5% sucrose. Results Imaging of the cell surface using SICM In order to access the apical surface of Cos-7 cells, we used a basic arrangement of the SICM. A schematic diagram of the microscope is presented in Fig. 1a (not to scale), while a detailed description of the instrument can be found in “Materials and methods” section. SICM [9, 13, 14] is a type of scanning probe microscopy that uses a glass nanopipette as a sensitive probe to acquire a topographical image of the surface. Distance feedback-control is based on the ion current that flows between electrodes in the nanopipette and the bath, and this current is reduced as the pipette approaches the surface. This allows non-contact imaging of live cells in a physiological buffer. Using the SICM technique, we are able to either examine the morphology of the whole cell or achieve high resolution images of the cell surface. Scanning the complete cell surface gives insight into the number of macrostructures present on the cell surface, for example microvilli, and can be used to probe cell surface dynamics [8]. The morphology of the cell also has an impact on the resolution of SICM images, since large structures and steep slopes make small features difficult to resolve. A typical SICM image of live untransfected (control) Cos-7 cell in physiological conditions is presented in Fig. 1b. Cos-7 cells have very few macrostructures visible on the surface, and although the highest part of the cell membrane above the nucleus (light area, Fig. 1b) generates a steep slope, there are also flat peripheral areas. High resolution scans of these peripheral areas generate images containing a number of indentations of the cell surface (Fig. 1c). Observed indentations range from 50- to 160-nm diameters (see an example in Fig. 1e) and could present endocytic pits formed by clathrin and/or caveolin. The mobility of these indentations was relatively high compared to the present scan rate of our SICM as shown later in the paper; therefore, we used fixed cells to identify particular endocytic pits and imaged them at a higher resolution. A high resolution image of a fixed Cos-7 cell presented in Fig. 1d reveals similar indentations as seen in Fig. 1c but at higher clarity and contrast. A digitally zoomed image is presented in Fig. 1f and shows two pits (top) with the corresponding topographical profiles (bottom). Fig. 2Topographical and fluorescent imaging of clathrin coated pits in fixed clathrin-GFP transfected Cos-7 cells by scanning surface confocal microscope. a Schematic diagram of SSCM. b Topographical image of cell. c Fluorescent image of the clathrin-GFP transfected cell shown in b. d 3D representation of overlaid topographical and fluorescent images shown in b and c, respectively. e High resolution topographical image of cell surface revealing numerous clathrin-coated pits. f Same topographical image as in e but inverted and presented in a red palette. g Overlaid inverted topographical and fluorescent images shown in f and g, respectively. The image reveals that the pit topography matches the clathrin-GFP fluorescence. h The distribution of clathrin-coated pit width calculated from the SICM topographical images Scanning surface confocal microscopy In order to identify what pit indentations detected topographically by SICM corresponded to what endocytic pits, clathrin or caveolin, we transfected Cos-7 cells with corresponding GFP construct and then used scanning surface confocal microscopy to study the transfected cells. A schematic diagram of the microscope is presented in Fig. 2a (not to scale). Basically, SSCM is a combination of SICM and scanning confocal microscope [7]. SSCM also uses a glass nanopipette to acquire a true topographical image of the surface and a combination of a laser, pinhole and photomultiplier to gather fluorescent information from the cell surface, removing any out-of-focus fluorescence. Because the laser beam is always focused on the very tip of the nanopipette, topographical and fluorescent data are spatially aligned, thus enabling correlation between topographical features and fluorescent signals to be found. Imaging clathrin-coated pits in membranes of fixed cells using SSCM Cells transfected with clathrin-GFP were scanned, at low and high resolution, to examine the distribution of clathrin-coated pits on the cell surface. A typical topographical image of clathrin-FGP transfected fixed Cos-7 cell, together with its fluorescent image, is presented in Fig. 2b and c, respectively. An overlay of topographical and fluorescent images is shown in Fig. 2d. Peripheral areas of the cell contain punctated GFP fluorescence and had a flat topography. A high resolution topographical scan of these cell surfaces at a 7 × 7-μm scan (Fig. 2e) reveals numerous indentations ranging from 58 to 159 nm with an average depth of 102 nm (see distribution in Fig. 2h). In order to identify what proportion of the imaged pits were real clathrin-coated pits, we overlaid the topographical and fluorescent data. To make pit indentations that appear as dark spots in topographical images, visible when overlaid with the fluorescence image, the topographical image was inverted and red palette was applied (Fig. 2f). An overlaid, inverted topography red palette and clathrin-GFP fluorescence image is presented in Fig. 2g. As can be seen, there is a ‘cloud’ of green fluorescent signal around almost every single red spot that corresponds to indentations in the topography, giving a high degree of co-localisation between these structures and fluorescent puncta. Therefore, we can conclude that the majority of observed indentations are clathrin-coated pits. Analysis of the images revealed that 102 out of 115 (88.7%) detected endocytic pits co-localised with clathrin-GFP fluorescent spots. Presumably, this is because we are also detecting other types of pits that are not clathrin-coated. Reverse analysis showed that 11 out of 113 (9.7%) detected clathrin-GFP fluorescence spots did not match pit indentations. This could reflect the fact that clathrin-coated pits become highly mobile and move away from the surface inside the cell as soon as they bud off the cell membrane [16]. At higher magnification, we could also see clustering of the clathrin-coated pits in round patches (data not shown) that is similar to observations made by Frankel et al. [5]. Imaging caveolae in membranes of fixed cells using SSCM Using SSCM, the surface of caveolin-GFP transiently transfected cells was then examined. Low resolution topographical, fluorescent and overlaid images are presented in Fig. 3a,b and c, respectively. Higher resolution studies (Fig. 3d and e) revealed indentations in the cell membrane. These pits were slightly smaller than the clathrin-coated pits, ranging between 52 and 90 nm and giving a mean depth of 69 nm (see distribution in Fig. 3f). Fig. 3Topographical and fluorescent imaging of caveolin pits in fixed caveolin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of caveolin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of cell surface. e High resolution fluorescent image of caveolin-GFP transfected cell shown in d. f Caveolin pit width distribution histogram calculated from SICM topographical images. g Digital zoom of the topographical image shown in d (dotted square) revealing numerous caveolin pits. h Same zoomed topographical image as in g but inverted and presented in a red palette. i Overlaid inverted topographical image shown in h and digitally zoomed fluorescent image from the area shown in e (dotted square). The image reveals that pits match the caveolin-GFP fluorescence On closer examination using digitally zoomed images [topographical (Fig. 3g), topographical inverted and in red palette (similar to one described for clathrin) (Fig. 3h) and an overlay of the topography and fluorescence image (Fig. 3i)], it could be seen that the indentations in the cell surface co-localise with fluorescent signal. However, compared to clathrin-coated pits, identification of caveolae is more problematic. In all experiments conducted, the fluorescent images have a punctate appearance. However, in only two out of 26 experiments did fluorescence and structure co-localise. In those experiments, 139 pits were observed on the cell surface and only 13 of them (9.35%) were identified as matching caveolin-GFP fluorescent signal. This is in good agreement with the previous measurement that 90% of the pits identified in the topographic image on the cell surface are clathrin-coated and indicates that the other 10% are caveolae. Our observation that we detect many fluorescence spots from caveolin-GFP that do not correspond to pits in the SICM image suggests that many caveolae are below the cell surface or present on the surface but not resolvable. By transmission electron microscopy, they have been identified as 50–100 nm in size [15]; however, this is often the size of the whole structure, with the neck being much smaller [25]. It has even been suggested that there is a proteinaceous cap covering the neck of the caveolae [25]. If this is the case, and the cap could influence the flow of ions, then we would not be able to image caveolae structures on the cell surface unless the cap is open. Imaging flotillin in membranes of fixed cells using SSCM To our knowledge, flotillin 1 and 2 has not been yet associated with cell membrane structure that could be detected topographically by any microscopy technique. However, it has been shown that this protein is present in lipid rafts [1] and is responsible for clathrin- and caveolin-independent endocytosis [6]. Here, we examined Cos-7 cells transfected with flotillin 1 and 2 by SSCM for the presence of topographically detectable membrane features that could potentially co-localise with flotillin-GFP fluorescent signal. Low resolution topographical and confocal images together with overlaid image of flotillin-GFP-transfected Cos-7 cells are presented in Fig. 4a,b and c, respectively. Higher-resolution topographical images of the cell surface (Fig. 4d and g) revealed 200 ± 20-nm indentations. Figure 4e presents inverted red palette topographical image of Fig. 4d. Inverted red palette topography and fluorescence overly reveals that some indentations co-localise with the flotillin-GFP fluorescent signal (Fig. 4f, arrows). Remarkably, we have found that larger and higher-intensity fluorescent spots correlated with smooth protrusions from the cell surface (Fig. 4g,h and i, hollow arrows). The image presented in Fig. 4h is a high-pass filtered topographical image of the same data as in Fig. 4g. Fig. 4Topographical and fluorescent imaging of fixed flotillin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of flotillin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of the cell surface revealing numerous indentations. e Same topographical image as in d but inverted and presented in red palette. f Overlaid inverted topographical image shown in e and high resolution fluorescent image of flotillin-GFP acquired from the same area. The image reveals that some indentations on the cell surface match flotillin-GFP fluorescence (white arrows). g High resolution topographical image of cell surface revealing numerous indentations (solid arrows) as well as two protrusions (hollow arrows). h Same topographical image as in g but high-pass filtered. i High resolution fluorescent image of caveolin-GFP transfected cell shown in g. The arrows point to indentations that match flotillin-GFP fluorescence. Hollow arrows point to protrusions that match flotillin-GFP fluorescence Imaging endocytic pits in membranes of living cells using SSCM In order to test whether SSCM can identify particular endocytic pits in membranes of live cells, we performed a series of experiments with live clathrin-GFP transfected Cos-7 cells. Figure 5a and b presents normal and inverted red palette topographical images of a live cell. When overlaid with fluorescence, the inverted red palette topography shows that almost all topographically detected pits co-localise with clathrin-GFP fluorescence. There are fluorescence spots that are not round, but elongated in shape that do not match pit indentations on the surface. These spots of fluorescence probably reflect fast-moving clathrin vesicles right under the cell membrane. Fig. 5Live topographical and fluorescent imaging of clathrin coated pits in clathrin-GFP transfected Cos-7 cells by SSCM. a High resolution topographical image of live cell membrane revealing numerous clathrin-coated pits. b. Same topographical image as in a but inverted and presented in red palette. c Overlaid inverted topographical image shown in a and fluorescent image of the same area. The image reveals that, on live cells, we can detect that the pits’ topography match clathrin-GFP fluorescence. d Sequence of topographical images of live cell membrane revealing dynamics of the clathrin-coated pits. The images are separated by 10 min Figure 5d shows a sequence of three topographical images acquired from the same area of a cell with 10-min intervals. As can be seen, the indentations that correspond to endocytic pits are highly mobile and appear on or disappear from the surface of the cell membrane. It is beyond the current time resolution of our SSCM to follow the dynamics of these pits. However, this is the first time that endocytic pits are resolved topographically on the surface of live cell. Discussion By combining high resolution ion conductance imaging of the cell surface topography with fluorescence confocal imaging, we can identify the molecular nature of endocytic pits on the surface of living cells and measure the topography of the pits. For the first time, we showed that flotillin 1 and 2 is involved in the formation of ~200-nm-size indentations in the cell membrane. This observation is important evidence in support of the involvement of this protein in clathrin- and caveolin-independent endocytosis. We have found on Cos-7 cells that about 89% of the detected pits are clathrin-coated and 9% are caveolae, leaving a small percentage to be presented by flotillin pits. In each particular case, cell preparation transfection could introduce some deviation of clathrin/caveolin/flotillin proportion comparing to untransfected control. The fact that clathrin-coated pit formation is dependent on multiple factors [11, 24] provides indirect evidence that transfection may not influence the amount of pit formation. In contrast, it has been shown that expressing the caveolin in cells that do not contain this protein is enough to form caveolae [3]. However, there are other studies indicating that, although in caveolin transfected cells the total amount of produced caveolin is increased, the concentration of caveolin in the cell membrane remains unchanged [12]. The sizes of the pits we have measured are in good agreement with those obtained by electron microscopy. We have also shown that it is possible to apply our method to live cells. The clathrin-coated pits show fast dynamics on the time scale of our current imaging but suggest that improvements in the speed of imaging should allow us to follow the dynamics of endocytic pits on living cells with the intriguing possibility of directly imaging the endocytic process in real time.

Sleep_Breath-3-1-1794626

Review of oral appliances for treatment of sleep-disordered breathing

Between 1982 and 2006, there were 89 distinct publications dealing with oral appliance therapy involving a total of 3,027 patients, which reported results of sleep studies performed with and without the appliance. These studies, which constitute a very heterogeneous group in terms of methodology and patient population, are reviewed and the results summarized. This review focused on the following outcomes: sleep apnea (i.e. reduction in the apnea/hypopnea index or respiratory disturbance index), ability of oral appliances to reduce snoring, effect of oral appliances on daytime function, comparison of oral appliances with other treatments (continuous positive airway pressure and surgery), side effects, dental changes (overbite and overjet), and long-term compliance. We found that the success rate, defined as the ability of the oral appliances to reduce apnea/hypopnea index to less than 10, is 54%. The response rate, defined as at least 50% reduction in the initial apnea/hypopnea index (although it still remained above 10), is 21%. When only the results of randomized, crossover, placebo-controlled studies are considered, the success and response rates are 50% and 14%, respectively. Snoring was reduced by 45%. In the studies comparing oral appliances to continuous positive airway pressure (CPAP) or to uvulopalatopharyngoplasty (UPPP), an appliance reduced initial AHI by 42%, CPAP reduced it by 75%, and UPPP by 30%. The majority of patients prefer using oral appliance than CPAP. Use of oral appliances improves daytime function somewhat; the Epworth sleepiness score (ESS) dropped from 11.2 to 7.8 in 854 patients. A summary of the follow-up compliance data shows that at 30 months, 56–68% of patients continue to use oral appliance. Side effects are relatively minor but frequent. The most common ones are excessive salivation and teeth discomfort. Efficacy and side effects depend on the type of appliance, degree of protrusion, vertical opening, and other settings. We conclude that oral appliances, although not as effective as CPAP in reducing sleep apnea, snoring, and improving daytime function, have a definite role in the treatment of snoring and sleep apnea. Introduction Treatment of sleep-disordered breathing (i.e. snoring, upper airway resistance syndrome, sleep apnea syndrome) can be divided into four general categories. These include: (1) lifestyle modification, i.e. weight loss, cessation of evening alcohol ingestion, sleep position training, (2) upper airway surgery, (3) oral appliances, and (4) CPAP. Although the latter category provides the most reliable therapeutic modality and is the most widely used method to treat sleep-disordered breathing today—it is also the most cumbersome one. Many patients, particularly young non-apneic snorers, find it unappealing, difficult to tolerate, and unacceptable. The only other non-invasive alternative, which can produce favorable results within a short time, is oral appliances. Although there are several reviews of oral appliances, which have appeared since the start of the new millennium [1–6], including a recent review and practice parameters for treatment of snoring and sleep apnea [124, 125], this treatment modality is still underutilized. Even the dentists who are primary providers of this treatment, lack education in this area. For example, Bian [7] surveyed 500 general dentists in the state of Indiana and found that 40% “knew little or nothing about oral appliances for treatment of obstructive sleep apnea”. Unarguably, the knowledge about oral appliances among dentists and physicians varies geographically, being higher in large urban centers, which provide more educational opportunities locally, but the results of the survey certainly indicate a need for more education in this area. This review will summarize our current state of knowledge of the efficacy of oral appliances for the treatment of snoring and obstructive sleep apnea. We shall not limit this review to a simple summary of the effect of oral appliances on nocturnal respiration, but will also examine other aspects of this therapy, such as the reduction in symptoms, vascular consequences, side effects, and compliance. However, the main objective of this review will remain to be the examination of the current data dealing with the efficacy of oral appliance therapy for the treatment of snoring and sleep apnea. Historical aspects George Cattlin [8] was probably the first person who seriously thought that the route of breathing may influence sleep quality and daytime function. He attributed good health of the native North American Indians, compared to their immigrant European counterparts, to the fact that they are taught, from the early age, to breathe through the nose rather than the mouth. He pointed out that breathing through the nose promotes more restful and better quality sleep, which translates into better daytime function and better general health. After the publication of his book, there appeared many patents describing devices designed to promote nasal breathing. Some of the early patented appliances are shown in Fig. 1. Fig. 1Examples of early oral appliances However, modern published clinical work begins in 1903, when Pierre Robin first described a device, called the “monoblock”, for the treatment of glossoptosis [9]. More than 30 years later, he used an oral appliance to reposition the mandible [10]. For the next 50 years, little work was done in this field. It took almost another 50 years to start using oral appliances for the treatment of snoring and sleep apnea when Cartwright and Samelson [11] described the tongue retaining device in 1982. This work stimulated further investigations, resulting in many subsequent studies, many of which will be summarized in this review. Types of appliances Although the type and number of specific appliances may be bewildering and is still growing (Table 1), all may be divided into three general groups: soft palate lifters (SPL), tongue retaining devices (TRD), and mandibular advancement appliances (MAA). The first category is virtually no longer in use today. The second category is used very seldom, mainly if there are dental reasons precluding the construction of MAA. The last category (MAA) is by far the most common type of dental appliance in use today. It protrudes the mandible forward, thus preventing or minimizing upper airway collapse during sleep. These devices can be either fixed (i.e. the protrusion distance cannot be changed), or variable (i.e. protrusion can be increased or decreased). The final protrusion distance represents a delicate balance between side effects and efficacy. For this reason, the construction and fitting of the appliance should be done by a dentist with an expertise in this area who is familiar with different appliances, is capable of selecting the appropriate one based on the dental examination and has access to a sleep laboratory where the objective efficacy of the appliance can be verified. Table 1Examples of oral appliancesOral appliancesThe EquilizerJasper jumperEsmarkThe SilencerPM PositionerTPEKlearwayTongue locking applianceSnoreExNAPAAdjustable soft palate lifterHAPTAPZ-training applianceTessiTOPSSnore-no-moreSnore GuardSNOARElastometricSilent NightHerbstSUADTheraSnore Mechanism of action of oral appliances Much was written on how and why oral appliances may improve snoring and sleep apnea. The results show that upper airway obstruction during sleep may occur at any site between the nasopharynx and the larynx. The most common sites of obstruction are behind the base of the tongue (retroglossal) and behind the soft palate (retropalatal). This partial or complete occlusion of the upper airway during sleep is a consequence of abnormal anatomy and physiology (i.e. the airway is narrow and “floppy”). There is still a lively debate as to the relative contributions of abnormal anatomy vs abnormal physiology in the pathogenesis of upper airway obstruction during sleep [12, 13]. This debate is partially fuelled by the fact that airway narrowing or even a complete occlusion is a normal physiological event during sleep. In patients with sleep apnea, this normal response is exaggerated. The question then becomes whether this exaggerated response is due to abnormal neuromuscular control superimposed on the otherwise anatomically normal airway, or is the airway anatomically narrower than normal without any abnormalities in the neuromuscular control. It is now an accepted fact that a combination of abnormal anatomy and physiology is necessary to produce pathological repetitive narrowing (or complete occlusion) of upper airway during sleep—i.e. sleep apnea. Given that sleep apnea and snoring are a consequence of abnormal anatomy and physiology of the upper airway, is there evidence that oral appliances can correct these abnormalities, at least in some patients? There are several studies, not only during wakefulness, but also during sleep, indicating that advancing the mandible forward can enlarge the airway and reduce pharyngeal collapsibility [14–19, 120, 123] in normal subjects and patients with sleep apnea. These investigations confirmed the effect of oral appliances on upper airway properties. Some authors suggested that measurements of airway pressures during sleep may even predict the beneficial response to oral appliances. For example, recently, Battagel et al. [19] performed sleep nasendoscopy in 27 patients with sleep apnea. The mandible was gently advanced by 4–5 mm to simulate the effect of the mandibular appliance. The authors suggested that this procedure may help to determine whether a particular patient is a candidate for oral appliance therapy. Similarly, Ng et al. [123] measured upper airway pressures during natural sleep in 12 patients with obstructive sleep apnea to identify the site of airway collapse. The authors found that oropharyngeal, rather than velopharyngeal collapse, was predictive of the beneficial response to oral appliance. However, another study of 25 patients with sleep apnea where esophageal pressure was measured during sleep, found no significant differences in nadir esophageal pressure or cephalometric parameters between the patients who responded to oral appliance therapy and those who did not [120]. It is safe to conclude at this time that anatomical changes in the oropharynx, produced by mandibular advancement, result in the alterations of the intricate relationships between different muscle groups controlling the upper airway caliber. In some patients with sleep apnea these alterations may prevent the obstruction, in others—worsen the obstruction, and yet in others, particularly in those with low level obstruction, the part of the airway where the obstruction occurs may be unaffected. There is currently no reliable way to predict the outcome of treatment with oral appliance in individual patients and therefore to select appropriate candidates for this treatment. Clinical features also do not seem to offer much help in trying to predict who will respond to oral appliances, or just the opposite, to identify patients who are not likely to respond to this therapy. When two expert maxillofacial surgeons examined (in a blind fashion) 100 patients with sleep apnea to determine if there were any contraindications to mandibular advancement devices, they found that primary contraindications were present in 34% of patients [20]. This relatively high rate of contraindications and disagreements between individual experts only point out that a team approach is necessary to select the proper treatment for patients with sleep apnea. Results of clinical trials The onset of the new millennium carried forward the momentum started by Cartwright and Samelson [11] resulting in the increased use of oral appliances for the treatment of snoring and sleep apnea. However, the emphasis on the type of appliances has changed. Tongue retaining devices are currently seldom used, being almost completely replaced by the mandibular advancement appliances. The latter are sometimes also called mandibular repositioners, protruders, devices, splints, prosthesis, etc.—but the common feature of all these appliances is their ability to adjust the degree of mandibular advancement to achieve resolution of snoring and sleep apnea. In what follows, we shall summarize the results of clinical trials employing oral appliances for the treatment of sleep apnea and snoring. In addition, we shall review and summarize the information regarding changes in daytime function as a result of using the appliance, clinical and dental side effects, and compliance with treatment. Oral appliances for the treatment of sleep apnea Table 2 summarizes the results of individual studies using oral appliances starting with the 1982 polysomnographic study of TRD by Cartwright and Samelson [11] until the present. The only criterion for the inclusion of a particular study into this table, and thus the only common feature of all studies listed, was the availability of at least partial results of nocturnal monitoring of respiration with and without oral appliance. Otherwise, the studies are highly variable in their design, methodology, data analysis, outcome definition and assessment and presentation of results. This makes the interpretation of individual results, and particularly any attempt to summarize all of them, very challenging. Before describing the methods of analysis and presenting the summary of the data, it is very important to keep in mind the following points. Table 2Clinical studies with oral appliances: 1982–2005ReferenceNTypeAHIbaseAHIapplSuccess rate (%)Response rate (%)Comments[11]14TRD56271414Case series; AI[22]16TRD54331932Case series; AI[23]16TRD–––56Case series; all patients had SMR or UPPP; AI[24]1TRD795100–Case report; AI[25]7MAA37125743Case series; Esmarch prosthesis; AI[26]44MAA5023–59Case series; Esmarch prosthesis; AI[27]5TRD4894060Case series; NAPA; AI[28]12MAA54362517Case series; modified functional appliance[29]1MAA359100–Case report; mandibular repositioning appliance[30]12TRD37175817Case series[31]1MAA572100–Case report; mandibular repositioning appliance[32]15TRD2711730Case series[33]14MAA3294357Case series; fixed splint; AI[34]20MAA47203540Case series; fixed dental orthosis[35]12MAA5019––Case series; fixed prosthesis; AI[36]2MAA3075050Two case reports; intra-oral fixed prosthesis; RDI[37]16MAA3796925Case series; NAPA; RDI[38]24MAA48127313Prospective case series; Herbst-like; RDI[39]19MAA3513––Case series of CPAP failures; Herbst[40]20MAA57262040Case series; Esmarch; AI[41]12MAA4530––Case series; Herbst vs MR (muscle relaxation) appliance; result for Herbst; ODI4[41]12MAA4541––Case series; Herbst vs MR (muscle relaxation) appliance; result for MR; ODI4[42]51MAA3218––Case series; mandibular advancement splint fixed 75% of maximum protrusion[43]4MAA200110–75Case series; fixed intra-oral prosthesis; apneas/night[44]30MAA6531––Case series; Esmarch; AI[45]21MAA34201924Crossover, comparing AMP device with CPAP[46]19MAA20106811Randomized, prospective crossover comparing the Snore-Guard with CPAP[47]23MAA37185230Case series; mandibular repositioning device;most patients were CPAP failures; RDI[48]1MAA343100–Case report; elastometric sleep appliance[49]14TRD383000Case series: SnorEx appliance; RDI[50]20MAA25145521Randomized, crossover comparing the AMP device with CPAP[51]8MAA44126313Case series; mandibular advancing positioner[52]1MAA534100–Case report; fixed dental appliance[53]14MAA44––Fixed mandibular splint; maximum protrusion; median AHI[53]9MAA71––Fixed mandibular splint; 70% of maximum protrusion; median AHI[54]44MAA2596416Case series, prospective; mandibular advancement device[55]18MAA42156111Case series of UPPP failures; Herbst[56]25MAA3397212Case series;mandibular positioning device; RDI[57]15MAA19320––Case series of snorers; mandibular advancement device; snores/h of sleep[58]14MAA3657121Case series; Serenox[59]41MAA1812783Prospective, randomized, parallel groups comparing dental appliance with UPPP[60]75MAA44125128Case series; TAP appliance[61]11MAA4510––Case series; dental appliance[62]15MAA288––Case series; Klearway appliance[63]28MAA53213236Case series; elastic mandibular advancement device[64]37MAA26115015Case series; three fixed appliances with 2, 4 and 6 mm protrusion; ODI4[65]8MAA72361350Case series comparing MAA (Snore-Guard) with TRD and with soft palate lifter (SPL)[65]5TRD5044––Ibid[65]2SPL4757––Ibid[66]10MAA41126030Case series; Herbst[67]38MAA33125518Case series; Klearway[68]24MAA23967–Randomized, crossover comparing Herbst vs monoblock; results given for Herbst[69]39MAA17859–Case series; SnorBan[70]256MAA43185414Case series; mandibular advancement device[71]22MAA40125923Case series; modified functional appliance[72]24MAA30143825Randomized, placebo-appliance-controlled, crossover; mandibular advancement splint[73]22MAA286––Case series; either Herbst or monoblock[74]72MAA43225322Case series; adjustable mandibular splints[75]25MAA3.41.8––Randomized, placebo-appliance-controlled, crossover series of snorers; snoring measured on a 0–4 scale[76]33MAA25958–Prospective case series; mandibular advancement device[77]23MAA2185222Randomized, crossover, comparing 4 mm with 14 mm inter-incisal opening; results for 4 mm opening[78]7MAA67204343Case series; Herbst-like[79]32MAA187639Randomized, parallel groups comparing UPPP and MAA[80]26MAA1888812Case series; Karwetzky activator[81]34MAA227––Case series; Karwetzky activator; median AHIs[82]73MAA27123627Randomized, crossover, placebo-appliance-controlled[83]6MAA136830Case series; titration study; results for maximum protrusion; Klearway[84]20MAA181430–Randomized, crossover, comparing CPAP with ISAD appliance[85]48MAA311547–Randomized, crossover, comparing CPAP with mandibular repositioning splint[86]20MAA382333–Randomized, crossover, placebo-appliance-controlled; mandibular advancement splint[87]34MAA294––Case series comparing patients on CPAP who switched to MAA[88]24MAA22870–Prospective randomized crossover, comparing MAS with CPAP; soft one-piece mandibular advancement splint[89]40MAA50165228Prospective, randomized, parallel groups comparing 75% and 50% of mandibular protrusion; results for 75% group[90]26MAA196734Prospective, randomized, parallel groups comparing 75% and 50% of mandibular protrusion; results for 75% group[91]25MAA38154424Case series; The Silencer appliance[92]80MAA2114––Randomized, crossover, controlled, comparing mandibular advancement splint with CPAP and with placebo tablet[93]44MAA46126418Case series; titration protocol; Herbst-like[94]19MAA34173711Case series; titration protocol; Klearway[95]277MAA21854–Case series; mandibular advancement devices[96]11MAA32––Prospective case series of non-apneic snorers; Herbst; ODI4[97]20MAA84––Case series; fixed mandibular advancement device[98]34MAA203940Case series of consecutive patients; TAP appliance[99]17MAA2515––Case series of patients with CHF; mandibular advancement device[19]19MAA3287911Case series; Herbst[100]16MAA4624––Prospective, randomized, crossover comparing Twin Block and Herbst; median AHI [101]251MAA2916––Mail survey of 544 patients; RDI; mainly Klearway, few mandibular repositioners, fewer TRDs[114]21MAA3425538Case series; Klearway appliance[115]92MAA18–––Case series; questionnaires; bed partners’ replies recorded[120]25MAA35.98.260–Median AHI; case series; 6 weeks use; split polysomnography[121]4MAA49.511.77525Case series[122]73MAA24.412.255–Prospective, randomized, placebo-appliance-controlled 4 weeks study[123]12MAA229.258–Case series[126]161MAA1865922Case series; OSA defined as AHI > 5 First, Table 2 contains the studies from the two extreme ends of the spectrum of scientific rigor. At the highest end of the spectrum there are prospective, randomized, crossover, controlled (either against placebo-appliance or another treatment modality) studies. At the lowest end of the spectrum are individual case reports. Some studies are prospective case series, but most studies are retrospective analysis of series of cases. Second, the investigations listed in Table 2 form a very inhomogeneous group with respect to several variables: time of follow-up study, type of polysomnography, which respiratory variables measured, presentation of results, type of oral appliance used, missing data, etc. The time interval between the diagnostic and “with appliance” polysomnography varied from a few hours to a few months. In some studies, split polysomnography (i.e. diagnostic part followed by “with appliance” part) was carried out, while in others, the two sleep studies were separated by a couple of days to several months. In some investigations, formal in-hospital polysomnography was performed, while in others, only at-home monitoring of oxygen saturation was carried out. In some of the earliest investigations, only the apnea index (AI) was measured. Later investigations reported the oxygen desaturation index (number of times per hour of sleep that oxygen saturation falls by more the 4% from the baseline—ODI4). Recent investigations all reported either the apnea–hypopnea index (AHI) or the respiratory disturbance index (RDI). Most investigations presented only the mean values, rather than the individual data, and some presented only the median values. Oral appliances used by the investigators included tongue retaining devices, soft palate lifters, and fixed and variable mandibular advancement appliances. In the vast majority of investigations, the mandibular advancement appliance was used, less than ten investigations employed the TRD, and only one investigation used the soft palate lifter. In many investigations, not all patients who had a diagnostic sleep study also had a follow-up “with appliance” study. Clearly, the above factors have a significant effect on the analysis of the pooled data presented in Table 2. The first step in analyzing the results of individual investigations is to decide on which outcome variables to analyze. Since we are interested in sleep apnea, the following four variables are an obvious choice—baseline index of respiration (we shall denote this as AHIbase), “with appliance” index of respiration (denoted as AHIappl), success rate defined as the reduction of AHIbase to a value less than the defining value for sleep apnea, and response rate defined as the reduction of AHIbase by greater than 50% while still remaining higher than the defining value for sleep apnea. Table 2 lists 89 distinct investigations, involving a total of 3,027 patients. Inspection of the individual results indicates the widest possible variability. Respiration is analyzed in terms of AI, ODI4, AHI, or RDI. The definition of sleep apnea was based either on AI < 5, AHI < 10, RDI < 10 or ODI4 < 10. Success and response rates were defined differently in many studies; however, whenever possible we extracted the information to calculate the success and response rates according to the standard definition given above. In other words, no matter what definition of sleep apnea was employed in a particular study, i.e. whether it was defined as AI < 5, AHI < 10, or ODI < 10, etc.—the success rate we calculated was based on the percent of patients in whom AHIappl was less than 10, and the response rate was calculated as the percent of patients in whom 10 < AHIappl < 0.5 × AHIbase. Clearly, it is not possible to carry out a meta-analysis of these studies because of the differences in study design, data collection, statistical analysis and presentation of the data. Even the simple descriptive statistics based on the pooled data must be interpreted with caution due to the methodological differences listed above. To obtain the general information about the efficacy of oral appliances, we analyzed the results of individual investigations in several different ways, as follows. First, we rejected all case reports [24, 29, 31, 48, 52] and all studies, which did not report the mean AI, the mean AHI, or the mean RDI [43, 53, 57, 64, 75, 81, 96, 100, 120]. These studies reported either the total number of apneas per night, snoring only, median AHI, or ODI4, etc. This procedure left a total of 75 studies involving 2,832 patients. We used this “pooled” data to calculate the “pooled” means for each outcome variable—AHIbase, AHIappl, success rate and response rate. If an individual study did not report this outcome variable—it was not used in the calculation of pooled means. For example, to calculate the AHIbase, we only had to reject 1 study [23] where this variable was not reported, thus leaving 74 studies with 2,816 patients. To calculate the AHIappl, we had to reject 2 studies [23, 115], thus leaving 73 studies with a total of 2,724 patients. Similarly, looking at Table 2, we can easily see which studies had to be rejected to calculate the success and the response rates. The results are shown in Table 3. We note that oral appliance reduced the AHIbase from 31 to 14. The success and response rates were 54% and 21%, respectively. Table 3Summary of the outcome variables for studies listed in Table 2VariableResultNo. of patientsNo. of studiesAHIbase312,81674AHIappl142,72473Response rate21%1,57751Success rate54%2,08759 A second way to analyze the data presented in Table 2 is to select only those investigations where all four outcome variables (AHIbase, AHIappl, success rate, response rate) were reported. This left 49 studies involving 1,517 patients. It is interesting to note that the results shown in Table 4, are almost identical to what was found in a larger dataset. Table 4Summary of results for complete studiesResults for complete studiesNo. of studies49No. of patients1,517AHIbase35AHIappl14Response rate20%Success rate54% A third way to analyze the data is to select only the randomized, crossover, placebo-controlled studies. There are only five such studies—all done after the year 2000 and all using the mandibular advancement appliance. Inactive appliance was used as placebo in four studies [72, 82, 86, 122], and a pill was used as placebo in one study [92]. In the latter study, only the mean AHIs were reported, but not the success and response rates. The results are shown in Table 5. We note that: (1) only patients with mild to moderate sleep apnea were studied, (2) in two out of five studies, the mean AHI was reduced by less than 50%, (3) the success and response rates differ depending on the cut-off value of the AHI (either five or ten), (4) for AHI = 10 cut-off, the success rate is very similar to that of the uncontrolled trials, but the response rate is lower. The overall conclusion from these placebo-controlled crossover trials is that oral appliances significantly improved sleep apnea by reducing AHIbase from 25 to 14 with the combined success and response rates of 64%. Table 5Summary of results if randomized, crossover, placebo-controlled studiesReferenceNAHIbaseAHIapplSuccess rate (%)Response rate (%)Comments[72]2430143825For AHI = 5 cut-off5417For AHI = 10 cutoff[82]7327123627For AHI = 5 cutoff[86]2038233010Identical results for AHI = 5 and AHI = 10 cutoffs[92]802114––Tablet used as placebo; CPAP arm was also present[122]73241236–For AHI = 5 cutoff55–For AHI = 10 cutoffSummary27025143524For AHI = 5 cutoff5014For AHI = 10 cutoff One of the issues facing the effective use of oral appliances is our ability to determine the appropriate degree of protrusion necessary to resolve apnea and snoring. Until recently, the only way to do so was to advance the mandible forward as much as is tolerated by the patient, to carry out polysomnography and hope that sleep apnea is eliminated. Unlike CPAP, which may be controlled remotely without waking the patient, the design of most appliances requires removing it from patient’s mouth to change the degree of protrusion. This wakes up the patient repeatedly, resulting in a high probability that the titration study will be unsuccessful, thus wasting valuable resources. However, several “titration” protocols were described recently to eliminate this problem. Fleury et al. [93] studied a protocol where this titration was done at home, over a period of several weeks, by advancing the mandible in 1 mm steps and recording symptoms and ODI4. The effective protrusion was defined as that which results in either resolution in symptoms or reduction in ODI4 to below ten. Using this protocol, they were able to determine the protrusion, which abolished sleep apnea in 64% of patients. Tsai et al. [94] described a remotely controlled mandibular advancement device, which could be titrated during the night in much the same way that the CPAP is titrated. The mandible was advanced remotely during the night in 1 mm increments until respiratory events were eliminated. The success was confirmed subsequently by carrying out all night polysomnography with the oral appliance set to the effective protrusion determined during the titration study. The positive predictive value of this titration protocol was 90%. On the other hand, Kuna et al. [114] found that the titration protocol was not predictive of the response during chronic use. In this investigation, 9 out of 21 patients with OSA achieved reduction in AHI to less than 10 during titration. However, none of them demonstrated the same beneficial response during longer use of the appliance at home with the effective protrusion determined during the titration night. These results indicate that oral appliance titration to predict the amount of mandibular advancement required to reduce AHI to less than ten is still imprecise and must be used with caution when determining the appliance settings for home use. Nevertheless, the application of titration protocols is a new and important development in this field, which may improve the success of oral appliance therapy by identifying patients who are likely to respond to this treatment. Predicting who will respond to the oral appliance therapy is not yet possible, although there are several studies where the differences in various parameters (mainly weight and measures of airway size and collapsibility) were studied in responders and non-responders [39, 116, 117]. The best correlates were always weight and oropharyngeal airway size. However, although these studies provide useful information, particularly with respect to the factors that determine airway occlusion, they do not as yet provide us with a method to predict who will respond to this therapy. Oral appliances vs CPAP Since CPAP remains as the “gold standard” treatment of sleep apnea, the objective success rate of any other treatment must be judged against it. There are seven randomized, crossover studies, which compared mandibular advancement appliances against CPAP. The results are summarized in Table 6. We note that the findings of all such studies are remarkably consistent—CPAP results in better improvement in AHI than oral appliances. In all studies except one, CPAP normalized the respiration, bringing AHI to less than ten. In one study where AHI with CPAP remained above ten, the highest pressure used was 10 cm H2O, which is probably too low. However, patients subjectively prefer oral appliances over CPAP. In five out of seven studies, the patients expressed preference for an oral appliance, in one study neither treatment was preferred and in another study CPAP was preferred. Table 6Randomized, crossover, CPAP vs oral appliance studiesReferenceNAHIbaseAHIapplAHI CPAPComments[46]192010468% of patients were satisfied with OA vs 62% with CPAP (p < 0.05)[45]21342011OA preferred[50]202514465% preferred OA, 30% preferred CPAP[84]2018144“Patients identified oral appliance as being easier to use”[85]4831158“Neither treatment was significantly preferred by patients”[88]242283“...17 out of the 21 subjects who completed both arms of the study preferred the MAS”[92]8021145“Although subjects reported that CPAP was the most difficult treatment to use, they felt that it was the most effective and overall preferred it to the MAS, which was in turn preferred to placebo”Summary23224146Oral appliance preferred overall There are 2 additional investigations [87, 102], which are not listed in Table 2 because of missing data regarding follow-up sleep study. Nevertheless, these investigations provide interesting information regarding the comparison of oral appliances and CPAP. Smith and Stradling [87] attempted to determine whether oral appliances can substitute CPAP at least for 1 month. The authors found that patients achieved similar reduction in ODI4 with CPAP (from 29 to 1) as with oral appliance (from 29 to 4). However, the patients did not like their oral appliance and were refusing to use it. Out of 50 patients on CPAP who were switched to oral appliance, only 11 were still using it by end of 1 month. Most patients discontinued its use because of discomfort, side effects, or treatment failure. This study therefore favors CPAP. On the other hand, McGown et al. [102] carried out a questionnaire survey of 126 patients treated with oral appliances. There were 41 patients who had tried both CPAP and oral appliance; 71% preferred oral appliance, 19% preferred CPAP, and 10% were unsure. This study favors oral appliance. We conclude therefore that CPAP is more effective than oral appliances in reducing AHI, but despite this, most patients prefer oral appliances, undoubtedly because they find them to be less cumbersome than CPAP. Oral appliances vs other treatments Since 1988 there were several studies [21, 53, 59, 64, 65, 68, 77, 79, 89, 90, 100], which compared either different types of appliances, different degrees of protrusion or different inter-incisal distance. The results are listed in Table 7. Columns labeled “base” and “appl” show the AHIbase and AHIappl, respectively. The column labeled “comp” gives the AHI measured when other, i.e. “comparison” treatment was used. The type of oral appliance employed and the precise variables that were compared are described in the last column. Since most studies employed parallel group design, baseline AHI before comparison treatment is also shown in this column. Table 7Studies comparing oral appliances to treatments other than CPAPReferenceNAHICommentsBaseApplComp[53]2343.50.8Parallel groups; OA=MAA with max protrusion, comp=MAA with 70% of maximum protrusion, but double inter-incisal opening; baseline AHI = 7 for comp group[59]4118610Parallel groups; OA=MAA, comp=UPPP; prospective, randomized, baseline AHI = 20 for UPPP group; results at 12 months[64]37261711Single group; OA=MAA with 2 mm protrusion, comp = 6 mm protrusion; ODI4 recorded[65]5503044Single group; OA=MAA, comp=TRD; only 5/8 patients agreed to try TRD[65]2473557Single group; comp=SPL; only 2/8 patients agreed to try SPL[68]242398Crossover, randomized; OA=MAA (Herbst), comp=monoblock[21]105510Parallel groups: OA=TRD, comp=somnoplasty; baseline RDI same for both groups[77]2321810Crossover, randomized; OA=MAA with 4 mm inter-incisal opening, comp=MAA with 14 mm opening[79]7218714Parallel groups, randomized; OA=MAA, comp=UPPP; baseline AHI for UPPP group = 20; results at 4 years[89]84471716Parallel groups, randomized; OA=MAA with 50% protrusion, comp=MAA with 75% protrusion; baseline AHI for 75% group = 50; results at 6 months[90]551666Parallel groups, randomized; OA=MAA with 50% protrusion, comp=MAA with 75% protrusion; baseline AHI for 75% group = 19; results at 12 months[100]16462534Crossover, prospective, randomized; OA=Herbst, comp=Twin Block; median AHIs reportedSummary392261112 Examination of the individual investigations reveals that when oral compliances are compared to each other, either two different appliances or the same appliance with different degrees of protrusion or opening—it is cleat that the efficacy (objective and subjective) is very much dependent on the type of appliance and the degree of advancement. This further emphasizes the point that oral appliance therapy should be carried out by a dentist with expertise in this field who is familiar with different types of appliances and can select the most appropriate one for the particular patient. There is no “best” appliance. The best one is that which is comfortable to the patient and achieves the desired efficacy. There are several studies, mainly case series, comparing oral appliances with surgical treatments. Comparisons with UPPP [59, 79] demonstrated the superiority of oral appliances. At 1 year follow-up, sleep apnea was resolved (AHI < 10) in 78% of the oral appliances group and 51% of the UPPP group. With longer follow-up, this success rate deteriorates. At 4 years follow-up, 63% of the oral appliance group and 33% of the UPPP group continue to have AHI < 10. One parallel group study of oral appliance (TRD) vs radiofrequency ablation (somnoplasty) found that both treatments significantly reduced sleep time spent with loud snoring. However, there was no difference between oral appliance and somnoplasty [21]. A recent small case series of 4 patients (out of 43 treated with oral appliances) who elected maxillomandibular advancement surgery [121] showed that initial AHI = 50 dropped to 12 with oral appliance and to 2 after surgery. Oral appliances for the treatment of snoring Although everyone can recognize snoring, it proved to be a very elusive entity to measure objectively. One can define the sound properties (i.e. frequency spectrum and intensity), relationship to breathing (i.e. waxing and waning sound, generally during inspiration), and measure this sound during sleep. However, subjective recognition of sound, which satisfies some pre-defined “snoring” criteria as de facto snoring depends very much on the listener. This is contrary to the case of apneas or even hypopneas, whose definition is independent of any subjective perception. These difficulties with the definition of snoring are the reasons why objective measurement of sound is seldom a routine part of polysomnography. However, snoring is the cardinal symptom of sleep apnea. In fact, it is frequently the only reason why these patients come to the sleep clinic in the first place. Consequently, when polysomnography does not reveal sleep apnea in these patients, the physician still has to deal with their snoring. Unfortunately, this is often ignored by physicians. The most frequent scenario is that a patient is referred to a sleep specialist because of snoring, polysomnography is carried out, no sleep apnea is found, the patient is reassured, advised to loose weight, stop smoking and drinking alcohol, embark on an exercise program, and discharged from the clinic. Sometimes this advice, dispensed in the form of preprinted sheets, is given also to non-obese non-smokers. Clearly, the patient leaves unhappy, the referring physician is dissatisfied with the help received from the specialist and nothing was accomplished to justify the expense incurred in the process of investigations. For apneic snorers, the problem is simpler because treatment with CPAP will abolish snoring. Non-apneic snorers without daytime symptoms do not tolerate CPAP well. Many of them will agree to try it, but the majority will stop using it after a short time (generally a few weeks to a few months). Oral appliances therefore constitute an attractive alternative for the treatment of snoring. In fact, they were originally invented precisely for that reason. Many, but not all investigations of oral appliances comment on their efficacy in reducing snoring. Recognizing that the objective measurement of sound during polysomnography may not correspond to the perception of this sound as being snoring—subjective assessment is generally employed. This assessment varies from simply asking an informal question “is your snoring improved?”, to employing a more formal method, which is usually a visual scale (analogue or digital) to rate snoring. Unfortunately, in almost all studies, the answers to these subjective questions are given by the snorers who of course are unaware of their snoring, rather than by the bed partner. Although snoring is recorded as the patient’s chief complaint, it is really not the patient’s complaint at all—it is the complaint of the bed partner. The implicit assumption in most investigations is that the patient’s responses reflect those of the bed partner. Nevertheless, the efficacy of snoring treatment must be assessed by the same bed partner, not by the snorer. This poses great logistical problems in carrying out appropriate investigations. However, some support for this assumption of equivalence between the responses of snorers and listeners is provided by a recent questionnaire data of Bates and McDonald [115] who found that 70% of snorers and 70% of the bed partners reported improvement in snoring after using a mandibular repositioning splint for 3 months. Table 8 lists 47 investigations, which utilized oral appliances. The only common feature among these investigations, and the reason why other investigations were not included—is that all of the listed ones contained a specific comment regarding snoring. The types of snoring assessment carried out in these investigations ranged from an informal question about snoring to rating the snoring using a visual scale and objective sound measurement together with subjective assessment. Very few investigations included objective measurement of snoring. Investigations with subjective assessment of snoring using questionnaires form a very inhomogeneous group because they used different types of questionnaires with different rating methods. Given these differences in methodology, it is not possible to rigorously summarize the results. However, certain generalizations can be made. Table 8SnoringReferenceNTypeSnoring measureCommentsBaseAppl[27]5TRD“Snoring decreased or completely disappeared”[28]12MAA“8/12 reported substantial reduction of sonorous sleeping[31]1MAA“After appliance insertion...immediate...reduction in snoring”[34]68MAA8.51.5Snoring severity assessed subjectively (max score = 10); snoring eliminated in 42%[35]12MAASnoring reduced, although never eliminated, in 79%[38]24MAA7.6Snoring on a scale from 0 to 10; improvement also on a scale from 0 to 10—result = 4.3[42]51MAA9.48.2No. of snores/min; snoring eliminated in 8/51, improved in 43/51[46]25MAASnoring less than “moderate” in 19/25 pts[47]23MAA“...20/23 patients (87%) reported subjective improvement...in snoring”[49]23TRD“Visual analog scores of snoring...were also reduced significantly” in 6/23 (23%) subjects[53]23MAA“...loud snore duration was reduced from a median of 27.1 min to 11.4 min”[54]44MAA“Snoring was satisfactorily reduced in” 37/44 patients (84%)[57]15MAA19320Median snores/h given; snoring loudness and time spent snoring also improved[102]132MAA“Snoring was reported...to be satisfactorily controlled in 107 (81%)...[58]14MAA6/14—no snoring; 8/14—mild snoring[59]41MAA0.70.5No. of snores/h of sleep at baseline and 12 months follow-up (NS)[60]75MAA“Dramatic reduction in the attributes of snoring was achieved”[66]112MAA76/112 (68%) snoring either eliminated or acceptable[68]24MAA5033No of snores/h sleep; results for Herbst appliance; 19/20 disturbed by snoring at baseline, vs 9/20 with appliance[21]10TRD113Percent of time spent in loud snoring[69]39MAA“Time with snoring dropped significantly from 16.3% to 6.6%”[71]22MAASnoring eliminated in 13/22, significantly reduced in 5/22; success rate = 18/22 (82%)[104]53MAAQuestionnaire survey; 27/53 were still using the device at 1 year, and 22 were satisfied (42%)[105]21MAAQuestionnaire survey; 22 patients fitted with appliance; 43% thought it reduced snoring, 48%—no benefit[102]126MAAQuestionnaire survey; “80 out of 94 patients reported improvement in snoring”[73]22MAA5924No. of snores/h of sleep; subjective improvement as well[75]25MAA3.41.8Randomized controlled vs placebo-appliance; non-apneic snorers; frequency of snoring (nights/week), p < 0.05; 15/25 greatly improved with MAA vs 2/25 with placebo[76]33MAA“19/33 had short-term satisfactory treatment results with the device”[72]28MAA402242Randomized, controlled vs placebo-appliance; snores/h of sleep (p < 0.005); mean snoring intensity significantly reduced; “the majority of patients reported substantial improvement in snoring (70%)[82]73MAA366207Randomized, controlled vs placebo-appliance; snores/h of sleep (p < 0.0001); mean and maximum snoring intensity significantly reduced; significant subjective reduction in snoring[86]16MAA3.12.6Randomized, placebo-controlled crossover; frequency of snoring (nights/week) (p = 0.07); no significant difference in loudness of snoring[79]32MAA0.70.5Duration of snoring/h of sleep (p < 0.01)[80]26MAA“The patients and their bed partners thought that...snoring...improved...”[84]20MAA5536Snoring epochs/h (p < 0.01); randomized crossover study vs CPAP[89]40MAA0.860.57Duration of snoring/h of sleep (p < 0.001); comparison of two protrusions; results for 75% protrusion group[90]26MAAComparison of two protrusions; results for 75% protrusion group; “problems with apneas and snoring...decreased by...79%...”[91]25MAA“...snoring...patients have benefited from oral appliance therapy and their spouses will testify to the same”[93]44MAASubjective assessment; “on average, a mean reduction of 90% of the intensity of snoring was reported by the patients”[95]619MAA“It is estimated that 50% of the 619 snorers and sleep apnea patients had treatment success or subjective beneficial effects...”[96]11MAA24075Noise level measured; “10 out of 11 subjects had a significant reduction in snore noise sound level...”[97]20MAA9.06.8VAS 0–10 scale (p < 0.05); result at 6 months follow-up; subjectively 14/20 were satisfied[106]110MAAQuestionnaire survey; 37 out of 77 patients who returned questionnaire thought snoring was satisfactorily controlled[99]17MAA5316p = 0.02; snoring time[19]25MAA15/25 snoring markedly improved[100]16MAA14464Snores/h; however, VAS 0–10 scale–no difference[101]251MAA75% of 191 users of appliance reported control of snoring; 43% of non-users of appliance also thought snoring was controlled[115]53MAASnoring was reported to be improved by 70% of the responding bed partners First, the majority of the investigations concluded that oral appliances are beneficial in reducing snoring in the majority of patients. Second, all of the randomized, placebo-appliance-controlled studies except one [86] found significant reduction in snoring, independently of whether it was assessed objectively or subjectively. Johnston et al. [86] did not find a significant difference in either the loudness (measured using the VAS 0 to 5 scale) or the frequency (nights/week) of snoring. However, an earlier study from the same group [75], employing similar methodology but different patient population (non-apneic snorers), did demonstrate significant reduction in snoring. This further illustrates the difficulties with subjective assessment of snoring in different patient populations. Even in the same patient population, there is a discrepancy between objective measurement and subjective perception, as found by Lawton et al. [100]. We shall present the summary of the individual investigations listed in Table 8 as follows. First, we selected only those investigations where a numerical value describing snoring with and without appliance was given. These results are shown in Table 9. There are 18 studies involving 529 patients. All, except one [21], employed the mandibular adjustment appliance. All of these investigations give a “number” to quantify snoring. We note the diversity of measurement of snoring in each study—the “number” in Table 9 represents either a VAS score, number of snorers/h (or min) of sleep, amount of time spent with loud snoring per hour of sleep or per night, number of nights per week spent with disturbing snoring or noise level, etc. However, we can calculate the percent change between the baseline night and the “with appliance” night, displayed in the last column of Table 9. We note that despite the diversity of snoring measurements, in all investigations the percent change is negative—which indicates that the investigators always found reduction in snoring with oral appliances. The mean reduction in snoring using the pooled data was 45%. Table 9Studies with measurement of snoringReferenceNSnoring measureExplanation of snoring measurementPercent changeBaseAppl[34]688.51.5Visual analogue scale 0–10−82[42]519.48.2Number of snores/min−13[57]1519320Number of snores/h−90[59]410.70.5Number of snores/h−29[68]245033Number of snores/h−34[21]10113Percent of sleep time spent in loud snoring−73[73]225924Number of snores/h−59[75]253.41.8Nights/per week with disturbing snoring−47[72]28402242Number of snores/h−40[82]73366207Number of snores/h−43[86]163.12.6Nights/per week with disturbing snoring−16[79]320.70.5Minutes of snoring/h of sleep−29[84]205536Snoring epochs/h of sleep−35[89]400.860.57Minutes of snoring/h of sleep−34[96]1124075Noise level−69[97]209.06.8Visual analogue scale 0–10−24[99]175316Total snoring time−66[100]1614464Number of snores/h of sleep−56Summary529−45 Effect of oral appliances on daytime function In assessing the effect of oral appliances on sleep apnea syndrome, it is not sufficient to focus only on the apnea/hypopnea index or snoring. We must also demonstrate the effect of this treatment on daytime function, which is almost always compromised in patients with sleep apnea and sometimes in non-apneic snorers also. There are several tools used to assess daytime function, but there is little consistency in using these tools in investigations involving oral appliances. Table 10 summarizes the results of investigations where some assessment of daytime function was performed, no matter how primitive. The methods of assessment differ in each investigation. Some relied on answers to questions regarding daytime sleepiness and tiredness, others utilized visual scales, yet other investigations measured response time to various tasks, etc. After 1995, the majority of investigations employed the Epworth sleepiness score (ESS). The results generally show improvement in daytime symptoms with oral appliances. Table 10Functional assessmentReferenceNTest or questionResultCommentsBaseAppl[11]14Daytime function––14/14 reported improvement[25]7Sleepiness––Improved[26]44Vigilance0.50.4Reaction time (p < 0.05)[27]5Daytime sleepiness––“Daytime somnolence was eliminated or diminished markedly”[28]12Daytime somnolence––“9/12 patients reported increased alertness and/or reduction in daytime sleepiness”[34]63Prevalence of daytime sleepiness––“51% of these patients reported no more sleepiness with orthosis use”[35]12Daytime sleepiness––“Daytime sleepiness was improved...in all but two patients”[33]14Symptom score (including sleepiness)5.51.1Significant(p < 0.001) improvement in daytime symptoms[38]24Sleepiness and improvement using 0–10 Likert scale6.4–Improvement on 0–10 Likert scale = 4.5 at 36 months[42]51Patients tired; patients sleepy4430[44]30No. of mistakes in vigilance test7.63.7p < 0.05[45]21EDS daytime symptoms2.41.61–5 scale; p < 0.0001) for all symptoms[46]25Prevalence of EDS84%40%p < 0.005; significant reduction in prevalence of other symptoms[47]23Patients with EDS2320[49]14EDS using VAS––Reduction in scores (p < 0.05) in 6 of 14 patients who were compliant with treatment[50]20ESS10.34.7p < 0.05; EDS improved in 13/20 patients[53]14ESS124.5Median score, p < 0.005[53]9ESS74Median score, p < 0.005[54]44Patients with daytime sleepiness4434[58]14Patients with moderate and severe daytime somnolence100[59]41Daytime sleepiness on 1–5 scale––Prospective, randomized, UPPP group and OA group; “in comparison with their baseline values...a significant (p < 0.001) reduction in subjective daytime sleepiness”[60]75ESS117p < 0.0005[107]90Quality of life (vitality+contentment+sleep)12994Significant improvement compared to baseline; two parallel groups—OA vs UPPP; no difference in vitality and sleep[66]112No. of patients “refreshed by sleep”–66/114“Most of the regular users had an improvement in their quality of sleep and day time somnolence...”[68]24ESS13.18.6p < 0.001; identical result for two different MAAs[71]22No. of patients whose sleepiness disappeared17/22“17 (85%) of 22 patients reported subjective improvement in excessive daytime sleepiness”[72]24ESS10.13.9p < 0.01[73]22ESS127.5p < 0.05 at 12 to 30 months follow-up[75]24ESS7.56.5p < 0.01; randomized placebo-controlled crossover trial of non-apneic snorers[76]19No. of patients reporting reduction in EDS13/19[77]23ESS1812p < 0.0001; identical result for two different MAAs[81]26Questionnaire: EDS1.61Scale from −3 (maximum deterioration) to +3 (maximum improvement)[87]34ESS137.7After 28 days, only 11 patients continued to wear MAA; initial ESS based on 34 patients, final—on 11[88]24ESS13.49.0p < 0.001; randomized crossover vs CPAP[85]48ESS1412NS; randomized crossover vs CPAP; extensive tests of daytime function; “these results do not support these MRS devices as first-line treatment for sleepy patients with SAHS”[82]73ESS119p < 0.0001); “the proportion of patients with normal subjective sleepiness was significantly higher with the MAS than with the control device (82 vs 62%, p < 0.01), but this was not so for objective sleepiness (48% vs 34%, p = 0.08)”[86]18ESS12.611.6NS; randomized placebo-controlled crossover trial of apneic snorers[89]42ESS11.57.5p < 0.001; prospective randomized comparing 50% and 75% protrusion; result for 75%; initial value—42 patients, final value—40[90]55Questionnaire: EDSRandomized comparison of two protrusions; “82% of patients in 50% group (n = 29) and 84% in 75% group (n = 26) reported a decrease in daytime sleepiness”[96]29ESS9.46.9p < 0.001[92]80ESS10.29.2p < 0.001; randomized vs placebo pill and CPAP[93]40ESS12.05.1p < 0.001[97]20ESS8.85.4p < 0.05[98]42ESS106p < 0.02; median values[19]27ESS96p < 0.001; median values[100]16ESS108Median values[101]161ESS117In users of OA; in 90 non-users—ESS fell from 11.1 to 8.1[115]67Concentration, energy levels, sleep quality, ESS9.7–ESS given; 29–59% of responders reported improvement[122]73ESS and full battery of neuropsychological measures5.04.2Total score of all self-report measures given; prospective, randomized, placebo-appliance-controlled 4 weeks study Because of the diversity of methods assessing daytime function, it is difficult to pool and summarize the individual data. One way of doing this is to select only those investigations, which employed identical methods of assessment and summarize the mean results, as was done in the previous tables. The most common single way of assessing daytime function in most investigations was the ESS. These investigations are listed in Table 11. There are 23 of them involving a total of 962 patients. However, not all investigations could be used in calculating pooled data. We rejected four investigations [19, 53, 98, 100], involving 108 patients where only the median and not the mean values of the ESS were reported. Consequently, we are left with 19 investigations involving 854 patients. As a group, these patients were only mildly sleepy with the mean ESS of 11.2. In all investigations, the ESS dropped with the use of oral appliances. For the entire group, there was a significant reduction in the ESS from 11.2 to 7.8. Table 11Functional assessment using ESSReferenceNESSCommentsBaseAppl[50]2010.34.7p < 0.05; EDS improved in 13/20 patients[53]14124.5Median score, p < 0.005[53]974Median score, p < 0.005[60]75117p < 0.0005[68]2413.18.6p < 0.001; identical result for two different MAAs; [72]2410.13.9p < 0.01[73]22127.5p < 0.05 at 12 to 30 months follow-up [75]247.56.5p < 0.01; randomized placebo-controlled crossover trial of non-apneic snorers[77]231812p < 0.0001; identical result for two different MAAs[87]34137.7After 28 days, only 11 patients continued to wear MAA; initial ESS based on 34 patients, final ESS—on 11[88]2413.49.0p < 0.001; randomized crossover vs CPAP[85]481412NS; randomized crossover vs CPAP; extensive tests of daytime function; “these results do not support these MRS devices as first-line treatment for sleepy patients with SAHS”[82]73119p < 0.0001); “the proportion of patients with normal subjective sleepiness was significantly higher with the MAS than with the control device (82% vs 62%, p < 0.01), but this was not so for objective sleepiness (48% vs 34%, p = 0.08)[86]1812.611.6NS; randomized placebo-controlled crossover trial of apneic snorers[89]4211.57.5p < 0.001; prospective randomized comparing 50% and 75% protrusion; result for 75%; initial n = 42 patients, final n = 40 patients[96]299.46.9p < 0.001)[92]8010.29.2p < 0.001; randomized vs placebo pill and CPAP[93]4012.05.1p < 0.001[97]208.85.4p < 0.05[98]42106p < 0.02; median values[19]2796p < 0.001; median values[100]16108Median values[101]161117Result for users of OA; in 90 non-users—ESS fell from 11.1 to 8.1[122]739.17.1Prospective, randomized, placebo-appliance-controlled 4 weeks studySummary85411.27.8References 19, 53, 98–100 were excluded (no mean values were given) In two investigations [85, 86], both randomized, crossover and controlled (one vs placebo and another one vs CPAP) reduction in ESS was not significant. Engleman et al. [85] carried out a very extensive study of daytime function comparing the effect of oral appliance to CPAP. Functional assessment included maintenance of wakefulness test, measures of daytime sleepiness and symptoms, measures of well-being (using the SF-36 questionnaire, HADS anxiety and depression score), and cognitive performance. The results favored CPAP in 7 out of 21 variables (including the ESS, AHI, effectiveness and symptoms), and showed no difference between CPAP and oral appliance in other variables (including the maintenance of wakefulness tests, cognitive performance and treatment preference). Johnston et al. [75, 86] compared oral appliance to placebo, but did not carry extensive investigations of daytime function—only the ESS and a 5-point scale describing how refreshed the patients felt in the morning. The authors found significant improvement compared to placebo, but only in non-apneic snorers [75], not in patients with sleep apnea [86]. In all other randomized, crossover, controlled studies there was a statistically significant improvement in the ESS, but not in other subjective measures of daytime performance. In fact, none of the studies demonstrated a significant improvement in all of the subjective outcomes studied. This is not surprising because almost all studies comparing placebo treatment with active treatment, no matter what it is, always demonstrate a significant placebo effect. Probably the most complete assessment of neuropsychological function was carried out by Naismith et al. [122] in a prospective, randomized, placebo-appliance-controlled study of 73 patients treated for 4 weeks. The authors demonstrated significant improvement in the measures of self-reported sleepiness, fatigue and energy levels, but no improvement in the measured speed/vigilance (except for the improved reaction time), attention/working/verbal memory or visuospatial/executive functioning. Walker-Engstrom et al. [107] compared the quality of life in two parallel groups of patients with sleep apnea 1 year after treatment with either oral appliance or UPPP. The ESS was not measured, but there was other extensive assessment of three quality of life dimensions (vitality, contentment and sleep). Both groups improved compared to the baseline. There was no difference in vitality and sleep dimensions between the two groups, but the UPPP group was more content than the oral appliance group. The effect of oral appliances on daytime function was not studied as fully and extensively as for CPAP. For example, there are no studies comparing driving simulator performance in patients treated with oral appliance, no studies comparing multiple sleep latency or maintenance of wakefulness. Recognizing the limited nature of the data—the conclusion from all of the investigations taken as a group must be that oral appliances improve daytime function, although they are not necessarily superior or consistently preferred than other treatments such as CPAP and UPPP. Effect of oral appliances on vascular disease Numerous investigations examined the relationship between sleep apnea and vascular events, such as coronary artery disease, hypertension, and cerebro-vascular disease. Fewer, but still many investigations were carried out to examine the effect of the treatment of sleep apnea with CPAP on changes in these conditions. Quite the opposite situation is seen regarding the effect of oral appliances on vascular disease. There are only three studies examining the effect of the treatment of sleep apnea with oral appliance on blood pressure. Both studies employed the randomized, controlled, crossover design. The first study was carried out by Gotsopoulos et al. [113] specifically for the purpose of examining the effect of 4 weeks treatment with a mandibular advancement splint on 24-h blood pressure in 67 patients with sleep apnea (mean AHI = 27). There was approximately 3.5 mmHg drop in the systolic and diastolic blood pressure with treatment, but only during wakefulness. There was no change in blood pressure during sleep. The second study by Barnes et al. [92] compared the effect of 3 months treatment with oral appliance to CPAP and placebo (a tablet). In 110 patients with sleep apnea (mean AHI = 21), the 24 h blood pressure was measured. Treatment with oral appliance (but not with CPAP or placebo tablet) resulted in the significant reduction in nighttime diastolic blood pressure by 2.2 mmHg. There were no changes in diastolic blood pressure during wakefulness and no changes in systolic blood pressure either during wakefulness or sleep. The third study was carried out by Yoshida [126] who measured blood pressure in 161 patients with sleep apnea before and after 60 days of treatment with oral appliance. There was a statistically significant drop in blood pressure from 132.0/82.1 to 127.5/79.2 mmHg. Regression analysis demonstrated weak, but significant correlation between the mean arterial and baseline blood pressures and the reduction in AHI. This area of investigation is still in its infancy, and undoubtedly, more results will be forthcoming in the future. There are no rigorous studies of the effect of oral appliances on other vascular diseases. Eskafi et al. [99] carried out a single night, unattended, home sleep study in 17 patients with sleep apnea (mean AHI = 25) and congestive heart failure with periodic breathing before and after intervention with a mandibular advancement device. The authors found improvement in sleep apnea (mean AHI reduced from 25 to 15), but no improvement in periodic breathing or left ventricular ejection fraction after 6 months of treatment. There is not enough evidence at the present time to draw any conclusions regarding the effect of oral appliance therapy on vascular disease. This remains a very interesting area of investigation. Given the differences in intra-thoracic pressure as a consequence of CPAP vs oral appliance, it is possible that results obtained with oral appliances therapy will be different from those obtained with positive pressure therapy. Side effects Almost every study describing oral appliances comments on the side effects voiced by patients. The type of side effects and their frequency depend on the questions asked, the rating scale, the number of patients in the study, etc. Some studies specifically focused on the side effects and compliance with treatment [101, 102, 106, 110], others simply asked a few questions about the side effects. Table 12 summarizes some of the common side effects; there is also a reference to the study, which reported the highest frequency of this particular side effect. Table 12Patient reported side effects of oral appliancesSide effectPercent of patientsReference reporting maximum percentDifficulty in chewing11–19[101]Excessive salivation9–60[106]Dry mouth14–86[73]Tooth discomfort11–59[73]Tongue discomfort6–8[101]Jaw discomfort8–41[73]Gum discomfort1–2[101]Headache2–27[89]Occlusive changes41[76]TMJ pain37[102]Masseter muscle pain45[66]No side effects at all100[39]; 14 subjects Excessive salivation, mouth, and teeth discomfort are the most common side effects reported. However, patients seldom consider these side effects troublesome. Provided that this complaint is addressed by the dentist and the appliance is adjusted, they continue to use it. In many cases the side effects are transient and disappear with continued use. When patients stop wearing the oral appliances it is mainly because of ineffectiveness, rather than because of side effects, although in some studies [95, 101] up to 40–50% of patients discontinued the use of the appliances because of the side effects. One study [39], involving only 14 patients all using the Herbst appliance, reported that none of the patients had any side effects! The conclusion, based on the results of most studies, is that when oral appliances are properly constructed by the dentist with expertise in this area, they are relatively comfortable in the majority of patients. There are also dental effects of oral appliances. It is still not entirely clear if long-term use of oral appliances will lead to permanent adverse dental changes, particularly when used in children. There are several studies addressing various dental–skeletal changes of oral appliances using various imaging techniques. One of the most common effects, commented upon in many studies, is the degree of vertical and horizontal overlap of the teeth (overjet and overbite, respectively). These results are summarized in Table 13. There are 11 distinct studies, involving 694 patients with mean follow-up time of 43 months. Two studies [78, 118] were rejected because only the median results were given, and one study [127] was rejected because no mean values for overjet and overbite were shown, only changes in these parameters over a period of 3 years. A summary of the remaining data involving 389 patients with mean follow-up of 39 months shows that the overbite is reduced from 3.8 to 2.4 mm and the overjet is reduced from 4.0 to 2.7 mm. It is clear that dental–skeletal effects of oral appliances are certainly present, but the long-term results and their clinical significance are unknown at this time. The recent studies of Marklund [118] and de Almeida et al. [119] described patients who were using mandibular advancement appliances for more than 5 years. Their results suggest that orthodontic changes (1) are variable (favorable in some and unfavorable in others), (2) are clinically relevant, and (3) might be predictable from the initial dental characteristics of the patients and the type of device. Table 13Dental effects: overjet and overbiteReferenceNF/U (months)OverjetOverbiteCommentsBaseF/UBaseF/U[39]19134.0−3.02.96.3Herbst appliance[108]32244.54.13.63.5[71]2265.971.083.97−8.01After correction for magnification error[109]87304.253.194.093.07Effects evident already at 6 months[73]22143.33.14.03.6Median results at follow-up[110]47283.93.63.22.8Significantly larger changes compared to reference group[110]28314.53.93.83.3Ibid[81]34304.43.13.62.5[111]30483.53.14.33.8NS; compared to UPPP[112]20303.842.634.432.61Effects evident at 6 months[90]2912––2.52.4NS; for 50% protrusion; same for 75% protrusion[118]187603.53.03.02.80Median values; overbite change NS; orthodontic side effect increase with treatment time and more frequent use[119]31892.120.452.70.4670 patients followed-up for 7.4 years; measurements made from models; “unfavorable change” group[119]10892.752.94.453.87As above; “no change” group[119]29893.952.724.472.52As above; “favorable change” group[127]6736––––Only changes, but not baseline values in overjet and overbite are given (−0.8 and −0.6, respectively over 3 years); small but significant reductions observed mainly during the first yearSummary389394.02.6References [73, 118] were excluded418373.72.4 Compliance Compliance with oral appliances depends strictly on the balance between the perception of benefit and side effects. Most patients treated with oral appliances have relatively mild sleep apnea and relatively few daytime symptoms; the main reason for treatment was snoring. Consequently, the perception of benefit is generally that of the bed partner, whereas the side effects are experienced by the wearer of the appliance. This is why the assessment of compliance is a complex issue. In some cases, although the appliance is quite comfortable, the patient may stop wearing it if the bed partner is no longer present or no longer complains of snoring. Table 14 shows the results of studies, which provide compliance data. All of them except one [49] employed mandibular advancement devices. There is a very wide variability between individual investigations—from as little as 4% to as high as 76% at the end of 1 year. Table 14Compliance with oral appliancesReferenceNF/U (months)Compliance (%)[34]71771–75[35]24124–5[38]243650–75[39]192468–93[47]294155–70[49]23621[103]1913152–76[107]451282[66]173945–70[70]2563190[71]226100[76]336258[102]1662242–56[79]454862[81]8618–2430–53[89]741272–76[106]1102240–57[95]6301275–76[101]5446830–64[115]92368[118]4506056Summary3,1073356–68 The largest study is that of de Almeida et al. [101]. It is based on a mail survey of 544 patients, of whom 251 returned the questionnaire on the average of almost 6 years after the construction of the appliance. The majority of patients were fitted with MAAs, although some had TRDs. At the time of follow-up, 161 patients continued to use the appliance. Assuming “the worse case scenario” (i.e. all those who did not return the questionnaire were no longer using the appliance) the compliance rate is 161/544 = 30%, while in the “best case scenario” the compliance rate is 161/251 = 64%. Among those who used the appliance, 82% of bed partners were satisfied with this treatment; even among the non-users of appliances, 46% of bed partners were satisfied. The main reasons for discontinuing the use of the appliance were discomfort (44%) and perception of little or no benefit (34%). Pooled data summarizing all 21 reviewed studies involving 3,107 patients, showed that at the end of 33 months, 56–68% of them continued to wear the appliance. Conclusions Oral appliances used to date constitute a relatively heterogeneous group of devices for the treatment of sleep apnea and non-apneic snoring. It is this heterogeneity, which partly accounts for the variability in their benefit and side effects. Another reason for variability is the diverse methodology employed in different studies. The evidence available at present indicates that oral appliances successfully “cure” mild-to-moderate sleep apnea in 40–50% of patients, and significantly improve it in additional 10–20%. They reduce, but do not eliminate snoring. Side effects are common, but are relatively minor. Provided that the appliances are constructed by qualified dentists, 50–70% of patients continue to use them for several years. Their effectiveness is inferior to CPAP. It is similar to surgical procedures, but these are invasive, (although not particularly dangerous) and irreversible. The effect of oral appliances on the vascular consequences of sleep apnea is not known. The place of oral appliances in the spectrum of treatment options for apneic and non-apneic snorers was extensively discussed in various reviews and guidelines, including the most recent report by the American Academy of Sleep Medicine [124, 125]. The current review does not alter those conclusions. It simply illustrates the marked variability of individual responses to oral appliance therapy, and therefore the necessity to approach each patient on an individual basis. Patients with sleep apnea should be informed about all treatment options. In some cases, the decision is simple; after informing patients about all available options, a strong and clear recommendation can be given by the health care practitioner. In other cases, the decision regarding treatment is arrived at only after individual consideration of all the factors—urgency of clinical situation, reimbursement plan available to patient, risk factors and the patient’s ability or motivation to modify them, patient’s preferences, and a possibility of having a trial of treatment with oral appliance and CPAP. There are patients with severe sleep apnea successfully treated with oral appliances, just as there are non-apneic snorers with or without upper airway resistance syndrome, successfully treated with CPAP. The decision regarding treatment in each individual patient is best made by medical practitioners with experience in sleep medicine who are aware of all options, and who are preferably a part of a specialized sleep disorders center. An important issue, not addressed in this review, is the underuse of oral appliances currently. This is due in part to the lack of qualified dentists working in this area and in part to reimbursement policies. At present, the majority of government-sponsored and private health care providers will cover (fully or partially) the cost of CPAP, whereas very few, if any, health care plans will cover the cost of oral appliances. Considering that this treatment approach is the only non-invasive alternative to CPAP, it is important to continue to lobby health care providers to enable this treatment for qualified patients.

Dig_Dis_Sci-3-1-1914298

Triggering for Submaximal Exercise Level in Gastric Exercise Tonometry: Serial Lactate, Heart Rate, or Respiratory Quotient?

Gastric exercise tonometry is a functional diagnostic test in chronic gastrointestinal ischemia. As maximal exercise can cause false-positive tests, exercise buildup should be controlled to remain submaximal. We evaluated three parameters for monitoring and adjusting exercise levels (heart rate [HR], respiratory quotient [RQ], and serial lactate measurements) in 178 tests in both healthy volunteers and patients suspected of gastrointestinal ischemia. Exercise levels above submaximal occurred in 20% of HR-, 2% of RQ-, and 5% of lactate-monitored tests (P<0.05 for HR vs. RQ and lactate). Low levels were seen in 5% of HR-, 10% of RQ-, and 41% of lactate-monitored tests (P<0.01 for lactate vs. HR and RQ). High levels resulted in 43% false-positive tonometry results compared to 19% of all tests (P<0.001); low levels did not result in more false negatives (5% vs. 6%). Although RQ monitoring yielded the greatest proportion of optimal exercise tests, serial lactate monitoring is our method of choice, combining optimal diagnostic accuracy, low cost, and simplicity. Introduction For diagnosis in patients with suspected chronic gastrointestinal ischemia, we have previously shown that gastric exercise tonometry can be used as a functional test providing information about the adequacy of the gastrointestinal mucosal perfusion [1, 2]. In these studies it was demonstrated that during 10 min of submaximal exercise, gastric ischemia occurred only in patients with splanchnic artery stenosis. An extreme exercise level may cause false-positive tests, as shown in a study in healthy volunteers, where lactate levels exceeding 8 mM resulted in gastric ischemia in 50% [3]. Furthermore, it may be conceivable that exercise of very low intensity can lead to false-negative results in exercise tonometry used for diagnosing chronic gastrointestinal ischemia. In order to prevent false-positive and false-negative tonometry tests, the exercise intensity should be monitored throughout the test, and if necessary, adjustments of the workload should be made in order to obtain an optimal exercise test. Exercise intensity can be monitored by various parameters including arterial plasma lactate concentration, decrease in serum arterial base excess (BE) or bicarbonate concentration (which are both directly related to lactate level), heart rate (HR), and respiratory parameters [respiratory gas exchange ratio (RQ) = carbon dioxide output (VCO2)/oxygen uptake (VO2)] [4]. In this study we evaluated and compared three consecutive time periods in each of which a different parameter was used for monitoring the exercise intensity and adjusting the workload in order to obtain a submaximal exercise test. Initially HR monitoring was used; in the second period exercise intensity monitoring and adjustment were guided by monitoring of RQ. In the final period exercise intensity was monitored by serial rapid lactate measurements. Subjects and methods In 10 volunteers (5 females, 5 males; mean age, 25 years; range, 23–28 years) and 157 patients (59 males, 98 females; median age, 55 years; range, 13–82 years), 178 tonometry exercise tests were performed. The volunteers were tested as part of a more extensive study investigating the effect of two different exercise levels on gastric tonometry [3]. All patients were suspected of having symptomatic chronic gastrointestinal ischemia. Their clinical presentation was that of unexplained abdominal pain, weight loss, diarrhea, or gastric ulcers. Gastric tonometry exercise testing was performed as a diagnostic function test in addition to duplex sonography and selective angiography of the splanchnic vessels. Some of the patients in this study have been previously described in a publication by our group investigating the diagnostic potential of gastric exercise tonometry [1]. The procedure of gastric tonometry exercise testing was described in detail in a previous study [3]. In short, a standard nasogastric tonometry catheter was inserted and connected to an automated air tonometry device that was set up to measure intragastric partial carbon dioxide pressure (PCO2) every 10 min. All subjects were studied after a fasting episode of 4 hr. Ranitidine, 100 mg, was given intravenously 90 min prior to exercise testing. A radial artery catheter was introduced in the nondominant arm to allow sequential arterial blood sampling. Exercise was performed on an electromagnetically braked bicycle ergometer (Lode, Groningen, Netherlands). Monitoring exercise intensity and adjusting the workload The maximal workload (Wmax) was estimated using standard criteria, i.e., age, sex, and weight [5]. The exercise episode was started at 10% of Wmax, and in the first 4–6 min the workload was increased every minute by 10% of Wmax. The workload was intended to remain constant thereafter at a submaximal exercise level. Three different approaches for monitoring the exercise intensity and—if necessary—guiding additional adjustments of the workload were evaluated in consecutive periods. First period: HR-triggered tests Exercise intensity was monitored by measuring HR, aiming for a HR in the last 4–5 min of 80% of maximal predicted HR (HRmaxpred). HRmaxpred was calculated by the following formula: HRmaxpred = 210 − 0.65, age [6]. Second period: Respiratory gas exchange ratio (RQ)-triggered tests The workload adjustments were guided by measurement of the RQ, aiming for an RQ of 1.0 in the last 4–5 min of the exercise episode. This RQ was chosen as it indicates the anaerobic threshold Third period: Lactate-triggered tests In 40 of the RQ-triggered tests serial rapid lactate measurements were done during exercise at t = 0, 4, 6, 8, and 10 min. These measurements were not used for guiding the exercise intensity, but to develop a lactate-based exercise triggering protocol. From the results of rapid serial lactate measurements in the RQ-triggered period, an algorithm was developed for triggering the exercise intensity (see Table 1). We aimed for a lactate concentration of 4 mM in the last 4 min of the test. In parallel with RQ triggering at an RQ value of 1.0, this lactate level resembles the anaerobic threshold, at which redistribution of blood flow patterns is initiated.Table 1Algorithm for lactate-guided exercise intensity adjustmentsStart at 10% of predicted WmaxIncrease workload by 10% of Wmax every minute (at t = 1, 2, 3, 4, 5, and 6 min)Lactate measurements at t = 4, 6, 8, and 10 minIf lactate >3 mM, decrease workload by 10% of WmaxIf lactate not >1 mM, increase 2 × 10% of Wmax In the first two periods (HR and RQ triggering) the tests were performed at the pulmonary function department of our hospital. During these exercise tests a 12-lead electrocardiogram was recorded (Case 12; Marquette Electronics Inc., Milwaukee, WI, USA). HR was recorded every minute. Breath-by-breath oxygen uptake (VO2) and RQ were measured by a respiratory gas analyzer system (Oxycon-α; Jaeger, Bunnik, the Netherlands) and recorded every 30 sec. In the third period (rapid serial lactate triggering) the test was performed at the gastroenterology function department; HR was monitored, but respiratory parameters were not monitored. Arterial blood samples for determination of base excess (BE), bicarbonate (blood gas analyzer; Radiometer ABL520, Copenhagen, Denmark), and lactate (enzymatic assay; Cobas Fara; Roche Diagnostics, Branchburg, NJ, USA) were drawn, in parallel with the 10-min tonometry interval, before and immediately at the end of the 10-min exercise episode. Rapid serial lactate measurements were performed using a small portable lactate analyzer (Accutrend; Roche Diagnostics, Almere, the Netherlands), specifically developed for use during exercise testing. Using this device, measurement results were available within 60 sec after blood sampling. The tests evaluated in the present study were all aimed at a submaximal level: lactate level (or BE decrease) at or just above 4 mM (the anaerobic threshold) and not exceeding 8 mM. After comparison with arterial lactate measurements (see Results), BE decrease during exercise was used as the parameter assessing the exercise intensity (Fig. 1). The optimal exercise level was a BE decrease of between 3 and 7 mM. Less than 3 mM and more than 7 mM were regarded as low and high exercise levels, respectively.Fig. 1Relationship between arterial lactate concentration after exercise and BE decrease at the end of the exercise period For comparing the results of HR and RQ measurements with BE decrease as the indicator of the exercise intensity reached, the following parameters were used: maximum RQ reached (RQmax), total time RQ>1 (RQt>1), maximum heart rate reached (HRmax) as a percentage of HRmaxpred, and total time of HR >80% of HRmaxpred (HRt>80). Statistics All values are given as mean ± SD unless otherwise stated. P values <0.05 were considered statistically significant. Correlations between lactate and BE and lactate and bicarbonate decrease, and between both HR and RQ parameters and exercise intensity as measured by BE decrease, were calculated using Spearman’s rank test. For comparison of HR and RQ parameters with exercise level (as divided into low, optimal, or high), one-way ANOVA with Bonferroni post hoc analysis was used. Group comparisons of the tonometry results of high and low exercise levels versus all tests and the diagnostic accuracy of tonometry in the different monitoring episodes were performed using chi-square testing. Results HR triggering In 39 tests (10 in healthy volunteers and 29 in patients) HR triggering was used. In 22 (56%) of these HR-triggered tests the mean HR in the last 4 min equaled the target HR of 80% ± 5% of HRmaxpred. In nine tests (23%) the target HR was not reached; in five of these the subjects were using β-blocking medication. In eight tests (20%) the HR was >85% of HRmaxpred. The resulting exercise levels as measured by BE decrease are listed in Table 2. In three tests the exercise intensity was too low. In two the target HR was not reached; one of the subjects used β-blockers. Two tonometry results were true negative; the other was false positive.Table 2Resulting exercise intensities for the three triggering regimesExercise level(BE decrease, <3 mM)(BE decrease, 3–7 mM)(BE decrease, >7 mM)Triggering methodLowTargetHighHR (39 tests)3 (8%)27 (69%)9 (23%)*RQ (84 tests)8 (10%)74 (88%)2 (2%)Lactate (55 tests)23 (41%)**29 (53%)*3 (5%)Note. Values are given as numbers (percentage of total for triggering method). *P < 0.05 and **P < 0.001 comparing the three triggering regimes. Of the eight tests with an erroneously high exercise level, in only one test was the HR too high. The other six tests in which the HR was too high resulted in the submaximal exercise levels aimed for. When analyzing the 110 tests in which both HR and RQ were recorded, only a weak correlation was observed both between HRmax and exercise intensity and between HRt>80 and exercise intensity as measured by BE decrease (r=0.23 and r=0.20, respectively; P<0.05). There was no significant difference for the HR-derived parameters comparing low, target, and high exercise intensity. Figure 2 displays the comparison of HRmax for the three exercise intensity groups.Fig. 2HRmax (95% confidence interval) at low, target, and high exercise levels HRmax is expressed as percentage HRmaxpred RQ triggering In 84 tests RQ triggering was used. In 67 (80%) of these tests the target RQ of 1.0 was reached while the mean RQ in the last 4 min was 1.0 ± 5%. In 11 tests (13%) the target RQ was not reached and in 6 tests (7%) the mean RQ in the last 4 min exceeded the target RQ by more than 5%. Of the eight tests with a low exercise level, the target RQ was not reached in four. In both tests with a too high exercise intensity, the mean RQ in the last 4 min was >1.05. When analyzing all 110 tests in which RQ was monitored, positive correlations were found for comparing RQmax and RQt>1 with BE decrease (r = 0.34 and r = 0.38, respectively; P < 0.0005). When divided into low, target, and high exercise intensity groups, both RQmax and RQt>1 showed significant differences among the three groups (P < 0.0005; one-way ANOVA with Bonferroni post hoc analysis). Figure 3 displays the comparison of RQmax for the three exercise intensity groups. Lactate triggering In 55 tests lactate triggering was used. In 26 of these tests the target lactate level of 4.0 mM in the last 2–4 min was not reached. This resulted in a BE decrease below 3 mM in 19 tests. In two tests the rapid lactate measurements exceeded 8 mM at t = 6 or 8 min. In one, the resulting exercise level was too high (BE decrease>7 mM). Of the 27 tests in which the target lactate levels were reached (at least 4 mM at t = 6 or 8 min, and remaining below 8 mM), the resulting exercise levels were too low in 7 and too high in 2. Arterial lactate vs BE decrease and bicarbonate decrease In 78 tests both arterial lactate levels and blood gas parameters were obtained. A high degree of correlation of both BE decrease and bicarbonate decrease with lactate level after exercise was found: r = 0.93 and r = 0.89, respectively (P < 0.0005). Figure 1 displays the relation between arterial lactate and BE decrease after exercise. Mean paired difference between lactate and BE decrease was 0.7 (SD, 0.7) mM, and that between lactate and bicarbonate decrease was 1.4 (SD, 0.9) mM. Comparison accuracy of exercise tonometry at low, target, and high exercise levels As shown in Table 1, lactate triggering resulted in significantly more low exercise levels compared to the other two regimes (P < 0.001).Fig. 3RQmax (95% confidence interval) at low, target, and high exercise levels False-negative tonometry results were found in 10 of all 178 tests (6%). Low exercise levels did not result in an increase in false-negative tonometry tests: in only 2 of 10 (5%) false-negative tests was the exercise level too low; 1 test was RQ-triggered, the other lactate-triggered. Tonometry results were false positive in 34 of all 178 tests (19%). Of the 14 tests with a too high exercise level, in 6 (43%) the tonometry tests were false positive (P < 0.001 comparing high exercise level vs. all tests). Three of these tests were HR-triggered (but HR did not exceed the target HR), two RQ-triggered, and one lactate-triggered. The sensitivity and specificity of tonometry exercise testing for gastrointestinal ischemia did not differ among the three groups (82% and 73%, respectively). Discussion For optimal diagnostic accuracy, the exercise level in gastric exercise tonometry can be monitored and adjusted by RQ measuring or, alternatively, by serial arterial lactate measurement. Although the latter resulted in more tests with lower than desired exercise levels, this had no influence on diagnostic accuracy. The greatest problem relating to exercise level in gastric exercise tonometry is the above-submaximal, or maximal, test, as this is associated with 43% false-positive tests in this study. In maximal exercise, splanchnic blood flow is severely reduced and can lead to reduction below the level required to maintain aerobic metabolism. Although data on the splanchnic vascular responses to maximal exercise have not been established, ischemia has been established frequently in these circumstances [3, 7]. In a study on splanchnic blood flow in trained volunteers, mean superior mesenteric artery blood flow fell by 38–49% after a 10-min exercise test at 70% of maximal workload [8]. It has been shown that the flow reduction in the celiac artery exceeds that of the superior mesenteric artery but still does not exceed about 50% [9]. Therefore the normal splanchnic flow after submaximal exercise remains well above the normal lower level of normal splanchnic blood flow, which is estimated as approximately 30% of the normal baseline value [10]. This explains why submaximal exercise does not result in ischemia in subjects with normal splanchnic vessels. Gastric ischemia during submaximal exercise can be seen in patients with splanchnic stenoses or in patients with reduced cardiac output [1, 11]. Thus, for practical clinical purposes 10 min of submaximal exercise seems adequate to distinguish between normal and pathological gastrointestinal vascular responses. Some exercise should be performed to allow for blood flow redistribution and reduction of the splanchnic blood flow and, thus, provocation of ischemia. However, the minimally required level is currently unknown. Even with very low-intensity exercise in healthy humans a significant shift in blood flow from the abdominal viscera to the exercising muscles was observed [12]. Thus submaximal exercise as currently advocated might not be necessary, and lower lactate levels may suffice for diagnostic testing. Triggering on RQ measurements resulted in the highest proportion of tests within the target range but has disadvantages. It requires a pulmonary function laboratory with specific devices for measurement of exhaled carbon dioxide and inhaled oxygen, making the test expensive and more complicated. The advantage of RQ monitoring in minimizing the proportion of below-target exercise level did not result in a lower number of false-negative tests in this study. The major advantages of the serial lactate measurement are its simplicity and low cost. This rapid test can be performed by paramedic personnel and enables determination of lactate within 60 sec. This enables adequate monitoring and triggering of exercise levels. A gastric tonometry exercise test, a routine procedure at our hospital in patients suspected of chronic gastrointestinal ischemia, takes 15 min of a doctor’s time and 2 hr for a registered nurse. Moreover, the currently available air tonometry device allows for accurate and reproducible measurements without the hassle and problems associated with classically used saline tonometry [13]. In conclusion, by using RQ and serial lactate measurements, adequate exercise levels for gastric exercise tonometry can be achieved. Rapid lactate measurements and the presented algorithm for gastric exercise tonometry are feasible for daily clinical practice.

Dig_Dis_Sci-3-1-1914244

Melanoma of the Anus Disguised as Hemorrhoids: Surgical Management Illustrated by a Case Report

Introduction Anal mucosal melanoma is a rare tumor that constitutes only 0.4–1.6% of all melanoma manifestations [1]. Prognosis is very poor, with a median survival of less than 2 years, despite curative surgery [2, 3]. Females are more likely to be affected than males and most patients present in the sixth or seventh decade of their lives [1]. Diagnosis is not always straightforward, and is often accidental after surgical treatment for presumed benign disease such as hemorrhoidectomy or lateral internal sphincterotomy, provided that tissue is submitted to the department of pathology [4]. Unfortunately, the tumor is often widely metastasized at the time of initial diagnosis [5, 6]. Here we present a patient who was referred to the surgeon for treatment of hemorrhoids but was diagnosed with anal melanoma. Case report A 62-year-old black man with a history of prostatism was referred to our hospital by his general practitioner with the diagnosis of bleeding hemorrhoids. For a 10-week period he noticed a swelling protruding from the anus with daily bleeding from the anus. Defecation was problematical because of pain, but the stool had a normal aspect. Inspection of the anus showed a painful swelling with a diameter of 1.5 cm with a dark necrotic aspect surrounding the anus below the dentate line. Rectal palpation was impossible because of pain. At the outpatient department the swelling was excised under local anesthesia of the entire anus (anal block). Because of the atypical aspect of the swelling, the material was submitted for pathologic examination, which revealed a melanoma of the anus. Further staging of the tumor was not possible based on this material because of fragmentation of the tissue. A sigmoidoscopy was performed but showed no further lesions. In addition, an abdominal and thoracic computed tomographic (CT) scan with contrast showed no signs of tumor around the anorectal region and no signs of intraabdominal, lymphatic or thoracic metastases. After the patient was diagnosed with having melanoma of the anus, examination under general anesthesia was performed. Inspection of the anal mucosa showed two additional lesions at 2 and 3 o’clock with the sacrum at 6 o’clock (Fig. 1). A wide local excision (WLE) including these lesions was performed with a margin of 2 cm, en bloc with 25% of the circumference of the rectal wall (Fig. 1). The total excised tissue was 6×7 cm. Pathologic examination revealed two small foci of the melanoma with a depth of maximal 3.5 mm. All margins were free of tumor. The patient recovered well from the operation. After 1-year follow-up by means of outpatient department visits and proctoscopy every 3 months, and thoracic and abdominal CT-scan twice a year, there were no signs of recurrent tumor.Fig. 1Melanoma of the anus: wide local excision. A=melanoma Discussion Patients with anorectal melanoma usually present with symptoms of rectal bleeding and an anorectal mass and are often misdiagnosed as having hemorrhoids, as was illustrated by the present case report [4]. Therefore, on any atypical anorectal lesion, biopsy should be performed in order to prevent delayed diagnosis. For the operative management of anorectal melanoma, two options are available: a wide local excision (WLE) or a more extensive abdominal perineal resection (APR). The choice between these surgical procedures is controversial. The main arguments in favor of APR are its ability to control lymphatic spread and to create bigger excision margins, resulting in an assumed lower local recurrence rate. But, in contrast to WLE, APR is associated with mortality, considerable postoperative morbidity (4% hemorrhage, 11–16% wound infection and 14–24% wound dehiscence) and the need for a permanent colostomy [7–11]. Despite attempted curative surgery, the median survival for anorectal melanoma is only 20 months and most patients die within 5 years regardless of the type of intervention used [12]. Therefore, quality-of-life issues must be given consideration when making treatment decisions. Droesch et al performed a systematic review of the literature, including 14 studies with a total of 301 patients [12]. Wide local excision was performed in 129 patients, and 172 patients underwent APR. Local recurrence developed in 47% of patients after WLE and in 23% of patients after APR. Although these data suggest a trend towards better locoregional control after APR, the difference was not statistically significant. In addition, there was no difference in overall survival between WLE and APR (median survival 21 months for WLE and 17 months for APR). Bullard et al found an opposite trend with a rate of 50% of local recurrence after APR, compared to 18% after WLE in 15 patients [5]. Postoperative radiotherapy may improve locoregional control after wide local excision. In the retrospective study of Ballo et al, actuarial 5-year local control was 74% and nodal control was 87% in 23 patients who received postoperative radiotherapy after wide local excision [13]. Definitive assessment of the efficacy of adjuvant radiation therapy requires further prospective studies. In general, it is assumed that the stage of the disease, especially the tumor thickness, is the main prognostic factor [13–15]. Therefore, Weylandt et al suggested that the decision between WLE and APR should be governed by the tumor thickness [16]. In patients with a tumor thickness below 1 mm, a local sphincter-saving excision with 1 cm safety margin would be appropriate; and in cases of a tumor between 1 and 4 mm, a wide local excision with a margin of 2 cm seems to be adequate. Patients with a tumor thickness above 4 mm or invasion of the internal sphincter muscle should be treated with APR. In the case report mentioned in this article, the tumor depth was 3.5 mm and excision margins were free of tumor, thus no additional APR was performed. In order to evaluate the deepness of the lesion preoperatively, endorectal ultrasonography can be performed [6]. Accuracy in evaluating tumor depth of rectal cancer ranges from 81 to 94%, and accuracy in detecting lymph node metastasis ranges from 58 to 80% [17]. However, its accuracy in evaluation of anorectal melanoma remains unproved and is still evaluated [5]. At the time of diagnosis, up to one-third (16–33%) of the patients have disseminated disease [3]. For these patients, palliative treatment with chemotherapy might be a treatment option. Kim et al reported a series of 18 patients with metastatic anorectal melanoma treated with cisplatin, vinblastine, dadabazine, interferon alpha-2b and interleukin-2 [18]. Major response was seen in 44% of the patients, and complete response occurred in 11%, with a median overall survival of 12.9 months. Yeh et al used a different treatment regimen of temozolomide, cisplatin and liposomal doxorubicin in a patient with stage IV anal mucosal melanoma [19]. This patient showed a remarkable response to chemotherapy, with minimal residual disease and excellent quality of life at 12 months after the start of treatment. However, this therapy still has to be further evaluated and currently no standard systemic therapeutic regimen exists for metastatic anorectal melanoma. In conclusion, anorectal melanoma represents both a diagnostic and therapeutic challenge to physicians given its non-specific presentation and rarity. It is associated with poor prognosis, regardless of the type of intervention used. Therefore, the overall treatment goal should be to optimize the quality of life. Since wide local excision is a more limited intervention associated with at least comparable survival compared to APR and no need for permanent colostomy, wide local excision is recommended as primary therapy if negative surgical margins can be achieved. APR should be reserved for patients in whom the tumor is thicker than 4 mm and/or involves the anal sphincter.

J_Urban_Health-2-2-1705510

Exploring Barriers to ‘Respondent Driven Sampling’ in Sex Worker and Drug-Injecting Sex Worker Populations in Eastern Europe

Respondent driven sampling (RDS) has been used in several counties to sample injecting drug users, sex workers (SWs) and men who have sex with men and as a means of collecting behavioural and biological health data. We report on the use of RDS in three separate studies conducted among SWs between 2004 and 2005 in the Russian Federation, Serbia, and Montenegro. Findings suggest that there are limitations associated with the use of RDS in SW populations in these regions. Findings highlight three main factors that merit further investigation as a means of assessing the feasibility and appropriateness of RDS in this high risk population: the network characteristics of SWs; the appropriate level of participant incentives; and lack of service contact. The highly controlled and hidden nature of SW organizations and weak SW social networks in the region can combine to undermine assumptions underpinning the feasibility of RDS approaches and potentially severely limit recruitment. We discuss the implications of these findings for recruitment and the use of monetary and non-monetary incentives in future RDS studies of SW populations in Eastern Europe. Introduction In many countries in Eastern Europe large scale social and economic transition over the past 15 years has been associated with widespread poverty and increasing levels of unemployment and population migration, particularly among women.1,2 Such transition has also been associated with shifts in patterns of sexual behaviour and sexual relationships, including increased commercial sex work, the growth of informal sex work economies and associated human trafficking.3 Additionally, a number of countries in the former Soviet Union have experienced a rapid or explosive spread of HIV associated with injecting drug use.4–7 These epidemics are potentially exacerbated by close links between sex work and injecting drug use.2,8–11 This has caused concerns that high levels of sexual mixing between injecting drug users (IDUs) and their sexual partners in settings of high HIV and sexual transmitted infection (STI) prevalence may transform HIV infection from concentrated epidemics among risk populations to generalised epidemics.9,10 It is therefore essential to assess the diffusion of HIV and related risk behaviour in hidden vulnerable populations, such as IDUs and sex workers (SWs), as accurately as possible. There has been increased and recent interest in an approach to sampling hidden populations known as respondent driven sampling (RDS).12,13 One of the claimed advantages of RDS over other methods of sampling hidden populations, such as time–location sampling, is that it requires little in-depth formative research among study populations.13,14 RDS begins with a set number of non-randomly selected seeds (members of the target population). Seeds recruit their peers (other target population members who make up their social network) who in turn recruit their peers into the study. This occurs through successive waves of recruits which, it is argued, become increasingly more representative of the underlying population as the recruitment progresses. Since RDS claims to generate a representative sample from non-randomly selected seeds, many researchers have assumed that sampling can proceed without detailed prior knowledge about the local target population.13,14 RDS has proved feasible and successful in recruiting hidden populations of IDUs in a variety of settings, resulting in the rapid acquisition of long and varied recruitment chains.11,14–19 RDS has also been used in several countries to collect both behavioural and biological data from SWs (e.g., Vietnam, India) and men who have sex with men (e.g., Bangladesh, Cambodia, Uganda, United States).20–24 However, little is known about the feasibility of RDS as a method of recruiting hidden populations of SWs in settings where the organisation of sex work and access to SWs is highly controlled and where there is little or no contact between the target population and local services. Drawing upon three case studies in Eastern Europe targeting SWs and IDUs involved in SW (IDU/SWs), we explore the feasibility of RDS, identifying some barriers to successful recruitment. These findings have implications for the direction of future research exploring the feasibility of RDS methods. Methods Using RDS, data on HIV prevalence and associated risk factors were gathered from SWs in Serbia and Montenegro and from IDUs involved in sex work in Russia (see Table 1). SWs in Serbia and Montenegro were given three uniquely numbered coupons with which to recruit other SWs. In Russia, although no RDS study directly targeting SWs was carried out, RDS was used to sample SWs/IDUs in an RDS study among IDUs. Incentives were provided to participants completing the survey (primary incentive) and for recruiting other eligible participants (secondary incentive). Participants in Belgrade were also offered a rapid HIV test and pre- and post-test voluntary and confidential counselling (VCT). Those with preliminary-positive tests for HIV were referred to a local clinic for confirmatory testing. The studies were undertaken by local fieldwork teams coordinated as part of larger internationally funded research initiatives. Table 1.Three RDS studies among sex workers and injection drug using sex workersCountryCity (population size)Formative researchRecruitment criteria for SWsNumber of seedsSample reached (target)Year, study durationSerbiaBelgrade (about 2 million)Literature search; questions on network characteristics and RDS study feasibility posed during individual interviews with SWs in 2005 (n=10)Exchanged sex for drugs, money or other goods in last 30 days (irrespective of gender or sexual orientation)Six initially, six added later209 (400)2005, 2 monthsMontenegroPodgorica (about 300,000)Literature search; questions on network characteristics and RDS study feasibility posed during in-depth interviews with SWs in 2005 (n=4)Exchanged sex for drugs, money or other goods in last 30 days (irrespective of gender or sexual orientation)One to start with, one added later2 (150)2005, 2 monthsRussian FederationTogliatti (740,636)Literature search [relied on previous qualitative study of IDUs conducted in 2001 (n=57) and epidemiological survey of IDUs including SWs (n=426)]Exchanged sex for drugs, money or other goods and injected drugs in last 4 weeksOne, 35 SWs recruited by a female non-SW IDU seed36 (not set)2004, 1.5 months All studies were approved by appropriate local ethical committees and international human subject review boards. Participants were required to agree to an informed consent before participating. Participants were not linked to the study with any personal identifiers, and all participant information was kept confidential and anonymous. Prior to data collection, formative research, comprising a combination of key informant interviews and a literature review, was undertaken to assess the local feasibility of the RDS study identifying the optimal study set up (Table 1, column 3). The formative research elicited data on size estimates of the sex work population; description of how sex work is organised (sectors of sex work, freedom of movement, average earnings and prices charged for services in each sector); and the legal situation surrounding sex work. In Serbia and Montenegro, researchers also asked SWs about the size of their social networks in order to fine-tune questions on which RDS analysis would be based. A social network was defined as the number of SWs, 18 years and older, each SW knows and has seen in the past week, month, three months, six months, or year. Results As shown in Table 1, a total of 209 SWs were recruited in Belgrade (Serbia), two in Podgorica (Montenegro) and 36 in Togliatti (Russian Federation). Case 1: Serbia RDS was conducted in the autumn of 2005 in Belgrade, Serbia’s capital, by a local fieldwork team with a limited access to SW networks. Estimates of the size of the SW population in the municipality, based on police records and anecdotal estimates, vary widely from 900 to 3,000 SWs.2,24,25 Sex work is illegal. Findings from in-depth interviews of SWs suggest that there are approximately ten areas located near the main transit routes in and out of Belgrade where street SWs operate in small groups. Qualitative data suggests that the majority of SWs operate independently, advertising their services in newspapers or through referrals via existing client networks, potentially making them harder to reach through RDS. Formative interviews also found that there was little mixing of SWs across different sex work sectors (street, agency, and SWs working independently), among street SWs themselves (transvestite, Roma and Serbian/Montenegrin heterosexual SWs) and among SWs working independently. However, in addition to a lack of strong connection among SWs and their independent working situations, the study team undertaking formative qualitative research associated SWs’ reluctance to participate in the study to perceptions of inadequate incentive (10 euros for 1 hour of their time) and a general mistrust associated with coming into contact with ‘official’ agencies. However, the same interviews suggested that in principle SWs would be interested in participating in a survey involving the collection of both behavioural and biological data through HIV testing. Interviewees indicated that they knew approximately 20 other SWs, which again suggested that RDS would be feasible. The RDS study had a target of 400 SWs, both male and female. The sample needed to be large enough for a statistically reliable analysis of risk factors and HIV prevalence, should RDS analysis show that the sample is reasonably representative. The interview site was on the premises of a non-governmental organisation (NGO) located in central Belgrade easily accessible by public transport. It was a part of a large apartment block with a separate entrance. The site was open everyday from 3 to 9 p.m. and could accommodate three to four participants an hour because of the computer administered questionnaire. After two months, the study ceased, having reached only 209 SWs. In total, 12 seeds were used during the study. Of the initial six seeds only three produced any subsequent waves. The rate of recruiting increased noticeably after an RDS study of IDUs being implemented at the same site had ceased. Due to the difficulties experienced by the research team in locating new seeds through other means, four participants with dense social networks were drawn from the seventh wave of two of the most productive recruitment chains and used as new seeds. This resulted in two more productive chains that ultimately reached seven waves. The average SW network size reported during the study was 12. In response to the question, “where do you most often solicit your clients?,” the majority of SWs replied that they solicited on the street (43%), followed by referral from an agency, pimps, friends or other SWs (28.9%) and by telephone (25.2%). Importantly, the take-up of VCT provided as part of the RDS study was high (71.8%), and SWs indicated that they participated because of the HIV testing, rather than the monetary incentive. Case 2: Montenegro There are no estimates of the number of SWs in Podgorica, the Republic’s capital. Sex work is ostensibly illegal. Through formative rapid assessment no street-based SWs were identified and the majority of SWs were found to work independently from their homes. Key informant interviews (IDUs, ex-IDUs, taxi drivers, public health officials, NGO representatives and journalists) identified the only semi-public sex work sites as being approximately six illegal brothels, registered as lap dancing venues, in which most of the SWs also lived. At least one such brothel was found not to allow SWs to leave the premises. Indications were that local police also ‘moonlighted’ as security guards in these brothels. Other links between brothels and public services included taxi drivers, beauty salons and hotels. The intersection of the police and sex work networks, the small size of the city and the high number of implicated actors suggested that SWs could be easily monitored and controlled. Previous attempts by the local NGOs to conduct research among SWs in Podgorica had failed. Key informants, interviewed during formative rapid assessment, reported knowing at least one SW or their pimps but in almost all cases introductions were denied or never materialized. Those conducting the formative rapid assessment received strong advice from local experts and key informants—including NGOs, police, and taxi drivers—against approaching brothel owners because of the highly controlled nature of SW venues in the city. After two weeks of trying to make a contact, four SWs were accessed via their drug user networks, the only entry points into SW networks available to researchers. These SWs were interviewed at their place of work (brothel and pimp’s flat). Each of the four interviewed SWs independently estimated that there were approximately 200 SWs in the city, many of them also drug injectors, most of whom worked independently, advertising their services via newspapers and television and via client recommendation but who were not open about their SW status. All four SWs interviewed indicated that they would participate in a RDS study and would invite others to do so, and each estimated they had personal regular contact with between five and ten SWs. The four SWs participating in the formative rapid assessment were invited to participate in the RDS study as seeds. Only one of the four participated but failed to recruit anyone else. She reported having had contact with ten SWs in 4 weeks, but no additional seeds were secured. Despite recruiting an additional SW (who required complete anonymity fearing being identified as a SW by others to his family and who had not seen other SWs in the past month), the RDS study failed to identify a sufficient pool of seeds willing to participate. Case 3: Russia The RDS study of IDUs and IDU/SWs was conducted in May 2004 in Togliatti City. Togliatti City is the second city of Samara Region situated approximately 1,000 km south of Moscow. Togliatti was selected as a study site because evidence suggests a recent increase in the number of IDUs and SWs and an explosive spread of HIV associated with injecting drug use.6,11 Approximately 2,000 SWs are estimated to operate in Togliatti, half of whom work from the street.6 Despite ambiguous Russian legislation towards sex work, it is not tolerated by the wider community or the police. The city has been noted for its intense street policing of drug users and SWs, often involving fines levied under administrative codes, such as possession of drugs, causing a public nuisance, or lack of official residency permit, rather than criminal codes.26,27 Street-based SWs in Togliatti operate openly along the main roads and highways. Two popular locations are Moskovsky Prospect where approximately 70 women are working at any one time, and Pobeda Street where about 100 women work. In a study conducted in Togliatti City in 2001, 50% of female IDUs (n=155) reported ever exchanging sex for goods or money, of whom 86% (n=66) were currently SWs.6 Although no stand-alone RDS studies of SWs have been conducted in Russia, attempts have been made to boost participation of IDU/SWs in RDS studies of IDUs motivated by the evidence of high proportion of SWs who also inject drugs in other cities.28–30 A cross-sectional survey of IDUs and female IDU/SWs was undertaken in Togliatti in 2004 as a follow up to the 2001 baseline survey. A total of 476 IDUs were sampled during a 5-week period in May 2004 using RDS. Among these, 55 (11%) reported ever exchanging sex for money, drugs or goods, and of these, 36 in the past month, which was lower than expected. At the start of the field work, seven seeds were selected, of which one seed was female who had exchanged sex in the past month. SWs were explicitly asked to recruit other SWs, and non-SWs were encouraged to recruit either SWs or non-SWs (Table 1, column 4). This first SW seed produced no recruits and was lost to follow up. Another female non-SW started a chain that recruited a total of 22 female IDUs who had ever exchanged sex, of whom 13 had exchanged sex in the past month. This female non-SW seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample, of which 15% (20) were SWs and who reported working on the street. Among the other seeds, only a handful of current SWs were recruited (n=4). Towards the end of the project more coupons were distributed to female SWs in an attempt to increase the SW sample (n=5), none of which produced any recruits. In order to increase the sample of SWs in the survey, 21 SWs were subsequently identified and interviewed without coupons. Data on the number of female SWs within an individual’s IDU network were unfortunately not provided. The mean IDU network size among SWs was 19.5 (SD 14.2) and among non-SWs 27.1 (SD 33.0). Only one SW recruited through RDS reported working from an apartment; all others were street-based SWs. We expected that RDS would enable us to recruit more hidden populations of SWs such as those working from apartments or hotels. The fact that SW seeds failed to produce any waves of SWs or were lost to follow up, indicates that SW networks may not be as well connected. Field notes show that initial attempts to access street SWs at Moskovsky Prospect and Pobeda Street had failed primary because of intensified police action in the area. A local officer of the Federal Security Service believed he had been recently infected with HIV by a SW from the area, and, in revenge, he and his colleagues enforced a repressive policy of fines and arrests among SWs. Recruiting SWs towards the end of the survey was more successful when policing practices returned to normal. Meanwhile, the sample target was reached for non-SW IDUs. Discussion Despite undertaking formative research in the planning of these RDS studies, we found that RDS methods were unable to attain sufficient samples of SWs in Serbia, Montenegro, and Russia. A number of inter-related factors emerged as important to the success of RDS: low motivation of SWs to participate due to their perception of inadequate incentive; low-density SW networks combined with high level of independence in the organisation of sex work; a tightly controlled small number of sex work venues; lack of contact between SWs and local services in the context of high levels of mistrust of ‘official’ agencies; and difficulties emerging in the organisation of the RDS studies of SWs at the same time as a parallel RDS study of IDUs. Whereas in other settings (e.g., Vietnam, north-eastern India, Brazil) SWs have reacted favourably to nominal monetary incentives in RDS studies, including incentives which fall below the rate charged for sex,1617 it is possible that the incentives offered were perceived as being too low. In order to earn primary and secondary incentives, which together in all three studies amounted to about a half-an-hour SW’s rate, they had to leave their work and travel to and from the fixed interview site at least twice plus spend at least an hour involved in the study. It is possible that SWs in Eastern Europe may be less interested in the incentives offered since they have comparatively higher earnings. Indications are that free HIV/STI health assessments and referral for treatment should be explored in addition to the incentive. The high acceptance of VCT in Belgrade, for example, is a good example of how provision of services can be more effective than monetary incentives in recruiting survey participants. If incentives are purely monetary, they should be adequate to entice SWs to participate; yet this is often not feasible given the risk of high incentives also encouraging the attempted participation of non-SWs. Unlike other RDS studies conducted in Vietnam,16 SWs in Belgrade felt uncomfortable being interviewed at a location where a concurrent study of IDUs was being conducted. Based on anecdotal information and the fact that SWs in Belgrade started participating in the study once the IDU study had finished, we believe that SWs were dissuaded from going to a location that was used by IDUs. This problem can be assessed during formative research using focus groups with SWs to assess whether they would feel comfortable sitting in a waiting room with a group of male and female IDUs. Perhaps the main reason for the poor success of RDS recruitment among SWs in these three settings is the characteristics of SW networks. RDS methodology requires that the target population form sufficiently large and dense networks. However, the information on the network sizes of a few SWs gathered through our formative research are not easy to interpret since there is no known cut-off point for determining an appropriate network size or density for RDS. Based on the results of our studies, SWs in our three study areas often work independently, thus forming smaller, more isolated, networks. Those working on the street and in brothels are also under tight control by managers, pimps and police thus restricting their movement and weakening network density. On the other hand, prevalent societal norms—a form of social control—prevent SWs who work independently from revealing their SW status and participating. The low recruitment rate encountered in our studies might be overcome by allowing studies to run for a much longer time—perhaps as long as six months or a year—allowing SWs to gain trust in the investigators. In the studies described above, interview sites were rented and staff employed for a fixed amount of time. A preferable setting is one with on-going services for SWs that can be used as an interview site for a long period of time allowing recruits to come at a slow rate. However, time was unlikely a major factor contributing to difficulties in RDS recruitment in the cities where our studies were conducted. The dearth of well-established projects targeting SWs in Eastern Europe means that greater effort should be invested prior to RDS studies to identify a greater number of seeds and engender SWs’ trust. In some cases existence of a well-established service with established contacts with local SW communities might be a decisive factor. For example, this was the case in a RDS study among SWs in Tbilisy, Georgia (January 2006, written communication, Johnston LG). In addition to promoting trust, such service organisations can provide additional networking opportunities for loosely connected SWs. Conclusion Given the cost of conducting second-generation sero-surveillance among highly vulnerable populations, formative research is important for establishing the appropriate type of sampling and reward system. We found the making available free and confidential HIV testing services was an effective incentive, and perhaps more so than monetary incentives alone. Although formative research would require some additional costs, it can increase efficiency in a more extensive and successful study. During formative research, we suggest that policy makers, service providers and target populations be consulted to determine the visibility of sex work, as low visibility can be associated with tight control of the sex work industry and weak networking among SWs, indicating difficulties using RDS. If researchers find it difficult to recruit SWs for formative study, the RDS study is likely to fail. Depending on the timescale, funding and interest among existing service providers, additional services may be provided well ahead of a RDS study in order to access SW respondents. Further investigation is needed into the ethnography of SW networks in this region, especially on network composition and service access. In the case of IDUs who exchange sex for money, goods, or drugs, it is important to understand their primary network linkage—injecting drug use or sex work—and how they represent their “other identity” within each network. Incentives need to be better identified in advance of RDS studies, specifically as they relate to level of income, opportunity costs, and trust in the investigators and seeds. Without this cultural and legal sensitivity, surveillance for HIV among this vulnerable population will not provide adequate information on epidemic spread or program effectiveness.

Ann_Hematol-4-1-2174523

Association between long travel and venous thromboembolic disease: a systematic review and meta-analysis of case-control studies

The term “economy-class syndrome” defines an infrequent episode of venous thromboembolism (VTED) related to a long travel, namely by plane. However, this relation has not clearly been demonstrated by investigators. We carried out a systematic review and a meta-analysis of cases-control studies that had studied this topic. We realised a systematic review of the literature and selected all the case-control studies published. Two authors carried out a methodological evaluation according to the Scottish Intercollegiate Guidelines Network items (concordance was analysed by weighted kappa index), and a systematic analysis of the potential biases of each study was assessed. We carried out the meta-analysis with the data extracted from the studies. We recovered eight cases-control studies. The relation between the antecedent of a long travel and subsequent VTED varied from OR = 1.1 to OR = 4.0 and was found to be significant in four studies. The studies were highly heterogeneous in methodology and so the results obtained about the relation between the long travel and the VTED and the score at SIGN50. Two meta-analysis were carried out: only with travels by plane in which the relation was not significant (OR = 1.21; CI 95%, 0.95–1.55) and with all types of transport, with a slightly significant relation (OR = 1.46; CI95%, 1.24–1.72). We may deduce from this systematic review that there does exist a weak association between episodes of VTED and a long travel, but not by plane specifically. The heterogeneity and the methodological quality of the studies published preclude of more robust conclusions. Introduction Venous thromboembolic disease (VTED) has been associated with many risk factors, one of which is the antecedent of a long travel, most often by plane. This association was previously suggested by Homans [1] and was believed so self-evident that it gave rise to the term “economy-class syndrome” [2]. Subsequently, the description of clinical cases related to other means of transport led to the coining of another more appropriate generic term, “traveller’s thrombosis” [3]. The scenario for this syndrome has varied from the alarm caused by the apparently high frequency of fatal pulmonary embolism (PE) occurring after a prolonged flight, as reported in the media, to the scepticism of a weak, although likely association, reported in various scientific studies. The published reports have ranged from descriptions of individual cases to cohort studies and even clinical trials. However, the low incidence of episodes of VTED after a long travel makes the design of a case-control study particularly useful for evaluating the suggested association [4]. Our objective was to carry out a systematic review and meta-analysis of case-control studies to analyse the association between long travels and the development of VTED. Materials and methods We carried out a bibliographic search using a combination of keywords and MeSH headings in the Medline, Embase and Cochrane Library bibliographic databases. We selected only case-control studies, in any language, with no limit on the date of publication, and performed a cross search of the references cited in these studies. The corresponding author named in each study was requested to inform us of any relevant data that had not been described in the original manuscript. Two authors (JTS and AJP) carried out a descriptive analysis of the studies identified in the search and evaluated their quality according to the checklist proposed by the Scottish Intercollegiate Guidelines Network [5]. Concordance in the evaluation of the items on this list was analysed using the respective weighted kappa indexes. We also analysed the limitations and potential biases that might reduce the validity of the studies examined. The sources of bias analysed were grouped into three categories: exposure misclassification (recall bias), selection bias (including detection bias, effect misclassification, survival bias, self-selection bias and Berkson’s bias) and confounding factors. Memory bias was defined as the fact that when subjects know they have suffered VTED, it makes them more likely to remember prior exposure to the antecedent of a long travel. Effect misclassification was minimised by considering only those studies in which VTED was diagnosed by objective complementary examination. Detection bias was suspected when the odds ratio (OR) in the days immediately after the travel was less than that corresponding to subsequent days. We only selected data published for VTED incidence at 30 days after the travel because was the time lapse most frequently analysed in the studies. Survival bias was considered to be that occurring when the study included prevalent cases, which could give rise to confounding of the variables related to the origin and those concerning the prognosis of the disease. Self-selection bias was considered to be present if the controls participated on their own initiative in the study and were not selected in a consecutive or random way; thus, they might be related to the results being sought. Berkson’s bias would be derived from the use of hospital patients as controls. The confounding factors were judged to be influential if no account were taken of the possibility that the exposure factor and the disease might be related through a third variable which was related to each of the other two. From the results included in the published studies and the data supplied by the different authors, we were able to perform a meta-analysis of the studies that had estimated the risk ratio of deep-vein thrombosis (DVT) and of DVT and/or PE either after a travel (by any means of transport) or only after a travel by plane. The OR was used as a measure of association, with a confidence interval of 95%. The hypothesis of homogeneity was evaluated using X [2] and Der Simonian–Laird tests, and fixed and random effects methods were used, respectively, for the cases of homogeneity and heterogeneity between the studies. We assessed possible publication bias using Begg and Egger tests and carried out a sensitivity analysis by evaluating the influence of the absence of each individual study on the global measure of association of all the other studies. The computer softwares used for this study were SPSS 11.5 (SPSS, Chicago, IL, USA) and EpiDat 3.1 [6]. Results Nine case-control studies were recovered; of these, we analysed the data from seven [7–14], as the other one [15] corresponded to a preliminary analysis for one of the seven. One of these [7] was made up of three sub-studies, but for reasons of consistency, we only included the data for two of these in the meta-analysis, as in the third one, the authors assessed patients outpatients and inpatients with PE. Table 1 shows the main characteristics of all these studies. The relation between the antecedent of a long travel and subsequent VTED varied from OR = 1.1 to OR = 4.0 and was found to be significant in four studies [8, 9, 11, 14]. In them, the OR ranged from 2.1 (1.1–4.0) to 4.0 (2.0–7.9). Table 1Principal characteristics of the case-control studies evaluating the association between a long travel and venous thromboembolic diseaseStudy, yearFerrari, 1999Samama, 2000Dimberg, 2001Arya, 2002Hosoi, 2002Ten Wolde, 2003Martinelli, 2003Cannegieter, 2006Period of study1992–19951990–19911995–19982000–20012000–20011997–20001999–20011999–2000Origin of casesPatients admitted with VTEDDVT, symptomatic outpatientsEmployees of the World Bank with confirmed DVTDVT, symptomatic outpatientsDVT, symptomatic outpatientsDVT outpatients; PE outpatients and hospital admissionsPatients with VTED during previous 12 months and examined for possible thrombophiliaVTED, outpatients and admissionsOrigin of controlsPatients admitted in Cardiology Department, age-matchedViral syndrome, matched by age and genderEmployees of the World Bank without DVT, matched by month and year of diagnosisHospital attention for compatible symptoms; DVT excludedHospital attention for compatible symptoms; DVT excludedHospital attention for compatible symptoms; DVT or PE excludedFriends or partners, volunteers, of hospital patientsPartners of casesNo. of travel/cases (%)39/160 (24.4%)62/494 (12.6%)3/17 (17.6%)20/185 (10.8%)15/101 (14.9%)8/130 (6.2%)31/210 (14.8%)233/1906 (12.2%)No. of travel/controls (%)12/160 (7.5%)31/494 (6.3%)163/489 (33.3%)31/383 (8.1%)13/106 (12.6%)38/959 (4.1%)16/210 (7.6%)182/1906 (9.5%)OR (95% CI)4.0 (2.0–7.9)2.1 (1.4–3.4)0.4 (0.1–1.5)1.4 (0.8–2.5)1.25 (0.56–2.7)1.6 (0.7–3.5)2.1 (1.1–4.0)2.1 (1.5–3.0)Type of transportVariousVariousOnly planeVariousVariousVariousOnly planeVariousDuration of travelAt least 4 h (not stratified)Not stated (“long travel”)Not statedStratified into 3 and 8 hours>3 hoursStratified from 3–5h to >16 hStratified into 8 hAt least 4 h, stratified into 4 hLapse between travel and VTED (weeks)43442448Principal limitationsBerkson’s bias, Controls age–matchedNot specific to assess risk of travel, Confounding, Controls matched by age and genderOnly international travels were evaluated (not duration of travel)Selection of controlsConfounding, Not differentiated by duration of travelBerkson’s biasSurvival bias, Matched by age, gender and academic level, Self–selection bias, Recall biasMemory bias, NO objective clinical information about controls With respect to means of transport, two studies [9, 12] only assessed travel by plane, while two [7] MEGA examined separately each means of transport. Another study [13] distinguished travel by plane from all other means of transport, while another two [10, 11] considered various means of transport jointly, but did not distinguish those corresponding to cases from controls, and in one study [8], any means of transport was eligible for consideration without further specification. The duration of the travel was addressed in different ways: two studies [8, 12] made no reference to this question; two studies [11, 15] only considered travels with a duration exceeding 4 h, and another, those exceeding 3 h [13]. The other three studies [7, 9, 10] considered different durations. The lapse of time between the travel and the diagnosis of VTED was 2 weeks in one study [13] and 4 weeks for the others, except that of Samama et al. [8] which did not specify the period exactly and MEGA study [14] that included 8 weeks before. In the study by Dimberg et al. [12], two periods were analysed (15 and 30 days), but in our own analysis, only the data corresponding to 30 days were taken into consideration, this period being equivalent to the 4 weeks used in the other studies. Four studies [7, 9, 11, 14] analysed cases of both DVT and of PE, while the others only considered patients with clinical presentation of VTED in the form of DVT. The selection of cases was generally that of patients with symptoms of DVT and/or PE. One study [12] used a register of days off work, kept by medical insurance companies for the staff of the company in which the study was carried out. The rate of prior history of a long travel, among the cases, ranged from 6.2% [7] to 24.4% [11]. Diverse methods were used for selection of controls. Three studies [7, 10, 13], selected outpatients who, after attending hospital with symptoms compatible with DVT and/or PE, were found not to have these diseases; in another study [11], the controls were hospital patients, matched by age, who had been admitted for other reasons during the same period of time as the cases. Two studies chose outpatient controls, matched by age and sex with the cases, one selecting patients with symptoms of respiratory viriasis [8] and the other using relatives and friends of patients who had suffered VTED [9]. Another study [14] chose the partners of the cases as controls. Finally, Dimberg et al. [12] chose ten control cases, matched by month and year of diagnosis of DVT, from among the staff of the same company that employed the cases. The study by Ferrari et al. [11] even used different exclusion criteria for cases and for controls such that anticoagulant treatment was an exclusion criterion for the former but not for the latter. The rate of prior occurrence of a long travel, among the controls, ranged from 4.1% [7] to 33.3% [12]. With respect to the possible biases present in each of the studies, we highlight the following: Recall or classification bias: the study by Martinelli et al. [9] could suffer from bias in this sense, as one of the exclusion criteria was the absence of a prior occurrence of VTED during the previous 24 months. In general, this bias is consubstantial with the studies of cases and controls such that the cases tend to relate the disease with the antecedent of interest.Detection bias: only one study [12] analysed two periods of time in which DVT had occurred after a long travel, namely at 15 and 30 days. The OR for the former period was greater than that for the latter, and thus, this form of bias was not present here.Misclassification bias: in all the studies, only cases of VTED that had been objectively diagnosed and for which the appropriate complementary examinations were taken into consideration, and thus, their correct classification was ensured. In three [7, 10, 13], the controls were derived from persons who, having sought medical treatment for symptoms compatible with DVT and/or PE, were found not to suffer either of these after the same objective examination used to confirm the cases. In others, the controls presented no symptoms of DVT and/or PE and so were also assumed to be true controls.Survival bias: this bias type could have occurred in the study by Martinelli et al. [9], as the cases chosen were those patients who had suffered an episode of venous thromboembolism during the previous 2 years and for whom a study of thrombophilia was performed; thus, there was, in fact, a selection of all the potential incidental cases of DVT during this period.Self-selection bias: in the study by Martinelli et al. [9], the controls were recruited from the relatives and friends of the cases, a circumstance that could have negatively influenced the relation being studied, as the sample was obtained from a population that was socially related to the cases, and thus, exposure factors could have been shared. In MEGA study [14], the controls were partners of the cases, and the evaluated relation might have been diminished.Berkson’s bias: this bias might have been present in the study by Ferrari et al. [11], as the controls chosen were patients admitted to hospital with chest pain, which could have meant they had a lower probability of prior travel; this pathology was, therefore, a potential limiting factor. Other studies [7, 10, 13] could also have been affected by this form of bias in that the controls they chose were patients who had attended hospital with symptoms compatible with those of DVT and/or PE; objective examination subsequently determined that neither pathology was present. The study by ten Wolde et al. [7] included cases of VTED present in patients who had been admitted to hospital for other reasons, which reduced the number of days before the episode of VTED in which the patient could have travelled. The methodological evaluation of the studies is shown in Table 2. Overall, the evaluation was appropriate in five studies [7, 10, 12–14], while results were less satisfactory in three [8, 9, 11] mainly because of the inadequate choice of controls and because the study design did not enable the authors to achieve a good estimate of the risk being studied. There was a high degree of agreement between the reviewers concerning the quality criteria of the cases and controls (weighted kappa index = 0.8). Table 2Methodological evaluation of the studies included in the systematic review in accordance with SIGN 50 criteriaCharacteristicFerrari 1999Samama 2000Dimberg 2001Arya 2002Hosoi 2002Ten Wolde 2003Martinelli 2003Cannegieter 2006Internal validityClear, appropriate questionsGAGGGGAGSelection of subjectsCases and controls from comparable populationsAAA-GA-GGAPGIdentical exclusion criteria for cases and controlsP-AAAGGGAGParticipation rate by cases and controls95-NS80NSNS74-79NS91-NS83-77Comparison between participants and non-participantsNSNSNSNSNSNSNSNSCases are defined and clearly differentiated from controlsA-GGPAGG-APG-AIt is clearly stated that the controls are non-casesAGPPG-AG-APPEvaluationKnowledge of exposure did not influence designation of casesAAAPG-APAAThe exposure is measured in a standard, valid wayAPA-PG-AG-AG-APAConfoundingIdentification of main confounding factorsP-APAP-AAA-PP-AAStatistical analysisIdentification of confidence intervalsYesYesYesYesYesYesYesYesOverall assessmentControl of bias and confounding factors+/++++/+++/++/++++++++/+++Confidence that the overall effect is due to the exposure being investigated++++++++++/++++++/+++The results are applicable to the target group of patients being studied+++++/++++++++/+++++++++++A single evaluation is shown when the two reviewers agree; otherwise, both evaluations are given.G Good, A adequate, P poor, NS not stated Results of the meta-analysis The studies were varied as regards the episode of VTED included (DVT and/or PE), the controls chosen, the means of transport evaluated and the estimated duration of the travels. These variations influenced the performance of the meta-analysis. Thus, we decided to carry out two meta-analyses, on the basis of the similarity between study designs, i.e., on the one hand, studies of patients with DVT (with or without PE), considering any type of transport, and on the other hand, those studies examining only cases in which the subjects travelled by plane (Table 3). Table 3Results obtained in the studies, by type of transport All types of transportOnly planeOther typesCasesControlsCasesControlsCasesControlsDVTSamama62/494 (12.6)31/494 (6.3)NSNSNSNSHosoi15/101 (14.9)13/106 (12.6)9/101 (8.9)12/106 (11.3)6/101 (5.9)1/106 (0.9)Aryaa20/185 (10.8)31/383 (8.1)16/185 (8.6)29/383 (7.6)4/185 (2.2)2/383 (0.5)Ten Woldeb8/130 (6.2)38/959 (4.1)NSNSNSNSDimbergcNANA17/30 (56.7)489/891 (54.9)NANADVT and/or PEFerrari39/160 (24.4)12/160 (7.5)NSNSNSNSMartinelliNANA31/210 (14.8)16/210 (7.6)NANACannegieterd233/1906 (12.2)182/1906 (9.5)86/1906 (4.5)72/1906 (3.8)147/1906 (7.7)110/1906 (5.8)NA Not applicable, NS not statedResults are expressed as number of patients travelling / total (%).aArya: Travels of more than 3 hbTen Wolde. Restricted to patients capable of travellingcDimberg: More specific case definition of the two analyseddCannegieter: Venous thromboembolism episodes within the 8 weeks after travel Meta-analysis of studies including patients with DVT or DVT+PE, all types of transport Six studies [7, 8, 10, 11, 13, 14] were examined. The Der Simonian–Laird test of heterogeneity gave a result of p = 0.011, with an estimated 75% total variance due to variance between the studies. The global estimator with a model of random effects produced an OR of 1.46 (95%CI, 1.24–1.72). No publication bias was recorded (the Begg and Egger tests results were not significant). The sensitivity analysis revealed that no study influenced significantly on the final OR. Figure 1a shows the forest plot graph corresponding to this meta-analysis. Fig. 1Forest plot for studies that evaluated the antecedent of any type of transport and only travels by plane. a Travels by any type of transport. b Travels by plane Meta-analysis of studies including patients with DVT or DVT+PE, only travels by plane This meta-analysis included five studies [9, 10, 12–14]. The results of the Der Simonian–Laird heterogeneity test were not significant, which suggests there was little heterogeneity between the variances of the studies. The OR estimator produced a non-statistically significant value of 1.21 (95%CI, 0.95–1.55). Again, no publication bias was detected, and the sensitivity analysis showed that none study was influential on the OR. Figure 1b shows the forest plot graph corresponding to this meta-analysis. Discussion The results from our analysis reveal a slight association between long travels and the development of DVT or PE, although this association disappears when only travels by plane are considered. The published case-control studies were characterised by considerable heterogeneity, especially in the selection of controls, and there were some errors in the study design that could have led to bias in estimating the effect of the association being studied. A meta-analysis of case-controls studies has recently been published. In that study, we did not find any relationship between a long-distance travel and venous thromboembolic disease when all the means of transportation were valued or only by plane, but not all published studies were included [14]. The pathogenic basis of the “economy class syndrome” lies in a series of predisposing environmental factors within aircraft (low humidity, relative hypoxia and low barometric pressure) that would facilitate the relative dehydration of passengers; these factors would be accompanied by mechanical ones, such as immobility, the narrowness of the seats and the prolonged maintenance of a seated position, with flexion and compression of the poplitea region [15–17]. The description of episodes of DVT related to other means of transport produces a loss of plausibility of the environmental factors associated solely with travels by plane and puts the prime focus on purely mechanical factors and on the characteristics of the patients themselves. Moreover, a recently published study [18] found no variation at all between different coagulation factors after exposure to the environmental conditions resembling in a prolonged travel by plane. Nevertheless, in one recent systematic review, Philbrick et al. [19] emphasised the presence of prothrombotic risk factors and a flight duration longer than 6 h as predisposing to suffer a VTE episode. The patient-related variables refer to a greater frequency of VTED risk factors. Thus, various studies have reported that a high percentage of the patients involved were obese, higher or smaller, aged more than 50 years, had antecedents of VTED, were taking oral contraceptives or presented a state of hypercoagulability or thrombophilia [2, 11, 15, 18, 19]. This result implies that there is a relation between transport-related factors and those concerning to the patients themselves, and that this is relevant to the development of an episode of VTED. Studies that have sought to analyse this association have obtained divergent results mainly because of the different study designs; this would explain the high variability in the OR obtained, which ranged from 1.1 to 4.0. Moreover, these studies were heterogeneous in various methodological respects, and this could have influenced the final results; some [8, 11] matched the cases and controls by sex and age, which limits the influence of these variables in determining the association under study; in the study by Martinelli et al. [9], the cases were a selection of all the possible incident cases within a given period; in another [11], the controls selected were not derived from the same population as that of the cases; the means of transport and the duration of the travel, as well as the type of VTED episode evaluated were different in all the studies; and, in general, no multivariate or stratified analysis was carried out to attribute the increased risk of developing an episode of VTED to the long travel. An interesting aspect from the methodological point of view is the possible selection bias that might have entered these studies: those patients (the cases) who travelled presented fewer VTED risk factors than did the controls, i.e. they were basically more healthy. Not only could this have led to the relation between the development of an episode of VTED and the antecedent of having made a long travel being underestimated, but it might even appear that the prior travel had a protective effect, as was the case in the study by Dimberg et al. [12]. One way of minimising this type of bias could have been to include only those patients who would physically have been capable of making a long travel; this was only actually done in the case of the study by ten Wolde et al. [7]. Another possibility could be to adjust for this variable (which might be considered that of comorbidity) by means of multivariate analysis. Given the methodological heterogeneity of the published studies of cases and controls, it was not possible to carry out a detailed meta-analysis of different aspects of the relation under study, and so its scope was limited to those studies that assessed the association between DVT or between DVT and PE and prolonged travels by plane or by any other form of transport. In any case, the association that was found in the latter case was only weak, and there was a large degree of variation in the methodological quality applied in the various studies. A noteworthy question is the antecedent of a prolonged travel made by the controls, with variations in all the studies of 6.3–8.1%, except in the case of the study by Dimberg et al. [12]; in the latter case, the scenario chosen for analysis led to the choice of a population in which a high proportion (33.3%) made international travels, an aspect that could have weakened any potential association. Similarly, the rate of incidence of a lengthy travel among the cases ranged from 6.7 to 14.8%, although in two studies, this figure was surprisingly high, 17.6% in that by Dimberg et al. [12] and 24.4% in the study by Ferrari et al. [11]. The methodological quality of the studies examined in this systematic review means that we must be cautious concerning the results reported. Although there does seem to be a likely relation between a long travel and the development of an episode of VTED, such an association must be of such a magnitude that a small bias or modification to the study could increase or decrease the strength of the association recorded. In conclusion, we may deduce from this systematic review that there does exist a real, but weak, association between episodes of VTED and the antecedent of a lengthy travel, and this relation with the travels by plane is only nearly significant. The heterogeneity and the methodological quality of the studies published on the question limit the robustness of the conclusions obtained.

J_Assoc_Res_Otolaryngol-3-1-1915593

Frequency-Dependent Properties of a Fluid Jet Stimulus: Calibration, Modeling, and Application to Cochlear Hair Cell Bundles

The investigation of small physiological mechano-sensory systems, such as hair cells or their accessory structures in the inner ear or lateral line organ, requires mechanical stimulus equipment that allows spatial manipulation with micrometer precision and stimulation with amplitudes down to the nanometer scale. Here, we describe the calibration of a microfluid jet produced by a device that was designed to excite individual cochlear hair cell bundles or cupulae of the fish superficial lateral line system. The calibration involves a precise definition of the linearity and time- and frequency-dependent characteristics of the fluid jet as produced by a pressurized fluid-filled container combined with a glass pipette having a microscopically sized tip acting as an orifice. A procedure is described that can be applied during experiments to obtain a fluid jet’s frequency response, which may vary with each individual glass pipette. At small orifice diameters (<15 μm), the fluid velocity of the jet is proportional to the displacement of the piezoelectric actuator pressurizing the container’s volume and is suitable to stimulate the hair bundles of sensory hair cells. With increasing diameter, the fluid jet velocity becomes proportional to the actuator’s velocity. The experimentally observed characteristics can be described adequately by a dynamical model of damped fluid masses coupled by elastic components. INTRODUCTION Sensory hair cells are the primary mechano-detectors in several mechano-sensory organs in vertebrates such as the hearing- and vestibular organ and the lateral line system in fish and amphibians. The hair bundle is the mechano-sensitive organelle of a hair cell and consists of a well-organized arrangement of villi on the apical side of the cell, called stereocilia, in some types of hair cells together with a true cilium, the kinocilium. In most mechano-receptor organs, the hair cells are covered by an overlying tectorial structure bathed in fluid often mechanically coupling many hair bundles (Pumphrey 1950; van Netten 1997). These tectorial structures, when driven by fluid flow, convey the mechanical signals present in the fluid flow to the hair bundles. In the mammalian cochlea, for instance, sound signals are converted to vibrations of the cochlear endo- and perilymph, which drive the cochlear partition, causing a motion of the stereocilia. Deflections of a bundle of stereocilia, which pivot at their base, induce changes in the open probability of mechanically gated ion channels located in the tips of the stereocilia (e.g., Hudspeth 2000; Fettiplace and Ricci 2006). The combined mechanics and hydrodynamics of the hair bundles and tectorial structure determine an organ’s sensitivity to a specific aspect of the excitatory fluid motion (e.g., van Netten 2006). To quantitatively study the mechanics of tectorial structures and individual hair bundles and the mechano-electrical transduction process of hair cells, a well-defined mechanical stimulus is required. Due to the small dimensions of most tectorial structures, manipulation at a microscopic spatial resolution is desirable, especially when stimulating an individual hair bundle. A commonly used mechanical stimulus for individual hair bundles consists of an electromagnetic or piezoelectric actuator driving an elongated microscopic fiber or glass probe pushing against the hair bundle (e.g., Strelioff and Flock 1984; Crawford and Fettiplace 1985; Howard and Ashmore 1986; Howard and Hudspeth 1988; Russell et al. 1992; Kennedy et al. 2003, 2005). The probe’s tip may adhere to the cell membrane of the kinocilium or tallest stereociliar row allowing for force application to the bundle in both directions. A stiff probe enables direct displacement of the bundle with virtually the same amplitude as the driving element, overruling the intrinsic bundle mechanics, and can be used to study transducer channel kinetics (e.g. Holton and Hudspeth 1986; Assad and Corey 1992; Vollrath and Eatock 2003; Kennedy et al. 2003). Alternatively, a more compliant probe with a stiffness comparable to that of the hair bundle can be used. Although under such conditions the application of displacement may become less direct, the stiffness of the probe can be designed as to not dominate the intrinsic mechanics of the hair bundle. Consequently, it allows for estimating the bundle’s intrinsic mechanical characteristics like its compliance or resistance (Crawford and Fettiplace 1985; Howard and Ashmore 1986; Howard and Hudspeth 1988; Jaramillo and Hudspeth 1993; Benser et al. 1996; Kennedy et al. 2005). Viscous forces do, however, attenuate the efficiency of a compliant probe at higher frequency stimuli, limiting their use up to a certain cut-off frequency (Crawford and Fettiplace 1985; Howard and Hudspeth 1987). As several types of hair cells and most mechano-detecting structures in mechano-receptor organs are fluid-driven, a fluid coupling between stimulator and mechano-receptor is an attractive natural alternative to direct-contact stimulation with a probe. Well-defined fluid displacements can be obtained using a stimulus sphere attached to a piezoelectric element. Submerged in water, a sphere vibrating with a constant displacement amplitude may generate a near-field water flow that has a constant displacement amplitude as a function of frequency up to the order of a kilohertz (van Netten 1991, 2006). It does, however, stimulate very inefficiently at larger distances, as the stimulus strength in the near field measured as water displacement amplitude (A) is proportional to the third power of the ratio of stimulus sphere radius (a) to the distance (r) to its centre (i.e. ; e.g. van Netten 2006). To compensate for this reduced efficiency at larger distances, a stimulus sphere with larger radius (a) may be used. Large spheres, however, are less practical in many situations in which small experimental chambers and objective lenses do not provide sufficient space. To obtain a more targeted but sufficiently strong mechanical stimulus, a fluid jet emerging from a glass pipette with a small opening at its tip may provide a practical alternative solution. The use of a fluid jet to mechanically excite individual hair bundles may have several advantages over stimulating with glass probes, noticeably yielding a better defined resting position of the bundle, a more even application of force, and in the case of lymph-driven hair cells, a more natural physiological stimulus. Also the asymmetry in force that may occur in pushing and pulling the cell membrane of the stereocilia, inevitably related to the direct contact of a glass probe, can be avoided. It is feasible that glass probes allow wider stimulating bandwidths although (lateral) resonances have been reported that may limit the high frequency response to the order of 10 kHz (e.g., Beurg et al. 2006; see also Discussion section). Fluid jet stimuli have been considered as effective force stimuli, whereas (stiff) probe excitation present displacement stimuli to the bundle (Vollrath and Eatock 2003). Such differences in stimulation may also lead to differences in speed of engagement of the transduction apparatus. In one of the first reports on the use of a fluid jet in mechano-reception research, it was applied to drive the cupula of a fish lateral line neuromast (Jielof et al. 1952). The 4-mm pipette opening diameter used in that study was relatively wide compared to later, more miniaturized models, which were designed to displace individual hair bundles (Flock and Orman 1983). A first step to calibrating the frequency dependence of a fluid jet was set by Saunders and Szymko (1989). They used glass micro-beads with a density close to that of water that were captured in the hydrodynamic flow field and monitored their motion by stroboscopic-illuminating microscopy. Later developments of fluid jet-producing devices were utilized to displace single inner or outer hair cell bundles or lateral line cupulae and were in some cases pressure-calibrated (Kros et al 1992; Denk and Webb 1992; Géléoc et al. 1997; Nicolson et al. 1998; Vollrath and Eatock 2003). In the present work, a calibration procedure of a newly designed piezoelectrically driven fluid jet device is presented. A compliant glass fiber with a resin sphere attached to its tip is used as a sense probe to measure the dynamic fluid displacement output of the fluid jet device. The motion of the sense probe, which is reflecting the dynamics of the fluid jet, is monitored using a displacement measurement method implemented in a transmitted-light microscope. The procedure, which can be applied in an experimental setting, enables the determination of the frequency dependence of the fluid jet. This is essential to correct experimental data obtained using the same fluid jet as a stimulus. To understand the physical factors of the fluid jet-producing device governing the frequency response of the produced fluid jet, a theoretical model of the device has been developed. The results of the model were compared to the measured data and show that the fluid resistance of the orifice at the pipette’s tip is a key parameter, influencing the amplitude and phase characteristics of the emerging fluid jet. As an example of its usefulness, the fluid jet system was applied to induce transducer currents in mouse cochlear hair cells, showing the importance to correct for the phase delay of the fluid jet, especially at high frequencies. This example also shows that fluid jet dynamics may lead to resonance, which in practice limits the use of the present design to a bandwidth of approximately 5.5 kHz. MATERIALS AND METHODS Fluid jet-producing device The device used for the generation of a fluid jet is shown in Figure 1A and its individual parts in Figure 1B. The main parts are a Perspex fluid container (C) and a brass rear end (R). Three screws (S) pull both parts together, sandwiching a piezoelectrically driven brass disc (P, Ø = 18 mm, thickness, 0.35 mm, resonance frequency ≈ 8.3 kHz, Conrad Electronic SE, Hirschau, Germany) in a configuration that is clamping the disc at its rim. The Perspex container is cone-shaped and is closed by the piezoelectric disc at the rear end. A rubber o-ring (O) between the Perspex and the piezoelectric disc provides a fluid-tight seal. When driven by a voltage signal, the piezoelectric disc produces forces that move the fluid in or out of the container. FIG. 1.The fluid jet-producing device. A Picture of the assembled fluid jet-producing device mounted on an x,y,z-micromanipulator. B Disassembled device showing its separate parts: R Rear brass element, P piezoelectric disc, O o-ring, S screws, C Perspex container, W rubber cone washer, Sp Perspex spacer, Sc Perspex screw cap, Pi glass pipette. The calibration bar indicates 1 cm. At the front end of the Perspex container, a glass pipette (Pi, outer Ø = 1.5 mm, inner Ø = 1.17 mm) is inserted. The screw cap (Sc) applies pressure on two cone washers (W) separated by a Perspex spacer (Sp) to produce a fluid tight seal and fixes the glass pipette in a mechanically stable way. In most applications, the pipette was narrowed at its tip using an electrode puller. Tip diameters were varied between 6 and 62 μm and tapering lengths were kept at approximately 3–4 mm. Also, in some experiments, untreated glass pipettes without tapered tips were used so that their effective opening equaled the inner diameter of the glass pipette (Ø = 1.17 mm). The total length of the glass pipette was about 5 cm of which about 1.5 cm was clamped inside the Perspex container. To facilitate horizontal application of the produced fluid jet in the experimental chamber, the glass was bent 1 cm from the tip over an angle of about 20° so that its tip could be aligned within the horizontal plane (Fig. 1A). For inducing transducer currents in hair cells (see Applications), straight glass pipettes were used. To obtain an air-free filling of the fluid container in the Perspex body part, it was filled with a low viscosity silicone fluid (200 Fluid 5 CS, Dow Corning, Midland, MI, USA), which has low surface tension properties. The silicone fluid was degassed under vacuum conditions for several hours before use to prevent dissolved gas from creating bubbles inside the fluid chamber. The glass pipette was filled with de-mineralized water, or in the case of physiological experiments, with extracellular fluid (bath solution). Inserting the filled glass pipette into the Perspex body part generated a pressure increase inside the fluid container, replacing a volume of the water in the back side of the pipette by silicone fluid. During this temporary overpressure, the fluid jet device was transferred to its position in the set-up, and the pipette tip was submerged in the bath solution preventing air from entering the tip. When prepared, as described above, the instrument also benefits from the high resistivity (≈1015 Ohm cm) of the silicone fluid creating an electrical resistance between the brass disc and the bath solution, thereby, preventing electrical cross-talk of the piezoelectric control signal to the possible electrophysiological recordings obtained from a preparation. Additional electrical shielding was obtained by connecting the brass disc, which is facing the preparation, to the electrical ground. The rear end of the fluid jet device, which together with the grounded brass disc enclosed the piezoelectric material, was also grounded, thereby, shielding off the applied voltages. A shaft connected to the rear end of the fluid jet device was used to mount the device on an x,y,z-micromanipulator (Fig. 1A). The tip of the jet pipette was lowered in the bath solution and positioned under visual or microscope guidance. All calibration measurements were done approximately 5 mm above the bottom and below the surface. Signal generation Sine wave stimuli at exponentially distributed frequencies ranging from 1 to 1,000 Hz were generated at 32 points per period using the full amplitude range of a 16-bit D/A converter (Ariel, DSP 16, Highland Park, NJ, USA). These signals were subsequently attenuated to the desired amplitude and filtered (8 pole Bessel, 3988, Krohn-Hite Corporation, Brockton, MA, USA) at eight times the stimulus frequency. Frequencies used to probe resonances exceeding 1 kHz were derived from a signal generator (PM 5129; Philips, Eindhoven, The Netherlands). Signals for exciting hair cells were generated using a 16-bit D/A converter with dedicated software (Power 1401 and Signal 2.0 software package, Cambridge Electronic Design Ltd, Cambridge, UK). Sense probes Sense probes used for the detection of fluid displacement consisted of compliant glass fibers (Borosilicate glass capillaries GC150T-10, Harvard apparatus Ltd., UK) produced with a micropipette puller (P-97 Sutter instrument Co., Novato, CA, USA) with a small sphere that was produced by dipping the glass fiber tip into a resin (Bison, Silicone universal transparent, Goes, The Netherlands). After curing the resin, sphere diameters ranged from 10 to 60 μm. The fibers were about 1 cm in length with a gradually decreasing diameter from about 100 μm to approximately 2 μm at their tips. The fibers were glued to a stiff glass capillary that was subsequently mounted on an x,y,z-micromanipulator. The mechanical frequency responses of the sense probes to fluid displacement were obtained by stimulating the sense probe with the fluid flow produced by a glass stimulus sphere (Ø 1.1 mm) attached to a piezoelectric element. The frequency response of this stimulus sphere was separately determined using a laser interferometer (see Motion detection). When stimulating the same sense probe with a fluid jet, the center of the fluid jet was aimed at the center of the resin sphere. The distance between the jet-producing pipette tip and the resin sphere of the sense probe was at least one diameter of the sphere. Based on the frequency responses of the sense probes, their overall stiffness coupling to the stiff glass tube held in the x,y,z-micromanipulator was estimated to be of the order of 10−4 N/m. Motion detection The motion of the stimulus sphere and the sense probe were measured with a heterodyne laser interferometer coupled to a fixed-stage transmitted light microscope mounted on a vibration isolated table. The objective lens (×40 WI, NA 0.8, Zeiss, Oberkochen, Germany) of the microscope (modified Axiotron, Zeiss, Oberkochen, Germany) focuses two coherent laser beams (HeNe 127, Newport Spectra Physics GmbH, Darmstadt, Germany) aligned in parallel so that their beam waists intersect in the plane of focus creating a measuring volume with microscopic dimensions (Ø ≈ 4 μm). A frequency difference of 400 kHz between the two laser beams produced by two Bragg cells (1201E, Isomet Corporation, Springfield, VA, USA) driven at 40.0 and 40.4 MHz, creates a moving fringe pattern in the measuring volume. Light scattered by a stationary object, therefore, fluctuates in intensity at a carrier frequency of 400 kHz (fhet). Additional motion of the object induces a phase modulation of these intensity fluctuations, which is displacement-dependent. The back-scattered laser light from an irregularity on the surface of the object was detected by a photomultiplier (model H6780 02, Hamamatsu Photonics, Japan) coupled to an I / V converter. The output, containing the carrier frequency, fhet, phase-modulated in proportion to the object’s displacement, was band-pass filtered with a centre frequency at fhet and electronically demodulated using a modified frequency demodulator (OFV 3000, Polytec GmbH, Waldbronn, Germany, equivalent r.m.s velocity noise was 0.1 μm/s). Time-varying phase modulations were thus treated as frequency modulations (i.e., Doppler shifts), which was appropriate, as the phase changes per time of the carrier were small compared to the carrier frequency itself. Under these conditions, the output signal of the demodulator is a calibrated linear measure of the velocity of the object within the range of 10−1 to 103 μm/s. For low frequency stimuli (<10 Hz), a digital phase demodulator (OVD-20, Polytec GmbH, Waldbronn, Germany) was used, which has an output proportional to the displacement of the object with a 32-nm resolution. The demodulator signals were low-pass filtered (3988, 8 pole Bessel, Krohn-Hite Corporation, Brockton, MA, USA) at eight times the frequency of stimulation and were amplified before being digitized using a 16-bit A/D converter (DSP 16, Ariel, Highland Park, NJ, USA) with a sample frequency at 32 times the frequency of stimulation. Displacement responses consisting of usually ten consecutive stretches, each 16 periods in length, were averaged on-line by the data acquisition board. The first 4 s of a response to a stimulus were not recorded to prevent effects of onset transients. The averaged response waveform was stored on hard disc. A fast-Fourier transform (FFT) was used to extract the amplitude and phase at the frequency of stimulation from this averaged waveform. In some experiments, a Position Sensitive Detector (PSD; PSM2-2 On-Trak, Photonics Inc, Lake Forest, CA, USA) was used to measure displacements of uncalibrated sense probes at frequencies beyond 1 kHz, using a A/D converter with dedicated software (Power 1401 and Signal 2.0 software package, Cambridge Electronic Design Ltd, Cambridge, UK). Hair cell electrophysiology Experiments were performed on apical-coil outer hair cells obtained from acutely isolated organs of Corti taken from neonatal C57Bl/6 mice (Harlan Netherlands B.V., Horst). Animal procedures conformed to Dutch governmental rules and the guidelines of the University of Groningen Institutional Animal Committee (RuG-DEC). Transducer currents in response to fluid jet stimulation were measured under whole-cell voltage clamp with an Axopatch 200B patch clamp amplifier (Axon Instruments Inc, Molecular Devices, Foster City, CA, USA) at −84 mV holding potential. Further details on the procedures and solutions used can be found elsewhere (Kros et al. 1992; van Netten et al. 2003). Model of fluid jet dynamics Schematics of a mechanical model illustrating the principal physical elements of the fluid jet-producing device are shown in Figure 2. The device is shown to work with only minor nonlinear distortion (e.g., Fig. 3) so that the system may be adequately analyzed in the frequency domain. In this section, all dynamic variables will therefore be considered to vary harmonically with angular frequency, ω. A piezoelectrically driven disc, with mass Mpiezo and stiffness Spiezo is used to pressurize the fluids in the jet-producing device. A force, F0, on the brass diaphragm of the piezoelectric disc is produced in proportion to the applied voltage, V0. This causes a displacement of the disc, Xc, so that the fluid in the Perspex container is pressurized. Compressibility of the fluid is neglected (see also Discussion section). FIG. 2.Mechanical model representation of the fluid jet-producing device. F 0 is the piezoelectrically induced force, proportional to the applied voltage, V 0. The piezoelectric element consists of a brass disc (dark gray) and a smaller ceramic plate. The spring, S piezo, and a resistive element, R c, together with the mass of the piezo, M piezo, and the mass of the container fluid, M c (cone-shaped), affect the fluid displacement amplitude, X c, in the Perspex container. The fluid displacement, X c, is amplified through a lever arm, with ratio L, into L·X c, which drives the fluid in the glass pipette. The same lever ratio applies to the forces F 1 and F 2. The displacement response of the fluid mass in the pipette, X p, is determined by the pipette’s spring, S p, the pipette’s tip resistive element, R p, and the mass of the pipette fluid, M p. X p is taken as a proportional measure of the displacement of the jet fluid.FIG. 3.Sense probe displacements induced by the fluid jet. A Sense probe displacement in response to a 106-Hz vibrational fluid jet stimulus as a function of time. An average of ten traces is shown. B Fast Fourier transform of the average waveform shown in A with a fundamental component at 106 Hz and a second harmonic component at 212 Hz, which is more than 40 dB less in amplitude than the fundamental component. Total harmonic distortion (THD) was calculated using , where a 1 is the response amplitude at the fundamental frequency and a n is the amplitude of the nth harmonic component, as determined from the FFT calculated from the averaged waveform of measured sense probe responses. The fluid in the Perspex container has a mass, Mc. Fluid mass appears in the dynamic equations (e.g., Eq. 1a) as a product of mass times its acceleration. The fluid mass in the cone-shaped Perspex container can be considered as the sum of a series of cross-sectional fluid slices with variable radius, so that the fluid mass of each slice is proportional to the square of its radius. Assuming that the equation of continuity holds in combination with incompressibility means that the fluid flux along the series of slices is conserved. The velocity of each fluid slice and also its acceleration must therefore be inversely proportional to the square of its radius, so that the product of mass and acceleration of each slice is independent of radius. This means that the overall product of mass and acceleration of the fluid in the cone-shaped Perspex container can be dynamically modeled with a cylinder with length L1 (7 mm), equal to the cone’s actual length and a diameter D1 (13.4 mm) equal to the cone’s base. Therefore, the mass of the fluid in the Perspex container is modeled as and its effective acceleration as . Here ρ (1,000 kg/m3) denotes the fluid density. The fluid mass in the container Mc (0.987 × 10−3 kg) sums with that of the piezoelectric disc Mpiezo (0.741 × 10−3 kg), resulting in Mtot = Mc + Mpiezo (1.728 × 10−3 kg). A resistive element, Rc, in series with mass Mc is included, which is related to friction of the fluid flow in the Perspex container and the motion of the piezoelectric disc. The mass in the cylindrical glass pipette is described by (53.8 × 10−6 kg), where L2 (50 mm) and D2 (1.17 mm) are the length and inner diameter of the glass pipette, while this mass has a displacement Xp. The overall effect of the conservation of flux in the conical Perspex container is the transformation of the effective displacement of fluid in the Perspex container into that of the glass pipette. The transformation factor equals the ratio of the (maximum) cross-sectional areas of the two fluid compartments (≈131). The force ratio of this transformation is governed by the same ratio, . The transformed fluid displacement, , is not completely transferred into displacement of the fluid mass in the pipette, Xp, as the elastic cone washers around the glass pipette absorb it partly. This is represented by the compliant element, Sp. A resistive element, Rp, describes the viscous friction in the pipette, which is dominated by the tip resistance. The resulting fluid displacement, Xp, is taken as a proportional measure of the fluid displacement of the jet emerging from the pipette tip. Again, assuming the equation of continuity, this proportion is related to the square of the ratio of the pipette’s tip diameter to the glass pipette’s cross-sectional diameter (D2). However, the absolute displacement of the jet is strongly dependent on the distance to the pipette tip (See also Absolute fluid jet velocity in the Discussion section). The governing equations relating the piezoelectric force, F0, to the fluid displacements Xc and Xp, utilizing the usual notation for time derivatives, are then: and Changing to the frequency domain, V0, F0, F1, F2, Xc, and Xp are all assumed to vary harmonically with time so that they are proportional to the factor exp(iωt). This factor will be omitted in the further analysis so that only (complex) amplitudes can be written for these variables. Displacement of the fluid emerging from the jet pipette, Xp, as a function of angular frequency, ω = 2πf, and piezoelectrically induced force, F0, is then described by: with a relative transformation factor, T(ω), between the displacement in the container and the pipette given by: Inspection of the denominator of Eq. 2a shows that two minima may occur as a function of ω = 2πf, corresponding with a low (fl) and high (fh) resonance frequency. The high resonance frequency, fh, can be found by minimizing the term between square brackets, assuming that T(ω) varies only slowly around fh. This frequency is therefore related to Mtot, the summed mass of piezoelectric disc and mass of the fluid in the Perspex container, and the parallel combination of Spiezo, and the up-transformed stiffness of the pipette, L2Sp. Together, they amount to the stiffness , in accordance with Mtot’s position in between these two parallel elastic elements (Fig. 2). The related high-resonance frequency is consequently given by: At relatively low frequencies the frequency dependent terms of the factor between square brackets (Eq. 2a) are relatively small so that this factor reduces to Spar. Then Xp can be approximated in the low frequency range by: where Sser is the series spring combination, of Sp and Spiezo / L2 given by: It then follows from Eq. 4a that the low-frequency resonance, fl, is given by: Equation 4c shows that the low frequency oscillation is due to the mass of the fluid in the pipette, Mp, that is elastically supported by the effective stiffness, Sser, which is formed by the series combination of Sp and the down-transformed piezo-stiffness, Spiezo / L2, in line with their series configuration (Fig. 2). The parameters Spiezo, Rc, Sp, and Rp were determined from fitting the full model (Eqs. 2a and 2b) to the measured data. The fluid masses were determined from their actual physical sizes (D1 = 13.4 mm; D2 = 1.17 mm) and lengths (L1 = 7 mm; L2 = 50 mm). RESULTS Linearity of the fluid jet Figure 3A shows a typical sinusoidal displacement of the sense probe driven by a fluid jet produced with a pipette tip diameter of 62 μm. The average sense probe displacement of 10 consecutive traces is given each containing 16 periods at a frequency of 106 Hz. The waveform clearly shows the 16 periods of the fundamental frequency with negligible harmonic distortion. The extent of the distortion was determined by the Fast Fourier Transform (FFT) calculated of the waveform shown in Figure 3A. Figure 3B shows the resultant frequency content of this waveform up to 600 Hz. The fundamental frequency is clearly visible at 106 Hz and rises about three orders of magnitude above the noise floor. The second harmonic component is visible, but has an amplitude more than 40 dB below the component at the fundamental frequency. Higher harmonics are even smaller. The calculated total harmonic distortion in this case was −32.7 dB (see legend Fig. 3). Fluid jet calibration To determine the frequency-dependent motion of a fluid jet produced with a pipette, we used the following procedure. The vibrational displacement of the sense probe induced by the fluid jet was measured as a function of stimulus frequency. The frequency-dependent amplitude and phase characteristics obtained this way are, however, contaminated with frequency-dependent characteristics of the sense probe and other possible frequency-selective components of the equipment. To isolate the fluid jet frequency response, three additional frequency responses were measured in this procedure, which are displayed in Figure 4. FIG. 4.Schematic representation of the fluid jet correction procedure. A Each column (1–4) represents a type of measurement needed to complete the correction. The frequency responses measured with the sense probe contain frequency-dependent properties of ED (equipment, demodulator), S (stimulus sphere), SP (sense probe), or FJ (fluid jet) as a function of frequency. Results represented in a column are used to correct the measured response in the next column, indicated by the long arrows and produce results pointed at with a short arrow. B Displacement amplitude and phase for each measurement (1–4) described in (A). Each solid line is the result of a correction (except for column 1) and is used in the next column to correct the measured frequency response (symbols), both for the amplitude and the phase response. The first step (Fig. 4A1) is to obtain the frequency-dependent properties of the demodulator (D) including the additional signal measuring and conditioning equipment (E) like (anti-alias) filters and amplifiers. For this step, a voltage-controlled oscillator (VCO) was used to generate a frequency-modulated 400 kHz carrier signal, which was sent to the demodulator. These signals of the VCO at various known modulation frequencies simulate a calibrated output of the photomultiplier similar to operating the laser interferometer. The resulting response, ED(f), characterizing demodulator (D) and equipment (E) is presented in Figure 4B1, as a function of frequency, f. It shows a nearly constant amplitude as a function of frequency and a clear phase change starting at about 300 Hz as a result of the used anti-alias filter. The second step characterizes the frequency response of the stimulus sphere, S(f). The laser interferometer is used to directly measure its displacement amplitude and phase as a function of input frequency with fixed voltage amplitude. This response, consisting of ED(f) × S(f) (Fig. 4B2; data points) can now be divided by ED(f), obtained in step 1, to isolate the pure stimulus sphere response, S(f) (Fig. 4B2; solid line). The resonance at approximately 900 Hz, originating from the mass of the stimulus sphere and its elastic attachment, can clearly be discerned. The fluid displacement produced by a vibrating stimulus sphere can be taken proportional to the displacement of the sphere S(f) (e.g., van Netten 2006). In the third step, the same stimulus sphere vibrating in water is used to hydrodynamically displace the sense probe, which has frequency characteristics SP(f) that subsequently will be used to probe the fluid jet displacement. The sense probe displacement resulting from sphere stimulation, ED(f) × S(f) × SP(f), as measured by the laser interferometer is thus contaminated with equipment, i.e., demodulator and stimulus sphere characteristics. This response is divided by the previously (step 2) isolated stimulus sphere response, S(f), resulting in the sense probe frequency response ED(f) × SP(f) (Fig. 4B3; solid line), which is intentionally still contaminated with the equipment response characteristics, ED(f). In the fourth step, this result, ED(f) × SP(f), is used to divide the fluid jet-driven sense probe response measured by the interferometer ED(f) × FJ(f) × SP(f), thus eventually isolating the amplitude and phase of the pure fluid jet displacement response, FJ(f), as a function of the frequency (Fig. 4B4; solid line). Effects of tip diameter Measurements The dimensions of the object that has to be stimulated largely determine the diameter of the tip of the glass pipette to be used for producing the fluid jet. Mechanically stimulating an individual hair bundle usually requires pipette tip (i.e., orifice) diameters smaller than 10–15 μm, whereas some tectorial structures like a lateral line neuromast may require tip openings of up to 1 mm. A narrow tip increases the outflow resistance and thus decreases the fluid flow of the jet as evidenced by the larger voltage amplitudes that have to be applied across the piezoelectric disc to obtain equal displacement amplitudes of the sense probe. Another important consequence of the tip diameter is its influence on the fluid flow frequency characteristics of the fluid jet. To illustrate this calibrated fluid jet characteristics obtained as described in the previous section are shown for different tip diameters in Figure 5. This figure also shows the amplitude and phase response of the resulting fluid displacement when using a pipette without a tip restriction (Fig. 5A). In this case, the glass pipette was cut at the same length as the pipettes used in Figure 5B and C, but was not tapered at its tip. Its outflow diameter therefore equals the inner diameter of the glass pipette (1.17 mm). The characteristics of the fluid jet generated using such an unrestricted tip in response to signals applied to the piezoelectric disc within a range of frequencies and with a fixed amplitude, have a constant displacement amplitude up to a certain frequency (about 100 Hz) and the jet thus effectively behaves as a displacement stimulus in this frequency range (Fig. 5A, data points). At higher frequencies, the amplitude starts increasing and approaches a resonance at about 150 Hz. Beyond this resonant frequency, the displacement amplitude decreases at a rate of about −40 dB/dec, which is reminiscent of a second-order system driven beyond resonance. Around the resonant frequency, the phase rotates over an angle of 180°, which is consistent with these second-order characteristics. FIG. 5.Effect of tip diameter on fluid jet response. Data points are the measured amplitude and phase responses as a function of frequency using a pipette without a tip restriction (A), a 56-μm tip (B), or a 7-μm tip (C). The solid lines are fits to the data with the model description (Eqs. 2a and 2b) of the jet-producing device (Fig. 2). Fixed parameters based on physical sizes and properties: L 1 = 7 mm; L 2 = 50 mm; D 1 = 13.4 mm; D 2 = 1.17 mm; ρ = 1,000 kg/m3. Fixed parameters: S piezo = 2.0 × 106 N/m; R c = 7 Ns/m. Varied parameters: A S p = 85 N/m, R p = 0.0165 Ns/m; F 0 = 7.0·10−3 N, (B) S p = 9 N/m, R p = 0.09 Ns/m; F 0 = 62·10-3 N, (C) S p = 11 N/m, R p = 2 Ns/m; F 0 = 52·10-3 N. A different jet response is produced with a pipette with an intermediate tip restriction (56 μm; Fig. 5B, data points). At low frequencies, the jet’s fluid displacement is proportional to the voltage applied to the piezoelectric disc, as evidenced by the constant amplitude and the almost zero phase. With this more restricted pipette tip, the displacement amplitude, however, starts declining beyond 15 Hz, changing towards a slope of about −20 dB/dec with an according −90° phase rotation in the mid-frequency range. At about 400 Hz, the slope of the amplitude starts to decrease further and also the phase lag changes to values more negative than −90°. With this more restricted tip diameter (56 μm), no clear resonance is evident as was the case with the larger diameter (1.17 mm). In the case of an extremely restricted tip with a diameter of 7 μm, the fluid jet device produces a displacement amplitude response, which declines with about −20 dB/dec over the measured frequency range (1–1,000 Hz; Fig. 5C data points), so that effectively, the velocity amplitude of the fluid jet is constant within this frequency range. Consistent with these characteristics is the almost constant phase lag of −90°, except at the higher frequencies where it starts to decline further. These frequency characteristics reflect an overdamped system. In the case of a small pipette tip diameter, therefore, the resulting dynamics lead to a fluid jet of which the velocity is proportional and in phase with the voltage applied to the piezoelectric disc within the measured frequency range. Model calculations To get a better understanding of the physical factors influencing the output characteristic of the jet-producing device, the measured data were described by a mechanical model of the masses of the fluids related to the two main compartments of the device, i.e., the Perspex container part and the pipette (see Materials and Methods). The mass of the piezoelectric disc (Mpiezo) is combined with the fluid mass contained in the Perspex fluid container (Mc), because they can be considered to move in unison, having a total mass . The fluid mass in the glass pipette is indicated with Mp. These two effective masses, Mtot and Mp, are mechanically coupled by a lever and a spring Sp (Fig. 2). The displacement amplitude of the mass in the pipette, Xp, is considered to be proportional to the displacement of the fluid jet emerging from the pipette tip and is calculated as a function of frequency of the force produced by the piezoelectric disc. The force is assumed to be proportional to the electrical signal applied to the piezoelectric disc. The results of this model were subsequently fitted to the measured data points (Figs. 5A-C, solid lines). The model results support the measurements in showing that the value of the resistance controlled by the tip of the pipette, Rp, strongly influences the fluid jet response characteristics. When fitting the model to the data obtained for different tip diameters, the value of the resistance, Rp, increases more than two orders of magnitude (Fig. 5A solid lines, Rp,small = 0.0165 Ns/m; Figure 5C solid lines, Rp,large = 2 Ns/m), when the pipette without tip restriction (Ølarge = 1.17 mm) is compared with the most restricted pipette (Øsmall = 7 μm). Fitting the flow produced by the pipette tip with intermediate opening (Ø = 56 μm) results in an intermediate resistance value (Fig. 5B, solid lines, Rp = 0.09 Ns/m). Besides these expected changes in Rp, the value of Sp needed to be modified (9 to 85 N/m) for obtaining proper model fits to the data. The cone washers (Fig. 1; denoted with W) are the most compliant structures in the assembly to which part of the elastic stiffness Sp can most likely be attributed. The variation in Sp found between individual fluid jets could be due to the variability with which the cone washers were positioned during the insertion of the pipette and subsequently tightened. In addition, we cannot exclude the variability in the amount of gas or air bubbles present in the silicone fluid causing these variations in Sp between individual fluid jets. Except for Sp and Rp, all other parameters of the model were kept fixed in fitting the results with different pipette tip dimensions (Fig. 5). Resonance frequencies Unless overdamped by resistive elements, a combination of a spring and a mass leads to a resonance phenomenon and is accompanied by a −180° phase rotation and an amplitude response slope change of −40 dB/dec around the resonance frequency. The present model, including two sets of masses and elastic elements, therefore predicts two possible resonant frequencies, fl, and fh (see Materials and Methods). The high resonance frequency, fh, is beyond the calibrated frequency range considered (1 kHz). This resonance is related to the summed mass (Mtot) of the fluid in the Perspex container (Mc) and that of the piezoelectric disc (Mpiezo) combined with the parallel stiffness (Spiezo) of the piezoelectric disc and the up-transformed stiffness of the cone washers (L2Sp), as effectively acting on the fluid mass in the Perspex container (Fig. 2; Eq. 3). The existence of a second resonance at higher frequencies was confirmed by applying both sinusoidal signals and voltage steps to the piezoelectric disc of the fluid jet device. The displacement of a (uncalibrated) sense probe in front of a small pipette tip (≈10 μm) of the fluid jet device driven by a voltage step clearly shows a resonance at about 5.65 kHz (Fig. 6). The same value was found using a series of sinusoidal voltages applied to the piezoelectric disc. The high-resonance frequency also coincides with the resonance frequency of the back side of the piezoelectric disc measured directly using laser interferometry, while the disc was being clamped and operated in the same fluid jet device (not shown). FIG. 6.High frequency fluid jet resonance. Response of the fluid jet to an unfiltered step driver voltage applied to the piezoelectric disc, showing a high resonance frequency, f h = 5.65 kHz. (solid line, calibration as indicated). Parameters used in the model (dashed line): S piezo = 2.12 × 106 N/m; R c = 4 Ns/m; S p = 6.4 N/m, R p = 0.402 Ns/m; F 0 = 2.5 × 10−3 N. In the case of the unrestricted pipette with small damping, the low resonant frequency, fl, was observed (Fig. 5A), together with the accompanying −180° phase rotation within the measured frequency range. This low-frequency resonance, fl, (see Eq. 4c) can be attributed to the mass of fluid in the pipette (Mp) and the combined series stiffness, Sser, that is associated with the compliant cone washers or air bubbles (Fig. 1, C) and the down-transformed piezo stiffness Spiezo/L2, which effectively also acts on Mp. The resonance frequency resulting from substituting the fitted parameters of Figure 5A in Eq. 4c yields fl = 152 Hz, similar to the measured value (≈150 Hz). Applications Mechanical excitation of sensory hair cells using the fluid jet To measure transducer currents at acoustic frequencies, whole-cell voltage-clamp recordings of individual outer hair cells were performed in acutely prepared organs of Corti of mice (e.g., Géléoc et al. 1997; van Netten et al. 2003). The hair bundles were stimulated by the present fluid jet device using narrow tips (Ø < 15 μm), so that fluid jet velocities were proportional to the voltage applied to piezoelectric disc (e.g., Fig. 5C). The pipette’s tip was aimed at the hair bundle, the mechano-receptive structure of a hair cell, at an angle of about 20° with the apical plate of the hair cell, and at a distance of approximately 15 μm from the hair bundle. Figure 7 shows examples of inward transducer currents measured in an apical mouse outer hair cell. The hair bundle was excited with 16 periods of a pure sinusoidal fluid stimulus at 100 Hz and at 1,000 Hz, as produced by the micro-fluid jet device. The driver voltage and resulting jet velocity are symmetric, whereas the measured transducer currents (Fig. 7B; 100 Hz; Figure 7C; 1,000 Hz) are asymmetric. This is expected as the transducer channels only gate ionic currents when they are deflected in the direction of the tallest hair bundles (e.g., Hudspeth 2000; Fettiplace and Ricci 2006). FIG. 7.Application of the fluid jet to excite sensory hair cells. A Schematic representation for recording transducer currents in cochlear outer hair cells. Indicated are the fluid jet’s pipette tip and the hair cell under whole-cell voltage-clamp configuration with a patch pipette at the lateral side. The sinusoidal driver voltage (DV) applied to the piezoelectric disc is indicated. Positive voltages correspond with the fluid moving out of the chamber towards the hair bundle so that it bends in the excitatory direction towards the tallest stereocilia. B Transducer currents in response to a fluid stimulus at 100 Hz. (C) Transducer currents in response to a fluid stimulus at 1,000 Hz from the same cell, and using the same fluid-jet pipette as in B. D Responses of transducer currents (dashed, 100 Hz; solid, 1,000 Hz) shown on an extended time axis in relation to two stimulus periods of the driver voltage (DV, thin black line upper trace). The two transducer currents have a comparable magnitude but show a different delay in activation with respect to the maxima of the driver voltage (vertical thin lines), which is caused by the different phase delays of the fluid jet at 100 and 1,000 Hz. The measured delays are 0.0194 and 0.171 periods, for respectively, the 100 Hz and the 1,000 Hz stimulus, and correspond to a phase delay of 7° and 62° respectively. E Step driver voltage (DV) applied to the piezoelectric disc of the device and the evoked transducer current showing a 5.65-kHz oscillation produced by resonance of the fluid in the Perspex container. In Figure 7D, two periods of the traces from Figure 7B and C are shown on an extended time scale. For both frequencies, there are delays in peak transducer current with respect to the maximum stimulus (vertical lines). The measured delays are 194 ± 155 μs and 171 ± 15 μs (mean ± SD, n = 16), for, respectively, the 100 Hz and the 1,000 Hz stimulus. This corresponds to a phase delay of 7 ± 6° at 100 Hz and to 62 ± 5° at 1,000 Hz, which can be attributed to the frequency-dependent phase delay of the fluid jet (Fig. 5C; see also Discussion). Hair cells also reliably transduce the (high-frequency) resonance of the fluid jet device in response to an unfiltered step applied to the piezoelectric disc as can be seen in Figure 7E. The hair cell response shows an initial step transducer current followed by an oscillation of 5.65 kHz. This frequency is identical to the resonance observed while applying an unfiltered fluid jet stimulus to the sense probe (Fig. 6) and thus illustrates the need for proper filtering of the voltage step to avoid fluid jet resonance. DISCUSSION Reliability of the calibration method The flow of microscopic fluid jets produced by several types of devices has been used in hair cell research for several decades (Flock and Orman 1983; Saunders and Szymko 1989; Kros et al. 1992; Rüsch et al. 1994; Géléoc et al. 1997; Vollrath and Eatock 2003; van Netten et al. 2003). In almost all cases, the mechanical output characteristics are assumed to be constant in terms of fluid displacement or velocity but generally lack detailed data on these actual output characteristics. In the present study, a method was developed to dynamically calibrate a microscopic fluid jet using a flexible sense probe that was independently calibrated by means of an additional stimulus device (stimulus sphere), providing its detailed frequency selectivity within a relevant frequency range. This information is necessary to properly analyze the displacements of objects induced by a fluid jet. Because changes in the tip diameter of the jet-producing pipette alter the frequency response, a calibration for each individual pipette is necessary especially when applied at higher frequencies (e.g., Fig. 7D). The developed calibration procedure using a sense probe has been shown to be practical within the frequency range tested (1–1,000 Hz). This calibration procedure intrinsically produces results for the flow of the jet in a free and unobstructed fluid field. This is related to the principle used, which is based on equivalent forces acting on a sense probe when stimulated with either a fluid jet or a sphere with known (free) flow field. The frequency-dependent characteristics of a particular sense probe can be determined well in advance of the actual physiological experiment, and the sense probes can be stored for later use. During an experiment, a sense probe can be positioned in close proximity of the preparation to enable calibration of the fluid jet directly after the stimulation of the preparation without having to move the pipette tip out of the water. Essential for this procedure is that the fluid jet’s frequency response does not change over time. A potential danger is that the pipette tip gets (partially) blocked by dirt floating in the bath solution. Such an additional restriction of the tip leads to changed jet response characteristics, precluding a proper correction of the measured data. However, in practice, such a blockage in the glass pipette’s tip can be monitored by microscopic visualization. Displacement or velocity output depends on tip size From the experiments on fluid jets produced by various glass pipettes, it appears that the pipette’s tip size is the dominant parameter determining the fluid jet output characteristics both in terms of the overall output and frequency selectivity (Fig. 5). One of the most relevant features of this stimulus device is whether it is the fluid displacement or velocity of the jet that follows the voltage applied to the device. Our results show that with a large tip diameter (unrestricted pipette) the displacement of the fluid jet is proportional to the displacement of the piezoelectric disc in a low frequency range (<100 Hz). This displacement mode is accompanied with resonance behavior (Fig. 5A). A restriction of the pipette’s tip diameter critically damps the system, preventing it from resonating. Under such conditions (e.g., Ø 56 μm; Fig. 5B), it produces a constant fluid displacement amplitude as a function of frequency of the input signal up to about 10–20 Hz. Beyond this frequency, the displacement amplitude falls off with −40 dB/dec and is accompanied by a phase rotation that may reach −180° at high frequencies. An even higher restriction of the tip diameter (Ø 7 μm; Fig. 5C) results in a fluid displacement that declines with about −20 dB/dec from low (1 Hz) to high (1,000 Hz) frequencies with a fixed phase delay of about 90°. This means that the velocity of the fluid jet follows the voltage applied to the piezoelectric disc. This is the most effective mode for producing a hydrodynamic stimulus to excite micro-sized objects such as sensory hair bundles. Comparison of measured fluid jet characteristics with model A physical representation of the fluid jet-producing device as two damped fluid masses coupled via a lever and loaded with springs describes the behavior of the fluid jet emerging from the pipette’s tip quite well (Fig. 5 solid lines). It follows from fitting the model response to the measured data that the dynamics of the fluid in the pipette, rather than that in the larger Perspex container, dominantly determines the overall frequency response characteristics below 1 kHz. In this frequency range the output can be approximated with Eq. 4a, which shows the importance of Rp and Mp in relation to the frequency selectivity. It also shows that the overall output at frequencies around and below the low resonance frequency () is inversely proportional to the stiffness of the piezoelectric disc . The high resonance frequency of the fluid jet was found at 5.65 kHz (Fig. 6). The value of this resonance frequency was confirmed by laser interferometric measurements on the back side of the piezoelectric disc under the same fluid load conditions. When the fluid was removed from the Perspex body and no pipette was inserted, the piezoelectric disc resonated at about 8.3 kHz. As in this configuration only the disc is moving, we could estimate its effective dynamic mass value in combination with the value of Spiezo to be Mpiezo = 0.741 × 10−3 kg. This appears to be 65% of the physical mass of the disc (1.14 × 10−3 kg), which seems to be a reasonable fraction if the circular disc is moving in a mode with a circular node imposed by its peripheral clamping support. The compressibility of the fluid in the Perspex container and that in the pipette has been neglected. This can be justified as the obtained maximum stiffness values of the elastic elements associated with these masses, Spiezo (∼2 × 106 N/m) and Sp (∼85 N/m), are significantly smaller than the effective stiffness values associated with their compressibility, 4.4 × 107 and 4.7 × 104 N/m, respectively. These effective stiffness values, Seff, were evaluated by considering the compressibility along the long axis of a fluid-filled cylinder having length, L, and cross sectional area, A, and can be shown to equal Seff = KA / L, with K (= 2.2 × 109 Pa) the bulk modulus of water (Lide 2005). Highly restricted pipettes with tip diameters of the order of 15 μm or less strongly overdamp the system and produce a velocity output which is proportional to the force produced by the piezoelectric disc and therefore to the voltage signal applied (Fig. 5C). The model therefore predicts the transition in fluid jet characteristics from a displacement stimulus in the situation of a large tip diameter (small tip resistance) to a velocity stimulus due to tip restriction. Figure 8 compares these model results in more detail for increasing tip resistance, Rp. At a relative low pipette resistance (Rp = 0.02 Ns/m), a constant displacement amplitude of the jet is produced at frequencies below about 80 Hz showing resonance around 150 Hz. Increasing the resistance to Rp = 0.07 Ns/m, critically damps the system making it a useful displacement stimulus device up to about 100 Hz. Gradually increasing the resistance Rp (2 to 20 Ns/m) changes the fluid jet apparatus into a velocity-producing device in the full frequency range considered (1–1,000 Hz) at the expense of the jet’s flow amplitude. This velocity output corresponds with a slope of −20 dB/dec in the displacement amplitude response. FIG. 8.The effect of increasing pipette resistance. Series of modelled frequency responses showing fluid displacement output as a function of the frequency of the voltage signal put across the piezoelectric disc. Parameter values: L 1 = 7 mm; L 2 = 50 mm; D 1 = 13.4 mm; D 2 =  1.17 mm; ρ  = 1,000 kg/m3; S piezo = 2.0 × 106 N/m, R c = 7 Ns/m, S p = 55 N/m, values of pipette resistance (R p in Ns/m) are given next to each curve. Absolute fluid jet velocity The calibration method described was focused on the relative frequency selectivity of the fluid jet emerging from the tip of the glass pipette. The absolute fluid velocity that can be obtained not only depends on the tip diameter, but it also varies strongly with the measurement position with respect to the pipette’s tip. This means that it is not useful to afterwards calibrate the absolute fluid velocity on a sense probe as done for determining the relative frequency sensitivity. Nevertheless, an estimate of the order of magnitude follows directly from curves like Figure 5C or alternatively by visualizing the trajectories of small (Ø = 1.0 μm) latex microbeads in the flow field (e.g., Sauders and Szymko 1989). Our observations, for instance, show that a sensitivity of about 1 mm/s per volt applied to the piezoelectric disc is obtained at a distance of about 30 μm in front of the pipette tip (Ø = 15 μm). From such calibrations, we estimate that the fluid flow velocity, required to produce an open probability of the transducer channels of 0.5, is about 2 mm/s. Linearly extrapolating this to the maximum voltages that may be applied to the piezo-electric element (100 V) without damaging it, leads to a maximal velocity in the order of 200 mm/s. Applications As shown in Figure 7, a fluid jet produced by the apparatus using a restricted tip diameter (<15 μm) can be adequately used to effectively evoke robust transducer currents in individual sensory hair cells. It is known from previous studies (van Netten et al. 2003; Ricci et al. 2005) that the speed of the transducer apparatus in mammalian outer hair cells is well exceeding the range of frequencies (1 to 1,000 Hz) considered here. This implies that the transducer apparatus is capable of following signals up into the kiloHertz range without delay. The observed delays in transducer currents (Fig. 7D) can therefore be attributed to the frequency selectivity of the fluid jet. Figure 5C illustrates that the difference in phase delay at 1,000 Hz (−130°) compared to that at 100 Hz (−80°) amounts to about 50°, when using restricted pipettes. The observed time delays of the hair cell responses (Fig. 7D) in stimulus periods (0.0194 at 100 Hz and 0.171 at 1,000 Hz) or in terms of phase delays (7° at 100 Hz and 62° at 1,000 Hz, a difference of 55°) are therefore in line with the measured phase difference of the fluid jet at these frequencies. As is also apparent from Figure 5C, the amplitude response of the fluid jet is quite comparable in terms of velocity of the jet at 100 and 1,000 Hz, in line with the equal magnitude of the measured transducer currents at these frequencies. These results support our notion that a proper calibration of the fluid jet characteristics is necessary for a correct interpretation of the (phase) measurements. A relevant issue related to the application to hair cell stimulation is the proper mechanical stimulus to excite a sensory hair bundle. Based on the micromechanics and hydrodynamics of an individual hair bundle, it has theoretically been argued that a hair bundle’s displacement response is flat as a function of the frequency of a fluid velocity stimulus up to a cut-off frequency determined by its stiffness and its size (Géléoc et al. 1997; van Netten 1997). The pipettes used to displace individual bundles have opening diameters that are matched to the dimensions of the bundle resulting in diameters of about 15 μm or below. When applying a (filtered) step voltage, the accompanying high resistance of the pipette tip results in a jet velocity proportional to the step and therefore produces an adequate stimulus to statically displace individual hair cell bundles inducing step displacements of the bundle. If unfiltered, the velocity output may, however, be contaminated with a ringing (5.65 kHz) caused by resonance of the fluid in the Perspex fluid container (Fig. 7E). Adequate low-pass filtering of the applied voltage signal can prevent these oscillations, but will inevitably put an upper limit to the displacement rise time of a hair bundle that can be achieved (see also next section). A second consequence of tip restriction is the decreased output response. Much larger voltages have to be applied to the piezoelectric disc to produce similar fluid jet displacements. This consequence of tip restriction was previously recognized by Saunders and Szymko (1989) who showed a square relation between fluid displacement amplitude and tip diameter. Remarkably, they did not report differences in the frequency response of their fluid jet due to tip diameter. Their calibrating method did not give precise information about the phase of the response and lacks relevant information on tip size. Their reported displacement frequency responses have a slope of −10 dB/dec, which makes their apparatus neither a true displacement nor a true velocity-producing device. When a preparation is only stimulated at a single frequency, the detailed frequency response of the fluid jet is less relevant as long as the desired displacement and related phase of the object can be established. Harmonic distortion of the produced fluid jet might, however, cause a problem in the investigation of a system’s nonlinearities. Although the harmonics produced by the described device are relatively small (e.g., Fig. 3; THD < −30 dB) and comparable to the fluid jet distortion reported by Saunders and Szymko (1989), they may nevertheless preclude the direct measurement of transducer-related hair bundle nonlinearities from harmonic analyses as predicted for specific types of hair cells (e.g. van Netten and Kros, 2000). Bandwidth limitations of the fluid jet-producing device The calibration method described here is restricted to about 1,000 Hz because of the frequency range of the stimulus sphere used to calibrate the sense probes, which resonates at about 900 Hz and produces only a limited fluid displacement beyond resonance (Fig. 4B2). However, it may be possible to design a method to mechanically calibrate the sense probes into the supra-kHz range so that the jet’s output can also be calibrated in an extended frequency bandwidth. In the velocity-producing mode, resonance of the fluid mass, Mc, in the Perspex container may limit the use of the device beyond the high resonance frequency (5.65 kHz), where the fluid jet velocity output declines with −20 dB/ dec. It may seem therefore that a fundamental restriction of the application of the fluid jet produced by the apparatus described lies in the restricted bandwidth imposed by the piezoelectric disc used. The piezoelectric disc’s intrinsic resonance behavior under dry unloaded conditions, i.e., while being clamped at its rim similarly as in the operational fluid jet device but without the fluid load imposed, is about 8.3 kHz. However, using the model, it can be shown that this is not a fundamental limit for its dynamic use, as the high-frequency resonance is also determined by the stiffness Sp, which contributes via up-transforming via the lever with a factor L2 to the stiffness of the moving fluid in the Perspex container determining the high-resonance frequency (Eq. 3). Enhanced (high) frequency characteristics, i.e., a higher resonance or cut-off frequency (fh) in the velocity mode using small pipette tip diameters, may thus be expected from designs with a smaller fluid mass (Mc) in the Perspex container, improved lever action (higher L), and overall stiffer elastic elements (Spiezo, Sp), although the latter will decrease the overall output of such devices. In practice, this means that at the expense of a smaller output faster fluid jet performance may be obtained by using smaller but stiffer (ultrasonic) piezo elements. Conclusions This work emphasizes the importance of the calibration of a jet-producing stimulus device and demonstrates the importance of the outflow restriction for the jet response characteristics. Not only the jet output is decreased with a higher tip resistance, but more importantly, its output characteristic changes from a displacement to a velocity fluid stimulus in relation to the input voltage of the device. A simple mechanical model that adequately describes the fluid jet’s characteristics can help understanding the physical parameters underlying these characteristics and can thus facilitate optimizing the design of this type of hydrodynamic stimulus device for particular applications.

Diabetologia-4-1-2235909

A randomised, 52-week, treat-to-target trial comparing insulin detemir with insulin glargine when administered as add-on to glucose-lowering drugs in insulin-naive people with type 2 diabetes

Aims/hypothesis This 52-week multinational, randomised, open-label, parallel-group, non-inferiority trial compared clinical outcomes following supplementation of oral glucose-lowering drugs with basal insulin analogues detemir and glargine in type 2 diabetic patients. Introduction People with type 2 diabetes inadequately controlled by oral glucose-lowering drugs can achieve clinically relevant improvements in HbA1c with the addition of insulin therapy [1–3]. However, patients and healthcare providers are often reluctant to initiate insulin due to concerns over injections, fear of hypoglycaemia and additional weight gain, and also because insulin treatment is perceived as complex and an added burden to diabetes management [4, 5]. Moreover, once insulin is initiated, recommended targets for glycaemic control (HbA1c < 6.5–7.0%) are often not met [5–7]. In recent years, the basal insulin analogues glargine and detemir have been introduced. These were developed to improve upon the limitations of NPH insulin (NPH) and other conventional basal insulins, which have an inadequate duration of action, a marked peak glucose-lowering effect and variability in response from one injection to another [8]. These analogues might help to overcome some of the barriers to insulin initiation and optimisation, including concerns over hypoglycaemia and weight gain. Several recent studies have assessed basal insulin as an add-on therapy to oral glucose-lowering drugs, comparing either insulin glargine or insulin detemir with NPH, and using titration algorithms based on glucose monitoring [1, 9–13]. These studies have demonstrated that simple regimens involving a once or twice daily injection of a basal insulin analogue can achieve clinically important improvements in glycaemic control similar to those achievable with NPH, but with less risk of hypoglycaemia. Insulin detemir has consistently shown less body weight gain than NPH when used in this way, as well as when used in basal plus mealtime insulin therapy [14, 15], whereas a weight advantage has been reported in only a few of the comparative trials of glargine vs NPH, as for example in the recent LANMET study [11]. Insulin glargine is licensed only for once daily use as a basal insulin for people with diabetes. Insulin detemir, in contrast, is available for once or twice daily use. Glucose clamp comparisons between these insulins have given contradictory information on whether their duration of effect is comparable [16, 17]. The only direct comparison in patients with type 2 diabetes suggests very similar pharmacodynamic profiles at clinically relevant doses [16], but methodological issues remain controversial. The objective of the current study was to compare treatment with insulin detemir and insulin glargine in line with their licensed indications as add-on therapy to oral glucose-lowering agents in insulin-naive patients with type 2 diabetes. Methods Study protocol This 52-week, parallel-group trial was conducted in 2003 and 2004 at 80 sites in Europe and the USA and included 582 insulin-naive people with type 2 diabetes, who were randomised (1:1) and treated with insulin detemir (Levemir; Novo Nordisk, Bagsværd, Denmark) or insulin glargine (Lantus; sanofi-aventis, Paris, France) as add-on therapy to oral glucose-lowering drugs. The trial was conducted in accordance with the Declaration of Helsinki and principles for Good Clinical Practice and was approved by ethics committees/review boards in all countries. All participants gave written informed consent.Concealed randomisation was carried out by an automatic telephone response system and was stratified according to oral glucose-lowering drug mono- or combination-therapy at entry. An open-label design was required to allow twice daily administration of insulin detemir if needed, according to the dosing algorithm targets for pre-dinner plasma glucose (PG) concentrations (Table 1). Glargine was only administered once daily at bedtime as per study protocol and in accordance with its licence [18]. To reduce potential bias, HbA1c results were only disclosed to investigators at randomisation and at trial end. Table 1Algorithm used for insulin dose titrationAlgorithmAdjustment of insulin dose (U)If positive response to previous dose adjustmentIf no response to previous dose adjustmentaEvening insulin dose adjustment Average pre-breakfast self-monitored PG  >10.0 mmol/l+12+12  9.1–10.0 mmol/l+8+10  8.1–9.0 mmol/l+6+8  7.1–8.0 mmol/l+4+6  6.1–7.0 mmol/l+2+2 If one self-monitored PG measurement  3.1–4.0 mmol/l−2–2  <3.1 mmol/l−4–4Morning insulin dose adjustmentb Average pre-dinner self-monitored PG  >10.0 mmol/l+8+8  9.1–10.0 mmol/l+6+8  8.1–9.0 mmol/l+4+6  7.1–8.0 mmol/l+2+4  6.1–7.0 mmol/l+2+2If one self-monitored PG measurement  3.1–4.0 mmol/l–2–2  <3.1 mmol/l–4–4a Non-responses: the average self-monitored PG level is increased and/or within the same range as at the last contactbSome insulin detemir-treated participants only+, insulin dose titrated up; –, insulin dose titrated down Participants Insulin-naive men and women with type 2 diabetes and the following characteristics were recruited: ≥18 years old, ≥12 months disease duration, BMI ≤ 40.0 kg/m2 and HbA1c 7.5–10.0%. For inclusion, they had to be receiving one or two oral agents (metformin, insulin secretagogues, α-glucosidase inhibitors) ≥4 months on at least one-half the maximum recommended dose, according to local guidelines. Exclusion criteria included treatment with thiazolidinediones (due to labelling restrictions in Europe), use of more than two oral agents within 6 months, hypoglycaemic unawareness or other medical conditions likely to interfere with trial conduct. Withdrawal criteria included pregnancy, HbA1c > 11.0% after the first 12 weeks of treatment and initiation of medication interfering with glucose metabolism. Study medications Oral glucose-lowering therapy, diet and physical activity were recommended to remain stable during the study; no meal-time insulin was allowed. Basal insulin was initiated once daily in the evening at a dose of 12 U and titrated according to a structured treatment algorithm. In line with its licence, people allocated to insulin detemir were allowed to receive an additional morning insulin dose if pre-dinner PG was >7.0 mmol/l, but only if pre-breakfast PG was <7.0 mmol/l or nocturnal hypoglycaemia (major episode or PG ≤ 4.0 mmol/l) precluded achievement of the fasting plasma glucose (FPG) target. Insulin detemir was administered with a pen-injector (FlexPen, Novo Nordisk) 1 h before to 1 h after dinner, and if needed within 30 min of breakfast. Glargine was given once daily at bedtime, via a pen-injector in the EU (OptiPen Pro 1, sanofi-aventis) and with syringes in the USA. The evening dose of insulin detemir was administered somewhat earlier than that of insulin glargine, to allow for the possibility that recipients might be switched to a twice daily schedule, in which case a more even distribution of the dose across 24 h would be achieved. Titration of basal insulin Participants attended 16 scheduled visits and nine telephone contacts over 1 year. Evening insulin doses were titrated throughout the trial to a FPG target ≤6.0 mmol/l in the absence of hypoglycaemia. An identical pre-dinner glucose target was applied for people administering a morning dose of insulin detemir (Table 1). Participants measured capillary PG using glucose meters (Medisense Xtra; Abbott, Wiesbaden, Germany). Dose adjustments were to be based on the average of three self-measurements before breakfast (and before dinner if on twice daily insulin detemir). During the first 12 weeks, participants had weekly investigator contact. A titration committee monitored the algorithm for insulin dose optimisation and reviewed prescribed insulin doses periodically. This committee was not informed of the treatment insulin being used, but this would have been evident for participants on twice daily insulin detemir. Outcome measures The primary endpoint was baseline-adjusted HbA1c at end of treatment. Secondary variables included clinic FPG, within-participant variation in PG, ten-point self-measured PG profiles, proportion of participants achieving HbA1c ≤ 7.0% with and without hypoglycaemia, change in body weight, incidence of hypoglycaemia, adverse events and standard safety parameters. Analyses and assessments HbA1c was analysed by HPLC (Bio Rad, Munich, Germany [EU analyses] and Bio Rad, Hercules, CA, USA [US analyses], DCCT-harmonised), with a reference range 4.3–6.1%. Clinic FPG was measured centrally by a hexokinase method (Gluco-quant; Roche, Mannheim, Germany). Body weight was measured using calibrated scales. Participants recorded ten-point PG profiles during the last week of treatment. Hypoglycaemia was classified as ‘major’ if assistance from another person was required, ‘minor’ if confirmed by PG < 3.1 mmol/l or ‘symptoms only’ if PG ≥ 3.1 mmol/l or no measurement was made. Statistical analyses The sample size was based on non-inferiority of insulin detemir relative to insulin glargine for HbA1c after 52 weeks. To achieve a power of 95% with an expected SD for change in HbA1c of 1.1% and allowing a dropout rate of 15%, it was originally planned to randomise 466 patients. However, this was extended to 566 patients to allow for potential regional differences between the EU and US. The possibility of a treatment × region interaction was tested, but eliminated from the model as no significant interaction was found. Non-inferiority was accepted if the upper limit of the two-sided 95% CI for the difference in HbA1c (detemir–glargine) was less than 0.4%-units, a value decided in discussion with the Food and Drug Administration (FDA) for the entire insulin detemir phase III study programme.Unless otherwise specified, all analyses were based on the intention-to-treat (ITT) population, this being all randomised patients exposed to insulin detemir or insulin glargine. A significance level of 5% was used for all analyses. Participants were regarded as being on once- or twice-daily insulin detemir according to the regimen used at trial completion. Statistical analyses were not made for these subsets of patients, as this was non-randomised and protocol-determined; results are given for observational comparison only.Analyses of HbA1c, clinic FPG and change in weight after 52 weeks were by ANOVA with treatment, region and oral glucose-lowering therapy as fixed effects and baseline (randomisation) value as covariate. The last observation carried forward (LOCF) principle was specified for HbA1c and clinic FPG in non-completers treated ≥12 weeks (time of the first on-treatment measurements). Change in weight was estimated based on all participants completing the trial. Additional weight analyses were prepared for the ITT population, applying the same LOCF principle or using imputed data, by treatment-specific linear regression, from week 12 onwards to account for continuing weight change and limit the influence of differences in withdrawal rates and times between treatments. Fisher’s exact test was used to compare the numbers of patients on insulin detemir and insulin glargine with an HbA1c ≤ 7.0% without symptomatic hypoglycaemia confirmed by PG <4.0 mmol/l or any single value <3.1 mmol/l during the last 3 months of treatment.The ten-point self-monitored PG profiles were analysed for parallelism using repeated measures ANOVA depending on treatment, region, oral glucose-lowering therapy, time and treatment × time interaction as fixed effects. Within-participant variation in self-measured fasting and pre-dinner PG was determined from four measurements for each during the last week of treatment. Hypoglycaemic episodes during the 52 week treatment period were analysed as recurrent events in a Cox regression analysis using a gamma frailty model (Splus 2000; Insightful, Seattle, WA, USA). Nocturnal episodes (2300 to 0600 hours) were analysed separately. Adverse events were summarised using descriptive statistics. All other analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC, USA). Results Participants Of 892 patients screened, 582 were randomised, while 289 did not fulfil the selection criteria and 21 withdrew consent. In each treatment arm, 291 patients were randomised and treated, of whom 231 (79%) and 252 (87%) completed the trial on insulin detemir and insulin glargine, respectively (Fig. 1). The higher withdrawal rate observed with insulin detemir was partly due to adverse events, ten of which were considered possibly or probably related to trial drug (Electronic supplementary material [ESM] Table 1). These included two cases of possible cutaneous allergy, five of injection site reactions and one each of injection site inflammation, hypoglycaemia and weight increase. Adverse event withdrawals with possible or probable relation to glargine included one case each of pruritus, myalgia, hyperglycaemia and hypoglycaemia. Details of all adverse events leading to withdrawal are provided in ESM Table 2. Other diabetes-related reasons for withdrawal with insulin detemir included HbA1c 12.0% (n = 1) and multiple hypoglycaemic episodes (n = 1). Other reasons for withdrawal included the closure of a trial site and various circumstantial events unrelated to the trial medications. Fig. 1Patient disposition during the trial and consequent analysis setsBaseline characteristics (Table 2) were comparable between the two treatment groups. Participants completing the trial on once or twice daily insulin detemir had a baseline HbA1c of 8.60 and 8.66%, respectively. Table 2Clinical characteristics of type 2 diabetic participantsCharacteristicsDetemirGlargineRandomised/exposed/ITT, n (%)291 (100)291 (100)Men/women, n166/125171/120Ethnicity (n)Black2212White250263Asian-Pacific islander77Other129Oral glucose-lowering drugs, n (%)Monotherapy73 (25)70 (24) Metformin32 (11)33 (11) Insulin secretagogues41 (14)37 (13)Combination therapy218 (75)221 (76) Metformin + secretagogue212215 Metformin + alpha glucosidase inhibitor31 Secretagogue + alpha glucosidase inhibitor34 Secretagogue + secretagogue (SU + glinide)–1Age (years)58.4 (10.2)59.4 (9.6)Weight (kg)a87.4 (16.6)87.4 (17.4)BMI (kg/m2)30.6 (4.8)30.5 (4.6)Duration of diabetes (years)9.1 (6.1)9.1 (6.4)HbA1c (%)a8.64 (0.78)8.62 (0.77)C-peptide (nmol/l)0.87 (0.56)0.85 (0.55)Values are n (%) or mean (SD)aBefore randomisationSU, sulfonylurea Insulin dosing Of participants treated with insulin detemir, 104 (45%) completed the trial on one daily injection, while 129 (55%) administered an additional morning dose. The majority of patients administering insulin detemir twice daily (n = 103) were transferred to this regimen within 12 weeks of treatment. At the time of transfer, about two-thirds of the patients adding a morning injection fulfilled the formal dose transfer guidelines, the other one-third were changed through investigator discretion. Participants using insulin detemir twice daily administered about 40% of their daily insulin dose before breakfast. After 52 weeks the mean daily dose of insulin detemir (n = 227) was 0.78 U/kg (0.52 U/kg on once daily [n = 102] and 1.00 U/kg on twice daily [n = 125]) and the mean daily dose of glargine (n = 248) was 0.44 IU/kg. Glycaemic control HbA1c decreased by 1.5% with both insulins and was comparable after 52 weeks at 7.2% (n = 268) and 7.1% (n = 275) for detemir and glargine respectively (Table 3), with an estimated difference of 0.05 (−0.11, 0.21)%, thereby meeting the criteria for non-inferiority for insulin detemir vs glargine. HbA1c was similar in participants completing the study on once or twice daily insulin detemir (7.1 and 7.1%). Clinic FPG was 10.8 mmol/l at baseline in both arms and was comparable at end of treatment with insulin detemir and insulin glargine (7.1 and 7.0 mmol/l) (Table 3). The profile of change in HbA1c and FPG was very similar (Fig. 2a). Mean self-monitored FPG was comparable between treatments, being 6.1 mmol/l with insulin detemir and 6.0 mmol/l with insulin glargine; self-monitored PG pre-dinner (6.8 and 7.0 mmol/l) was also similar. Fig. 2Change in HbA1c with time (a). Black circles, insulin detemir; white circles, insulin glargine. b Mean ten-point self-monitored PG profiles during the last week of treatment. Triangles, insulin detemir (once daily); squares, insulin detemir (twice daily); black circles, insulin detemir (all patients); white circles, insulin glargineTable 3Glycaemic control in type 2 diabetic patients treated with insulin detemir or insulin glargine DetemirGlargineDifference (95% CI) (detemir/glargine)p valueCompleters on once dailyCompleters on twice dailyAllAllParametersnMeannMeannMeannMeanHbA1c % (SE)a1047.12 (0.11)1277.06 (0.10)2687.16 (0.08)2757.12 (0.08)0.05 (–0.11, 0.21)–Clinic FPG (mmol/l)a1047.27 (0.31)1276.73 (0.25)2687.14 (0.21)2726.98 (0.21)0.16 (–0.26, 0.58)–HbA1c ≤7.0%, n (%)10352 (51)12769 (54)248129 (52)259135 (52)–1.00HbA1c ≤7.0% without hypoglycaemia, n (%)10331 (30)12748 (38)24882 (33)25990 (35)–0.71Within-participant variation (mmol/l)Pre-breakfast (mmol/l)b1030.93 (15.0)1251.15 (19.6)2381.06 (17.5)2571.03 (17.3)–0.45 Pre-dinner (mmol/l)b1031.27 (19.8)1251.84 (26.4)2381.60 (23.6)2581.55 (22.0)–0.41aMean (SE); n=number of patients for whom data are availablebWithin-participant SD (CV%)Within-participant variation in self-monitored PG pre-breakfast and pre-dinner did not differ significantly between insulin detemir and insulin glargine (Table 3). The overall shape of the ten-point self-monitored PG profiles during the last week of treatment was parallel for insulin detemir (n = 218) and insulin glargine (n = 246) (NS) and appeared identical, regardless of treatment regimen (Fig. 2b).Of participants in both arms, 52% achieved HbA1c ≤ 7.0%, while 33% treated with insulin detemir and 35% treated with insulin glargine did so in the absence of hypoglycaemia. Fasting and pre-dinner PG targets of ≤6.0 mmol/l were achieved by 25 and 20% of participants treated with insulin detemir and insulin glargine (NS), respectively; the pre-breakfast target alone was achieved by 46 and 58% (p < 0.01) and the pre-dinner target by 38 and 30%, respectively (p < 0.05). Hypoglycaemia The risk of hypoglycaemia of any type was comparable between treatments (Table 4). The overall rate of hypoglycaemia was low at 5.8 vs 6.2 episodes per patient-year with insulin detemir versus insulin glargine (relative risk: 0.94 [96% CI 0.71, 1.25]), while the rate of nocturnal hypoglycaemia was only 1.3 episodes per patient-year with both insulins. Adjustment for HbA1c did not affect the outcome of the analyses. Major hypoglycaemic episodes were rare with both insulins, especially at night, and could not be statistically analysed. Table 4Hypoglycaemic episodes in type 2 diabetic patients treated with insulin detemir or insulin glargineEvents  Insulin detemir (n = 291)Insulin glargine (n = 291)Relative risk (detemir/glargine) (95% CI)Participants, n (%)Episodes (n)Rate (per patient-year)Participants, n (%)Episodes (n)Rate (per patient-year)All182 (63)15215.8191 (66)16706.20.94 (0.71–1.25) Nocturnal95 (33)3521.393 (32)3501.31.05 (0.69–1.58)Major5 (2)90.08 (3)80.0– Nocturnal3 (1)50.04 (1)40.0–Minor135 (46)7372.9151 (52)7862.91.05 (0.75–1.46) Nocturnal73 (25)2120.871 (24)1920.71.17 (0.75–1.83)Symptoms only137 (47)7603.0133 (46)8663.20.88 (0.61–1.25) Nocturnal48 (17)1280.549 (17)1510.60.88 (0.50–1.54)No statistical analyses were performed on the small numbers of major events. Body weight Weight gain in participants completing 52 weeks of treatment was lower with insulin detemir (n = 230) than with insulin glargine (n = 252) (3.0 [SE = 0.4] vs 3.9 [SE = 0.4] kg, p = 0.01). Change in weight analysed by LOCF (detemir n = 268, glargine n = 275) confirmed this lower weight gain (2.7 vs 3.5 kg, p = 0.03), as did analysis with imputation for non-completers (2.8 vs 3.5 kg, p = 0.04). Patients completing the study on once daily insulin detemir (n = 104) had a weight gain of 2.3 (SE = 0.5) kg, whereas those treated twice daily (n = 126) gained 3.7 (SE = 0.4) kg, similar to glargine (Fig. 3). Fig. 3Mean weight change from baseline at week 52 in patients completing treatment on insulin detemir once or twice daily (and overall) and insulin glargine. ***p < 0.001; †p < 0.012 Adverse events and other safety measures Serious adverse events were less frequent with insulin detemir (42 patients with 47 events) than with glargine (53 patients with 73 events). However, only five serious events with insulin detemir (two hypoglycaemia, one each of hypothyroidism, injection-site reaction and a motor vehicle accident) and four with glargine (three hypoglycaemia, one hyperglycaemia) were considered by local investigators to be probably or possibly related to trial products. A participant treated with insulin detemir was found dead in bed (no autopsy report available), but had a history of myocardial infarction. A patient using glargine was hospitalised after the study for pulmonary fibrosis and died of cryptogenic fibrosing alveolitis. Adverse events recorded as serious tended to be of a wide-ranging disparate nature, with no clear pattern of between-treatment differences. Full details of all serious adverse events and events recorded as possibly or probably related to the study insulins are given as in the ESM Tables 1, 2, 3, and 4).The only differences in adverse events, judged as possibly or probably related to trial drugs, between treatments were: injection-site disorders (13 patients [4.5%] on insulin detemir compared with four [1.4%] on glargine), allergic reactions (three patients on detemir vs one on glargine) and skin disorders including pruritus and rash (six patients on insulin detemir vs one on glargine). There were no differences in standard safety parameters between treatments. Discussion The results of this first head-to-head comparison of insulin detemir and insulin glargine as add-on for treatment with oral glucose-lowering agents in people with type 2 diabetes using forced insulin titration suggest that clinically significant and similar improvements in glycaemic control can be achieved with both analogues, together with a similarly low risk of hypoglycaemia. Insulin doses were higher with insulin detemir overall, partly as a result of the doses used by those taking it twice daily. The withdrawal rate appeared to be higher with insulin detemir (21 vs 13%), which was partly accounted for by adverse events (8 vs 4%), this excess being primarily due to injection site reactions. Insulin detemir was associated with a modest relative reduction in weight gain, consistent with observations in previous comparisons of this analogue with NPH in type 2 diabetes [12, 13, 19, 20]. In the present study, the between-treatment difference in weight was primarily accounted for by those patients completing the study on once-daily insulin detemir. It is, however, possible that differences in eating pattern were contributory factors in both the insulin detemir dosing schedule and the different levels of weight gain observed between the two insulin detemir dosing schedules. The mechanism(s) responsible for relatively lower weight gain observed here and previously with insulin detemir remain the object of speculation [14, 15, 21, 22]. The present study was designed to compare the two basal insulin analogues as they had been used in previous studies and according to label. Thus, insulin glargine was given once daily at bedtime regardless of glycaemic profile, while for insulin detemir an option was provided for adding a second dose, primarily based on pre-dinner blood glucose level following a structured insulin titration protocol. The decision to use twice-daily insulin detemir could not be attributed to hypoglycaemia, as patients who were switched to a twice-daily regimen had on average 3.7 hypoglycaemic episodes per patient-year prior to transfer, compared with 4.7 episodes per patient-year in patients completing treatment on once-daily insulin detemir. The frequency of nocturnal hypoglycaemia was also lower in relevant patients before transfer (0.7 vs 0.9 episodes per patient-year). Interestingly, the initiation of insulin with once-daily insulin detemir has recently been tested by Philis-Tsimikas et al. in type 2 diabetes patients on oral glucose-lowering agents, resulting in HbA1c reductions of 1.5% (morning injection) and 1.6% (evening injection) from baseline values of 9.1 and 8.9%, respectively [13]. These improvements in glycaemic control approach the magnitude of HbA1c reduction shown in the current study and in previous studies of once-daily insulin glargine [9–11], although the insulin detemir doses were higher at trial-end and the absolute HbA1c values achieved at trial-end were relatively high. The design of the current study does not allow definitive conclusions for comparison of once and twice daily dosing with insulin detemir, although these post hoc observations do suggest that once daily administration can be an appropriate starting regimen for people using insulin detemir as add-on to oral glucose-lowering drug therapy. However, with dose optimisation, a significant proportion of patients may eventually need a twice-daily regimen, guided by structured glucose monitoring. Further studies are required to better define the differences between these two basal insulin analogues on the basis of a similar insulin administration regimen, once daily only, for both insulins. Alternatively, if the option of adding a second insulin dose is available for both insulins, then a properly designed study protocol should allow addition of this second insulin injection only when a normal or much lower FPG target is achieved. Indeed, the use of lower FPG targets (e.g. ≤5.5 mmol/l, as has been used previously) should also enable a higher proportion of patients to reach guideline HbA1c targets. In conclusion, the use of insulin detemir or insulin glargine as add-on to oral glucose-lowering therapy resulted in comparable HbA1c improvements and a similarly low risk of hypoglycaemia. Further comparisons of detemir and glargine are required to fully understand how each may relatively benefit defined groups of patients starting on insulin. Electronic supplementary material Below is the link to the electronic supplementary material. ESM 1 (PDF 12.4 kb) ESM Table 1 Treatment emergent adverse events probably or possibly related to trial product by system organ class, ITT cohort (PDF 20.2 kb) ESM Table 2 Adverse events leading to withdrawal (PDF 17.2 kb) ESM Table 3 Treatment emergent serious adverse events by system organ class, ITT cohort (PDF 23.1 kb) ESM Table 4 Treatment emergent serious adverse events preferred term, occurring in more than one subject, ITT (PDF 14.6 )

Matern_Child_Health_J-2-2-1592143

Social Marketing: Planning Before Conceiving Preconception Care

Social marketing approaches can help to shape the formation of and to create demand for preconception care services. This article describes four components of social marketing, often referred to as the 4 P’s, that should be carefully researched and set in place before a national effort to launch and sustain preconception care services is pursued. First, the product or package of services must be defined and adapted using the latest in scientific and health care standards and must be based on consumer needs and desires. Second, the pricing of the services in financial or opportunity costs must be acceptable to the consumer, insurers, and health care service providers. Third, the promotion of benefits must be carefully crafted to reach and appeal to both consumers and providers. Fourth, the placement and availability of services in the marketplace must be researched and planned. With the application of market research practices that incorporate health behavior theories in their exploration of each component, consumer demand for preconception care can be generated, and providers can take preconception care to the market with confidence. Introduction Preconception care (PCC) is a critical component of maternal and child health that does not currently have a strong foothold in our health care delivery system [1–3]. A more detailed discussion of how preconception care supports preconception health (PCH) and ultimately healthy pregnancies and babies is found in the introduction of this volume. In order to market a health product or service, it is important to understand what people want or need before it is developed, and then to provide them with the opportunity to meet those needs in a way that is compelling to them and also improves a mutually beneficial outcome for patients and their providers: a healthy baby. Of course, unlike commercial products, the outcome of preconception care services (a healthy pregnancy and a healthy baby), cannot be guaranteed; there are many factors that influence pregnancy and birth outcomes that are outside a mother or healthcare provider’s realm of control. Despite the risk of adverse outcomes, there is much a woman, her partner, and her health care provider can do together preconceptionally to optimize pregnancy and birth outcomes. Social marketing is defined as “the application of commercial marketing technologies to the analysis, planning, execution, and evaluation of programs designed to influence voluntary behavior of target audiences in order to improve their personal welfare and that of society” (4). The social marketing process involves identifying an effective “marketing mix” of product, price, promotion, and place with the goal of offering an exchange that has clear and compelling benefits, minimal barriers, and an advantage over the competition. In plain language, that means setting up a situation so that future parents, health care professionals, and health insurers can all benefit and support a common goal of healthy women, healthy pregnancies, and healthy babies. To accomplish this, resources first need to be dedicated to conducting audience and market research using a systematic approach at all levels. Often, health behavior theories can be used to shape the development of new products or services as well as to evaluate their effectiveness in achieving intended outcomes. Table 1 lists some of the theories and frameworks that can be applied to addressing the 4 P’s of preconception care services (5). Table 1Theories and frameworks applicable to guiding research on the 4 P’s of social marketing preconception care servicesMarketing componentTheory or frameworkFocus of the theory or frameworkRelevant constructs or conceptsProductSocial marketingThe application of marketing techniques to the design, implementation, and evaluation of programs designed to facilitate voluntary exchanges of mutual benefit and improved personal well-being and societal welfareFormative research; Market segmentation; Competitive analysisDiffusion of InnovationThe spread of new ideas, products, or services within a society or from one society to another; focus on innovationRelative advantage; Compatibility; Complexity; Trialability; ObservabilityPriceSocial cognitive theoryThe interaction between personal factors, environmental factors, and human behaviorReciprocal determinism; Behavioral capability; Expectations; Self-efficacy; Observational learning; ReinforcementsHealth belief modelThe perceptions of threat posed by a health problem, the benefits of avoiding the threat, and factors influencing the decision to actPerceived benefits; Perceived barriers; Cues to action; Self-efficacyPromotionDiffusion of innovationThe spread of new ideas, products, or services within a society or from one society to another; focus on diffusionCommunication channels; Social systems/networksPrecaution adoption process modelThe journey from lack of awareness to action and maintenance in dealing with hazards; acknowledge inherent riskUnawareness; Unengaged; Decision-making; ActingPlaceCommunication theoryThe different types of communication that affect health behaviorMedia Exposure; Media effects; Agenda setting; FramingNote. For easy-to-understand information about the theories, frameworks, constructs, and concepts listed in this table, go to http://www.nci.nih.gov/theory/pdf for a downloadable copy of NCI’s “Theory at a Glance—A guide for health promotion practice” Viewing health care services as a business, and using language such as “marketing” and “sales,” may be uncomfortable to some who prefer to view the process of having a baby as a natural, emotional and deeply personal part of the human experience. However, the notion of providing preconception care as a service could very well benefit from a well-conceived and constructed social marketing research plan and execution that acknowledges and embraces the emotions of the patient and her partner. Thus, thinking about developing a social marketing plan to support preconceptional health care could help to ensure that babies are born with the best start in life. One example of how the business market has taken advantage of this type of opportunity is the development of a bank that preserves umbilical cord blood (UCB). The original idea in 1988 was a not-for-profit resource bank of donated cord blood for those who might need it for transplantation purposes (6). However, since that time, enterprising companies have created a market and generated the “need” for cord blood storage for a couple’s own child—even though the chances they will personally need it are roughly 1 in 10,000. Companies who forecast a profit in this service use direct marketing strategies to expectant couples through diaper companies and magazine subscriptions. Attempts to patent the cryopreservation processes and limit competition have raised legal, ethical and clinical questions—but the market to sell the idea of cord blood collection and preservation is lucrative and the demand is increasing (7). Advocates of preconception care can learn important lessons from the UBC preservation business marketing executives who market, sell and provide services to a similar audience. Product Preconception care (PCC) is the product or concept we are trying to “sell” as a precursor for assuring PCH: The need for PCC is well-supported by research in the health field (1, 8, 9). The product has been described in a number of publications as including a “bundle” of services most often including screening of the following elements: reproductive history; medications and potential teratogens; environmental exposures; age, family medical and genetic history; infections and immunizations; social habits and risk behaviors; diet and exercise; and any chronic medical conditions (10, 11). Preconception care is supported by a number of practitioner specialties (1, 12, 13) and was related to a Healthy People 2000 goal (14). Whereas some aspects of PCC have more evidence linking them to improved outcomes (e.g., folic acid for NTDs and glycemic control for diabetes) and others have less evidence (e.g., genetic counseling and smoking cessation), the need for a more integrated approach to health prior to conception enjoys a broad range of support (15–18). Nevertheless, there has been little support of PCC in a systematic manner in the US or other countries as a standard of care (2, 19–21). A social marketing approach can provide an important support for that uptake by beginning with a critical first step–defining the attributes of the “product” we are selling from a potential consumer’s point of view—which involves formative research with consumers. Key questions for the research may include: what do women and their partners want, when are PCC services optimally delivered, and what needs would have to be met in order for women or couples to value PCC services? This approach dictates that it is important to understand the perspective of the patient (and her partner) as more than just as consumers of health care services, but also by understanding their other perceived needs and desires in order to provide a satisfactory exchange. Couples may not view the decision to start a family as a medical decision, so this may be one reason the health care delivery system is more likely to be engaged some time after conception. In fact, couples may assess their financial readiness to start a family before their physical, emotional, or relationship readiness for this life-altering decision. The research that is an integral part of the social marketing process can help us to understand the decision-making dynamic (or lack thereof) that couples may engage in prior to conception. While only 50% of pregnancies are planned in the U.S., understanding how couples think about pregnancy may be a good place to start. Frishman suggests other “windows of opportunity” that could be explored among women including when women inquire about birth control, obtain negative pregnancy tests, or have recently delivered a baby (22). Wallerstedt et al. suggest that preconceptional health promotion and interconceptional counseling may be even more beneficial for parents who have had previous perinatal or infant loss (23). Social marketing research can help provide an understanding of the motivations and barriers for and against preconception care. A social ecological perspective (24) encourages program planners to look at multiple levels of influence: 1) intrapersonal or individual level; 2) interpersonal or social level; 2) institutional or organizational level; 4) community level; 5) and public policy or societal level. A major corporation preparing to launch a new product for women who might become pregnant (e.g., a new home pregnancy test) would never begin without spending substantial time and resources on understanding the target market and identifying the product attributes that will appeal to that market, while also knowing what the competitive environment is before they make investments in product development that might not have a profitable return. Product attributes can have a major impact on both the speed and extent of a product’s uptake by a target population. Rogers’ diffusion of innovation theory (25) identified a number of product attributes including relative advantage (is the product better than what it will replace?), compatibility (does the product fit with the intended audience?), complexity (is the product easy to use?), trialability (can the product be “tried out” before making a decision to purchase it?), and observability (are the results of the product visible and measurable?). Program planners can position their products effectively in the marketplace if they maximize their product’s appeal by addressing these important product attributes. Price The next aspect of social marketing research to increase the supply of and demand for preconception care is price—defining the costs to patients, providers and insurers, measured in time and/or money, in order to have quality and timely preconception care. Once the product attributes have been defined and developed to address PCC services in a mutually satisfactory way for women, health care professionals and insurers, then appropriate pricing is vital to allow for uptake. Lowering the intangible (non-financial) costs to patients can be facilitated by using some common standards of social marketing: the most effective health messages often incorporate emotional connections and values to make behavior change more appealing. Also, the best social marketing approaches make it easy for people to make changes that the service providers want them to—in this case in order to be healthy, and have a healthy pregnancy and baby. In addition to appeal and ease, some marketers would say that a new product would have to have relative advantage over products currently in the marketplace. “New and improved” labeling does matter when a product is introduced. A product’s relative advantage can often be framed as more appealing and/or easier, but at its core it must meet a customer need or desire. Appealing and easy actions to improve health are perceived as less costly by the patient or customer. Social cognitive theory (26) which addresses the interaction between personal, environmental, and behavioral factors is commonly used to understand many facets of a person’s expectations of outcomes from performing a behavior, environmental contexts (helps and hindrances) related to the behavior, experiences in performing the behavior (positive or negative), and efficacy (ability/confidence) in performing the behavior. These are just a few of the ways in which the pricing can build on the positive product attributes developed in the first step. It also helps that the outcome—a healthy pregnancy and baby—is a highly valued and very tangible benefit for those who want it. The pricing for a health care organization is more complicated. Cost-effectiveness studies and business case studies need to document how the organization’s PCC service costs compare with treatment of preventable health conditions. Balancing financial overhead and time pressures in these times of managed care may be a real challenge—and particularly so for reaching women who may have unplanned pregnancies. The greatest challenge may be weighing the intangible costs and benefits against the financial costs: How much preconceptional folic acid counseling is worth a child being born without a lifelong disability like spina bifida? How many smoking cessation sessions balance out the costs of a premature/low birth weight baby who might spend his first months of life in the NICU? How many fewer patients might a health care professional have time to see and bill for when pregnancy counseling they do for people with genetic conditions takes up more time? And what additional patient education messages, addressing chronic or acute health issues, are not being delivered during a regular well visit because the health care professional’s time is being used discussing preconception health issues? The health care provider medical office may well not be the best and/or most efficient way to market preconception care services to consumers in many cases. The practicing physician may also not be the first or best person to deliver the preconception care message. The goal of a good pricing strategy is to find an effective and innovative way to reach the target market while reducing the costs to both the health care delivery system and the consumer. Promotion After the product has been defined as a generally acceptable package of PCC services, and the price to individual and organization is determined to be mutually beneficial, how can a social marketing approach diffuse it into common practice and social norms? This is where the promotion comes into play among two key audiences: 1) people who would like to be providers (or sellers) of the product and 2) people who would be consumers (or buyers) of the product. Often, promotion is thought to focus solely on creating demand among consumers for a particular product, and the promotion process is what most people typically think of as marketing or advertising. However, whereas most businesses prefer to have products “on the shelf” to meet the needs of consumers proactively, consumers are more influential than ever before in demanding that a product appears on their vendor’s shelves. PCC could very well become a product that consumers themselves create demand for—especially since the health care industry already sees the value of preconception care services but has not built a product offering that delivers what they believe to be important. Once the product and pricing attributes are developed, then promoting the product among potential buyers and sellers could be done using a number of communication theories that provide guidance on what types of communication messages and media vehicles (e.g. communication channels) can achieve in terms of exposure and effects. In fact, trade shows and product expositions are a major venue for new products to be introduced to businesses who sell products. Health care provider/insurer conferences, continuing medical education efforts, and medical school training could be venues for promoting PCC services among potential providers (27). Because the product attributes have already been defined as a benefit, and pricing has been addressed in the previous step, promotion should be a matter of convincing both buyers and sellers that they are acting in their own best interests. After promotion has helped to generate consumer demand for the product, PCC is an opportunity for current health care professionals, insurers and those in the business of health to expand their services—as in a classic new product offering. An important way for organizations to justify offering these services is to demonstrate an immediate benefit (perhaps in sales of other products and services) in addition to longer term benefits (perhaps fewer complicated pregnancies, lower malpractice premiums, fewer patients who need life-long care). And of course, the actual benefits would be determined through a process of market research with stakeholders—such as patients, health professionals and insurers (Table 2). Addressing current barriers and how to overcome them is a major contribution of the promotion activities. Using innovative ways to promote preconception care services not only can be modified from general market promotions but also by taking advantage of existing opportunities to deliver tailored “gateways/pathways” to PCC services. These could vary from fertility treatment centers to other family planning arenas. Whether a woman is seeking to become pregnant or trying to avoid becoming pregnant (but preserving her options for the future), she is a consumer who may be fertile ground, so to speak, for preconception care services. A challenging part of promoting PCC services will be figuring out how to move the thinking of a “now-oriented” consumer towards more “future-oriented” events; especially because future outcomes could happen earlier than planned/anticipated. This promotion could take advantage of additional opportunities such as involving male partners and other influential people in women’s lives—and also taking a lifelong approach to health (13, 28). Another challenge to promoting PCC services will be addressing the ethical aspects of promoting a product that cannot guarantee outcomes: Not every woman who chooses and uses preconception care services will be able to have a healthy pregnancy or a healthy baby. In fact, it might even be the case that unhealthy women (e.g. those with chronic diseases or other medical conditions) are the first to want and use preconception care services because they know that their poor health status makes pregnancy a hazardous situation for themselves and their baby. The importance of recognizing hazards and taking precautionary actions to protect health, which are addressed in the precaution adoption process model (29), may be relevant in promoting PCC services in a manner that acknowledges pregnancy as having inherent risk for both the mother and the unborn baby. Table 2Sample social marketing questions for selected stakeholders of preconceptional care servicesConsumersHealth care providersInsurersProduct – What action must be taken by each audience (e.g., product, behavior, or policy)What does PCC services need to look and feel like for consumers to want/use/demand them?What does PCC services need to look and feel like for busy health care providers to want to offer them?What does PCC services need to look and feel like for health insurers to want to cover this service?Pricing –Why would they want to do it?What is the value of PCC services and what would they be willing to pay for them?What is the value for offering PCC services to patients and what would it cost to offer them?What is the return on investment for including PCC services as a covered service?Promotion –How will each audience be told about the what, why, where, and how?How will PCC services be publicized and made available to consumers in a way that they are attractive, affordable, and accessible?How will PCC services be presented to health care providers so that they are willing and able to offer them?How will PCC services be presented to insurers so that they are willing and able to reimburse for them?Place –Where can they do the action needed?What settings/locations are most acceptable, appropriate, and accessible for providing PCC services?What settings/locations are most acceptable, appropriate, and accessible for offering PCC services?What forums or conferences can instigate productive discussions about including PCC services as a covered expense? Place It is useful to ask how PCC services could be placed, or distributed, to women across their reproductive lifespan in a manner that addresses social marketing’s call toward mutually satisfactory exchanges, and how the placement of PCC services could affect their delivery. What components of PCC might change across the life span (e.g. early health promotion/contraception/STD infection/vaccinations/others)? The high level of unintended pregnancies in the US—40–60% for adults, 95% for teens (30)—is also a concern, because any approach that requires a self-selection into the audience will miss about half of the pregnancies. Some authorities have suggested a lifelong approach to preconceptional health—throughout the lifespan of a women’s potential fertility (say, 10–50 years of age) and an approach that may move women from one set of services and questions into another (2, 31). Social marketing can help by determining in the early phases what women and health care professionals want, and thus ensuring that supply can be ready when the demand is articulated. Health care professionals need to be involved in the design of those PCC services that they would deliver, and must do so in the context of what they would want to deliver—and those they would benefit from. Women report that one of their most influential sources of health information is their own health care professional, who knows them, their medical history and their concerns (32). Health care professionals and their medical staff may be the front line “sales people” for preconceptional care; if doctors never offer their patients the opportunity to “buy” this product, it will go nowhere. Medical professionals who are trained on the benefits of the product (e.g., by inclusion in medical school training and on medical board exams) and rewarded for using it (by reducing malpractice premiums for example) will find it more acceptable and worthwhile to integrate the practice into the time pressures of the busy medical office. Once a physician selection is made, patients visit for an acute problem, management of a chronic condition, and for routine checkups. Placement market research will help to define how PCC services may fit into each of these areas. This integrated vision of PCC could become part of the core training curricula for various medical professionals—more than an option during continuing education. This strategic preparation for placement also ensures that when the increased demand from the consumer begins to be evident at the health care provider office, the medical professionals in that office will know how to respond to that demand, and have the knowledge and systems in place to do so. Existing “bundles” of services that can be prepackaged to respond to this demand have in some cases been developed (33, 34). If the research in the first phase shows that the busy medical office is not the best location for providing PCC services, then alternate placement sites must be identified. Conclusion The best marketing and sales happen when the product has the qualities that a consumer wants and that a seller wants to provide. PCC must meet the needs and desires of patients, health professionals and insurers in order to be successfully adopted in the United States. For women and their families, that may mean believing that a healthy, wanted baby is in their future. For doctors, that may mean finding a way to give healthy women the preventive services they want and need while still dealing with the urgent care of their sick patients. For insurers it may mean a balance in service provision to meet both short and long term financial goals. But market research is needed to actually define exactly what the benefits are to each of those groups. And it is likely to be that the satisfactory exchange is more than just a purely medical application of clinical services. Consumers across the US are willing to spend $3.00 to drink what is essentially a 50 cent cup of coffee, but they are experiencing something more than the product’s primary attributes (e.g., flavor, caffeine) but also the positive associations with the product (e.g., funky, cool). In the same way, consumers can tell us how they would like to experience preconception health care services (35). All of us need to understand that if we market and sell it as the best experience possible, PCC could contribute to healthier babies, fewer medical treatment costs, and a healthy next generation that thinks about pregnancy in a whole different way.

Clin_Oral_Investig-3-1-1797073

Deposition of fluoride on enamel surfaces released from varnishes is limited to vicinity of fluoridation site

The aim of the in-situ study was to determine fluoride uptake in non-fluoridated, demineralized enamel after application of fluoride varnishes on enamel samples located at various distances from the non-fluoridated samples. All enamel samples used were demineralized with acidic hydroxyethylcellulose before the experiment. Intra-oral appliances were worn by ten volunteers in three series: (1, Mirafluorid, 0.15% F; 2, Duraphat, 2.3% F and 3, unfluoridated controls) of 6 days each. Each two enamel samples were prepared from 30 bovine incisors. One sample was used for the determination of baseline fluoride content (BFC); the other was treated according to the respective series and fixed in the intra-oral appliance for 6 days. Additionally, from 120 incisors, each four enamel samples were prepared (one for BFC). Three samples (a–c) were placed into each appliance at different sites: (a) directly neighboured to the fluoridated specimen (=next), (b) at 1-cm distance (=1 cm) and (c) in the opposite buccal aspect of the appliance (=opposite). At these sites, new unfluoridated samples were placed at days 1, 3 and 5, which were left in place for 1 day. The volunteers brushed their teeth and the samples with fluoridated toothpaste twice per day. Both the KOH-soluble and structurally bound fluoride were determined in all samples to determine fluoride uptake and were statistically analyzed. One day, after fluoridation with Duraphat, KOH-soluble fluoride uptake in specimen a (=next) was significantly higher compared to the corresponding samples of both the control and Mirafluorid series, which in turn were not significantly different from each other. At all other sites and time points, fluoride uptake in the enamel samples were not different from controls for both fluoride varnishes. Within the first day after application, intra-oral-fluoride release from the tested fluoride varnish Duraphat leads to KOH-soluble fluoride uptake only in enamel samples located in close vicinity to the fluoridation site. Introduction It is well established that fluoride products play an important role in the prevention and remineralization of carious lesions. Topical fluoride applications in the form of varnishes and varnishes may lead to appreciable acquisition of fluoride on and in both enamel and dentin samples, which were directly treated with the agent [4, 6, 14, 21, 25]. Especially, the KOH-soluble fluoride deposit on the surfaces of dental hard tissue might be elevated depending on the fluoride concentration and composition of the fluoride varnish applied. However, the KOH-soluble fluoride deposit is continuously dissolved in the oral cavity due to the influence of saliva. This leads to a drastic reduction of the deposit within a period of about 1 week after application of the fluoride agent [2, 5, 14]. After the use of fluoride-containing products, also, salivary fluoride levels are increased for a certain period of time depending on the concentration and application form used. This effect is important because fluoride levels above 0.04 ppm in the surrounding solution of the dental hard tissue have been shown to be related to lower risk of caries progression in clinical studies [16, 17]. The fluoride distribution in the oral cavity showed site-specific variations, which was exemplarily demonstrated by Weatherell at al. [24] after dissolving a fluoride tablet in the oral vestibulum. With oral fluoride-containing mouthrinses, salivary fluoride levels remain elevated for up to 12 h after application [8, 13, 23, 26]. The use of fluoride mouthwashes twice daily leads to a sustained elevated fluoride level even between the daily applications [12]. Fluoride dentifrices can cause increase in salivary fluoride content for a period of about 60–90 min [3, 11, 26]. Also, after application of fluoride varnishes onto tooth surfaces, salivary fluoride levels remain increased for up to 32 h [13, 18, 22]. The above-mentioned studies had made clear that fluoride-treated tooth surfaces and salivary fluoride levels are increased after administering local fluoride regimes. Besides these effects, it may be speculated that fluoride applications also lead to fluoride uptake at tooth surfaces that were not directly treated or covered with fluoridation agents such as varnishes. This fact could especially be important for demineralized surfaces, which were inaccessible to direct fluoride application such as inter-proximal areas. Due to toxicological and practical reasons, application of fluoride varnishes is mostly limited to some sites of the dentition. Therefore, the aim of the present study was to determine fluoride transfer from fluoride-varnished enamel surfaces to demineralized enamel surfaces located at various distances from the fluoridation site. Materials and methods Enamel samples Enamel cylinders (4 mm in diameter) were prepared from the buccal surface of bovine incisors. The cylinders were ground flat and polished, thereby removing about 200-μm depth of the enamel as controlled with a micrometer. Afterwards, all specimens were sterilized with gamma irradiation (25 kGy). For rehydration, the sterilized samples were stored in synthetic saliva [1] for a minimum of 2 weeks. Then, all samples were demineralized with acidic hydroxyethylcellulose (pH 4.8, 3 days). According to this procedure, each two enamel samples were prepared from 30 bovine incisors and each four samples were gained from additional 120 incisors. One sample from each tooth was used for the determination of baseline fluoride content of the respective tooth. The remaining samples of the 30 bovine incisors were later treated according to series 1–3. The remaining samples of the 120 bovine incisors were used for the determination of intra-oral fluoride transfer and acquisition. Set-up of the study The study was designed and conducted according to the guidelines of Good Clinical Practice. Ethical approval of the study was granted by the Ethics Committee of the University of Göttingen (2/9/01). Ten panellists (aged 21 to 29 years) participated in the study. They were all residents of Göttingen (fluoride content in domestic water: <0.2 ppm F). The subjects had been instructed to avoid fluoride uptake. The participants had a stimulated salivary fluoride rate within the normal range of 1.0–5.0 ml/min. Further inclusion criteria were that they had at least 20 teeth, no faulty dental restorations, no glass ionomer fillings and no current dental caries activity. Intra-oral, lower jaw appliances with two buccal aspects were worn by ten volunteers in three series of 6 days each. In each series, one enamel sample was placed for 6 days in one buccal aspect of the appliance after treatment according to the respective series (=central, fluoridated sample). With each five of the participants, the fluoridated samples were placed in the right buccal aspect; with the remaining subjects, the fluoridated samples were fixed in the left aspect of the appliance. A washout phase of 1 week elapsed between the three series. In series 1 and 2, 0.1 g of the fluoride varnish Mirafluorid® (1) or Duraphat® (2) was either applied on the central enamel sample (Table 1). Care was taken to avoid contamination of the remaining enamel samples of the intra-oral appliances with the varnish applied. After application of the varnish, the intra-oral appliances were immediately inserted into the oral cavities. In series 3, the respective samples were not fluoridated with varnish and served as controls. Table 1Ingredients and composition of the fluoride varnishes Mirafluorid® and Duraphat® according to manufacturers Mirafluorid®Duraphat®Fluoride content0.12%2.26%Fluoride formSodium fluorideSodium fluorideCetylaminehydrofluorideBis(hydroxyethyl)-aminpropyl-N-hydroxyethyl-octadecyl-amindihydrofluoridepH4.6NeutralSolventSolvent-freealcohol (33.14%)BaseWater-soluble polymerNatural resins (colophonium, mastix, shellac)Other ingredients–Wax, saccharine, flavourManufacturerHager & Werken, Duisburg, Germany Colgate-Palmolive, Hamburg, Germany Each three unfluoridated samples were placed into each appliance at different sites (a–c): (a) directly neighboured to the central, fluoridated specimen (=next), (b) at 1-cm distance (=1 cm) and (c) in the opposite buccal aspect of the appliance (=opposite). These three samples were replaced after 1, 3 and 5 days by new unfluoridated samples, which were removed after 1 day intra-oral exposition. Thus, each one sample was worn at the respective site for the following periods: days 0–1, days 1–2, days 3–4 and days 5–6. The central samples, which were fluoridated on day 0, were not removed from the appliances before completion of the experiment. The volunteers brushed their teeth and the samples with a pea-size amount of fluoridated toothpaste (0.125% fluoride as amine fluoride) twice per day (elmex®, GABA, Lörrach, Germany) during the experiment to simulate the common practice of using a fluoridated dentifrice and to follow the recommendations of the Ethical Committee, which did not allow detaining a fluoride dentifrice to the participants. They began to use the dentifrice 3 days before the start of the trial. After toothbrushing, the enamel samples in the appliances were carefully cleaned with ten brushing strokes using the toothbrush (impregnated with the remaining dentifrice slurry) that had been used for brushing of the teeth before. A minimum of 8 h elapsed between application of the respective varnish onto the central specimen and first brushing of the samples. During the experiment, the appliances were worn night and day except during meals, during which, they were stored in 0.9% saline. Fluoride analysis Immediately after removal from the intra-oral appliances, the enamel samples were cleaned with a soft toothbrush under running tap water and analyzed for KOH-soluble and structurally bound fluoride. The fluoride was assayed by a specific fluoride electrode (Orion Research, Cambridge, USA). The structurally bound fluoride was determined in two successive layers of 30 μm each. The layers were removed by means of a special grinding technique described in detail previously [15]. The amount of KOH-soluble fluoride was determined by the method of Caslavska et al. [9]. The assays were prepared as described in those studies, resulting in a 0.9-ml sample solution for KOH-soluble fluoride and 3-ml solution for structurally bound fluoride determination, respectively. Also, fluoride content of the fluoridated samples was assessed after the experimental period. Statistics According to the manufacturer, sensitivity of the fluoride electrode is given as 1 μmol/l in a solution. Bioanalytical guidelines recommend disregarding the measurements that are close to the sensitivity of an assessment method [20]. An international cooperation study [7] determining the lowest level of sensitivity for measurements with a fluoride electrode showed that measurements below 4 μmol/l (equivalent to 0.544 μg/cm2 KOH-soluble fluoride and 604.8 μg/cm3 structurally bound fluoride) were unstable, not reproducible and showed high standard deviations and coefficients of variation (>20%). Therefore, these readings were regarded as to be “below detection limit”. Thus, fluoride uptake was not calculated by subtracting the baseline fluoride content of the respective tooth, which was below detection limit, from the measured value recorded for the specimen. All groups were compared regarding their number of readings above detection limit using chi-square tests. For those groups, in which more than 50% of the readings were above the detection limit, discriminations were conducted using the rank-sum test according to Wilcoxon and Mann–Whitney. The level of significance was set at P < 0.05. Results In Table 2, the percentages of fluoride measurements in the respective groups, which were above the detection limits, are given. It is obvious that most of the groups had more than 50% of their readings below the detection limit. Chi-square tests proved that the sample groups with more than 50% readings above detection limit were statistically significantly different from the remaining groups, which in turn did not differ significantly from each other. Table 2Percentages of measurements above detection limitsa for KOH-soluble and structurally bound fluoride in two successive layers in enamel specimens worn in intra-oral appliances at different sites and for different periods (days 0–6, days 0–1, days 1–2, days 3–4 or days 5–6) after the application of either Duraphat, Mirafluorid or no fluoride varnish (control) on the central specimen on day 0  KOH-soluble F−1st Layer (0–30 μm)2nd Layer (31–60 μm)PeriodSiteMirafluorid (%)Duraphat (%)Control (%)Mirafluorid (%)Duraphat (%)Control (%)Mirafluorid (%)Duraphat (%)Control (%)0–6Central90100010100101010000–1Next10600030003000–11 cm10300050001000–1Opposite00001000001–2Next20100101000001–21 cm20000000001–2Opposite10000000003–4Next100001000003–41 cm10000000003–4Opposite10000000005–6Next0000000005–61 cm10000000005–6Opposite000000000aDetection limits (KOH-soluble F = 0.544 μg/cm2, structurally bound fluoride = 604.8 μg/cm3) In Table 3, KOH-soluble fluoride and structurally bound fluoride data of the experimental groups are given, for which more than 50% of the readings were above detection limit. As mentioned above, chi-square tests had proven that these values were statistically significantly different from baseline content and from the remaining groups. For the remaining groups, fluoride contents are not given because statistical data such as mean value or median are not reasonable to calculate for values, which are not reliably measurable by definition. Acquisition of KOH-soluble fluoride was detectable in the central, fluoridated samples of both groups, Mirafluorid and Duraphat, with the latter once accumulating statistically significantly more KOH-soluble fluoride (P = 0.00015). The central, fluoridated Duraphat samples also showed significant uptake of structurally bound fluoride in both enamel layers with significantly higher uptake in the first layer compared to the second one (P = 0.02836). One day after fluoridation, the samples located next to the Duraphat-varnished central specimens also accumulated measurable amounts of KOH-soluble fluoride. The KOH-soluble precipitate on these samples was statistically significantly less pronounced compared to the central specimens (P = 0.00015). The samples located next to the Duraphat-treated showed measurable KOH-soluble fluoride acquisition at days 0–1. They, therefore, differed significantly from both the control and Mirafluorid series, which did not show measurable fluoride acquisition at this time point and were, in turn, not significantly different from each other. At all other sites and time points, fluoride uptake in the enamel samples was not different from controls for both fluoride varnishes. Table 3Mean ± standard deviation (median; minimum–maximum) of KOH-soluble fluoride and structurally bound fluoride (in 1st and 2nd layer) given for the experimental groups with more than 50% of the respective measurements above detection limit  KOH-soluble F− (μg/cm2)1st Layer (μg/cm3)2nd Layer (μg/cm3)PeriodSiteMirafluoridDuraphatDuraphatDuraphat0–6Central0.67 ± 0.19a (0.62; 0.54–1.1)8.38 ± 3.18a,c (8.82; 3.65–14.5)2237 ± 576b (2,282; 1,233–3,315)1636 ± 487b (1,503; 1,110–2,646)0–1Next–*0.83 ± 0.56c (0.76; 0.16–2.12)–*–**Less than 50% readings above detection limitSignificantly different values in the same line (aKOH-soluble F−, bstructurally bound F−) or ccolumn are marked. Discussion As stated in “Introduction”, several investigations had demonstrated that intra-orally applied fluoride regimes may cause fluoride release into saliva. In a previous study, Dijkman et al. [10] had demonstrated that no measurable fluoride enrichment could be detected in untreated sound enamel controls located in a distance of 3 mm from fluoride varnish-treated samples after a period of 1 week. The present study made clear that within the first day after application the highly concentrated fluoride varnish Duraphat KOH-soluble fluoride deposition in close vicinity to the fluoridation occurs. However, in consistence with the investigation by Dijkman et al. [10], no measurable fluoride transfer could be revealed after the first day. Although, when interpreting the results of the present study, it should be noted that a more sensitive method for fluoride determination might have shown slightly different results. In the present study, no fluoride acquisition could be detected in samples located at a distance of 1 cm from the fluoridated site or fixed in the opposite vestibulum of the participants. This means that fluoride transfer via saliva was strictly limited to the close neighbourhood of the fluoridation site. This finding corresponds to a previous study, which demonstrated that significant elevation of salivary fluoride concentrations is more or less restricted to the fluoridation site [24]. With exception for the samples directly treated with Duraphat, no measurable acquisition of structurally bound fluoride was observed in any other sample group. In a previous study, application of Mirafluorid did not also result in uptake of structurally bound fluoride [2]. However, also the samples which were located next to the specimens treated with Duraphat for 1 day and which showed a KOH-soluble fluoride deposit did not show increase of structurally bound fluoride. This finding could be explained by the fact that formation of structurally bound fluoride is a time-dependent process during which some of the KOH-soluble deposit is dissolved, allowing fluoride to diffuse into the underlying enamel and to be structurally incorporated into the enamel crystallites [19]. However, in these samples, structurally bound fluoride was assessed already 1 day after insertion into the appliance. It might be speculated that with longer exposition in the oral cavity, structurally bound fluoride might have been formed by dissolution from the KOH-soluble fluoride deposit. In the present study, demineralized enamel specimens were used for assessing fluoride acquisition. Recent studies had proven that fluoridation measures lead to significantly higher uptake of fluoride in demineralized samples as compared to sound enamel [1]. It could, therefore, be assumed that fluoride acquisition of sound enamel surfaces due to fluoride transfer in the oral cavity might be actually more limited as compared to the present results. Under clinical conditions, a varnish is often applied onto more sites, which might elevate salivary fluoride to a presumably higher extent as in the present study, where the varnishes were applied to a single site only. Due to the results of the present study, it could be concluded that within the first day after application, intra-oral fluoride release from the tested fluoride varnish Duraphat leads to KOH-soluble fluoride uptake only in demineralized enamel samples, which are located in close vicinity to the fluoridation site. Owing to the negligible transfer of fluoride from the varnish to more remote sites, this finding means, for the clinical situation, that all sites in the oral cavity requiring fluoridation have to be directly treated with the varnish.

Acta_Neuropathol_(Berl)-3-1-2039857

MLC1 is associated with the Dystrophin-Glycoprotein Complex at astrocytic endfeet

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease with onset in childhood, caused by mutations in the MLC1 gene. MLC1 is a protein with unknown function that is mainly expressed in the brain in astrocytic endfeet at the blood–brain and cerebrospinal fluid–brain barriers. It shares its localization at astrocytic endfeet with the dystrophin-associated glycoprotein complex (DGC). The objective of the present study was to investigate the possible association of MLC1 with the DGC. To test this hypothesis, (co)-localization of DGC-proteins and MLC1 was analyzed by immunohistochemical stainings in gliotic brain tissue from a patient with multiple sclerosis, in glioblastoma tissue and in brain tissue from an MLC patient. In control tissue, a direct protein interaction was tested by immunoprecipitation. Results revealed that MLC1 is co-localized with DGC-proteins in gliotic brain tissue. We demonstrated that both MLC1 and aquaporin-4, a member of the DGC, were redistributed in glioblastoma cells. In MLC brain tissue, we showed absence of MLC1 and altered expression of several DGC-proteins. We demonstrated a direct protein interaction between MLC1 and Kir4.1. From these results we conclude that MLC1 is associated with the DGC at astrocytic endfeet. Introduction Megalencephalic leukoencephalopathy with subcortical cysts (MLC, MIM 604004) is a childhood leukoencephalopathy with autosomal recessive inheritance [21]. Patients develop macrocephaly during the first year of life, followed by slow motor deterioration with cerebellar ataxia and spasticity [21]. Magnetic resonance imaging (MRI) shows diffusely abnormal, mildly swollen cerebral white matter and subcortical cysts in anterior temporal and frontoparietal areas [21]. Histopathologic and electron microscopic examination of brain tissue reveals countless intramyelinic vacuoles [22]. From early on, the striking similarity in MRI features of MLC and congenital muscular dystrophy (CMD) with merosin deficiency (CMD type 1A, MDC1A) was noted [21]. Patients with MDC1A also have diffusely abnormal, mildly swollen cerebral white matter and sometimes anterior temporal cysts (Fig. 1) [23]. Microscopic examination of the brain of a patient, who was later genetically confirmed to have MDC1A (B. Echenne, personal communication) also revealed myelin vacuolation [8]. MDC1A is caused by mutations in the LAMA2 gene, encoding the laminin-α2 chain of merosin [9]. In contrast to MDC1A, MLC is not associated with muscle disease [21]. We hypothesized that MLC could be the “brain-only” variant of MDC1A. We could, however, not confirm linkage of MLC to the LAMA2 locus and instead demonstrated that mutations in another gene, MLC1, cause MLC [10]. MLC1 encodes a plasma-membrane protein of unknown function. Recently, the MLC1-protein was found to be located in astrocytic endfeet at the blood–brain and cerebrospinal fluid–brain barriers [3, 19]. Fig. 1MRI of the brain of a patient with MLC (a–c) and MDC1A (d–f). The axial T2-weighted images (a, b, d, e) reveal extensive signal abnormalities and mild swelling of the cerebral white matter in both MLC (a, b) and MDC1A (d, e). The corpus callosum and internal capsule are relatively preserved in both cases (a, d). The sagittal T1-weighted images reveal a large cyst in the anterior temporal white matter and smaller cysts in the frontal subcortical white matter in the MLC patient (c). There are small cysts in the same regions in the MDC1A patient (f). Both MLC and MDC1A patients had a genetically confirmed diagnosis Other proteins localized at astrocytic endfeet are those of a multi-subunit complex called dystrophin-associated glycoprotein complex (DGC) [12, 20]. The DGC is expressed in various tissues and has been characterized best in skeletal muscle where it connects the cytoskeleton of muscle fibers to the extracellular matrix [14]. Mutations in different DGC components lead to various muscular dystrophies. In a subset of muscular dystrophies, the congenital muscular dystrophies (CMDs), the muscular phenotype is often combined with brain involvement, including white matter abnormalities, most notably in MDC1A [23]. The brain DGC is involved in the adhesion of perivascular glia to the extracellular matrix [20]. At the astrocytic endfeet, the DGC consists of four main components [6, 16, 29]: (1) α- and β-dystroglycan, which form the backbone of the DGC by forming a transmembrane link between the actin cytoskeleton and the extracellular matrix. α-Dystroglycan is a heavily glycosylated peripheral membrane protein that binds to extracellular ligands such as merosin and agrin. β-Dystroglycan is a transmembrane protein that anchors the extracellular α-dystroglycan to the plasma membrane and interacts intracellularly with dystrophin (and/or utrophin, which can replace dystrophin) and syntrophin. (2) Dystrophin and/or utrophin, are linker molecules that can form a bridge between actin and β-dystroglycan. (3) α-Dystrobrevin, which is linked to dystrophin and/or utrophin binds to syntrophin. (4) Syntrophin, which binds to the water channel aquaporin-4 and the potassium channel Kir4.1 is responsible for the polarized expression of these proteins in astrocytic endfeet. The striking similarities in brain MRI features and pathology between MLC and MDC1A, together with the specific localization of both MLC1 and merosin at astrocytic endfeet, brought us to the hypothesis that there may be an association between MLC1 and DGC in the brain. In the present paper we report on our studies to test this hypothesis. We investigated the co-localization of MLC1 and DGC-proteins in gliotic white matter, because MLC1 staining is more intense in gliotic brain tissue than in normal tissue [3]. Previous studies in humans and mice have shown that if members of the DGC are mutated or missing, the complex may be disrupted, which affects the localization of other protein members [1, 13, 16, 27]. We therefore also tested our hypothesis by studying the localization of MLC1 and several DGC-proteins in glioblastoma and MLC brain tissue. Besides, we assessed a direct association between MLC1 and DGC-members by co-immunoprecipitation experiments. Materials and methods Immunohistochemical staining of human brain tissue Human control brain tissue specimens, both frozen and paraffin-embedded, consisting of neocortex and white matter, were obtained at autopsy from patients without neurological disease and neuropathologic abnormalities. Additionally, frozen brain tissue was obtained from a multiple sclerosis patient to study gliotic white matter in which MLC1 has a normal localization but MLC1 expression is increased [3]. During life, all patients or their next of kin had given consent for autopsy and the use of brain tissue for research purposes. Human glioblastoma multiforme tissue (frozen) and brain biopsy tissue from MLC patients (both frozen and paraffin-embedded) were obtained for diagnostic purposes and used for research purposes with informed consent of patients and families. Light-microscopy of glioblastoma tissue was used to select areas that display characteristic glioblastoma features, including necrosis, giant cells and endothelial proliferation, for further studies. Brain biopsies were obtained in the following MLC patients: EL18 (decreased MLC1 expression [2], frozen tissue), EL649 (homozygous for the c.733G > C (p.Ala245Pro) mutation; paraffin-embedded tissue), and EL746 [compound-heterozygous for the c.268-1G > A and c.597 + 1G > A mutations, leading to splice-defects and truncation of the protein (p.Cys90-Val107del and p.Val200-Ala238del); paraffin-embedded tissue]. Immunohistochemistry was performed as described previously [7] with antibodies against the following proteins: MLC1 (polyclonal; 1:200 [3]); merosin (monoclonal; 1:500 [11]); aquaporin-4 (polyclonal; 1:200, Chemicon, Huissen, The Netherlands); Kir4.1 (polyclonal; 1:200, USBiological, Swampscott, Massachusetts), β-dystroglycan (monoclonal; 1:100, Santa Cruz, Heidelberg, Germany), α-dystroglycan (monoclonal; 1:100, UpstateCellSignaling solutions), agrin (monoclonal; 1:2500 [26]); glial fibrillary acidic protein (GFAP, polyclonal; 1:500, DAKO, Heverlee, Belgium) and NeuN (monoclonal; 1:1600, MAB377, Chemicon). In all slides, except the slides used for double stainings, the nuclei are counterstained with Haematoxylin. Double immunofluorescence staining was performed as described [3] with agrin, β-dystroglycan (monoclonal; 1:100) or syntrophin (monoclonal; 1:100, Affinity BioReagents, Raamdonksveer, The Netherlands) versus MLC1. In negative controls the primary antibodies were omitted. Double-stainings were performed as described previously [25]. In frozen tissue double-stainings were done with agrin (red) antibodies versus AB4 (blue, polyclonal; 1:200, Lab Vision–NeoMarkers, Fremont, CA, USA) antibodies and in paraffin tissue with agrin or α-dystroglycan antibodies versus NeuN or GFAP. For the agrin (brown)/NeuN (blue) double-stainings, agrin was visualized with biotinylated secondary antibodies (DAKO) and NeuN with poly-HRP (Immunologic, Duiven, The Netherlands). For the agrin (red)/GFAP (blue) double-stainings, agrin was detected with poly-HRP (Immunologic) and GFAP with biotinylated secondary antibodies (DAKO). In negative controls the primary antibodies were omitted. Immuno-precipitation and western blotting Preparation of brain tissue and immunoprecipitation experiments were performed as previously described [5] without antibodies as a negative control, with 1–15 μg mMLC1-C (home made), with the nonspecific CHOP (Santa Cruz, Heidelberg, Germany) as an extra negative control, and with Kir4.1 (USBiological, Swampscott, Massachusetts) antibodies. Western blots were incubated with the Kir4.1 antibodies overnight at 4°C and complexes were visualized using ECL Western Blot detection reagents (Amersham, Buckinghamshire, United Kingdom). Results Co-localization of MLC1 and DGC-members To test whether MLC1 co-localizes with DGC-proteins at astrocytic endfeet, we performed double-immunofluorescent staining experiments in gliotic brain tissue with the transmembrane protein MLC1 (Fig. 2a, d, g; green) versus the extracellular matrix protein agrin (Fig. 2b; red), the intracellular protein syntrophin (Fig. 2e; red), and the transmembrane protein β-dystroglycan (Fig. 2h; red). There was partial overlap (yellow) between all three DGC-proteins (red) and MLC1 (green). The co-localization (Fig. 2i; yellow) of MLC1 (Fig. 2g; green) and β-dystroglycan (Fig. 2h; red) was most striking. If MLC1, agrin, β-dystroglycan or syntrophin was omitted, fluorescent labeling was only visible for the appropriate isotype, thereby excluding cross-reactivity (not shown). On omission of the primary antibody no staining was observed (not shown). Fig. 2Co-localization of MLC1 and DGC-proteins. a–i Double immuno-fluorescence labelling of MLC1 (a, d, g, green), agrin (b, red), syntrophin (e, red), and β-dystroglycan (h, red) in gliotic human brain tissue. There is partial overlap (c, f, i, yellow) between MLC1 and these DGC-proteins. The overlap (i, yellow) between the transmembrane proteins MLC1 (green) and β-dystroglycan (red) is most striking. In all panels the ocular magnification and 10 μm scale bars are indicated in the lower right hand corner MLC1 and aquaporin-4 are redistributed in glioblastoma cells Staining normal human brain tissue with anti-MLC1 and anti-aquaporin-4 antibodies confirmed their specific expression around blood vessels, consistent with the previously described perivascular localization (Fig. 3a, c) [3, 15]. In glioblastoma tissue, both MLC1 and aquaporin-4 had altered expression patterns. The proteins were no longer confined to astrocytic endfeet, but were redistributed over the entire glioblastoma cell (Fig. 3b, d). Anti-agrin staining showed expression of the protein in the extracellular matrix around blood vessels of all sizes in normal brain tissue (Fig. 3e), whereas it was exclusively expressed around larger blood vessels in glioblastoma tissue (Fig. 3f). Double staining of agrin (red) with AB4 (blue), an endothelial marker, in glioblastoma tissue confirmed agrin and AB4 staining around larger blood vessels (Fig. 3g) and absence of agrin around small vessels (Fig. 3g, boxed area). This is in line with previously published results [27]. On omission of the primary antibody no staining was observed (not shown). Fig. 3Localization of MLC1 in glioblastoma tissue. Immunohistochemical staining of MLC1, aquaporin-4 (AQP4) and agrin in control brain (a, c, e) and tumor tissue (b, d, f, g). a MLC1 has a perivascular localization in normal tissue. b In glioblastoma tissue, the restriction of MLC1 to perivascular areas is lost and the protein has an intracellular location in glioblastoma cells. cAQP4 is expressed in perivascular astroglial endfeet in control brain. dAQP4 is localized in glioblastoma cells. e, f Agrin is expressed in the extracellular matrix around blood vessels of all sizes in normal tissue (e) and only around large vessels in glioblastoma tissue (f). g Double-staining of agrin (red)/AB4 (blue) in glioblastoma tissue shows agrin and AB4 (endothelial marker) staining around larger blood vessels. The boxed area in panel g shows that agrin staining is absent around small vessels. In all panels the ocular magnification and 10 μm scale bars are indicated in the lower right corner Absence of MLC1 and altered expression of DGC-proteins in MLC brain tissue Anti-MLC1 staining revealed perivascular expression of the protein in control brain tissue (Fig. 4a) [3] and absence of staining in MLC tissue (Fig. 4b). The distribution of merosin, β-dystroglycan and aquaporin-4 was similar in control (Fig. 4c, e, g) and MLC tissue (Fig. 4d, f, h). In control tissue, Kir4.1 showed a perivascular localization in configuration suggesting presence in distal atroglial processes (Fig. 4i), while in MLC patient tissue Kir4.1 additionally showed a diffuse cytoplasmic staining of astrocytes (Fig. 4j). In control tissue, agrin (Fig. 4k) and α–dystroglycan (Fig. 4m) were seen in the perivascular extracellular matrix, whereas these proteins were observed both in the perivascular extracellular matrix and within cells in MLC tissue (Fig. 4l, n). Fig. 4Localization of proteins associated with the DGC in MLC patients. In control tissue the perivascular staining of MLC1 is visible (a), but no staining is observed in MLC brain tissue (b). Normal perivascular basal lamina staining with merosin is seen in control (c) and MLC (d) brain tissue. β-dystroglycan has a normal perivascular distribution in both control (e) and MLC (f) brain tissue. Aquaporin-4 (AQP4) has a normal perivascular distribution in both control (g) and MLC (h) brain tissue. Kir4.1 is present in distal astrocytic processes around blood vessels in control tissue (i). In MLC tissue (j) Kir4.1 is expressed both in distal astrocytic processes around blood vessels and in cell bodies of astrocytes (boxed area). Agrin is expressed in the extracellular matrix around blood vessels in control tissue (k). In MLC tissue (l) agrin is expressed in both the extracellular matrix around blood vessels (boxed area) and in cells, as indicated by arrows. Immunostaining for α–dystroglycan shows a normal perivascular distribution in control tissue (m) and expression of α–dystroglycan around blood vessels (boxed area) and in cells in MLC tissue (arrows in n). o Double-staining of NeuN (blue)/agrin (brown) in MLC tissue shows cytoplasmic agrin staining in NeuN positive cells (arrows). p Double-staining of Agrin (red)/GFAP (blue) in MLC brain tissue. The picture on the left shows an astrocyte that is only GFAP positive (arrow), while the picture on the right shows an astrocyte that is both GFAP and agrin positive (arrow, brown staining in cell body). In all panels the ocular magnification, 10 μm scale bars and patient numbers are indicated in the lower right corner The cells expressing these latter two DGC-proteins had the morphological characteristics of neurons. Double stainings of agrin and α-dystroglycan with NeuN, a neuronal marker, in MLC brain tissue confirmed that both agrin (brown) (Fig. 4o) and α-dystroglycan (not shown) co-localized with NeuN (blue). Although, most redistribution appeared to be in neurons, double stainings of agrin (red) and GFAP (blue) showed that also some astrocytes displayed positive cell body staining for agrin (Fig. 4p). On omission of the primary antibody no staining was observed (not shown). Direct protein interaction between MLC1 and Kir4.1 To verify interaction of DGC-proteins with MLC1, immunoprecipitation experiments were performed using human control brain lysates. Figure 5 shows that after immunoprecipitation with both anti-MLC1 and anti-Kir4.1 antibodies and probing the precipitate for the presence of Kir4.1, a band at 200 kDA was detected, the size expected for Kir4.1. This band was not present in a pull-down without antibodies or with an unrelated antibody (anti-CHOP). Of concern in this experiment was the use of cross-linking reagents, since this might cause nonspecific interactions. When cross-linking reagents were left out, we failed to detect interactions, which is in agreement with previous studies on interaction between DGC-members [5]. Fig. 5Co-immunoprecipitation (IP) of human brain lysates. Antibodies used for IP are indicated above the figure. The dash indicates pull-down without antibodies; anti-CHOP was used as a nonspecific antibody. The resulting Western blot was probed with antibodies against Kir4.1. The arrowhead shows the position of Kir4.1 at 200 kDa and the asterisk indicates a nonspecific band that is pulled down with all the antibodies used Discussion The DGC is important in linking cytoskeletal proteins to the extracellular basal lamina. It is present in several tissues including muscle, heart, nerve and brain. The composition of this complex shows some differences for different tissues. Structural defects in a number of DGC-members cause muscular dystrophies. Best known is Duchenne muscular dystrophy, related to mutations in dystrophin. Another muscular dystrophy is the CMD MDC1A, in which the MRI features are strikingly similar to those of MLC. MDC1A is caused by mutations in the laminin-α2 chain of merosin [9]. A specific group of CMD variants is known as “dystroglycanopathies” and includes Walker–Warburg syndrome, Fukuyama type of CMD, muscle–eye–brain disease, CMD type 1C and type 1D. They are characterized by hypoglycosylation of α-dystroglycan [14]. MDC1A and most of the dystroglycanopathies lead to both muscle disease and brain involvement, including cerebral white matter abnormalities. We hypothesized that MLC1 could be associated with the DGC in the brain. To strengthen our hypothesis, we showed co-localization between MLC1 and DGC-proteins around blood vessels by immunofluorescent stainings. A common feature of the DGC is that mutations affecting one of its components often lead to destabilization of the complex with reduced or altered expression of other DGC-members. For instance, in muscle and brain tissue of patients with a dystroglycanopathy, immunostaining is reduced for α-dystroglycan as well as merosin and agrin [13]. In patients with Duchenne muscular dystrophy and Fukuyama type of CMD, decreased expression of multiple DGC-proteins has been found in muscle and brain [4, 17, 18]. In brain tissue of syntrophin, knock out mice altered expression of aquaporin-4 has been reported [16]. In glioblastoma tissue, abnormal expression of aquaporin-4 and syntrophin over the entire surface of glioblastoma cells and absence of α-dystroglycan from glial structures have been reported [27, 28]. Agrin was only present around larger vessels but absent around small vessels in glioblastoma tissue [27, 28]. We decided to use these DGC destabilization phenomena to test our hypothesis. Because of the co-localization of MLC1 and DGC-members at perivascular regions and the disassembly of the DGC in glioblastoma tissue we were interested in whether MLC1 is redistributed in this type of brain tumor. We, therefore, tested the expression pattern of MLC1 by immunohistochemical staining in glioblastoma tissue. We confirmed previous findings for agrin and aquaporin-4 [27, 28] and demonstrated that MLC1, like aquaporin-4, is redistributed in glioblastoma cells. We were also interested whether MLC1 mutations would lead to destabilization of the complex with reduced or altered expression of DGC-members in MLC brain. We demonstrated the absence of MLC1 and altered expression of Kir4.1, agrin and α-dystroglycan in brain tissue of MLC patients, whereas merosin, β-dystroglycan and aquaporin-4 retained their normal perivascular localization. Agrin and α-dystroglycan were redistributed in both neurons and astrocytes, although most positive staining cells appeared to be neurons. All the above results provide circumstantial evidence for an association between MLC1 and the DGC. To show a direct protein–protein interaction we performed co-immunoprecipitation experiments that revealed an association between MLC1 and Kir4.1. All together, the above findings provide strong evidence for an association between MLC1 and the DGC. So far almost all defects in DGC-proteins have been associated with a muscular dystrophy, which is absent in MLC. This difference can be ascribed to a difference in DGC composition for muscle as compared to brain. Whereas merosin is expressed both in muscle and brain, MLC1 is not expressed in muscle (http://biomed.ngic.re.kr/cgi-bin/cards/carddisp?MLC1&search = KIAA0027) Both in MLC and MDC1A, the water content of the affected white matter is abnormally high due to intramyelinic vacuole formation [8, 22, 24]. The DGC is crucial for anchoring of water and potassium channels at the perivascular endfeet [15]. Destabilization of the DGC may lead to changes in glial polarity, disturbances of the blood–brain barrier and alterations in ion and water homeostasis of the brain [27, 28], and result in an increased myelin water content. Our findings may, therefore, have implications for the pathophysiology of white matter abnormalities observed in MLC, MDC1A and the dystroglycanopathies.

Pediatr_Nephrol-3-1-1805046

Clinical predictors of neurocognitive deficits in children with chronic kidney disease

The purpose of the study was to explore associations between neurocognitive function and chronic kidney disease (CKD)-related clinical characteristics. Twenty-nine children, ages 7 to 19 years, with an estimated creatinine clearance (eCrCl) of 4–89 ml/min per 1.73 m2 body surface area were enrolled. Intellectual function (IQ), memory, and attention were measured and expressed as age-based standard scores. Clinical data were obtained by physical examination, laboratory testing, parental questionnaires and medical chart review. Pearson correlations and standard Student’s t-tests were used to identify significant (P < 0.05) relationships between targeted clinical variables and neurocognitive scores. Increased CKD severity correlated with lower IQ (P = 0.001) and memory function (P = 0.02). Memory function was lower in children with longer duration of disease (P = 0.03). Similarly, IQ scores were lowest when kidney disease had started at a younger age (P = 0.03) and with a greater percent of life with CKD (P = 0.04). Our findings provide preliminary evidence that increased disease severity, longer duration of disease, and younger age of onset of kidney disease potentially place children with CKD at increased risk of neurocognitive deficits. Additional investigation is required to better quantify these risk factors, particularly regarding how much variability is accounted for by these specific risk factors. Introduction Neurocognitive difficulties have long been observed in the chronic kidney disease (CKD) population [1]. While identification and treatment of specific comorbidities of CKD (e.g., anemia) have yielded improvement in the overall functioning of this population, reports of neurocognitive deficits in children with CKD continue to appear. These neurocognitive deficits undoubtedly will have significant lifelong implications for the CKD population as they move into adulthood. A recent review by Groothoff [2] examined the long-term outcomes of children diagnosed with CKD and identified persistent difficulties with a limited knowledge base, memory, and concentration into adulthood, as well as lower educational attainment. A better understanding of potential risk factors for cognitive impairment and decline in the pediatric CKD population could allow for earlier intervention and, possibly, less morbidity, particularly for certain cognitive deficiencies. Studies aimed at investigating neurocognitive impairment in children with CKD have identified a wide range of delays in motor and cognitive development. Measures of general intelligence, memory and attention have been used most frequently to look at cognitive function in the pediatric CKD population. Most studies have demonstrated lower IQ scores among children with end stage renal disease (ESRD) than in unaffected siblings [3] or the general population [4, 5] and also when pre- and post-transplantation performances are compared [6]. Memory deficits also have been identified in children with mild CKD as well as ESRD [7]. Fennell et al. [7] also reported in this same study that there was no significant decline in measures of attention among these children; however, improved attention performance has been demonstrated in children with ESRD after transplantation [6]. Observations of hyperactivity at school also have been noted in 50% of a study population assessing cognitive outcomes following dialysis during infancy [4]. The measures of cognitive performance in each of these studies varied widely but, when viewed collectively, suggest that children with CKD are vulnerable to cognitive deficiencies in IQ, memory and attention. One thing to note, however, is that most studies to date have focused primarily on children with ESRD, leaving unanswered whether children with mild to moderate CKD also suffer this same vulnerability. Cognitive deficiencies in children with CKD could arise through a gradual process, proportional to the level of kidney dysfunction, or may develop once a filtration threshold has been passed. It seems logical that the severity of CKD might be proportional to the degree of cognitive impairment, but the data are not yet available to support this. The stage of neurologic development at the time of disease onset or the cumulative time children spend with CKD may also impact on the degree or type of cognitive impairment experienced. To our knowledge, no published study has yet focused specifically on isolating risk factors to identify which children with CKD are most at risk for cognitive decline. The primary goal of this study was to explore associations between clinical aspects describing CKD and selected neurocognitive test scores in a sample of children and adolescents with CKD. In addition, this study should provide the foundation for identifying specific clinical risk factors for subsequent neurodevelopmental dysfunction. Methods Participants Study participants were recruited from a university-based pediatric nephrology practice. The participants included children between 7 and 19 years of age, with an estimated creatinine clearance (eCrCl) by Schwartz formula [8] of less than 90 ml/min per 1.73 m2 body surface area for at least 3 months duration. Renal transplant recipients and children with profound developmental delays were excluded. Following parental consent and patient assent, 29 children meeting these criteria underwent neurocognitive testing at a single point in time. A standardized physical examination and laboratory sample collection were also performed within 2 weeks of cognitive testing. A questionnaire providing demographic and historic information was completed by a parent or legal guardian. The study was reviewed and approved by the University of North Carolina Institutional Review Board. Included among the study sample were 17 children with obstructive uropathy or structural congenital renal anomalies, seven with glomerulonephritis, one with calcineurin inhibitor toxicity, one who had sustained ischemic injury, one with renal insufficiency of undetermined etiology, one child with Alport’s syndrome, and one with cystinosis. Participants were evenly divided with respect to gender and disease severity. Approximately half the study sample had mild to moderate CKD with eCrCl ranging from 31–89 ml/min per 1.73 m2 body surface area. Kidney disease onset was documented from birth in 59%. The majority (59%) were hypertensive, and, among these children, 70% were on antihypertensive therapy, and 65% had measured blood pressure above the 95th percentile at the time of examination. The mean hemoglobin level was 12.7 mg/dl, with only three (10%) below 11 mg/dl, with values of 10.8 mg/dl, 10.7 mg/dl and 10.1 mg/dl. The general characteristics of our study sample are described in Table 1. All the participants attended school in a regular classroom setting, and two reported having an individualized education plan (IEP) at the time of participation. Table 1Demographic characteristics and medical variables of the study sample (PD peritoneal dialysis, HD hemodialysis)Study sample characteristicsNumber (n = 29)aRangeAge (years)12.5 (3.2)7–19Male (%)52Caucasian (%)52eCrCl (ml/min per 1.73 m2 body surface area)32 (29)4–89ESRD (13 PD, 1 HD) (%)48Age at disease onset (years)4.4 (5.9)0–16Duration of CKD (years)6.7 (4.7)0.2–15Percent of life with CKD69 (39)1–100Hypertensive (%)59Hemoglobin (mg/dl)12.7 (1.5)10–17aMean (standard deviation) or percent Neurocognitive instruments All participants completed a battery of intellectual, attention, and memory tasks as part of a larger neuropsychological evaluation. To gain an estimate of overall intellectual functioning, we administered the Wechsler Abbreviated Scale of Intelligence [9]. The Gordon Diagnostic System [10], a computerized continuous performance test, was also administered to gain an estimate of attention functioning. Within the memory domain, the Wide Range Assessment of Memory and Learning [11] was employed to assess short-term memory across verbal and visual domains. Age-based standard scores (mean = 100, standard deviation = 15) were generated for variables from each of these tasks. In all instances, higher scores represented higher function for each cognitive test result. All tasks were administered by trained examiners who were supervised by a child neuropsychologist. The Wechsler Abbreviated Scales of Intelligence (WASI) is built on the well-known Wechsler measures of global intellectual functioning [9]. It was designed for ages 6 years through adulthood, and employs a fluid-crystal model of intelligence. Four subtests comprise the WASI and include vocabulary, block design, similarities, and matrix reasoning. All four subtests were administered to gain a brief full scale IQ score. The Gordon Diagnostic System (GDS) is a computerized continuous performance test that measures various aspects of visual attention [10]. For this study, the vigilance task was administered, which requires the individual to respond to specific visual stimuli (e.g., the number 1 followed by the number 9) from a series of numbers presented at a rate of approximately one per second. This task yields standard scores for the number correct, correct variability, commissions, and commission variability. For this study, the total correct standard score was employed. The Wide Range Assessment of Memory and Learning (WRAML) is a comprehensive memory battery of tests [11]. It includes nine different subtests that measure short-term verbal memory (story memory, sentence memory, number/letter), short-term visual memory (picture memory, design memory, finger windows), and new learning capabilities (sound symbol, visual learning, verbal learning). A general memory index is also computed from the nine subtests. Normative data are available for ages 6 through 18 years. The WRAML was administered and scored according to standardized procedures, with the general memory index being employed for this study. Clinical measures Disease severity, age of onset, and duration of renal disease, coded as time in years and as percent of life with CKD, as well as the presence of anemia or hypertension were explored as potential risk factors of interest, given their association with detrimental cognitive outcomes in the CKD literature. Clinical data were collected through physical examinations and laboratory tests performed within 2 weeks of cognitive testing. Age of disease onset, as well as duration and percent of life with CKD, were determined through record reviews and were based on the date of primary disease diagnosis. The Schwartz formula [8], using creatinine and height measurements, was used to estimate the eCrCl for non-dialysis patients. Creatinine clearance and urea kinetics were calculated for peritoneal dialysis patients from a 24 h fluid collection. All but three of the peritoneal dialysis patients exceeded Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines for maintaining a Kt/V of at least 2.0, and those not achieving this level ranged from 1.8 to 1.9 [12]. For our continuous analysis, we represented the eCrCl from the 24 h fluid collection from dialysis patients (incorporating residual renal function for the six dialysis patients who still produced urine) on a continuum with the eCrCl from the Schwartz formula that had been calculated for CKD patients. The one hemodialysis participant had a Kt/V of 1.34 and received dialysis four times a week. For analysis, we imputed this participant’s eCrCl, designating the mean creatinine clearance value from anuric peritoneal dialysis participants. For the dichotomous analysis, “mild to moderate” disease was categorized as an eCrCl of 30 to 89 ml/min per 1.73 m2 body surface area, representing those patients with stage 2 or 3 CKD as defined by K/DOQI guidelines [12]. Those with an eCrCl of less than 30 ml/min per 1.73 m2 or those on dialysis, consistent with stage 4 or 5 CKD, were categorized as “severe CKD/ESRD”. Anemia was defined as a hemoglobin level of less than 11 mg/dl, reflecting the normative ranges of hemoglobin for children aged 9 years and above and the K/DOQI guidelines that specify a target hemoglobin level of 11–12 mg/dl in CKD [13]. Hypertension was defined as either the regular use of antihypertensive medications or the presence of a blood pressure greater than the 95th percentile for age, height, and gender [14]. Data analysis Initial summary statistical analysis was used to describe the total study sample. This included both descriptive analysis and graphical examination of all relevant variables. To address the primary research question for this study, we used bivariate analysis comparing neurocognitive scores and the clinical characteristics of the study sample, using Pearson’s correlations for all continuous variables. Trend analysis based on linear regression was also used when continuous variables were compared graphically. For those variables that, by definition, were dichotomous (e.g., hypertension), Student’s t-tests were used to compare the mean neurocognitive scores between categories. For those clinical variables for which an argument could be made either to express continuously or to dichotomize on the basis of a normal range (e.g., hemoglobin), both correlations and t-tests were used. Results An initial inspection of the neurocognitive test scores showed that the sample of children and adolescents with CKD was mildly variable, with most of the participants falling within the low average to average range on most tasks. For the full study sample, the mean IQ score was 91 (range 61 to 117, SD 16), and the mean memory score was 88 (range 65 to 128, SD 16), both falling within the low average to average range when compared with normative expectations. The mean attention score of 96 fell well within the average range but did show much more variability (range 37 to 120, SD 23). Disease severity Disease severity, represented by the eCrCl, yielded the strongest relationship with both IQ (r = 0.57, P = 0.001) and memory (r = 0.45, P = 0.02), demonstrating that higher eCrCl correlated with higher scores in these cognitive domains. Figures 1 and 2 illustrate the linear trend in the relationship between the continuous representation of disease severity and both IQ and memory scores. The correlation between attention and eCrCl was not significant. Additionally, lower IQ measures among severe CKD/ESRD participants in our study were evident when compared dichotomously with those with mild to moderate CKD (P = 0.0008), and the same relationships held for memory scores (P = 0.009) and attention scores (P = 0.012), despite the fact that attention was not linearly associated with eCrCl. Figure 3 illustrates the mean cognitive scores for categories of disease severity. While a downward trend with increasing severity is observed across all cognitive domains, statistical analysis confined to only the 15 non-dialysis patients yielded similar but less robust correlations among all areas of cognition, with IQ being the strongest (r = 0.46, P = 0.08). Fig. 1Relationship between IQ and estimated renal functionFig. 2Relationship between memory and estimated renal functionFig. 3Mean cognitive scores by level of renal disease Age at disease onset and CKD duration Increased CKD duration in years was associated with lower memory scores (r = −0.40, P = 0.03). Age at disease onset did not correlate significantly with IQ, memory, or attention scores in our original analysis. However, it was noted that many participants in our study sample had presented with already severe disease, making the date of disease diagnosis less reflective of the actual age at which reduced renal function began. In a secondary exploratory analysis, we repeated our analysis using only those subjects (n = 10) for whom we had a clearly documented date on which the eCrCl dropped below 90 ml/min per 1.73 m2. Among these patients were six with obstructive uropathy or structural congenital renal anomalies, three with glomerulonephritis, and one with cystinosis. In this secondary analysis we observed that the direction of prior relationships was preserved, with a stronger relationship between IQ and both age at disease onset (r = 0.69, P = 0.03) and percent of life with CKD (r = −0.66, P = 0.04). This may indicate that younger age at onset of CKD and longer percent of life with CKD correlate with lower IQ scores. Hypertension and anemia Table 2 summarizes the results of our analysis of hypertension and anemia. The dichotomous separation of these two variables did not produce significant group differences. These findings suggest that the presence of anemia or hypertension at the time of testing did not correlate with differences in neurocognitive test results between study participants with or without these clinical features. In a secondary analysis we also explored designation of hypertension, defined initially as either the use of antihypertensive medications or the presence of hypertension at the time of examination, on the basis of the presence of each criterion separately, and we found no differences. Additional examination of anemia revealed that only three patients had a hemoglobin level below the anemia definition of < 11 mg/dl, with values of 10.8 mg/dl, 10.7 mg/dl and 10.1 mg/dl. Consequently, we were unable to assess anemic patients across a wide range of hemoglobin (Hb) values. Within our sample of hemoglobin ranging between 10.1 mg/dl and 17.0 mg/dl, no association between our cognitive domains and hemoglobin was observed. Table 2Relationships between hypertension, anemia and cognitive measuresNumber (n = 29)IQaGeneral memoryaAttentionaTotal cohort91 (16)88 (16)96 (23)Non-hypertensive90 (18)87 (16)89 (24)Hypertensive92 (15)89 (17)100 (21)Non-anemic91 (16)89 (16)98 (22)Anemic (<11 mg/dl)92 (17)81 (23)76 (20)aFor each represented test among the normal population, the mean is 100 with a standard deviation of 15. No significant differences on Student’s t-tests for hypertension or anemia. The use of a contemporary blood pressure or hemoglobin value to represent the hypertension or anemia status of our participants evaluated the potential effect of these factors on their cognitive performance on the day of testing. Day-to-day changes in blood pressure could cause changes in cognitive function. Another approach is to use time-averaged hemoglobin and blood pressure values as markers of long-term strain on cognitive skills. Therefore, we also did a retrospective chart review of all blood pressure and hemoglobin values recorded in the 6 months prior to the study visit and generated a mean value for each of these two variables. On the basis of these time-averaged values, we repeated our analysis. In the blood pressure range represented, hypertension remained unrelated to any of the cognitive variables. Hemoglobin levels, however, demonstrated a subtle correlation with memory performance in the continuous analysis (r = 0.41, P = 0.03). Dichotomous analysis of anemia yielded a significant difference in cognitive scores only when defined as a time-averaged hemoglobin level less than 10.5 mg/dl (mean memory score of 67 among anemic patients versus 91 among non-anemic participants, P = 0.01). However, this must be interpreted with caution, since only three participants fulfilled this criterion for anemia. We should also acknowledge that there is a high degree of correlation among the results of the individual cognitive domains tested. Among the children included in our study, 12 (41%) had no cognitive scores greater than one standard deviation below the normative mean of 100, eight (28%) deviated in this fashion in a single cognitive domain, three (10%) did so in two areas, and six (21%) did so in all three areas. The level of overlap is, in part, likely due to the nature of cognitive testing, whereby testing in one area is, to some degree, dependent upon function in another area. The fact that there is not complete overlap, however, indicates that cognitive testing can distinguish among these deficits with some level of specificity. Discussion Neurocognitive deficits among children with ESRD have been demonstrated in the literature, with some suggestion that key clinical variables can be associated with the level and, perhaps, the pattern of these deficits. The primary purpose of this study was to examine the relationship between targeted clinical variables, based on the available literature, and selected neurocognitive functions as defined by IQ, memory, and attention. Further, the current study examines these relationships across the full spectrum of CKD, including the mild to moderate stages of CKD. While it is recognized that some of the etiologies leading to CKD in our patients, namely cystinosis, calcineurin inhibitor toxicity, and ischemic injury, could potentially be independently associated with some level of cognitive dysfunction, in all three cases the children did not demonstrate evidence of other neurologic signs that would indicate central nervous system damage from their underlying disorders. Additionally, these children are representative of a typical pediatric CKD population, and any contribution from the presence of their comorbid conditions would not compromise the validity of our findings. In the current study, several key findings were evident. First, disease severity significantly correlated with IQ and memory, while duration of disease significantly correlated with memory in the expected direction. Age at onset and percent of life with CKD did not initially correlate with any of the neurocognitive indices. When the age at onset and percent of life with the disease were examined in a secondary analysis restricted to a group of participants with more precisely defined onset of true decreased renal function, there was a clear trend that suggested that the younger the age at onset and the greater the percent of life with CKD, the lower the IQ score. The finding that younger age at renal insufficiency onset is significantly related to lower IQ complements previous observations in two prior studies of children with ESRD [3, 15]. Lawry et al. [15] found that among 24 children, including both dialysis and transplant patients, younger age at ESRD onset correlated with lower IQ scores. Brouhard et al. [3] also found that younger age at renal diagnosis was associated with lower achievement scores, although not specifically with lower IQ scores. Also interesting to note is the wide range of IQ and memory scores among the ESRD subset of our study sample, as can be observed in Figs. 1 and 2. This might reflect the presence of other clinical variables, such as duration on dialysis, dialysis adequacy, or presence of residual renal function, which could also influence cognitive performance. A similar wide range of results among children with ESRD has been observed in other study samples, such as that of Brouhard et al. [3], in which children with renal disease had scores that included some in the average range but still had mean IQ and achievement scores significantly lower than their healthy sibling controls. These findings support the hypothesis that disease severity plays a strong role in predicting neurocognitive vulnerability, yet also suggest that there might be additional contributing variables characterizing end stage disease that warrant further investigation. In an effort to explore this issue further, the sub-analysis confined to the relationship between disease severity and cognitive outcomes in the non-dialysis participants demonstrated a more subtle downward trend of scores across all cognitive domains that did not reach statistical significance. This was likely, in part, due to the reduction in power resulting from our limiting the analysis to only 15 participants. It also might suggest that therapy with dialysis itself contributes to cognitive dysfunction. The literature has been more ambiguous with regard to attention, and our findings did not demonstrate a significant deficit in attention. Our sample did, however, demonstrate significantly more variation in the attention measure among children with CKD than one would anticipate in the general population, as exhibited by the large standard deviation in our study sample. This suggests the possibility that there may be a subset of children with CKD who are more vulnerable to attention-related problems. More extensive exploration of the various components of attention may be required to explore this observation further. Hypertension, as a single measure or time-averaged, was not associated with the neurocognitive outcomes evaluated. Hypertension in the general pediatric population has been correlated with decreased performance in tasks requiring memory, attention, and concentration [16]. Using school-aged children from a population-based US survey who were diagnosed with hypertension on the basis of systolic or diastolic blood pressure above the 90th percentile for age, height and gender, Lande et al. [16] showed that hypertension was significantly associated with lower neurocognitive scores representative of short-term memory, attention and concentration problems. In our study sample of children with CKD, the majority of our participants were on antihypertensive therapy, so any existing hypertension in our study sample was at least partially treated, thus significantly restricting the range of variance available for correlation analysis. Furthermore, some antihypertensive therapies, for instance clonidine, have the potential to cause sedating effects in children but can actually help concentration in other children. The effects of medications on just a few children could appear amplified in our small study sample, and, thus, a true relationship could be obscured. The hemoglobin level at the time of testing did not predict cognitive function. Time-averaged hemoglobin concentrations less than 10.5 mg/dl were associated with lower memory performance. It has also been well established that anemia is associated with decreased cognitive function in adult patients with renal disease [17, 18]. Stivelman’s review on the topic highlights several studies investigating the use of erythropoietin in uremic adult patients. Increasing hematocrit was associated with improved neurocognitive measures [17]. For example, Marsh et al. [18] used erythropoietin in 24 adult hemodialysis patients to improve their average hematocrit from 23% to 36%, resulting in significantly improved neuropsychological test scores reflecting memory, learning and attention. In our study sample of children with CKD, only three children had a low hemoglobin level, and these were between 10.1 mg/dl and 10.8 mg/dl. The mean scores for memory and attention among those three children were 8 points and 22 points lower, respectively, than those for non-anemic children, suggesting that there could be a relationship that would require a larger sample size to identify. In the normal or near-normal range, detrimental effects of lower hemoglobin levels are likely more subtle and, thus, less likely to be captured in analysis of a small sample. Our time-averaged hemoglobin analysis supported this as well. This is the first study to date that focuses specifically on identifying potential risk factors for neurocognitive decline in children and adolescents with CKD. Its value is further enhanced by the inclusion of children with mild to moderate CKD, thus extending findings to a wider range of pediatric CKD patients. Increased disease severity, duration of CKD, and younger age at CKD onset were identified as potential risk factors for the targeted neuropsychological functions of IQ and memory. Our results further suggest a significant linear relationship with disease severity, with IQ scores continuously declining as disease severity worsens. Not only does this finding argue against a threshold effect, it implicates disease severity as an important risk factor for neuropsychological dysfunction. These findings also suggest the possible utility of these variables in a cumulative risk model to predict neurocognitive dysfunction in CKD and lay the foundation for exploration of such a model. Finally, these findings emphasize the importance of conducting further research in neurocognitive development among children with CKD. This includes research both to more clearly predict which children with CKD are most vulnerable to neuropsychological dysfunction and to identify specific correlates of this dysfunction. While there have been great strides in improving quality and length of life among children with CKD, a better understanding of those processes that interfere with normal neurodevelopment is a critical element of providing optimal care to these children.

Breast_Cancer_Res_Treat-3-1-2001225

A decade of letrozole: FACE

Third-generation nonsteroidal aromatase inhibitors (AIs), letrozole and anastrozole, are superior to tamoxifen as initial therapy for early breast cancer but have not been directly compared in a head-to-head adjuvant trial. Cumulative evidence suggests that AIs are not equivalent in terms of potency of estrogen suppression and that there may be differences in clinical efficacy. Thus, with no data from head-to-head comparisons of the AIs as adjuvant therapy yet available, the question of whether there are efficacy differences between the AIs remains. To help answer this question, the Femara versus Anastrozole Clinical Evaluation (FACE) is a phase IIIb open-label, randomized, multicenter trial designed to test whether letrozole or anastrozole has superior efficacy as adjuvant treatment of postmenopausal women with hormone receptor (HR)- and lymph node-positive breast cancer. Eligible patients (target accrual, N = 4,000) are randomized to receive either letrozole 2.5 mg or anastrozole 1 mg daily for up to 5 years. The primary objective is to compare disease-free survival at 5 years. Secondary end points include safety, overall survival, time to distant metastases, and time to contralateral breast cancer. The FACE trial will determine whether or not letrozole offers a greater clinical benefit to postmenopausal women with HR+ early breast cancer at increased risk of early recurrence compared with anastrozole. Introduction The aromatase inhibitors (AIs) have proven to be a powerful drug class for use in hormone-sensitive breast cancer and have shown superiority over the selective estrogen-receptor modulator (SERM) tamoxifen in preclinical models of hormone-dependent breast cancer [1] and in randomized controlled trials in patients with advanced breast cancer [2] and early breast cancer [3, 4]. Initial adjuvant therapy with either letrozole (Femara®) or anastrozole (Arimidex®) was shown to be significantly more effective than tamoxifen in both the Breast International Group (BIG) 1-98 and Anastrozole and Tamoxifen Alone or in Combination (ATAC) randomized controlled trials in postmenopausal women with localized breast cancer [3, 4]. In the BIG 1-98 primary core analysis, patients with hormone receptor-positive (HR+) tumors randomized to receive letrozole initially were compared with those assigned to receive tamoxifen initially (N = 8,010). After a median follow-up of 25.8 months, 351 events had occurred in the letrozole group (n = 4,003) and 428 events in the tamoxifen group (n = 4,007), with 5–year disease-free survival (DFS) estimates of 84.0% and 81.4%, respectively. Letrozole significantly reduced the risk of breast cancer recurrence (hazard ratio = 0.81; 95% confidence interval [CI] 0.70, 0.93; P = 0.003), especially the risk of distant recurrence (hazard ratio = 0.73; 95% CI 0.60, 0.88; P = 0.001) [3]. An analysis limited to patients randomized to either letrozole-only or tamoxifen-only arms (N = 4,922) was recently published and allows for more direct comparisons with results from other trials of continuous therapy with a single endocrine agent [5]. Results from the letrozole-only or tamoxifen-only arms were consistent with those published for the primary core analysis and showed that letrozole significantly reduced the risk of DFS events (hazard ratio = 0.82; 95% CI 0.71, 0.95; P = 0.007) [5, 6] and the risk of distant metastases. After a median follow-up of 51 months, 352 DFS events (14.3%) were observed in the letrozole-only group (n = 2,463), compared with 418 DFS events (16.9%) in the tamoxifen-only group (n = 2,459) [5, 6]. The ATAC trial, which compared anastrozole with tamoxifen for 5 years, did not have receptor positivity as a study requirement. After a median follow-up of 68 months (n = 6,186), anastrozole significantly prolonged DFS (575 events with anastrozole vs. 651 events with tamoxifen; hazard ratio = 0.87, 95% CI 0.78, 0.97; P = 0.01) and time-to-recurrence (402 vs. 498 events; hazard ratio = 0.79, 95% CI 0.70, 0.90; P = 0.0005), and significantly reduced the risk of developing distant metastases (324 vs. 375 events; hazard ratio = 0.86, CI 0.74, 0.99; P = 0.04) and contralateral breast cancers (35 vs. 59 events; 42% reduction, 95% CI 12, 62; P = 0.01) in the intent-to-treat (ITT) population [4]. However, neither the time to distant recurrence nor distant DFS (DDFS) were significantly improved with anastrozole in the HR+ population [7]. While both letrozole and anastrozole have been evaluated extensively in early breast cancer, no head-to-head trial of these two AIs has been conducted in this setting. This report will focus on the design of the Femara versus Anastrozole Clinical Evaluation (FACE) trial, and describe how it will prospectively address potential efficacy and safety differences between the two AIs. Rationale for head-to-head trial An American Society of Clinical Oncology technology assessment concluded that AIs should be included in the adjuvant treatment of postmenopausal women with HR+ breast cancer [8] but did not recommend one AI over another. The National Comprehensive Cancer Network has also recommended initial adjuvant therapy with an AI (specifically letrozole or anastrozole) as an alternative to tamoxifen [9]. Some evidence suggests that AIs may not be equivalent, even though they belong to the same pharmacologic class of agents; differences have been reported in terms of potency, suppression of aromatization, antitumor effects, and pharmacogenomics. However, whether or not one AI is superior in treating early breast cancer is not known. Relative potency Bhatnagar and colleagues compared the aromatase-inhibiting potency of letrozole and anastrozole in a variety of aromatase-containing cellular endocrine and tumor models [10, 11]. While letrozole and anastrozole were approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole was found to be more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells (50% inhibitory concentration [IC50] 20 vs. 600 nM, respectively), normal human adipose fibroblasts (0.8 vs. 14 nM), and human cancer cell lines (MCF-7Ca 0.07 vs. 0.82 nM and JEG-3 0.07 vs. 0.99 nM). Miller and colleagues reported that letrozole and anastrozole were more potent than aminoglutethimide in vitro against tumor samples obtained from postmenopausal women with breast cancer, with letrozole demonstrating the lowest IC50 (2 nM, 8 nM, and 20 μM, respectively) [12]. Letrozole was compared with anastrozole in vivo in athymic mice inoculated with MCF7 cells [13]. Tumor volumes increased to 145.9% in controls and decreased to 22.4% with letrozole 10 μg, and to 95.6% or 78.2% with anastrozole 10 or 60 μg, respectively. These results are consistent with a higher in vitro potency of letrozole in cell-based assays [13]. Suppression of aromatization The effects of letrozole and anastrozole on suppression of total-body aromatization and plasma estrogen concentrations have been compared in patients with metastatic breast cancer [14, 15]. Levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (> 99.1% suppression in all patients; Wilcoxon, P = 0.0022, comparing the two drug regimens). Suppression of estrone and estrone sulfate was found to be significantly greater during treatment with letrozole compared with anastrozole (P = 0.019 and P = 0.0037, respectively). Another study conducted in 54 postmenopausal women with invasive breast cancer showed that more complete inhibition of aromatase was achieved by 2.5 mg of letrozole than 1 mg of anastrozole, resulting in significantly greater suppression of estradiol (P < 0.0001) [15]. Thus, letrozole reduces estradiol levels to a greater degree than anastrozole, but it is not known whether this difference is clinically relevant. Breast cancer proliferation Biological changes in breast tumors occurring within 14 days of starting treatment may predict the efficacy of different endocrine agents in the adjuvant setting and could prove to be useful surrogate markers to compare drug efficacy [16]. A study of neoadjuvant endocrine therapy compared the effects of letrozole and anastrozole on the expression of HRs and markers of tumor proliferation in postmenopausal women with estrogen receptor (ER)-positive breast cancer [16]. Neoadjuvant letrozole and anastrozole decreased overall ER expression (Allred score) after 14 days, but more cases showed a reduction in progesterone receptor (PgR) expression following letrozole treatment (75/106) than with anastrozole treatment (65/102). Furthermore, only letrozole significantly reduced proliferation at lower Allred ER expression levels (scores 2–5). This is a potentially important finding, because it has been suggested that a greater suppression in proliferation may lead to improved DFS [17]. Clinical activity Letrozole and anastrozole have not been directly compared in the adjuvant setting, but data from a randomized, head-to-head trial in patients with advanced breast cancer are available [18]. Postmenopausal women with advanced breast cancer (N = 713) that had progressed either during antiestrogen therapy or within 12 months of completing that therapy were randomized to receive letrozole (2.5 mg per day) or anastrozole (1 mg per day). Letrozole was significantly superior to anastrozole in terms of overall response rate (19.1% vs. 12.3%, P = 0.013), but there were no significant differences in median time to progression, the primary end point of the trial. Both agents were well-tolerated, and there were no significant differences in safety. Anastrozole and letrozole in the adjuvant setting have demonstrated superiority to tamoxifen in significantly reducing the risk of recurrence. In the ATAC trial, at 68 months’ median follow-up, anastrozole significantly reduced the risk of distant metastases in the ITT population by 14% (P = 0.04) but not significantly in the HR+ patient subgroup (hazard ratio = 0.84; P = 0.06) [4]. A recent study of the recurrence rates after 2.5 and 5 years from the ATAC study showed that there were fewer recurrence events with anastrozole at these time points due to reductions in contralateral, primary, loco-regional, and distant recurrences [19]. In addition, at 25.8 months’ median follow-up in the BIG 1-98 trial, letrozole significantly reduced the risk of distant metastases by 27% (P = 0.001) in the HR+ population, and another analysis of the early risk of relapse in 5,980 patients, with a median follow-up of 25 months, showed that letrozole reduced distant recurrences early on [3, 20]. The recently reported analysis of letrozole-only and tamoxifen-only arms in the BIG 1-98 trial showed that the time to distant metastases advantage for letrozole was consistent with these findings from the primary core analysis [5]. These data are potentially important, because the development of distant metastases directly translates into decreased survival. The ATAC trial showed a 3% relative improvement in overall survival (OS) (P = 0.7) with anastrozole at 68 months of follow-up, while a 9% relative improvement in OS (P = 0.35) was seen with letrozole at 51 months of follow-up [3, 5]. Subset analyses of randomized trials comparing letrozole or anastrozole with either tamoxifen or placebo demonstrated differences between these AIs and suggested that specific patient populations may derive differing degrees of benefit from a particular AI. In the trial, retrospective subgroup analyses with a median follow-up of 33 months revealed no significant benefit of anastrozole over tamoxifen in patients with node-positive tumors and with prior chemotherapy [21], and these findings were confirmed in the 4-year update of the ATAC trial. Thus, the hazard ratio for risk of recurrence in patients with four or more positive nodes was 0.96 (95% CI 0.72, 1.25), and in patients with prior chemotherapy, it was 0.98 (95% CI 0.76, 1.28), indicating no differences between treatments [22, 23]. No analyses in similar subgroups were presented in the 68-month update [4]. Prospectively planned subgroup analysis revealed a benefit of letrozole over tamoxifen in patients who had received chemotherapy and in those with node-positive tumors [3, 5]. In the former subset, letrozole reduced the risk of recurrence after 5 years (hazard ratio 0.70; 95% CI 0.54, 0.92; P = 0.01). In the node-positive subset, letrozole reduced the risk of an event ending a period of DFS by 29% (hazard ratio 0.71, 95% CI 0.59, 0.85; P < 0.001). The advantage for letrozole in these patient subsets was confirmed in the recent analysis of the letrozole-only and tamoxifen-only arms of BIG 1-98 [5]. Interestingly, with longer follow-up of 51 months in this monotherapy analysis, there was an emerging benefit in the node-negative group, as letrozole reduced the relative risk of recurrence by 12% in this patient population. The MA.17 trial, evaluating the efficacy of extended adjuvant letrozole therapy, although not positive for OS in the overall population, demonstrated that OS was statistically significantly improved with letrozole among lymph node-positive breast cancer patients compared with placebo (hazard ratio 0.61; 95% CI 0.38, 0.98; P = 0.04) [24]. The question of whether one third-generation AI is superior for the adjuvant treatment of postmenopausal women with HR+ breast cancer remains, as does the question of whether there are any specific patient populations who derive particular benefit from a specific AI. Patients with early breast cancer can be assigned to risk groups on the basis of clinical and pathological characteristics. In the St. Gallen Guidelines [25], node-positive patients are considered to be in the intermediate- or high-risk group depending on the number of positive nodes and human epidermal growth factor receptor-2 (HER2) expression (see Table 1). The guidelines state that endocrine therapy with an AI is a recommended option for patients with node-positive tumors who are in the intermediate- or high-risk groups. As high-risk patients are at greater risk of relapse, a drug specifically effective in this patient population would provide the oncology community with valuable information that may alter the outcomes of these patients. Table 1Risk categories for early breast cancer according to the St. Gallen Guidelines. Reprinted from [25], with permission from the European Society for Medical OncologyLow risk Steroid hormone receptors expression, node-negative, and all of the following features:pT ≤ 2 cmGrade 1No peritumoral vascular invasionHER2/neu gene neither overexpressed nor amplifiedAge ≥35 yearsIntermediate riskNode-negative and at least one of the following features:pT > 2 cmGrade 2–3Peritumoral vascular invasionHER2/neu gene either overexpressed or amplifiedAge < 35 yearsNode positive (1–3 involved nodes) and HER2/neu gene neither overexpressed nor amplifiedHigh riskNode positive (1–3 involved nodes) and HER2/neu gene either overexpressed or amplifiedNode-positive (4 or more involved nodes)HER2 human epithelial growth factor receptor 2pT pathological tumor size (i.e. size of the invasive component) Is one AI superior in early breast cancer? Microarray analysis was used to study the effects of various hormone therapies on ER+ MCF-7 cells, stably transfected with the aromatase gene (MCF-7aro cells) [26]. The study found that hormonal stimulation of gene expression can be counteracted by treatment with AIs (letrozole and anastrozole) and an anti-estrogen (tamoxifen), but that each agent had its own unique effects on gene expression (see Fig. 1), suggesting possible differences between letrozole and anastrozole [26]. Although differences between letrozole and anastrozole have been demonstrated in preclinical models, it is widely recognized that preclinical findings do not always translate into clinical results, and that comparisons in one treatment setting or subpopulation cannot be extrapolated to another. A prospective trial is therefore needed to address the question of whether one AI is superior to another. Fig. 1Changes in inhibitor-responsive genes after treatment with letrozole, anastrozole, or tamoxifen. The Venn diagrams show the numbers of genes responsive to individual inhibitors in hormone-regulated genes. Reprinted from [26], with permission from the American Association for Cancer Research FACE was designed to test whether there is a preferable AI for the adjuvant treatment of postmenopausal women with HR+ and lymph node-positive cancer [27]. Node-positive patients were selected, because this population has a higher risk of relapse, and recurrence events occur earlier than in node-negative patients [20, 28, 29]. Thus, conducting the FACE trial in patients with lymph node-positive early breast cancer will provide an answer more quickly than conducting a trial in a broader population that includes patients with node-negative tumors. FACE trial design FACE is a phase IIIb open-label, randomized, multicenter trial [30]. The primary objective of the trial is to compare DFS at 5 years for letrozole and anastrozole. Secondary objectives are to assess safety, OS, time to distant metastases, and time to contralateral breast cancer [27]. Patients The trial is recruiting 4,000 patients from up to 250 international sites. Eligible patients are postmenopausal women with HR+ and lymph node-positive tumors who have recently undergone surgery for primary breast cancer (pathologic or clinical stage IIA, IIB, or IIIA). All patients must provide written informed consent. HR+ tumors are defined as tumors with any detectable ER or PgR expression by institutional standards. Patients who are PgR+ and ER− are eligible for the trial. Pathologic assessment of axillary lymph nodes is determined by sentinel node biopsy and/or axillary lymph node dissection. Patients are stratified according to the number of involved lymph nodes and HER2 tumor status. Adjuvant trastuzumab is permitted in patients with HER2+ tumors. Other inclusion criteria include World Health Organization performance status of 0 or 1, lipid panel (fasting total cholesterol and triglycerides) ≤ grade 1 (National Cancer Institute Common Terminology Criteria for Adverse Events v3.0), and adequate hematologic, hepatic, and renal function. Patients with T4 tumors, metastatic disease, contralateral breast cancer including ductal carcinoma in situ, or evidence of disease progression are excluded. Other exclusion criteria include prior neoadjuvant endocrine therapy; hormone replacement therapy (except intravaginal estradiol preparations) not stopped at least 4 weeks before randomization; adjuvant anti-estrogen therapy for > 1 month immediately following surgery, radiotherapy, and/or chemotherapy; breast cancer chemoprevention with anti-estrogens if < 18 months between stopping and diagnosis of breast cancer; and therapy with any hormonal agent, such as raloxifene, for management of osteoporosis. Randomized trial design and treatments Eligible patients are randomized to receive either letrozole 2.5 mg or anastrozole 1 mg daily for up to 5 years (see Fig. 2). The date of randomization must be no more than 12 weeks from completion of surgery or 4 weeks after completion of adjuvant chemotherapy. Treatment assignments are balanced based on the number of lymph nodes (1–3, 4–9, 10+) and HER2 status (positive, negative, or unknown). Treatment will commence within 30 days of randomization and following the completion of standard chemotherapy (if given) and concurrently with radiotherapy (if given). Patients receive treatment with the allocated AI for up to 5 years or until disease recurrence/relapse. Recurrence and survival will be assessed every 12 months. Fig. 2FACE randomized trial design Efficacy end points The primary end point is DFS, defined as the time from the date of randomization to the date of the first documentation of re-occurrence of invasive breast cancer in local, regional, or distant sites; new invasive breast cancer in the contralateral breast; or death from any cause. Secondary efficacy end points include OS, defined as the time from the date of randomization to date of death from any cause; breast cancer-free survival, defined as the time from date of randomization to the date of death due to breast cancer; time to development of distant metastases, defined as the time from date of randomization to the date of the first development of any recurrent or metastatic disease in sites other than the local mastectomy scar, the ipsilateral breast in case of breast conservation, or the contralateral breast; and time to development of contralateral breast cancer, defined as the time from date of randomization to the date of the first development of any disease in the contralateral breast. Although the FACE trial, co-chaired by Drs. Ian Smith and Joyce O’Shaughnessy, is an open-label trial, analysis of the data in a blinded fashion will make the data from this trial comparable with that obtained in a single-blinded trial. Both patients and their physicians will know which drug is being taken, but the analysis of the data will be conducted blinded to study treatment. The sponsor of the trial will not have access to the database, and all efficacy analyses will be conducted by an independent academic organization (the Instituto Nazionale Tumori, Milan, Italy), which will receive the data in a blinded manner. The data will be reviewed by an Independent Data Monitoring Committee, chaired by Professor Martine Piccart. The Independent Data Monitoring Committee will then make recommendations to a Trial Steering Committee chaired by Dr. Kathy Pritchard. The Independent Data Monitoring Committee will decide when the data will be released. The final analysis will be performed after the expected total number of DFS events have occurred. This is anticipated to be 7 years after the start of the study, following an accrual period of about 2 years and a minimum of 5 years of further follow-up. There are two planned interim analyses, scheduled to occur after one third and subsequently after two thirds of the maximum number of events have been observed. The interim analyses will be conducted after 320 and 639 events, respectively, have been recorded. In addition, analyses of secondary end points will be conducted at the interim time points. FACE is powered to detect a 3.5% absolute difference between the two treatment arms in DFS at 5 years. The 3.5% difference corresponds to a hazard ratio of 0.83 in favor of letrozole, corresponding to 5-year DFS values of 80.0% and 76.5% for letrozole and anastrozole, respectively. Safety end points General patient safety and drug tolerability will be evaluated. Adverse events are recorded at every visit and graded for severity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0. A checklist of adverse events is used to solicit adverse event information from patients. Safety analyses specifically include cardiovascular events and bone fracture events. All patients are evaluated clinically for osteoporosis and fracture risks. Bone mineral density testing is recommended at least every 2 years for all patients during study therapy by dual X-ray absorptiometry, peripheral dual X-ray absorptiometry, or ultrasound densitometry. Osteoporosis may be managed as clinically indicated using calcium supplements, vitamin D, or bisphosphonates. Measurements of fasting serum lipids are obtained at 6 and 12 months and then annually thereafter for the duration of the study treatment. Other laboratory assessments include hematology and blood chemistry. Other head-to-head studies Other trials that are directly comparing AIs are also under way. A randomized phase III trial [31] is comparing neoadjuvant therapy with exemestane, letrozole, or anastrozole in postmenopausal women undergoing surgery for stage II or stage III breast cancer. Another ongoing randomized trial, MA.27 [32], has been designed to compare the event-free survival of postmenopausal women with HR+ primary breast cancer when treated with exemestane or anastrozole. Results from these trials and ongoing pharmacogenomic studies [26] will also help individualize AI therapy for early breast cancer. The study of inherited genetic polymorphisms that affect drug response and toxicity promises to help physicians individualize hormone treatment. For example, “slow metabolizers” of tamoxifen may have a worse outcome in the adjuvant setting with tamoxifen treatment than “fast metabolizers,” suggesting that these patients might be better treated with an AI [33–35]. Polymorphisms in tamoxifen metabolizing cytochrome P (CYP) 2D6 gene affect the plasma concentration of tamoxifen active metabolites: women with the CYP2D6 *4/*4 or wt/*4 genotype could have lower benefit of tamoxifen treatment and tend to have a higher risk of disease relapse [35, 36]. Genetic polymorphisms in the aromatase gene, CYP19, have recently been characterized [37]. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies revealed that polymorphisms may lead to changes in aromatase activity and altered affinity for AIs. These findings indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease. Clinical trials have been initiated to study the impact of genetic differences on response to AI therapy and may eventually lead to patient-specific selection of therapy based on optimizing efficacy and toxicity. Conclusions Letrozole and anastrozole have both demonstrated superior efficacy compared with tamoxifen as initial therapy for early breast cancer [3, 4]. Preclinical and clinical evidence suggests that AIs do not have identical pharmacodynamic profiles, but it is not known whether one agent may be more effective as adjuvant therapy for early breast cancer. Differences in potency in preclinical studies, and the reduction in distant metastases in the BIG 1-98 study, suggest the potential for clinical efficacy differences between AIs. Based on the results of these trials, international guidelines now recommend adjuvant hormone therapy with an AI [9, 25] in patients with an increased risk of early recurrence. The FACE trial is addressing an important medical question in the oncology community: whether or not letrozole offers greater clinical benefit to postmenopausal women with HR+ early breast cancer at increased risk of early recurrence compared with anastrozole. Results from the FACE trial may refine the treatment strategies for treating breast cancer in postmenopausal women.

Photosynth_Res-3-1-1769344

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm. Introduction When light energy is absorbed in vivo by purple bacterial light-harvesting (LH) complexes it is rapidly transferred to the reaction centers (RC) where the light energy is efficiently used to drive chemical reactions (Ke 2001). In most types of purple bacteria there are two types of antenna complexes: peripheral LH2 complexes and the LH1 complexes (Ke 2001). The structure of the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been resolved to a resolution of 2.0 Å (McDermott et al. 1995). This LH2 complex consists of a ring of nine heterodimeric subunits. However, such high-resolution structure has not been determined for the LH1 complex yet. There are, however, low-resolution projection structures produced by transmission electron microscopy (TEM) (Karrasch et al. 1995) of two-dimensional (2D) crystals of the LH1 complex and a 4.8 Å X-ray crystal structure of the LH1-RC core complex (Roszak et al. 2003). TEM analysis of the LH1 complexes revealed two types of complex, monomeric complexes from Rhodospirillum rubrum (R. rubrum) (Karrasch et al. 1995) and dimeric complexes from Rhodobacter sphaeroides (R. sphaeroides) (Jungas et al. 1999). The recent crystal structure of the LH1-RC ‘core’ complex from Rhodopseudomonas palustris (Rps. palustris) reveals that the LH1 complex surrounds the contours of the RC so that the ‘core’ complex has an overall oval rather than a circular shape (Roszak et al. 2003). This structure showed the RC surrounded by the LH1 complex which consisted of 15 pairs of transmembrane helical α- and β-polypeptides and their coordinated BChls. The complete closure of the RC by the LH1 is prevented by a single transmembrane helix called W. Atomic force microscopy has also been used to observe antenna complexes in both natural and reconstituted membranes (Scheuring et al. 2001, 2003, 2004; Fotiadis et al. 2004; Bahatyrova et al. 2004a, b; Stamouli et al. 2003). Scheuring et al. observed the LH1 complex as a minor component together with the major LH2 complexes from Rubrivivax geratinosus (Scheuring et al. 2001) in reconstituted membranes and intact LH1-RC core complexes in native photosynthetic membranes (chromatophore) from Rhodopseudomonas viridis (Scheuring et al. 2003) and Rhodospirillum photometricum (Scheuring et al. 2004). Fotiadis et al. observed the LH1-RC core complex from R. rubrum in membranes formed from Escherichia coli lipids (Fotiadis et al. 2004). Bahatyrova et al. showed that the LH1 complexes of a mutant lacking the RC from R. sphaeroides which form circular, elliptical, and even polygonal ring shapes as well as arcs and open rings (Bahatyrova et al. 2004b) and that the LH1 complexes are positioned to function as an energy collection hub from the LH2 complexes in native membranes (Bahatyrova et al. 2004a). Our understanding of charge separation and energy transfer in these LH2 and LH1-RC core complexes has enabled the first steps to be taken towards generating artificial systems that convert light energy into usable electrical current. Previous attempts to produce an artificial, energy-converting electrode system used either the LH1 complexes (Ogawa et al. 2002) or RC (Blankenship et al. 1995) immobilized on the electrodes. Until now, there have only been a few attempts to immobilize intact ‘core’ complexes, consisting of both the LH1 and the RC components together, onto an electrode (Ogawa et al. 2004; Das et al. 2004). We have recently developed a procedure to create a self-assembled monolayer (SAM) of reconstituted LH1 complexes on a transparent indium tin oxide (ITO) electrode modified with 3-aminopropyltriethoxysilane (APS) using electrostatic interactions or hydrogen bonding between the electrode surface and the anionic LH1 polypeptides at pH 8.0 (Ogawa et al. 2002). The near infra-red (NIR) absorption spectrum showed that the LH1 complex was stable when immobilized onto the electrode. Our current work extends this approach to the native LH1-RC core complexes. LH1-RC ‘core’ complexes isolated from R. rubrum and Rps. palustris were successfully assembled on an ITO electrode modified with APS (APS-ITO). Efficient energy transfer and photocurrent responses could be observed upon illumination at 880 nm. Materials and methods Growth of R. rubrum and Rps. palustris bacterium The photosynthetic bacteria, R. rubrum strain S1 and Rps. palustris strain 2.1.6 were grown anaerobically in the light in modified Hutner’s media as previously described (Roszak et al. 2003; Visschers et al. 1991). Purification of the LH1 complex of R. rubrum Chromatophores of R. rubrum were prepared as previously described (Visschers et al. 1991). Carotenoid was extracted from chromatophores using benzene. Usually approximately 30 mM OG (1 mM Tris, pH 7.5) was added to dissolve the chromatophores until the near IR absorption band shifted from 873 to 820 nm. Then the resultant aqueous solution was applied to a Sephadex G-100 column (1.5 cm i.d. × 75 cm) to separate the RC and, LH1 complexes with carotenoids and subunit LH/BChl a complexes without carotenoid (B820 complexes). The RC was eluted immediately after the void volume and then the LH1 complexes with carotenoid. Finally the B820 complexes were collected. Absorbance (λmax) of the LH1 complex in the absence of carotenoid (15 mM OG at 25°C): 372 nm (0.86), 586 (0.23), 870 (1.14). Absorbance (λmax) of the LH complex in the presence of carotenoid (20 mM OG at 25 °C): 372 nm (0.86), 475 (0.19), 512 (0.22), 546 (0.19), 586 (0.235), 875 (0.9). Isolation and purification of the core complex of R. rubrum and Rps. palustris The LH1-RC core complexes isolated from R. rubrum and Rps. palustris were purified essentially as described previously (Roszak et al. 2003). These LH1-RC core complexes from R. rubrum and Rps. palustris were initially solubilized by the addition of LDAO to 0.4% or 1% v/v in 20 mM Tris–HCl pH 8.0, respectively. The LH1-RC core complex of Rps. palustris was then separated from the LH2 complexes by sucrose density centrifugation, and was further purified by ion exchange chromatography by DE52 cellulose column. The OD880 of the isolated LH1-RC core complexes was adjusted to 0.3. Preparation of the core complex assembled on APS-ITO electrodes The basic methods for this have been reported previously (Ogawa et al. 2002). Transparent indium tin oxide electrodes were cleaned by immersion in piranha solution (H2O2:H2SO4 = 3:7). We obtained APS-ITO electrodes by reacting 3-aminopropyltriethoxysilane with the surface of ITO electrodes in dry benzene at 80–90°C for 4 h. The OD880 of the isolated core was adjusted to 0.3. APS-ITO electrodes were immersed in the LH complex and the core complex solutions in Tris–HCl pH 8.0 for 6 h at 4°C, and then rinsed with Tris–HCl pH 8.0. The core complexes were immobilized, as a self-assembled monolayer on an APS-ITO electrode. Near IR, FT-IR, and fluorescence spectra Near IR spectra were recorded with Hitachi U-2000 and U-3500. Fluorescence spectra were measured with a Nippon Roper fluorometer by using a halogen tungsten light bulb (TS-428 DC), a single monochromator (SP-150M) for selection of the excitation wavelength, a monochromator (SP-306) and a CCD detector (Spec 10-100 BR/LN) to detect the emitted fluorescence. The slits were set at 0.50 mm for the LH1 complexes in OG. Slits were set at 1.00 mm for the LH1 complex on the electrode. The samples were measured at 25°C. FT-IR spectra were recorded with Perkin-Elmer Spectrum 2000. Photocurrent measurements Photocurrents were measured at −0.2 V (versus Ag/AgCl) in a home made cell that contained three electrodes; an APS-ITO electrode incorporating the core complex as a working electrode, an Ag/AgCl (saturated KCl) as a reference electrode, and a platinum flake as a counter electrode. The working electrode was illuminated with a halogen lamp unit, AT-100HG, through a monochromator, SPG-120S (Shimadzu). The solution consisted of 0.1 M phosphate buffer (pH 7.0), containing 0.1 M NaClO4 and 5 mM methyl viologen. Results and discussion Figure 1 shows the NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20 mM Tris–HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line), respectively. These spectra show that these core complexes have the absorption maximum at 880 nm with two smaller peaks at 800 and 760 nm. The former peak is attributable to the overlap of bacteriochlorophyll a (BChla) in the LH1 complex (880 nm) and the reaction center BChla dimer ‘special pair’ (870 nm) and the latter two peaks to the BChla called ‘accessory’ (800 nm) and bacteriopheophytin (760 nm) in the RC, respectively (Blankenship et al. 1995). The NIR absorption spectra of these core complex on the electrode indicate that these complexes were not denatured when assembled onto an APS-ITO. In the previous study it was apparent that when the RC of R. rubrum was assembled, by itself, on the electrode it was relatively labile (Matsumoto et al. 1999). In present study, the complete core complex proved to be quite stable, when assembled onto the electrode. The enhanced stability of the RC surrounded by the LH1 complex probably results from supportive interactions between the two complexes. Fig. 1NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20 mM Tris–HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line) Table 1 shows the NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris in OG micelles and on an APS-ITO electrode. The fluorescence bands of these core complexes on the electrode are identical to those in the OG micelles, again indicating that these complexes are stable when assembled onto the APS-ITO electrode. Interestingly, when illuminating at 880 nm the fluorescence emission of BChla molecules in the LH complex of R. rubrum on the APS-ITO was strongly quenched, due to the presence of the RC of R. rubrum. This indicates that an efficient energy transfer from BChla in the LH1 complex to the RC in the core complex is still occurring on the electrode (data not shown) (Ogawa et al. 2004). FT-IR spectra of the LH complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris assembled on the APS-ITO show the absorptions at 1650 and 1550 cm−1. These bands can be assigned to the amide I (C=O stretching vibration) and amide II (N–H deformation vibration) bands of α helical conformation, respectively (Miura et al. 1998). These results indicate that the LH polypeptides are in the same α helical configurations on the ITO electrode as in OG micelles (Parkes-Loach et al. 1988; Kashiwada et al. 2000). Table 1NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustrisIn OG micelleOn APS-ITOAbsorbance (nm)Fluorescence (nm)Absorbance (nm)Fluorescence (nm)R. rubumLH1870890870890R. rubumLH1-RC880900880900Rps. palustrisLH1-RC878900878900 In summary, all of these results indicate that the LH1 and LH1-RC core complexes were not denatured by binding to the APS-ITO surface. Similar results were obtained using Langmuir–Blodgett (LB) films to lay down LH1 complex membranes as a layer on a glass substrate (Iida et al. 2000). The LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris on the APS-ITO electrode were stable enough to handle at 4°C in the dark condition least 24 h. Figure 2 shows the time course of the photocurrent generated from the LH1-RC core complex, LH1 complex or the RC of R. rubrum assembled onto an APS-ITO when the electrode was illuminated with a pulse of light at 880 nm. It is clear in Fig. 2 that an enhanced photocurrent was observed for the LH1-RC core complex. In contrast no photocurrent was observed for either LH1 complex or the RC. Under our experimental conditions a cathodic photocurrent was observed, implying that one-way electron transfer from pigments in the LH1-RC core complex to methyl viologen was occurring as shown in Fig. 3 (Nagata et al. 2003; Imahori et al. 2000). Fig. 2Time course of the photocurrent of the LH1-RC core complex, LH1 complex or the RC complex of R. rubrum on an APS-ITO electrode when the electrode is illuminated with pulsed light (880 nm) firing continuously for 30 sFig. 3Schematic drawing of LH1-RC core complexes on an APS-ITO electrode generated cathodic photocurrent which shows the electron flow from the complex to methyl viologen (left) according to the cathodic photocurrent as shown in Fig. 2. Energy diagram for cathodic photocurrent generation by the LH1-RC core complex (right) Figure 4 shows excitation spectrum of the photocurrent density (dots) and the NIR absorption spectra (solid line) from R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes assembled onto an APS-ITO, respectively. These photocurrent responses showed a maximum at the wavelength corresponding to the absorption bands of the complex. Interestingly, an enhanced photocurrent was observed especially upon illumination at 880 nm for both R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes. The quantum yield of the photocurrent was 0.05% for both the R. rubrum and Rps. palustris complexes (Imahori et al. 2000). When the LH1 complex of R. rubrum alone, was immobilized on the electrode, the observed photocurrent was mainly generated by light absorbed at 770 nm, i.e. from monomeric BChla (Nagata et al. 2003). Furthermore, when the RC complex of R. rubrum only was immobilized on the electrode, an efficient photocurrent was not observed upon illumination at 880 nm as shown in Fig. 2. Thus, the enhanced photocurrent observed at 880 nm in the assembled LH1-RC core complex can be ascribed to energy transfer from the LH1 to the RC and then electron transfer from the electrode to the RC as shown in Fig. 3 (Ogawa et al. 2004). This data indicates that the LH1-RC core complex was well organized on the ITO and the photocurrents were driven by light that was initially absorbed by the LH components. Fig. 4Photocurrent density (dots) and NIR absorption spectrum (solid line) of LH1-RC core complexes form: (a) R. rubrum and (b) Rps. palustris assembled on an APS-ITO electrode In conclusion, the SAM method is clearly successful in allowing assembly of functional LH1-RC core complexes on the electrode. This has been confirmed by NIR absorption spectroscopy, demonstrating that the photocurrent response, which is derived from electron transfer between the RC and the electrode, is enhanced by illumination at 880 nm.

Int_Arch_Occup_Environ_Health-4-1-2413126

Perceived work-related stress and early atherosclerotic changes in healthy employees

Objective This study was conducted to investigate the relationship between perceived work-related stress and preclinical atherosclerosis. Introduction Stress can no longer be recognized as a problem of an individual only. Due to its versatile presence in every human’s life, often resulting in health complications, stress has become a social problem and the role of work-related stress in this puzzle should not be underestimated. Coronary heart disease (CHD) is on the list of stress-related health problems. Atherosclerosis, the basis of CHD, develops in a long-lasting, multifactorial and complex process. In the etiopathogenesis of atherosclerosis, apart from traditional factors like lipids, carbohydrate metabolic disorders, hypertension and lifestyle (smoking, alcohol use and limited physical exercise), immunological disorders, as a consequence of reciprocal interactions in the psycho-neuro-immunological system, have been recently brought up (George and Shoenfeld 1997; Bednarska-Makaruk and Pasierski 2000; Kiecolt-Glaser and Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; Jędryka-Góral et al. 2002; Jędryka-Góral 2003; Dinan 2005). Early atherosclerosis can even occur in young healthy people (Pasierski 1999). For a long time now, occupational medicine has been interested in the relationship between work-related stress and CHD. Studies undertaken in this field have been based on western as well as eastern european populations (Jonsson et al. 1995; Hammer et al. 1998; Bosma et al. 1998, Kristenson et al. 1998; Kivimaki et al. 2002; Smith et al. 2005; Malinauskiene et al. 2005). Although there is a large body of literature on clinically overt CHD and its relation to work-related stress, there have been very few studies on preclinical CHD in those circumstances (Lynch et al. 1997; Nordstrom et al. 2001; Rosvall et al. 2002; Hintsanen et al. 2005). The current study aimed to fill this gap and to assess the relationship between work-related stress and preclinical atherosclerotic changes in healthy employees. We considered carotid artery intima-media thickening and/or plaque presence as a surrogate of coronary atherosclerosis. To detect these changes “gold-standard” methodology, i.e., B-mode carotid ultrasound examination, was applied. Because of the complex nature of the pathogenesis of CHD, other contributing factors have not been disregarded. Methods Organization of the study First, all participants took part in a psychological study in which the level of stress and coping were evaluated with relevant questionnaires (see below). Then, the participants consecutively presented themselves for a clinical visit in which a medical examination took place and a blood sample was taken. Tests for biochemical parameters (see below) were performed on the same day as the blood sample was taken, according to routine laboratory procedures. Sera for immunological parameters (see below) were collected and stored according to international laboratory standards, then tested in runs (up to 40 sera per day) to minimize day-to-day variations. Participants One hundred healthy managers and 50 office workers (F:M = 50:50) aged 35–65 were studied. All of them were investigated for the presence of individual (age, obesity, hypertension, diabetes, smoking, alcohol use and low physical activity) and family (obesity, hypertension, diabetes, heart attack and stroke) risk factors for atherosclerosis. The studied individuals’ detailed characteristics are presented in Table 1. Table 1Characteristics of studied groupsManagers (n = 100)Office workers (n = 50)Total (n = 150)Number50 (50%)25 (50%)75 (50%) Females Males50 (50%)25 (50%)75 (50%)Age47.2 ± 6.850.16 ± 6.4648.68 ± 6.63 Mean (years)  Minimum343534.5 Maximum656464.5Individual risk factors26 (26%)23 (46%)49 (32%)  Hypertension  Obesity19 (19%)17 (34%)36 (24%) Diabetes1 (1%)1 (2%)2 (1.3%)Alcohol use Every day3 (3%)03 (2%)  2–3×a week23 (23%)2 (4%)25 (16.7%)  Less69 (69%)47 (94%)116 (77.3%)  Never5 (5%)1 (2%)6 (4%) Smoking cigarettes49 (49%)30 (60%)79 (52.7%) Hormone replacement therapy8 (8%)4 (8%)12 (8%)Physical activity  2–3×a week26 (26%)12 (24%)38 (25%)  1×in 7–10 days32 (32%)14 (28%)46 (30.7%)  Less42 (42%)24 (48%)66 (44%)Family risk factors41 (41%)31 (62%)72 (48%) Hypertension Obesity45 (45%)23 (46%)68 (45.3%) Diabetes31 (31%)12 (24%)43 (28.7%) Heart attack36 (36%)21 (42%)57 (38%) Stroke30 (30%)17 (34%)47 (31.3%) Instruments Work-related stress was operationalized as perceived pressure caused by inappropriate working conditions. It was measured by the “Sources of pressure in your job” scale that is a part of the short version of the Occupational Stress Indicator (OSI-2) by Cooper et al. Widerszal-Bazyl’s (2001) Polish adaptation of OSI-2 was used in the study. The scale consists of 40 items on potential sources of pressure at work; respondents are required to rate each source on six-point response scales, in which 1 = it is very definitely not a source (of pressure) to 6 = it is very definitely a source. Those 40 items were divided into eight subscales concerning the following stress factors: workload (e.g. “Having to work very long hours”, “Taking work home”), relationships (e.g. “Inadequate guidance and backup from superiors”, “Feeling isolated”), home–work balance (e.g. “Pursuing a career at the expense of home life”), recognition (e.g. “Underpromotion—working at a level below my level of ability”), organizational climate (e.g. “Characteristics of the organization’s structure and design”, managerial role (e.g. “Managing or supervising the work of other people”), personal responsibility (e.g. “Having to take risks”), hassle at work (e.g. “Attending meetings”). The scale made it possible to assess the global level of work-related stress (the sum of scores for all items) as well as to assess the level of a given kind of work stress, e.g. workload (the sum of scores for items related to workload). The reliability of the Polish version of the total scale was very high: Cronbach’s α = 0.95. It was also satisfactory for most subscales: workload α = 0.95, relationships α = 0.89, home–work balance α = 0.81, recognition α = 0.75, organizational climate α = 0.63, managerial role α = 0.57, personal responsibility α = 0.78, hassle at work α = 0.54. (Widerszal-Bazyl 2001). Coping was measured with the “How you cope with the stress you experience” scale that is also part of OSI-2, too. It consists of 10 items on potential coping strategies; respondents are required to rate the extent to which they actually use them as ways of coping with stress. Answers are given on six-point scales, from 1 = I never use it to 6 = I use it very extensively. Some coping strategies included into the scale dealt with coping through control (e.g. “Effective time management”), and some with coping through social support (e.g. “Talk to understanding friends”). The reliability of the Polish version of the total coping scale was Cronbach’s α = 0.70, and reliability of its subscales: 0.70 (for coping through control) and 0.50 (for coping through social support). Therefore, the total index of coping was used in the analysis, as well as the index of coping through control. Prior to the study, appropriate approval from the Local Ethics Committee was obtained. Participants were provided with detailed written information on the study’s objectives and the methods that were going to be used; subsequently they signed their informed consent. Serum levels of biochemical (total cholesterol, LDL, HDL, TG, glucose) and serological risk factors of atherosclerosis (anticardiolipin, anti-β2 GPI, anti-oxLDL, anti-HSP and anti-hsCRP antibodies) were evaluated. Anticardiolipin antibodies Patients’ sera were tested with ELISA for the presence of anticardiolipin antibodies, according to the modified Harris method (Luft et al. 1990). The results were presented as OD units. Reference ranges of aCL were established by studying sera from 100 healthy donors. The cut-off value was established as OD = x + 4SD estimated for sera of healthy blood donors; it was 0.109 for the IgG class and 0.156 for the IgM class. Anti-β2 glycoprotein I (β2 GPI) antibody levels were measured with a commercial DIASTAM—FBGP 200 from Axis-Shield (Cat. No. FBG200) test. The results were expressed as OD units. Reference ranges of anti-β2 glycoprotein I antibodies were established by studying sera from 152 healthy Caucasian donors (Axis-Shield Diagnostics Ltd. examination). The cut-off value was established as OD = 0.195. Anti-HSP antibody levels (IgG class) were estimated with non-commercial methodology, after necessary modification, according to Tsoulfa et al. (1989), with the use of McLean reagents. The cut-off value was established as x + 4SD estimated for sera of healthy blood donors; it was OD = 0.423. The estimation of IgM and IgA class antibodies was performed in the same manner. Anti-oxLDL IgG antibody levels were measured with ELISA with the use of a commercial test from OLAB (Cat. No. BI-20032). The cut-off value was established as 200 arbitrary units. hsCRP concentration was measured with ELISA with the use of commercial tests from EUCARDIO Laboratory Inc. (Cat. No. ZZ C4011E). The cut-off value was established as 4000 ng/ml. Intima-media thickness (IMT) and atherosclerotic plaque in carotid arteries were searched using a computer analysis of B-mode ultrasound images. Both carotid arteries were measured with a 7.5-MHz linear-array transducer, part of Hewlett Packard Sonos. Plaque was defined as intima-medial thickening larger than 50% in comparison to the surround area (Pignoli et al. 1986) Statistical analysis of data was performed with SPSS v. 11.5. Means comparisons were carried out with a t test for independent groups. In bivariate correlation analysis Pearson’s r coefficients were calculated. The level of significance was set at α < 0.05. Results Assessment of work-related stress The level of work-related stress measured with OSI-2’s Sources of pressure scale was as follows: managers—mean = 154 (σ = 25), for office workers—mean = 145 (σ = 28), for both groups—mean = 151(σ = 25). According to Polish sten norms, the sten scale (“sten” stands for standard ten) consists of ten units. Each unit equals 0.5 of a standard deviation. It is, assumed that stens five and six means average results, stens seven to ten high results, and stens one to four low results (Canfield 1951). Developed for Polish managers (Widerszal-Bazyl 2001), the managers’ mean falls on sten six, whereas the office workers’ mean falls on sten five (Table 2). As a rule, stens five and six are interpreted as average results. Table 2Results of psychological examinationStudied groupsWork-related stressCopingMeanStensMeanStensManagers154664056Office workers145553855Total151654056 Assessment of coping The level of coping measured with OSI-2’s Coping scale was as follows: managers—mean = 40, office workers—mean = 38, both groups—mean = 40. According to Polish sten norms the managers’ mean falls on sten five for men and on sten six for women. The office workers’ mean falls on sten five (for both genders). Thus, it can be said that the coping results were in the average range. Laboratory findings The abnormality most frequently disclosed in biochemical findings were elevated levels of total cholesterol, LDL and triglycerides (in 52, 39 and 22% of the studied individuals, respectively). Decreased levels of HDL and increased levels of glucose were less frequent and were found in 12% of cases each. Of the immunological parameters studied, oxLDL antibodies were most frequently found (in 26% of cases), followed by an elevated level of hsCRP (in 11% of cases). In a few cases antibodies to β2 GPI and HSP-65 (IgG class) were found (in 1% and 3% of studied cases, respectively). None of the studied individuals was positive for aCl. For six sera which were borderline or positive for HSP-65 IgG class, IgM and IgA class antibodies were searched. Only in three cases were the results weakly positive (two in IgM and one in IgA class). Ultrasound examination of carotid arteries Changes in the ultrasound examination of carotid arteries confirming the presence of early atherosclerosis are shown in Table 3. Plaque was found in 43 (29%) cases: in 26 managers (26%) and in 17 office workers (34%); groups did not differ statistically. The distribution of the mean values of the IMT measure was slightly skewed to the right. The mean values of IMT in managers and office workers were 0.0620 ± 0.014 and 0.0610 ± 0.012 mm, respectively; groups did not differ statistically. Table 3Results of ultrasound examination of carotid arteries in studied groupsManagers (n = 100)Office workers (n = 50)Total(n = 150) Number of individuals without changes74 (74%)33 (66%)107 (71%) Number of individuals with plaque26 (26%)17 (34%)43 (29%) Intima-media measurement values 0.0620 ± 0.0140.0610 ± 0.0120.0618 ± 0.013 Correlation between IMT and the presence of plaque in carotid arteries with atherosclerosis risk factors Positive correlation was found between IMT and age (r = 0.42; P < 0.001), diastolic blood pressure (r = 0.20; P < 0.05), LDL (r = 0.28; P = 0.001), anti-HSP antibodies (r = 0.24; P < 0.05), whereas negative correlation was revealed with aCl—IgG (r = –0.20; P < 0.05) and global work-related stress level (r = −0.26; P < 0.01). Among the stressors studied, statistical significance was shown for interpersonal relations (r = −0.23; P < 0.00), work−home balance (r = −0.19; P < 0.05), managerial role (r = −0.24; P < 0.01) and organization climate (r = −0.22; P < 0.01). The presence of plaque correlated positively with age (r = 0.42; P < 0.001), smoking (r = 0.18; P < 0.05), LDL (r = 0.30; P < 0.01) and negatively with global work-related stress level (r = −0.28; P < 0.01). Statistical significance was shown for the following stressors: work load (r = −0.24; P < 0.01), interpersonal relations (r = −0.18; P < 0.05), work−home balance (r = −0.33; P < 0.01) and responsibility (r = −0.25; P < 0.01) (see Table 4). Table 4Correlations between IMT and the presence of plaque in carotid arteries and atherosclerosis risk factors (Pearson’s r)Risk factorIMTPlaqueAge 0.42**0.43**BMI 0.120.04Systolic blood preasure 0.130.06Diastolic blond preasure 0.20*0.10Cigarette smoking 0.130.18*Physical activity−0.020.07LDL0.28**0.30**HDL −0.16−0.05Triglycerides0.04 0.05Glucose 0.080.02hsCRP 0.050.02Anti-HSP 0.160.19aCl–IgG −0.20*−0.10aCl–IgM −0.14−0.15Anti-oxLDL −0.10−0.17Workload −0.15 −0.24**Relationships−0.23** −0.18*Home–work balance −0.19*−0.33**Managerial role −0.24*−0.14Personal responsibility −0.14−0.25**Hassle at work −0.11−0.14Recognition −0.25**−0.15Organizational climate −0.22**−0.15Total index of coping −0.12−0.01Coping through control −0.050.02Global level of stress−0.26**−0.28*** Correlation is significant at 0.01 (2-tailed)** Correlation is significant at 0.05 (2-tailed) Correlation between LDL/smoking and global stress level Pearson’s analysis showed a negative relation between LDL and global stress level (r = −0.17; P < 0.05), and between smoking and global stress level (r = −0.17; P < 0.05). Work-related stress, coping, lifestyle and IMT Pearson’s analysis was performed in two triangles: (a) work-related stress–coping–IMT, and (b) work-related stress–healthy lifestyle–IMT. No statistically significant relations were shown either between work-related stress and coping, or between coping and IMT (P > 0.05), or between work-related stress and healthy lifestyle (no smoking, no excessive use of alcohol, high physical activity), or between healthy lifestyle and IMT (P > 0.05). Discussion A multidisciplinary approach to assessing risk factors for atherosclerosis was considered by us in an earlier study (Jędryka–Góral et al. 2006). On the basis of a deep multilevel analysis we were able to show that in healthy individuals, as in CHD patients, individual and biochemical risk factors were likely to play a leading role in the development of early atherosclerosis. Psychosocial stress should also be considered; however, inflammatory-immunological factors have not been found to be an independent predictor. As inflammatory-immunological factors, antibodies to oxLDL and HSP-65, hsCRP as well as anticardiolipin and anti-β2 GPI antibodies were considered. In the extensive literature of the subject, these parameters are recognized as directly involved in the pathogenesis of atherosclerosis (George and Shoenfeld 1997; Kiecolt-Glaser, Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; Jędryka-Góral et al. 2002; Jędryka-Góral 2003). The present study was undertaken to extend the investigation of psychosocial stress and atherosclerosis. We looked in depth for correlation between preclinical atherosclerosis and work-related stress; correlations with other risk factors of CHD were done in parallel. As expected, we found correlation between IMT and age, diastolic blood pressure, LDL and anti-HSP antibodies, and correlation between plaque and age, smoking and LDL. Surprisingly, our results revealed that early atherosclerotic changes negatively correlated with the level of global job strain and some of its components (interpersonal relations, work–home balance, managerial role, organization climate—for IMT; work load, interpersonal relations, work–home balance, responsibility—for plaque). Ambiguous results have been previously reported in other, although very few, studies where work, psychosocial factors and carotid atherosclerosis were studied with ultrasound. Rosvall et al.’s (2002) hypothesis that work-related stress characterized by high psychological demands and low decision latitude was associated with increased carotid atherosclerosis could not be confirmed either for women or for men. On the other hand, Lynch et al.’s (1997) prospective study showed that men who experienced work-related stress (demanding work and low economic rewards) had significantly greater progression of carotid atherosclerosis than more advantaged men. Hintsanen et al. (2005) provided data that job strain (a joint effect of job demands and job control) was associated with increased IMT in men but not in women. Similarly, Nordstrom et al. (2001) showed that in men with greater work-related stress (workplace demands and intrusion of work concerns into home life) the risk of focal lesions or intima-media thickness in coronary artery increased, whereas in women stress was not related to the prevalence of early atherosclerosis. Hlatky et al. (1995) disclosed that job strain (high psychological demands and low decision latitude) did not correlate with the presence of coronary disease in angiography, either for men or for women. By searching for a relationship between work-related stress and atherosclerosis, we wanted to prove that the correlation found between early atherosclerotic changes and LDL and smoking is, at least partly, caused by a higher stress reaction in the studied individuals. It was assumed that individuals with a level of high stress undertook unhealthy behaviour: smoking or excessive food intake. However, for both LDL and smoking the correlation with stress was negative. One should keep in mind that only 39% of the studied individuals had elevated levels of LDL and 47% reported smoking. Therefore, these results should be interpreted causally. We explored in depth the negative correlation we found between IMT and the presence of plaque, and the level of work-related stress. Being aware that OSI-2 measured perceived stress, another hypothesis was formulated; persons with a high level of work-related stress (conscious stress) undertake preventive activities at the level of coping or healthy lifestyle. Perceived work-related stress as a risk factor for a clinically overt CHD has been investigated in multiple studies. Some prospective studies showed an association of work-related stress with an incidence of CHD (Bosma et al. 1998; Kivimaki et al. 2002; Kivimaki et al. 2005); whereas others did not (De Bacquer et al. 2005; Rosvall et al. 2002; Eaker et al. 2004). The largest INTERHEART study covering 11 119 cases and 13 648 controls from 52 different countries all over the world confirmed this association with regard to work, home, financial and major life stress (Rosengren et al. 2004). There are studies which, indicated that perceived work-related stress had profound impact on internal organs, sensual organs, the locomotor system and skin health problems (Cheng et al. 2001; Lindgren et al. 2002). In the literature of the subject, coping is closely linked to stress. The most common meaning of coping is, an effort to solve problems and to seek reduction of tolerance to stress. The CATS theory (cognitive activation theory of stress) offered a new approach to coping (Ursin, Eriksen 2004; Eriksen et al. 2005). This theory assumed two cognitive reformulations of the learning theory (stimulus–stimulus learning = classical conditioning and response learning = instrumental conditioning). Both reformulations are essential to understanding the relationship between learning, activation and relations between stress and health. The stress response (an alarm in the homeostatic system) results in behaviors that aim to cope with a situation. The level of alarm depends on what outcome of a stimulus is expected and the specific responses available for coping. Response outcome expectancy might be positive (coping), negative (hopelessness) or none (helplessness). Hence, coping defined as acquired positive outcome expectancy has some predictive value for stress and health. According to Weidner and Cain (2003) research on how people cope with stress situations disclosed avoidant mechanisms (denial, distraction, excessive alcohol consumption) in men, whereas more cardioprotective strategies (depression, asking for help)—in females. Stress management goes far beyond coping strategies. Toobert et al.’s (2002) results disclosed success of a complex intervention program to reduce CHD risk, which included improvement of diet, stress management, social support, smoking and physical activity. In our study on preclinical atherosclerosis and work-related stress, we were not able to confirm the hypothesis about the above-mentioned prophylactic activities. An analysis of correlation showed no significant relations between work-related stress and coping, between coping and IMT, between work-related stress and healthy lifestyle, or between healthy lifestyle and IMT. A relatively novel conception was presented by de Lange et al. (2005), who claimed that the relationship between stressful work and psychological well-being may be reciprocal. Their results showed that mental health might influence employees’ perceived work characteristics. It is likely that the employees in our study had been recruited from that part of working society who felt fit to undertake ambitious tasks of managers and office workers and who developed job adaptive mechanisms over time. The most probable interpretation of the negative correlation between perceived work- related stress and preclinical atherosclerosis is that in the case of individuals with a low level of perceived work-related stress, somatization of stress took place, i.e., stress is not perceived at the conscious level but it leads to somatic effects (e.g., IMT). We are aware of some limitations of our study. Firstly, the results regarding work-related stress were based on self-reported data, which per se could always be a matter of some bias. Secondly, the number of employees studied was limited to 150. Voluntary participation of employees and fully unselected method of recruitment can guarantee the objectivity of the results. Thirdly, it was a cross-sectional study. A prospective type of research on risk assessment is preferable nowadays. However, our study did not aim to assess risk of CHD but to find correlation between work-related stress and early atherosclerosis, so we believe the measure taken for this purpose was appropriate. We are more than convinced that to achieve full understanding of negative correlation between work-related stress and early atherosclerosis requires further interdisciplinary studies and we would be happy to continue exploration into this intriguing field.

Purinergic_Signal-4-2-2377318

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases. Introduction Monociliated ductal epithelial cells are receiving much attention due to their remodeling in polycystic kidney diseases, role in other cystic diseases of the kidney and other tissues, and in sensory physiology [1–12]. Cilia and flagella from lower organisms have important roles in sensory physiology in response to flow, touch, chemical and osmotic stimuli [1–5, 15, 16]. MDCK cells and other cell and tissue models of the renal collecting duct have been essential in characterizing cilium-derived cell calcium (Ca2+) signals [17–30]. It appears likely that this cilium-affected Ca2+ signal is derived from Ca2+ entry from extracellular stores and Ca2+ release from intracellular stores; the latter, perhaps, an ER cisternae near the basal body immediately beneath the monocilium [17–30]. Previously thought to be a vestigial organelle [1–5] or morphological marker for the cortical collecting duct (CCD) principal cell (PC) [31], the apical central monocilium is likely a sensor for ductal epithelia [5]. Recently, our laboratory has shown that epithelial sodium channel (ENaC)-mediated sodium (Na+) absorption is upregulated in mutant cilium-deficient orpk CCD PC monolayers versus genetically rescued cilium-competent controls [32]. ENaC activity is present under open-circuit voltage and short-circuit current measurements in rescued cell monolayers with a well-formed cilium, but the electrical signals were three- to sixfold less than mutant monolayers [32]. One of our working hypotheses is that an inhibitory signal is lost (when the monocilium is not well formed) that is responsible for tonic attenuation of ENaC function [32]. Indeed, flow-induced Ca2+ signals have been shown recently by Praetorius and Leipziger not to be due to the cilium of MDCK cells per se but to autocrine ATP signaling that is stimulated by pressure pulses and responsible for Ca2+ spark and wave signal formation [33]. Immature MDCK cells without discernible cilia and mature MDCK cells with cilia responded similarly [33]. Alternatively, Satlin and coworkers have compelling data that monocilia do confer flow-based Ca2+ signals in isolated perfused CCDs from control mice versus mutant Tg737orpk mice [20]. In multiple different preparations from tissue to renal epithelia to heterologous cells, flow-induced Ca2+ signals have been observed [17–30]. However, the concept that an autacoid might mediate these cilium-specific effects has not been addressed. Herein, we show that the fully formed monocilium does confer a more robust Ca2+ signal in rescued cell monolayers versus mutant cell monolayers that are deficient in well-formed cilia. This finding agrees with the majority of the literature examining this phenotype [17–30]. However and more importantly, we show that stimulated or regulated ATP release is impaired when the monocilium is malformed. Ionomycin-, hypotonicity-, and mechanically induced ATP release are more robust in cilium-competent monolayers versus cilium-deficient monolayers. Varying the order of stimuli also revealed that cell Ca2+ influences the mechanically induced secreted ATP pool and that hypotonic cell swelling and other mechanical stimuli trigger the release of the same ATP pool. Finally, the flow-induced Ca2+ signal in this cell model requires autocrine ATP release and signaling, as it was blocked by the ATPase/ADPase scavenger, apyrase. These data may reconcile the different conclusions within the Praetorius and Leipziger study [33] and the study of Satlin and coworkers [20]. To our knowledge, this is the first report linking the sensory apical central and nonmotile cilium to ATP secretion. Materials and methods Cell culture The collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk) mouse model of autosomal recessive polycystic kidney disease (ARPKD) and the genetically rescued cells with the wild-type orpkTg737 gene were a generous gift from Dr. Bradley Yoder (University of Alabama at Birmingham). The “mutant 1” cell clone (94D pcDNA 3.1 cells), the “rescued 1” cell clone (94D BAP737-1 cells), and the “rescued 2” cell clone (94D 737-2 cells) were handled identically and were grown under G418 selection on 6.5-mm diameter (Corning Costar) or 12-mm diameter filter supports (Millicell CM) and bathed in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 HAM with L-glutamine and 15 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) (Sigma) [32]. This medium was supplemented with 5% fetal bovine serum (FBS) and contained (per 1,000 ml) dexamethasone (100 μl/l of 2 mg/ml stock), interferon-γ (25 μl of 800 U/μl stock), T3 (10 μl/l of 13 mg/ml stock), G418 (500 μl of 400 μg/ul stock), penicillin-streptomycin (10 ml of 100X stock), and ITS (10 ml of 0.5 mg/ml insulin transferrin selenium concentrated stocks). The cells continued to be bathed on both sides of the filter until a monolayer tight to fluid formed. Measurement of the resistance of the filters was used as an indicator of the formation of a mature monolayer. Once the monolayers were formed for at least 12 h, the cells were then acceptable for the experimental assay. In addition to these original clones developed in the Yoder laboratory, we also generated two new clonal lines from the original 94D mixed mutant CCD cell cultures, namely “mutant 2” and “mutant 3.” The only difference between these two subsequent clones and the original “mutant 1” clone is the fact that the “mutant 1” clone is stably transfected with an “empty” pcDNA 3.1 vector that confers G418 resistance. “Mutant 2,” “mutant 3,” and the mixed mutant cultures are grown in a medium without G418. We also studied a “rescued B2” clone that was stably transfected and rescued with the wild-type Tg737 gene but we derived from a different mutant CCD originally, 94E. The generation of these additional clones was described in a previous publication [32]. Bioluminescence detection of ATP released from epithelial monolayers In every assay performed, there was an initial basal ATP measurement followed by subsequent ATP measurements in response to different stimuli added in different sequences. ATP release assays were performed mainly on well-polarized cell monolayers grown in clear polyester 6.5-mm diameter filter supports. Mutant versus rescued cell monolayers were studied side by side in each ATP release protocol. The use of luciferase-luciferin to indirectly measure the ATP concentration has been published previously in detail [34]. Every drug prior to experimentation was tested to ensure that they did not interfere with the luciferase enzyme activity [34–37]. There was also no change in cell viability with any of these maneuvers as has also been reported previously [34–37]. Each experiment began with a basal ATP measurement or the addition of Opti-MEM I medium (GIBCO-BRL) with 1 mg/ml luciferase-luciferin reagent (Sigma) being added to the apical or basolateral side of the filter’s monolayer. Basolateral ATP release was negligible; therefore, all data reported are apical or luminal ATP secretion. Basal levels of ATP release were measured for 6 min in 15-s, nonintegrated photon collection periods in a TD-20/20 Luminometer (Turner Designs) [34–37]. In different orders of addition, we used the following stimuli: (1) ionomycin (2 μM) (to increase the intracellular calcium concentration), (2) distilled water with 1 mg/ml luciferase-luciferin (to dilute the osmotic strength of the Opti-MEM I medium), and (3) intentionally harsh pipetting in the center of the apical surface of the cell monolayer (to induce a mechanical stimulus on the apical membrane). Normally, addition of drug or distilled water (or the same volume of medium as a volume addition control) is performed or dispensed very slowly along the wall of the plastic support that holds the permeable filter so as not to disrupt the tight, confluent monolayer. Therefore, by quickly pipetting the medium up and down onto the monolayers, a mechanical stimulation is induced. Luminescence was measured for 6 min after each stimulus. All experiments ended with the addition of hexokinase to eliminate any ATP left in the medium. All assays were performed at room temperature. Fura-2/AM imaging of cytosolic free calcium in a cell monolayer-based perfusion system Fura-2/AM imaging was performed as described previously [38–40]. However, to remain faithful to the study of polarized cell monolayers, we designed a cell monolayer-based perfusion chamber system where 12-mm diameter Snapwell Transwell filter supports are inserted into a homemade perfusion chamber designed to accommodate this special filter support. Apical and basolateral perfusion are then performed separately through independent injection ports and separate ejection ports are subjected to vacuum. Response to changes in apical flow from 1 ml/min (a “slow” flow) to 5 ml/min (“high” flow) were performed to induce a flow-induced Ca2+ transient akin to that observed by many other laboratories. We assessed mutant and rescued cell monolayers in parallel during all protocols. We also assessed the flow-induced Ca2+ transient signal in the absence of apical extracellular Ca2+ and in the presence of gadolinium chloride and apyrase. It was not possible to calibrate the Fura-2 fluorescence signal to real free cytosolic Ca2+ values because of ionomycin contamination of the flow chamber. As such, fluorescence ratio values are shown. We give estimates of what the free Ca2+ may be based on previous calibrations of the same cells grown as nonpolarized cells. Materials All reagents and drugs were purchased from Sigma. Larger 12-mm diameter filter supports were obtained from Millipore. Smaller 6.5-mm diameter filter supports were obtained from Corning Costar. It should be noted that we are using less luciferase:luciferin detection reagent than in past studies [34–37]. Results Basal ATP release or secretion is not different between mutant and rescued CCD PC monolayers Basolateral ATP release was near background and was negligible in both mutant and rescued cell monolayers. This result does not mean that ATP is not released across the basolateral cell surface. ATP may, in fact, be released but “trapped” between the cell monolayer and the filter support and/or the detection reagent may not gain sufficient access to this “trapped” space. Therefore, only apical-directed ATP release was studied exclusively in the detection experiments below. Regardless of the filter support used to form cell monolayers and the amount of detection reagent included in the real-time experiments, basal or unstimulated or constitutive ATP release was low and not different between the panels of mutant and rescued cell monolayers studied. There was agreement between all mutant clones and all genetically rescued clones in all protocols below. As such, the data are pooled for mutant cell monolayers and rescued cell monolayers. Basal ATP release data are summarized in the bar graph in Fig. 1 as bioluminescence in arbitrary light units (ALU). In total, the amounts of ATP released were below 50 nM. A standard curve was run with each series of ATP release experiments. Thus, basal ATP secretion is not different across the apical membrane of mutant and rescued cell monolayers and the amount of ATP released is barely sufficient to engage the most sensitive P2Y or P2X cell surface receptors. Fig. 1Basal ATP release across the apical cell surface is not different between mutant cilium-deficient and genetically rescued cilium-competent orpk kidney cell monolayers. In three separate preparations of mutant versus rescued cell monolayers on different filter supports and exposed to different concentrations of luciferase:luciferin detection reagent, ATP release was not different between the cilium-deficient and cilium-competent cell monolayers (n = 12 for left set of bars; n = 6 for center and right datasets). In this study, we had a breakthrough with regard to what filter support to use for our ATP release experiments studying cell monolayers on filter supports. We found that the 6.5-mm diameter filter support (the same as used in open-circuit and Ussing chamber experiments studying ENaC upregulation [32]) was ideal for these real-time ATP release assays into the apical medium because the apical cell surface was as close as it could be and fully exposed to the luminescence detector in the base of the chamber. The left set of bars is data derived from this filter support and procedure. All other data in subsequent figures derive from this preparation. A diagram shows how the filter support was handled and placed into the luminometer chamber. After washing of serum-containing medium away from the monolayer, a small volume of Opti-MEM 1 medium (15 μl) is added in a drop to the lid of a 35-mm dish to hydrate the preparation and to promote adherence to the lid of the dish; 200 μl of Opti-MEM 1 medium containing 1 mg/ml lyophilized luciferase:luciferin reagent are placed within the cup in contact with the apical surface of the cell monolayer. Detection is then begun for bioluminescence due to secreted ATP from the cell monolayer into the apical space in continuous 15-s collection periods delayed only by the time to push the “start” button on the luminometer and to record the luminescence to an Excel spreadsheet. The standard curve with which approximations were made as to the secreted ATP concentrations was performed with each dataset, was compiled, and is as follows: 10−10 M ATP, 0.052 ± 0.005 ALU...10−9 M ATP, 0.476 ± 0.042 ALU...10−8 M ATP, 4.95 ± 0.51 ALU...10−7 M ATP, 51.2 ± 4.8 ALU...10−6 M ATP, 455 ± 23 ALU...10−5 M ATP, 3956 ± 311 ALU...10−4 M ATP and 10−3 M ATP, >9999 ALU (exceeded limit of detection of luminometer). It is important to note that these are estimates of secreted ATP because we do not add inhibitors of ecto-ATPases and it is likely that our ATP release signal is diminished significantly by ATP degradation before detection. The left set of bars show basal ATP secretion from the 6.5-mm diameter clear polyester filter support without feet that is immediately above the light sensor and bathed in 15 μl of serosal medium. The middle set of bars show ATP secretion from a 12-mm polyester clear filter support with feet and bathed in 200 μl of serosal medium. The third set of bars at right show ATP secretion from a polycarbonate filter support with feet and bathed in 200 μl of serosal medium Ionomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers We first assessed the effect of an increase in intracellular free calcium (Ca2+) on ATP release. This first study was done in part because of the emerging role of the primary central monocilium in mediating Ca2+ transients, sparks, and waves in cell monolayers derived from extracellular and intracellular stores [17–30]. This cilium-derived Ca2+ signal is triggered by touch or flow across this organelle and is mediated, at least in part, by the polycystin proteins, PC-1 and PC-2 [17–30]. Polycystin-2 is a part of the TRPP subfamily of TRP genes and is a distant relative of the transient receptor potential or TRPC gene family of Ca2+ entry channels [17–30]. Ionomycin (2 μM) applied to the apical surface of well-polarized cell monolayers triggered a slow, monophasic rise in ATP release over 3–5 min (see typical time courses below in Figs. 6 and 8). This response was a similar phenotype to that observed in previous studies by our laboratory in human vascular endothelial cell monolayers [37]. In mutant monolayers deficient in monocilia, an increase in cell Ca2+ increased extracellular ATP from below 50 nM to 0.4 μM. Figure 2 shows data as bioluminescence in arbitrary light units (ALU) with corresponding estimated ATP for each step of the protocol based upon parallel standard curves with known amounts of ATP. In sharp contrast, rescued monolayers responded to ionomycin stimulation with a rise in secreted ATP from approximately 50 nM to 2 μM, three- to fivefold higher sustained amounts on average than in mutant monolayers. The data in Fig. 2 also group the data from the panels of mutant and rescued clones which were in agreement. Taken together, these data show that cell Ca2+-stimulated ATP release is more robust when a well-formed central monocilium is present. In fact, the “releasable” pool of ATP that is sensitive to Ca2+ appears intact in cilium-competent monolayers and deficient in cilium-deficient monolayers. Fig. 2Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2 μM, added in a 2-μl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n = 6 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P < 0.05 by paired Student’s t-test; the cross reflects P < 0.05 significance by analysis of variance (ANOVA) and Tukey’s ad hoc test. The statistical analysis and results are given similarly in all other figures Hypotonicity-induced ATP release is more robust in rescued versus mutant cell monolayers Hypotonic challenge and resultant cell swelling and regulatory volume decrease (RVD), a shared mechanism of cell volume regulation by all cells [41–44], is a robust stimulus for ATP release [41–46]. It is a shared sentiment by most that ATP release precedes and triggers, at least in part, RVD mechanisms such as opening of parallel K+ and Cl− channels to mediate rapid KCl efflux and to lower cell volume back to basal levels [41–46]. As such, we used hypotonic challenge to assess ATP release in mutant cilium-deficient cell monolayers versus rescued cilium-competent cell monolayers. Figure 3 summarizes these data. These stimuli followed the ionomycin treatment. We show other typical real-time courses below when hypotonic challenge was performed in the absence of ionomycin (see Fig. 7). In all monolayers, hypotonic challenge of the apical surface (50% dilution of the medium osmolality with distilled water containing 1 mg/ml detection reagent) triggered an increase in ATP release that had transient and sustained components. In mutant monolayers, hypotonic challenge increased ATP detected in the medium from approximately 0.4 to 3 μM during the peak of stimulation and to a slightly lesser value at the end of the hypotonicity challenge phase. Addition of the same volume of isotonic medium (a “volume addition” control) increased ATP secretion slightly from 0.4 to 0.5 μM (data not shown). Uniformly, rescued cilium-competent cell monolayers responded more vigorously to the hypotonic stimulus. This stimulus again followed the ionomycin treatment. ATP release increased from approximately 2 to 5–10 μM at the peak of the response and to a lesser sustained level at the end of the hypotonic challenge phase. In volume addition control experiments, ATP release was triggered that was only transient and increased from 2 to 4 μM on average (data not shown). Taken together, these data show that rescued cell monolayers respond more vigorously to hypotonic challenge than mutant cell monolayers with regard to ATP secretion, a signal required for cell volume regulation. Moreover, increases in cell Ca2+ prime swelling-induced ATP release and allow a larger ATP secretion signal to visualize. Fig. 3Hypotonic cell swelling-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. Hypotonic challenge was induced by adding an equal volume of distilled water (200 μl with 1 mg/ml L:L reagent) down the wall of the apical chamber into the apical medium. The fold difference in three other preparations is graphed at right and was relatively constant again (n = 6–9). Statistics are shown like in Fig. 2 Mechanically-induced ATP release is more robust in rescued versus mutant cell monolayers Harsh and fast pipetting of an 50 μl volume of isotonic medium directly down on the center of the cilium-competent monolayers (the “mechanical stimulus”) in three repetitions triggered an immediate ATP secretion that had transient and sustained phases like the swelling-induced ATP release from 2 to 3–4 μM in rescued monolayers. This response was greatly attenuated in the mutant monolayers where ATP release was augmented only slightly from 0.4 to 0.5 μM. These experiments were performed after ionomycin challenge and the summary data are presented in Fig. 4b. In the absence of ionomycin pretreatment (Fig. 4a), mutant monolayers failed to respond to this stimulus altogether, whereas rescued monolayers responded significantly with an ATP release transient. Again, it is noted that increases in cell Ca2+ also appear to prime this mechanically sensitive ATP release mechanism. Taken together, these data suggest that the presence of a well-formed apical central monocilium is a prerequisite for robust ATP secretion to provide a meaningful autocrine ATP signal on the luminal surface of cortical collecting duct principal cells. Fig. 4Mechanically induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. In a where no ionomycin pretreatment was performed, four mutant and four rescued cell monolayers were compared. In b where mechanical stimulation was performed after ionomycin challenge, six mutant and six rescued cell monolayers were compared. Without ionomycin, there was no response from the mutant cell monolayers. With ionomycin, there was a response from mutant monolayers that was barely significant. In contrast, rescued cell monolayers responded robustly to the addition of 50 μl of Opti-MEM 1 medium with detection reagent pipetted vigorously onto the center of the monolayer. In two additional repetitions, 50 μl of apical medium was drawn up and ejected onto the center of the monolayer before luminescence readings continued. Figure 5 shows the nature of the response versus the hypotonic challenge and the ionomycin stimulation. Statistics are shown like in Fig. 2 Figures 5, 6, 7, and 8 provide typical real-time courses showing the luminescence data (in arbitrary light units) for mutant and rescued monolayers illustrating the summary data presented in Figs. 1, 2, 3, and 4. With the exception of basal ATP release, all phases of ATP secretion are markedly more robust in cilium-competent models versus cilium-deficient models across the apical cell surface. These data also revealed that there may be shared and different pools of “releasable” ATP within the cell. These data also began to reveal that increases in cell Ca2+ influence ATP secretion markedly. Fig. 5Typical real-time course of mechanically induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. In this and three subsequent figures, the green (closed or solid) balls reflect data from cilium-competent cell monolayers and the white (open) balls reflect data from cilium-deficient cell monolayers. Lines do not connect the points because there are delays of a few seconds between readings. One can observe that there are clear ATP release transients induced in rescued cell monolayers by the mechanical insults. The signals observed from mutant cell monolayers are greatly attenuatedFig. 6Typical real-time course of ionomycin and mechanically induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. Ionomycin induces a slow monophasic sustained increase in ATP secretion that is three- to fivefold more robust in rescued cell monolayers. In the presence of ionomycin stimulation, the mechanical insults produce even more pronounced ATP secretion that is more sustained in nature when cell Ca2+ is elevated. Nevertheless, the signals observed from mutant cell monolayers remain greatly attenuatedFig. 7Typical real-time course of hypotonic challenge-induced ATP release across the apical cell surface in mutant versus rescued cell monolayers. Hypotonicity induced a marked increase in ATP release that was fivefold more robust in rescued cell monolayers versus mutant cell monolayers. Of interest, however, mechanical stimulation via repeated pipetting near the center of the cell monolayers had no effect on ATP release after hypotonic cell swelling. These data suggest that both stimuli are indeed mechanical in nature and that the same pool of “releasable” ATP is being affected by each stimulus. The slight drop in signal with the pipetting stimulus is the addition of 50 μl of isotonic medium to the 400 μl of 50% diluted medium and the resultant change in osmotic strength at the apical cell surface. The hypotonicity-induced signal observed from mutant cell monolayers is again greatly attenuatedFig. 8Typical real-time course of all three stimuli given in an order where each stimulation can be observed in mutant versus rescued cell monolayers. Although the large scale diminished the mechanically induced signal and data, an ATP release transient is observed in rescued cell monolayers and not in mutant cell monolayers. The monophasic ionomycin response is then observed, again more robust in rescued cell monolayers. Then, hypotonic challenge is performed in the presence of ionomycin. Here, the largest values of secreted ATP are observed that approach ∼15–20 μM in the apical medium bathing cilium-competent cell monolayers. In these plots, the effects of the broad specificity anion transport inhibitor, DIDS, are shown. DIDS diminishes ATP release in both types of monolayers and in other epithelial cell monolayer preparations to different degrees. The inhibition is partial and DIDS does not affect the detection reagent. Hexokinase is added at the end of each experiment to eliminate secreted ATP and to diminish the luminescence signal to low levels An original physiological role for the apical central monocilium was that of a “mechanosensor.” Figure 5 validates this idea using ATP secretion as the biological endpoint. Repeated pipetting of isotonic medium containing the same amount of detection reagent revealed repeated stimulation of ATP release transients in cilium-competent monolayers. The responses in mutant cilium-deficient cell monolayers were greatly attenuated. This comparison was performed on the same day with the same preparation of luciferase:luciferin detection reagent. Figure 6 shows a similar experiment but with ionomycin pretreatment. Here, the classic ionomycin-induced ATP secretion phenotype is shown. It is a slow and monophasic increase in secreted ATP that is robust; however, the response of rescued monolayers is fourfold greater than mutant monolayers. Subsequent mechanical stimulation via repeated pipetting showed a more vigorous response in the cilium-competent monolayers versus the cilium-deficient monolayers. Figure 7 provides multiple compelling illustrations. First, the response to hypotonic challenge is shown and it is much more robust in rescued versus mutant cell monolayers. Interestingly, however, the repeated harsh pipetting stimulus is without effect in both rescued and mutant cells after the hypotonic challenge. In fact, the luminescence decreases modestly. These data suggest that these two mechanical stimuli may mobilize the same “releasable” pool of ATP. Figure 8 shows the relative effects of the three stimuli used in this study on rescued cilium-competent cell monolayers and on mutant cilium-deficient cell monolayers. Although greatly diminished because of the Y-axis scale, the mechanical stimulus triggered a fourfold greater ATP secretion transient in the rescued cell monolayers versus the mutant cell monolayers. Ionomycin also produced a fourfold greater sustained ATP release in rescued versus mutant monolayers. Hypotonic challenge also triggered a more robust response in the mutant cell monolayers. A large degree of the stimulated ATP release was inhibited by the broad specificity anion transport inhibitor, DIDS, suggesting that an ATP transport process may be mediating the release of ATP from intracellular pools. In the presence of DIDS inhibition, release is inhibited while degradation of released ATP proceeds unabated, leading to a sharp decline in the signal. In contrast, ionomycin-stimulated ATP release is attenuated by performing the luminescence experiment at 4°C (data not shown), suggesting a vesicular mechanism of release. Hexokinase is added at the end of every protocol to scavenge the ATP and abolish the luminescence signal. Taken together, these data suggest that cell Ca2+ is critically important for priming the ATP release machinery. These data also suggest that the “releasable” pools of ATP are present in cilium-competent cell monolayers beneath the apical cell surface, while they may be impaired or missing in cilium-deficient cell monolayers. These ideas will be revisited and discussed below. Flow-induced calcium signals are attenuated in cilium-deficient mutant monolayers versus cilium-competent monolayers An original physiological role for the apical central monocilium was that of a mediator of flow- or touch-induced Ca2+ signals in MDCK cells by Praetorius and Spring [17–19]. Subsequent studies in heterologous cells, renal collecting duct cell models, and renal collecting ducts showed that an intact cilium is required for the flow-induced Ca2+ signal [20–30]. To be faithful to a polarized epithelial cell monolayer system used routinely for this study and other studies [32, 34–40], we devised a flow chamber where the apical versus basolateral sides of the monolayer could be perfused independently and at different flow rates. The 12-mm diameter Snapwell transwell filter can then be inserted into this chamber for selective perfusion and fluorescence imaging. With a constant low rate of perfusion of the basolateral surface of the monolayers, low versus high rates of perfusion were performed on the apical surface in rescued versus mutant monolayers. Figure 9 shows the response to modifications in saline Na and Mg and to high flow (5 ml/min) versus low flow (1 ml/min). When Na and Mg are lowered to mimic the amount left in collecting duct tubular fluid after the bulk of each cation is reabsorbed along the nephron (10 and 0.1 mM, respectively), an increase in cell Ca2+ is observed that is due to Ca2+ entry from extracellular stores. The modifications in extracellular saline have augmented Ca2+ entry and cytosolic free Ca2+ in previous experience [39, 40, 47]. Ca2+ competes with Na+ through all Ca2+ entry channels that are, by definition, Ca2+-permeable nonselective cation channels [39, 40, 47]. Mg2+ blocks or impedes the pore of many different Ca2+ entry channels [39, 40, 47]. The response was greater in cilium-competent cell monolayers versus cilium-deficient cell monolayers to this Na+ and Mg2+ removal. When flow is increased, a flow-induced calcium signal is observed in cilium-competent monolayers that is reminiscent of such signals seen in isolated perfused renal collecting duct [20] and in other systems [21–29]. In cilium-deficient monolayers, this signal is present but impaired markedly. Taken together, these data show that cilium-competent cell monolayers are more able to respond to flow stimuli than cilium-deficient cell monolayers. Fig. 9Typical real-time courses of flow-induced Ca2+ transient signals across the apical cell surface in mutant versus rescued cell monolayers. Fluorescence ratio values were shown for Fura-2. It was very difficult to perform ionomycin-based calibrations to derive real cytosolic free Ca2+ values in this monolayer preparation that could be trusted. Moreover, the ionomycin used contaminated the perfusion lines for subsequent experiments. The flow-induced responses are observed in the center of each trace and the flow effect was reversed by returning to low flow. The use of the low Na+, low Mg2+ Ringer allowed us to view this transient more easily; it was more robust and long-lived under these ionic conditions where the Ca2+ entry channel inhibitors, Na+ and Mg2+, were minimized Summary data are also shown in Fig. 10 that shows the responses under different saline conditions alone and responses to high flow in different conditions. Several nuances emerge from these data that agree with previous work in which our laboratory and our collaborators have been involved [20, 38]. It is important to note that our main intent was not to reproduce previously published data but to link cilium-derived Ca2+ signals to ATP secretion studied above. First, basal cell Ca2+ is indeed significantly higher in mutant cells versus rescued cells in this monolayer imaging preparation (Fig. 10a). These results agree with those performed by another collaborator in a study by Siroky et al. [38]. Second and curiously, however, the basal cell Ca2+ phenotype switches in experiments performed in the absence of extracellular Ca2+ (Fig. 10b). In nominally Ca2+-free solutions, mutant cell Ca2+ is driven quite low, while rescued cell Ca2+ is maintained if not slightly augmented. These data with regard to resting cell Ca2+ indeed suggest that Ca2+ entry under basal or unstimulated conditions is actually heightened in mutant cell monolayers versus rescued cell monolayers due to a deregulated Ca2+ entry pathway [38]. A valid explanation for the reversal in phenotype under Ca2+-free conditions may result from the loss of ER cisternae immediately beneath apical central monocilia, causing mutant cell Ca2+ to decrease markedly under these conditions. In contrast, cilium-competent monolayers may have robust intracellular Ca2+ stores tied immediately beneath the monocilia to maintain basal cell Ca2+ in the absence of Ca2+ entry. Fig. 10Summary data under different experimental conditions for flow-induced Ca2+ transient signals across the apical cell surface in mutant versus rescued cell monolayers: requirement for autocrine secreted nucleotides. a Summary data from the experiments shown in Fig. 9. b Responses to flow in the absence of extracellular calcium. A flow-induced Ca2+ signal is still present in the cilium-competent cell monolayers. It is as robust in the peak values of Ca2+ increase (the tip of the spike is used for the summary data); however, the shoulder is absent from the transient in the absence of extracellular Ca2+. In the cilium-deficient cell monolayers, there is a slow increase in Ca2+ that is not significant and appears unrelated to flow. The asterisks in a and b refer to a P < 0.05 significant change from the previous stage of the experiment by paired Student’s t-test. The crosses show significant difference by ANOVA between mutant and rescued cell monolayer Ca2+ levels. c The asterisks refer to a P < 0.05 significant change from the previous stage of the experiment by paired Student’s t-test. Only data from rescued cell monolayers are shown where the Ca2+ signal is much more robust. Gadolinium and apyrase blocked the flow-induced Ca2+ transient signal, suggesting that Ca2+ entry channels and an autocrine extracellular ATP signal are required to observe the Ca2+ signal In the presence of extracellular Ca2+, modification of the Ringer to mimic collecting duct tubular fluid and to disinhibit Ca2+ entry channels augmented cell Ca2+ significantly only in cilium-competent cell monolayers (Fig. 10a). A change in the flow rate from 1 ml/min to 5 ml/min again only augmented cell Ca2+ in the form of a brief Ca2+ transient with a “spike” and a “shoulder” (Fig. 10a). A typical phenotype is shown in Fig. 9. Mutant cell monolayers failed to respond to flow with a significant change in cell Ca2+ (Fig. 9 shows the typical degree of response to flow). In the absence of extracellular Ca2+, the same magnitude and type of Ca2+ entry “spike” was observed in rescued cilium-competent cell monolayers (Fig. 10b). However, the sustained “shoulder” of this response was missing in the absence of extracellular Ca2+. Any response from the mutant cell monolayers was insignificant and sluggish. In this light, the rescued cell monolayers showed a complete reversal when flow was slowed; however, the mutant cell monolayers did not. The reason for this lack of reversal is unclear, but we speculate that the cells can no longer “sense” flow and, therefore, display deregulation with regard to cell Ca2+ in this manner as well. In Fig. 10c, responses in the rescued cell monolayers were assessed in the presence of apical gadolinium chloride, a broad spectrum inhibitor of mechanosensitive ion channels and Ca2+ entry channels [30, 38]. Infusion of gadolinium chloride during the course of the apical perfusion in the presence of extracellular calcium and in low and high flow also quieted the flow-induced calcium signal (Fig. 10c). The rise in cell Ca2+ in response to the reduction of Na+ and Mg2+ was not blocked, suggesting that this Ca2+ entry mechanism is insensitive to gadolinium. These data suggest that a mechanosensitive Ca2+ entry channel is sensitive to gadolinium and plays a role in flow-induced Ca2+ entry. These data agree with the literature [17–30]. Many studies, however, have also implicated autocrine and paracrine purinergic signaling as a major player in mechanically induced Ca2+ sparks and waves in monolayer and tissue preparations [33, 48–54]. Indeed, in immature and mature MDCK cells lacking or bearing monocilia, a pressure pulse-induced Ca2+ signal was observed in each preparation and the signal was abolished by antagonists to purinergic signaling [33]. To test whether cilium-conferred autocrine ATP release and signaling was important for the cilium-dependent Ca2+ signal, we performed a similar apical flow protocol in the presence of the ATPase/ADPase, apyrase (Fig. 10c). Apyrase blocked the flow-induced Ca2+ signal. Apyrase also attenuated the rise in cell Ca2+ induced by lowering apical Na+ and Mg2+. Taken together, these data suggest that an autocrine ATP signal, released by the monolayer itself, contributes directly to the flow-induced and cilium-derived Ca2+ signal. With the more robust stimulated ATP release phenotypes and the flow-induced Ca2+ signals in the rescued monolayers versus mutant monolayers, we speculate that each may require the apical central monocilium as an integrating sensory organelle. Discussion Figure 11 provides a cartoon that illustrates our current working hypothesis concerning the requirement of an autocrine ATP signal for monocilium-specific signaling. Our data suggest that the secreted ATP near the apical surface of principal cells of the renal collecting duct with well-formed monocilia is robust enough to engage cell surface P2X receptor channels or P2Y G protein-coupled receptors. In cilium-deficient mutant cell monolayers, the amount of ATP secreted under basal conditions or in response to stimuli may be insufficient. P2Y and P2X receptors may be present on or near the monocilium to transduce this autocrine ATP signal. Recently, in a P2X4-deficient mouse [55], flow- or shear stress-induced Ca2+ signals and resultant nitric oxide production were attenuated markedly in vascular endothelial cells versus wild-type controls. These transgenic mice also have blood pressure regulation problems [55]. Ando and colleagues had implicated ATP release and P2X4 previously in endothelial cell Ca2+ entry signaling [56–58]. Our laboratory has emerging data that P2X4 is expressed on the basal half or just beneath motile respiratory cilia in human respiratory tissues (L. Liang, Z. Bebok, and EM Schwiebert, unpublished observations). Silberberg and colleagues have long postulated that a “P2X cilia” is critical in mediating extracellular ATP regulation of ciliary beat in freshly isolated epithelial cells from airway and esophagus [59–61]. Our laboratory has found that P2X4, P2X5, and P2X6 have shared and robust expression in epithelia from the respiratory tract, gastrointestinal (GI) tract and renal system [35, 36], and from vascular endothelia from multiple blood vessels [37]. Work in progress is examining the expression of P2X and P2Y purinergic receptors on cilia. Fig. 11Working model: the monocilium is required for a mature ATP secretion apparatus at the apical cell surface and both the monocilium and an autocrine ATP signal are required for flow-induced Ca2+ signals As introduced, it is becoming abundantly clear that the monocilium in particular and cilia and flagella in general are sensory organelles [1–5]. In tissues where high flow is present (large airways, proximal tubule, arteries, arterioles, etc.), the monocilium or cilia may be flow sensors for the cell on which they are present. In tissues where low flow is present (bile duct, pancreatic duct, renal collecting duct, small airways, capillaries, venules, veins, etc.), the monocilium may be a chemosensor or an osmosensor. Our data suggest that it may both influence ATP secretion as well as be a chemosensor for the secreted ATP. Arguably, secreted nucleotides and nucleosides are most potent in a local microenvironment within a cellular or tissue microenvironment. Burnstock and colleagues have described this concept elegantly in past review where they described purinergic signaling as being potent and essential in the lumen of tubules and ducts of kidney and gut [62, 63]. A semiclosed system such as the lumen of a renal tubule or duct is ideal in this regard. Along these lines, the effect of changes in flow has been studied extensively and elegantly. Touch, flow, shear stress, and cell swelling are potent regulators of ion transport [64–67]. Ca2+ entry as well as Na+, K+, and Cl− transport are governed by mechanical stimuli [17–30, 64–67]. Mechanical stimuli are also well-known to trigger ATP release in many cell and tissue systems [48–54, 68]. The propagation of Ca2+ sparks and waves triggered by mechanical stimuli are thought to be mediated by at least two cellular mechanisms: (1) Ca2+-permeable gap junctions that link the cells together and (2) paracrine extracellular purinergic signaling that allows cells to communicate in a local environment [68]. In fact, our data show that an increase in cell Ca2+ primes the “releasable” pools of ATP that are mobilized by hypotonic cell swelling and other mechanical stimuli. However, one can still observe both pipetting- and hypotonicity-induced ATP release without ionomycin pretreatment that is more robust in cilium-competent versus cilium-deficient cell monolayers. Moreover, we also found that we could not observe a mechanically induced ATP release signal after hypotonic cell swelling. This finding suggests that these two “mechanical” stimuli (albeit different) affect the same pool of releasable ATP. Unfortunately, we still need better tools to define each ATP release mechanism and pool. However, our work with low temperature inhibition of vesicle traffic and inhibition of anion transport properties with the broad specificity inhibitor, DIDS, suggests that both ATP transport mechanisms and exocytosis of ATP-filled vesicles contribute to secreted ATP phenotypes (EM Schwiebert et al., unpublished observations). Recently, we found that ENaC-mediated Na+ absorption is upregulated markedly in cilium-deficient CCD PC monolayers derived from the Tg737orpk mouse [32]. Of many hypotheses put forward to explain this pathophysiological phenotype, one prominent postulate was that the malformed cilium caused the loss of key inhibitory signals that are normally cilium-derived that limit ENaC activity. Indeed, in several different cellular systems, there is agreement that purinergic signaling inhibits ENaC function [69–74]. Modulation of purinergic receptor-driven cell Ca2+ signaling may be a future putative therapeutic target (along with ENaC itself) to control hypertension that occurs in the majority of human ARPKD patients, especially the children that escape respiratory insufficiency soon after birth [6]. Finally, it is our hope that this work connects the seemingly disparate conclusions of Satlin and coworkers [20] and those of Leipziger, Praetorius, and colleagues [33]. Taken together, our data suggest that a well-formed monocilium central to the apical membrane of a collecting duct principal cell is essential for a mature ATP secretion apparatus. One could conceive of this apparatus as a well-formed pool of ATP poised to be secreted in response to different stimuli or the appropriate presence of ATP release machinery (ATP-filled vesicles and/or ATP transport mechanisms). Our studies also suggest that the cilium-derived Ca2+ transient, induced by flow in our study or by other modes in other studies, requires an underlying autocrine ATP signal that is likely transduced by P2X and P2Y ATP receptors on or near the monocilium. Without a well-formed cilium at the apical surface, autocrine purinergic signaling, cilium-derived signaling, and modulation of downstream effectors become disrupted.

Antonie_Van_Leeuwenhoek-3-1-2140094

New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati

Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology, extrolite production and partial β-tubulin, calmodulin, and actin gene sequences. The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences. Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology, short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface. Aspergillus turcosus can be distinguished by velvety, gray turquoise colonies and short, loosely columnar conidial heads. The four new taxa also have unique extrolite profiles, which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis. A. turcosus produced kotanins. N.denticulata sp. nov., N. assulata sp. nov., N. galapagensis sp. nov., and A. turcosus sp. nov. are described and illustrated. Introduction Aspergillus section Fumigati and its teleomorph Neosartorya include many species which are important because they can be pathogenic or allergenic to man (Brakhage and Langfelder 2002), cause food spoilage and produce mycotoxins (Cole and Cox 1981). Certain species also produce interesting bioactive extrolites that are potential drug candidates (Turner and Aldridge 1983). Section Fumigati currently includes now 26 Neosartorya species and nine anamorph species (Pitt et al. 2000; Samson 2000; Horie et al. 2003; Hong et al. 2005, 2006). During a survey of Aspergillus and Penicillium species from Korea, many isolates belonging to section Fumigati were isolated. These isolates were compared to known taxa and those present at the CBS and IBT culture collections which were atypical or unidentified, using a polyphasic approach (Frisvad and Samson 2004). We have examined the macro- and micromorphology, extrolite profiles and β-tubulin, calmodulin, and actin gene sequences of the isolates, and based on the above data, here we describe four new species in Aspergillus section Fumigati. Materials and methods Morphological examinations For macro-morphological observations, isolates were cultivated on Czapek yeast autolysate (CYA), malt extract agar (MEA), CZ agar (CZA), and oatmeal agar (OA) (Samson et al. 2004). The isolates were inoculated at three points on each plate of each medium and incubated at 25°C in the dark for 7 days, and additionally at 37°C on CYA. For microscopic observations, mounts were made in lactic acid from MEA colonies; a drop of alcohol was added to remove air bubbles and excess conidia. Scanning Electron Microscopy (SEM) was performed using a Hitachi S570 electron microscope. For SEM, mature cleistothecia were transferred to aluminum stubs with double sided adhesive tape. A small drop of 10 mM ACES buffer containing 0.05% Tween-80 was added and the cleistothecia crushed. The suspension was air dried and coated with platinum. DNA analyses Isolates used for sequence analyses are listed in Table 1. Genomic DNA was extracted according to the procedure described by Lee and Taylor (1990). The 5′ portion of the β-tubulin gene (benA) was amplified using primers bt2a and bt2b (Glass and Donaldson 1995). Amplifications of the partial calmodulin and actin genes were set up as described previously (Hong et al. 2005). The amplified DNA fragments were purified by QIAquick PCR purification kits (Qiagene, Hilden, Germany). DNA sequences were determined using BigDye Terminator v3.1 Cycle Sequencing kit (ABI 0401041, Foster, CA, USA) and ABI 3100 DNA sequencer. Both strands of each fragment were sequenced using the same primers. Table 1Aspergillus section Fumigati isolates used in this studySpeciesIsolate numberGenBank accession numberSourceβ-tubulinCalmodulinActinA. brevipesCBS 118.53TAF057311Soil, AustraliaA. duricaulisCBS 481.65TAF057313Soil, Buenos Aires, ArgentinaA. fumigatiaffinisIBT12703TDQ094885Soil, USAA. fumigatusCBS 133.61AY685150Chicken lung, USAA. fumisynnematusIFM 42277TAB248076Soil, VenezuelaA. lentulusCBS 117887AY738513Man, USAA. novofumigatusIBT 16806TDQ094886Soil, EcuadorA. unilateralisCBS 126.56TAF057316AY689366DQ094847Rhizosphere, AustraliaA. viridinutansCBS 127.56TAF134779Rabbit dung, AustraliaA. turcosus sp. nov.KACC 42090 = IBT 27920DQ534142DQ534147DQ534178Air conditioner, Inchen, South KoreaKACC 42091T = IBT 27921DQ534143DQ534148DQ534179Air conditioner, Seoul, South KoreaKACC 41955 = CBS 117265 = IBT 3016DQ534144DQ534149DQ534180Car air conditioner, Seoul, South KoreaN. assulata sp. nov.KACC 41691TDQ114123DQ114131DQ534189Tomato soil, Buyeo, North KoreaN. aurataCBS 466.65TAF057318AY870685DQ534112Jungle soil, BruneiN. aureolaCBS 105.55TAF057319Soil, Tafo, GhanaN. coreanaKACC 41659TAY870758Tomato soil, Buyeo, North KoreaN. denticulata sp.nov.CBS 652.73T = KACC 41183DQ114125DQ114133DQ534181Soil under Elaeis guineensis, SurinameCBS 290.74 = KACC 41175DQ114126DQ114134DQ534182Acer pseudoplatanus, NetherlandsN. fennelliaeCBS 598.74TDQ114127DQ114135DQ534121Eye ball of Oryctolagus cuniculus, USAN. fischeriCBS 544.65TAF057322Canned applesN. galapagensis sp. nov. CBS 117522T = IBT 16756 = KACC 41935DQ534145DQ534151DQ534190Soil, EcuadorCBS 117521 = IBT 16763 = KACC 41936DQ534146DQ534152DQ534191Soil, EcuadorN. glabraCBS 111.55TAY870734AY870693DQ534183Rubber scrab from old tire, Iowa, USAN. hiratsukaeCBS 294.93TAF057324AY870699DQ534184Aloe juice, Tokyo, JapanN. laciniosaKACC 41657TAY870756Tomato soil, Buyeo, North KoreaN. multiplicataCBS 646.95TDQ114129DQ114137DQ534185Soil, Mouli, TaiwanN. nishimuraeIFM 54133AB201360Forest soil, KenyaN. nishimuraeCBS 116047DQ534075DQ534150DQ534186Cardboard, NetherlandsN. pseudofischeriCBS 208.92TAY870742AY870702DQ534187Human vertebrate, USAN. quadricinctaCBS 135.52TAF057326Cardboard, York, UKN. spathulataCBS 408.89TAF057320Soil under Alocasia macrorrhiza, TaiwanN. spinosaCBS 483.65TAF057329Soil, NicaraguaN. strameniaCBS 498.65TAY870766AY870726DQ534188Soil from maple-ash-elm forest, Wisconsin, USAN. tatenoiCBS 407.93TDQ114130DQ114139Soil of sugarcane, Timbauba, BrazilN. udagawaeCBS 114217TAF132226Soil, BrazilCBS Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, IBT Institute for Biotechnology, Lyngby, Technical University of Denmark, IFM Institute for Food Microbiology (at present, the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University), Chiba, Japan, KACC Korean Agricultural Culture Collection, Suwon, South Korea, T type strain DNA sequences were edited with the DNASTAR computer package. Sequence alignments were performed by using CLUSTAL W (Thompson et al. 1994) and improved manually. The neighbor-joining (NJ) method was used for the phylogenetic analysis. Evolutionary distances between the sequences were calculated by Kimura’s formula (Kimura 1980) using the program DNADIST of the PHYLIP program package (Felsenstein 1995). Phylogenetic trees were prepared by the NJ method (Saitou and Nei 1987) using the program NEIGHBOR of the PHYLIP package. Bootstrap values were calculated from 1,000 replications of the bootstrap procedure using programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the package (Felsenstein 1995). For parsimony analysis, the PAUP* Version 4.0 software was used (Swofford 2002). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1,000 bootstrap replications (Hillis and Bull 1993). An Aspergillusclavatus isolate was used as outgroup in these experiments. The β-tubulin, calmodulin and actin gene sequences, determined in this study, have been deposited in GenBank and the accession numbers are listed in Table 1. Analysis for extrolites The isolates were grown at 25°C for 1 week on CYA and YES in the dark and extracted according to Smedsgaard (1997). Extrolites were analyzed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad and Thrane (1987), as modified by Smedsgaard (1997). Results and discussion Molecular studies For the phylogenetic analysis of β-tubulin sequences, most accepted species in section Fumigati except Neosartorya indohii, N. sublevispora, and N. tsurutae were included to determine the phylogenetic positions of the putatively new species (Fig. 1). For the calmodulin and actin datasets only sequences of closely related species were included (Suppl. Figs 1, 2). 468 nucleotides of the β-tubulin gene were analyzed. Among the 225 polymorphic sites, 126 were found to be phylogenetically informative. The topology of the NJ tree is the same as one of the 28 most parsimonious trees inferred by the PAUP program (length: 441 steps, consistency index: 0.7143, and retention index: 0.6993). The calmodulin data set included 538 characters, with 87 parsimony informative characters (tree length: 272, consistency index: 0.8051, and retention index: 0.7706). The actin data set included 394 characters, with 66 parsimony informative characters (tree length: 200, consistency index: 0.7900, and retention index: 0.7801). Fig. 1Taxonomic position of some new species in Aspergillus section Fumigati inferred from Neighbor-Joining analysis of partial β-tubulin gene sequences. The numbers above/below the nodes represent bootstrap values of >60% (out of 1,000 bootstrap replications). The number of nucleotide changes is represented by branch length The cladograms based on β-tubulin, calmodulin, and actin gene sequences revealed that isolates CBS 652.73 and CBS 290.74, which had identical sequences at each loci, were related to the heterothallic species N. fennelliae, but the similarity between this species and the two isolates was quite low (96.5% in the β-tubulin gene partition and 97.8–98.4% in the calmodulin gene partition). These two strains had unique ascospore ornamentations, with denticulate convex surfaces and a prominent equatorial furrow (Fig. 2) and could be easily microscopically differentiated from any other Neosartorya species (Samson et al. 1990; Horie et al. 2003). Both isolates produced gliotoxin, while CBS 652.73 also produced viriditoxin. Gliotoxin is also produced by A. fumigatus and N. pseudofischeri, but there were several differences in the profile of extrolites in N. pseudofischeri and these isolates (data not shown). A. fumigatus and N. pseudofischeri are among the most divergent species in the group (Geiser et al. 1998; Horie et al. 2003; Hong et al. 2005, 2006; Varga et al. 2000), yet they share the production of this mycotoxin. Here we describe CBS 652.73 and CBS 290.74 as N. denticulata sp. nov. Fig. 2Neosartoryadenticulata sp. nov. (A) colonies on OA after 28 days of incubation, (B) aspergillum, (C) ascoma, (D) ascospores under a light microscope, (E) and (F) ascospores by SEM Isolate KACC 41691 did not show a clear relationship to any species in the β-tubulin phylogeny, but was closest to CBS 116047 based on calmodulin and actin sequence data (Suppl. Figs 1, 2). CBS 116047 is best accommodated as N. nishimurae. However, isolate KACC 41691 is homothallic, whereas N. nishimurae is heterothallic. This isolate has similar morphological characteristics to N. pseudofischeri, but our genotypic analyses indicate that they are phylogenetically distinct. KACC 41691 produces ascospores with several large flaps and two distinct equatorial crests (Fig. 3). These characteristics are similar to those of N. pseudofischeri in which the ascospores also have triangular flaps on a convex surface (Peterson 1992), but in KACC 41691 the flaps are more pronounced. Furthermore, KACC 41691 differs from N. pseudofischeri by its growth rates on MEA and CZA (after 7 days at 25°C colonies were 49–58 and 24–42 mm, respectively, for KACC 41691, and 90, and 60–70 mm for N. pseudofischeri). The ascomatal initials in N. pseudofischeri are characterized by many coiled hyphae whereas the initial in KACC 41691 is simpler. Ascospores are larger (5.1–6.0 μm in KACC 41691, while 4.5–5 μm in N. pseudofischeri). We could not detect any known extrolites in KACC 41691 but it produced partially characterized apolar compounds in common with several Neosartorya species. Here we propose the name N.assulata sp. nov. for isolate KACC 41691. Fig. 3Neosartoryaassulata sp. nov. (A) colonies on CYA after 7 days of incubation, (B) aspergillum, (C) ascoma, (D) ascospores under a light microscope, (E) and (F) ascospores by SEM Isolates CBS 117522 and CBS 117521, both isolated from soil from the Galapagos Islands, were phylogenetically distinct from all other species within section Fumigati (Fig. 1, Suppl. Figs 1, 2). The colony texture of these two isolates is funiculose with conidiophores which arise from bundles of aerial hyphae. These conidiophore structures resemble as described by Horie et al. (1993) for A. fumisynnematus, but in this species the conidophores are longer, up to 210 μm, with larger vesicles (16–20(25) μm in diam.). Ascomata were produced in 2 weeks-old colonies and ascospores were released after about 3 weeks. Ascospores resemble those of N. glabra and N. laciniosa, and have two conspicuous equatorial crests with a microtuberculate convex surface (Fig. 4). Isolates CBS 117522 and 117521 produced gregatins and several other extrolites not yet found in other Neosartorya or Aspergillus species, and appeared to be chemically unique. Gregatins have previously been found in A. panamensis in section Sparsi (Anke et al. 1980, 1988; Peterson 2000). Fig. 4Neosartoryagalapagensis sp. nov. (A) colonies on CYA after 7 days of incubation, (B) ascoma, (C) and (E) stipes and conidial heads arisen from hyphal bundle, (D) conidia under a light microscope, (F) ascospores under a light microscope, (G) ascospores by SEM Here we describe isolates CBS 117522 and CBS 117521 as N.galapagensis sp. nov. Isolates KACC 42090, KACC 42091, and KACC 41955 also showed a distinct taxonomic position within section Fumigati in the three gene phylogenies. The closest taxon to these three isolates in the β-tubulin and calmodulin gene phylogenies was the heterothallic species N.nishimurae (Fig. 1, Suppl. Fig. 1). However, the similarity of β-tubulin sequences between the two species was only 96.9% which is close to that observed between N. fischeri and N. spinosa (data not shown). Although A. fumigatus, A. lentulus, A. viridinutans, A. fumigatiaffinis, and A. novofumigatus share similar morphological characteristics with these three isolates, these species showed comparatively low β-tubulin gene sequence similarities of 89.8, 91.6, 93.6, 92.3, and 92.7%, respectively. Isolates KACC 42090, 42091, and 41955 did not produce any teleomorph structure after incubation for 28 days on CYA, MEA, CZA, and OA at 25°C. During mating experiments, all of the pairings with N.fennelliae, N.nishimurae, N. spathulata, N. udagawae, and between conidial strains failed to yield cleistothecia. Some conidiophore characters suggest a similarity to A. fumigatus and A. lentulus, but these isolates are different from A. fumigatus by the vesicles which are fertile over the upper two-thirds and has short, loosely columnar conidial heads. These isolates are different from A. lentulus by their velvety and gray turquoise colonies. These isolates grow at 10 and 50°C on MEA and CZA. On the contrary, A. fumigatus does not grow at 10°C, while A. lentulus, A. fumigatiaffinis, and A. novofumigatus are unable to grow at 50°C (Hong et al. 2005). Isolates KACC 42091, 42090, and 41955 were also chemically unique. The extrolites produced by the isolates described here are typical for Aspergillus section Fumigati (data not shown). The three isolates also produced kotanins, previously found in species in less obviously related groups of Aspergillus, such as A. niger from section Nigri and A. clavatus from section Clavati (Turner and Aldridge 1983). Here we describe isolates KACC 42090, KACC 42091, and KACC 41955 as A. turcosus sp. nov. The list of 26 known species of Neosartorya and nine anamorph species from the section Fumigati (Horie et al. 2003; Hong et al. 2005, 2006) is still expanding. With the species proposed here, there are now 29 Neosartorya species and 10 Aspergillus species in this group, 39 species in total. Unfortunately, some of the recently described species are not available for the scientific community, such as N. indohii, N. nishimurae, N. otanii, N. sublevispora, N. takakii, and N. tsurutae. Taxonomy Neosartoryadenticulata Samson, S.B. Hong and Frisvad. sp. nov. (Fig. 2). Species homothallica; ascomata superficialia, luteo-alba vel dilute lutea, globosa vel subglobosa, 140–230 μm in diam., hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 12–14 μm diam. Ascosporae lenticulares, 4–5 μm diam, denticulatae. Conidiophora ex hyphis aeriis oriunda, 3–4.5 μm lata; conidiorum capitula columnaria sed brevia, uniseriata, vesiculae spathulatae vel subclavatae, 7–12 μm diam; phialides 7.5–9 × 2–3 μm, didimidium superius vesiculae occupantes. Conidia subglobosa vel late ellipsoidea, levia, 2–3 μm diam. Holotype of N.denticulata, here designated as CBS 652.73T (dried culture), isolated from soil in Suriname. Homothallic, cleistothecia superficial, yellowish white to pale yellow, globose to subglobose, 140–230 μm in diam., surrounded by a loose covering of hyaline to yellowish white hyphae. Asci 8-spored, globose to subglobose 12–14 μm, evanescent at maturity. Ascospores, 4–5 μm, denticulate with a prominent equatorial furrow. Mycelium composed of hyaline, branched, septate, smooth-walled hyphae. Conidial heads short, columnar. Conidiophores arising from aerial hyphae, uniseriate, stipes 3–4.5 μm wide; vesicles spathulate to subclavate, 7–12 μm in diam.; phialides 7.5–9 × 2–3 μm, covering the upper half of vesicle. Conidia subglobose to broadly elliptical, smooth, 2–3 μm. Colonies on MEA growing rapidly, 35–40 mm in 7 days at 25°C, white. Conidial heads produced only in colony margins. Colonies on CYA, 22–24 mm in 7 days at 25°C, 35–38 mm in 7 days at 37°C, white, loosely overgrown by aerial hyphae in center, weakly sulcate in marginal area. Conidial heads few in number. Reverse yellowish white to pale yellow (12A23) (Kornerup and Wanscher 1978). Extrolites: The two isolates produced the mycotoxin gliotoxin. CBS 652.73 was a particularly strong producer, and also produced the mycotoxin viriditoxin. Furthermore, the two isolates produced some unique, yet unelucidated secondary metabolites. Additional isolates: CBS 290.74 = KACC41175, from Acer pseudoplatanus, The Netherlands. Distinguishing features: Denticulate ascospores with a prominent equatorial furrow and the production of gliotoxin. Neosartorya assulata S.B. Hong, Frisvad and Samson. sp. nov. (Fig. 3). Species homothallica; ascomata superficialia, alba vel luteo-alba, globosa vel subglobosa, 150–250 μm diam, hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 14–16 μm diam. Ascosporae lenticulares, 5–6 μm diam, duabus cristis distantibus praeditae, valvis nonnullis distinctis longis intumescentii ornamentatae. Conidiophora hyalina, 3–7.5 μm lata; conidiorum capitula columnaira, brevia, uniseriata; vesicula subclavata, 10–18 μm diam. Phialides 7–9 × 2–3 μm, didimidium superius vesiculae occupantes. Conidia subglobosa vel late ellipsoidea, levia, 2–3 μm diam. Holotype of N. assulata, here designated as KACC 41691T (dried culture), isolated from soil, tomato field, Buyeo, North Korea. Homothallic, cleistothecia superficial, white to yellowish white, globose to subglobose, 150–250 μm in diam. Asci 8-spored, globose to subglobose 14–16 μm, evanescent at maturity. Ascospores lenticular, spore body 5.0–6.0 μm, with two well-separated equatorial crests and convex surface decorated with several large, round flaps. Mycelium composed of hyaline, branched, septate, smooth-walled hyphae. Conidial heads short, columnar. Conidiophores arising from aerial hyphae and substrate, 3–7.5 μm wide; vesicles subclavate, 10–18 μm in diam., uniseriate, phialides 7–9 μm, covering the upper half of vesicles. Conidia, subglobose to broadly elliptical, ovoid, smooth, 2–3 μm. Colonies on MEA, 49–58 mm in 7 days at 25°C, white, radially weak sulcate. Conidial heads aerial, numerous. Colonies white on CYA, 37–41 mm at 25°C, 64–68 mm at 37°C in 7 days. Radially and roundly sulcate, with some clear exudates. Conidial heads aerial, abundant. Reverse yellowish white (1A2) to pale yellow (1A3) (Kornerup and Wanscher 1978). Extrolite profile: This species is characterized by relatively weak production of secondary metabolites. It does produce some indole alkaloids and some apolar metabolites. Distinguishing features: Large, round flaps on convex surface of ascospores with two distinct equatorial crests. Neosartorya galapagensis Frisvad, S.B Hong and Samson. sp. nov. (Fig. 4). Species homothallica; ascomata luteo-alba, globosa vel subglobosa, 90–200 μm diam,. hyphis hyalinis vel luteolis laxe textis circumdata. Asci 8-spori, globosi vel subglobosi, 12–15 μm diam; ascosporae late lenticulates, ca. 5 μm diam, duabus cristis distantibus 1–2 μm latis praeditae, valvis exigue tuberculatis. Conidiophora singula vel funiculosa, levia, 2–4 μm lata; conidiorum capitula columnaria, brevia, uniseriata; vesiculae subclavatae, 4–11 μm diam. Phialides lageniformes, 5–7 × 2–3 μm, dimidium superius vesiculae occupantes. Conidia globosa vel subglobosa, levia vel exigue asperulata, 2.3–3.0 μm diam. Holotype of N.galapagensis, here designated as CBS 117522T (IBT 16756 = KACC 41935) (dried culture), isolated from soil, Galapagos Islands, Ecuador, D. Mahooney. Colonies on MEA 28–35 mm in diam. after 7 days at 25°C and more than 70 mm after 7 days at 37°C, funiculose in texture, yellowish white (3A12). Conidiophores sparse, cleistothecial initials produced after ca. 10 days of incubation. Colonies on CYA 27–40 mm in diam. after 7 days at 25°C and 61–65 mm after 7 days at 37°C, strongly funiculose in texture and white with a golden yellow (5B78) reverse without diffusible pigment. Conidial heads columnar. Conidiophores arising from bundles of aerial hyphae or the basal mycelium, smooth walled, up to 100 μm long, 2–4 μm in width; vesicles 4–11 μm (sub)clavate with 5–7 μm flask-shaped phialides which are fertile on the upper half to two-thirds of the surface. Conidia 2.3–3.0 μm, globose to subglobose and the surface usually smooth. Cleistothecia yellowish white (4A2), globose to subglobose, 90–200 μm in diam., surrounded by a loose covering of aerial hyphae. Peridium consisting of angular cells, 3–8 μm in diam.; asci 8-spored, globose to subglobose, 12–15 μm in diam.; ascospores broadly lenticular, spore body ca. 5 μm in diam. with two distinct equatorial crests 1–2 μm wide, convex surface of ascospores microtuberculate. Extrolite profile: All isolates examined in the species produce several gregatins and several partially characterized secondary metabolites. This species is chemically very distinct and different from the other species in section Fumigati or Neosartorya species. The gregatins have also been found in A. panamensis (Anke et al. 1980, 1988). The latter species was placed in Aspergillus section Usti by Raper and Fennell (1965). Additional isolates: CBS 117521 = IBT 16763 = KACC 41936, ex soil, Galapagos Islands, Ecuador. Distinguishing features: The Aspergillus anamorph arising in bundles of aerial hyphae and the ascospores with two wide conspicuous equatorial crests and microtuberculate convex surface. Aspergillus turcosus S.B. Hong, Frisvad and Samson. sp. nov. (Fig. 5). Fig. 5Aspergillus turcosus sp. nov. (A) and (B) colonies on MEA (A) and CYA (B) after 7 days at 25°C, (C) conidia under a light microscope, (D) conidial heads Coloniae in agaro maltoso ad 42–51 mm diam post-seven dies 25°C, 70 mm diam 37°C. Coloniae velutinae, griseo-glaucae vel griseo-virides, plerumque planae; reversum luteo-aurantium vel griseo-aurantium. Conidiophora levia, 4–7 μm lata. Conidiorum capitula columnaria, brevia, uniseriata; vesiculae globosae vel subclavatae, 15–25 μm diam. Phialides lageniformes, 6–8 × 2–3 μm, duo tertia superiora vesiculae occupantes. Conidia subglobosa vel ovoidea, levia, 2.5–3.5 μm diam. Holotype of A. turcosus, here designated as KACC 42091T (=IBT 27921) (dried culture) isolated from home air conditioner, Seoul, South Korea. Colonies on MEA 42–51 mm in diam. after 7 days at 25°C and more than 70 mm after 7 days at 37°C. Colony texture velvety, gray-turquoise to gray-green (24-25B3-5) and usually plane. In reverse, colonies are yellowish orange (4B6) to grayish orange (5B6). Colonies on CYA attain a diam. of 38–41 mm after 7 days at 25°C and more than 70 mm after 7 days at 37°C. Colony texture is velvety. Colony texture and color similar to that on MEA. In reverse, colonies are deep yellow (4A78). Conidial heads loose and short columnar. Conidiophores smooth-walled, up to 80 μm long and 4–7 μm wide. Vesicles are 15–25 μm in diam., flask-shaped to globose, bearing 6–8 μm flask-shaped phialides over two-thirds of the surface. Conidia are subglobose, ovoid and smooth, 2.5–3.5 μm in diam. Extrolite profile: Kotanins and several unique compounds but not yet elucidated secondary metabolites. Additional isolates: KACC 42090 = IBT 27920, KACC 41955 = IBT 3016. Distinguishing features: Velvety colony, gray-turquoise (green) color on CYA, phialides over two-third of the vesicle and growth at 10 and 50°C are distinctive characteristics of the species. Electronic supplementary material Below is the link to the electronic supplementary material. (DOC 19 kb)

Ann_Surg_Oncol-4-1-2277449

Isolated Hypoxic Hepatic Perfusion with Retrograde Outflow in Patients with Irresectable Liver Metastases; A New Simplified Technique in Isolated Hepatic Perfusion

Background Isolated hepatic perfusion with high-dose chemotherapy is a treatment option for patients with irresectable metastases confined to the liver. Prolonged local control and impact on survival have been claimed. Major drawbacks are magnitude and costs of the procedure. We developed an isolated hypoxic hepatic perfusion (IHHP) with retrograde outflow without the need for a heart-lung machine. Approximately 50–60% of colorectal cancer patients will develop liver metastases during follow-up. In nearly a quarter of these patients the liver is the only site of disease.1 If hepatic metastases of colorectal cancer are resectable, 5-year survival rates are reported between 25% and 45% depending on several prognostic factors.2 Patients with irresectable hepatic metastases have a 0–2% 5-year survival rate and a median survival of 10 months without treatment.3 Therefore, aggressive, selective treatment of the liver seems justified since control of hepatic metastases translates into improved overall survival. Despite the increasing response rates with systemic chemotherapeutic agents such as oxaliplatin and irinotecan in combination with 5-FU and the promising results with the addition of angiogenesis inhibitors,4 overall survival remains poor.5,6 In order to improve responses and survival, locoregional chemotherapeutic regiments have been developed. These therapies include hepatic arterial embolization, intratumoral injections of ethanol, acetic acid, biological agents, stereotactic or intra-arterial radiotherapy, intralesional laser therapy, cryotherapy, radiofrequency ablation, and regional infusion or perfusion of chemotherapeutic drugs. The best approach for regional infusion of chemotherapeutics in the liver is unknown. Hepatic artery infusion (HAI), hepatic artery ligation with hepatic artery and portal vein infusion, or portal vein infusion have all been attempted. Of these modalities, HAI is the most widely applied form. A number of studies have been conducted comparing systemic chemotherapy with HAI, and a modest but significant improvement of survival was demonstrated by HAI in a meta-analysis.7–9 In a leakage-free perfusion setting, isolated hepatic perfusion (IHP) shields the systemic compartment to drug exposure, and in combination with a washout procedure it protects against systemic toxicity. Clinical studies using melphalan with or without tumor necrosis factor alpha (TNF) have shown promising results.10–17 However, IHP is a major, complex, expensive, and time-consuming operation. These features in combination with hepatic and systemic toxicity are major drawbacks toward wide clinical application. Our aim was to develop an IHP that is easy to perform, with limited morbidity or mortality, not time consuming, and with minimal costs of the operation without affecting the antitumor efficacy. We started an isolated hypoxic hepatic perfusion (IHHP) technique with percutaneously placed balloon catheters and retrograde outflow without a heart-lung machine and extracorporeal venovenous bypass in pigs.18 In a recent study, we demonstrated the feasibility of this technique (retrograde outflow of the liver) in a clinical setting.17 A major drawback of the technique was 38% systemic leakage rate. On the basis of these results, we modified the technique by applying an “open” surgical procedure; balloon catheters are replaced by clamps and all tributaries to the vena cava such as the adrenal, lumbar, and diaphragmatic veins are dissected and ligated. Here we present the results of the first 24 patients with irresectable liver metastases who underwent an IHHP with melphalan. The major difference between this new technique and the classic IHP is that there is no veno-veno bypass and heart-lung machine involved and the drainage of the liver is retrograde through the portal vein. MATERIALS AND METHODS Patient Selection Criteria Inclusion criteria were as described previously.17 In all patients a radical resection of the primary tumor was performed prior to entering the study protocol except for one patient with hepatic metastasis of unknown primary. The liver metastases were considered irresectable, on the basis of multiple lesions in multiple segments of the liver and/or location near vascular structures. Tumor involvement had to be less than 50% of the total liver volume to prevent massive necrosis in case of a response. Absence of extra hepatic tumor growth was evaluated by computed tomography (CT) scan of thorax and abdomen. All patients had a Karnofsky performance score of at least 90, liver enzymes (ALAT, ASAT and AF) not higher than five times the normal values, and bilirubin not higher than two times the normal values. Exclusion criteria included: age younger than 18 or older than 70, portal hypertension, significant central nervous system disease, significant cardiovascular, pulmonary or renal disease, uncontrolled infections, presence of organ grafts, and chemotherapy or radiation therapy within 4 weeks prior to the IHHP. Routinely, an angiography was performed to exclude aberrant hepatic arteries or to visualize other anatomic anomalies. The study protocol was approved by the Medical Ethical Committee of the Erasmus University Medical Centre, and written informed consent was obtained from all patients. Perfusion Circuit Perfusion sets (PfM, GmbH, Cologne, Germany) consisted of a tubing set with a volume of 220 mL, containing a bubble trap. All IHHPs were performed with inflow via the hepatic artery and outflow via portal vein. In the perfusion circuit, flow was maintained by a roller pump and pressure was measured via a sideline. Drugs A dosage of 1 mg/kg melphalan (L-Pam, Alkeran, Wellcome Ltd. London, UK) was used in 20 of the 24 patients and infused through a sideline into the perfusion circuit. Two times 1.5 mg/kg melphalan and two times 2.0 mg/kg was used. In the first eight patients, a bolus infusion was used. In the last 16 patients, a 10-minute pump infusion was used. The total perfusion (10-minute infusion included) with melphalan was conducted for 20 to 30 minutes. Surgical Procedure of the Retrograde Flow IHHP A right subcostal incision was performed, and the liver was mobilized from its retroperitoneal and diaphragmatic attachments. A prophylactic cholecystectomy was not performed routinely. Tributaries to the vena cava such as the adrenal, lumbar, and diaphragmatic veins were dissected and ligated. The vena cava was isolated and clamped above and below the liver, respectively, to prevent venous leakage. The portal vein, proper hepatic artery, and gastroduodenal artery were dissected, and the hepatic artery cannulated via the gastroduodenal artery with an 8 F catheter for inflow of the perfusate. In two patients, an aberrant left hepatic artery, coming from the left gastric artery was cannulated together with the proper hepatic artery for inflow. The portal vein was cannulated with a 14 F catheter for outflow. Patients subsequently received 2 mg/kg heparin. The hepatic artery catheter and the portal vein catheter were connected to the perfusion circuit primed with 220 mL Haemaccel (Behring Pharma, Amsterdam, The Netherlands). After clamping of the caval vein superior and inferior of the liver, clamping of the aorta just beneath the diaphragm, clamping the portal vein just above the pancreas, clamping the proper hepatic artery just proximal of the gastroduodenal branch, and clamping of the common bile duct with its surrounding tissue, the retrograde isolated perfusion was performed. The retrograde perfusion setup is depicted in Fig. 1. FIG. 1.The retrograde perfusion setup. The perfusate was circulated by a constant flow. Stable perfusion was monitored by pressure measurement and the perfusate level in the bubble trap. Hereafter, melphalan was infused into the circuit and the perfusion was conducted for 20 to 30 minutes. Thereafter, a washout procedure was performed by 1 L of Haemaccel collecting the venous effluent. Total liver ischemia time never exceeded 60 minutes. The isolation was terminated by relief of the clamps on the caval vein followed by the relief of the aortic clamp, controlling systemic blood pressure. The catheter in the hepatic artery could be removed and the gastroduodenal artery could be ligated followed by decanulation and closing the venotomy of the portal vein. In case of an aberrant left hepatic artery, this artery was ligated after removal of the catheter. Leakage Monitoring During IHHP, potential leakage of drugs was monitored using a radioactive tracer. A small calibration dose of human serum albumin radiolabeled with 131I was injected into the systemic circulation prior to the perfusion and 10-fold higher dose of the same isotope was injected into the isolated hepatic perfusion circuit. Continuous monitoring was performed with a precordial scintillation probe. Systemic leakage is expressed quantitatively as a percentage (100% leakage representing a homogeneous distribution of the isotope in the body).14 Blood Sampling Before, during, and after the perfusion blood samples were taken and collected to study pharmacokinetics of melphalan and hematological, renal, hepatic, and gastrointestinal toxic side effects. Toxicity is graded according to the standardized WHO common toxicity criteria.19 Measurement of Melphalan Concentrations Melphalan was measured in plasma by gas chromatography–mass spectrometry (GC-MS). P-[Bis(2-chloroethyl)amino]-phenylacetic acid methyl ester was used as an internal standard. Samples were extracted over trifunctional C18 silica columns. After elution with methanol and evaporation, the compounds were derivatized with trifluoroacetic anhydride and diazomethane in ether. The stable derivates were separated on a methyl phenyl siloxane GC capillary column and measured selectively by single ion monitoring GC-MS in the positive EI mode described earlier by Tjaden et al.20 Assessment of Tumor Response This was done by comparing pre-perfusion CT and/or MRI scans of the liver with scans made at 8–10 weeks after IHHP. The tumor marker carcinoembryonic antigen (CEA) was monitored (when indicated) preoperatively and 8–10 weeks postperfusion, but was not used for response assessment. Clinical responses are assessed by standardized WHO criteria.19 RESULTS Patient Characteristics (Table 1) In a total of 24 consecutive patients, 13 males and 11 females with a median age of 57 years (range, 41–70) were included in the protocol. Eighteen patients had irresectable metastases of colorectal origin, four ocular melanomas, one sarcomas, and one unknown primary hepatic metastases. TABLE 1.Characteristics of 24 patients with irresectable liver metastases treated by IHHP with retrograde outflow via portal vein with melphalanPTTumor primaryProgressive on chemotherapyToxicityResponse after perfusionTime to progr. (months)StatusMonths after perfusionHepatoBloodRenal1CRCNo100CR10Alive462CRCNo100PR24Dead383CRCYes140SD12Dead424CRCNo100PR6Dead295CRCNo130PR20Dead326EMNo100PR5Dead97SARCNo130PR13Dead178CRCNo200PR9Dead99CRCYes100SD12Dead3710CRCYes100PD2Dead1011CRCNo100PR9Dead2912CRCNo100PD9Dead1813CRCNo100PD2Dead814CRCYes100PR4Dead1815EMNo100PR5Dead616CRCNo120PR9Alive2917UPYes130PD2Dead518EMNo100PR9Dead2019CRCNo220PR4Dead620CRCNo101PR5Dead521EMNo120PR13Dead1522CRCYes100PD4Dead623CRCYes100SD5Dead624CRCYes100PD9Alive11PT, patient number; CRC, colorectal cancer; CR, complete response; SARC, sarcoma; PR, partial response; EM, eye melanoma; SD, stable disease; UP, unknown primary; PD, progressive disease. Operation Characteristics Median operation time was 240 minutes (150–300 minutes), including perfusion time. Median blood and fluid loss was 900 mL (300–3200 mL). In the first eight patients, melphalan was administered as a bolus into the isolated liver circuit. The last 16 patients received a 10-minute pump infusion of melphalan. The perfusion (10-minute infusion included) with melphalan was conducted for 20 to 30 minutes. Perioperative mortality (<90 days) was 0%. Median hospital stay was 9 days (5–29 days). Leakage Control All IHHPs were leakage free during the perfusion except in one patient, where 2% systemic leakage was measured. When this perfusion was finished, it appeared that in this patient there was an insufficient clamping of the suprahepatic caval vein. After washout of the isolated circuit with Haemaccell, a median of 7% (4–10%) leakage into the systemic circuit was demonstrated by continued monitoring until the end of the operation. Toxicity Study (Table 1) Hepatic toxicity consisted mainly of a transient rise of liver enzymes during the first week after IHHP. WHO grade I occurred in 92% of the patients (22 of 24) and WHO grade II in 8% of the patients (2 of 24). Three patients experienced bile duct necrosis (discussed in detail in the section “Complications”). No other hepatic toxicities occurred (no coagulopathy was observed). Systemic toxicity was mainly leucopenia, WHO grade I–III in 25% (6 of 24), and severe grade IV leucopenia in one patient (4%) after perfusion. Two of these patients received G-CSF (Neupogen, Amgen B.V., Breda, The Netherlands). One patient received prophylactic G-CSF. Eighteen patients (71%) did not develop leucopenia. No renal (except for one patient grade 1) or gastrointestinal toxicity was observed. Melphalan Pharmacokinetics Figure 2 shows a drug concentration vs time curve in the isolated circuit in a patient with a pump infusion. The area under the concentration versus time curve (AUC) calculation showed a regional concentration of 2382 (μg × min/mL) versus undetectable systemic that makes the ratio AUC regional/systemic infinite (Fig. II). The median peak regional melphalan concentration was 68.04 μg/mL (range, 42.3–256.99) and negligibly low to undetectable systemic concentrations. After washout and subsequent releasing of the clamps, median peak systemic 4.2 μg/mL (range, 3.00–18.33) melphalan concentrations were observed. The AUC calculation showed a systemic concentration of 275 μg × min/mL after washout. Thus, intrahepatic melphalan concentrations during the IHHP are >9 fold higher than the post-IHHP systemic melphalan concentrations. FIG. 2.Drug-concentration versus time curve. Complications In three patients, bile duct necrosis occurred (Fig. 3). One of these patients was asymptomatic, and bile duct necrosis was found on routine CT scan during follow-up. Two of the patients with bile duct necrosis developed fever. No other explanation could be found for the fever, besides the intrahepatic “bilomas,” and therefore one patient was treated with percutaneous drainage and endoscopic stent placement in ductus choledochus. The other patient with fever received antibiotics. Both patients recovered, and slight elevations of bilirubin persisted, without complaints. In the first eight patients melphalan was administered as a bolus into the isolated liver circuit. After four patients melphalan dose was increased to 1.5 mg/kg in two patients and to 2 mg/kg melphalan in another two patients. Three of these patients (37.5%) developed bile duct necrosis, which is why the other 16 patients were treated with 1 mg/kg melphalan using a 10-minute pump infusion into the circuit. Bile duct necrosis did not occur in these patients. Postoperative chemical cholecystitis was not demonstrated. FIG. 3.Intra-hepatic bile duct necrosis after IHHP Tumor Response and Patient Survival (Table 1) In one patient a complete response was observed (4%). Partial response (PR) was seen in 14 patients (58%), and stable disease (SD) was demonstrated in three patients (13%). Overall, a clinical response rate of 62% and a clinical benefit of nonprogression in 75% were observed. Six patients developed progressive disease (25%). Progressive disease occurred with median interval after IHHP of 9 months (range 2–24 months). Sixteen patients had progression of the liver tumors during follow-up. Fourteen patients developed extrahepatic disease during follow-up. To analyze the response rates and survival, it may be more opportune to include only the 18 patients with colorectal liver metastases. Median follow-up was 18 months. Median patient survival for patients with colorectal liver metastases (n = 18) was 18 months (range, 5–46 months). Median time to progression was 9 months (2–16 months). Seven of these 18 patients experienced failures of systemic chemotherapy. Two patients (PtNo 3 and 23) were progressive on the combination of 5-fluorouracil (5FU) and oxaliplatin (FOLFOX) (10 and 6 cycles, respectively), two patients (PtNo 9 and 10) were progressive on 5-FU and leucovorine (22 and 16 cycles, respectively), one patient (PtNo 14) was progressive after Xeloda (four cycles) and irinotecan (four cycles), one patient (PtNo 24) after six cycles of FOLFOX and four cycles of irinotecan, and one patient (PtNo 22) was progressive after Xeloda (six cycles), irinotecan (six cycles), and combination of Xeloda and oxaliplatin (XELOX) (eight cycles). Median survival for patients with colorectal liver metastases, not treated with chemotherapy before IHP (n = 11), was 29 months (range, 5–46 months). DISCUSSION Isolated liver perfusions are only performed by a few centers worldwide, demonstrating promising antitumor effects; however, the technique is a major, complex, expensive, and time-consuming operation. In the present study we describe an alternative technique using a retrograde hepatic flow in an isolated hypoxic hepatic perfusion (IHHP) system. The technique was performed in 24 patients and demonstrated to be safe, with minimal morbidity and no morbidity. Overall response rate was 62% with no progression in 75%, and median time to progression of disease was 9 months. The magnitude of the standard isolated liver perfusion technique is well demonstrated in two recently published series by the centers with the largest experience in IHP (NCI, Bethesda, MD, USA, and LUMC, Leiden, The Netherlands, respectively).11,15 Median IHP operation time was reported to be more than 8 hours (range, 5.8–14.7 hours). In the study of Bartlett et al.11 the estimated blood loss was 2.2 L (range, 0.8–4.5 L), and Rothbarth et al.15 reported a range of 0.9–10 L (median not given, older series mean of 4.4 L). Mortality (<30 days) occurred in both series with 5.6% in the Leiden series (n = 71) and 4% in the NCI series (n = 124). Classical IHP uses a venovenous bypass and a heart-lung machine, which is a time-consuming procedure necessitating a specialized perfusion team. In the present study a modification was developed, allowing a safe perfusion in a shorter operation time (median duration, 4 hour), with less blood loss (median, 900 cc), reducing morbidity and without mortality, without the need of a heart-lung machine or venovenous bypass. The absence of mortality (<90 days) in the present study can be explained by the smaller number of patients and/or patient selection. However, it may well be related to the smaller magnitude of the operation and the corresponding decreased operation time and blood loss. Median hospital stay was 9 days (range, 5–29), which was also shorter than the 12 and 11 days in the aforementioned studies.11,15 Hepatic toxicity is remarkably low in the present series except for three patients who developed bile duct necrosis (Table 1). High peak concentrations of melphalan in the isolated circuit might explain the bile duct necrosis that was observed in patients who were perfused with higher melphalan concentrations. In a preclinical study, the occurrence of severe cholangiofibrosis was strongly dependent on the melphalan concentration.21 The study also demonstrated that tumor response was not affected by melphalan peak concentration, but by the total dose of melphalan. Therefore, to prevent high peak concentrations keeping the same concentration of melphalan, bolus infusions were replaced by a 10-minute pump infusion. This resulted in the absence of bile duct necrosis in the last 16 perfused patients. The maximum period of 60-minute hypoxia did not seem to result in extrahepatic and/or systemic toxicity. Moreover, clamping of the aorta just underneath the diaphragm for a maximum of 60 minutes did not lead to renal and/or gastrointestinal morbidity. To overcome the use of a venovenous bypass and heart-lung machine, we performed a hypoxic perfusion. The use of hypoxia might also enhance the antitumor effect. Hypoxia renders tumor cells more sensitive to cytostatic agents in general and enhances the antitumor effects of drugs such as melphalan.22,23 Changing the perfusion direction may reduce liver toxicity, without affecting the antitumor efficacy as described by Rothbarth et al.24 An animal study demonstrated an unaffected tumor uptake and a reduced liver uptake by 80% in an IHP model with arterial inflow and retrograde portal outflow.25 This may explain our remarkably low hepatic toxicity in the present series using retrograde outflow in a clinical setting. In the large IHP series of patients with colorectal liver metastasis, response rates of 74% and 59%,11,15 respectively, are reported, which is in line with the response rate in the present study. The median time to progression in these IHP studies was 14.5 and 7.7 months, respectively. The time to tumor progression of 9 months after IHHP seems to be comparable to these numbers. Alexander et al. published their results in patients with progressive disease after irinotecan-based therapies, and still a response rate of 60% was obtained after IHP with melphalan.16 The 2-year survival was 28% with a 12-month median survival. Although interpretation and comparison of the results from these different studies are difficult, the results of the retrograde IHHP technique described here are similar. In the past 10 years, new anticancer agents (oxaliplatin and irinotecan) have increased the response and survival rates. The addition of targeted therapies (cetuximab and bevacuzimab) may increase the response rates more than 70%. Especially in this era of exciting developments, tailored treatment to increase individual survival rates while keeping the toxic effects to a minimum is challenging. The role of IHP in colorectal liver metastases has been discussed in recent reviews.24,26,27 The promising results of the IHP studies in the past years cannot be disregarded. It is conceivable to combine IHP with adjuvant treatment strategies to reduce the recurrence rate, which is the major determinant of the survival rate after IHP. Continuing locoregional treatment by HAI after an IHP procedure is technically feasible and appears to prolong the duration of the response and survival in patients with colorectal hepatic metastases.11 Of the 18 patients with colorectal liver metastases in the present series, 10 developed extrahepatic disease, suggesting the need for additional systemic treatment. Duration of tumor control needs to be improved. To achieve this, the addition of vasoactive drugs, identified in our laboratory to have synergistic activity in combination with melphalan by significantly increasing drug uptake in the metastases, will be explored in further studies.28,29 At this time, IHHP should be reserved for those patients who are progressive on standard chemotherapy protocols. When responses in future trials are improved, this technique might be optional for patients who prefer a single procedure, which could potentially lead to similar results as multiple courses of systemic or intra-arterial chemotherapy. Leaving behind at the end of the IHHP a port-a-cath system for subsequent HAI in case of (re)progression should also be considered as a strategy to further improve prolonged locoregional tumor control. In ocular melanoma, the liver is the only organ site involved in 70–80% of patients and is usually responsible for death.30,31 The prognosis of a patient with hepatic metastases from uveal melanoma is extremely poor, with a median survival between 2 and 5 months.32 Partial liver resection is seldom possible. The therapeutic options are not very effective with systemic chemotherapy, providing 9% partial responses and no survival benefit.33,34 There is limited experience with IHP for hepatic metastases from uveal melanoma, but the response rates are promising.35–37 Despite the fact that the experience with hepatic metastases from uveal melanoma is small in the present study (n = 4), IHP seems to be an effective treatment. All four patients died because of progressive tumor growth and consequent liver failure after 6, 9, 15, and 20 months. Therefore, especially in this group of patients, local tumor control is likely to result in a survival benefit. CONCLUSIONS Isolated hypoxic hepatic perfusion with retrograde outflow via portal vein is a promising technique that is a relatively simple procedure with reduced blood loss, no mortality, very limited toxicity, and response rates comparable to classic, extensive IHP. The operation time is reduced to 4 hours, without the need of a heart-lung machine and perfusionists, which reduces the costs.

Pharm_World_Sci-3-1-2082656

When pharmacotherapeutic recommendations may lead to the reverse effect on physician decision-making

For long the medical literature has shown that patients do not always receive appropriate care, including pharmacotherapeutic treatment. To achieve improved patient care, a number of physician-oriented interventions are being delivered internationally in an attempt to implement evidence based medicine in routine daily practice of medical practitioners. The pharmacy profession has taken an active role in the delivery of intervention strategies aimed at promoting evidence based prescribing and improved quality and safety of medicine use. However, the medical literature also supports the notion that valid clinical care recommendations do not always have the desired impact on physician behaviour. We argue that the well-established theory of psychological reactance might at least partially explain instances when physicians do not act upon such recommendations. Reactance theory suggests that when recommended to take a certain action, a motivational state compels us to react in a way that affirms our freedom to choose. Often we choose to do the opposite of what the recommendation is proposing that we do or we just become entrenched in our initial position. The basic concepts of psychological reactance are universal and likely to be applicable to the provision of recommendations to physicians. Making recommendations regarding clinical care, including pharmacotherapy, may carry with it implied threats, as it can be perceived as an attempt to restrict one’s freedom of choice potentially generating reactance and efforts to avoid them. By identifying and taking into account factors likely to promote reactance, physician-oriented interventions could become more effective. It is well known that the use of medicines often results in avoidable complications or adverse events. In the United States a study showed that approximately 60% of iatrogenically related admissions to intensive care units were drug related and 75% were preventable [1]. In Australia, adverse medicine events are responsible for 1.6% of all hospital admissions [2]. To achieve improved quality and safety of medicine use, a number of physician-oriented interventions are being delivered internationally in an attempt to improve evidence based prescribing in routine daily practice of medical practitioners—e.g., distribution of educational materials, decision support systems, outreach visits, medication reviews, etc. The pharmacy profession has taken an active role in the delivery of intervention strategies related to pharmacotherapy. For example, in medication review programmes, pharmacists interview patients, conduct assessments of information gathered on patients’ use of medicines and prepare reports for physicians, which may contain pharmacotherapeutic recommendations [3]. Pharmacists also are typically involved in educational outreach programmes. In such programmes it is common for pharmacists to take up the role of educator, visiting physicians in their office to provide educational interventions on specific pharmacotherapeutic topics. As pharmacists and physicians share expertise in pharmacotherapy, their involvement in intervention strategies aimed at promoting quality and safety of medicine use makes sense. Indeed, the literature has reported positive results on patient health outcomes arising from such interventions [4, 5]. However, despite the delivery of interventions designed to foster quality and safety of medicine use, the literature provides support for the notion that, at times, despite physicians agreeing to the value of evidence based clinical recommendations, intervention strategies do not always have an impact on physician behaviour. For example, in a study in which both physicians and community pharmacists had negotiated actions based on medication reviews, the researchers were only able to document a rate of implementation of 42% of the proposed actions [6]. These findings are supported by our own experience working with community pharmacists involved in medication management programmes in Australia. Some of these pharmacists report that sometimes their recommendations have the desired impact on physician choice of pharmacotherapy, but at times they have no impact at all and in some instances they even cause physicians to respond negatively. As expected, failure to change physician behaviour is not restricted to the prescribing area. Indeed, non-compliance to evidence based recommendation is a consistent finding in clinical care research [7]. For example, Grol and Grimshaw [7] note that hospital-acquired infections affect one in every 11 patients with 13% mortality rate and that 15–30% of infections are considered to be preventable by handwashing [8]. Still, compliance with handwashing by health care providers, in particular medical practitioners, is known to be poor [7]. Indeed, with surprising frequency, and to the considerable dismay of interventionists, physician-oriented interventions often fail to improve handwashing behaviour [7, 9]. Without a doubt while there are certainly circumstances that may lead physicians to justifiably question clinical care recommendations, in general non-compliance to evidence based recommendations proves costly to patients and society alike. Currently, there is a lack of theoretical framework for understanding instances when valid recommendations do not lead to the desired impact on physician behaviour. The key question is: What are the barriers that impede physician acceptance of valid recommendations? We argue that the theory of psychological reactance [10] might at least partially explain instances when evidence based recommendations fail to impact on physician behaviour. Psychological reactance Psychological reactance is a tendency to resist perceived attempts by others to control the individual’s behaviour [10]. Most of us like to perceive ourselves as being in control; we value our sense of freedom and autonomy and like to project an image of being in control [11]. In general, when we perceive that our freedom of choice is being restricted by the action of others, we react in a way that affirms our ability to choose. Often we choose to do the opposite of what the recommendation is proposing that we do or we just become entrenched in our initial position. This phenomenon is called psychological reactance [10]. Reactance is directed towards reattaining the restricted freedom; a motivational state compels us to reassert that freedom [10]. In many circumstances, reactance results in an increase in the attractiveness of the constrained behaviour and an increased sense of confidence in the ultimate decision made [10]. Particular noteworthy is the finding that reactance appears to be a biological tendency, an inherent human characteristic. At the very least, it can be said that reactance manifests itself at a very young age [12]. For the last four decades the theory of psychological reactance has been widely tested and applied with reactance being widely recognised as a powerful determinant of behaviour. To date, studies across a vast array of behaviours have consistently demonstrated that recommending that individuals take a certain course of action often produces the opposite effect, consistent with this tendency to resist perceived attempts by others to restrict freedom of choice. For example: in the marketing area research indicates that expert recommendations are desirable as long as they are consistent with the choice tendency of individuals. When expert recommendations contradict the consumer’s choice tendencies, a reactant state is activated on the part of the decision maker leading to a behavioral “backlash” that results not only in consumers ignoring the recommendations but in intentionally contradicting them [13]. Not only there is an increase in choice of the non-recommended option, but also a significant increase in confidence in the non-recommended option [13]. In the healthcare literature, psychological reactance has been primarily used to explain patient non-compliance, i.e., why some patients refuse to cooperate with medical treatment [14, 15]. Fogarty and Young [15] conducted research that indicates that individual variability in compliance to a medication regimen is related to individual variability in reactance as a personality trait [15]. Psychological reactance has also for long been observed in the natural setting in response to perceived attempts to restrict one’s freedom of choice. A classic example of psychological reactance in the everyday life took place when a city in the United States banned the possession and use of laundry detergents containing phosphates for environmental reasons. It was found that residents stockpiled an enormous quantity of detergents containing phosphates before the ban went into effect. Others organised carpools to neighbouring counties to purchase detergents containing phosphates [16, 17]. As a result of the ban, the perceived value of the prohibited products was increased; compared with residents in neighbouring counties, who were free to choose whatever detergent they liked, residents subjected to the ban rated phosphate detergents as gentler, easier to pour, better whiteners and stain removers, and more effective in cold water. It is noted that phosphates have no impact whatsoever on the cleaning effectiveness of detergents. Psychological reactance and physician-oriented interventions The long history of successful support for reactance theory in diverse settings and areas of behaviour may also have implications for physician-oriented interventions in which evidence based clinical recommendations are provided, as health care providers may not be immune to the phenomenon of psychological reactance. It is true that the basic concepts of psychological reactance are universal and likely also to be applicable to the provision of evidence based clinical recommendations to physicians. In the context of physician-oriented interventions, the theory and research on psychological reactance would predict that at times when opposing recommendations are provided, physicians’ perceptions of threats to their freedom to select an appropriate course of action could produce the opposite reaction leading them to become more likely to ignore the recommendation or to choose the non-recommended option. This would be the case even in circumstances where physicians may agree, in essence, to the value of the recommendation....to the extent that it is perceived by the individual that the communicator is trying to make him change, his freedom to decide for himself will be threatened and he will experience reactance... Information and arguments can be quite helpful to the individual and may result in positive influence, but the perception that the communicator is attempting to influence will tend to be seen as a threat to one’s freedom to decide for oneself ([10], p. 94 cited in [14]).Indeed, making recommendations regarding clinical care, including pharmacotherapy, may carry with it implied threats, as it can be perceived as an attempt to restrict one’s freedom of choice generating reactance and efforts to avoid them. Furthermore, the inherent “veto” power that physicians have over the treatment regimen may facilitate manifestation of reactant-style responses making non-compliance to evidence based recommendations more likely to occur. It is also important to recognise that even in circumstances where physicians agree to participate in interventions, they may not necessarily expect their practice to be challenged. For example, there are several possible reasons for a physicians agreeing to a medication review. In some cases, physicians may wish to monitor the individual patient’s self-medication habits or they may be concerned about possible patient non-compliance with the medication regimen. It is also true that physicians have for long subscribed to a body of ethical rules created above all for the benefit of the patient [18]. Professional behaviour of medical practitioners is guided by these ethical rules, which dictate what is morally approved and disapproved conduct specifying what ought to be done [18]. It is reasonable to expect medical ethics to have a considerable impact on physician decision-making in situations that involve potential harm to the patient. For example, the use of a non-steroidal anti-inflammatory drug (NSAID) in a patient taking the anticoagulant warfarin puts the patient at an increased risk of bleeding. In such situations, which involve potential harm to the patient, recommendations arising from intervention strategies most certainly will be recognised by the physician as an injunctive professional norm (a norm of “what ought to be done”) and most certainly will be implemented. However, it is possible that providing physicians with valid opposing clinical care recommendations that do not involve potential harm to the patient may at times be counterproductive. For example, in a patient with low gastrointestinal and cardiovascular risk, recommendation to use a non-selective NSAID if the patient is responding well to a COX-2 selective NSAID, may be perceived as an unwarranted attempt to restrict one’s action, increasing the likelihood of a reactant-style response. Although no study appears to have specifically addressed reactance from a health care providers’ perspective, a recent qualitative study has provided some indication that reactance might play a role in physician decision-making in response to pharmacotherapeutic recommendations [19]. This study investigated determinants of successful collaboration between community pharmacists and physicians in medication review. Participating pharmacists discussed the need to be diplomatic in their recommendations, as they believed physicians’ misperception that they were “telling them what to do” could induce dismissal of their recommendations. Possible determinants of reactance It is possible that reactant responses may be more likely to occur in situations where the physician has not requested the information/recommendation. Experiments outside the medicines area have demonstrated that unsolicited information can evoke psychological reactance even when recommendations provide a substantial amount of information beyond what the decision maker knows [20]. Interestingly enough, in situations where the recommended action went contrary to the individual’s choice tendency, this reactance to unsolicited information was significantly more likely to occur when recommendations came from a source perceived to be an expert [20]. The authors suggest that unsolicited recommendations may be perceived as more of a threat or an intrusion when they come from credible sources. It is noted that although unsolicited information is typically provided to physicians through written materials and computerised decision support systems, in some programmes medicines use review can be initiated by the pharmacist and report sent to the patient’s physician in an unsolicited fashion [21]. It is also possible that the perception that the particular interventionist is directing recommendations specifically to the particular physician, as opposed to the treatment of the patient’s condition in general, also could make reactance more likely to occur. Brehm [10] demonstrated that reactant responses are more likely to occur when individuals perceive the constraint as being specifically directed at them as opposed to when it is perceived to be impersonal [10]. If indeed, this is found also to apply to physician-oriented interventions, it may have implications for how pharmacists and other health care professionals provide recommendations to physicians. Sense of medical professionalism might also be implicated in reactant responses. We do value our sense of freedom and autonomy and we like to project an image of being in control [11]. Indeed, physicians are trained to make sound decisions with regard to treatment regimen and it is reasonable to expect them to believe that they are in control and have the knowledge and skills required to make the best possible decisions. The acceptance of a contrary recommendation to what is being used to treat the patient’s illness could undermine the physician’s own sense of competence, reducing the cognitive basis of professionalism. On the other hand, dismissal of the recommended option could have the opposite effect reinforcing physician’s ability to choose the most appropriate treatment. We also know that once aroused, reactance may heighten individual’s sensitisation to additional threats to freedom of control [22]. It is also reasonable to believe that at a time when governments are encouraging greater involvement of allied healthcare professionals in patient care, issues related to professional territoriality may sensitise physicians to reactance in response to recommendations from allied healthcare professionals, including the pharmacist. Although the literature reports positive medical profession’s attitudes to pharmacists and their clinical role, some sentiments of encroachment are supported by studies that found that not all physicians view favourably greater involvement of pharmacists in patient care [23–25]. Thus, the physician-pharmacist interaction in the intervention process could be burdened with social and personal pressures, which can increase the possibility of a reactant response. Indeed, psychological reactance may offer a theoretical framework that could enhance our understanding of the, at times, difficult relationship between pharmacists and physicians. It is also possible that the nature of the physician relationship with the allied healthcare professional is also a determinant of reactance. For example, it has been argued that the most successful pharmacist interventions have been those in which the pharmacist works in close liaison with the physician [26]. Indeed, in the United States much of the evidence supporting pharmacist involvement in collaborative drug therapy management is derived from the ambulatory care setting [27] where routine contact with physicians may allow for the development of the trusting relationship needed for interprofessional collaboration [28]. Finally, another factor that may determine reactant responses to recommendations include individual differences, as some people are more likely to have a reactant response in general to any perceived constraint [29]. Individual variations in susceptibility to reactance can explain why identical situations may lead to markedly different perceptions of threat and magnitudes of reactant responses in different persons. Indeed, scales developed to measure reactance as a trait demonstrate that individual reactance levels lie along a continuum from very low to very high [29]. Naturally, in the context of physician-oriented interventions, the nature and intensity of the reactant response is also likely to be a function of how much the particular physician values his/her freedom to select the treatment option him/herself. Again, a recent qualitative study provides some support to the notion that some physicians are more prone to psychological reactance than others [19]. In this study, personality of medical practitioners was discussed as a factor influencing the outcome of medication review. Participating pharmacists distinguished between physicians who they thought were amenable to discussing their prescribing decisions and those who were not. These results indicate that interventionists do assess the reactance potential of the individual physician although it is unlikely that they label the assessment as such. It is noteworthy that when interventionists sense reactance in their interaction with the physician, they may be faced with a difficult dilemma, as they are aware of the possibility that the provision of evidence based clinical recommendation may trigger the opposite response. What can the interventionist do in such situation? Dealing with reactance A phenomenon like psychological reactance, which is likely to impede the desired outcome of physician-oriented interventions, needs to be adequately addressed when training healthcare professionals to deliver such interventions. We must be mindful that the process of delivering physician-oriented interventions can be difficult as psychological reactance is not a reasonable or rational response; it can cause people to become highly emotional and motivated to defend their behaviour. Prior research on reactance suggests that individuals’ ability to exercise some choice (freedom) in relation to recommendations may impact on compliance to the advice provided. That is, maximising the individual’s perception of free choice can minimise reactance [22]. It is possible that the degree to which the physician is incorporated in the selection of evidence based clinical care practices may affect the extent to which the intervention will induce reactance. Indeed, it is possible that intervention strategies which give physicians the impression they have little or no say on their own clinical practices will be less likely to be effective, as they are perceived as more threatening, compared to those where physicians perceive themselves as exercising some choice. We are now training pharmacists on how to deal with psychological reaction in the context of medication review. We also expect such training to enhance commitment to the pharmacy lead intervention from the interventionist’s perspective, as after being equipped with tools for dealing with reactance, pharmacists may experience a greater sense of self-efficacy in dealing with all types of physicians, including those who are highly reactant. In addition, awareness of reactant responses and its causes may assist pharmacists and other healthcare professionals in altering their emotional response to reactant behaviour avoiding a decreased sense of self-efficacy. Concluding remarks The current paper addressed an intrinsic human characteristic, psychological reactance, as a factor that may influence the outcome of physician-oriented intervention strategies, including those typically delivered by pharmacists. Although psychological reactance has not been tested from a healthcare provider’s perspective, there is enough research to suggest the basic cognitive process represented is universal and also valid in the context of clinical care recommendations in the healthcare setting. Indeed, reactance theory fits well as it offers some explanation of why at times valid recommendations fail to have the desired impact on physician behaviour. It is possible that as human beings, even healthcare professionals are subjected to the same human emotions and weaknesses as humans in general. However, a role for reactance in clinical care recommendations needs to be investigated empirically. If this role is established, factors likely to increase the potential for reactance may need to be taken into account when implementing intervention strategies that attempt to influence physician behaviour. It also is acknowledged that physician non-compliance to valid recommendations is more likely to be a complex, multi-faceted phenomenon. Physicians may not adhere to evidence based recommendations for a number of reasons unique to the individual patient, and the circumstance. It is most likely that reactance, although a powerful determinant of behaviour in general, is only one of the many factors that influence decision-making in relation to clinical care/pharmacotherapeutic recommendations.

Oecologia-4-1-2270359

Impacts of savanna trees on forage quality for a large African herbivore

Recently, cover of large trees in African savannas has rapidly declined due to elephant pressure, frequent fires and charcoal production. The reduction in large trees could have consequences for large herbivores through a change in forage quality. In Tarangire National Park, in Northern Tanzania, we studied the impact of large savanna trees on forage quality for wildebeest by collecting samples of dominant grass species in open grassland and under and around large Acacia tortilis trees. Grasses growing under trees had a much higher forage quality than grasses from the open field indicated by a more favourable leaf/stem ratio and higher protein and lower fibre concentrations. Analysing the grass leaf data with a linear programming model indicated that large savanna trees could be essential for the survival of wildebeest, the dominant herbivore in Tarangire. Due to the high fibre content and low nutrient and protein concentrations of grasses from the open field, maximum fibre intake is reached before nutrient requirements are satisfied. All requirements can only be satisfied by combining forage from open grassland with either forage from under or around tree canopies. Forage quality was also higher around dead trees than in the open field. So forage quality does not reduce immediately after trees die which explains why negative effects of reduced tree numbers probably go initially unnoticed. In conclusion our results suggest that continued destruction of large trees could affect future numbers of large herbivores in African savannas and better protection of large trees is probably necessary to sustain high animal densities in these ecosystems. Introduction Savanna ecosystems are characterized by a continuous grass layer interspersed with trees and shrubs. The density and diversity of large herbivores is very high in African savannas and many pastoral communities depend on semi-natural savannas through livestock grazing (Prins and Olff 1998; Olff et al. 2002). The effects savanna trees have on understorey grass productivity have been a focus of research for decades, and both negative and positive effects have been described (Belsky et al. 1989; 1993; Mordelet and Menaut 1995; Ludwig et al. 2001; 2004a). Trees reduce grass growth by competing with grasses for water, light and nutrients (Scholes and Archer 1997; Anderson et al. 2001; Ludwig et al. 2001, 2004b), but trees can also improve grass production through hydraulic lift (Caldwell et al. 1998; Ludwig et al. 2003), reduced evapotranspiration (Amundson et al. 1995: Ludwig et al. 2001) and increased nutrient availability (Scholes and Archer 1997; Ludwig et al. 2004a). Although total plant production affects animal biomass, forage quality is of much more importance for understanding herbivore performance and food limitation (Van Soest 1994) and the interplay between forage quality and quantity determines to a large extent the composition of the herbivore assemblage (Prins and Olff 1998; Olff et al. 2002). Two previous studies which compared forage quality under trees and in the open field both report higher forage quality under Acacia (Belsky 1992) and Eucalyptus (Jacskon and Ash 1998) trees. Increased forage quality is probably caused by higher soil nutrient concentrations under tree canopies (Kellman 1979; Belsky et al. 1989; Scholes and Archer 1997). Also water competition and shade could increase forage quality because nutrients taken up by grasses are less diluted due to a lower biomass production. Currently, large tree cover is decreasing in several African savannas due to high elephant pressure and frequent fires (Eckhardt et al. 2000). In Tarangire National Park (NP) (northern Tanzania) the cover of large trees declined from 20% in 1970, to only 5% in 1996 (Van de Vijver et al. 1999). A recent study in Namibia showed that the number of large trees was reduced by 50% over the last 36 years while the extent of shrubland dramatically increased (Mosugelo et al. 2002). Also in Kruger NP (South Africa) and in the Serengeti-Masai Mara ecosystem (Tanzania and Kenya) the number of large trees in savannas has decreased over the last decades (Ruess and Halter 1990; Eckhardt et al. 2000). Outside protected areas the situation is often even more dramatic, with most of the large trees being removed by local people for production of charcoal (Kituyi et al. 2001; Luoga et al. 2002). Reductions in tree cover could have serious consequences if trees have a positive effect on herbivore food quality and availability. The objective of this study was to investigate the impact of trees on forage quality and how a reduction in the number of large trees could affect herbivore populations in African savannas. Specifically, we focussed on the impact of the dominant East African savanna tree, Acacia tortilis, on nutrient concentrations, protein content and digestibility of grasses. We included small, large and dead trees in our study to be able to determine how large tree destruction affects forage quality through time. A linear programming model was used to test the hypothesis that forage growing under trees is necessary for the long-term survival of wildebeest, the dominant large herbivore in East African savannas. Materials and methods Site description Data were collected in Tarangire NP (4°S, 37°E, 1,200 m above sea level) which is located at the eastern side of the Great Rift Valley in northern Tanzania and encompasses an area of about 2,600 km2. Mean rainfall is 650 mm/year (Van de Vijver 1999). The wet season is from November until May with most of the rain typically falling during March and April. The Tarangire River runs through the park, and in the dry season this river is the main permanent fresh water supply within the entire 35,000 km2 Masai ecosystem (Prins 1987). Northern Tarangire NP is in the dry season range of large migratory herds of Burchell’s zebra (Equus burchelli) and wildebeest (Connochaetes taurinus) and to a lesser extent Cape buffalo (Syncerus caffer). Towards the end of the dry season Northern Tarangire has one of the highest concentrations of large herbivores in the world. At the beginning of the wet season wildebeest and zebra leave Tarangire NP to graze on the Simanjiro Plains. During most years both species give birth and lactate on the Simanjiro plains and return to Tarangire NP in the dry season (Voeten and Prins 1999). The park is also famous for its large herds of elephants (Loxodontaafricana) which primarily reside in the park during the dry season and sometimes disperse to surrounding areas in the wet season. The number of large elephants has increased sharply over the last 20 years due to destruction of habitat outside the park which forced more elephants into Tarangire NP. Two wooded savanna types dominate the park: Acacia savanna, which can be found in the riverine area with lacustrine soils in which A. tortilis is the dominant tree species, and the deciduous savanna, which is concentrated on the ridges and upper slopes on Precambrian well drained red loamy soils (see Van de Vijver et al. 1999). Here, the dominating trees are Combretum and Commiphora species. Most of the herbivores concentrate in the Acacia savanna (Tarangire Conservation Project 1997, Voeten 1999), presumably because of the higher forage quality, so we focused our work on this savanna type. As A. tortilis accounts for 90% of the large trees in this savanna type (Van de Vijver et al. 1999) we focused our study on grass vegetation growing under and around this tree species. Total woody cover, including bushes, is about 20%, and especially the cover of large trees has declined over the last 30 years (Van de Vijver et al. 1999). Grass sample collection and chemical analyses A study site of 2 × 2 km was selected with a vegetation which was representative of the Acacia savanna in Tarangire NP. The study site was located between the main entrance of the Tarangire NP and the park headquarters. For this study the same four large trees were used as described in Ludwig et al. (2004a). Trees were selected on the basis that they were separated from other large trees by at least 100 m and 50 m from any bushes or small trees. Trees close to termite hills were avoided. Measurements and calculations by Prins and Van der Jeugd (1993) suggest that these trees are ca. 100 years old. Trees have a canopy diameter between 15 and 25 m. A previous study showed that around large trees, three different vegetation zones of the herbaceous layer can be distinguished, namely: (1) under large tree canopies, (2) just outside tree canopy projections (called “around tree canopies”), and (3) in open patches more than 50 m from any tree (Ludwig et al. 2004a). Species composition was recorded at peak standing biomass during the early dry season in June 2000 in 20 different 2 × 2-m plots in each of the three different communities. For each plot the cover of all grass species was visually estimated. Nomenclature followed Clayton and Renvoize (1982). Under tree canopies, plots were located in between the tree stem and canopy edge. Plots around tree canopies were selected at twice the distance from the stem, as the edge of the tree crown. For example, if the canopy edge was at 10 m from the trunk the centre of the plot under the canopy was located at 5 m from the trunk and the plot “around canopies” at 20 m from the trunk. Open grassland plots were at least 50 m from any tree. To analyse grass nutrient concentration, grass samples were collected in the early dry season in June 2000, because this is the time of year when migratory herds of wildebeest and zebra return to Tarangire NP and start grazing at the study site. At each of the three different vegetation zones around large trees, samples of the most abundant grass species were collected. Different species were sampled in the three zones because of differences in species compositions. See Table 1 for species sampled in each zone. Three Cynodon species, Cynodon dactylon, Cynodon nlemfluensis and Cynodon plectostachius, were treated as one group because they are very similar, rarely flower and it is very hard to distinguish between the species without uprooting them and when they are not flowering (see Clayton and Renvoize 1982). Table 1Average (SD in parentheses, n = 4) vegetation cover, live and dead stems and leaves and leaf/stem ratio of grass species growing under and around canopies of large Acacia tortilis trees and in open grassland patchesLocationSpeciesCover of vegetation at specific zone (%)Live stem (%)Live leaf (%)Dead stem (%)Dead leaf (%)Leaf/stem ratioDataUnder tree canopyPanicum maximum933.1 (5)51.1 (13)4.5 (6)11.3 (4)1.81 (0.73)Cynodon spp.6247.4 (7)42.0 (8)4.4 (2)6.2 (3)0.95 (0.24)Cenchrus ciliaris940.8 (15)28.3 (9)6.8 (3)24.0 (13)1.22 (0.54)Around tree canopyCynodon spp.750.4 (6)25.4 (3)5.4 (3)18.8 (7)0.80 (0.14)Cenchrus ciliaris640.9 (19)27.2 (3)15.4 (10)16.5 (10)0.83 (0.37)Digitaria macroblephera1439.0 (15)25.2 (8)7.9 (15)27.8 (3)0.70 (0.24)Chloris virgata943.7 (7)30.7 (8)9.0 (6)16.6 (6)1.14 (0.12)Urochloa mosambicencis2152.4 (3)34.7 (11)4.9 (5)8.0 (10)0.91 (0.20)Heteropogon contortis1743.8 (6)27.2 (9)15.9 (4)13.1 (3)0.77 (0.25)Open grasslandH. contortis3848.3 (4)32.9 (7)8.4 (5)10.4 (5)0.77 (0.11)Sehima nervosum3650.1 (12)29.2 (9)12.0 (7)8.7 (14)0.62 (0.16)StatisticsadfLocationF2 ,381.766.57**1.861.848.60***Tree (block)F3, 38 1.600.421.180.081.15** P < 0.01, *** P < 0.001aVariance of data was analysed with a general linear model (GLM) with grass species and location (open grassland and under and around canopies) as fixed factors Grass samples were collected following the “hand plucking method” (Wallis de Vries 1995). Samples were plucked between thumb and backward-bent index finger to simulate large herbivore grazing as closely as possible. One sample of every species was collected within each zone. One sample consisted of between 15 and 25 pickings collected in small mono-specific stands at different spots within the same zone. Samples were dried in the sun and sorted into leaves and stems and live and dead material. After drying at 70°C for at least 48 hours, live leaves were analysed for total N, P, K, Ca, Mg and N using a modified Kjeldahl procedure with Se as a catalyst (Novozamski et al. 1983). After digestion, N and P concentrations were measured colorimetrically with a continuous flow analyser (Skalar SA-4000; The Netherlands) and K, Ca, Mg and Na were analysed with an atomic absorption spectrophotometer (Varian Spectra AA-600; The Netherlands). Neutral detergent fibre (NDF) was determined according to Goering and Van Soest (1970) and digestibility of organic matter (DOM) according to Tilley and Terry (1963). Crude protein was calculated as 6.25 × total N concentration. DOM, NDF and protein concentration of the vegetation per zone were calculated by multiplying the nutrient concentration in a specific species by its relative abundance within the vegetation. Dead and smaller trees have a different effect on the understorey vegetation than large trees or open grassland (Ludwig et al. 2004a). To study how large-tree removal affects forage quality through time, grass samples were also collected around dead and smaller A. tortilis trees. Samples of the dominant grass species were collected under bushes, small trees and around dead trees. Well-developed “bushes” of A. tortilis were ca. 4 m high and had no signs of tree or umbrella shape development. “Small trees” had a clear tree form and were about 10 m tall and had started to form an umbrella shape. “Dead trees” were large trees which had died 4–8 years ago. For detailed description of tree stages see Ludwig et al. (2004a). Samples were collected as described above and analysed for protein, NDF and DOM. Linear model The problem of having to comply with several different requirements can be analysed with a linear programming model (Belovsky 1978; Voeten 1999). This is an optimization model in which one variable is maximized or minimized, while subjected to different constraints. Here, it is used to study whether herbivores can meet all their nutritional and energy requirements by selecting either food from under or around trees or from open grassland or a combination of food sources. As a basis the model described by Voeten (1999) is used. We used wildebeest as model species because it is the most abundant herbivore in Tarangire NP (Tarangire Conservation Project 1997) and nutrient and energy constraints of this species have partly been described by Murray (1993, 1995). It would be ideal to include more herbivore species but only data for wildebeest are available. Each constraint was formulated as a general linear equation: where C is a constraint value which stands for either nutrient, energy or fibre intake. We formulated minimum requirements for nutrient, protein and energy intake, and a maximum value for fibre intake. Iiis the amount of food consumed by class i; in this study grass leaves from under or around tree canopies or open grassland. The parameter ciconverts Ii into the same unit as C and is based on the nutrient, energy and fibre content measured in the grass leaves of the different zones. Murray (1995) calculated, from a feeding trial in the Serengeti, northern Tanzania, that wildebeest need an energy intake of 22.32 MJ/day for maintenance. This was based on an average body mass of 143 kg. The metabolic energy of grasses equals the digestible energy multiplied by 0.82 (Van Soest 1994). The digestible energy can be calculated from the DOM multiplied by the gross energy of grass. The energy content of grasses averages 19 MJ/kg DOM (Crampton and Harris 1969), and this number was used for all grass species. Thus the constraint equation for energy intake is: where DOM is the digestibility of organic matter as determined in vegetation. I is the intake rate [kg dry weight (DW)/day] and i stands for the foraging zone. Digestible protein (DP) requirements at maintenance for ruminants can be calculated as (Voeten 1999): where W stands for metabolic weight (kg). We used an average weight of wildebeest of 143 kg (Murray 1993) which means they need 130 g DP per day for maintenance. DP for tropical grasses can be calculated from crude protein using a formula proposed by Prins (1987): The second constraint equation used in the model is: For ruminants the daily intake rates are often constrained by rate of digestion and passage through the rumen (Voeten 1999). The digestibility rate of food is often correlated with the cell wall content, measured in the vegetation as NDF (Van Soest 1994). Reid et al. (1988) calculated from a feeding trial of cattle on a C4 grass diet that maximum daily NDF intake can be calculated as: For wildebeest of 143 kg, the maximum intake is thus 2.76 kg NDF so the third constraint equation is: In addition, two equations were defined based on requirements of the two most important nutrients for wildebeest as determined by Murray (1995). The constraint equation for maintenance levels of Ca and P are: where P and Ca are the concentrations measured in the vegetation. Na is not considered in this model because wildebeest can satisfy their daily requirements through drinking water from the Tarangire River (Voeten 1999). The different constraints were used to calculate whether wildebeest can meet all nutritional and energy requirements for maintenance (to maintain a stable body weight) by selecting forage from different foraging zones. As an input value for each of the three zones a weighted average, depending on plant cover, of the dominant grass species was used. Plant cover instead of biomass was used because only plant cover data were available. Statistical analysis Percentages of dead and live leaves and stems, nutrient concentrations, NDF and DOM of grass samples were analysed with a general linear model (GLM) with vegetation zone (under and around tree canopies, and open grassland) as fixed factors and trees as a random factor. Differences in NDF, DOM and protein concentrations of grasses from different tree stages (open grassland, bushes and small, large and dead trees) were analysed with a GLM with tree stage as a fixed factor. All data were statistically analysed with SPSS 11.0. Results Cynodon species covered 62% of the vegetation under tree canopies (Table 1). The other two abundant species under trees were Panicum maximum and Cenchrus ciliaris. The remaining 20% was covered mainly by herb species. Around tree canopies the vegetation was more species rich with six more or less abundant grass species (see Table 1). In open grassland, the vegetation consisted almost exclusively of the grass species Heteropogon contortis and Sehima nervosum. Percentage of live and dead stem and dead leaves was similar under and outside trees. Percentage of live leaves, however, was higher under trees compared to around trees and open grassland. Forage quality was much higher under large tree canopies than in open grassland. This was indicated by higher leaf/stem ratios, higher concentrations of protein and DOM and lower NDF concentrations (Table 2, Fig. 1). For example, digestibility of green leaf organic matter (DOM) was highest in Cynodon grasses growing under the canopy where 70% of green leaves was digestible (Table 1) and DOM was lowest in H. contortis growing in open grassland. For NDF a reversed pattern was observed with the lowest values for Cynodon spp. and highest for H. contortis and S. nervosum. Protein contents of grasses were highest under tree canopies and Cynodon spp. and Cenchrus ciliaris showed a higher protein content under than around tree canopies. Forage quality of grasses growing around tree canopies was also higher than in grasses from open field but lower than under canopies. Leaf/stem ratios, protein concentration and DOM were all higher around trees canopies than in open field. P concentrations showed a different pattern: concentrations were higher in grasses growing around canopies than under canopies and in open grassland (Table 1). Table 2Average (SD in parentheses) of digestibility of organic matter (DOM), neutral detergent fibre (NDF), protein and nutrient concentrations of grass species growing under and around canopies of large A. tortilis trees and in open grassland patchesLocationSpeciesDOM (%)NDF (%)Protein (mg/g)P (mg/g)K (mg/g)Ca (mg/g)Mg (mg/g)DataUnder tree canopyP. maximum65.7 (6.5)70.6 (3.3)141 (19.7)1.77 (0.17)40.6 (2.2)4.12 (0.26)3.25 (0.79)Cynodon spp.70.3 (7.2)61.5 (4.9)165 (8.4)1.80 (0.32)38.4 (2.4)6.91 (0.71)2.44 (0.57)Cenchrus ciliaris59.8 (3.2)70.1 (5.0)137 (5.0)1.74 (0.18)44.8 (3.8)4.34 (2.19)1.95 (0.43)Around tree canopyCynodon spp.65.1 (12.4)66.5 (8.6)127 (26.2)2.38 (0.42)36.1 (11.2)6.70 (2.18)2.58 (0.38)Cenchrus ciliaris58.0 (9.0)71.5 (6.1)106 (15.4)2.43 (0.07)41.9 (7.0)3.30 (0.65)1.87 (0.38)D. macroblephera62.6 (8.0)72.9 (6.2)75 (13.5)2.85 (0.52)38.6 (1.5)3.75 (0.76)2.26 (0.55)Chloris virgata66.8 (5.9)70.4 (4.1)105 (5.0)2.59 (0.61)37.6 (1.8)4.91 (0.30)3.03 (0.45)U. mosambicencis69.1 (5.4)64.8 (3.5)104 (10.3)3.59 (0.78)44.3 (3.9)5.28 (0.56)4.00 (0.40)H. contortis57.0 (4.8)70.2 (3.0)82 (11.8)1.46 (0.13)17.2 (0.5)3.30 (0.3)1.64 (0.18)Open grasslandH. contortis51.7 (2.2)74.0 (2.2)68 (9.3)1.65 (0.19)15.1 (0.7)3.31 (0.31)1.22 (0.15)S. nervosum54.7 (1.0)74.6 (1.0)61 (9.7)1.46 (0.13)12.0 (0.8)4.67 (0.36)1.47 (0.06)StatisticsaLocationF10.4***5.55**49.9***11.7***28.2***1.347.77**Tree (block)F7.08***5.45***1.361.071.360.640.71 ** P < 0.01, *** P < 0.001aVariance of data was analysed with a GLM with grass species and location (open grassland and under and around canopies) as fixed factorsFig. 1Indicators of forage quality of grasses growing under different sized Acacia tortilis trees, dead trees and in open grassland. Mean (+SD) protein, digestibility of organic matter and neutral detergent fibre (NDF) concentrations of green grass leaves. Bars with the same letter are not significantly different (LSD test, P > 0.05) Protein concentration, NDF and DOM differed significantly between different tree stages (P < 0.05). Protein concentrations were highest under large trees and lowest in open grassland (Fig. 1). Smaller Acacia (bushes and small trees) showed intermediate values. Under dead trees grass protein concentrations were higher than in open grassland but lower than under large trees. DOM percentages showed a similar trend to protein concentrations; under small Acacia trees and bushes DOM of grasses was higher than in open grassland but lower than under large trees. NDF values showed the opposite trend with lowest values under large trees and highest values in open grassland (Fig. 1). NDF values of grasses growing under bushes and small and dead trees were lower than in open grassland but not significantly different from those of grasses growing under large trees. In general, these patterns show that forage quality increased with tree age and size. Under dead trees forage quality was higher than in open grassland but lower than under large trees. Results from the linear programming model show that by selecting forage from under large tree canopies wildebeest can meet all their energy, nutrient and protein requirements before reaching their maximum intake determined by the grass fibre concentration (Fig. 2). Wildebeest can also satisfy their requirements for nutrients, protein and energy by selecting grass around tree canopies. However, the results of the linear programming model indicate they cannot do so by selecting food exclusively from open grassland. Due to the relatively high fibre content in forage from open grassland the maximum intake for wildebeest is about 3,700 g/day. Due to the low nutrient and protein concentrations in grasses from the open field, this amount is not enough to satisfy all the dietary requirements for wildebeest. All requirements can be satisfied by combining forage from open grassland with either forage from under or around tree canopies. Especially if forage from open grassland is combined with grass from under large tree canopies only a relatively small amount (10–20%) of forage is required from under trees and the rest of the forage can be selected from open grassland (Fig. 2). Fig. 2Results of a linear programming model, predicting whether wildebeest can meet their daily requirements for energy, nutrients and protein by selecting forage from under tree canopies, around tree canopies, or in open grassland patches. Within this model the minimum daily amount of energy, protein, P and Ca needed by wildebeest is defined. The lines indicate the minimum food intake needed to meet these requirements for a combination of two of the three food sources available. For fibre the maximum food intake is shown. The maximum and minimum intakes rates are calculated using Eqs. 2, 5, 7, 8 and 9. The shaded part indicates all possible combinations of food sources which meet nutrient, energy and protein requirements without exceeding maximum intake rates determined by the fibre content. DW Dry weight Discussion Our results show that the forage quality of the herbaceous layer is much higher under trees than in open grassland. Grasses growing under tree canopies contained a lower fibre content and the highest concentrations of protein and Ca and had a higher digestible organic matter content. The higher forage quality under trees is partly caused by the different species which dominate the vegetation under large trees. However, Cynodon spp. growing under trees also had higher protein concentrations and DOM contents than the same species growing around tree canopies. This shows that a higher below-crown forage quality it is not only a species effect but also due to a different environment under trees. The positive effect of trees on forage quality did not stop at the canopy edge. Grasses around tree canopies still had higher nutrient concentrations and leaf/stem ratios and a higher DOM than the two species dominating open grassland. The grasses we sampled around tree canopies were growing about 7–10 m from the canopy edge. So one mature Acacia tree with a canopy diameter of 15 m can potentially increase forage quality over an area of more than 600 m2. Our results could have been affected by the fact that we only sampled once. However plant samples taken in 2 previous years during different periods within the season show much higher nutrient concentration in grasses under trees compared to open grassland (Ludwig et al. 2001, 2004a). So it is likely that forage quality is higher under trees throughout the season. Increased forage quality under savanna trees can be caused by a range of different effects because savanna trees influence the availability of all major resources used by grasses. Trees reduce light availability through shade, often increase soil nutrient concentrations, and compete with grasses for belowground resources, especially water (Belsky 1994; Scholes and Archer 1997; Anderson et al. 2001). Shade alone can already increase forage quality (Cruz et al.1999). For example, two independent studies showed that N concentrations were increased in P. maximum growing under artificial shade (Deinum et al.1996; Durr and Rangel 2000). However, probably most important is the higher soil nutrient availability under savanna trees. In a previous study, under the same Acacia trees, we found that concentrations of all major nutrients were higher under trees compared to open grassland (Ludwig et al. 2004a). Higher soil nutrient concentrations increase plant nutrient concentrations which improve the forage quality. Higher soil nutrient concentrations usually also increase grass productivity which would result in reduced plant nutrient concentrations due to dilution. However, grass productivity under these Acacia trees is mainly limited by water. Previous studies showed that soils under Acacia trees had lower soil moisture contents and soil water potentials than soils beyond the edge of canopies (Ludwig et al. 2003, 2004a). Higher forage quality under these savanna trees is probably caused by a combination of reduced soil moisture and increased nutrient availability. The results of the linear model show the importance of the higher forage quality from under and around trees for large herbivores. When wildebeest forage only in open grassland, they cannot satisfy all their nutritional and energy requirements to maintain a stable body weight. The grass species growing in open grassland have a high fibre content which limits the maximum daily intake. Due to this limited intake insufficient amounts of protein and P can be extracted. Most of the available data on energy and nutrient requirements originate from agricultural studies and still few data are available for wild herbivores. For our model, we adjusted daily intake requirement for wildebeest from agricultural data in combination with experimental work on wildebeest (Murray 1993). These adjustments could have resulted in some errors in the model. For example the maximum NDF intake used originated from cattle and could be different for wildebeest. Also only one standard value for energy contents of grasses was used because no other data were available. Due to the large differences in forage quality of grasses from under trees compared to the open field, it is unlikely that a small change in model parameters or input values will significantly affect the conclusion that wildebeest need at least some forage from under trees. For example, wildebeest would have to increase their fibre intake by more than 35% to satisfy their protein needs in open grassland, and only if their protein needs were 30% less than assumed in our model could wildebeest satisfy their needs without grazing under or around trees. Another indication that large herbivores would need to forage under trees is that the crude protein concentration is below 7% in grasses from open grassland. At these protein concentrations the digestion rate is severely limited for all herbivores (Van Soest 1994) which suggests that large herbivores cannot survive when foraging from open grassland only. These results show the importance of trees for the functioning of savanna ecosystems and many large grazers probably depend indirectly on large trees for their long-term survival. Trees are not the only source of spatial heterogeneity in forage quality. For example also termite hills, faeces concentrations and rivers can increase nutrient availability and thus improve forage quality (Grant and Scholes 2006). So in the absence of trees, large herbivores could forage in these “hot spots” to satisfy their nutrient requirements. However, trees cover a much larger part of the landscape than other sources of spatial heterogeneity and are thus particularly important in improving forage quality. In the absence of trees, herbivores can increase forage quality through positive feedbacks of grazing (Augustine and McNaughton 2006). For example through the formation of grazing lawns (McNaughton 1985) However, these positive feedbacks are mainly observed in nutrient-rich savannas and grasslands such as the Serengeti (Grant and Scholes 2006). Forage quality of grasses growing around dead trees was lower than under large trees but still higher than in open grassland. Grass protein concentrations, for example, were almost twice as high around dead trees as in open grassland. So, forage quality does not dramatically decline immediately after trees die. This might explain why negative effects of tree removal have largely been ignored. In a previous study, we also showed that grass biomass production is higher around dead trees than in open grassland and under large trees due to the lingering positive effect of trees (increased soil nutrient availability persists for some years), combined with the removal of negative effects such as shade and competition for water (Ludwig et al.2004a). Thus, the first impression after trees are cut down/killed is that trees were suppressing grass production. When the positive effect of higher soil nutrient concentrations around dead trees has disappeared, grass forage quality and productivity will be as currently found in open grassland, where both productivity and quality are lower than under trees that died recently. So only after a time lag of at least 8 years (Ludwig et al. 2004a), when the effect of trees on forage quality has disappeared, does it become clear that tree felling only temporarily increases grass biomass and ultimately reduces forage quality, thereby jeopardising animal productivity. Large trees killed by elephants or human pressure can be replaced by regenerating small trees or bushes; however, their impact on grass quality and availability is different from that of large trees. Bushes tend to reduce grass production (Ludwig et al. 2004a) and have a smaller positive effect on forage quality than large trees. Grass forage quality under the small trees was also still significantly lower than under large trees. The small trees we used for our study were about 20 years old so it will take more than two decades before forage quality will be as good as before clearing. Another problem is that the contradictory effects of different sized trees on grass quality and production can encourage bush-clearing. Increased grass production after large tree removal suggests that trees negatively affect grass production. After clearing the large trees, regenerating bushes start to repress grass production which again encourages removal of all woody species. Bush-clearing is, however, ultimately counterproductive because it will result in savannas with few or no large trees and thus a much lower forage quality. Over the last 20 years, numbers of elephants have increased in several NPs in Africa (Van de Vijver et al.1999; Eckhardt et al.2000; Skarpe et al.2004). This increase can have serious effects on other large herbivores. For example, the increase in the number of African elephants in Kruger NP which took place over the last 50 years, and the consequent reduction in the number of large trees (Eckhardt et al.2000) could have caused the demise of rare selective antelopes such as the roan. Roan largely depend on forage from P. maximum (Heitkonig 1994), a grass species which dominates the vegetation under trees in southern African savannas (Ruess and Halter 1990; Scholes and Walker 1993; Scholes and Archer 1997; Eckhardt et al.2000). As the effects of tree removal only become clear years after trees die, impacts of increased elephant pressure might not be clear at the moment but could become a serious problem in the near future. In conclusion, our results presented here show that savanna trees have an important role in affecting the quality of forage available for wildebeest in Tarangire NP. Some recent work shows that forage quality is also higher under trees in other African savannas and more future work should show how general our results are (Treydte et al.2007). As trees increase food quality for herbivores the reduction in the number of large trees observed in savannas across Africa (Van de Vijver et al.1999; Eckhardt et al.2000; Mosugelo et al.2002) can have serious consequences for the survival of large herbivores. These negative effects of tree removal are probably usually not recognised because forage quality only slowly reduces after trees die. However, better protection of large trees in savanna ecosystems could be necessary to sustain current numbers of large herbivores.

Clin_Oral_Investig-4-1-2238790

A survey of failed post-retained restorations

Survival of endodontically treated, post-restored teeth depends on a multitude of factors, all of which are practically impossible to include in a randomized, controlled clinical study. The purpose of this survey was to characterize and analyze reported failures of post-retained restorations to identify factors critical to failure and to type of failure. A questionnaire was mailed to private practitioners in Denmark with a request to complete the questionnaire whenever a patient presented with a failed post-retained restoration. Information was gathered on factors related to the patient, the tooth, the restorative materials, and the techniques. Two-hundred and sixty questionnaires were collected from 171 practitioners over a 3-year period. Functioning time until failure varied between 3 months and 38 years. Mean survival time until failure was 11 years. Of the failed restorations, 61% had functioned for 10 years or less. Fracture of the tooth was the most common type of failure reported, followed by loosening of the post and fracture of the post. Tapered posts implied an increased risk of tooth fracture compared to loosening or fracture of the post, and the relative risk of tooth fracture increased with the functioning time until failure. Fracture of the post was more common among male than female patients. On the basis of this survey of failed post-retained restorations, it was concluded that tapered posts were associated with a higher risk of tooth fracture than were parallel-sided posts. Introduction Endodontically treated teeth have been reported to have shorter survival time than vital teeth [14, 20, 29, 33, 35]. This is believed to reflect that endodontically treated teeth are often structurally compromised because of a series of events: caries or trauma, endodontic therapy, and preparation for restorative procedures [12, 19, 32, 34, 40, 41, 47–49]. Thus, to minimize the negative influence on longevity, the restorative procedure of endodontically treated teeth calls for careful consideration of treatment alternatives. In the case of minimal-remaining tooth substance, the retention of a crown has traditionally been provided by a core and the retention of the core by a post. Two fundamentally different techniques find widespread use for the construction of a post and core: (1) a post and core cast as a single unit and (2) a prefabricated post upon which a core is directly produced in, e.g., amalgam or resin composite. The first prefabricated posts on the market were made of stainless steel or gold-plated brass. Later, posts of titanium alloys became popular, and recent developments have introduced posts made of fiber-reinforced resin composite or ceramic. Simultaneously, adhesive dentistry, offering reliable bonding to tooth substance and to restorative materials via various pretreatments, has markedly progressed to widen the possibilities also for post and core treatment. As mentioned, the post serves to retain the restoration. However, the post should also serve to protect the remaining tooth structure. These two functions of endodontic posts may be evaluated by determining the retentive ability of the posts and the fracture resistance of endodontically treated teeth provided with post-retained restorations. The latter may also be indirectly determined by analysis of the stress-distributing characteristics of the posts. Post-related factors that have been found to exert influence on retention of posts and protection of tooth structure include: shape, length, diameter, surface design, and stiffness of the post as well as the type of luting cement used. As regard to post shape, parallel-sided posts provide better retention and less stress formation and increased fracture resistance than do tapered posts [2, 5, 18, 28, 37, 38, 42, 46]. An increase in post length has a similar, positive effect [2, 5, 7, 8, 17, 42]. However, some restrictions apply to post length. First, the apical seal of the endodontic restoration must be ensured through maintenance of 5–6 mm of apical gutta-percha [1, 24, 53]. Second, dentin in the apical third of teeth is often very thin. This not only increases the risk of parietal perforation, but it also weakens the tooth to a relatively high degree, thereby increasing the risk of root fracture. Little evidence is available as regard to the influence of post diameter. Consensus regarding retention seems to be that an increase in post diameter cannot be relied on as a measure to improve retention [5]. A study based on finite element analysis found that stress formation in the dentin decreased when post diameter was increased [2]. Conversely, another study showed that thinner posts provided increased resistance to fracture [48]. A consensus regarding fracture resistance seems to be that the root canal should not be uncritically enlarged, as decreasing the bulk of dentin weakens the tooth and reduces the resistance to fracture [5, 12, 30]. As regard to surface design, serrated posts provide better retention than smooth-sided posts, and threaded posts provide even better retention [5, 7, 37]. However, the superior retention of threaded posts, obtained by engaging in dentin, simultaneously is accompanied by increased stress formation within the root [36, 43] and thus by an increased risk of root fracture [5, 10, 12, 43]. Post stiffness, which is a combination of post diameter and elastic modulus of the post material, is another important characteristic, but an unsettled discussion prevails as to optimal stiffness. On the one hand, insufficient stiffness will allow excessive distortion of the restoration at the margins during function, leading to breakdown of cement and risk of secondary caries. Moreover, a decrease in the elastic modulus of the post material has been found to increase stress formation in the root and to decrease fracture strength of the restored tooth [2, 23, 38, 39]. Thus, low-modulus posts fail sooner or at lower stress values than do high-modulus posts. On the other hand, several studies have found that low-modulus posts display failures that cause little damage to the remaining tooth structure (loss of marginal seal, loss of retention, core fracture, and post fracture), while high-modulus posts are associated with a higher incidence of root fractures when they finally fail; that is, they cause more damage to the remaining tooth structure and often result in the extraction of the tooth involved [22, 23, 39, 47]. Retention of posts in the root canal is primarily sought through the use of a luting agent. Zinc phosphate cement has been the luting agent of choice for many years. The progress within adhesive dentistry and the advent of ceramic- and fiber-reinforced resin composite posts have prompted questions of the usefulness of resin cement for the luting of posts. Zinc phosphate cement provides retention by mechanical interlocking in irregularities in the surface of the root canal on one side and in the surface of the post on the other. After appropriate pretreatment of the respective surfaces, resin cements provide retention not only by mechanical interlocking but also by micromechanical and chemical adhesion. In vitro studies have found improved retention, less and more favorable stress formation, and improved fracture resistance with resin cements than with zinc phosphate cement [2, 6, 25, 31, 51]. Other factors from those related to the post are of importance for success of post-retained restorations. Thus, researchers agree that the extent of remaining tooth structure is a key issue for fracture prognosis [9, 12, 30, 42, 47]. Another paramount factor is the ferrule effect: Teeth that allow preparation of a 1.5–2-mm ferrule show superior fracture resistance [44, 45, 54]. The foregoing has shown that numerous factors influence the longevity of endodontically treated teeth provided with post-retained restorations and that it would be practically impossible to carry out a randomized controlled clinical trial that includes all relevant variables. Previous studies have shown annual failure rates related to post and core treatment of up to 5% [4, 9, 11, 15, 41, 46]. The purpose of this survey of failed post-retained restorations was to analyze reported failures in an attempt to identify factors that are critical to failure and to type of failure. Materials and methods A questionnaire was developed and piloted, and in June 2000, it was posted, in five copies, to the members of the Danish Dental Association who worked in private practice (3,444 practitioners). The practitioners were asked to complete the questionnaire in case a patient presented with a failed post-retained restoration. Questions were asked about age and gender of the patient, tooth involved, number of posts, type, design, length, and diameter of the post(s), type of core, type of luting agent, type of restoration, height of any ferrule, degree of tooth destruction, functioning time until failure, and type of failure. After 3 years, 260 questionnaires had been received, and the collection of questionnaires was terminated. Data were transferred to a computer and analyzed by use of the Statistical Analyzing System (SAS). Basic descriptive statistics including frequency distributions were performed. Selected data were analyzed by chi-squared tests and Fischer’s exact probability tests (α = 0.05). Cross tabulations were carried out to further characterize the data and to identify codeterminants of functioning time until failure and codeterminants of type of failure. Results The 260 questionnaires had been completed by 171 practitioners. The response rate per question varied between 26 and 98%. Of the 260 patients, slightly more were female (58%) than male (42%). The age of the patients varied between 18 and 91 years, and the age distribution was statistically similar among female and male patients. The sample involved nearly three times as many maxillary teeth (74%) than mandibular teeth (26%). In the maxilla, there were fewer molars (15%) than premolars (38%) and incisors and canines (47%), and in the mandible, there were fewer incisors and canines (8%). Table 1 shows the distribution of the types of posts and cores: Slightly fewer teeth had been restored with cast post and cores than with prefabricated posts and direct cores. Of the 114 cast post and cores, 57% had been cast in a gold alloy, 25% in a silver–palladium alloy, and 18% were unaccounted for. Of the 130 prefabricated posts used in combination with a direct core, 51% were titanium posts, 20% were stainless steel posts, 8% were carbon fiber-reinforced resin composite posts, and 21% posts were either of an alternative or unknown material. Table 1Distribution of type of post and cores among failed restorationsType of post and coreNumberCast post and core114Prefabricated post with direct core130Prefabricated post with cast core5One-piece post crown7Total256 Of the 260 failed post-retained restorations, 54% comprised tapered posts, 22% comprised parallel-sided posts, 20% comprised posts of a combined tapered-parallel design, and 4% comprised posts with a design that was not accounted for. The majority of the cast posts (76%) and of the stainless steel prefabricated posts (73%) were tapered. The titanium posts were equally distributed between the three designs (38, 31, and 31%), and the carbon fiber-reinforced resin composite posts were either parallel sided (50%) or of a combined design (50%). Of the 176 failed post-retained restorations that were specified as regard to the maximum diameter of the post, 68% comprised posts with a diameter equal to or less than one third of the diameter of the root, and 32% comprised posts with a diameter of more than one third. Of the 237 failed post-retained restorations that were specified as regard to length of post, 76% comprised posts with a length of more than 50% of the total length of the root. Table 2 shows the distribution of the posts as regard to type, material, and length of posts: For all posts, except those of stainless steel, the majority of the posts were of a length that was equal to or more than half the length of the root. For the stainless steel posts, 50% were shorter than half the length of the root. Table 2Distribution of posts of failed restorations as regards post type, post material, and post length in percent of root lengthTypeMaterialLengthTotal15–49%50–74%75–90%CastGold9391260Silver–palladium318829PrefabricatedTitanium14301761Stainless steel1311125Carbon fiber0729Total3910540184 Of the 130 teeth with a failed restoration that had been treated with a prefabricated post and a direct core, 29% had a core made in amalgam, 59% a core made in resin composite, 3% a core made in glass ionomer cement, and 9% a core made in a material that was not accounted for. Amalgam had been used more often than resin composite for stainless steel posts (70 vs 30%), whereas resin composite had been used more often than amalgam for titanium posts (84 vs 16%) and, in all cases, for carbon fiber-reinforced resin composite posts. Of the 260 failed post-retained restorations, 40% had been luted with zinc phosphate cement, 17% with glass ionomer cement, 10% with resin cement, and 33% posts with an agent that was not accounted for. The majority of the cast posts (68%) and of the stainless steel prefabricated posts (93%) had been luted with zinc phosphate cement. Most titanium posts had been luted with zinc phosphate cement (48%) or glass ionomer cement (37%). Of the carbon fiber-reinforced resin composite posts, 90% had been luted with resin cement and 10% with glass ionomer cement. Of the 260 failed restorations 69% had a ferrule, 29% did not have a ferrule, and 2% were not accounted for. As regards the degree of tooth destruction before luting of the now failed post-retained restoration, only 25% restored teeth out of 260 were accounted for. In 47 cases, the whole clinical crown had been missing at the time of restoration, and in 18 cases, between one half and three fourths of the clinical crown had been missing. Table 3 shows for how long the restorations had functioned in the mouth at the time of failure. The time of functioning of the 178 restorations that were accounted for varied between 3 months and 38 years. The mean survival time until failure was 11 years, and the median survival time was 8 years. Of the 178 restorations, 61% had functioned 10 years or less at the time of failure. Of the 260 restorations, 51% displayed fracture of the tooth, 30% displayed loosening of the post, 17% displayed fracture of the post, and 2% displayed a type of failure that was not accounted for. Table 3Distribution of failed post-retained restorations as regards functioning time until failureFunctioning time (years)Number0–1152–5506–104311–204321–3827Total178 Among the post-retained restorations that failed after a relatively short functioning time (0–10 years) as compared to a relatively long functioning time (11–38 years) were: (1) significantly more parallel-sided and combined tapered-parallel posts than tapered posts, (2) significantly more carbon fiber-reinforced resin composite prefabricated posts than titanium and stainless steel prefabricated posts, (3) significantly more cast silver–palladium posts than cast gold posts, (4) significantly more resin composite cores than amalgam cores, and finally (5) significantly more posts luted with resin cement or with glass ionomer cement than with zinc phosphate cement. These results are unexpected, and as the “Discussion” will argue, the results do not truly reflect factors critical to functioning time. Several factors were significantly related to type of failure. (1) The frequency of tooth fracture was similar for female and male patients (51 vs 50%) as was the frequency of loosened posts (36 vs 25%), but the frequency of fractured posts was higher for male than for female patients (24 vs 13%). (2) The shorter the post, the lower the frequency of post fracture (Table 4). (3) Tapered posts more often than parallel-sided and combined tapered-parallel posts displayed fracture of tooth (61 vs 39 and 41%) and less often fracture of post (9 vs 30 and 31%). (4) Stainless steel prefabricated posts more often than titanium prefabricated posts displayed fracture of the tooth (81 vs 36%). (5) Teeth restored with amalgam cores displayed mainly tooth fractures (70%), teeth restored with cast gold cores displayed mainly tooth fractures (51%) or loosening of post (35%), and teeth restored with resin composite cores displayed all three types of failure with almost similar frequency (35, 25, and 40%). (6) More teeth in which the post had been luted with zinc phosphate cement displayed tooth fracture than teeth for which glass ionomer cement or resin cement had been used (54 vs 34 and 36%). (7) Compared with the frequencies of post fracture and loosening of the post, the frequency of tooth fracture increased with increasing functioning time until failure, and correspondingly, restorations that failed early on showed an equal frequency of the three types of failure (Table 5). Table 4Distribution of failed post-retained restorations as regards length of post and type of failureLength (mm)FailureTotalFracture of toothFracture of postLoosening of post2–520211336–1077204714411–201212731Total1093465208Table 5Distribution of failed post-retained restorations as regards functioning time of restoration and type of failureFunctioning time (year)FailureTotalFracture of toothFracture of postLoosening of post0–1546152–5141511406–10188174311–20272144321–38183526Total823253167 Discussion Five percent of the private practitioners that had been invited to participate in the survey did so. Possible explanations for this low response rate include the open invitation design, the relative extensive questionnaire, and the fact that the questionnaire could not be completed once and for all but had to be procured whenever a patient presented with a failed post-retained restoration. Considering that prosthodontics is not a registered specialty in Denmark, there is no reason to believe that the treatments provided by the participating practitioners were of another type or quality than those provided by private practitioners in general. The fact that the questionnaires were filled out by individual practitioners in their everyday environment and not by calibrated investigators in an optimal research facility explains that the response rate to each question was not 100% and also implies some uncertainty as to the validity of the answers, especially those that included an element of estimation, e.g., of post diameter. The abovementioned factors call for some caution when interpreting the results of the survey. As regards age and gender, the patient sample is not only representative of the private practice patient population in Denmark as such but also similar to patient samples in previously published clinical studies of post-retained restorations [9, 27, 46]. These clinical studies also supply explanations for the present finding that more maxillary teeth, especially incisors and canines, than mandibular teeth presented with failed post-retained restorations: Not only are more maxillary teeth than mandibular teeth treated with posts [9, 11, 26, 46], but post-retained restorations in maxillary teeth have also been reported to fail relatively more frequently than similar restorations in mandibular teeth [46]. In particular, post-retained restorations in anterior maxillary teeth have been found to show high failure rates, presumably because of higher functional horizontal forces acting on the anterior teeth than on posterior teeth [13, 26, 27, 46]. The types of posts, cores, and cements used for the failed restorations, not only reflect that preferences vary among practitioners but also that the age of the restorations vary widely, as did the materials and treatments available at the time of restoration. Given the design of this survey, i.e., we have only considered restorations that have failed, we have no knowledge of the entire population of post-retained restorations. Thus, the survey does not allow distinction between an over-representation of a given material or treatment caused by outspread use and over-representation caused by poor longevity. The length of time that the post-retained restorations had functioned before failure varied from 3 months to 38 years with a mean survival time until fracture of 11 years. In agreement with the findings of previous surveys of failed post-retained crowns [21, 50], there was a tendency to a relatively high initial failure rate. However, the proportion of restorations that failed during the first 10 years was lower in the present survey (61%) than in the survey by Lewis and Smith (94%) [21]. In the survey of Turner [50], only 17% had survived more than 5 years. The longer lifespan found in the present survey may reflect that the samples of the previous surveys included posts that were generally shorter and more often smooth and/or tapered than the posts of the present survey. These features are likely to have increased the prevalence of loosened posts at an early stage. A number of specific types of posts or materials were found to have significantly high prevalence in the group of post-retained restorations that failed after a relatively short functioning time: carbon fiber-reinforced resin composite posts, posts cast in silver–palladium, parallel-sided and combined tapered-parallel posts, resin composite cores, and resin cement and glass ionomer cement. Some of these posts and materials are probably found in this group of restorations that failed after a short functioning time, not because they in fact are associated with short functioning time but because they have been on the market for a relatively short time compared with the alternatives and thus are over-represented in the total group of restorations with a short functioning time. The most commonly reported type of failure was tooth fracture followed by loosening of the post and, finally, fracture of the post. This relative distribution is at odds with that of previous, retrospective studies, which found loosening of the post to be the most common type of failure [3, 21, 46, 50]. Failures in the form of loss of retention are most often restorable, whereas tooth fractures are most often nonrestorable and lead to extraction of the tooth. It is possible that it was easier for the practitioner to remember to submit a questionnaire when a more drastic tooth fracture occurred than when a post had lost retention and had to be recemented. Such a situation would tend to underestimate the proportion of loosened posts. An alternative explanation is that the distribution between types of failure has indeed changed to reflect a decrease in the use of smooth and tapered posts. Several factors were found to significantly influence the distribution between types of failures. One factor was gender of the patient: Whereas there were, relatively speaking, equally many male and female patients in the group of post-retained restorations that displayed tooth fracture or loosening of post, there were more male patients in the group of restorations that displayed post fracture. It may be that the greater bite forces exerted by men [16] not only increase the failure rate of post-retained restorations as such [46] but also predispose for fracture of the post. Another factor was post length: Failed shorter posts were associated with a lower prevalence of post fracture than were longer posts. This finding is not readily explained on the basis of stress analyses but may reflect that there was an over-representation of stainless steel posts and/or of tapered posts in the group of short posts: Stainless steel has a higher elastic modulus and is a stronger material than gold alloys and titanium alloys [52] and may thus be expected to withstand higher stresses. Tapered posts were associated with a higher prevalence of tooth fracture than were parallel-sided and combined tapered-parallel posts and with a correspondingly lower prevalence of post fracture. This significant tendency to fail because of tooth fracture confirms results of previous studies [42, 46]. Likewise, failed stainless steel posts were associated with a higher prevalence of tooth fracture than were titanium posts. This could be an indirect result of most stainless steel posts being short, having high elastic modulus, and having a tapered shape in contrast to most titanium posts being parallel sided. Teeth that failed and had been restored with amalgam cores displayed significantly more tooth fractures than teeth restored with cores of resin composite and cast gold post and cores. Because of intermediate mechanical properties, e.g. elastic modulus, of amalgam as compared to resin composite and gold alloy, mechanical properties cannot easily explain the high prevalence of tooth fracture. Instead, it is likely to be caused not by the amalgam core itself but by other factors that characterize the posts with which amalgam was used. Amalgam was the preferred core material for stainless steel posts, whereas resin composite was the preferred core material for titanium posts. As just mentioned, stainless steel posts were more often than other posts tapered, a factor that has also been shown to predispose for tooth fracture. The fact that zinc phosphate cement also seemed to predispose for tooth fracture may similarly be explained, not by inferiority of the material itself but by the posts with which it was used: A relatively higher percentage of posts cast in silver–palladium and of prefabricated stainless steel posts had been luted with zinc phosphate cement than with any other cement, and these two types of posts were associated with a high prevalence of tooth fracture. The final factor that was found to be significantly associated with type of failure was the length of time that the post-retained restorations had functioned until failure: The prevalence of tooth fracture increased with increasing functioning time until failure. This finding is in contrast to that of previous surveys of failed post-retained crowns in which no correlation between type of failure and length of functioning time was found [21, 50]. The discrepancy can be explained by the fact that the present survey included teeth of a much wider range of functioning time until failure and higher mean age at failure. This survey did not confirm the generally accepted, paramount influence on fracture prognosis of extent of remaining tooth structure and of a ferrule [9, 12, 30, 42, 44, 45, 47, 54]. With regard to extent of remaining tooth structure, the reason is that an exceptionally low response rate did not allow statistical analysis of this parameter on a possible influence on functioning time and type of failure. The fact that the survey also did not find a significant influence of the existence of a ferrule calls for the following comments. First, the fact that most restorations were reported to have a ferrule reduced the probability that the statistical analysis would find a significant effect. Second, it is very positive to note that clinicians are aware of the importance of including a ferrule in the preparation. Third, the fact that restorations failed despite that the crowns had a ferrule points to the relevance of optimizing other elements in the restoration of an endodontically treated tooth. Conclusions The following are the conclusions derived from the study: Fracture of the tooth was the most common type of failure among the failed post-retained restorations followed by loosening of the post and fracture of the post.Compared with parallel-sided posts, tapered posts more often displayed fracture of the tooth and less often fracture of the post.Post-retained restorations that had functioned for a long time before failure showed an increased risk of tooth fracture compared to loosening of the post and fracture of the post.Fracture of the post was more common among male patients than among female patients.

Int_J_Cardiovasc_Imaging-3-1-2121118

Alterations in aortic elasticity in noncompaction cardiomyopathy

Background Noncompaction cardiomyopathy (NCCM) is a recently recognized disorder frequently associated with systolic and diastolic heart failures. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls. Introduction Noncompaction cardiomyopathy (NCCM) is a recently recognized rare disorder [1, 2]. It is characterized by prominent myocardial trabecularizations, and deep intertrabecular recesses leading to the spongy appearance of the myocardium [3]. The disease is frequently associated with systolic and diastolic heart failure (HF), ventricular arrhythmias, and systemic embolization [4]. Alterations in arterial function have been demonstrated in patients with HF [5, 6]. With two-dimensional transthoracic echocardiography (TTE), ascending aortic diameter changes during a heart cycle can be measured. When blood pressure data are also available, aortic elasticity can be characterized [7, 8]. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls. Methods Study population A total of 20 patients with typical echocardiographic features of NCCM were investigated [3, 9]. Their clinical and demographic data are presented in Table 1. Clinical assessment included medical and family history, physical examination, electrocardiography, two-dimensional echocardiography, and, in most cases, contrast echocardiography. Their results were compared to 20 age- and gender-matched controls without apparent cardiovascular disease. Informed consent was obtained from each patient, and the study was approved by the institutional review board and complied with the Declaration of Helsinki. Table 1.Clinical and demographic data of NCCM patientsNCCM patientsAge (years)38 ± 16Male (%)8 (40)Diabetes (%)1 (5)Index eventArrhythmia (%)6 (30)Heart failure (%)8 (40)Screening (%)6 (30)ElectrocardiogramAtrial fibrillation (%)2 (10)LV hypertrophy (%)3 (15)Left bundle branch block (%)6 (30)NCCM noncompaction cardiomyopathy Diagnostic criteria for NCCM Previously proposed echocardiographic diagnostic criteria for NCCM by Jenni et al. [3] were used: (1) absence of coexisting cardiac anomalies, (2) segmental, excessive thickening of the left ventricular (LV) wall with a two-layered structure: a thin, compacted epicardial layer and a much thicker, noncompacted layer with the characteristic appearance of numerous, prominent trabeculations (meshwork) and deep intertrabecular recesses, (3) color Doppler evidence of deeply perfused intertrabecular recesses, and (4) predominant localization of thickening in the LV apical, midlateral, and midinferior walls. Hypertensive heart disease was excluded by clinical and echocardiographic examinations (septal thickness < 13 mm). Blood pressure measurement Systolic and diastolic blood pressures (SBP and DBP, respectively) were measured in the supine position with an automatic mercury cuff sphygmomanometer from the left arm after 10 min of rest. The first and the fifth Koratkoff sounds were taken for the SBP and DBP. Blood pressure values were averaged from three consecutive measurements. None of the patients or controls used coffee or tea within 1 h before blood pressure measurements. Transthoracic echocardiography All patients underwent a complete two-dimensional TTE and Doppler study using a Philips Sonos 7500 echocardiography equipment (Philips, Best, The Netherlands) in the left lateral decubitus position from multiple windows. The LV wall segments were analyzed according to the 9-segment model as described by Jenni et al. [3]. Systolic and diastolic ascending aortic diameters (SD and DD, respectively) were recorded in M-mode at a level of 3 cm above the aortic valve from a parasternal long-axis view (Fig. 1). Gain, depth, and sector angles were individualized for the best measurement. The SD and DD were measured at the time of maximum anterior motion of the aorta and at the peak of the QRS complex, respectively. Fig. 1Measurements of systolic (SD) and diastolic (DD) diameters of the ascending aorta are shown on the M-mode tracing obtained at a level 3 cm above the aortic valve Evaluation of aortic stiffness Aortic stiffness index (β) was defined as ln(SBP/DBP)/[(SD - DD)/DD], where ln is the natural logarithm. Statistical analysis Data are reported as means ± standard deviation. For variables, Student’s t test and analysis of variance (ANOVA) test were used where needed. A value of p < 0.05 was considered statistically significant. SPSS 12.0 software was used for statistical calculations. Reproducibility of echocardiographic measurements The reproducibility of the aortic diameter measurements was tested in all NCCM patients at both systole and diastole by two independent, blinded observers. Agreement between the two observers was verified using the Bland–Altman method [10]. Results Patient population The presenting symptoms were HF in eight (40%) and arrhythmias in six (30%) patients. The remaining six (30%) asymptomatic patients were NCCM relatives and were diagnosed after family screening (Table 1). Three HF patients were in NYHA-class III HF and the other five were in NYHA-class II. Cardiac medication used in NCCM patients were: angiotensin-converting enzyme inhibitors in 13 (65%), beta-blockers in 11 (55%), oral anticoagulant therapy in 10 (50%), diuretics in 8 (40%), digitalis in 2 (10%), and nitrate in 1 (5%). None of the controls received any cardiac medication. Transthoracic echocardiography The number of noncompacted segments in the NCCM patients was 4.6 ± 2.0 in the NCCM group and (as seen in Table 2) these patients had significantly increased LV dimensions and reduced LV ejection fraction. Aortic stiffness index (β) was significantly increased in NCCM patients compared to controls. For controls, NCCM patients with moderate HF, and NCCM patients with severe HF, β values were 3.5 ± 1.1, 7.9 ± 5.5, and 10.4 ± 1.8, respectively (p < 0.001). Aortic stiffness indices of all individual patients and controls are presented in Fig. 2. Table 2.Transthoracic echocardiographic and blood pressure data in NCCM patients and normal subjectsGroup 1 (normal subjects)Group 2 (NCCM patients)LV end-diastolic diameter (mm)47.4 ± 3.661.0 ± 10.9*LV end-systolic diameter (mm)29.9 ± 2.948.2 ± 12.3*LV ejection fraction (%)67.5 ± 5.936.0 ± 17.6*Systolic aortic diameter (mm)26.7 ± 4.126.6 ± 4.4Diastolic aortic diameter (mm)23.3 ± 3.824.6 ± 4.3Pulsatile change in aortic diameter (mm)3.4 ± 1.12.0 ± 1.2*Systolic blood pressure (mmHg)125.2 ± 12.9120.4 ± 17.4Diastolic blood pressure (mmHg)77.8 ± 8.574.1 ± 9.6Aortic pulse pressure (mmHg)47.4 ± 8.746.3 ± 11.7Aortic stiffness index (β)3.5 ± 1.18.3 ± 5.2*Continuous variables are given as mean ± standard deviationLV left ventricular, NCCM noncompaction cardiomyopathy*p < 0.001Fig. 2Individual β indices of NCCM patients and control subjects. NCCM noncompaction cardiomyopathy Reproducibility The mean ± standard deviation difference in values obtained by two observers for the measurements of aortic diameter at systole was −0.7 ± 2.2 mm, with a correlation coefficient between these independent measurements of 0.88 (p < 0.01) (Figs. 3a and 4a). At diastole, the difference between these observations was 0.05 ± 1.95 mm, with a correlation coefficient between observations of 0.9 (p < 0.01). The difference in values that were detected by observers was within twofold of the standard deviation of the mean (Figs. 3b and 4b). Fig. 3a Interobserver correlation (r = 0.88, p < 0.01) between aortic systolic diameters in NCCM patients. b Interobserver correlation (r = 0.9, p < 0.01) between aortic diastolic diameters in NCCM patients. NCCM noncompaction cardiomyopathyFig. 4a Reproducibility of systolic aortic measurements in NCCM patients. b Reproducibility of diastolic aortic measurements in NCCM patients. NCCM noncompaction cardiomyopathy Discussion NCCM is a recently recognized disorder characterized by a loosened, spongy myocardium associated with a high incidence of progressive systolic and diastolic HF. In recent studies, alterations have been demonstrated in arterial function in patients with chronic HF [11–14]. To the best of authors’s knowledge, this is the first time that the aortic distensibility in a series of NCCM patients was examined. In this study, increased aortic stiffness index (β) was found in NCCM patients compared to age- and gender-matched controls. In prior studies, it has been shown that pulsatile changes in ascending aortic vessel diameter can be indirectly measured during routine TTE. Stefanadis et al. [7] found that noninvasive measurements of aortic distensibility were as accurate as invasive methods. Aortic stiffness index (β) relies on aortic and blood pressure data and is one of the most frequently used parameters to characterize arterial stiffness [7, 8]. Increased aortic stiffness is an independent risk factor, predictor of cardiovascular mortality, and a contributor to LV afterload [15, 16]. Increased aortic stiffness occurs early during the development of pacing-induced congestive HF in animals [17, 18] and has also been described in clinical patients with HF [5, 6], with comparable changes in arterial distensibility in ischemic and idiopathic dilated cardiomyopathy [19]. Rerkpattanapipat et al. [11] demonstrated that the distensibility of the proximal aorta is markedly reduced in older patients with HF due to LV systolic dysfunction beyond the changes from the aging process. They also described a correlation between exercise intolerance and reduced aortic distensibility. Giannattasio et al. [12] described that arterial compliance is impaired in congestive HF, and although more marked in severe congestive HF, the impairment is manifested in mild congestive HF as well [12]. Nakamura et al. [13] described vascular hypertrophic remodeling and endothelial dysfunction-associated alterations in vascular wall elastic properties in limb muscle conduit arteries in patients with congestive HF. Poelzl et al. [14] described an intriguing relationship between phenotype changes and functional impairment in peripheral conduit arteries of HF patients. This process is very similar to what is known for ventricular remodeling in HF. There are several potential factors affecting aortic function in patients with HF [11]. Neurohormonal changes associated with HF include an increase in sympathetic drive and an activated renin–angiotensin system resulting in increased plasma norepinephrine levels causing vasoconstriction and sodium retention [20–23]. Angiotensin II has a hypertrophic effect on smooth muscle cells of the vascular wall [24]. Early atheromatous changes and endothelial dysfunction can also be associated with HF [25], and can affect vascular elasticity. However, there is also an interaction between aortic stiffening and HF. Aortic stiffening can increase LV load causing LV stiffening with increased wall tension, early impairment in diastolic LV relaxation, and contractility inducing LV hypertrophy and fibrosis [26, 27]. These results suggest that aortic stiffness may contribute to the progression of systolic and diastolic LV dysfunctions. Study limitations During this study, brachial cuff pressure measurement instead of a direct assessment of aortic pulse pressure by catheter was used. However, previous studies demonstrated an excellent correlation in aortic distensibility measured by invasive and noninvasive methods [7]. Coronary artery disease is correlated with increased aortic stiffness and was not excluded in most but not in all our patients by coronary angiography. The aortic elastic properties of control patients were somewhat larger than those described in the literature. The reason for this can be the larger body mass index (29.0 ± 5.1 kg/m2) in our controls. However, a relatively higher β in the normal subjects in our study only strengthens the abnormal findings in the NCCM patients. Conclusions Increased aortic stiffness can be observed in patients with NCCM with moderate to severe HF. These alterations may be due to HF-induced neurohormonal changes.

Surg_Endosc-4-1-2292805

The effects of laparoscopic cholecystectomy, hysterectomy, and appendectomy on nosocomial infection risks

Background Recent reviews of the literature have concluded that additional, well-defined studies are required to clarify the superiority of laparoscopic or open surgery. This paper presents precise estimates of nosocomial infection risks associated with laparoscopic as compared to open surgery in three procedures: cholecystectomy, appendectomy, and hysterectomy. With the realization of smaller incisions, better cosmesis, less postoperative pain, same-day surgery, speedier postoperative recovery, and the potential for reduced complications, laparoscopic approaches have all but replaced the traditional laparotomic alternatives for certain commonly performed surgical procedures. However, the widespread adoption of laparoscopic techniques into the overall surgical armamentarium has been slowed by a variety of factors, including the learning curves required to integrate new levels of depth perception and fine dexterity, longer operating times without commensurate economic reward, and the nullification of savings from earlier hospital discharge by the cost of disposable surgical instrumentation. Nevertheless, the laparoscopic approach is now widely accepted as the gold standard for cholecystectomy and the surgical treatment of gastroesophageal reflux [1, 2]. Laparoscopic appendectomy has been controversial since its introduction in the early 1980s, particularly for cases of complicated appendicitis. Although laparoscopic appendectomies now account for almost half of appendectomies in the United States [3], several recent reviews of the literature have concluded that additional, well-defined studies will be required to clarify the superiority of laparoscopic or open approaches [4–6]. Despite the demonstration that abdominal hysterectomy is associated with higher morbidity and worse outcomes when compared to the vaginal or laparoscopic approach, the majority of hysterectomies worldwide are still performed in this fashion. For the remaining cases, laparoscopic hysterectomy is least apt to be performed [7, 8]. Although laparoscopy facilitates vaginal hysterectomy for the larger uterus, allows for the concurrent diagnosis and treatment of benign pelvic conditions such as endometriosis or pelvic adhesions, permits concomitant adnexal surgery, and provides the ability to secure and reaffirm intraperitoneal hemostasis at the end of the procedure [9], a meta-analysis of randomized controlled trials comparing different types of hysterectomy published by the Cochrane Collaboration failed to clearly demonstrate the superiority of laparoscopic hysterectomy over vaginal hysterectomy [10]. Some studies have reported that laparoscopic approaches are associated with lower risks of surgical site infections than their open counterparts [11–13], but the effects of laparoscopic surgery on overall nosocomial (hospital-acquired) infection risks have not been established. Since a significant number of nosocomial infections in surgical patients occur after discharge [14, 15], it is likely that comparisons of laparoscopic and open surgeries have underestimated risks. Nosocomial infections are a leading cause of death in the United States, affecting two to three million patients annually [16]. Starting in 2009, Centers for Medicare and Medicaid Services (CMS) will stop reimbursing hospitals for certain complications including surgical-site infections, catheter-associated urinary tract infections, and central-line associated bloodstream infections [17]. Therefore, the establishment of nosocomial infection risks in general is important. The objective of this study is to obtain more precise estimates of nosocomial infection risks associated with laparoscopic and open approaches for cholecystectomy, appendectomy, and hysterectomy. We hypothesize that laparoscopic surgery will reduce the risk of nosocomial infections for each of these surgical modalities. To test these hypotheses we performed a retrospective analysis of more than 11,000 admissions, each with one of the procedures of interest, from 22 hospitals that have implemented a nosocomial infection monitoring system that can detect nosocomial infections up to 30 days post discharge. Methods The Nosocomial Infection Marker (NIM) The Nosocomial Infection Marker (NIM, patent pending, Cardinal Health) monitors and tracks nosocomial infection rates for hospitals and communities. Cardinal Health extracts data from client facilities on an ongoing basis using a secure, Health Insurance Portability and Accoutability Act- (HIPAA) compliant method. Data are cleaned and mapped in real time as they arrive at the Cardinal Health data center by proprietary software systems. Rare exceptions that are not electronically modeled are modeled by technical and clinical experts, processed and loaded. The new models are then used by the systems to process like data in the future. The Nosocomial Infection Marker is a computer algorithm that identifies the existence of nosocomial infections at the microbiological level. Specifically, the NIM algorithm distinguishes likely pathogens from contaminants, identifies duplicate isolates, and temporally determines hospital versus community-acquired pathogen acquisition [18]. In a multihospital study using comprehensive medical records review and gold-standard infectious disease physician discrepancy resolution, the NIM algorithm identified nosocomial infections with 86% sensitivity and 98.5% specificity hospital-wide, statistically outperforming Centers for Disease Control (CDC) case finding methods [18]. Unlike the NIM, traditional CDC case finding methods are subjectively applied with inconsistent results and are only used for certain types of infections, mostly in ICUs [18, 19]. Like the NIM, the performance characteristics of CDC methods have only been formally evaluated in one study [19]. Data The Nosocomial Infection Marker (NIM) was used to identify nosocomial infections during hospitalization and post discharge. Data were extracted from the Cardinal Health (CAH) data repository for the period September 1, 2004 through December 31, 2006 from 22 hospitals in 15 states. Hospitals with matching International Statistical Classification of Diseases and Related Health Problems (ICD-9) procedure codes and more than 100 admissions with a primary ICD-9 procedure for cholecystectomy, appendectomy, or hysterectomy were eligible. These hospitals had a median number of beds of 359, with an interquartile range from 191 to 483 beds; one hospital exceeded 1,000 beds and two had fewer than 150. Mean hospital CMI was 1.56 with a standard deviation of 0.22. Admissions with primary Diagnosis Related Group (DRGs) listed in Table 1 comprised more than 95% of eligible admissions. When these admissions were restricted to adults 18 years and older for cholecystectomy and hysterectomy, and patients 2 years and older for appendectomy, 11,662 admissions were available for analysis. Table 1DRGs included in the analysis% admissions NIM rate% laparoscopySimple presentations (complexity = 0)166Appendectomy W/O complicated Principal Diag W Cc3.162.7167.21167Appendectomy W/O Complicated Principal Diag W/O Cc14.531.1271.66195Cholecystectomy W C.D.E. W Cc0.5214.7516.39196Cholecystectomy W C.D.E. W/O Cc0.210.0025.00197Cholecystectomy Except By Laparoscope W/O C.D.E. W Cc3.649.4311.79198Cholecystectomy Except By Laparoscope W/O C.D.E. W/O Cc2.012.5640.60358Uterine & Adnexa Proc For Non-Malignancy W Cc11.433.3036.53359Uterine & Adnexa Proc For Non-Malignancy W/O Cc25.721.2047.92493Laparoscopic Cholecystectomy W/O C.D.E. W Cc14.864.15100494Laparoscopic Cholecystectomy W/O C.D.E. W/O Cc11.420.68100Complex presentations (complexity = 1)164Appendectomy W Complicated Principal Diag W Cc2.969.2847.25165Appendectomy W Complicated Principal Diag W/O Cc3.393.0454.43354Uterine, Adnexa Proc For Non-Ovarian/Adnexal Malig W Cc2.248.055.36355Uterine, Adnexa Proc For Non-Ovarian/Adnexal Malig W/O Cc2.042.1019.33357Uterine & Adnexa Proc For Ovarian Or Adnexal Malignancy1.8910.005.00Diag = Diagnosis; W/O = Without; C.D.E. = Common Duct Exploration; Proc = Procedure; Malig = Malignancy; W = With; Cc = Complication and comorbidities Data elements included NIMs, age, gender, insurance type (Medicaid, Medicare, private, other), hospital case mix index (CMI), primary DRG, whether or not the admission was through emergency department (ED), and ICD-9 procedure codes. The primary ICD-9 procedure code was used to identify both procedure (cholecystectomy, appendectomy, or hysterectomy) and type of approach (open or laparoscopic) and the primary DRG was used to differentiate simple from complex presentations in an attempt to account for intrinsic infection risks and biases towards open approaches. DRGs indicating malignancy or other complex presentations were assigned a complexity value of 1, as shown in Table 1. Hospital CMI was used to control for differences between and clustering within hospitals. Statistical Analysis Single and multiple logistic regression analyses were performed to quantify the associations between NIM rate and procedure, approach, patient age, gender, insurance type, complexity of presentation, ED admission status, and hospital CMI. The first model pooled all three procedures and included binary variables to adjust for the influence of each procedure on the acquisition of NIMs. Then separate models for cholecystectomy, appendectomy, and hysterectomy were constructed. Finally models were constructed for procedure and approach for wound, urinary tract, bloodstream, and respiratory tract NIMs. Results Hysterectomies comprised 43.3% of all procedures, cholecystectomies 32.7%, and appendectomies 24.0%. The percentage of cholecystectomies, appendectomies, and hysterectomies that were laparoscopic was 84.7%, 65.6%, and 39.5%, respectively. Unsurprisingly, fewer than one-quarter of all patients were male. Approximately 19.3% of admissions were covered by Medicare, 7% by Medicaid, 58.8% by private health insurance, and the remaining 14.8% by other types of insurance. NIM rates were defined as the number of admissions with at least one NIM divided by the total number of admissions. Of the 11,662 admissions, 337 (2.89%) had at least one NIM (Table 2). Overall, NIM rates were higher for open approaches (4.09%) than laparoscopic ones (2.11%). NIM rates were highest for cholecystectomy (3.57%), followed by appendectomy (2.60%), then hysterectomy (2.53%). Table 2Nosocomial infection rates by approach and procedureAdmissionsAdmissions with ≥1 NIMRate (%)11,6623372.89Approach  Laparoscopic70611492.11  Open46011884.09Procedure  Cholecystectomy38081363.57  Appendectomy2803732.60  Hysterectomy50511282.53Approach by procedure  Laparoscopic    Cholecystectomy3226842.60    Appendectomy1840422.28    Hysterectomy1995231.15  Open    Cholecystectomy582528.93    Appendectomy963313.21    Hysterectomy30561053.44 There were 399 NIMs identified in 337 admissions. Of all NIMs identified, 118 (30%) were from surgical wounds, 122 (31%) were from the urinary tract, 37 (9%) were from the blood, 29 (7%) were from the respiratory tract, and 93 (23%) were from other sources. At least one post-discharge NIM was identified in 136 admissions, accounting for 40% of all admissions with a NIM. Of the 147 post-discharge NIMs, 39% were from surgical wounds, 31% were from the urinary tract, 7% were from blood, and 22% were from other sources. Of the 136 total admissions with at least one post-discharge NIM, 92 patients had NIM-associated readmissions. Univariate Analyses Simple logistic regressions examined associations between NIM rates and surgical approaches (laparoscopic or open), and the following covariates: gender (male, female), age (<18 y, 18–34 y, 35–49 y, 50–64 y, 65–74 y, ≥75 y), surgical procedure (cholecystectomy, appendectomy, hysterectomy), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0/1), admitted through ED (0/1), and hospital CMI. The results, summarized in Table 3, show significantly higher NIM rates for males, adults aged ≥65 years, patients undergoing cholecystectomy, complex admissions, and admissions covered by Medicare; and significantly lower NIM rates for laparoscopic surgery, females, adults 18–49 years old, patients undergoing hysterectomy, and those covered by private insurance. NIM rates were positively correlated with hospital CMI, but were unaffected by ED admission status. Table 3Univariate analyses of factors associated with NIMVariable CategoryNIM rate (%)OR95% CIGenderMale3.841.501.19–1.90Female2.59Age<18 years2.410.820.49–1.3918–34 years1.500.460.32–0.6535–49 years2.200.660.52–0.8450–64 years3.411.250.97–1.6165–74 years3.891.411.02–1.95≥75 years7.102.962.25–3.90InsurancePrivate2.110.510.42–0.65Medicare5.242.321.85–2.92Medicaid3.191.120.74–1.68Other2.770.950.70–1.29ApproachLaparoscopic2.110.320.21–0.52Open4.09ProcedureCholecystectomy3.571.411.13–1.76Appendectomy2.600.870.67–1.13Hysterectomy2.530.790.64–0.97CMI2.281.59–3.27ComplexityComplex6.312.742.14–3.50Not complex2.40Emergency department admissionEmergent3.031.060.72–1.55Nonemergent2.88CI: confidence interval; NIM: nosocomial infection marker; OR: odds ratio Multivariable analyses Since ED admission status was insignificant in the univariate analysis, it was excluded from the multivariable analyses. Pairwise correlations of all remaining covariates were performed, and all pairs were reasonably uncorrelated. Therefore, all were included in the multivariable analyses. Results of multiple logistic regression models, which controlled for gender (male, female), age (<18 yr, 18–34 yr, 35–49 yr, 50–64 yr, 65–74 yr, ≥75 yr), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0/1), and hospital CMI, show that laparoscopic procedures reduced the odds of acquiring a nosocomial infections by half, but that the effect is entirely attributable to reduced infection risks in laparoscopic cholecystectomy and hysterectomy with odds ratios (ORs) of 0.34 (p < 0.01) and 0.48 (p < 0.01), respectively. No change in nosocomial infection risk was found for laparoscopic appendectomy. Estimates for individual procedures are shown in Table 4. Table 4Multivariable logistic regression analyses of factors associated with NIMVariableOdds ratio for NIMPooled (n = 11,662)Cholecystectomy (n = 3,808)Appendectomy (n = 2,803)Hysterectomy (n = 5,051)Laparoscopy0.48**0.34**0.970.48**Type of procedure    Cholecystectomy1.87**–––    Hysterectomy1.05 –––Age    <18 years0.83–0.90–    18–34 years0.64*0.470.840.72    50–64 years1.222.13*1.000.96    65–74 years1.02*2.21*0.580.51    ≥75 years1.92**4.04**3.310.61Male1.4*1.111.89*–Type of insurance    Medicare1.421.331.122.09*    Medicaid1.453.47**1.110.79    Others1.291.531.221.32    CMI1.63*1.091.312.88*    Complexity2.45**NS3.952.54**** Statistically significant at the 1% level* Statistically significant at the 5% level Consistent with results of the univariate analyses, multivariable regression showed a significantly higher risk of NIMs for males, patients aged ≥65 years, and complexity of presentation at the time of admission. CMI was found to be significant in both univariate and multivariable analyses [OR = 1.63 (CI95 1.11–2.40), p < 0.05], and this risk was significantly higher for hysterectomy patients, for whom the odds of acquiring a NIM were as high as 2.88 times that of not acquiring NIM. Analysis of the same dataset by source of infection (urinary tract, wounds, respiratory tract, bloodstream, and others) revealed that the overall infection rates at each of these sites were all statistically significantly lower for laparoscopic approaches, as summarized in Table 5. The odds of acquiring a site-specific infection were statistically significantly lower for all sites in laparoscopic cholecystectomy and for wound sites in hysterectomy. Table 5Odds ratios by sourceUrinary tractWound Respiratory tractBloodstreamOthersOverall OR (95% CI)0.61 (0.38–0.96)0.41 (0.27–0.62)0.20 (0.08–0.49)0.31 (0.14–0.65)0.52 (0.33–0.82)By procedure    Cholecystectomy0.48 (0.24–0.97)0.20 (0.11–0.39)0.17 (0.06–0.45)0.23 (0.10–0.55)0.34 (0.18–0.64)    Appendectomy0.83 (NS)1.06 (NS)0.27 (NS)Too few NIMs0.91 (NS)    Hysterectomy0.76 (NS)0.27 (0.09–0.79)Too few NIMs 0.48 (NS)0.62 (NS)OR: odds ratio, CI: confidence interval, NS: not significant There were no significant differences between laparoscopic versus open appendectomy for all sources of nosocomial infections (urine, blood, wound, respiratory, and other). However, laparoscopic appendectomy is associated with a statistically significantly higher risk of abscess (p < 0.05), a finding consistent with the literature [12]. Forty-one percent of patients with a nosocomial infection had at least one post-discharge nosocomial infection, and 58 of 115 (50%) of surgical wound infections were post discharge. There were 118 readmissions associated with at least one post-discharge NIM, and post-discharge NIMs associated with readmission were significantly lower for laparoscopic approaches (p < 0.001). Excluding appendectomy, the odds ratio for laparoscopic versus open NIM-associated readmission was 0.346 (CI95 0.19–0.63). Discussion This study demonstrates that laparoscopic cholecystectomy and hysterectomy reduced the overall odds of acquiring nosocomial infections from all sources by more than 50% and reduced the odds of readmission with nosocomial infection by two-thirds. Laparoscopic appendectomy showed no differences in overall nosocomial infection risks compared to open surgery. The findings for wound infections are consistent with results from randomized trials, which have reported statistically significantly lower surgical site infection rates for laparoscopic approaches [10–12, 20]. This study also demonstrates statistically significant differences in source-based infection risks by procedure and approach. Specifically, wound, bloodstream, respiratory tract, urinary tract, and other nosocomial infections were all statistically significantly less likely to occur in association with laparoscopic cholecystectomy. Risks of wound infections in laparoscopic hysterectomy were also significantly lower than in open procedures. However, no differences in infection risks were found between laparoscopic and open appendectomy. It was important to stratify admission by complexity of presentation to avoid biases associated with complex presentations, higher intrinsic infection risks, and surgical approaches; for example, primary DRGs indicating malignancy or complex presentations (Table 1) were associated with open surgical approaches in 69% of admissions, whereas simple presentations were associated with open approaches in only 35% of admissions. Complex presentations are also commonly believed to be at higher risk of infections, an association that is also demonstrated in this analysis. Therefore, by controlling for presentation complexity, this analysis accounts for some of the intrinsic risk of infection as well as a bias towards open surgical approaches. The difference in patient severity between hospitals was accounted for by using CMI, and indeed CMI is significant in the univariate and multivariable models, with CMI contributing to nosocomial infection risks. Interestingly, admission through the emergency department was not significant in determining differences in nosocomial infection risks. One possible explanation is that emergency department use is a crude measure of patient severity because it may also be related to other factors such as time of day of admission and socioeconomic status. However, other variables associated with comorbidity, like age and certain payer types, were significant. Limitations While a variety of confounders were controlled for, this study is limited by the absence of certain data; for example, antibiotic use, anesthesia scores, wound class, body mass index, prior hospitalization, and certain comorbidities (i.e., cardiovascular status, diabetes mellitus, and immunodeficiency) were unavailable. These variables could explain additional NIM risk. Although omitted-variable bias is often a concern in multivariable modeling using retrospective databases, the similarity of findings in our univariate and multivariable analyses suggest that these results are robust. This study documents for the first time that laparoscopic hysterectomy and cholecystectomy are associated with statistically significantly lower overall risks of nosocomial infections (p < 0.01). Laparoscopic hysterectomy and cholecystectomy were also associated with statistically significantly fewer readmissions with nosocomial infections (p < 0.01). Differences in infection risks between laparoscopic and open appendectomy were not found to be statistically significant, suggesting that these differences are likely small or nonexistent. Where differences in risks have been demonstrated, future studies should be performed to quantify their effects on health care costs and length of stay. Other important directions for future research include controlling for potentially important confounders to test the robustness of our results and extending the analysis to examine the effect of laparoscopic versus open surgery on the risk of nosocomial infections for additional surgical procedures. Another interesting avenue for future research would be to examine the effect of hospital volume on the rate of nosocomial infections.

Anal_Bioanal_Chem-3-1-2117336

Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm−2 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL−1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening. Introduction A demand for smaller enzyme reactors has emerged in recent years, as a consequence of ongoing miniaturization in the biochemical and analytical sciences. These micro-reactors have been used in biocatalysis and biosensing. In the field of proteomics the reactors are a tool in peptide mapping, in which proteins are identified via peptide fragment identification after proteolysis. Currently, in spite of its limitations, most of these analyses are conducted by means of 2D gel electrophoresis followed by digestion of the proteins, liquid chromatographic (LC) separation, and mass spectrometric (MS) identification of the peptides [1–3]. The most time-consuming step in this procedure is digestion of the protein using a protease. In general, every protein to be investigated is individually incubated with the protease at a concentration of approximately 1–2% protein weight for 2 to 18 h at an elevated temperature (typically 37 °C). In addition to the long incubation time needed, a certain level of auto-digestion of the protease can be expected. To reduce sample handling, digestion time, and the risk of sample contamination, methods for the on-line digestion of proteins have been developed that use proteases immobilized on a solid support. Immobilized enzyme reactors have been developed and used over the years for several industrial and analytical purposes [4–6]. An obvious benefit for immobilizing biocatalysts is the fact that the enzyme can be used in several catalytic cycles and that both catalyst and reaction mixture can easily be separated. Moreover, immobilized enzymes generally show an improved stability even at more extreme reaction conditions. Several procedures have been developed for immobilization of enzymes, e.g. adsorption or encapsulation in a matrix or membrane. Alternatively, and more often used, is the covalent attachment of biocatalysts to carrier materials, which allows the immobilization of a large amount of enzyme for a high activity per surface area. Generally, particulate large-pore carrier materials are used, such as controlled-pore glass [7, 8], silica [9], or polymers like the commercially available poroszyme [10–12]. Current research in the production of immobilized enzymes is focused on the use of monolithic materials, as they enable efficient fragmentation of proteins [13–17]. Although both commercially available and self-prepared reversed-phase capillary monolithic columns have successfully passed reproducibility assessment [18, 19], synthesis of monoliths suitable for small-scale enzyme reactors can still be troublesome. Materials suitable for the fabrication of larger-scale enzyme reactors are commercially available from BIA Separations (Ljubljana, Slovenia). Although it is possible to apply an immobilized enzyme reactor (IMER) positioned after the separation column [20], most papers dealing with on-line digestion of protein samples position the IMER upstream of the separation column. In these cases the sample is first digested and the resulting peptide fragments are separated and identified by LC–MS. This approach is often employed in multi-dimensional LC methods [13, 21, 22], and has also found application in peptide mapping using capillary electrophoresis [23, 24]. Alternatively, as recently shown by Zhao et al. [25] and Krenkova et al. [26], who covalently coupled trypsin to the wall of fused-silica nanoelectrospray emitters, a protein sample can be analyzed by direct infusion into a mass spectrometer. This paper describes the development of trypsin-modified open-tubular micro-reactors. The chemistry was controlled and optimized using surface plasmon resonance (SPR), a technique allowing sensitive and real-time monitoring of surface reactions such as protein binding [27]. The surface modification resulting in the highest enzyme immobilization yield, was used to covalently immobilize the trypsin on the inside wall of a fused-silica capillary. The constructed trypsin micro-reactor, which is compatible with micro- and nano-LC, was further characterized. The influence of reaction time, pH, temperature, and reactor stability were investigated with the model substrate insulin B-chain. The reactor was also applied to digestion of the proteins cytochrome C and myoglobin. The produced peptides were analyzed by liquid chromatography–mass spectrometry. Experimental Materials The SPR equipment used was from IBIS Technologies (currently available from Eco Chemie, Utrecht, The Netherlands) equipped with a 200-μL polycarbonate cuvet. The gold-sensor disks, purchased from SSENS (Hengelo, The Netherlands), were positioned on the IBIS-prism using index-matching oil from R.P. Cargille Laboratories (Cedar Grove, USA). PEEK nuts, unions, tubing, and loops were from Upchurch (Santa Monica, USA). Manual injections during the preparation of the reactors were performed using a Rheodyne 7010 injector (Inacom, Veenendaal, The Netherlands) equipped with a 1-mL PEEK loop. Model 10ADvp HPLC pumps from Shimadzu (Kyoto, Japan) were used for reactor preparation and activity determinations. The water used for washing and to prepare buffers was produced by a Sartorius Arium 611 ultrapure water system (Nieuwegein, The Netherlands; conductivity >18.2 MΩ cm). The model ABS759A UV absorbance detector was equipped with a capillary flow-cell (75-μm i.d.) and was obtained from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). On-line digestion experiments with micro-HPLC separations were conducted using LC-Packings instruments and columns (Amsterdam, The Netherlands). The equipment consisted of an injector (Famos), nanovalve column switcher (Switchos), nanopump (Ultimate), and a photodiode-array detector (PDA) equipped with a micro flow cell (45 nL). The reversed-phase pre-columns were 5 × 0.3 mm with 5-μm 100 Å C18 particles. The 150 × 0.3 mm reversed-phase micro-column contained 3-μm 100 Å C18 PepMap particles. The mass spectrometer was an Agilent LC/MSD XCT ion trap (Amstelveen, Netherlands). Acetic acid, boric acid, calcium chloride (CaCl2), ethanol, ethanolamine (EA), hydrochloric acid (HCl), hydrogen peroxide, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide (NaOH), and sulfuric acid were purchased from Merck (Darmstadt, Germany). HPLC grade acetonitrile and ethanol were from Biosolve (Valkenswaard, The Netherlands). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris-hydroxymethylaminoethane (TRIS), urea, and acetone were purchased from Acros Organics (Geel, Belgium). Benzamidin, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), benzoyl-arginine ethyl ester (BAEE), iodoacetamide, N-hydroxysuccinimide (NHS), and carboxyl-modified dextran (CMD) were purchased from Fluka (Buchs, Switzerland). Horse-heart cytochrome C, insulin B (oxidized), polyoxyethylenesorbitan monolaurate (Tween 20), and porcine pancreas trypsin were purchased from Sigma (St Louis, USA). The specific activity of trypsin was determined according to the method of Schwert and Takenaka [28] using BAEE as substrate and turned out to be 14700 U mg−1. Aminopropyltriethoxysilane (APTES), carbonyl diimidazole (CDI), glycidoxypropyltrimethoxysilane (GOPS), and mercaptoethanol (ME) were from Aldrich Chemical Company (Milwaukee, USA). Amino-modified dextran (AMD) was from Unavera ChemLab (Mittenwald, Germany). Fused-silica capillaries were purchased from Bester (Mijdrecht, The Netherlands). Piranha solution was prepared by mixing 1 part 30% hydrogen peroxide and 6 parts concentrated sulfuric acid (caution, aggressive solution). TRIS digestion buffer consisted of 50 mmol L−1 TRIS and 1 mmol L−1 CaCl2 adjusted to pH 8.2 by use of 1 mol L−1 HCl solution. Methods Preparation of dextran-coated SPR sensor disks (Fig. 1) Fig. 1 Surface modifications used to immobilize the enzyme in fused-silica capillaries. a, GOPS-modified; b, GOPS/AMD-modified; c, APTES/CMD/AMD-modified surface. In the last case the CDI activation and enzyme (H2N–enzyme) coupling are as in b The SPR sensor disks were extensively cleaned in freshly prepared piranha solution. After 1 h the disks were thoroughly rinsed with water, dried in a stream of nitrogen gas, and immediately incubated for 6 h in a 10 mmol L−1 solution of ME in ethanol in order to produce a self-assembled monolayer (SAM) containing hydroxyl functionalities. After SAM formation, the disks were washed with ethanol and water, and dried with nitrogen gas. The resulting ME disks were incubated for 1 h with a 10% GOPS solution in 98% ethanol after which the disks were washed with ethanol, dried with nitrogen gas and stored overnight at 50 °C. Dextran-modified sensors were prepared by incubating GOPS-modified sensor disks for 20 h at room temperature with a 10% AMD solution in a 50 mmol L−1 borate buffer, pH 9.5. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use. Alternatively, ME disks were incubated for 1 h with a 10% APTES solution in acetone. After washing, drying, and storing overnight in the oven, similar to the GOPS disks, the surfaces were incubated for 1 h with a solution containing 5% CMD in water containing 200 mmol L−1 EDC and 50 mmol L−1 NHS. After removing the solution, the disks were washed with water and ethanol, and incubated for 15 min in a solution containing 100 mmol L−1 CDI in acetone. These activated surfaces were washed with ethanol, blown to dryness with nitrogen, and incubated overnight with a solution of 10% AMD in water. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use. SPR experiments The GOPS-modified SPR sensor disks were incubated overnight with 200 μL of a solution containing 2.5 mg mL−1 trypsin and 50 μg mL−1 benzamidin in 50 mmol L−1 borate buffer, pH 9.5 (Fig. 1a). Both types of dextran-modified sensor disk were activated for 60 min with 200 μL 100 mmol L−1 CDI in dry acetone. After washing the surface with water to remove the last traces of acetone, the sensors were incubated overnight with 200 μL 2.5 mg mL−1 trypsin and 50 μg mL−1 benzamidin in 50 mmol L−1 borate buffer, pH 8.5 (Fig. 1b and c, respectively). The remaining esters were inactivated by incubating the disks for 10 min with 1 mol L−1 EA in 50 mmol L−1 borate buffer, pH 8.5. The successive steps in the immobilization were monitored with SPR and the amount of covalently coupled enzyme was calculated from the recorded angle shift. During the experiments the SPR system was thermostatted at 25.0 °C. Preparation of dextran-coated fused-silica capillaries In order to generate a proper surface for silanization, fused-silica capillaries were cleaned for 30 min with 2 mol L−1 NaOH solution at a flow-rate of 5 μL min−1. The capillary was then washed for 30 min with 0.1 mol L−1 HCl, for 5 min with water, and finally for 5 min with ethanol. In order to prepare dextran-coated capillaries, the capillaries were flushed for 60 min with a 10% GOPS solution in ethanol. After this step, the capillaries were closed with silicon plugs and dried overnight at 50 °C. After silanization, the capillaries were flushed with methanol at a flow-rate of 10 μL min−1 after which the capillaries were chemically modified in flow, using injections with a Rheodyne 7010 manual injector equipped with a 1-mL PEEK loop, in a way similar to that described above in the SPR section. Washing steps were conducted at flow rates of 10 μL min−1, overnight incubations at a flow rate of 1 μL min−1. All chemistries are outlined in Fig. 1. Enzyme activity determination Insulin B chain was used as a substrate to determine the activity of trypsin. Injections of 1 μL of a concentration of 20 μmol L−1 in digestion buffer were introduced into the reactor that was kept at the indicated temperature using a water bath. To determine the enzyme activity in solution, trypsin was incubated in these solutions also (final trypsin concentration 0.4 μg mL−1). At time intervals from 0.5 to 30 min, 100-μL samples were taken and the activity was stopped by the addition of 5 μL 20% TFA in water. Buffers that were used to determine the effect of pH on activity were MES (pH 5.5 to 6.5), MOPS (pH 6.5 to 7.8), TRIS (pH 7.5 to 9), and CHES (pH 8.6 to 10.1). The buffers were adjusted to pH by use of 1 mol L−1 NaOH and were prepared at a 50 mmol L−1 concentration, also containing 5 mmol L−1 CaCl2. To determine the effect of temperature, the temperature during incubation was varied between 10 °C and 60 °C. Both the on-line and off-line samples were analyzed using micro-HPLC with PDA detection, as outlined below. The insulin B conversion of both immobilized trypsin and the enzyme in solution was calculated from the peak areas of substrate and products. On-line protein digestion in micro-HPLC For the on-line peptide and protein digestion experiments the trypsin-modified capillaries were positioned between the LC injector and a 10-port valve, as shown in Fig. 2, and were immersed in a thermostatted water bath set at 37 °C unless mentioned otherwise. Protein samples were prepared in digestion buffer and were transported through the capillary towards a 10-port valve using a 5% acetonitrile solution containing 0.05% TFA. The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed. By switching the valve the trapping column is in series with the RP micro column and an acetonitrile gradient started. The gradient was composed of two solutions: (A) 5% acetonitrile in water containing 0.05% TFA and (B) 80% acetonitrile in water containing 0.04% TFA. In 30 min the gradient changed linearly from 0 to 50% B, followed by 10 min at 90% B and 20 min at 0% B. The eluent was monitored with the PDA detector in the range from 200 to 595 nm. Fig. 2Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used ESI-MS was conducted in the positive-ion mode with the capillary voltage set at 3500 V. The flow rate and temperature of the nitrogen drying gas were 5 L min−1 and 325 °C, respectively. The sequence of the peptide fragments was determined by using the mass spectrometer in auto-MS–MS mode fragmenting the two peptides that were most abundantly present when the signal reached threshold. The MS–MS result was analyzed by a Mascot database search (http://www.matrixscience.com). Results and discussion Surface chemistry and enzyme immobilization As shown before [29], modification of an SPR sensor surface with a dextran hydrogel leads to less non-specific adsorption of proteins compared with unmodified surfaces. The presence of such a layer also enhances the immobilization capacity of biomolecules compared with monolayer-based coatings. Due to the flexible nature of the dextran chains, the accessibility also is often improved compared with molecules immobilized on a flat surface. As the amount of protein present in a capillary after immobilization cannot be determined easily, SPR sensors were used as a model to investigate the effect of the different surface modifications on the amount of trypsin that could be attached covalently. Hydroxyl functionalities necessary to enable silanization are introduced using mercaptoethanol (ME), but all other surface modifications are carried out in exactly the same way, both on the SPR sensor surfaces and in the fused-silica capillaries. Trypsin immobilization after silanization with GOPS by reaction of the trypsin primary amines and the glycidyl function of GOPS results in an SPR angle shift of 350 ± 20 m° (n = 3), which equals an amount of trypsin of 2.9 ± 0.2 ng mm–2 covalently attached to the surface, which is close to monolayer coverage of trypsin. When the GOPS-silanized surface is modified with AMD, resulting in a dextran hydrogel, the amount of trypsin that can be immobilized after CDI activation increases to 9.5 ± 0.3 ng mm−2 (n = 3). To further increase the amount of trypsin, an intermediate dextran layer was added. Therefore, fused-silica capillaries were APTES silanized and modified with CMD and AMD, subsequently. The amount of trypsin that could covalently be attached to these layers was determined with SPR and proved to be 11.1 ± 0.5 ng mm−2 (n = 2). As this amount is significantly more than obtained with the GOPS/AMD surface, all further experiments have been conducted with fused-silica capillaries the surface of which is modified with dextran in this way. Assuming that the investigated surface modification allows a similar amount of trypsin to be immobilized per surface area in fused-silica capillaries, the quantity of enzyme immobilized on the dextran-modified capillaries is more than five times larger per surface area compared with other open-tubular, trypsin micro-reactors described in the literature [23, 30–32]. These reactors, first described by Amankwa and Kuhr [30], are based on immobilized avidin and, at saturation, 6.5 pmol biotinylated trypsin was immobilized in a 50-cm long capillary of 50-μm i.d.. This amount equals 2 ng trypsin mm−2 capillary surface, which is less than monolayer coverage. Characterization of the enzyme reactor Fused-silica capillaries were modified with dextran as described above and the activity and characteristics of the trypsin micro-reactor were assessed with the oligopeptide insulin B chain, a 30 amino acids long insulin fragment. When insulin B is exposed to the enzyme present in the reactor, hydrolysis at the arginine present at position 22 and, to a lesser extent, the lysine at position 29 is observed. Using the aforementioned substrate insulin B, the pH optimum was determined for the enzyme in solution and the immobilized enzyme. As can be observed in Fig. 3a, the optimum pH value for both free and immobilized trypsin is pH 8.5. Therefore, all further experiments are conducted at this pH value. Fig. 3Effect of (a) pH and (b) temperature on the relative activities of trypsin in solution (circles) and trypsin immobilized on an APTES/CMD/AMD-modified fused-silica surface (triangles). In b the maximum insulin B conversion rate determined under experimental conditions is 4.41 ± 0.02 pmol min−1 μg−1 for the enzyme in solution and 12.32 ± 0.35 pmol min−1 μg−1 for a 3-μL, 50-μm i.d. microreactor operated at a flow rate of 1 μL min−1 Also the effect of temperature on the activity of both the immobilized trypsin and the enzyme in solution was determined in the range 10 °C to 60 °C. As can be observed in Fig. 3b, the activity of trypsin increases with temperature to reach a maximum around 40 °C and decreases at higher temperatures. The immobilized enzyme shows a higher activity compared with the enzyme in solution, which may be due to an often observed higher stability of immobilized enzymes, also under more extreme conditions. From the data the activation energy can be determined. For the immobilized enzyme a value of 12.2 ± 0.3 kJ mol−1 can be calculated and for the enzyme in solution a value of 17.2 ± 0.5 kJ mol−1. This means that for the immobilized enzyme the temperature has less effect on the activity than for the enzyme in solution. As the highest activity is observed at 37 °C, all further experiments are conducted at that temperature. For the enzyme in solution a specific activity of 4.41 ± 0.02 pmol min−1 μg−1 is determined at a concentration of 20 pmol μL−1 insulin B and pH 8.5 and 37 °C. Under similar conditions, in a microreactor of 3 μL with a 50 μm i.d. and operated at a flow rate of 1 μL min−1, an amount of 12.32 ± 0.35 pmol insulin B is converted. In such a reactor a total of 2.67 μg trypsin might be present taking the earlier SPR observations into account, which means that the immobilized enzyme is both accessible and still active after immobilization. The specific activity after immobilization is higher than observed in recent papers for the protease pepsin immobilized on beaded chitosan [33, 34] and trypsin photoimmobilized in a fused-silica capillary [34]. The stability during operation of the open-tubular reactor was tested at 37 °C and pH 8.5. Activity tests using the model substrate show that the activity of a reactor that is continuously in operation, is constant for at least two weeks. The enzyme trypsin dissolved in digestion buffer and incubated for 24 h at 37 °C loses 60% of its activity, and after three days no activity is measured. On-line digestion of proteins The on-line digestion of horse cytochrome C is accomplished as described in the Methods section. Samples are submitted to on-line digestion in a 22-cm long trypsin-modified APTES/CMD/AMD-coated capillary with an i.d. of 75 μm (total volume 1 μL) or with an i.d. of 50 μm and a length of 51 cm (1 μL) or 140 cm (2.75 μL). When the protein is not reduced, the heme-moiety will remain covalently attached to the peptides containing the protein sulfhydryls. By monitoring the heme-containing peptides at 395 nm, the progress of the digestion and the amount of undigested protein can be determined. The results are summarized in Table 1. Table 1Summary of the on-line digestion experiments for cytochrome C. The experiments were conducted at pH 8.5 and 37°C (n = 3)capillary ID (μm)reactor volume (μL)amount injecteddigestion time (s)% undigested protein (SD)(μg mL−1)(pmol)751248206065.9 (4.8)751124106035.8 (3.475112.4106023.6 (1.3)501248206014.0 (1.9)50112410602.3 (2.0)502.75248201650502.75124101650502.7512.4101650The experiments were conducted at pH 8.5 and 37 °C (n = 3) As expected when a limited amount of enzyme activity is present in a reactor (22 cm  ×  75 μm), for increasing concentrations cytochrome C a larger amount of protein is undigested. Nevertheless, many tryptic peptides are still generated. As can be expected, an increase in exposure time of the substrate with the immobilized enzyme will result in an improved digestion yield. A longer contact time is achieved by increasing the reactor volume by using a longer enzyme-modified capillary. Additionally, the enzyme-to-substrate ratio is increased, which is accomplished by changing the surface-to-volume ratio by using a capillary with a smaller internal diameter. As can be observed in Table 1, decreasing the i.d. of the capillary leads to improved digestion for a reactor of equal volume due to a higher surface-to-volume ratio and hence a higher amount of enzyme. The use of a 140 cm ×50 μm capillary allows the complete digestion of up to 20 pmol (248 μg mL−1) of cytochrome C in less than 5 min including the sample concentration and removal of salts by the trapping column. Chromatograms for the on-line digestions obtained with capillaries with an i.d. of 50 μm are shown in Fig. 4. Figure 4a shows a blank run in a capillary containing no enzyme and Fig. 4b and c present on-line digestions for a micro-reactor of 2.75 μL operated at 5 μL min−1 and 1 μL min−1, respectively. As discussed above, incomplete digestion of the injected cytochrome C will lead to the presence of multiple peptide fragments containing the heme group, as is visible in Fig. 4b. The digestion is complete when the sample exposure time is 165 s (flow rate 1 μL min−1) as both intermediate products and the undigested protein (retention time 44 min in Fig. 4a), which are visible at a wavelength of 395 nm as outlined above, are no longer observed. An injection of off-line-digested cytochrome C showed a similar chromatogram as is shown in Fig. 4c. Fig. 4Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated The on-line digestion of horse cytochrome C is also monitored with mass spectrometry. The effect of flow rate and hence incubation time on the digestion of the protein and the number of peptides identified with a Mascot database search is determined and summarized in Table 2. In this table the undigested amounts of protein which have been determined using the PDA detector are also shown. At a flow rate of 1 μL min−1 protein digestion is complete and many peptides are matched, resulting in high sequence coverage and Mascot score. With higher flow, and hence decreasing incubation time, the amount of protein that remains undigested increases and consequently fewer peptides are produced and observed. However, even at relatively high flow rates an adequate amount of peptides is still formed and the protein can be identified on the basis of the fragments present. Nevertheless, the ion intensity is low and some peptides are not retrieved as they are below the threshold for auto-MS–MS. Table 3 summarizes the peptides observed and matched using the MS–MS data and a Mascot.database search of proteins digested at a flow rate of 1 μL min−1. A base-peak chromatogram (BPC) of the digestion of cytochrome C under these conditions is shown in Fig. 5a. The MS–MS fragmentation of one of the peptides is presented in Fig. 5b. Table 2General overview of the on-line digestion experiments for cytochrome C (n = 3)flow rate (μL min-1)digestion time (s)sequence coverage (%)total no peptides matchedMascot scoreundigested protein (%)1017661134760.3533662150131.8283662364918.5116587278330Table 3Peptide fragments observed by MS in the on-line digestion of cytochrome C and myoglobin The experiments were conducted at pH 8.5 and 37 °C at a flow rate of 1 μL min−1 using a 2.75-μL trypsin reactorPeptide sequenceTheoretical m/zExperimental m/zPositionMissed cleavageCytochrome C (sequence coverage 87%)1....IFVQKCAQCHTVEK1632.8816.9 (2+)9–2212....HKTGPNLHGLFGR1432.8717.1 (2+)26–3813....TGPNLHGLFGR1167.6584.6 (2+)28–3804....TGPNLHGLFGRK1295.7648.6 (2+)28–3915....KTGQAPGFTYTDANK1597.8533.4 (3+)39–5316....TGQAPGFTYTDANK1469.7735.6 (2+)40–5307....TGQAPGFTYTDANKNK1711.8571.4 (3+)40–5518....GITWKEETLMEYLENPKK2208.1736.8 (3+)56–7329....EETLMEYLENPK1494.7748.0 (2+)61–72010 EETLMEYLENPKK1622.8812.1 (2+)61–73111...YIPGTK678.4678.1 (1+)74–79012...MIFAGIKK906.5454.0 (2+)80–87113...KTEREDLIAYLKK1477.8739.6 (2+)88–99214...TEREDLIAYLKK1349.7675.5 (2+)89–99115...EDLIAYLK963.5482.6 (2+)92–99016...EDLIAYLKK1091.6546.6 (2+)92–1001Myoglobin (sequence coverage 88%)1....GLSDGEWQQVLNVWGK1814.9908.7 (2+)1–1602....VEADIAGHGQEVLIR1605.8803.6 (2+)17–3103....VEADIAGHGQEVLIR1605.8536.1 (3+)17–3104....LFTGHPETLEK1270.7636.0 (2+)32–4205....HLKTEAEMK1085.6543.5 (2+)48–5616....HGTVVLTALGGILK1377.8689.7 (2+)64–7707....HGTVVLTALGGILKK1505.9502.7 (3+)64–7818....KKGHHEAELKPLAQSHATK2109.1703.7 (3+)78–9629....KGHHEAELKPLAQSHATK1981.0661.1 (3+)79–96110 GHHEAELKPLAQSHATK1853.0618.8 (3+)80–96011...YLEFISDAIIHVLHSK1884.0628.8 (3+)103–118012...HPGNFGADAQGAMTK1500.7751.5 (2+)119–133013...ALELFRNDIAAK1359.8680.6 (2+)134–145114...YKELGFQG940.5471.0 (2+)146–1531Fig. 5On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions In further experiments horse myoglobin was digested on-line. This protein is generally regarded as difficult to digest [15, 35]. When 1 μL of a 10 μmol L−1 solution in buffer is injected at a flow rate of 1 μL min−1 (165 s exposure time), the injected protein is completely digested, as was observed with UV detection (data not shown). Using mass spectrometric analysis, 13 different peptides are observed and matched using the MS–MS data and a Mascot database search, resulting in a sequence coverage of 88%. The matched peptides are summarized in Table 3 and a BPC of the on-line digestion of myoglobin is shown in Fig. 6a, with an MS–MS spectrum of one of the tryptic peptides. Both the degree of digestion and the sequence coverage are adequate compared with other systems that often use a high percentage of modifier to enhance digestion, as the absence of denaturing agents during digestion leads to little or no digestion of myoglobin [35]. Therefore these reactors are generally used for direct infusion into MS or off-line protein digestion as the presence of high concentrations of methanol or acetonitrile in the digestion buffer will seriously impede on-line protein digestion in combination with RP-LC. Fig. 6Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7 Amankwa and Kuhr [30] reported that proteins and peptides adsorb to the capillary surface of their trypsin reactor. Reactor fouling, and as a result sample carry-over, is also observed when trypsin-modified particulate polymers or monoliths are used [36]. Adsorption of proteins and peptides to these relatively hydrophobic columns is prevented by the addition of up to 20% methanol to the digestion buffer. For dextran-coated surfaces, it is known that the non-specific adsorption of protein is minimal and depends on the character of the oligosaccharide layer [29, 37, 38]. During protein-digestion experiments on SPR sensor surfaces modified identically with the capillary reactors no adsorption during incubation is observed (data not shown). Similarly, during on-line digestion using the dextran coated capillaries, sample adsorption and carry-over are not observed, as repeated sample injections result in identical chromatograms and blank buffer injections do not show peptide fragments. On basis of the latter result and the long lifetime of the reactor, trypsin auto-digestion is probably absent. The developed IMER has been applied for the digestion of horse cytochrome C and myoglobin in buffer without prior pretreatment followed by separation and MS–MS sequencing. The time needed to obtain complete protein digestion and sample trapping is 5 min with an average sample residence time of 165 s. The total analysis time, digestion and LC separation, is 65 min. The developed micro-reactor is competitive when compared with other systems used for on-line protein digestion described in literature [13, 14, 39–41]. In the capillary IMERs described by Amankwa and Kuhr [30] and Bossi et al. [34] the complete digestion of native proteins was only obtained when the protein resided in the trypsin-coated capillary for 15–25 min. The peptide separation was carried out with CE in these latter cases. Many reactors use a protein denaturation step to make the protein more susceptible to proteolytic action. The digestion efficiency can be enhanced by partial denaturation by addition of 35–45% acetonitrile to the buffer [11, 15, 35, 36] or by sonication [42, 43]. However, the latter has not yet been used in an automated set-up and the presence of high amounts of modifier will make reversed-phase chromatography in a subsequent step for the separation of the fragments produced during protein digestion very difficult. The described reactors were efficient in terms of time needed for digestion and the digestion result, but were, as a rule, used for direct infusion into the MS or used for off-line digestion. Alternatively, protein denaturation is accomplished using chemicals such as SDS, guanidine HCl or urea. A recent example is the miniaturized on-line proteolysis–capillary LC system as described by Samskog et al. [12], who employed a 10-μL column packed with poroszyme. The sample contained guanidine.HCl for protein denaturation, and was diluted with buffer, also containing methanol, prior to injection. The total time needed for digestion, trapping, and removal of the high concentration of salt was 15 min. The system needed periodic regeneration to counteract the effects of residual salts in the analyte. Conclusions To study the immobilization of the protein trypsin in a fused-silica capillary, a number of surface modifications was tested. In order to investigate the amount of protein that can be covalently attached to these surfaces, SPR sensor disks are modified with these coatings to mimic the capillary surface. The SPR measurements show that the best results were obtained using a dextran coating with an intermediate layer. The resulting open-tubular trypsin reactors having a pH optimum of pH 8.5 display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. The capillary reactors show flow-dependent catalysis. For a capillary IMER, the conversion of the insulin B chain increases with decreasing flow and hence a longer residence time. The same is observed for the digestion of horse cytochrome C. The complete digestion of 20 pmol μL−1 horse cytochrome C is observed without the need of protein denaturation, reduction, or alkylation when the average residence time of the protein sample in a 140 cm  ×  50 μm capillary IMER is 165 s. For the proteins used in this study trypsin-reactors were described that were capable of faster digestion using denaturing agents like acetonitrile to enhance the digestion process. However, the presence of such agents would seriously hamper direct analysis of the peptides formed using RPLC and MS. Identification of the proteins cytochrome C and myoglobin is possible by the tryptic peptides that are produced on-line, separated by micro-RPLC, and analyzed using mass spectrometry with auto MS–MS. The open-tubular reactor can be produced easily, reproducibly, and inexpensively, and can be used for other applications such as enzyme-inhibitor studies. Future research will focus on the development of miniaturized multi-dimensional analysis systems employing on-line digestion using these IMERs. The capillary enzyme reactors show no backpressure and seem promising for coupling to other analytical techniques such as capillary electrophoresis and surface plasmon resonance.

J_Gastrointest_Surg-3-1-1852378

Management of Hepatic Angiomyolipoma

Preoperative diagnosis of hepatic angiomyolipoma is difficult, and the treatment for it remains controversial. The aim of this study is to review our experience in the treatment of hepatic angiomyolipoma and to propose a treatment strategy for this disease. We retrospectively collected the clinical, imaging, and pathological features of patients with hepatic angiomyolipoma. Immunohistochemical studies with antibodies for HMB-45, actin, S-100, cytokeratin, vimentin, and c-kit were performed. Treatment experience and long-term follow-up results are summarized. During a period of 9 years, 10 patients with hepatic angiomyolipoma were treated at our hospital. There was marked female predominance (nine patients). Nine patients received surgical resection without complications. One patient received nonoperative management with biopsy and follow-up. One patient died 11 months after surgery because of recurrent disease. We propose all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5 cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis virus carrier. Introduction Hepatic angiomyolipoma (AML) is a rare mesenchymal tumor of the liver composed of smooth muscle cells, adipose tissue, and proliferating blood vessels. Since its first description by Ishak in 1976, approximately 200 cases have been reported in the English literature.1 This type of tumor is usually seen in kidneys associated with tuberous sclerosis.2 Definite pathologic diagnosis is made by identification of the three different components and HMB-45 positive staining.3 In the past, this tumor has been considered an entirely benign and slow-growing lesion without the possibility of malignant transformation. Therefore, several authors have suggested that this disease can be managed with conservative treatment.4–7 However, since 2000, several reports have revealed that this kind of tumor can be malignant with evidence of recurrence.8–10 Although the combination of ultrasonography, computed tomography (CT), magnetic resonance (MR) imaging, and angiography increases the accuracy in diagnosis of hepatic AML, the correct preoperative diagnostic rate of imaging studies has been reported to be less than 50%.6,10–14 Even the postoperative pathologic diagnosis has been easily mistaken as hepatocellular carcinoma (HCC).14,15 Many patients have been treated with surgical resection of the tumor. Therefore, the proper treatment of hepatic AML has remained controversial. The purpose of this study is to retrospectively review the clinical, imaging, and pathological features of patients with hepatic AML treated at our hospital and to summarize our experience in the diagnosis and treatment of this disease. We also review the literature to highlight the important questions concerning hepatic AML: (1) Is hepatic AML a pure benign tumor? (2) What is the natural course of this tumor? Does the tumor size enlarge frequently during observation? (3) What difficulties exist in preoperative diagnosis with imaging studies and fine needle aspiration biopsy (FNAB)? (4) Is it proper for a hepatitis-carrier patient with hepatic AML to be treated with conservative management? (5) What are the criteria for patients with hepatic AML to be treated with surgical resection or conservative management? Materials and Methods The clinical, imaging, and pathological features of 10 patients with hepatic AML treated at the authors’ institute were retrospectively reviewed. The follow-up information was obtained in each case. All tumor tissue was paraffin-embedded for routine hematoxylin and eosin (H&E) staining. Immunohistochemical assays were performed using a three-step indirect peroxidase complex technique with the following antibodies: HMB-45 (DAKO, dilution 1:40), actin (DAKO, dilution 1:50), S-100 (DAKO, dilution 1:800), cytokeratin (Biogenix, dilution 1:80), vimentin (DAKO, dilution 1:50), and c-kit (MBL, dilution 1:200). Results Patients and Clinical Data Ten patients with hepatic angiomyolipoma were diagnosed at National Taiwan University Hospital from July 1995 to June 2004. There was marked female predominance (9/10). The median age was 44 years old with a range from 34 to 64 years. Most patients (60%) presented no symptoms and were detected incidentally by health check-ups or during medical exams for other diseases. Four of 10 patients had symptoms caused by the space-occupying effect of the tumors such as abdominal pain, abdominal fullness, and palpable mass, or other nonspecific symptoms such as fever, general malaise, or body weight loss (Tables 1 and 2). None of them had a history of renal AML or tuberous sclerosis. Two patients were hepatitis B-virus (HBV) carriers. The plasma levels of α-FP and CEA were within normal limits in all patients. Table 1Clinical Presentation of Hepatic AngiomyolipomaClinical FeatureNo. of PatientsAge34–64 years (median 44 years)Gender (female: male)9:1Symptoms No symptom6 Abdominal pain2 Abdominal fullness2 Palpable mass1 Body weight loss2 Malaise1 Fever2Tumor location Right lobe5 Left lobe4 Caudate1Tumor size (cm) <53 5–101 >106Preoperative diagnosisAngiomyolipoma4(40%) Based on radiological images2 Based on tumor biopsy2Hepatocellular carcinoma3(30%)Angiosarcoma1(10%)Hemangioma1(10%)Metastasis1(10%)Associated liver diseaseHBV carrier2Table 2Clinical Profile of Patients with Hepatic AngiomyolipomaCaseSex/AgeTumor Size (cm)/lobeSymtoms/SignsIncidental FindingTreatmentOutcome/F/U Months1F/3418/RNilH/CAtypical hepatectomyWell/39 mon2F/3410/REpigastralgiaRight lobectomyWell/59 mon3F/3713/LPalpable mass, abdominal fullness, BW loss, feverExtended left lobectomyDead/14 mon recurrent, liver and lung mets4F/4020/REpigastralgiaRight lobectomyWell/109 mon5F/427/RNilH/CFNAB and F/ULost F/U/6mon6F/4611/LAbdominal fullness, malaise, BW loss, feverLeft lateral segmentectomyWell/40 mon7F/4915/RNilExam of appendicitisS56 segmentectomyWell/37 mon8F/513/CNilH/CCaudate lobectomyWell/40 mon9F/532.5/LNilF/U echo due to colon cancer s/pLeft lateral segmentectomyWell/33 mon10M/644/LNilH/CLeft lobectomyWell/32monH/C = health check-up, BW = body weight, F/U = follow-up, FNAB = fine needle aspiration biopsy, mon = month, mets = metastasis, s/p = postoperation Imaging Studies Based on the combined imaging studies of abdominal ultrasonography, CT, MR imaging, and angiography, the diagnostic accuracy of hepatic AML in this series was only 20% (Table 1). Other preoperative imaging impressions included hepatocellular carcinoma, angiosarcoma, hemangioma, and metastatic lesions. Two other cases were diagnosed by fine needle aspiration biopsy (FNAB). The accurate preoperative diagnostic rate was 40% (4/10) after imaging studies and FNAB (Table 1). Pathologic Study All 10 patients had a single tumor. Five tumors were in the right lobe of the liver and four were in the left lobe. One tumor was located in the caudate lobe. Most tumor sizes were larger than 5 cm (70%). The median tumor size was 10.5 cm, ranging from 2.5 cm to 20 cm (Tables 1 and 2). Gross pathology identified all tumors as a well-circumscribed, nonencapsulated tumor masses consisting of soft to elastic tissue. The cut surface in tumors varied from yellow to dark brown. Histopathologic studies of these 10 tumors showed a picture of hepatic angiomyolipoma composed of myoid and vascular components with a variant content of fatty tissue. Hematopoiesis was noted in two cases. Immunohistochemical studies were performed in all patients except one (case 5). Most tumors were found positive for HMB-45 (10/10), SMA (4/9), S-100 (7/9), Vimentin (6/9), but negative for cytokeratin (0/9). Only three tumors were found positive for c-kit (Table 3). Table 3Immunohistochemical StudyCaseHMB-45ActinS-100CytokeratinVimentinc-kit1++−++−−+2+++++−++3++−++−+−4++++−−+−5++6+++++−+−7++−++−+−8++−−−+−9++−++−−−10++++−++++: strongly staining, >30% positivity; +: weakly staining, 10∼30% positivity; − no staining, or <10% positivity Treatment and Follow-up One patient (case 5) was confirmed with AML through fine needle aspiration biopsy. Nonoperative management with close follow-up was performed. However, this patient was lost after 6 months of follow-up. The other nine patients underwent hepatectomy with tumor resection. These nine patients, except for one patient (case 3), had no postoperative complications or disease recurrence, and were regularly followed up at our outpatient department, follow-up ranging from 32 to 109 months (Table 2). The very unusual patient (case 3) was a 37-year-old woman with a 13 × 9 × 9 cm, large tumor at the left lobe of the liver, receiving extended left lobectomy (Fig. 1a,b). Pathology revealed a picture of hepatic AML (Fig. 2a,b). Figure 1A 37-year-old woman (case 3) presented with fever and palpable abdominal mass. (a) The axial view of contrast-enhanced CT scans on portal venous phase shows a huge hepatic tumor at the left hepatic lobe with heterogeneous enhancement. Notice the engorged vessels within the tumor are vividly identified (arrow). (b) The MR coronal Tru FISP, fast imaging with steady-state precession. (TR/TE/FA = 4.3/2.1/72°) shows engorged vessels in the tumor. The right portal vein (arrow) is displaced by the tumor. (c) After 6 months of extended left lobectomy, the abdominal ultrasonography reveals a huge recurrent tumor (arrows) in the previous location of left hepatic lobe, and numerous smaller tumors in the right lobe. (d) Celiac angiography also demonstrates the recurrent huge tumor and other multiple smaller ones in the right lobe of liver. Note the early drainage vein (arrow).Figure 2Microscopic appearance of the hepatic angiomyolipoma in case 3. (a) The primary tumor is composed of polygonal to spindle cells arranged in solid sheets or trabecular pattern with endothelial lining. Some of the tumor cells have eosinophilic cytoplasm, and some have large fat vacuoles. Some of the nuclei are bizarre, and some have large eosinophilic nucleoli (H&E stain, original magnification ×100). (b) The tumor cells are strongly immunoreactive for HMB-45 (original magnification ×100). Recurrent tumor was noted 6 months later, and the patient received fine needle aspiration biopsy. (c) Microscopically, it shows tumor cells with clear to ample eosinophilic cytoplasm arranged in trabecular pattern (H&E stain, original magnification ×40). (d) Immunohistochemical staining shows the tumor cells are also positive for HMB-45 (original magnification ×200). Unfortunately, 6 months later, ultrasonography showed recurrent hepatic lesions at the right lobe of the liver (Fig. 1c). MRI also confirmed a large tumor in the caudate lobe and numerous smaller nodules in the right lobe of the liver. Angiography also revealed multiple tumor stains (Fig. 1d). Fine needle aspiration biopsy was performed. The biopsy specimen was immunoreactive to HMB-45 antibody (Fig. 2c,d). The clinical and histologic picture demonstrated recurrent malignant hepatic angiomyolipoma. At the 11th postoperative month, chest CT scans revealed multiple metastatic nodules. Three months later, the woman died due to hepatic failure and renal failure. Discussion In the past, hepatic AML has been considered as a “benign” mesenchymal tumor. However, in 2000, Dalle reported the first case of malignant hepatic AML with vascular invasion and recurrence with multiple liver metastases and suspected portal vein thrombosis 5 months after primary tumor resection.8 Another two cases have been reported with hepatic recurrence after operation. One case was a 16-year-old girl with hepatic AML, receiving left lobectomy, with late recurrence noted 6 years after operation.9 The other one was in the Flemming’s report. Recurrent hepatic tumors were noted 3 years after operation.10 Flemming also suggested that a proliferation index exceeding 3% and multicentric growth indicate a propensity for recurrence. In this study, we reported a 37-year-old woman with left hepatic AML. A recurrent hepatic mass was noted 6 months after tumor resection, and multiple lung metastases were noted later. The patient died 14 months after diagnosis. To our knowledge, this case is the fourth reported case of recurrence in the literature, and the tumor in this case behaved as the most malignant one.8–10 Therefore, hepatic AML should not be considered as an entirely benign tumor; at least, it has malignant transformation potential. Accordingly, conservative treatment should be performed carefully, especially for patients with poor compliance, who are unable to undergo a strict follow-up regimen. There were few reports concerning the growth velocity of hepatic AML in long-term follow-up. In one retrospective study of 26 patients, there were six patients who were followed up for more than 1 year and finally decided to receive operation because of the enlargement of the lesions. In that study, the tumor size of one patient increased from 4 to 10 cm during the 5-year follow-up. Another patient had a tumor increasing from 1.5 to 5 cm in 13 years follow-up.13 In another case report of a 38-year-old patient, the tumor size enlarged from 8 to 14.4 cm over a 3-year follow-up period.16 Irie also reported that a 40-year-old woman had hepatic AML with tumor size increasing in size from 4 cm to 7 cm during a 14-month follow-up period.16 Although hepatic AML seems slow-growing, the probability of tumor enlargement and hence an induced mass-compression effect is not uncommon in the long-term follow-up period. In the present series, the median age of patients was 44 years old, and 70% of patients were below 50 years. If all of these patients had received nonoperative management, the mass effect of tumor enlargement might have been presented during a long-term follow-up period, especially in younger patient groups with longer remaining years of life. Moreover, the difficulties and complications of operation at later years would increase when the tumor enlarges, especially for those patients with an original larger tumor (>5 cm). Similar to the patients presented in this series, most patients with hepatic AML are not symptomatic.12–14 Usually, these patients are diagnosed during health check-ups. Most symptoms are mass-compression effects including upper abdominal pain, abdominal fullness, and palpable mass. There are also some vague symptoms such as body weight loss, general malaise, and fever. In one review article with a collection of 52 patients, the incidence of symptoms or signs dramatically increased when tumor size was larger than 5 cm.11 Twenty-one percent (4/19) of patients with a tumor smaller than 5 cm present symptoms/signs; however, the incidence increases to 64% (7/11) when tumor size is between 5 and 10 cm. The incidence increases to 89% when tumor size is larger than 10 cm. In our series, 40% (4/10) of patients were symptomatic, and all four of these patients had a tumor larger than 10 cm. Accordingly, we suggested that patients with tumor larger than 5 cm should receive tumor resection, because most patients in this group were predisposed toward being symptomatic. The typical findings in imaging studies of hepatic AML are as follows: (1) heterogeneously hyperechoic mass in US, (2) heterogeneously low density with low attenuation value (less than −20 HU) in plain CT, (3) high intensity on T1 and T2 weighted MRI, and (4) hypervascularity and tumor stain on angiography.12 Although a combination of US, CT, MRI, and angiography is able to increase the accuracy in preoperative diagnosis, hepatic AML usually shows various patterns in imaging studies. The differences in imaging studies occur because the relative proportions of vessels, muscles, and fatty tissue vary widely from one tumor to another. Consequently, hepatic AML is sometimes difficult to diagnose based on imaging studies.17 Therefore, fine needle aspiration biopsy has been reported to be useful in the preoperative diagnosis of this tumor.4,5,17,18 However, more attention should be paid to the tumor’s various morphologic appearances when minute samples are interpreted. With the combined tools of imaging studies and FNAB, the preoperative diagnostic accuracy has been smaller than 32% (ranging from 0 to 32%) in larger series.6,10–15 In a collaborative study reported by Tsui, including 30 cases from nine international hepatology centers, 50% were primarily misdiagnosed as carcinoma or sarcoma, either by imaging studies or by needle biopsy.15 In Flemming’s series, only one preoperative case was diagnosed correctly.10 In the present series, only four preoperative cases (40%) were correctly diagnosed by combined imaging studies and FNAB. Definite pathologic diagnosis of this tumor is usually made by identification of the three different components of smooth muscle cells, adipose tissue, and blood vessels. HMB-45 positive staining of myoid cells has been used as a pathologic characteristic of hepatic AML.3,19 Because of the rarity and pleomorphism of the histological features of hepatic AML, histologic diagnosis may be difficult, especially with needle biopsy. Many features in AML can mislead the unwary pathologist to a diagnosis of hepatocellular carcinoma: polygonal cells in trabecular arrangement, peliosis, nuclear pleomorphism, prominent eosinophilic nucleoli, deficient reticulin framework, presence of glycogen, eosinophilic globules, and tumor necrosis.14 In Zhong’s series of 2000, none of the 14 cases were correctly diagnosed before operation. Furthermore, five cases were misdiagnosed as hepatocellular carcinoma or sarcoma by pathologists, even after operation. Therefore, we should be cautious when using FNAB as a diagnostic tool. In an endemic area of hepatocellular carcinoma such as Taiwan,20 conservative management is risky because cases of fat-rich minute hepatocellular carcinoma will make the differential diagnosis more difficult. Furthermore, Chang reported one case with hepatic AML and concomitant hepatocellular carcinoma.21 In this series, two patients were carriers of hepatitis B virus with a high risk for hepatoma formation. Not only would these hepatitis-carrier patients bear more risk, but physicians would also bear more risk and psychological pressure during a long-term follow-up period if conservative management were adopted. Because of the small patient number, we could not get definitely conclusive management suggestions solely from the results of this retrospective study. But a combination of our experience and a review of the literature, we suggest all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5 cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis-virus carrier.

Appl_Microbiol_Biotechnol-4-1-2271084

Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol−1, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol−1, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community. Introduction The anaerobic digestion of organic material is accomplished by the concerted action of various trophic groups of bacteria (Schink et al. 1992). The soluble organic compounds are degraded to CH4, CO2, and H2S via a syntropic interaction of fermentative and acetogenic bacteria with methanogens or sulfate-reducing bacteria (SRB). The electrons for CO2 reduction to methane or acetate, or sulfate reduction to sulfide are derived from the fermentative metabolism. The fermentative metabolism may include homoacetogenic or heteroacetogenic microorganisms depending upon whether acetic acid is the only major metabolic product (Drake 1994; Thauer et al. 1977) or other organic acids, such as propionic acid, in addition to acetic acid are produced (Seeliger et al. 2002). The products of fermentative acetogenesis and CO2-dependent homoacetogenesis can serve as substrates for acetate- and propionate-oxidizing SRB or acetoclastic methanogens depending upon the presence or absence of sulfate as electron acceptor. SRB are believed to outcompete methanogens in the presence of nonlimiting sulfate concentrations because they compete better for common substrates. This is explained on the basis of their kinetic properties (Ks and μmax) and given the favourable thermodynamic conditions (Ward and Winfrey 1985; Widdel 1988). In contrast, methanogens tend to dominate in low-sulfate environments (Stams 1994). In most anaerobic environments, hydrogen is present as an intermediate for which SRB, hydrogenotrophic methanogens and homoacetogens will compete. Under standard conditions, sulfate reduction and methanogenesis are thermodynamically more favourable than homoacetogenesis. However, due to the interspecies transfer of fermentation products, such as hydrogen, the real thermodynamic values may be far from standard conditions and even less to reverse reactions. Studies with the sludge from the bioreactors have indicated that hydrogen is mainly consumed by SRB when H2 is limiting and sufficient sulfate is present (Isa et al. 1986). However, when H2/CO2 was added as the sole substrate, a coexistence of heterotrophic SRB and homoacetogens was reported by Weijma et al. (2002), suggesting that when SRB are dependent for growth on homoacetogens, it is likely that the growth kinetics of homoacetogens determines the competition (Stams et al. 2005). The composition of the influent chemical oxygen demand (COD; electron donor) to ratio tends to have a significant effect on the competition for electron flow between different microbial communities (McCartney and Oleszkiewics 1993). The theoretical mass ratio needed to achieve complete removal of organic matter is 0.67, assuming that eight electrons are transferred per molecule of sulfate (Lens et al. 1998). In general, low and high mass ratios should favor sulfidogenesis and methanogenesis, respectively. However, values reported in the literature vary considerably (Annachhatre and Suktrakoolvait 2001; Choi and Rim 1991; Mizuno et al. 1994). Although the effect on the major processes like sulfidogenesis and methanogenesis on the whole has been well studied (McCartney and Oleszkiewicz 1993), our understanding of its influence on the diversity and dynamics of the microbial communities is still limited. This is partially due to the limitations of the traditional microbiological techniques, including pure culture isolations, most probable number estimation, selective enrichments, etc. Fortunately, molecular techniques, like denaturing gradient gel electrophoresis (DGGE; Muyzer et al. 1993) and fluorescence in situ hybridization (FISH; Amann et al. 1995), have provided alternative approaches to overcome the limitations associated with culture-based analyses. DGGE profiling of 16S rRNA gene fragments amplified from both genomic DNA and reverse-transcribed RNA, i.e., cDNA not only determines the presence and distribution but also the actively growing members in the mixed microbial communities (Dar et al. 2007a). FISH, on the other hand, allows for the identification and quantification at the level of populations and even single cells (Amann et al. 1995). In the present study, the structure and function of anaerobic communities maintained in lab-scale continuously stirred tank reactors (CSTR) under sulfidogenic and nonsulfidogenic conditions was investigated. An integrated approach of molecular techniques including DGGE and FISH together with chemical analysis was used to link the microbial population dynamics with changes in the lactate (electron donor) concentration and sulfate availability. The focus of our study was the competition and interaction between the dominant microbial communities of SRB, acetogens and methane-producing Archaea. Materials and methods Reactor operation The schematic representation of the experimental design is given in Fig. 1. The start-up reactor (R-0) was inoculated with sludge from a methanogenic wastewater treatment plant treating lactate-containing wastewater. The reactor was operated in a continuous mode for about five to six volume changes at a dilution rate of 0.02 h−1 with lactate as the only electron donor and maintained under excess of sulfate. The lactate (12 electron) to sulfate (8 electron) molar ratio (r) was 0.65 mol mol−1. The culture in the reactor R-0 was used as an inoculum for the reactors, R1 and R2. The reactors R1 and R2 were operated at 30°C with a working volume of 2 l and at two different concentrations of lactate, 3.5 and 20 mM, respectively. Sulfate was fed to the reactors at a concentration of 10.32 mM, i.e., at ratios far below and far above the theoretical ratio of 0.67. Nonsulfidogenic conditions were applied to reactor R2 by further reducing influent sulfate feed and reducing the lactate concentration to 6.7 mM (r = 20.9). The composition of influent mineral medium was as follows (concentration in mM): KH2PO4, 3.6; Na2SO4, 10 or 0; NH4Cl, 6; MgSO4·7H2O, 0.3; CaCl2·2H2O, 0.4; FeSO4·7H2O, 0.02; a stock trace element solution, 1 ml/l; a stock selenite–tungstate solution, 1 ml/l; and a stock vitamin solution, 1 ml/l. The stock trace element solution contained (mg/l): H3BO3, 30; MnCl2·4H2O, 100; CoCl2·6H2O, 190; NiCl2·6H2O, 24; CuCl2·2H2O, 2; ZnCl2, 140; Na2MoO4·2H2O, 36. The stock selenite–tungstate solution contained (mg/l): Na2SeO3·5H2O, 6 and Na2WO4·2H2O, 8. The stock vitamin solution was composed of (mg/l): biotin, 10; vitamin B12, p-aminobenzoic acid, and calcium D (+) pantothenate, 50; thiamine, pyridoxine–HCl, and nicotinic acid, 100. The medium was autoclaved, and the dissolved oxygen removed from the hot media by sparging it with oxygen-free nitrogen. The medium was maintained in an oxygen-free atmosphere by continuously flushing it with nitrogen. The pH in the reactors was maintained at 7.5 by automatic titration with 1 M NaOH and 1 M HCl. An automatic level controller maintained the working volume of 2 l inside the reactor vessels. The cultures were continuously sparged with oxygen-free nitrogen to flush the gaseous H2S. Sulfide precipitated as ZnS after letting the gaseous H2S through a zinc acetate trap connected to the gas exhaust of the reactor. Cultures were run at D = 0.02 h−1 for at least five to six volume changes (one volume change is 50 h) and until chemical analysis of effluent showed constancy over at least two consecutive days. Fig. 1Schematic representation of the experiment. Reactor R-0 was the start-up reactor. The culture in R-0 was used as an inoculum for the reactors R1 and R2a. The cultures were run at a dilution rate of 0.02 h−1 for five to six volume changes with each volume change of 50 h Chemical analysis Acetate and other volatile fatty acids were analyzed either by gas chromatography (GC) or by high-performance liquid chromatography (HPLC). Sulfate was analyzed by ion chromatography as described previously (Scholten and Stams 1995). Sulfide was measured quantitatively by a colorimetric assay (Cline 1969). As sulfide was stripped from the culture liquid with nitrogen to reduce sulfide toxicity, the sulfide produced was calculated from the sulfate consumed. Hence, the given sulfide values indicate minimum values. Nucleic acid extraction, reverse transcription of RNA, and PCR amplification Nucleic acid extraction was carried out as described previously (Dar et al. 2007b). One microliter (80–100 ng) of RNA template was used to carry out the reverse transcription (RT) of the isolated RNA into cDNA. Reverse transcription was performed using the iScript cDNA Synthesis Kit (BioRad, California, USA) according to the manufacturer’s protocol. Amplification of the 16S rRNA gene fragment was performed using the primer pair 341F-GC and 907R and the protocol as described previously (Muyzer et al. 1995). Nested amplification with group-specific primers for SRB was also performed as described previously (Dar et al. 2005). We used 1 μl of genomic DNA and 2 μl of cDNA as template for the amplification reactions. The quality of the PCR products was examined on 1% (w/v) agarose gel, and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies, Delaware, USA). DGGE of 16S rRNA gene fragments and phylogenetic analysis DGGE was performed as described by Schäfer and Muyzer (2001) using the D-Code system (BioRad Laboratories, California, USA). After electrophoresis, the gels were incubated for 30 min in a solution containing ethidium bromide (0.5 μg/ml), rinsed for 20 min in Milli-Q water, and photographed using a BioRad GelDoc station (BioRad, California, USA). Individual bands were excised, resuspended in 20 μl of Milli-Q water, and stored overnight at 4°C. A volume of 3 to 5 μl of the supernatant was used for reamplification with the original primer sets. The reamplified PCR products were run again on a denaturing gradient gel to check their purity. Before sequencing, the PCR products were purified using the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany). The obtained 16S rRNA gene sequences were first compared to the sequences stored in publicly accessible database using the NCBI BLAST search tool (http://www.ncbi.nlm.nih.gov/BLAST; McGinnis and Madden 2004). Subsequently, the sequences were imported into the ARB software program (Ludwig et al. 2004) and aligned using the automatic aligner function. The alignment was further corrected manually, and an optimized tree was calculated using the neighbor-joining algorithm with Felsenstein correction. Design of oligonucleotide probes Specific oligonucleotide probes for the 16S rRNA of the dominant anaerobic communities in the reactors were designed using the probe design tool of the ARB software package (Ludwig et al. 2004) as described previously (Dar et al. 2007a). The oligonucleotides used for in situ hybridisation are given in Table 1. Table 1Oligonucleotides used in this studyProbe nameTarget organismProbe sequence (5′–3′)ReferenceEUB338IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. 1990EUB338IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. 1999EUB338IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. 1999ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann 1991SPS770Genus SporomusaATC CCG TTC ACT CCC CTGThis studySRB385Most DeltaproteobacteriaCGC GTC GCT GCG TCA GGAmann et al. 1990SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. 1996DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. 1992DSM1265Genus DesulfomicrobiumAGA TTC GCT CGA CCT CGCThis studyDSV139Desulfovibrio sp. strain L7CGC TGT TAT CCC GAT CACDar et al. 2007aDSCOC814Desulfococcus groupACC TAG TGA TCA ACG TTTDevereux et al. 1992 Fluorescence in situ hybridization Fixed cell samples from the reactors were immobilized on Teflon-coated multiwell microscopic slides as described previously (Dar et al. 2007b). Hybridization was carried out according to the protocol as described previously (Manz et al. 1992) using a formamide concentration of 35% (v/v). Quantification of the hybridized cells was performed as described previously (Neef et al. 1996). The hybridized cells were analyzed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualized with general probes for bacteria (EUB338 I, II, and III), sulfate-reducing bacteria (SRB385 and SRB385Db), or with the general DNA stain DAPI (4′,6′-diamidino-2-phenylindole). In addition, a general probe specific for members of the domain Archaea (ARC915) was used. The hybridization experiments were done in duplicate, using different fluorochromes for each probe. Different microscopic fields on each slide were analyzed to confirm the results. Hybridization stringencies of the newly designed probes were determined by performing hybridizations with increasing formamide concentrations as described previously (Manz et al. 1992) using target organism(s) and nontarget organism displaying three mismatches within the target region. Sequence accession numbers The sequences determined in this study were submitted to GenBank under accession numbers EU276620–EU276626. Results Microbial community dynamics at different lactate/sulfate ratio Figure 2 summarizes the effluent composition in the bioreactors, after a minimum selection period of five to six volume changes, as a function of the different lactate to sulfate ratios imposed. The start-up reactor R-0 had a lactate/sulfate ratio (r = 0.65 mol mol−1) theoretically sufficient to allow full lactate oxidation to CO2 with sulfate (also see Table 2). The results for R1 with excess sulfate (r = 0.34 mo/mol) indicate that after five to six volume changes only sulfate, sulfide, and acetate, and no other organic acids were detected in the effluent of the bioreactor (Fig. 2). Nearly all lactate (3.5 mM) had been converted to acetate (3.2 mM) with the equivalent conversion of sulfate to sulfide. These measurements indicate partial oxidation of lactate to acetate, coupled to sulfate reduction was an important reaction in R1 (reaction 2, Table 2). At a high lactate to sulfate ratio (r = 1.94 mol mol−1) in the reactor R2a, propionate was detected in the effluent besides acetate (Fig. 2), demonstrating that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table 2). In this sulfate-limited reactor, nearly all sulfate had been converted to sulfide. At a very low sulfate concentration (r = 20.9), lactate fermentation to acetate and propionate was observed as a dominant reaction (reactor R2b). The shift in the microbial population structure due to different lactate to sulfate ratios after five to six volume changes was recorded by DGGE profiling of 16S rRNA gene fragments, which were amplified either from genomic DNA or from reverse-transcribed RNA, i.e., cDNA. Fig. 2Effluent concentrations of sulfide, sulfate, acetate, and propionate in reactors R1, R2a, and R2b. The sulfide data are minimum numbers due to N2 strippingTable 2Stoichiometry of anaerobic degradation reactions relevant to this studyNo.ReactionΔG01 kJ reaction−11−128.52−80.83−55.74−37.75−85.46−31.1 The comparative analysis of the profiles based on the DNA and RNA of the same sample reflect not only the presence but also the actively growing populations. Figure 3 illustrates the results of the bacterial 16S rRNA–DGGE reflecting the shift in the predominant populations with the changing lactate to sulfate ratios. For the three lactate to sulfate ratios used (i.e., r = 0.34, 1.94, and 20.9), the DGGE profiles based on 16S rRNA were not identical to the 16S rDNA-based profiles of the same sample. The phylogenetic affiliation of 16S rRNA gene sequences is presented in Fig. 4. A neighbor-joining tree was generated using the sequences of the DNA fragments excised from the denaturing gel. Band B3, representing the most persistent species of all the microbial communities in the DNA based profiles, showed high sequence similarity to the Sporomusa–Pectinatus–Selenomonas group with the closest relative being Dendrosporobacter quercicolus. The DGGE profile of the samples taken from R2a to R2b showed an increase in the intensity of fragment B3. Fragment B5 was abundant in reactors R1 and R2a but with varying degrees of intensity. The fragment was not only persistently present but also belonged to an actively growing population as suggested by its simultaneous presence in the RNA-based profiles. However, the fragment was not present in the DGGE profile when the sulfate feed to the reactor R2b was brought down (r = 20.9). Comparative sequence analysis showed that band B5 clustered with the genus Desulfovibrio with Desulfovibrio mexicoense as the closest relative among the cultured representatives. Fragment B6, which was also significantly present, especially in the RNA-based profiles of R1 and R2a, was related to the members of genus Desulfobulbus with the closest relative being Desulfobulbus rhabdoformis. Band B4 was observed only in R1 maintained at a limiting lactate concentration (r = 0.34 mol mol−1). The fragment was found closely related to Desulfomicrobium apsheronum. Two other fragments excised, band B1 and band B2, although present, did not form a significant proportion of the active microbial communities in R1 and R2 as indicated by the RNA-based profiles. The two fragments were affiliated to an uncultured group of Cytophaga and Spirochetes, respectively. Nested amplification with group-specific primers (results not shown) resulted in the identification of an acetate-utilizing SRB represented by band B7. The sequence of the fragment was found closely related to Desulfococcus biacutus. Fig. 3DGGE analysis of 16S rRNA gene fragments using DNA and RNA samples from reactors R-0, R1, R2a, and R2b. Lanes 1 (DNA) and 2 (RNA) sample from reactor R-0 (influent lactate 6.7 mM); lanes 3 (DNA) and 4 (RNA) sample from reactor R1 (influent lactate 3.5 mM); lanes 5 (DNA) and 6 (RNA) from reactor R2a (influent lactate 20 mM); lanes 7 (DNA) and 8 (RNA) from reactor R2b (in the near absence of sulfate). Bands indicated with a dot were excised and sequencedFig. 4Phylogenetic tree based on 16S rRNA gene sequences obtained from the DGGE bands (see Fig. 3). Sequences determined in this study are in boldface. Black dots on the nodes indicate bootstrap values of 90% and higher (1,000 replicates). The scale bar indicates 10% sequence difference The archaeal 16S rRNA gene-targeted DGGE indicated that the diversity was limited to a single species (results not shown) and it was found at all lactate to sulfate ratios except the lowest ratio (r = 0.34) with 3.5 mM lactate. Sequence analysis of the excised fragment indicated its close relatedness to a group of uncultured Archaea distantly related to Methanosaeta sp. Microbial community dynamics using FISH The FISH results were obtained using oligonucleotide probes that targeted dominant microbial communities present under different substrate concentrations. The results are presented as relative abundances of populations targeted by specific probes and are expressed as the percentage of the total positive signal obtained by general probes for bacteria (EUB338 I, II, and III) or with the general DNA stain DAPI (4′,6′-diamidino-2-phenylindole). In addition, the general probe specific to Archaea (ARC915) gave a measure of Archaea. The percentage abundance of the specific probes relative to the general probes is summarized in Fig. 5a and b. In general, probe EUB338 stained more than 90% of all the cells in the samples. Probe SPS770 specifically targeted the species that was closely related to D. quercicolus. At the influent lactate concentration of 3.5 mM (reactor R1), the relative abundance of this organism was <2% of the total bacterial population (Fig. 5a and 6b). When compared to the start-up reactor R-0, the relative abundance of the cells targeted by probe SPS770 was 8–10% of the total bacterial cells (Fig. 5a and 6a). The relative percentage of cells that hybridized with this probe was significantly higher (40–45% of the total bacterial cells), in samples taken from reactor R2a (lactate excess, 20 mM; Fig. 5a and 6c) and increasing even higher to more than 70% (Fig. 5a and 6d), when the sulfate feed to reactor R2b was minimized. Probes DSR660, DSV139, and DSM1265 were used to target different sulfate-reducing communities (Table 1) that were identified in the reactor samples using DGGE. Desulfomicrobium-like SRB targeted by probe DSM1265 were present in high proportions at excess sulfate (reactor R1, r = 0.34 mol mol−1). The percentage abundance relative to the total bacteria was more than 65% compared to 15–20% in the inoculum (R-0; Fig. 5b). The numbers were about 5–8% at limiting sulfate concentrations in reactor R2a (r = 1.94) and decreased further to undetectable levels in the virtual absence of sulfate in reactor R2b (r = 20.9). Desulfovibrio-like SRB targeted by probe DSV139 were the most resilient of all the SRB. Their numbers did not change significantly, fluctuating between 10% and 7% of the total bacteria in the two reactors R1 and R2-a, respectively (Fig. 5b). They were reduced to undetectable levels when the sulfate supply to reactor R2-b was virtually stopped. Fig. 5a Relative abundance of Archaea, sulfate-reducing bacteria, and acetogenic bacteria. b Relative abundance of Desulfomicrobium-, Desulfovibrio-, Desulfobulbus-, and Dendrosporobacter-like bacteria. The percentage abundance is relative to the signal obtained with probe EUBmixFig. 6Whole-cell hybridization of samples from reactors R-0, R1, R2a, and R2b probe EUBmix labeled with Cy5 (blue), probe SRB385 and 385Db labeled with Fluos (green), and probe SPS770 labeled with Cy3 (red). a Reactor R-0 (influent lactate 6.7 mM); b reactor R1 (influent lactate 3.5 mM); c reactor R2a (influent lactate 20 mM); d reactor R2b (in the near absence of sulfate) The relative abundance of Desulfobulbus-like SRB targeted by probe DSR660 showed the most significant increase among all SRB detected, responding to an increase in lactate concentration combined with sulfate limitation. The percentage abundance increased from about 10–12% in the inoculum (reactor R-0; Fig. 5b) to about 20% of the total bacteria in reactor R2a maintained at limiting sulfate concentration (r = 1.94). In reactor R1 (i.e., at sulfate excess conditions), the Desulfobulbus-like SRB were <5% of the total bacteria (Fig. 5b). The change to a low sulfate concentration in the reactor resulted in a considerable increase in the methanogenic population. FISH with probe ARCH915 indicated an increase of the methanogenic population from 5% in the inoculum (R-0) to more than 10–15% under limiting sulfate (R2-a) and nonsulfidogenic (R2-b) conditions (Fig. 5a). The relative abundance of the methanogenic population decreased to undetectable levels after the shift from R-0 to the lactate-limited reactor R1. Discussion This study evaluates the shift in microbial structure in lab-scale CSTR reactors as affected by changing lactate to sulfate ratios using a combined approach of molecular methods (PCR–DGGE and FISH) and chemical analysis. The combination of different methods allowed the establishment of a link between the population structure and function of the anaerobic communities in the reactors under sulfidogenic and nonsulfidogenic conditions. PCR–DGGE profiles produced using DNA templates may not reflect the dynamic changes in community structure. Metabolically active cells have a much higher ratio of rRNA to DNA than dormant cells (Weller and Ward 1989). Therefore, the use of rRNA templates may emphasize the actively growing populations in the community as opposed to those that are relatively inactive but persistent in the community. Because PCR-based approaches for the analysis of microbial diversity in mixed populations can be influenced by several constraints (von Wintzingerode et al. 1997), our results based on PCR–DGGE do not necessarily reflect the abundance of target sequences in the reactor samples. We, therefore, tried to confirm the relevance of the sequence data by whole-cell hybridization using fluorescently labeled oligonucleotide probes. Depending on different lactate to sulfate ratios, theoretically, various scenarios involving SRB in the degradation of lactate are possible as shown in Table 2. Complete oxidation of lactate to CO2 and sulfide by SRB (reaction 1, Table 2).Incomplete oxidation of lactate to acetate by SRB (reaction 2, Table 2).Fermentative degradation of lactate to propionate and acetate and the utilization of fermentation products by SRB and acetoclastic methanogens (reactions 3, 4, 5, and 6, Table 2).In the absence of sulfate the fermentative degradation of lactate coupled to acetoclastic methanogens (reactions 3 and 6, Table 2). The net metabolic pathway of the degradation of lactate by a mixed culture inoculum used in this study suggests its dependence upon the relative quantities of lactate and sulfate. Theoretically, the complete oxidation of lactate to inorganic carbon requires a minimum lactate to sulfate ratio of 0.67 mol mol−1 (reaction 1, Table 2) and if the ratio is below 0.67, additional electron donor is required for complete sulfate reduction. At lower molar ratio of lactate to sulfate (0.34 mol mol−1, reactor R1), only sulfate, sulfide, and acetate were detected in the effluent. These results directly show the quantitative oxidation of lactate to acetate coupled to sulfate reduction as a dominant reaction (reaction 2, Table 2). This is consistent with high percentage abundance of Desulfomicrobium- and Desulfovibrio-like SRB (known, incompletely oxidizing, SRB capable of performing reaction 2, Table 2; Figs. 5a and 6b; Widdel 1988). Complete lactate oxidation to carbon dioxide with sulfate as electron acceptor would be energetically favorable (reaction 1, Table 2). However, the kinetics of this complete oxidation may not enable efficient competition with the incomplete lactate oxidizers. This is evident from the observation that in batch culture in the presence of excess sulfate, incompletely oxidizing SRB outcompete completely oxidizing species due to their superior maximum specific growth rate on fermentation intermediates like lactate, ethanol, propionate, etc. (McCartney and Oleszkiewicz 1993; Widdel 1988). The actual competition between partial and complete lactate oxidizing SRBs will be determined by the affinity for lactate (μmax/Ks). Given the fact that the nested amplification suggested that the numbers of the complete oxidizers were very low in the inoculum, a long period of time would have been required before such SRB would have grown to a significant number. Therefore, we cannot rule out that eventually the complete oxidizers might have become dominant under lactate-limiting conditions in R1. In the near absence of sulfate, lactate fermentation to acetate and propionate (reaction 3, Table 2) was observed as a dominant reaction in the reactor R2b. This was reflected by a significant increase in the relative abundance of Dendrosporobacter-like species and a near washout of SRB species (Fig. 5a and 6d). D. quercicolus is an obligate anaerobe that clusters with the Sporomusa–Pectinatus–Selenomonas group (Strompl et al. 2000). The members of this group are known to perform acetogenic and propionic acid fermentation (reaction 3, Table 2; Biebl et al. 2000). Major fermentation products of D. quercicolus and other closely-related members of the group are acetate and propionate. Theoretically (reaction 3, Table 2), the fermentation of 6.7 mM of lactate should yield approximately 4 mM of propionate and 2 mM of acetate assuming that 0.5 mM is assimilated into the biomass. However, the acetate concentration in the effluent of the reactor R2b (Fig. 2) indicated a net loss of 15% of the electron donor. The plausible explanation for this loss can be acetoclastic methanogenesis (reaction 6, Table 2) that can be assumed from an increase in the methanogenic population to 15% under nonsulfidogenic conditions (Fig. 5a). The lactate to sulfate molar ratio in reactor R2a (1.94 mol mol−1) was increased by a factor of three compared to the inoculum, reactor R-0 (0.67 mol mol−1) and a factor of about six compared to reactor R1 (0.34 mol mol−1). The conditions in reactor R2a were changed to sulfate limitation with a substantial increase in influent lactate concentrations. Indeed, the sulfate concentration had dropped to <0.2 mM, showing that the SRBs could no longer compete for the remainder of the substrates. The products in the effluent included propionate and acetate besides sulfide, suggesting that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table 2). This indicates a change in the net metabolic pathway for the degradation of lactate compared to the reactor R1. This change may be attributed to the lactate fermenting bacteria catalyzing reaction 3 (Table 2). The same is suggested by a significant increase in the population of D. quercicolus-like species, 5 times increase relative to reactor R-0 and even 20–25 times relative to reactor R1 (Fig. 5a and 6c). This is in agreement with the previous studies performed (McCartney and Oleszkiewicz 1993; Mendez et al. 1989; Pichon et al. 1988; Ueki et al. 1986). In all these studies, an increase in the buildup of propionate was observed with increasing organic substrate to sulfate ratio. McCartney and Oleszkiewicz (1993) attributed this change to the net metabolism of lactate, the combined influence of the increased lactate to sulfate ratio, and the time for which the culture was exposed to excess sulfate. This is clearly also the case in our experiments: if a steady state has been reached, the DNA and RNA bands in the DGGE are expected to be similar, if not identical. As demonstrated in Fig. 3, this was not the case. Again, if growth rates would have been only marginally different, a true steady state might have taken several weeks to become established, and hence, we cannot rule out that we have looked at a snapshot during a shift in the population. In this study, the exposure of the original inoculum from the anaerobic wastewater plant to excess sulfate was approximately 13 days during start-up reactor, R-0, before an increase in the lactate to sulfate ratio was imposed in reactor R2a (1.94 mol mol−1). This exposure time was enough to enrich SRB in sufficiently high numbers for the development of substantial sulfidogenesis leading to sulfate depletion. As pointed out above, the consequence was that the culture runs into sulfate limitation and, under this condition, acetogens and methanogens experience no competition for the remaining substrate. It is interesting to note that the buildup of propionate in reactor R2a was concurrent to the increase in D. rhabdoformis-like SRB (Fig. 5b), which are known to degrade propionate (reaction 4, Table 2). This increase in the relative abundance of D. rhabdoformis-like SRB, suggesting a synergistic association between heteroacetogenic bacteria and SRB, is a feasible scenario in the presence of limiting sulfate. An overall electron balance over the system was established, assuming a biomass yield of 0.07°C-mol C-mol−1. In the experiment, at a lactate to sulfate ratio of 0.34 mol mol−1 (reactor R1), the electron balance was nearly 95%. However, in the other experiments with R2, a net loss of reduction equivalents between 10% and 50% is observed. This may be attributed to methane or H2 formations, which were stripped out with the sulfide. Assuming the dominance of partial lactate oxidation coupled to sulfate reduction in reactor R1 (0.35 mol mol−1) and a partial lactate fermentation to propionate and acetate in reactor R2-a (1.94 mol mol−1), both H2 and acetate are the most plausible precursor for methane formation. However, the observation that relatively high concentrations of acetate were found in the reactor effluent (1–4 mM) and the detection of methanogenic Archaea which clustered with Methanoseata sp. suggests that, given the dilution rate of 0.02 h−1, only a low affinity acetoclastic methanogen catalyzing reaction (reaction 6, Table 2) could be maintained in the system. These results were consistent with the increase in the percentage abundance of methanogenic Archaea to more than 10–15% of the total DAPI-stained cells. Because the conditions in the reactors did not favor the growth of nonmethanogenic Archaea (extreme halophiles, thermoacidophiles, the Archaeoglobales, and thermophiles placed in Crenarchaeota kingdom; Woese 1987), cells targeted by probe ARCH915 can be assumed to be the total methanogenic representation in this environment. In conclusion, this study points to different metabolic routes being followed by a mixed anaerobic community, even in the mineralization of simple substrates like lactate. We could establish a link in microbial population dynamics to major perturbations caused by changing influent lactate to sulfate ratios. This study demonstrated that at low lactate to sulfate molar ratios in the influent, SRB had a competitive advantage over acetogens and methanogens and that, in the near absence of sulfate, heteroacetogens formed a syntropic association with methanogens. Higher lactate to sulfate ratio resulted in a pathway that had propionate and acetate as products, and the majority of sulfidogenesis and methanogenesis was dependent on the fermentation products. All these results were substantiated by corresponding shifts in relative abundance of the microbial communities present as analyzed by DGGE and FISH.

Arch_Dermatol_Res-3-1-2064945

Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction (DEJ) separation, an inflammatory cell infiltrate in the upper dermis, and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180. Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation, mast cell degranulation, neutrophil infiltration, and proteinase secretion. This model is not compatible with study of human pathogenic antibodies, as the human and murine antigenic epitopes are not cross-reactive. The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies, and presents an ideal in vivo system to test novel therapeutic strategies for disease management. Etiology of bullous pemphigoid In 1953, Lever [28] described bullous pemphigoid (BP) as a subepidermal blistering disorder primarily seen in the elderly. Lesional/perilesional skin of BP patients exhibits detachment of the basal keratinocytes of the epidermis from the dermis at the level of the lamina lucida [55], resulting in tense, fluid-filled vesicles. BP is both an inflammatory disease and an autoimmune disease, characterized by an inflammatory infiltrate at the site of the dermal–epidermal junction separation and by the deposition of autoantibodies and complement components along the basement membrane zone (BMZ). A number of inflammatory cells are present in the upper dermis and bullous cavity, including eosinophils (the predominant cell type), neutrophils, lymphocytes, and monocytes/macrophages. Both intact and degranulating eosinophils, neutrophils, and mast cells (MC) are found in the dermis. Local activation of these cells may occur via the multiple inflammatory mediators present in the lesional skin and/or blister fluids, including (a) granular proteins derived from degranulated leukocytes, such as eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), and neutrophil-derived myeloperoxidase (MPO) [1, 4, 8] and (b) chemoattractants and cytokines, such as C5a fragments, histamine, leukotriene B4, interleukin-1, -2, -4, 5, -6, -8, -15, TNF-α, IFN-γ, RANTES, and eotaxin [9, 10, 21, 22, 46, 47, 48, 58, 62]. Additionally, several proteinases are found in BP blister fluid, including plasmin, collagenase, elastase, and 92-kDa gelatinase [2, 14, 24, 27, 44, 45, 52, 57]. These proteolytic enzymes may play a crucial role subepidermal blister formation in BP via their ability to degrade extracellular matrix proteins. BP patients generate a polyclonal repertoire of autoantibodies that bind to the BMZ and activate complement, as well as circulating autoantibodies [20]. These autoantibodies target two major hemidesmosomal antigens of 230 kD (BP230 or BPAG1) and 180 kD (BP180, BPAG2, or type XVII collagen) [25, 40, 56, 57]. BP230, a component of the hemidesmosomal plaque, is an intracellular protein, while BP180 is a type II transmembrane protein [19, 23, 56]. Like BP230, BP180’s amino-terminal portion localizes to the intracellular hemidesmosomal plaque [15, 18, 19]. Its carboxyl-terminal region extends into the extracellular milieu of the BMZ, making it the preferred target for pathogenic BP autoantibodies. This antigenic extracellular region consists of 15 collagen domains separated from one another by non-collagen sequences. The largest of these non-collagen domains is referred to as NC16A. Epitope mapping studies indicate that BP autoantibodies of IgE and IgG isotypes and IgG1 and IgG4 subclasses recognize multiple epitopes that cluster within BP180 NC16A [3, 11, 16, 26, 63]. Serum levels of these autoantibodies are correlated with disease severity [11, 17, 49]. Most BP patients elicit a cell mediated autoimmune response in addition to the humoral response described. Autoreactive CD4+ T lymphocytes recognize epitopes within the extracellular region of BP180, primarily in the NC16A domain [5, 29]. These T cells express memory cell surface markers and exhibit a Th1/Th2 mixed cytokine profile. These studies suggest that BP is a T and B cell-dependent and antibody-mediated skin autoimmune disease. Development of murine IgG passive transfer model of BP The strong correlation between BP disease severity and serum BP180-specific autoantibody levels suggests that BP blister formation is mediated by autoantibodies. Early attempts to demonstrate the pathogenicity of patient autoantibodies via a passive transfer mouse model were unsuccessful because BP autoantibodies that react with an immunodominant and potentially pathogenic epitope in BP180-NC16A fail to cross-react with the murine form of this autoantigen (mBP180 NC14A) [30]. In 1993, Liu et al. [30] devised a strategy to overcome this difficulty and generated rabbit polyclonal antibodies raised against a cloned segment of mBP180 NC14A and passively transferred the purified rabbit anti-mBP180 IgG into neonatal BALB/c mice. The injected animals developed a disease that exhibited the following hallmarks of human BP: (a) clinical skin lesions; (b) in vivo deposition of rabbit IgG and mouse C3 at the basement membrane by direct IF; (c) dermal-epidermal separation and an extensive inflammatory cell infiltration by H&E staining [30]. This infiltrate includes neutrophils, lymphocytes, and monocytes/macrophages, with neutrophils being the predominant cells [7, 30]. Immunopathogenesis of experimental BP in the murine model Development of an in vivo system to study an experimental BP model has allowed for great progress in defining the etiopathogenesis of disease. Specifically, the roles of pathogenic antibodies, the complement system, inflammatory cells, and proteolytic enzymes have all been elucidated in the context of the murine IgG passive transfer model. Injection of anti-mBP180 IgG initiated subepidermal blister formation, and the levels of circulating anti-mBP180 antibodies completely determine disease onset and severity [30, 34]. Epitope mapping studies demonstrated that pathogenic anti-BP180 antibodies recognize a 9–12 amino acid stretch within the murine BP180 NC14A region of the antigen [31]. This epitope overlaps the region of the human BP180 NC16A that contains the immunodominant epitopes recognized by human BP autoantibodies, supporting the relevance of this murine system as a model for human disease. Using mice deficient in different inflammatory cell types, it has been demonstrated that mast cells, macrophages, and neutrophils, but not T and B lymphocytes, play a direct role in subepidermal blistering in experimental BP [7]. Anti-mBP180 autoantibodies trigger skin blistering in wild-type mice and mice deficient in T, B, or both T and B cells, but fail to induce lesions in mice deficient in mast cells, macrophages, or neutrophils. Quantification of disease severity in these mice demonstrated that mast cells and neutrophils play a key role in experimental BP. Autoantibodies bind to basement membrane antigens and activate complement in human BP. In the rabbit anti-mBP180 IgG-induced BP, complement activation is absolutely required. Both BALB/c mice depleted of complement (by pretreatment with cobra venom) and C5-deficient mice are resistant to experimental BP, and reconstitution of the C5-deficient mice with C5a restores susceptibility [32]. F(ab’)2 fragments generated from the pathogenic anti-mBP180 IgG cannot induce subepidermal blisters in C5-sufficient mice [32]. Mice deficient in complement component C4 (specific for the classical and lectin pathways), but not in factor B (specific for the alternative pathway), are resistant to BP [41]. Wild-type mice depleted of complement component C1q (specific for the classical pathway) fail to develop BP skin lesions when injected with pathogenic IgG [41]. These data suggest that the classical pathway of the complement activation plays a major role in development of disease, namely through the generation of C5a that in turn activates MCs [6]. Mice injected with pathogenic anti-mBP180 antibodies exhibit extensive MC degranulation in the lesional skin, similar to that observed in human BP [6, 59]. MC-deficient mice are resistant to experimental BP, but reconstitution of these mice with MCs restores their susceptibility to disease. MC activation precedes neutrophil infiltration, and either the absence of MCs or the inhibition of MC degranulation prevents neutrophil infiltration and blister formation. However, MC-deficient mice reconstituted locally with neutrophils, or injected locally with neutrophil chemoattractants IL-8 or TNF-α form blisters in response to anti-mBP180 IgG. These results suggest that mast cells release proinflammatory cytokines critical for neutrophil recruitment [6]. Experimental BP requires neutrophil infiltration, and there is a direct correlation between disease severity and number of infiltrating neutrophils [33]. Depletion of neutrophils, or disruption of the events upstream of neutrophil infiltration (complement activation and MC degranulation), renders mice resistant to BP blister formation. However, complement system and MC deficiencies only confer protection from the pathogenic effects of BP180 autoantibodies if neutrophil infiltration is blocked as a consequence; that is, complement activation and MC degranulation are only critical to disease progression in their roles of neutrophil recruitment. This suggests that infiltrating neutrophils are the cells that directly cause tissue injury in the dermal–epidermal junction, leading to BP skin blisters. Upon activation through molecular interactions between the FcγIIIR on the neutrophil cell surface and the Fc portion of pathogenic anti-mBP180 IgG [61], infiltrating neutrophils secrete proteolytic enzymes known to degrade the extracellular matrix, including neutrophil elastase (NE) and gelatinase B (GB). A deficit of either of these two enzymes blocks blister formation in mice [35, 36]. In vitro, although both GB and NE are capable of degrading the recombinant BP180 protein, only NE produces DEJ separation when incubated with skin sections [36, 52]. In vivo, the degradation of BP180 depends on NE activity. Taken together, these findings suggest that GB acts upstream of NE. Specifically, GB proteolytically inactivates the physiological inhibitor of NE (α1-proteinase inhibitor), which allows for unmitigated NE cleavage of extracellular matrix proteins (including BP180), resulting in DEJ separation [37]. Development of the humanized murine passive transfer model While the rabbit anti-mBP180 IgG passive transfer model has provided invaluable insight to the key steps in BP disease development, it does not allow for experimentation with BP autoantibodies isolated from human clinical samples. A second animal model, in which rabbit antibodies directed against the extracellular domain of hamster BP180 (NC16A region) are transferred into neonatal hamsters, also lacks cross-reactivity with antibodies directed against human BP180 [59]. To assess the pathogenicity of human anti-BP180 autoantibodies, we generated a novel mouse strain in which the murine BP180NC14A was replaced with the homologous human BP180NC16A epitope cluster region [38]. The humanized NC16A (NC16+/+) mice injected with anti-BP180NC16A autoantibodies develop BP, and the disease development depends on complement, mast cells and neutrophils (Fig. 1). Fig. 1Proposed mechanism of subepidermal blister formation in humanized NC16A mouse model of BP. Subepidermal blistering is an inflammatory process involved in following steps: a anti-BP180 autoantibodies are injected into NC16A mice; b pathogenic antibodies bind to the pathogenic epitope of BP180 antigen in basal keratinocytes (BK); c the molecular interaction between BP180 antigen and anti-BP180 IgG activates the complement system (C′) as evidenced by BMZ deposition of mouse C3; d C′ activation products C3a and C5a cause mast cells (MC) to degranulate; e MC activation leads to neutrophil (PMN) recruitment; f infiltrating PMNs bind to the BP180-anti-BP180 immune complex via the molecular interaction between Fcγ receptors on neutrophils and the Fc domain of anti-BP180 IgG. Activated PMNs release proteolytic enzymes; g proteolytic enzymes degrade BP180 and other extracellular matrix proteins, leading to dermal–epidermal junction separation; h pathogenic antibody-injected mice develop clinical blisters Using the NC16A+/+ mice and BP patients’ sera, we mapped the pathogenic epitope of BP180 in vivo [39]. IgG passive transfer experiments identified one pathogenic epitope (referred to as BP180NC16A2.5). BP180NC16A2.5-specific autoantibodies induced subepidermal blisters in the NC16A+/+ mice, and these blisters were blocked by pretreatment with recombinant BP180NC16A2.5. We also found that the NC16A2.5 pretreated mice had significantly reduced levels of BMZ-bound and circulating pathogenic antibodies and showed reduced complement activation, mast cell degranulation and neutrophil infiltration. These results suggest that targeting the pathogenic epitope specifically could be a new therapeutic strategy to treat BP. A second humanized animal model has been developed by Nishie et al. [42, 43] that replaces the mouse BP180 (COL17) with the human analogue. Upon injection of human anti-BP180 autoantibodies, these mice reproduce the DEJ separation at the lamina lucida, the deposition of human IgG along the DEJ, and an inflammatory cell infiltrate consisting of neutrophils and eosinophils seen in human disease. The pathogenic effects of the autoantibody injection are ablated by pretreatment with a 77-amino acid peptide fragment of COL17 NC16A, referred to as R1. These studies further support the therapeutic potential of employing decoy peptides to block the pathogenic epitope. Relevance of murine and humanized passive transfer models to human BP Both in vitro and in vivo data demonstrate that BP180 is the target for pathogenic autoantibodies in BP (Table 1). While clinical human BP and experimental murine and humanized murine BP closely mimic each other at the clinical, histological, and immunological levels, the IgG passive transfer models do not reflect the large number of eosinophils typically found in the inflammatory infiltrate of human BP lesional skin. Some patients do exhibit neutrophil-rich pemphigoid, indicating that the neutrophil-mediated blistering observed in these mouse models may be one of several disease mechanisms that contribute to the formation and persistence of subepidermal blisters. The presence of a neutrophil-rich inflammatory infiltrate in the two humanized mouse models, as well as in the aforementioned hamster model, lend credence to a neutrophil-mediated disease mechanism. Furthermore, findings that in vitro DEJ separation induced by human BP autoantibodies specific for BP180NC16A depends on neutrophils [51] and neutrophil-derived elastase and gelatinase B [50], and that BP180 degradation by human BP blister fluid depends on neutrophil elastase activity [57] support this theory. Taken together, these findings strongly suggest that like the IgG passive transfer model of BP, neutrophils may be responsible for subepidermal blister formation in human BP, at least in those patients who show neutrophil infiltration in their lesional/perilesional skin. The humanized mouse models do not yet offer insight to an eosinophil-mediated mechanism of disease progression, leaving the role of these inflammatory cells, if there is any, yet to be discovered. It is possible that these established mouse models, without additional experimental manipulations, may not be able to fully duplicate human BP pathology related to eosinophil infiltration; therefore, they may not be appropriate to study the role of eosinophils in BP. Table 1In vitro and in vivo evidence of pathogenicity of anti-BP180 antibodiesSystemAntibodies usedReferenceIn vitro Human skin sectionBP sera[13]Anti-BP180NC16A autoantibodies[51]Rabbit anti-BP180NC16A IgGIn vivo Wild-type miceRabbit anti-murine BP180 IgG[30] HamsterRabbit anti-hamster BP180 IgG[60] Humanized BP180 miceAnti-BP180NC16A autoantibodies[38, 39] Humanized NC16A miceAnti-BP180NC16A autoantibodies[42, 43] Zone et al. [64] reported that IgE hybridoma to LABD97 antigen, a component of the shed ectodomain of BP180, when injected subcutaneously in SCID mice with engrafted human skin, induced eosinophil infiltration and histological blisters in engrafted human skin. Fairley et al. [12] also reported that total IgE isolated from BP sera, when injected into human skin grafted onto athymic nude mice, triggered a dermal infiltrate composed of neutrophils, eosinophils, and histological separation of the epidermis from the dermis. These findings implicate eosinophils in BP; however, a direct link between eosinophil recruitment and blistering in these human skin graft models needs to be established. Conclusions The murine IgG passive transfer model of BP developed by Liu et al. [30] has provided an invaluable tool for the dissection of the mechanism of disease progression. The development of humanized murine models of BP now provides unprecedented insight to the pathogenic role of the autoantibodies that mediate disease. Initial observations with these humanized models indicate that humanized mouse BP exhibits phenotypic and immunological similarities with murine and human BP. Additionally, these humanized models enabled the first identification of pathogenic epitopes in human disease. Blocking of the pathogenic epitope with decoy peptides, if they do not enhance the autoimmune responses, presents a novel clinical therapeutic strategy that may be very effective in abolishing the pathogenic effects of disease.

J_Med_Internet_Res-5-4-1550574

Generic Design of Web-Based Clinical Databases

Background The complexity and the rapid evolution and expansion of the domain of clinical information make development and maintenance of clinical databases difficult. Whenever new data types are introduced or existing types are modified in a conventional relational database system, the physical design of the database must be changed accordingly. For this reason, it is desirable that a clinical database be flexible and allow for modifications and for addition of new types of data without having to change the physical database schema. The ideal clinical database would therefore implement a highly-detailed logical database schema in a completely-generic physical schema that stores the wide variety of clinical data in a small and constant number of tables. Introduction Clinical databases may contain a large variety of data from different domains, eg, patient visits, test results, laboratory reports, diagnoses, therapy, medication, and procedures. Clinical databases may have different purposes, eg, patient management, electronic patient records, clinical research, and quality control. Clinical databases usually have a large number of users with different requirements for views of the database. The administrator does not want to view data per patient, while the nurse must be able to lookup current medication for a specific patient. The researcher may want to do data mining on clinical information for thousands or millions of patients, and the clinician should be able to see his or her ambulatory schedule. Most clinical databases comprise only a part of these functionalities, but these examples illustrate the challenge that designers of clinical databases face. Furthermore, in contrast to schemas from many other domains (eg, finance and public administration) the logical data schemas of clinical data are always incomplete and developing. In databases, an entity is a single person, place, or thing (eg, patient or diagnostic test) about which data can be stored. In conventional relational database design, each entity is mapped to one or more tables using values of one or more rows to uniquely identify each record. That means that for each entity there exists at least one table. This strategy works well for most databases even if the number of concepts involved in a domain may be high. As long as the domain of interest remains relatively unchanged, the table layout (ie, the physical schema) should work well for many years. The domain of clinical science in particular (and biology in general) is, however, under constant development as new concepts appear and old concepts are modified or deferred. In a conventional database (that is, in a conventional relational database), new tables must be created to record new concepts. To give users access to the new tables, new forms must be designed and links to these forms must be provided in the user interface. If a table that is already in the database needs to be modified care must be taken not to destroy existing data and not to break any constraints. Accordingly, user-interface forms must be redesigned to reflect changes (eg, fields that have been added or removed) in existing tables. The complexity and the rapid evolution and expansion of the domain of clinical information thus require a large maintenance overhead if data are laid out using a conventional design. For this reason, it is desirable that a clinical database be flexible and allow for modifications and for addition of new types of data without having to change the physical database schema. The ideal clinical database would therefore implement a highly-detailed logical database schema in a completely-generic physical schema that stores the wide variety of clinical data in a small (and constant) number of tables. The aim of this project was to provide an overview of techniques and problems in generic design of Web-based clinical databases. Methods Medline was searched through PubMed [1]. Searching was done by trial-and-error using combinations of keywords to get the best match of articles covering the problem. Furthermore a search strategy was devised for Google [2] using a similar trial-and-error strategy. Results The final PubMed search was done on July 11, 2003 using the search term: (generic database design clinical) OR (entity attribute value). This term was translated by PubMed into: (((entity[All Fields] AND attribute[All Fields]) AND value[All Fields]) OR (((generic[All Fields] AND ("databases"[MeSH Terms] OR database[Text Word])) AND design[All Fields]) AND clinical[All Fields])). Thirty-three papers were found and 13 were selected based on their title. Of these, 7 were selected based on their abstract and the full-text papers [3- 9] were either downloaded or ordered from the Danish National Library of Science and Medicine. Google was searched on the same day using the search term: clinical database generic design. The search was restricted to the first 30 hits. One additional paper [10] and 1 Web resource [11] of interest were found. The 9 resources were all from either of 2 research groups: Department of Medical Informatics, Columbia University, New York, NY and Center for Medical Informatics, Yale University, New Haven, Conn. Three production databases were the basis of the 2 group's research: The Clinical Data Repository at Columbia-Presbyterian Medical Center (CPMC), the Adaptable Clinical Trials DataBase (ACT/DB), and SENSELAB. CPMC [8- 10] is a large clinical repository for millions of patients dating back to the beginning of the nineteen nineties. Several front-end applications offer access to the database giving different views for health care professionals, administrators and researchers. ACT/DB [3,4,6,7,11] is a clinical-trials database built upon the same design principles as CPMC. Nadkarni et al introduce the term "entity-attribute-value (EAV) design" for generic structuring of data in a relational database [7]. The database is accessible through a generic Web-based interface (WebEAV) [4]. Web forms for displaying and editing data are generated automatically during run time from metadata stored in the database. SENSELAB [5] is a database for heterogeneous neuronal data. As such it is not a clinical database. However, the SENSELAB architecture uses an object-oriented approach to the EAV model by defining classes and relations (EAV/CR). The EAV/CR architecture is useful for scientific data in general, but it is of special interest for clinical databases. The principles and design issues involved in these databases are the focus of the remainder of this paper. I will not go into details about the specific implementations of these systems, rather I will present techniques involved in the design of generic database systems. For design details about the 3 database systems the reader is encouraged to consult the references. Entity-Attribute-Value Design In conventional database design, each parameter of interest is represented in a separate column in a table. As new kinds of data need to be managed, the number of columns and/or tables needs to grow. To add a new attribute for patient description (eg, phone number) to a conventional relational database design (Table 1), another column has to be added to the table. Table 1 Conventional relational database design (example) PatientID Name Date of Birth 1 Jens Hansen 1956-Aug-01 2 Hans Jensen 1974-Sept-04 In EAV design, however, data may be stored in a single table with (conceptually) 3 columns: 1 column for entity identification, 1 for attribute, and 1 for the value of the attribute (Table 2). Table 2 EAV (Entity-Attribute-Value) database design PatientID Attribute Value 1 Name Jens Hansen 1 DateOfBirth 1956-Aug-01 2 Name Hans Jensen 2 DateOfBirth 1974-Sept-04 To add a phone number attribute in the EAV table (Table 2), all that is required is to define a new code for phone number to be stored in the attribute column. No change to the table schema is needed. Theoretically, most of the facts that are stored in a database can be stored in a single EAV table. The EAV design has several advantages: Flexibility: There are no limits to the number of attributes per entity. The logical database schema can grow without affecting the physical schema. Storage: In a clinical database thousands of parameters are available while only a few may be recorded for each patient. In a conventional design this may lead to empty (NULL) fields. The EAV design does not need to reserve space for attributes with NULL values. Efficient entity-centered queries: If, for example, all information for a single patient is needed, it is necessary to query all data tables looking for information about this patient. In a conventional database this may be a time-consuming task that requires looking through hundreds of tables each of which may or may not have information for this patient. As the number of tables and columns grow, the query must be reprogrammed. In an EAV database only 1 table needs to be queried, no joins are necessary, and no change of code is required as the domain evolves. (A join combines data from 2 or more tables based upon a common attribute.) The EAV design has, however, some drawbacks: Data display: As discussed later, the user naturally regards data as being organized conventionally in tables and columns regardless of the physical layout of data. Consequently it may be necessary to transform ("pivot") EAV data into a conventional layout when displaying data. This and other tasks that a conventional database would do automatically (eg, referential integrity checking or form-to-subform linkage) require considerable front-end programming in EAV designs. (Referential integrity checking is checking that values in one table that are intended to be used as keys to another table are indeed found in the second table.) Less-efficient attribute-centered queries: In contrast to entity-centered queries, complex attribute-centered queries, which are based on attribute values, are significantly less efficient and technically more difficult in an EAV database than in a conventional database. The query "show me all patients whose name starts with J and whose date of birth is earlier than 1970" is straightforward in a conventional database. To achieve the same result in an EAV database, set operations (for example, INTERSECT) or joins on multiple versions of the EAV table would have to be performed. (INTERSECT is an operation that compares 2 queries to identify records that are found in both.) Set operations and joins are considerably slower than simple select operations. As the number of attributes increase the execution time increases exponentially. Querying EAV data will be discussed in greater detail later. Constraint checking: In a well-designed conventional database, constraint checking is either unnecessary or trivial. For example, in a conventional table non-null constraints may be placed on columns to prevent incomplete records from being saved. An incomplete record would appear if, for example, the user forgets to fill in a field on a form. In an EAV table a missing attribute-value pair would normally result in a missing record. For example, if no record for one patient's last name is saved in the EAV table this will—from a logical point of view—lead to data that is inconsistent, in the sense that the data for this patient will not be similar to the data for other patients. To prevent this from happening in an EAV database, checking of such constraints should be programmed into the user interface. Metadata EAV design is a way of simplifying the physical schema of the database, making it domain-independent. Regardless of the physical schema, the user naturally perceives the data as conventionally structured in tables and columns. The logical schema of the database reflects the user's perception of the data. In an EAV database the logical schema differs greatly from the physical schema. In a conventional database the two are similar. Therefore, an EAV system must have some means of translating the physical schema into a logical schema that reflects the user's understanding of data. This is achieved through metadata (or dictionary) tables whose content defines the semantics of the domain being modeled. An example of a metadata table could be a table listing the attributes available to the data in Table 2. In this example the metadata table would have 2 records, Name and DateOfBirth. If it is necessary to record further information about patients, eg, sex and phone number, that information should simply be added to the metadata table. Thus, in this case, metadata represent what would be the column names of a conventional data table. The metadata model may be enhanced considerably by, eg, adding more descriptive attributes to the metadata table. These attributes may have several purposes—eg, definition of an attribute's data type, constraints, or display layout (text field, select box, etc). These issues will be discussed in greater detail in the next section. Evolution of the EAV Model In the following sections, I give examples of different EAV schemas going from the most-simple, least-flexible to the most-advanced, most-flexible schema. The term "simple" is not to be interpreted as inadequate. The simple solution may be the right solution for a specific task. The examples reflect the systems described in the literature but are simplified for pedagogical reasons. A Simple EAV Model A simple EAV schema for a clinical database is outlined in Figure 1. Figure 1 Simple EAV schema for a clinical database. (The crows-foot symbol—3 small lines at the end of a relationship line—illustrates a one-to-many relationship between patient and data, and between attribute and data. Text in each ellipse identifies table type.) Table 3 shows the database tables depicted in the schema of Figure 1. Data have been arranged in a conventional table for patient demographics, an EAV table for clinical events, and a metadata table defining the attributes available to the EAV table. Table 3 represents the patient from Table 1 after a course of influenza that started July 1, 2003 and ended July 11, 2003: The entity part of the Data table is defined by the combination of patientID and date. The attributeID column holds a reference to the Attribute table, which defines the name and type of available attributes. In a real-world production database there would probably be another table to hold the definition of data types. Values may of course be of any type, for example, text, number, or Boolean (true/false). In the example in Table 3, the Value field of the Data table is text type. Such a design achieves simplicity by storing all simple types as text values. This approach has, however, some drawbacks. First, not all data types will fit into a text field. Binary objects, eg, x-ray pictures or ECG (electrocardiogram) curves, are too large as are long texts (memo-fields). Second, queries based on values will be less efficient for nontextual values. The text "12" is less than the text "2" even though it is numerically greater, because text is sorted character by character, from left to right. Table 3 Database tables for the simple EAV schema in Figure 1 Patient table* patientID name Date of Birth gender 1 Jens Hansen 1956-08-01 Male Data table † patientID date attributeID Value 1 2003-07-01 1 Influenza 1 2003-07-01 2 2003-07-11 Attribute table ‡ attributeID attributeName dataType 1 Diagnosis Text 2 EndDate Date * Conventional table for patient demographics. † EAV table for clinical events (data). ‡ Metadata table defining attributes available to the EAV table. Different strategies have been used to store binary data and to increase the efficiency of value-based queries. The simple solution is to ignore the problem and accept that all values be stored as text. This approach may be fully acceptable if it is not necessary to store binary data and if fast value-based queries of large data sets are not required. Another approach is to add a column to the Data table for each data type necessary. For each record, only 1 value-field will be filled in (Table 4). Table 4 Data table with a column for each data type, as a strategy for storing binary objects patientID date attributeID textValue numericValue longValue dateValue 1 2003-07-01 1 Influenza 1 2003-07-01 2 2003-07-11 This approach, of course, does not comply with rules for good database design as empty fields are recorded for each record. It may, however, be acceptable in small "quick-and-dirty" applications [12]. The most solid and, from a database designer's perspective, correct solution is to segregate the data table into a number of tables based on the data type of the attribute (Table 5). Table 5 Data table segregated into multiple tables based on the data type of the attribute, as a strategy for storing binary objects Data table patientID date dataID 1 2003-07-01 1 1 2003-07-01 2 DataDate table dataID attributeID value 1 2 2003-07-11 DataText table dataID attributeID value 2 1 Influenza This approach is used in CPMC, ACT/DB, and SENSELAB. For simplicity I chose to show only 1 data table in the illustrations. The modeling of patient demographic data in a separate conventional table rather than in the EAV table is deliberate (although not necessary). For a schema that is not expected to change often, as is the case with patient demographics, the advantages of an EAV layout do not exceed its disadvantages; and conventional tables and EAV tables can coexist happily together. Furthermore, this design makes it easy to model the one-to-many relation between patient and clinical events. Relations between entities in an EAV table are complicated to model in the simple EAV design. In an electronic patient-record system, for example, it should be possible to record relationships between clinical events (eg, infection leads to a course of penicillin or myocardial infarction leads to death). The enhancement of the EAV design to handle complex relationships between classes will be described later with the EAV/CR schema. For a simple application intended mainly for data entry, the simple EAV schema may suffice. With the need for a more-advanced user interface for data-display and input purposes, however, some means of grouping attributes becomes necessary. With the simple EAV schema, grouping attributes together on display forms may be done only by entity (patientID and date) or attribute. The application has no way of telling how EAV data records are related and should be displayed together—eg, multiple values from the same blood chemistry panel. Enhancing the EAV Model Grouping related attributes for display purposes may be accomplished in several ways. One or more descriptive columns may be added to the "entity part" of the Data table, or the metadata schema may be enhanced. An example of a combination of both methods is shown in Figure 2. Figure 2 Enhanced EAV schema with grouping of attributes for form display. (Text in each ellipse identifies table type.) A group table and a form table have been added to the metadata schema. Attributes may now be grouped and attribute groups may be part of forms. To the entity part of the Data table a new field, formID, has been added telling the application to which form a data record belongs. Now any medical event recorded in the Data table belongs to a form and then may be displayed together with all the other attributes on that form. Furthermore, this design facilitates reuse of attribute groups on different forms. Depending on the domain being modeled and the requirements of the users, other metadata schemas may be suitable. The simple and the enhanced EAV schemas discussed above are examples of the use of generic EAV tables in clinical database applications. Although to some degree generic, the proposed schemas will need adjustment to the actual domain in question. To achieve total domain-independence more refined models must be created. An Object-oriented Approach to EAV Modeling The EAV/CR model adds an object-oriented framework to the EAV model by definition of classes and relations. The EAV/CR model was developed for scientific data in general but is useful for clinical data [5]. Figure 3 shows a simplified example of the EAV/CR table layout used in the SENSELAB database. The class and the attribute tables hold the definitions of classes and their fields. The ClassHierachy table records relations between classes. In this example a subclass can have any number of superclasses, and a superclass can have any number of subclasses. The attribute table records the class to which the attribute belongs and the type of attribute. An attribute can be of any simple type and may even be of class type. Class instances (objects) are recorded in the Object table and instance fields are recorded in the Data table, which is similar to the data table in the simple EAV models. Figure 3 EAV schema with classes and relations (EAV/CR). Simplified from Nadkarni et al [5]. (Text in each ellipse identifies table type.) The example in Table 6 depicts 2 classes, patient and doctor, which are subtypes of a common person class. The patient class has an attribute of object type referring to the patient's responsible doctor. For readability IDs are presented as names instead of numbers. This example illustrates the use of inheritance and composition in database design. Inheritance and composition are two important concepts in object-oriented programming. Inheritance can be regarded as an "is-a" relationship between objects—a patient is a person, and a doctor is a person. Composition is often referred to as a "has-a" relationship—a patient has a doctor. Thus, with this simple layout with (conceptually) just 5 tables, any real-world object can be recorded. Furthermore, objects may be part of other objects; and objects may be related through inheritance. Ad hoc relations between objects (eg, penicillin leads to rash) may be recorded as objects themselves. For this purpose, a class, ObjectRelation, could be defined with 2 attributes, objectID and relatedObjectID. More descriptive attributes may be added to this class if required—eg, causality. Obviously, considerable up-front programming is required to drive an ergonomic user interface for the EAV/CR model in a real-life production environment. On the other hand, this is a one-time-only job. Another drawback of the EAV/CR design is that the system administrator must have a solid understanding of the object-oriented framework in order to design useful classes. An EAV/CR database is therefore hardly an end-user tool for the average clinician or researcher. As always, flexibility comes with a price. Table 6 Database tables as an example of the EAV schema with classes and relations (EAV/CR) in Figure 3 Class table className Person Patient Doctor ClassHierachy table superClassID subClassID Person Patient Person Doctor Attribute table classID attributeName dataType Person Name Text Person Date-of-birth Date Patient Doctor Class: Doctor Patient Gender Text Doctor Position Text Object table objectName classID Patient01 Patient Doctor01 Doctor Data table objectID attributeID value Patient01 Name Jens Hansen Patient01 Date-of-birth 1956-08-01 Patient01 Doctor Doctor01 Patient01 Gender Male Doctor01 Name Doc Doctor01 Date-of-birth 1960-03-12 Doctor01 Position Head Querying EAV Data From a database perspective, querying EAV data is not different from querying conventional data. As mentioned earlier, however, in an EAV database, the physical layout differs greatly from the logical layout, and the user generally wants to see data displayed in a conventional format. As an example, querying Table 1 for facts about patients whose names start with Jens and who were born before 1970 is straightforward: SELECT * FROM table1 WHERE name LIKE 'Jens%' AND dob < '1970'; To achieve the same result from querying Table 2 requires executing a rather-complex SQL (Structured Query Language) statement: SELECT d1.patientID AS patientID, d1.value AS name, d2.value AS dob FROM table2 AS d1 INNER JOIN table2 AS d2 USING (patientID) WHERE d1.attribute='name' AND d1.value LIKE 'Jens%' AND d2.attribute = 'dob' AND d2.value < '1970'; The same result may be obtained in several ways, but in any case the query must include set operations (INTERSECT) or as in this example a self join for each attribute. (A self join is a join of a table with itself.) Aside from being complex and out of reach for most end users, these operations are far slower than simple select statements. I did an experiment using data for one million patients described by 3 attributes: name, date of birth, and gender. These facts were duplicated in a conventional table and in an EAV table in a MySQL database. Three queries were performed on each table with 1, 2, and 3 attributes respectively. Execution time was approximately 2 seconds for the conventional table irrespective of the number of attributes. For the EAV table execution time was 7, 14, and 24 seconds respectively. Thus execution time increases linearly with the number of rows (1 million in the conventional table and 3 million in the EAV table) and—in the EAV table—with the number of joins involved in a query. In the conventional table, however, the number of joins did not affect query time. Some strategies have been suggested to deal with this problem: There may not be a problem. Attribute-centered queries are important for research questions; their performance is not critical for the care of individual patients. If the need for cross-patient data is infrequent the advantages of EAV design probably exceeds the disadvantages. Any need for regular cross-patient data access could be met by making backups of the production database and restoring them onto separate hardware. Resource-intensive queries run on the backup data will not affect the production server. Additionally, the EAV data schema could be transformed into numerous conventional tables after backup thus easing query design by end users with modest SQL skills [6]. If complex, attribute-centered, user-defined, ad hoc queries are important to an application, steps should be taken to facilitate this. First, a user interface, whether graphical or not, should be built to help users retrieve data. The user should be able to freely select any combination of attributes and criteria. The interface should then translate user requests into semantically-valid and syntactically-valid SQL statements; and from the user's point of view, it should not matter whether data are stored in conventional tables or EAV tables. This approach was taken by Nadkarni and Brandt in the development of the ACT/DB Query Kernel [6]. Optimization of queries may increase the efficiency considerably. Breakdown of complex SQL statements into smaller parts run sequentially may increase query speed. Each part accesses 1 or 2 tables to create a temporary table (or view). These (smaller) temporary tables are then joined [3]. Depending on the ability of the database engine to devise an efficient search strategy, the overall query speed may benefit from creating and joining smaller temporary tables compared to self-joining the full EAV table. An efficient database engine should, however, itself be able to optimize the original query, so that little is gained from this approach. In the MySQL database described above, the creation of a single temporary table took longer (more than 30 seconds) than the execution of the full 3-attribute search (24 seconds). Johnson et al [10] suggest an extension to the SQL-query language to facilitate "pivoting" of attribute-centered data into a conventional layout—the Extended Multi-Feature (EMF) SQL. Extended Multi-Feature SQL processing time is linearly proportional to number of attributes. In summary, querying EAV data is a more complex task than querying data in a conventional layout; and attribute-centered queries are less efficient with EAV data compared to conventional data. Graphical User Interface The challenge for the user-interface designer of an EAV database is to display data and to let the user manipulate data simulating a conventional layout irrespective of the physical layout—in other words: to bridge the physical and the logical schemas. The World Wide Web offers an opportunity to simplify database deployment and maintenance. In a typical Web database application, the user's browser requests data from a remote Web server, which sends the request to a database server. After receiving data back from the database server, the Web server formats it into a Web page and sends it to the client browser. There are several advantages of Web deployment: Problems of form deployment are eliminated since all forms reside on the Web server. Deployment costs are reduced because Web browsers are available free. Also, hardware costs are reduced since browsers usually have smaller hardware requirements than desktop database-management systems do. The form-rendering model of Web pages is simpler and smarter than that of traditional software platforms. Objects on a Web page can be automatically reformatted when the browser win­dow is resized or the user changes the font size. Traditional software developers must put much effort into physical screen size issues. This is not necessary with Web forms. Web browsers use clever caching algorithms. That means that when the browser visits a particular page, its contents are cached on the client. On revisit, only components that have changed are downloaded again. This reduces download time and network load. For these reasons, Web deployment is becoming more and more popular for multi-user applications. However, Web database applications are significantly more complex to develop than traditional database applications for several reasons: Web-development tools are less mature than tools for traditional software development; and development of Web database applications still requires much "coding-by-hand." As an example, simple errors such as misspelled variable names, which would be trapped at edit or compile time in a traditional environment, will not be detected until runtime in a Web application. Browser-server communication is inherently stateless; when the server has sent a Web page to the client, it "forgets" about the client. Tracking information (eg, user authentication) through several Web pages therefore involves extra programming. To maintain information, the developer must store data either in (hidden) form fields on Web pages or in session variables, which can be accessed as long as the session lasts. Both approaches complicate development and may compromise security because other users (or processes) may gain access to these data intentionally or accidentally. Designing Web forms requires much more programming than does designing forms in traditional client-server environments. Web form fields are typeless and input masks for formatting user inputs are not inherent parts of Web forms. (In typeless fields the user may accidentally enter numbers in text-only fields or accidentally enter text in numbers-only fields.) This puts pressure on the programmer to put much effort into both client-side and server-side data validation. In a traditional environment, form fields may be typed; thus, eg, the programmer does not need to worry about users entering letters in number fields or invalid dates in date fields. In a Web form, all validation procedures must be hand coded. Finally, population of select boxes (drop-down menus) and radio buttons (option buttons) with dynamic data is usually much easier in a traditional environment than on a Web form. Programming Web forms is tedious and error prone, and automation is highly recommended. Nadkarni et al have studied a generic framework for automatic generation of Web forms for display and manipulation of EAV data (WebEAV) [4]. The main objective was to automate the generation of Web forms based on metadata in an EAV database. When details about an event are requested, a form is generated from the metadata of the attributes involved. Each form field has a unique name, which is constructed such that the field name contains its own metadata. When data is sent back to the server, the server creates the correct SQL statements by parsing field names, and data are updated accordingly. WebEAV makes extensive use of client-side validation of data. Standard validation code in the form of JavaScript is built into the Web page. Validation relies on the use of form field events (eg, OnChange, OnFocus, and OnBlur) and metadata for the attributes in the form (eg, data type, maximum and minimum bounds, and non-null requirements). Discussion Based on searching the literature, it appeared that the Entity-Attribute-Value model is useful for generic design of clinical databases. The most advanced model uses an object-oriented approach and gives tremendous flexibility, allowing the designer to model any type of concept and any relation between concepts in the domain of interest without ever having to worry about changing the table layout or maintaining the user interface. With the ever changing and evolving domain of clinical information, generic design is of special interest for clinical databases, because changes to the logical schema will not affect the physical schema. However, database designers from other areas (eg, biology or literature) may also find the EAV approach useful. Historically, EAV was introduced into clinical databases in TMR (The Medical Record), built at Duke in the 1970s [13,14]. In addition to the ones mentioned in this paper, production databases using EAV components include TrialDB [14], the HELP system [15], the Cerner and 3M repositories, ClinTrial, and Oracle Clinical. Pros and Cons of EAV Design The advantages of generic design are obvious. The disadvantages, however, may be less obvious and depend on the objectives of the specific application in question. From a performance point of view, the strength of the EAV design lies in effective entity-centered queries since no joins are necessary to retrieve all facts about entities (eg, patients or medical events) as would be the case in a conventional design with facts spread over hundreds of tables. The drawback lies in inefficient attribute-centered queries, since a (self) join is necessary for each attribute that is requested. Performance of EAV tables may not be an issue for small databases, but for large clinical repositories with hundreds of concurrent users, query time may be a critical factor. Also, the need for complex attribute-centered data retrieval differs greatly between applications. An electronic patient-record system, for example, is usually aimed at displaying patient-centered (ie, entity-centered) facts, while a research database usually must have some means of aggregating data across a large number of patients. In the latter, however, query efficiency may not be a problem, since data summaries are retrieved only intermittently and may be stored on separate hardware. These issues warrant careful design of the database schema and cautious decisions about when to use conventional tables in place of generic EAV tables. As a rule of thumb, conventional table design is appropriate for entities whose schemas are not expected to change often (eg, people or institutions). Metadata Preserves Information The simplicity and flexibility of the database schema also increases the complexity of collecting and displaying information from data. The user needs to see and enter related data on the same form. Often single values do not make sense unless coupled with other values. Take as an example a body weight of 176 lb (80 kg). This would be perfectly normal (and desirable for some of us) for an adult male with a height of 5 ft, 10 in (182 cm). For a 10-year-old girl, 176 lb would be highly disturbing. Using a simple EAV data table layout, relations between data are lost unless steps are taken to store these as well. This is the whole idea of metadata—to conserve information about relationships between atomic data values. The metadata schema is the only thing that differs between the different EAV models presented in Figure 1, Figure 2, and Figure 3 and between the actual implementations of the EAV model presented in the articles. The data parts are for practical purposes the same. It appears that metadata schemas themselves may be more or less generic depending on how closely related they are to the actual domain being modeled. The more specific the metadata schema is, the less flexible it will be. On the other hand, a specific metadata schema will require less programming to drive the user interface than a highly generic one. To summarize this part, the choice of model depends on the domain and the requirements for flexibility. The object-oriented approach is by far the most flexible solution and in many ways an elegant solution. On the other hand, the complexity introduced by this model may not be justified unless the domain requires the fine-grained control over objects and relations. A simple model may well be the right solution for a simple job. Databases and Objects Much effort has been put into generalizing clinical databases. The most flexible and generic models take an object-oriented approach to data modeling the mapping of objects to tables in a relational database. There is no doubt that object-oriented design is "hot" in the medical area. But porting of object-oriented generic databases from traditional relational databases to produce object-oriented database management systems (OODBMSs) does not seem to be just around the corner. One reason for this may of course be that object-oriented database management systems are still lagging behind relational database management systems with respect to efficiency and availability, although extensive research is going on in this field. Furthermore, object orientation is still a new concept to most clinicians who design databases. But even with modest skills in an object-oriented programming language such as Java, the similarities between object-oriented programming and object-oriented data management seem striking. Object-oriented databases come in two flavors [16]: Systems that provide object-oriented extensions to relational systems by adding composite attributes, class hierarchies, and extensions to a data manipulation language such as SQL. These systems are called object-relational systems. Systems that extend an existing object-oriented programming language like C++ or Java to deal with databases. Such languages are called persistent programming languages. The term "persistent" refers to the fact that the programming language must devise some means of storing objects even when the program is not running. Databases built upon persistent programming languages are called object-oriented databases. The former approach has similarities to the approaches described in this project in that these build upon conventional relational database management systems. The SENSELAB database allows for composition and inheritance, and CPMC has explored the extension of SQL to facilitate attribute-centered querying EAV data. The latter approach to generic database design has to my knowledge not been described in the medical literature. The idea of encapsulating all data and functionality relevant to an object within each object opens up a plethora of possibilities of interest for the developer and manager of clinical information systems: The object-oriented paradigm ("everything is an object") is a means of describing real-world concepts, and objects may be easier to understand for a clinician than complex relationship sets in a relational database. One could say that object-oriented design brings together the logical and the physical schema. Even if this may not be completely true, the user should not have to worry about how to design tables for storing of objects. The database will take care of this. Object-oriented languages handle complex attributes and inheritance much more elegantly than do even the most cleverly-designed relational database. When referring to an object in an object-oriented programming language, the object's fields and methods are available to the user immediately, through the object's interface. To mimic an object in a relational database, the database must be queried for all attributes of interest, and each value must be accessed separately. Objects may contain methods. For example, a person object may contain a print() method, which outputs all information related to the objects in a suitable format. The client programmer, who builds the user interface, does not have to worry how this information is gathered. This programmer only needs to grab the information and present it in a nice layout on a form. Furthermore, different subtypes of the person class, eg, patient or doctor, may have different implementations of the print() method. This is an example of polymorphism and is one of the most powerful features of object-oriented programming languages. Classes may be reused. If a class has been designed, it may be reused in other applications; and if a class is redesigned (eg, to improve execution speed) the client programmer does not need to know this, as long as the class' interface is unchanged. A detailed discussion of object-oriented programming is outside the scope of this article. However, the power of object-oriented programming may be summarized in the terms encapsulation and polymorphism. Encapsulation means that an object knows all about itself and that it interacts with the surroundings only through a well-defined interface. Encapsulation facilitates reuse and safe programming. Polymorphism means "having many forms." A polymorphic reference is one that can refer to objects of different (sub) types at different times, which is exactly what we need in a generic database. It is obvious that these (and other) facilities of object-oriented programming languages would be of immense value in the creation of generic clinical databases. It is, however, important to realize that a database management system, whether object-oriented or not, comprises much more than a programming and query language—important issues being storage management, transaction management and concurrency control—and these issues are still under development in object-oriented database management systems. (Concurrency control involves locking parts of the database to prevent unintentional overwriting of data.) Conclusions The objective of generic database design is to provide a robust physical database schema that does not need to change as the domain evolves. Generic databases are of special interest for clinical information systems, and several approaches to generic design have been exercised. They have in common the use of Entity-Attribute-Value tables for storing data and a number of metadata tables to describe the semantics and the relations between data. An object-oriented approach to generic modeling of metadata is by far the most flexible and domain-independent approach. However, the overhead in taking this approach may not be justified for less-advanced applications. Further studies regarding the implementation of object-oriented database management systems for the purpose of generic clinical databases are suggested.

Osteoporos_Int-4-1-2277446

Efficacy of different doses and time intervals of oral vitamin D supplementation with or without calcium in elderly nursing home residents

Summary The effect of equivalent oral doses of vitamin D3 600 IU/day, 4200 IU/week and 18,000 IU/month on vitamin D status was compared in a randomized clinical trial in nursing home residents. A daily dose was more effective than a weekly dose, and a monthly dose was the least effective. Introduction Vitamin D deficiency is common in older persons, in particular in residents of homes for the elderly and nursing homes and in patients with hip fracture [1–3]. In these groups the prevalence of vitamin D deficiency, defined at that time as serum 25(OH)D < 30 nmol/L based on values in healthy blood donors, was reported to be 75% [3]. This is mainly explained by the fact that older persons do not often go outside in the sunshine and dietary vitamin D intake is low. Vitamin D deficiency causes secondary hyperparathyroidism, which leads to cortical bone loss, osteoporosis and fractures [4]. It may also cause fatigue, muscle weakness, increased body sway and falls [5, 6] . Vitamin D supplementation in vitamin D deficient elderly increases the serum concentration of 25-hydroxyvitamin D (25(OH)D) and decreases the serum concentration of parathyroid hormone (PTH) [3]. It also decreases wintertime bone loss from the lumbar spine [7] and increases bone mineral density of the femoral neck [8]. Vitamin D supplementation combined with calcium decreased body sway and falls in a German study [5] and decreased hip as well as other non-vertebral fractures in French nursing home residents [9], whereas the results in more healthy elderly, living independently in the community were equivocal [10–14]. Vitamin D status in the elderly may be improved by ultraviolet irradiation [15] or by vitamin D supplementation [3, 7, 8–14]. Some controversy exists on the required serum 25(OH)D level, but most investigators agree that the level should be at least 50 or even 75 nmol/l [16, 17]. The Dutch Health Council advises vitamin D 600 IU daily for elderly of 70 years and older who do not come outside in the sunshine [18]. Oral vitamin D3 can be taken once a day but also with longer intervals because of its long half life, being around 25 days. It is not known whether equivalent doses once a week or once a month are equally effective. A low calcium intake aggravates vitamin D deficiency by increasing the turnover of vitamin D metabolites by secondary hyperparathyroidism [4]. On the other side, a high calcium intake does not completely protect against secondary hyperparathyroidism, and thus cannot compensate for vitamin D insufficiency [19]. The calcium requirement for skeletal maintenance is raising with age whereas the capacity for compensating a low calcium intake declines with age [20]. In the Netherlands the mean daily calcium intake of independently living elderly in homes and apartments of the elderly is about 900 mg [2]. In the guidelines of the Dutch Health Council, the advised daily amount of calcium for elderly 70 years and older is 1200 mg [18]. The aim of the present study was to investigate, in a Dutch nursing home population, whether there is a difference in efficacy of different doses and intervals of oral vitamin D3 supplementation with the same total dose. A second aim was to assess the additional effect of calcium supplementation following vitamin D supplementation on serum PTH and markers of bone turnover. Subjects and methods Subjects Ten somatic and psychogeriatric nursing homes participated and 1,006 subjects were invited. Of these, 146 did not respond, 386 refused to participate and 136 did not meet inclusion criteria. Participants were 338 (76 male and 262 female) patients of 70 years or older with a mean age of 84 years (SD 6.2). Exclusion criteria were going outside in the sunshine more than once a week, the use of vitamin D or calcium supplementation, the use of more than one vitamin D-fortified food or drink per day, complete immobilisation and a very poor life expectancy. Poor cognition was not an exclusion criterion. This did not affect adherence. Nursing homes were enrolled in the study throughout the year. Participants living together in the same nursing home started the study during the same season. The dietary vitamin D intake was estimated at about 100 IU/day, based on fish and margarine consumption. In the Netherlands only margarine is fortified with vitamin D3 (3 IU/g) and the diet does not contain vitamin D2. Written informed consent was obtained from participants or their proxies. The protocol as well as the patient information letters were approved by the Ethical Review Board of the VU University medical centre. Randomisation Participants were randomised in blocks of six, to receive, during the study period of four and a half months, either oral vitamin D3 600 IU/day (one tablet) or placebo, 4200 IU/week (seven tablets once a week) or placebo or 18,000 IU/month (one powder once a month) or placebo. (Solvay Pharmaceuticals, Weesp, Netherlands). After four months, participants in every group were randomised again to receive during 14 days either calcium carbonate or placebo. The first 156 participants who were randomised received 800 mg calcium carbonate (320 mg Ca2+) or placebo, the subsequent 120 participants received 1,600 mg calcium carbonate (640 mg Ca2+) or placebo. The study medication was centrally distributed to ensure compliance. The study was completed by 269 patients. Measurements At baseline co-medication was registered and a questionnaire for dietary calcium intake was used to calculate the mean daily calcium intake from dairy products, underestimating calcium intake by 200–300 mg/day [2, 21]. The ability of standing and walking was assessed by a standing score, ranging from 1 (cannot stand alone) to 5 (can easily get up and remain standing without help) and a walking score ranging from 1 (cannot do one active step) to 5 (completely independent walking). Both scores have previously been described [22]. During the study all falls and fractures were registered by the nursing staff on special forms and checked with the routine incident registration. At the end of each study period in a nursing home, every ward was asked to complete a questionnaire on the opinion of the nursing staff about the suitability of each distribution form, compliance, the risk of making mistakes, time investment and preferences. Random samples of the returned medication were counted in order to verify compliance. Adequate compliance was defined to exist when more than 80% of the study medication was ingested. Twice a quality check was made on the research medication by taking random samples for determining the vitamin D3 content of tablets and powders. Fasting blood samples were obtained at baseline, at two and four months. Serum 25(OH)D was measured by radioimmunoassay (Diasorin, Stillwater, MN) with an inter-assay coefficient of variation (CV) of 10% at 30 nmol/L. Serum PTH was measured by radioimmunoassay (Incstar, San Juan Capistrano, CA) with an inter-assay CV of 10% at 3.5 pmol/L. Serum carboxy-terminal collagen crosslinks or CTX, a marker for bone resorption, was measured by immuno-assay (CrossLaps, (Roche) with an interassay CV of 5%. For these parameters the sera of a single participant were all measured within the same run to decrease variation. Serum calcium, phosphate, albumin, creatinine and alkaline phosphatase (APh) were measured using standard laboratory procedures, immediately after obtaining the blood samples. Serum calcium was corrected for serum albumin using the formula: Statistical analysis Statistical analysis was performed using SPSS 12.0.1. Data are presented as means (and standard deviation [SD]) or – in case of skewed distributions – as medians (and interquartile range [IQR]). Associations between baseline serum 25(OH)D and PTH, PTH and AF, and AF and CTX were examined by means of the Pearson correlation coefficient or – when one or both outcome variables had a skewed distribution – the Spearman rank order correlation coefficient. Baseline characteristics of dropouts and completers were compared by logistic regression analysis. Linear multilevel analysis with SPSS Mixed Models was used to investigate: (1) the effect of vitamin D supplementation on change (from baseline (t0) to 4 months (t2)) in biochemical outcome variables (serum 25(OH)D, serum PTH, bone turnover markers) and (2) the effect of additional calcium supplementation on change (from 4 months (t2) to 4.5 months (t3)) in biochemical outcome variables, adjusting for possible clustering of observations. The included levels were repeated measures (i.e., time), respondent, and nursing home. Nursing home was included in the final analyses only in case of a change of the effect size of more than 10%. Separate models were created with 25(OH)D, phosphate, corrected calcium, CTX, PTH, and APh as the respective dependent variables. We examined the potential confounding effect of season, age, sex, mean daily calcium intake, creatinine, standing and walking score at t0. For PTH and APh, logarithmic transformations were performed to normalize variance to allow parametric tests. For these log-transformed outcome variables, the estimated mean difference between two intervention groups was transformed back using an antilog transformation. The resulting estimate is the ratio of the geometric means of the outcome variable in both intervention groups. The geometric mean resembles the median. The level of significance was set at P < 0.05. Results Baseline characteristics Table 1 shows the baseline characteristics of the 338 participants enrolled in the study. These were very similar for the different intervention groups. In the total group, baseline mean serum 25(OH)D was 25.0 nmol/L (SD 10.9). In 55% of the participants, serum 25(OH)D was lower than 25 nmol/L while 77% had levels below 30 nmol/L and 98% below 50 nmol/L (data not shown). Baseline median serum PTH was 7.2 pmol/L (IQR 5.1–10.5) (ref. values: 1–11 pmol/L). There were statistically significant correlations at baseline between serum 25(OH)D and serum PTH values (r = −0.25; P < 0.001), serum PTH and serum APh values (r = 0.16; P < 0.01), and serum APh and serum CTX values (r = 0.23; P < 0.001) (data not shown). The median daily calcium intake from dairy products was 750 mg (IQR 560–1035). Table 1Characteristics of 338 participants at baseline (t0) by intervention group at t0 (Pl D = placebo vitamine D, D = vitamin D) and t2 (Pl Ca = placebo calcium, Ca = calcium) TotalD totalD dailyD weekyD monthlyPl D totalCabPl Cab( = 338)( = 166)( = 55)( = 54)( = 57)( = 172)( = 68)( = 71)% female77.576.583.672.273.778.576.578.9VariableMean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Age (yr)84.2 (6.2)84.2 (6.5)84.3 (6.3)84.3 (6.4)83.9 (6.9)84.2 (5.9)83.3 (6.2)84.5 (6.8)25(OH)D (nmol/L)25.0 (10.9)24.9 (10.1)24.0 (8.6)26.7 (12.6)24.1 (8.8)25.0 (11.7)25.3 (10.6)24.1 (9.6)Calcium corrected (mmol/L)2.42 (0.10)2.42 (0.09)2.42 (0.09)2.41 (0.08)2.42 (0.10)2.43 (0.10)2.41 (0.08)2.42 (0.10)Phosphate (mmol/L)1.03 (0.14)1.02 (0.14)1.02 (0.13)1.02 (0.16)1.02 (0.13)1.04 (0.14)1.01 (0.14)1.03 (0.13)CTX (ng/L)592 (277)571 (274)594 (274)626 (311)496 (218)613 (280)552 (288)565 (255)Albumin (g/L)33.4 (3.3)33.4 (3.3)33.0 (3.5)33.1 (3.4)34.2 (2.8)33.4 (3.2)34.0 (3.2)33.0 (3.4)Standing score (1–5)3.3 (1.6)3.3 (1.6)3.5 (1.6)3.1 (1.7)3.5 (1.6)3.3 (1.6)3.2 (1.7)3.5 (1.6)Walking score (1–5)3.0 (1.4)3.0 (1.4)3.0 (1.4)3.0 (1.6)3.0 (1.3)3.0 (1.4)2.9 (1.4)3.1 (1.4)Skewed variableMedian (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Median (IQR)Calcium intake (mg/day)b750 (560–1035)750 (594–1015)725 (623–1039)755 (550–1028)788 (583–955)750 (550–1053)773 (614–1024)730 (565–1003)PTH (pmol/L)7.2 (5.1–10.5)7.2 (5.2–10.4)7.4 (5.2–10.4)6.7 (5.3–9.9)7.2 (5.1–10.5)7.1 (5.1–11.1)6.5 (5.2–9.4)7.3 (5.1–10.9)AF (U/L)85 (69–102)86 (71–104)82 (73–100)90 (68–106)87 (67–105)85 (69–99)89 (68–105)82 (67–98)Creatinine (μmol/L)93 (81–103)92 (81–103)87 (80–95)96 (85–104)94 (81–105)93 (82–105)91 (80–102)92 (81–102)IQR = interquartile rangeaFrom dairy productsbWithin participants completing the vitamin D intervention ( = 276) treated with vitamin D ( = 139) Trial schedule Figure 1 shows the trial schedule as well as the results of the randomisation procedure of both the vitamin D and the calcium intervention. Of the 341 participants originally enrolled, three were enrolled incorrectly because of hypercalcemia (corrected serum calcium: 2.69; 2.83; and 2.85), leaving 338 participants eligible for the study. Fig. 1Flow diagram of progress through the randomized clinical trial of vitamin D supplementation followed by the randomized clinical trial of calcium supplementation Vitamin D intervention The 338 enrolled participants were randomised to treatment with vitamin D3 one tablet of 600 IU each day (n = 55), a placebo in the form of one tablet each day (n = 57), vitamin D3 in the form of seven tablets (4200 IU) once a week (n = 54), a placebo in the form of seven tablets once a week (n = 58), vitamin D3 in the form of one powder once a month (n = 57), or a placebo in the form of one powder of 1,800 IU once a month (n = 57). The treatment period of four months was completed by 276 participants. Of the 62 drop-outs, 41 died during the study period and there were 21 withdrawals: nine participants became terminally ill; five participants became uncooperative to donate a blood sample; one participant showed signs of discomfort during blood sampling; one participant became immobile; 3 participants were moved elsewhere; and one participant dropped out for unknown reasons. Finally, one participant was excluded from the analyses due to extremely high levels of alkaline phosphatase (278, 1025, 2661 U/L at, respectively, t0, t1, t2) for unknown reasons (further analysis was refused by the patient). The number of drop-outs did not differ significantly between the placebo (n = 35) and the vitamin D group (n = 27). Dropouts were similar to completers with respect to most baseline characteristics (sex, age, 25(OH)D, corrected calcium, phosphate, albumin, standing score, walking score, mean daily calcium intake, and creatinine), but had higher serum levels of CTX, and APh (P < 0.05) (data not shown). Calcium intervention The 276 participants who completed the vitamin D intervention study were randomised to treatment with calcium one tablet each day (n = 138), or placebo one tablet each day (n = 138). The treatment period of 14 days was completed by 269 participants. Of the seven drop-outs, three died, three became terminally ill and one participant dropped out for unknown reasons. Only those treated with vitamin D were included in the analysis of the calcium intervention (n = 68 for calcium; n = 71 for placebo); there was only one drop-out in each group. Effectiveness of Vitamin D supplementation Nursing home and potential confounding variables at baseline were not included in the final models since the effect sizes were hardly influenced by adding these variables. Serum 25-hydroxyvitamin D Effects of vitamin D supplementation on serum 25(OH)D in the various treatment groups are shown in Table 2 and Fig. 2. The mean difference in increase of serum 25(OH)D was 38.5 nmol/L (95% confidence interval (CI) 25.6–41.5) in favour of vitamin D when compared to placebo. Daily, weekly and monthly administration of vitamin D resulted in increase of serum 25(OH)D when compared to placebo (P < 0.001). The mean difference in increase of serum 25(OH)D was highest after 4 months with daily administration of vitamin D (mean 47.2 nmol/l) when compared to weekly (mean 40.7 nmol/l, P < 0.01) and monthly (mean 27.6 nmol/l, P < 0.001) administration. Weekly administration of vitamin D resulted in a greater increase of serum 25(OH)D than monthly administration (P < 0.001). The percentage of patients with serum 25(OH)D below cut offs of 25 nmol/l, 50 nmol/l and 75 nmol/l is shown in Table 3. At 4 months, the percentage of patients with serum 25(OH)D < 50 nmol/l was 10.9, 10.6 and 36.4% in the daily, weekly and monthly groups of vitamin D supplementation respectively. Table 2Mean and standard deviation (SD) or median and interquartile range (IQR) for biochemical measurements at baseline (t0), after 2 months (t1) and after 4 months intervention (t2) with placebo (Pl) or vitamin D (D) daily, weekly, or monthly in 276 participants Mean (SD) or median (IQR)GroupsaMean differenceb or ratio geometric meansc (95% CI)P valuebGroupsaMean differenceb or ratio geometric meansc (95% CI)P valuebt0t1t2t0→t2t0→t2t0→t2t0→t2Serum concentration25(OH)D (nmol/L)Pl25.2 (12.1)24.3 (11.2)25.5 (12.0)D/Pl38.5 (35.7–41.3)0.000D daily23.0 (8.3)59.9 (16.5)69.9 (17.8)Dd/Pld47.2 (42.3–52.1)0.000Dd/Dw6.6 (1.7–11.4)0.009D weekly27.3 (12.7)58.8 (12.8)67.2 (14.0)Dw/Plw40.7 (35.8–45.6)0.000Dw/Dm11.2 (6.3–16.1)0.000D monthly23.8 (8.0)44.8 (14.1)53.1 (15.9)Dm/Plm27.6 (22.8–32.5)0.000Dm/Dd−17.8 (−22.7–−12.8)0.000Phosphate (mmol/L)Pl1.04 (0.12)1.05 (0.14)1.01 (0.14)D/Pl0.057 (0.025–0.088)0.001D daily1.01 (0.14)1.07 (0.12)1.05 (0.11)Dd/Pld0.088 (0.033–0.143)0.002Dd/Dw0.022 (−0.033–0.076)0.434D weekly1.03 (0.15)1.06 (0.13)1.04 (0.14)Dw/Plw0.065 (0.01–0.12)0.022Dw/Dm−0.001 (−0.055–0.054)0.981D monthly1.02 (0.13)1.07 (0.16)1.04 (0.12)Dm/Plm0.017 (−0.037–0.072)0.533Dm/Dd−0.021 (−0.076–0.034)0.454Calcium corrected (mmol/L)Pl2.42 (0.10)2.40 (0.09)2.42 (0.09)D/Pl0.029 (0.008–0.05)0.007D daily2.42 (0.10)2.42 (0.10)2.45 (0.10)Dd/Pld0.036 (0–0.071)0.050Dd/Dw0.006 (−0.03–0.042)0.736D weekly2.41 (0.08)2.41 (0.10)2.43 (0.10)Dw/Plw0.019 (−0.018–0.055)0.307Dw/Dm−0.001 (−0.036–0.035)0.974D monthly2.42 (0.09)2.42 (0.09)2.44 (0.10)Dm/Plm0.033 (−0.003–0.068)0.072Dm/Dd−0.005 (−0.041–0.03)0.762PTH (pmol/L)cPl7.2 (5.0–11.8)7.8 (5.6–10.8)7.5 (5.1–11.0)D/Pl0.77 (0.7–0.85)0.000D daily7.3 (5.0–10.3)5.7 (4.3–7.45.1 (3.7–7.7)Dd/Pld0.66 (0.56–0.78)0.000Dd/Dw0.83 (0.7–0.99)0.037D weekly6.5 (5.3–9.5)6.5 (4.6–8.7)5.9 (5.2–7.6)Dw/Plw0.85 (0.72–1.01)0.067Dw/Dm1.02 (0.86–1.22)0.773D monthly7.2 (5.1–10.9)6.6 (4.4–9.3)5.6 (4.3–8.9)Dm/Plm0.81 (0.68–0.96)0.019Dm/Dd1.17 (0.99–1.39)0.061CTX (ng/L)Pl598 (270)612 (274)624 (256)D/Pl−14 (−57–29)0.491D daily578 (267)574 (265)579 (279)Dd/Pld−34 (−109–41)0.341Dd/Dw1 (−73–75)0.976D weekly606 (316)599 (314)595 (304)Dw/Plw−27 (−102–48)0.445Dw/Dm−36 (−111–39)0.314D monthly490 (207)528 (229)523 (230)Dm/Plm19 (−56–93)0.589Dm/Dd35 (−40–110)0.329AF (U/L)cPl84 (67–99)82 (69–99)82 (67–101)D/Pl0.97 (0.92–1.03)0.363D daily82 (72–99)88 (69–101)82 (67–97)Dd/Pld0.96 (0.87–1.06)0.412Dd/Dw0.96 (0.87–1.06)0.394D weekly88 (66–107)81 (64–105)80 (69–104)Dw/Plw1.03 (0.93–1.14)0.553Dw/Dm1.1 (1–1.21)0.057D monthly86 (67–103)80 (64–96)74 (63–100)Dm/Plm0.94 (0.85–1.03)0.180Dm/Dd0.95 (0.86–1.05)0.270aDifferences in mean change between following groups: D/Pl = vitamin D versus placebo; Dd/Pld = vitamin D daily versus placebo daily; Dw/Plw = vitamin D weekly versus placebo weekly; Dm/Plm = vitamin D monthly versus placebo monthly; Dd/Dw = vitamin D daily versus weekly; Dw/Dm = vitamin D weekly versus monthly; Dm/Dd = vitamin D monthly versus dailybMean difference of for example 38.5 for 25(OH)D (D/Pl) means that the mean increase of 25(OH)D over 4 months was 38.5 in the vitamin D group compared to zero (set as a reference) in the placebo groupcFor PTH and AF the ratio of geometric means (resembling the ratio of medians) is presented in stead of the mean difference. Ratio of geometric means of 0.77 for PTH (D/Pl) means that the median PTH decreases with a ratio of 0.77 over 4 months in the vitamin D group compared to a ratio of 1.00 (set as a reference) in the placebo groupFig. 2Mean (± 1.96 × SD) serum 25(OH)D concentrations at baseline, 2 and 4 months during treatment with vitamin D daily, weekly, or monthly, or placeboTable 3Percentage of participants with 25(OH)D levels below a certain cut-off point at baseline and after vitamin D or placebo supplementationGroup:TotalPlaceboVitamin D totalVitamin D dailyVitamin D weeklyVitamin D monthly25(OH)D < 25 nmol/LBaseline (t0)55.356.953.660.948.951.12 months (t1)30.157.82.92.20.06.84 months (t2)27.452.62.22.20.04.525(OH)D < 50 nmol/LBaseline (t0)97.596.498.6100.095.7100.02 months (t1)65.494.836.526.119.165.94 months (t2)58.097.119.010.910.636.425(OH)D < 75 nmol/LBaseline (t0)99.699.3100.0100.0100.0100.02 months (t1)95.299.391.287.089.497.74 months (t2)88.3100.076.663.072.395.5 Secondary outcome measures: serum phosphate and corrected calcium Serum phosphate and corrected serum calcium values increased significantly more in the vitamin D group than in the placebo group. However, no differences between daily, weekly, or monthly administration were found (Table 2). Serum parathyroid hormone and bone turnover markers Effects of vitamin D supplementation on serum PTH, serum APh and CTX in the various treatment groups are shown in Table 2. Serum PTH (P < 0.000) decreased in the vitamin D group from 7.2 to 5.5 pmol/l when compared to placebo, which is a decrease of 23% (ratio 0.77, 95% CI 0.70–0.85,  < 0.001). The decrease of serum PTH was greater with daily administration of vitamin D when compared to weekly (P < 0.05) and monthly (P < 0.10) administration. The difference between weekly and monthly administration of vitamin D was not significant. The serum concentrations of alkaline phosphatase and CTX did not change following vitamin D supplementation. Effectiveness of calcium supplementation There was no effect of calcium supplementation on any of the six biochemical outcome variables when compared to placebo. Also after stratification by administration of vitamin D (daily, weekly or monthly), an effect of calcium supplementation was not observed except for corrected calcium levels which increased more in the calcium group than in the placebo group in the daily dose vitamin D group only (P < 0.05). No effect was found of calcium doses (800 mg vs 1,600 mg calcium carbonate). Adding nursing home and other potential confounding variables at baseline did not influence the results. Fractures The number of falls and fractures did not differ between the intervention groups and the control groups, which was expected given the short study period of four and a half months. Compliance The compliance assessed within 96 random samples of the returned medication was good. In the daily administration group, all 33 participants were compliant–i.e., used at least 80% of the tablets–(median = 97.0; IQR 94.5–100). For weekly administration, 80% of the 35 participants were compliant–i.e., used at least 80% of the tablets (median = 98.5; IQR 84.0–100). For monthly administration, 93% of the 28 participants were compliant–i.e., used at least four out of five powders (median = 100; IQR 85.0–100). Survey nursing staff A survey among the nursing staffs of the participating nursing home wards showed a distinct preference (72%) for daily administration compared to weekly and monthly. Thirty-ning percent of the nursing staffs reported the impression that fewer mistakes were made using daily administration instead of weekly or monthly administration. Discussion The results of this study confirm the poor vitamin D status often observed in institutionalised elderly. In this study, baseline serum 25(OH)D levels in these nursing home residents was comparable to those observed in other studies in institutionalized elderly in the Netherlands [2, 3], resulting in median serum PTH levels in the upper normal range. A negative correlation between serum PTH and serum 25(OH)D was observed, confirming other studies [4]. In all treatment groups oral vitamin D supplementation appeared to be effective, resulting in increasing serum 25(OH)D levels and decreasing serum PTH levels as observed in other studies [3, 4, 23]. However, no effect was seen on serum alkaline phosphatase and CTX levels. Daily administration of vitamin D3 was significantly more effective than weekly and monthly administration. This could be due to more regular absorption in the gut or better compliance. The percentage of participants with serum 25(OH)D < 50 nmol/l after four months of supplementation was about 10% in the daily and weekly groups, but was more than 35% in the monthly group. An option would be to increase the dose when vitamin D is supplemented only once per month. The dose of 600 IU/day was chosen according to the Dutch and US recommendations [4, 18]. In order to attain a higher percentage of people with serum 25(OH)D > 50 nmol/l, the dose of vitamin D should be 700–800 IU/day as was recommended for patients with osteoporosis in a recent review [24]. The overall decrease of serum PTH was 23% with vitamin D supplementation, which corresponds to our previous vitamin D supplementation study in a nursing home [15] and which is a larger decrease than that observed in healthy independently living elderly women where the decrease of serum PTH was 15% [8]. This is consistent with the more severe vitamin D deficiency and the greater degree of secondary hyperparathyroidism observed in these, mainly psychogeriatric, nursing home residents. Improvement of vitamin D status and suppression of PTH secretion may reduce bone turnover and bone loss, increase bone mineralization and thereby reducing fracture risk, although this was not the subject of this study. For calcium supplementation, calcium citrate, lactate or carbonate can be used. In this study, calcium carbonate was used based upon bioavailability, cost and clinical efficacy [25]. The absorption of calcium from dairy products is about similar to that from calcium carbonate [26, 27]. Because of the side effects of calcium carbonate (gastrointestinal irritation, constipation, belches), possibly more pronounced in a population of frail elderly with substantial comorbidity and comedication, one should not choose a too high supplementation dose. Given the expected dietary calcium intake of about 900 mg per day, two supplementation doses 800 mg calcium carbonate (320 mg Ca2+) and 1,600 mg calcium carbonate (640 mg Ca2+), respectively) were used. The median calcium intake in these Dutch nursing home residents (750 mg per day from dairy products, total estimated dietary intake 950–1,000 mg per day) was slightly lower than the guidelines recommend and relatively high compared to institutionalised elderly in other countries, probably due to a higher dairy intake. This was expected since every Dutch nursing home has its own dietician. Calcium supplementation combined with vitamin D in the last part of the study did not lead to a decrease of biochemical markers of bone turnover. An explanation might be that immobility is a cause of high bone turnover, which is not suppressed by calcium supplementation. The nursing staff’s preference for daily supplementation of vitamin D is probably due to the fact that it fits better in a regular distribution routine, is less time consuming and less susceptible for making mistakes. In conclusion, 98% of the participants had a baseline serum 25(OH)D lower than 50 nmol/L. Oral vitamin D3 supplementation, administered daily was more effective than weekly doses in nursing home residents, while monthly administration was the least effective, 35% still having a serum 25(OH)D < 50 nmol/l after 4 months treatment in this group. Calcium supplementation did not augment the effect of vitamin D supplementation.

Qual_Life_Res-4-1-2275305

Quantification of the level descriptors for the standard EQ-5D three-level system and a five-level version according to two methods

Objectives Our aim was to compare the quantitative position of the level descriptors of the standard EQ-5D three-level system (3L) and a newly developed, experimental five-level version (5L) using a direct and a vignette-based indirect method. Introduction The EQ-5D is a widely used instrument to describe and value generic health (status) in terms of five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension comprises three levels, indicating no problems, some or moderate problems, and extreme problems, resulting in a total of 243 (35) unique health states [1]. The condensed format of the EQ-5D has undoubtedly contributed to its global dissemination, as it is easy to include in existing surveys by questionnaire designers, easy to fill out by respondents, and easy to report by analysts. However, compared with other generic preference based instruments such as the Health Utilities Index Mark 2 and Mark 3 (HUI2 and HUI3) and the Short Form 6D (SF-6D), which define respectively 24,000, 972,000, and 18,000 unique health states, the EQ-5D is lacking descriptive richness [2–5]. Although the EQ-5D descriptive system has demonstrated strong psychometric properties in general, its restricted ability to discriminate (clinically relevant) small to moderate differences in health status between individuals or within individuals over time is recognized [6–9]. Moreover, several studies have reported on the ceiling effect of the EQ-5D in the general population as well as in patient populations [10–15]. A straightforward way of improving the discriminatory potential of the EQ-5D descriptive system is to increase the number of response options. In most health-status classification systems, the response options are ordered in terms of severity along a hypothetical measurement continuum. Since the exact position of the response options defines the discriminatory abilities of the descriptive system [16, 17], it is important to know where on the measurement continuum the level descriptors are quantitatively positioned. Previous research in which a five-level (5L) version of EQ-5D was compared with the standard three-level (3L) EQ-5D demonstrated increased discriminatory power, increased reliability, and satisfactory validity [18, 19]. This paper presents a head-to-head comparison of the quantitative positioning of the level descriptors of the standard 3L EQ-5D descriptive system versus a newly developed, experimental 5L system, which covers 3,125 unique health states (55). Two independent methods were used. The first method directly compared the nonextreme level descriptors (for 3L: the level two midcategory; for 5L: the level two, level three, and level four categories) for each dimension separately on a visual analogue scale (VAS). The second, indirect, method required respondents to score complete health scenarios (vignettes) on dimension-specific VAS scales and subsequently to classify the same vignettes on the two EQ-5D instruments (3L and 5L). Methods Instruments Three instruments were used in this study: the standard EQ-5D3L version, an adapted Dutch 5L version developed in 1993 [20], and a set of five dimension-specific VAS scales. The version of the 5L EQ-5D used in this study was an experimental version, since at the time of this study, no official five-level version had been advocated by the EuroQol Group. We chose to test a five-level EQ-5D system, even though we also could have chosen four or six levels. An increase in the number of levels is always an increase of discriminatory potential at the cost of a more complex descriptive system (which might compromise the robustness of the value function). Five levels appears to be an optimal number of response options concerning reliability [21, 22]. Furthermore, Preston et al. (2000) investigated feasibility for 11 different rating formats (ranging from 2 to 11 and a 101 point scale) and found that feasibility peaked at five levels [23]. We chose to add two in-between levels to the existing 3L descriptive system (between levels 1 and 2 and levels 2 and 3) because we considered this the most obvious option in regard to the objective of refining the EQ-5D instrument. In any preference-based instrument, level descriptors are practically required for valuation research in which generic profiles are to be valued. A small focus group was assigned to determine the wording of the level descriptors. The level descriptors presented here were translated from Dutch. The one-, three-, and five-level descriptors in 5L were the same as the one-, two-, and three--level descriptors in the standard EQ-5D3L. The grading terms that were used for the intermediate levels two and four in the 5L-system were “a little” for level 2 (5L-2) in Anxiety/Depression and “mild problems” for the remaining dimensions; and “severe” for level 4 (5L-4) in Pain/Discomfort, “very” for Anxiety/Depression, and “many problems” for the remaining dimensions. One further alteration was made to both the 3L and 5L systems: the most severe response category in Mobility was changed from “confined to bed” to “unable to walk about”, so it would be analogous to the extreme response categories of the other dimensions. Table 1 displays the exact wording of the descriptors in the 3L and 5L systems, respectively. Table 1Direct quantification of three- and five- level (3L, 5L) descriptorsNumberMeanMedian95% CI3LMobility    No problems in walking abouta––––    Some problems in walking about7426.702222.82−30.59    Unable to walk abouta––––Self-care    No problems with self-carea––––    Some problems washing or dressing self7430.182826.10−34.25    Unable to wash or dress selfa––––Usual activities    No problems with performing usual activitiesa––––    Some problems with performing usual activities7729.742525.95−33.53    Unable to perform usual activitiesa––––Pain/Discomfort    No pain or discomforta––––    Moderate pain or discomfort6632.333128.56−36.10    Extreme pain or discomfort6686.368983.75−88.98    Worst imaginable pain or discomforta––––Anxiety/Depression    Not anxious or depresseda––––    Moderately anxious or depressed6733.943429.89−37.99    Extremely anxious or depresseda6788.829086.88−90.77    Worst imaginable anxiety or depressiona––––5LMobility    No problems in walking abouta––––    Mild problems in walking about7511.31119.73–12.88    Some problems in walking about7538.394035.39–41.39    Many problems in walking about7579.808276.81–82.79    Unable to walk abouta––––Self-care    No problems with self-carea––––    Mild problems washing or dressing self7611.24109.72–12.76    Some problems washing or dressing self7637.143834.14–40.15    Many problems washing or dressing self7680.618177.81–83.40    Unable to wash or dress selfa––––Usual activities    No problems with performing usual activitiesa––––    Mild problems with performing usual activities7711.08109.29–12.87    Some problems with performing usual activities7739.014036.12–41.90    Many problems with performing usual activities7780.818377.70–83.91    Unable to perform usual activitiesa––––Pain/Discomfort    No pain or discomforta––––    Mild pain or discomfort538.8587.43–10.26    Moderate pain or discomfort5332.323129.58–35.06    Severe pain or discomfort5367.946864.98–70.90    Extreme pain or discomfort5391.269488.96–93.57    Worst imaginable pain or discomforta––––Anxiety/Depression    Not anxious or depresseda––––    A little anxious or depressed599.4687.97–10.94    Moderately anxious or depressed5932.563330.01–35.11    Very anxious or depressed5967.376664.55–70.20    Extremely anxious or depressed5991.349289.42–93.25    Worst imaginable anxiety or depressiona––––CI confidence intervalaLevel descriptor used as anchor in visual analog scale To obtain quantitative values for each level descriptor of 3L and 5L, the VAS was used. We used five VAS scales, one for each EQ-5D dimension. Each VAS consisted of a horizontal hashmarked line without corresponding numbers, with the extreme-level descriptors belonging to that dimension as anchors. Respondents were asked to indicate their score on the VAS by marking the line. For the most severe category of Pain/Discomfort and Anxiety/Depression, the original descriptor was labeled “extreme”. Because the study was part of a larger process of choosing the definite level descriptors for the official five-level version of the EQ-5D, we decided to use the entire continuum of disability (extreme included), and used “worst imaginable” as upper VAS anchor for these two dimensions. This is analogous to the other three dimensions, which ranged from “no problems” to “unable to”. Study design Data collection took place in the form of one of two panel sessions and a follow-up postal survey 2 weeks later. A convenience sample of 82 laypeople from an existing general population panel (N = 560) participated. All participants were familiar with the vignette presentation form used in the indirect method. All participants completed both the direct and the indirect quantification task. For the direct method, all 3L answers were obtained during the panel sessions and all 5L answers as part of the postal survey to avoid memory effects. For the indirect method, participants scored ten health states in the panel sessions (acute pharyngitis, exacerbation of eczema, hip fracture, cerebrovascular accident/stroke with moderate impairments, moderate gastritis, low spinal cord lesion, mild depression, back and neck pain, severe dementia, and acute multiple injury) and the remaining five in the survey (otitis externa, severe stable brain injury, irritable bowel syndrome, acute large burn, and posttraumatic stress disorder), because we expected that more than ten health states within one session could lead to concentration problems. The two sets of health states were balanced according to severity and duration. Following this design, the indirect method provided 225 responses for each respondent: 15 diseases × 5 dimensions × 3 response scales. Direct quantification of level descriptors In the direct method, respondents were asked to project the 3L and the 5L descriptors on the VAS scales for each dimension separately. As the extreme levels were used as anchors of the VAS, for 3L only, the midcategory (3L-2) level descriptor needed to be scored, except for Pain/Discomfort and Anxiety/Depression, which needed additional scoring of 3L-3 (extreme). Similarly, the midcategories 5L-2, 5L-3, and 5L-4 descriptors were scored for each dimension, except for Pain/Discomfort and Anxiety/Depression, which included the scoring of 5L-5. Indirect quantification of level descriptors As an alternative to the direct method, we developed an indirect method that we believe lies closer to the actual use of the EQ-5D instrument, as it uses a (hypothetical) health state as a calibrator or medium to derive a VAS score. In contrast to the direct method, the object of measurement in the indirect method is not a 3L or 5L descriptor but a complete health scenario (vignette). Each vignette was scored with the 3L and 5L descriptors and on a VAS, one for each separate dimension, independently. Consequently, an indirect head-to-head comparison of 3L and 5L scores could be made, calibrated via the common VAS score. Figure 1 shows one of the vignettes. Each vignette was designed to present a disease as close to clinical reality as possible, therefore also including information on disease duration. All 15 diseases were presented on a standardized sheet (vignette) that contained (1) a disease label with a naturalistic description of the disease; (2) the course of the disease over a 1-year period using a calendar (the grey scales represent the duration of the disease); (3) the location of the disease with, if relevant, a visual representation; and (4) the EQ-5D dimensions, of which the levels were left unspecified, as the respondents were invited to select the appropriate EQ-5D level (according to his or her own view) for each dimension. Respondents were asked to read each vignette carefully and to select the level of each dimension of the EQ-5D descriptive system that best described the presented health state in their view using three response scales: the standard 3L response scale, the new 5L scale, and the VAS scale (similar to the VAS used in the direct method). Fig. 1Disease vignette with empty EQ-5D descriptive system The 5L and 3L response scales were presented on the left and the right side of one page (per dimension), respectively. The respondents were first invited to score the 5L descriptors for all dimensions and all vignettes while covering the right side of the page that showed the 3L descriptors. Next, they were instructed to return to the first vignette, asked to cover the left side with the 5L scores, and provide the 3L response for all vignettes. Pilot testing revealed that when respondents scored 3L first, there was a tendency to avoid the in-between levels 2 and 4 of 5L, and for this reason, all respondents were asked to score 5L first. Adequate instruction was critical, stressing that 3L and 5L were two independent ways of scoring (in the postal survey, these instructions were repeated in writing). Subsequently, VAS scores were obtained on a separate form without respondents having access to the 3L and 5L scores. The demanding task of first providing 5L classifications on all five dimensions of all 15 vignettes minimized possible memory effects when the participants were instructed to return to the first vignette to score the 3L classifications while covering the 5L responses. Analysis Results of the direct and indirect methods are presented with conventional descriptive statistics. Results of the indirect method were derived by grouping 3L-VAS pairs and 5L-VAS pairs for each respondent per vignette and subsequently by calculating level means over all vignettes and all respondents combined. For each respondent, scorings were removed for the combined 3L, 5L, and VAS scores if at least one of the 3L, 5L, or VAS scores was missing, equalizing the number of VAS observations between 3L and 5L. Characteristics For both the direct and indirect methods, the 3L–5L extension of EQ-5D was investigated in terms of three characteristics. First, equidistance addresses the degree to which 3L and 5L level descriptors are distributed evenly over the VAS continuum, either without or with transformation. Equidistance is determined for each dimension and each instrument (3L and 5L) separately. Untransformed equidistance implies that level descriptors are distributed according to VAS ratings of 0–50–100 for 3L and 0–25–50–75–100 for 5L. There is evidence that the precision of the VAS might be illusory, as respondents mentally divide the VAS continuum in a smaller number of segments, which is nine or ten at maximum [23, 24]. Therefore, we defined a deviation of 5 VAS points as the maximum acceptable deviation (which makes a segment of 10 VAS points, as the deviation can be either way). Furthermore, a deviation of 5 VAS points has been used before [16]. If untransformed equidistance is rejected, equidistance using power [y  = (ax)b] transformation is considered. A power relation of, e.g., y = (5.38*x)1.5 for 5L would result in a VAS rating distribution of 0–12–35–65–100. Note that transformation is only possible for 5L, as there is only one 3L observation apart from the anchors. Part of the evaluation of equidistance is analysis of the position of the extreme levels according to the indirect method: are the VAS ratings for the extreme level descriptors close to the supposed anchor values for the indirect method? Ideally, 3L-1 and 5L-1 scores would equal 0 and 3L-3 and 5L-5 scores would equal 100, except for Pain/Discomfort and Anxiety/Depression in which the 3L and 5L extreme level descriptors were not identical to the VAS anchors. Second, isoformity is the degree to which the positions of 3L-2 and 5L-3 level descriptors (and also 3L-3 versus 5L-5 for Pain/Discomfort and Anxiety/Depression) are similar. Isoformity directly compares the 3L and 5L descriptive systems for each separate dimension between instruments. For the indirect method, all 3L level means, including 3L-1 and 3L-3, can be compared with 5L. Analysis of isoformity is based on paired 3L–5L response means for each dimension separately. For the direct method, isoformity was tested with a paired t test between the 3L and 5L scorings. For the indirect method, a deviation of 5 VAS points was defined as the maximum acceptable deviation. Finally, consistency between dimensions is the degree to which the positions of the same level descriptors differ across dimensions. Consistency, between dimensions was tested for each instrument (3L, 5L) separately. The first three dimensions (Mobility, Self-Care, and Usual Activities) were distinguished from the last two (Pain/Discomfort and Anxiety/Depression), as these—in Dutch—share identical level descriptors, e.g., some problems for Mobility, Self-Care, and Usual Activities. For the direct method, analysis of variance (ANOVA) was used for each identical level descriptor for the first three dimensions combined (one comparison for 3L and three for 5L) and Pain/Discomfort and Anxiety/Depression combined (two comparisons for 3L and four for 5L), resulting in a total of ten comparisons . For the indirect method, consistency is tested with a generalizability study (G-study). In a G-study, one is able to separate multiple sources of error variance [25]. Generalizability coefficients (G-coefficients) can be constructed as functions of the estimated variance components, expressing consistency on a 0–1 scale, with 1 expressing perfect consistency [26, 27]. We used a variance components analysis based on the restricted maximum likelihood method and identified four possible sources of variance: label, vignette, dimension, and respondent. Four separate G-studies were conducted, one on the first three dimensions and one on the remaining two dimensions, for each instrument (3L, 5L) separately. A G-coefficient expressing consistency between dimensions was calculated on the basis of these variance components (“Appendix A”). We regarded transformed or untransformed equidistance to be a desirable characteristic for the new 5L system as opposed to no systematic relation between the quantitative position of the level descriptors at all. Consistency between identical-level descriptors across dimensions was also regarded as a desirable property because this expresses that respondents have a consistent conceptualization of the grading terms used over different dimensions of health. When consistency is achieved, this does not imply that utility values would also be expected to be consistent over dimensions, because utility values are an expression of an entire EQ-5D profile, whereas we investigated VAS scores within each dimension separately. Furthermore, a choice-based method presumably leads to different results than the dimension-specific VAS scales we used. We investigated isoformity to see whether the new 5L system was a refinement or a new system, and whether isoformity was achieved or not does not tell us anything about the 5L system in itself. Results The mean age of the participants was 53.6 years, with 42.7% being men. Of the 82 respondents who attended in the panel sessions, 81 returned the survey. Three respondents (4%) were of Turkish nationality, two (2%) were of Moroccan nationality, and the remaining 75 (94%) were of Dutch origin. In the Pain/Discomfort and Anxiety/Depression dimensions, respondents often failed to score the extreme-level descriptor when using the direct method (8 and 9 for 3L, respectively, and 22 and 16 for 5L, respectively). For these respondents, the remaining scorings were deleted for that dimension because of possible context effects (i.e., spreading out the VAS scores of the remaining 3L descriptors over the VAS scale). For the direct method, missing responses for 3L ranged from 6.1% (Usual Activities) to 19.5% (Pain/Discomfort) and for 5L from 4.9% (Usual Activities) to 34.6% (Pain/Discomfort). For the indirect method, missing responses ranged from 1.1% (Usual Activities) to 2.5% (Pain/Discomfort) for the three response scales (3L, 5L, and VAS) combined. Characteristics: direct method Results for the direct method are shown in Table 1 and Fig. 2. Untransformed equidistance was rejected for all level descriptors except 5L-4 in Mobility (80), although Self-Care and Usual Activities were only 1 VAS point away for Mobility. Regardless of dimension, level descriptors were positioned systematically lower than the expected value for equidistance for 3L-2 (16–23 VAS points lower), 5L-2 (14–16 points lower), and 5L-3 level (11–18 points lower), whereas 5L-4 was sometimes higher (4–5 points) and sometimes lower (7–8 points). Transformed equidistance (power function) provided an excellent fit for all dimensions of 5L (R2 ≥ 0.99). Fig. 2Direct quantification of the three- and five-level (3L, 5L) descriptors. Visual analog scale (VAS) means by dimension Isoformity could not be established except for the middle-level descriptors (3L-2 vs. 5L-3) for Pain/Discomfort and Anxiety/Depression (Table 2). Relatively large gaps appeared between 3L-2 and 5L-3 for Mobility (11), Self-Care (8), and Usual Activities (9), with 5L-3 showing systematically higher values. Although there was a statistically significant difference between the extreme level descriptors (3L-3 vs. 5L-5) for Anxiety/Depression, the absolute difference was 3 VAS points. Table 2Isoformity of identical three-and five-level (3L, 5L) descriptors for the direct quantification methodDimensionComparisonMean differenceP valueMobility3L-25L-3−11.4<0.001Self-care3L-25L-3−8.00.002Usual activities3L-25L-3−9.4<0.001Pain/Discomfort3L-25L-3−1.40.501Pain/Discomfort3L-35L-5−4.90.012Anxiety/Depression3L-25L-32.80.276Anxiety/Depression3L-35L-5−3.00.025 Consistency between dimensions gives supportive results for both 3L and 5L, as none of the ten comparisons (ANOVA) showed significant differences (see Fig. 2). Generally, VAS means are similar among the first three dimensions as well as among Pain/Discomfort and Anxiety/Depression. Characteristics: indirect method Results of the indirect method are shown in Table 3 and Fig. 3. Untransformed equidistance of 3L-2 was rejected for all dimensions (systematically 7–14 VAS points too low) as well as for 5L-2 (systematically 8–13 points lower) and 5L-3 (systematically 8–17 points lower). Untransformed equidistance was achieved only for the 5L-4 level for all dimensions (systematically 1–5 points lower), with VAS scores ranging from 70 (Mobility and Usual Activities) to 74 (Anxiety/Depression). Transformed equidistance (power function) provided an excellent fit for all dimensions of 5L (R2 ≥ 0.99). Table 3Indirect quantification of three- and five-level (3L, 5L) descriptorsNumberMeanMedianCI3L Mobility    No problems in walking about5991.6901.31–2.07    Some problems in walking about40342.944040.24–45.64    Unable to walk about18091.709989.28–94.12Self-care    No problems with self-care4823.2402.40–4.08    Some problems washing or dressing self43539.183436.58–41.78    Unable to wash or dress self27385.479582.77–88.16Usual activities    No problems with performing usual activities2354.5023.49–5.51    Some problems with performing usual activities58236.553034.40–38.71    Unable to perform usual activities37888.549586.87–90.22Pain/Discomfort    No pain or discomfort24612.6449.94–15.34    Moderate pain or discomfort64335.763133.92–37.60    Extreme pain or discomfort27583.218980.82–85.61Anxiety/Depression    Not anxious or depressed4336.2915.01–7.57    Moderately anxious or depressed47842.454040.26–44.63    Extremely anxious or depressed27084.809082.73–86.865LMobility    No problems in walking about5471.3000.92–1.69    Mild problems in walking about14715.331112.64–18.02    Some problems in walking about15936.483133.00–39.97    Many problems in walking about21769.827666.72–72.92    Unable to walk about11297.3610095.24–99.48Self-care    No problems with self-care3982.4501.43–3.48    Mild problems washing or dressing self20412.70910.76–14.64    Some problems washing or dressing self18436.093333.00–39.17    Many problems washing or dressing self25771.207868.33–74.06    Unable to wash or dress self14791.379987.80–94.94Usual activities    No problems with performing usual activities1363.2201.49–4.95    Mild problems with performing usual activities26812.39910.68–14.10    Some problems with performing usual activities22832.533029.97–35.09    Many problems with performing usual activities35169.547567.18–71.90    Unable to perform usual activities21295.3510093.74–96.96Pain/Discomfort    No pain or discomfort1458.3405.32–11.37    Mild pain or discomfort27417.271215.13–19.41    Moderate pain or discomfort36736.833534.91–38.76    Severe pain or discomfort26371.727969.05–74.39    Extreme pain or discomfort11592.769889.86–95.65Anxiety/Depression    Not anxious or depressed3054.7503.07–6.43    A little anxious or depressed24116.481014.33–18.63    Moderately anxious or depressed27141.984139.72–44.25    Very anxious or depressed24874.198071.69–76.70    Extremely anxious or depressed11692.339789.61–95.04CI confidence intervalFig. 3Indirect quantification of the three- and five-level (3L, 5L) descriptors. Visual analog scale (VAS) means by dimension VAS results for the extreme-level descriptors show that the lower extreme is close to 0, except for Pain/Discomfort (3L-1 = 13; 5L-1 = 8). VAS results for the upper extreme values are systematically higher for 5L than for 3L (range of difference: 6–10). Noticeable are large deviations in Self-Care (3L-3 = 85; 5L-5 = 91) and Usual Activities (3L-3 = 89). Isoformity was accepted for 3L-1 versus 5L-1 for all dimensions and for 3L-2 vs. 5L-3 for all dimensions except Mobility (showing a gap of 7 points). Isoformity was rejected for the upper extreme comparison 3L-3 versus 5L-5 for all dimensions. Consistency between dimensions gave supportive results for both 3L and 5L. Table 4 shows the G-study results. Most variance is attributed to the label component, whereas less than 2% of variance is attributed to the components including dimension, which is reflected in high G-coefficients for all comparisons. Consistency for 5L is somewhat higher (0.87; 0.86) than for 3L (0.86; 0.81). Table 4Consistency between dimensions for the indirect quantification method. Variance components estimates (percentages) and generalizability coefficients (G-coefficients) for comparable dimensions of three- and five-level (3L, 5L) instruments3L5LMobility/Self-care/Usual activitiesLabel66.12Label71.52Vignette8.05Vignette6.35Dimension0.26Dimension0.04Respondent0.33Respondent0.79Label × vignette5.60Label × vignette2.91Label × dimension0.22Label × dimension0.12Label × respondent2.20Label × respondent2.59Vignette × dimension0.60Vignette × dimension0.17Vignette × respondent3.77Vignette × respondent2.57Dimension × respondent0.76Dimension × respondent0.60Residual12.09Residual12.34G-coefficient0.86G-coefficient0.87Pain/Discomfort; Anxiety/DepressionLabel65.25Label73.58Vignette4.95Vignette2.73Dimension0.00Dimension0.00Respondent0.65Respondent0.77Label × vignette1.91Label × vignette1.02Label × dimension0.04Label × dimension0.00Label × respondent2.96Label × respondent3.36Vignette × dimension1.06Vignette × dimension0.17Vignette × respondent5.52Vignette × respondent4.50Dimension × respondent0.88Dimension × respondent0.45Residual16.78Residual13.42G-coefficient0.81G-coefficient0.86 Discussion In this study, we compared the quantitative position of the level descriptors of the standard EQ-5D3L and a new five-level version using two independent methods. The study showed that the extension of the EQ-5D3L to a five-level version by inserting two extra levels, leaving the existing descriptors unaltered, is not a simple refinement but a redesign. The inserted levels pushed the extreme levels closer to the anchors, which indicates that 5L makes better use of the measurement continuum, contributing to superior descriptive power of the 5L version. In both the 3L and 5L versions, the position of the 3L or 5L descriptors, reassuringly, was independent of dimension. Equidistance was not achieved for both systems, in most cases showing values lower than the equidistant values. Both methods revealed a large gap between the 5L-3 and 5L-4 levels, regardless of dimension. This could be caused by the wording of 5L-3 [some and moderate(ly)] being interpreted as fairly mild. In Pain/Discomfort, respondents tended to avoid the lower anchor of the scale, indicating some pain or discomfort on VAS while scoring no problems on 3L and 5L. This indicates that respondents preferred a more refined response scale for scoring pain or discomfort, maybe a scale with even more than five response options (as is the case of, e.g., the HUI3 or SF-36). Also noticeable were the gaps observed for the upper extreme in Self-Care, for which we cannot provide an explanation. Isoformity between 3L and 5L showed mixed results. The 3L-1 vs. 5L-1 descriptors showed isoformity (indirect method only), as expected, as these both indicated the upper ceiling (no problems). Isoformity was also established for the middle level descriptors of Pain/Discomfort and Anxiety/Depression for both methods. This could be due to the wording of the middle level descriptors, as the descriptor some problems represented a wider range and hence more potential variation, than moderate(ly), as used in Pain/Discomfort and Anxiety/Depression. Assuming that the descriptor some problems was a well-considered choice in the development of the original EQ-5D3L system in order to cover the entire range between the two extremes, it is questionable whether that descriptor is still suitable in a 5L version. Direct quantification is a well-known method of estimating the magnitude of level descriptors or response labels [16, 17, 28, 29]. This approach, however, ignores the fact that the VAS values expressed for the level descriptors did not necessarily reflect the self-report use of such descriptors (and the use in subsequent valuation studies) in a similar way, because the valuation of an abstract level descriptor might lead to different results than self-reported health. The indirect method is novel: to our knowledge, this is the first time a quantification of level descriptors is estimated with this method. The indirect method has several advantages. First, we believe it is a better representation of the hypothesized measurement continuum of EQ-5D, as the medium of the vignette (disease) was used to calibrate 3L and 5L descriptors on a VAS scale. Second, it is closer to the general use of the EQ-5D instrument as a self-report health status assessment measure and is therefore likely to be more valid. Classifying a vignette can be regarded similarly to a health status classification by proxy assessment. Other advantages of the indirect method are analytical: values can be calculated for all level descriptors, including the anchors, and it is possible to investigate explained variance for various components (G-study). Furthermore, the indirect method proved to be much more feasible than the direct method, considering the lower number of missing responses. Disadvantages are that no direct comparison (e.g., paired t test) between 3L and 5L is possible, as there is only one VAS value for each 3L–5L response pair, and that the indirect method is more time consuming. A potential weakness of the study procedure is that 3L and 5L were presented on one sheet, and panelists were asked to score 5L dimensions first while covering 3L and vice versa. We cannot be sure that respondents actually complied to the blinding procedure in the follow-up measurement. Also, there might have been an order effect, as 5L always preceded 3L. The 5L instrument presented here obviously improves the discriminatory potential of the EQ-5D descriptive system, as the level descriptors generally capture a larger part of the measurement continuum and broaden the measurement space. Furthermore, 5L showed slightly better consistency between levels. In a previous study, we demonstrated increased discriminatory power of the same 5L version of EQ-5D, as well as superior reliability (interobserver and test–retest) and face validity when compared with the standard EQ-5D3L [18]. Awaiting a valuation study for an official version of 5L, a set of preference weights was developed for this 5L version of EQ-5D using item response theory (IRT) methodology [30]. An officially sanctioned five-level descriptive system will become available within a short period [31] and is expected to be in use alongside the standard three-level EQ-5D. The experimental five-level EQ-5D version presented here is likely to demonstrate a less severe ceiling effect. Assuming that milder states are more common in the general population, we expect increased benefit in the detection of mild problems and in measuring and monitoring general population health, although the extra 5L-4 level is expected to also lead to better differentiation and detection of more severe health states. The methodology presented here can be of use in the development of generic or disease-specific health status measures.

Surg_Endosc-4-1-2262145

Virtual reality training for endoscopic surgery: voluntary or obligatory?

Introduction Virtual reality (VR) simulators have been developed to train basic endoscopic surgical skills outside of the operating room. An important issue is how to create optimal conditions for integration of these types of simulators into the surgical training curriculum. The willingness of surgical residents to train these skills on a voluntary basis was surveyed. Endoscopic surgery requires dedicated skills such as three-dimensional orientation in a two-dimensional representation of the operating field and complex instrument handling [8, 7, 5]. Training of these skills in the operating room (OR) is under pressure due to planning issues and ethical considerations. Virtual reality (VR) simulators have been developed to train basic endoscopic surgical skills outside of the OR. Several simulators have been validated and found adequate for the transfer of skills from the simulator to the OR [2, 6, 9, 13, 14]. However, discussion arises on how to integrate these simulation-based training modalities in the surgical training curriculum. A questionnaire was distributed to 245 Dutch surgical residents to explore the perspective of the trainee on this issue. Approximately 75% of residents felt that endoscopic skills training outside the OR is useful [12]. In another study, sixty Dutch gynaecology residents responded positively (3.9 on a five-point Likert scale) with regard to training in laparoscopic skills before real surgery [10]. Fifty-five percent of these 60 residents did not have the opportunity to train laparoscopic skills. However, those that did appeared to train only once or twice a year and 33% did not use available skills trainers voluntarily at all. We hypothesized that insufficient simulator access might be the reason for this contradiction. Therefore, we investigated the willingness of surgical residents to train in endoscopic skills on a voluntary basis when VR simulators were indeed readily available. We also evaluated the effect of competitive incentives on the frequency and duration of simulator training. Materials and methods Equipment, tasks and scoring system This study is performed with the LapSim virtual reality simulator, which uses the Virtual Laparoscopic Interface (VLI) hardware, (Immersion Inc., San Jose, CA, USA) The VLI has an interface with a 2600 MHz hyperthreading processor Pentium IV computer running Windows XP and is equipped with 256 random-access memory (RAM), a GeForce graphics card and an 18-inch thin-film transistor (TFT) monitor. The systems feature LapSim Basic Skills 3.0 software (Surgical Science Ltd, Göteburg, Sweden), from the LapSim Basic Skills package, consisting of nine tasks. A training program was designed that included all nine tasks: camera navigation, instrument navigation, coordination, grasping, lifting and grasping, cutting, clipping and cutting, suturing and fine dissection [4]. The computer stores and displays between seven and eleven parameters of performance per task. These parameters are related to time, errors or efficiency of handling. Tasks can be adjusted to different levels of difficulty. The training program for this study was set at an advanced level with thresholds that are based on the performance of 30 experienced endoscopic surgeons (more than 100 endoscopic procedures). The scoring system is two-tiered. First, for any given parameter the system determines whether or not the participant passes or fails the test. Secondly, if a participant passes, a score of between 0% and 100% is attached to his or her performance on that particular parameter. The overall score per task is determined by the sum score of the parameters, divided by their number. Hence, an overall score of 100% can only be obtained by scoring 100% on each of the individual parameters measured during performance of the particular task. An outcome score of 100 points is given to those participants who score a 100% on the task performed. Logically, a score of 85% thus translates into 85 points. A maximum overall score of 900 could be obtained (i.e., 100 points on each of the nine tasks measured). Participants Twenty-one surgical residents, ranging from postgraduate year (PGY) -one level to PGY 6 level, with different endoscopic surgical experience, were given unlimited access to the simulator. Seven residents were at the beginning of their surgical educational program (PGY 1 and PGY 2) and therefore inexperienced in endoscopic surgery. Seven residents were in the middle (PGY 3 and PGY 4) and eight residents were at the end (PGY 5 and PGY 6) of their surgical educational program. Setting and incentives In the period May 2005 to January 2006 a simulator was placed in the general room for surgical residents at the surgical ward of the University Medical Centre in Utrecht. Before the study, residents were instructed on how to operate the simulator, and allocated a personal login number for the simulator. By placing the simulator in the general residents’ room, it was readily and easily accessible 24 hours a day. The room is secured by a code-locked door and accessible by residents only. During the first four months, there were no additional incentives other than the permanent (24-hour) accessibility to the residents for training on the simulator. After these four months, a competitive element was introduced in which the frequency of training was also rewarded (bi-weekly). The overall end-score was calculated every other week by adding this frequency bonus to the highest scores for each task. These overall end-scores for each resident were publicly announced to the complete department of surgery and the winner (the resident with the highest score) was awarded a prize. Questionnaire After eight months all residents were requested to fill out a questionnaire. Ten questions were presented on a five-point Likert scale, concerning their perception of their own experience level in endoscopic procedures, their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum, and their opinion about the application of virtual reality as a means to training endoscopic skills. Value 1 was assigned to “totally agree”, value 5 to “totally disagree”. In addition, the residents were asked about their frequency of usage of the simulator. If a participant indicated little usage, he or she was questioned why, and what could motivate increased usage. Results In the first four months only two of the 21 residents (10%) trained on the simulator, for a total of 163 minutes. One resident was a PGY 2, the other one a PGY 5. In the second period of four months the number of trainees increased to seven residents (33%, two PGY 2, two PGY 3, one PGY 5 and two PGY 6). The duration of training increased to 738 minutes, thereby constituting an average increase of 23.9 minutes per subject. Fifty-eight percent of training was performed during night shifts. All 22 residents (100%) replied to the questionnaire. The total training time, as an accumulation of estimation on individual training time, was 4140 minutes. The actual cumulated training time for all residents was 901 minutes (22%). Thirteen out of 15 residents who did not train at all (86%) stated that this was due to a lack of time during the day. One resident (7%) stated he had been not interested enough to train and indicated that he had alternative priorities. Another resident (7%) stated that she was fully occupied due to an intensive-care traineeship and maternity leave. Residents suggested that the use of the VR trainers could be enhanced by incorporating a mandatory VR training into the surgical curriculum (9x = people agreeing), to oblige certain skills level on VR simulator before starting endoscopic surgery in the OR (3x), to implement competitive training with coaching (2x), to diminish working pressure (2x), to have more-advanced exercises available on the simulator (3x), to place the VR simulator in a location other than in the residents’ room (1x). Only two stated that more initiative of residents was required to improve outcome. Figure 1 refers to the perception of residents’ own experience level in endoscopic procedures, their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum and their opinion about the application of virtual reality as a means to training endoscopic skills. Fig. 1.Results of questionnaire In general, the opinion of the residents on their own experience level, on the possibility to develop and train endoscopic skills during their training and on the role of virtual reality varies considerably [standard deviation (SD) 0.44 to 1.39]. Their opinion on obligation of VR training to improve endoscopic skills and having VR training as a mandatory part of the basic skills training is most uniform. Residents do not have a marked positive, nor a marked negative opinion on the presented statements on receiving enough training for acquiring basic skills (mean 2.63, SD 1.19), on receiving sufficient training time in the OR to train in endoscopic skills (mean 3.47 SD 1.17) and on acquiring a satisfactory level of basic psychomotor skills (mean 2.42, SD 1.16). The same applies to their opinion on the representation of their training results on the simulator (mean 2.98, SD 0.83); as well as on the statement that thresholds should be reached before training in the OR is allowed (mean 2.47, SD 1.39). There is one statement they do not agree with; I will not train unless it is obligatory (mean 4.26, SD 1.10). Discussion Virtual reality training has the potential to improve and professionalize the training in endoscopic basic psychomotor skills [2, 6, 9, 13, 14]. Training results can be shown instantly to demonstrate objective performance and progress of performance assessment. However, one of the main concerns in acquisition of expensive equipment for educational purposes is its effectiveness. While initial enthusiasm about new innovative equipment is usually high among the surgical community, actual usage tends to be disappointing [3]. This study was undertaken to evaluate the aptitude for training on a voluntary basis when a VR simulator was readily available. Free unlimited access to a VR simulator without obligation or assessment in our setting did not seem to motivate surgical residents to use the simulator for improvement of their psychomotor endoscopic skills level. The addition of a competitive element and a desirable prize had only a marginal effect on the frequency and duration of training. We believe that the effort required to provide this incentive is disproportionate to its marginal effect. The majority of residents (86%) stated that ‘lack of time due to high working pressure’ is the most important reason for not using the simulator. Following a recent European guideline, as set by the European Commission, a working week for a resident in training is being reduced from 70 to 48 hours [1, 11]. This may have led to an increase in pressure during working hours, with little time available for voluntary training. However, spare time has increased vastly compared to the former curriculum. Residents did not use personal free time for VR simulator training to improve their skills. The perception of their own experience level in endoscopic procedures and the possibility of developing and training in endoscopic skills within the current surgical curriculum was in general neutral. Therefore no conclusions can be drawn from this. There is favorable, uniform opinion on the desirability of integration of skills training into the curriculum. In addition, residents believe skills training ought to be mandatory for marked improvement of their psychomotor skills. Interestingly, the disagreement on the statement of not training unless it is obligatory (mean 4.26, SD 1.10) appears to have no bearing in reality, because our study shows very limited use of the simulator. This incongruence might be caused by political correctness or by a discrepancy between intentions and actions. It must be said that our result reflects the quantity of training on a voluntary basis of 22 residents in a single institute only, and might therefore not represent the attitude of national or international surgical residents. In conclusion, the acquisition of expensive devices to train basic psychomotor skills for endoscopic surgery is probably only effective when it is a mandatory part of the curriculum.

Pflugers_Arch-3-1-1915642

Carbohydrate supplementation during prolonged cycling exercise spares muscle glycogen but does not affect intramyocellular lipid use

Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fiber-type-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63 ± 4% of maximal O2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg−1 h−1) or without (CON placebo trial; water only). Continuous infusions with [U-13C] palmitate and [6,6-2H2] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (Ra) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38 ± 19 and 57 ± 22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA Ra and subsequent plasma FFA concentrations were lower, resulting in a 34 ± 12% reduction in plasma FFA oxidation rates during exercise (P < 0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men. Introduction During exercise, there is a complex interaction between carbohydrate and fat metabolism to provide the required adenosine triphosphate (ATP) to sustain the necessary power output. The relative utilization of endogenous fat and carbohydrate stores during exercise can vary enormously and strongly depends on exercise intensity [28, 38], duration [28, 41, 45], and training status [19, 36]. During moderate intensity exercise, the oxidation of plasma-derived free fatty acids (FFA) in conjunction with other fat sources (muscle- and/or lipoprotein-derived triacylglycerol) contributes approximately half of the total energy required for contracting skeletal muscle. As the exercise duration lengthens, there is an increasing contribution of fat oxidation to total energy expenditure, which is primarily attributed to an increase in the release, uptake, and oxidation of plasma FFA [28, 41, 45]. Studies utilizing more prolonged (≥2 h) moderate-intensity exercise protocols have consistently shown that the progressive increase in plasma-FFA delivery to working skeletal muscle late in the exercise coincides with an inhibition of intramyocellular lipid (intramyocellular triacylglycerol [IMTG]) mobilization and/or oxidation [28, 41, 45]. In accordance, pharmacological inhibition of adipose tissue lipolysis has been shown to stimulate the use of intramyocellular lipids during the latter stages of prolonged exercise [42, 44, 47]. Carbohydrate supplementation can also serve to decrease FFA delivery and lower whole-body fat-oxidation rates during exercise [9, 10]. Therefore, it could be hypothesized that the reduced FFA availability via carbohydrate supplementation stimulates IMTG use during the latter stages of prolonged exercise. In contrast, other studies have reported an inhibitory effect of glucose ingestion on hormone-sensitive lipase activity [48], which would actually reduce IMTG hydrolysis [11]. It therefore remains to be established how carbohydrate supplementation during exercise modulates IMTG utilization. Carbohydrate supplementation during exercise has been reported to improve endurance performance by maintaining euglycemia during the late stages of prolonged exercise and/or by sparing the muscle glycogen stores [9]. However, the latter remains equivocal, as most studies have failed to report muscle glycogen sparing after carbohydrate supplementation during prolonged cycling exercise [5, 9, 13, 17, 24, 25]. The inconsistent findings of the effects of carbohydrate supplementation on subsequent muscle glycogen sparing may likely be attributed to differences in the applied research design, with specific reference to the mode of exercise (running vs cycling) and/or specific muscle group and/or fiber type recruitment [33]. The present study applies continuous infusions of [U-13C] palmitate and [6,6-2H2] glucose with muscle biopsy sampling before and after exercise to quantify both whole-body and muscle-fiber-type-specific IMTG and glycogen use. The purpose of this study was to assess the impact of carbohydrate supplementation on skeletal muscle glycogen and intramyocellular lipid use during prolonged cycling exercise in endurance-trained men and, as such, to determine whether carbohydrate supplementation reduces muscle glycogen use and/or augments the use of the IMTG pool as a substrate source. Materials and methods Subjects Ten endurance-trained male cyclists were selected to participate in this study. Subjects’ characteristics are provided in Table 1. Subjects were informed about the nature and risks of the experimental procedures before their written informed consent were obtained. This study was approved by the local Medical Ethical Committee of the Academic Hospital Maastricht. Table 1Subjects’ characteristics (n = 10)Characteristics Age (years)23 ± 3Height (m)1.83 ± 0.04Body mass (kg)72.5 ± 9.6BMI (kg m−2)21.7 ± 1.9Body fat percentage (%)9.8 ± 2.4Fat free mass (kg)65.3 ± 7.9Basal plasma glucose (mmol l−1)5.1 ± 0.3Plasma glucose120 min (mmol l−1)3.9 ± 1.1Basal plasma insulin (mU l−1)9.0 ± 2.6HbA1c (%)5.2 ± 0.2VO2max (ml kg−1 min−1)59.8 ± 5.6Wmax (W)398 ± 41Values are expressed as means ± SD. Body mass index (BMI) is calculated by dividing body mass by the square of the height.Plasma glucose120 min Plasma glucose concentration 120 min after ingesting 75 glucose in the oral glucose tolerance test Pretesting Subjects initially performed a continuous incremental cycling test on a cycle ergometer (LODE Instrument, Groningen, The Netherlands) to exhaustion to determine peak pulmonary oxygen uptake (VO2max; Oxycon-β, Mijnhart, The Netherlands) and maximal workload capacity (Wmax). After an overnight fast, body composition was assessed using the hydrostatic weighing method, with corrections made for residual lung volume via the helium dilution technique (Volugraph 2000, Mijnhart, Bunnik, Netherlands). Body-fat percentage was calculated using Siri’s [31] equation. To assess whole-body insulin sensitivity an oral glucose tolerance test was performed in each subject according to the World Health Organization criteria [2]. In addition, insulin resistance was estimated using the homeostasis model assessment for insulin resistance or HOMA-IR index [23]. Diet and activity before testing All subjects were instructed to maintain their usual dietary and physical activity patterns throughout the entire experimental period. In addition, they filled out a food intake diary for 2 days before the first exercise trial to keep their dietary intake as identical as possible before the other trials. Subjects refrained from heavy physical labor and exercise training for 3 days before each trial. Dietary analyses showed no differences in energy intake during the days before each of the trials with an average energy intake of 12.7 ± 2.4 MJ, with 57 ± 7, 28 ± 5, and 15 ± 3% of the energy intake (energy%) derived from carbohydrate, fat, and protein, respectively. The evening before each trial, subjects received the same standardized meal (41.2 kJ kg bw−1; containing 72, 11, and 17 energy% carbohydrate, fat, and protein, respectively). Experimental trials Each subject performed three trials, separated by at least 1 week: two experimental trials (CON: control vs CHO: carbohydrate) and an acetate correction trial (Fig. 1). Each trial consisted of 60 min of resting measurements, followed by 180 min of cycling at 50%Wmax. In the main trials, an [U-13C] palmitate and [6,6-2H2] glucose tracer were infused continuously at rest and during exercise with breath and blood samples collected at regular intervals. Muscle biopsy samples were collected before and immediately after exercise. In the carbohydrate supplementation trial (CHO), plasma FFA availability was reduced, and plasma glucose availability was augmented through carbohydrate ingestion. To allow for a continuous supply of glucose from the gut, subjects ingested an 8% CHO solution every 20 min during exercise at the rate of 0.7 g CHO kg−1 h−1, as recommended by the American College of Sports Medicine [1]. In the control trial, a placebo water drink was provided (CON). In a third trial, [1,2-13C] acetate was infused continuously at rest and during exercise, and only breath samples were collected. Both the CON and acetate trials were performed in an overnight fasted state. The acetate recovery factor was used to accurately correct [U-13C] palmitate oxidation rates for carbon label retention in the bicarbonate pool(s) and by way of isotopic exchange reactions in the TCA-cycle for each subject [40]. Fig. 1Schematic of study protocol. After an initial resting blood, breath, and muscle biopsy sample, a continuous infusion with [U-13C] palmitate and 6,6-2H2 glucose was started for 240 min. At rest and during 3 h of cycling at 50% of Wmax substrate use was assessed with (CHO trial) and without (CON trial) carbohydrate supplementation Protocol After an overnight fast, subjects arrived at the laboratory at 8:00 a.m. by car or public transportation. After 30 min of supine rest, a percutaneous muscle biopsy was taken from the vastus lateralis muscle [3]. A Teflon catheter (Baxter, Utrecht, The Netherlands) was inserted into an antecubital vein of one arm for blood sampling, another catheter was inserted in the antecubital vein of the contralateral arm for isotope infusion. Thereafter, a resting blood sample was taken and expired breath samples were collected into vacutainer tubes. Subsequently, subjects were administered a single intravenous dose of NaH13CO3 (0.06375 mg kg−1), to prime the bicarbonate pool(s), followed by a [6,6-2H2] glucose prime (13.5 μmol kg−1). Thereafter, a continuous infusion of [6,6-2H2] glucose (0.3 μmol kg−1 min−1) and [U-13C] palmitate (0.01 μmol kg−1 min−1; or [1,2-13C] acetate in the acetate recovery trial) was started (t = 0 min) via a calibrated IVAC pump (IVAC 560, San Diego, CA) and continued for 4 h. At t = 60 min, an initial drink bolus (7 ml kg−1) was given, and then nine more drink boluses (2.5 ml kg−1) were given at t = 80, 100, 120, 140, 160, 180, 200, 220, and 240 min of either an 8% carbohydrate solution or a placebo. At t = 60 min, subjects started to exercise on a cycle ergometer at a workload of 50%Wmax for 3 h. Expired VO2 and VCO2 were measured (Oxycon-β) at rest and throughout exercise for 5 min every 20 min before sampling of blood and expired breath collection. Immediately after cessation of exercise, a second muscle biopsy was taken (t = 240 min). Breath and blood samples were collected at t = 0, 30, and 60 min (during rest) and at t = 80, 100, 120, 140, 160, 180, 200, 220, and 240 min (during exercise). Beverages In the CHO trial, subjects received a beverage volume of 2.5 ml kg−1 every 20 min to ensure a given dose of 0.7 g CHO kg−1 (50% as glucose and 50% as maltodextrin) every hour. Repeated boluses were administered to enable a continuous supply of glucose in the circulation, preventing perturbations in [6,6-2H2] glucose enrichment. In the CON trial, a sweetened placebo drink, which contained no carbohydrate, was provided. Glucose and maltodextrin were obtained from AVEBE (Veendam, The Netherlands). To make the taste comparable in all experiments, beverages were uniformly flavored by adding 0.2 g sodium–saccharine solution (25% w/w), 1.8 g citric acid solution (50% w/w), and 5 g of cream vanilla flavor (Numico Research, Wageningen, The Netherlands) for each liter of beverage. Experiments were performed in a randomized order, with test drinks provided in a double-blind fashion. Tracer infusion Infusion rates of [U-13C] palmitate and [6,6-2H2] glucose averaged 7.3 ± 0.8 and 817.5 ± 30.4 nmol kg−1 min−1, respectively, in the CON and CHO trial. At the onset of exercise, [U-13C] palmitate infusion rates were doubled (14.6 ± 1.6 nmol kg−1 min−1). In the acetate recovery trial, a corresponding amount of 13C was infused, resulting in an average [1,2-13C] acetate infusion rate of 61.6 ± 2.4 and 123 ± 5 nmol kg−1 min−1 at rest and during exercise, respectively. Palmitate, glucose, and acetate tracer concentrations in the infusates averaged 1.03 ± 0.07, 64.8 ± 7.2, and 4.74 ± 0.18 mmol l−1, respectively. Blood- and breath-sample analysis Blood samples (7 ml) were collected in ethylenediamine tetraacetic acid (EDTA)-containing tubes and centrifuged at 1,000×g for 10 min at 4°C. Aliquots of plasma were frozen immediately in liquid nitrogen and stored at −80°C. Plasma glucose (Uni Kit III, Roche, Basel, Switzerland), lactate [16], FFA (NEFA-C, Wako Chemicals, Neuss, Germany), free glycerol (148270, Roche Diagnostics, Indianapolis, IN), and triglyceride (TG; GPO-trinder 337B, Sigma Diagnostics, St. Louis, MO) concentrations were analyzed with a COBAS semi-automatic analyzer (Roche). Expired breath samples were analyzed for 13C/12C ratio by gas chromatography continuous flow isotope ratio mass spectrometry (GC-IRMS; Finnigan MAT 252, Bremen, Germany). For determination of plasma palmitate and FFA kinetics, FFA were extracted from plasma, isolated by thin-layer chromatography, and derivatized to their methyl esters. Palmitate concentration was determined on an analytical gas chromatograph with flame ionization detection using heptadecanoic acid as an internal standard and on average comprised 26 ± 2 and 24 ± 3% of total FFA for the CON and CHO trials, respectively. Isotope tracer/tracee ratio of [U-13C] palmitate was determined using GC-combustion IRMS (Finnigan MAT 252). Plasma glucose was first extracted with chloroform–methanol–water and derivatization was performed with butylboronic acid and acetic anhydride as described previously [27]. After derivatization, plasma [6,6-2H2] glucose enrichment was determined by electron ionization GC-MS (Finnigan INCOS-XL). Glucose (Uni Kit III, Roche) and acetate (Kit 148261, Boehringer) concentrations in the infusates were determined with the COBAS FARA. Calculations From respiratory measurements, total fat and carbohydrate oxidation rates were calculated using the nonprotein respiratory quotient [26]. with VO2 and VCO2 in liters per minute (l min−1) and oxidation rates in grams per minute (g min−1). Breath and plasma enrichments are expressed as tracer/tracee ratios (TTR); in which sa indicates sample and bk indicates background value. Rate of palmitate and glucose appearance (Ra) and disappearance (Rd) were calculated using the single-pool nonsteady-state Steele [32] equations adapted for stable isotope methodology as described elsewhere [49]. As such, plasma palmitate and glucose Rd were calculated by correcting the Ra for the time-dependent changes in plasma metabolite concentration. where F is the infusion rate (μmol kg−1 min−1); V is the distribution volume for palmitate or glucose (40 and 160 ml kg−1, respectively); C1 and C2 are the palmitate or glucose concentrations (mmol l−1) at time 1(t1) and 2(t2), respectively, and E2 and E1 are the plasma palmitate or glucose enrichments (TTR) at time 1 and 2, respectively. 13CO2 production (Pr13CO2; mol min−1) from the infused palmitate tracer was calculated as: where TTRCO2 is the breath 13C/12C ratio at a given time point, VCO2 is the carbon dioxide production (l min−1), k is the volume of 1 mol of CO2 (22.4 l mol−1), and Ar is the fractional 13C label recovery in breath CO2 observed after the infusion of labeled acetate [30] and calculated as: where F is the infusion rate of [1,2-13C] acetate (mol min−1). Plasma palmitate oxidation (Rox; mol min−1) can subsequently be calculated as: where Rd palmitate is the rate of palmitate disappearance (mol min−1); F is the palmitate infusion rate (mol min−1), and 16 is the number of carbon atoms in palmitate. Total plasma FFA oxidation was calculated by dividing palmitate oxidation rates by the fractional contribution of plasma palmitate to total plasma FFA concentration. Muscle derived TG use was estimated by subtracting plasma FFA oxidation from total fat oxidation. However, it should be noted that the indirect stable isotope methodology does not differentiate between muscle- and lipoprotein-derived TG use. However, the contribution of lipoprotein-derived TG oxidation to total energy expenditure is assumed to be of relative minor quantitative importance, especially in an overnight-fasted state [37]. In a previous study, where we applied both an [U-13C] and [6,6-2H2] glucose tracer [18] during moderate intensity exercise, it was shown that the percentage of plasma glucose Rd that was oxidized varied between 96–100%. Therefore, plasma glucose oxidation rate during exercise was calculated as: Therefore, muscle glycogen oxidation was calculated by subtracting plasma glucose oxidation from total carbohydrate oxidation. Muscle-sample analyses Muscle tissue samples were freed from any visible nonmuscle material and rapidly frozen in liquid nitrogen. About 15 mg of each muscle sample was frozen in liquid nitrogen-cooled isopentane and embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands). Multiple serial sections (5 μm) from all biopsy samples were thaw mounted together per subject on uncoated, precleaned glass slides and stained with either acid-Schiff (PAS) [29] or immunolabelled oil red O [21] to measure fiber-type-specific glycogen or intramyocellular lipid content, respectively. To determine the muscle-fiber typing, (type I vs type II), we performed a myosin adenosine 5′-triphosphatase stain [22]. As previously described, the applied fluorescence and bright field microscopy techniques represent semiquantitative methods that can be used to compare fiber-type-specific IMTG and glycogen content [39, 41, 43, 44]. Fiber-type-specific IMTG content was expressed as the fraction of the measured area that was stained with oil red O. For IMTG quantification, average lipid-droplet size was calculated by dividing the total number of lipid aggregates by the total area measured. Mixed muscle-lipid content, lipid-droplet size, and lipid-droplet density were determined by calculating the average value in the type I and type II muscle fibers, with a correction for the relative area occupied by each fiber type within each field of view of each muscle cross-section, within each individual subject. The oil red O epifluorescence signal was recorded for each muscle fiber resulting, on average, in a total of 71 ± 19 muscle fibers analyzed for each muscle cross-section (45 ± 14 type I, 26 ± 10 type II) per subject. The PAS bright-field images were converted post hoc into eight-bit grayscale values to quantify glycogen. The mean optical density of the PAS-stained muscle fibers were determined by averaging the optical density measured in every pixel in the cell, corrected for the mean optical density of the background stain, containing no muscle fibers. For PAS analysis, on average per subject, a total of 151 ± 61 muscle fibers were analyzed for each muscle cross-section (92 ± 32 type I, 59 ± 26 type II). Statistics All data are expressed as mean ± SD. Before statistical analysis, data normality was confirmed at P > 0.05 using the Kolmogorov–Smirnov test on log-transformed raw data. To compare tracer kinetics, substrate utilization rates, IMTG contents, and/or plasma metabolite concentrations over time between trials, a two-way repeated measures analysis of variance was applied. When a significant F ratio was obtained, post hoc analyses were completed using a Student–Newman–Keuls test. For nontime-dependent variables, a Student’s t test for paired observations was used. Statistical significance was accepted at P < 0.05. Results Plasma metabolite and hormone concentrations Plasma FFA, triacylglycerol (TG), glycerol, glucose, lactate, and insulin concentrations over time during exercise are shown in Fig. 2. There were no differences in plasma FFA and glycerol between trials at rest or during the first 40 min of exercise (Fig. 2a,c). However, at all time points after 60 min, plasma FFA and glycerol concentrations increased over time in both trials, but levels remained significantly lower in the CHO vs CON trial (P < 0.01). Plasma TG levels declined throughout exercise in both trials but were significantly higher in the CHO vs the CON trial (P < 0.05; Fig. 2b). Plasma glucose concentrations increased in the CHO trial at the initiation of carbohydrate ingestion and were significantly higher in the CHO vs CON trial (P < 0.05; Fig. 2d). Over the last hour of exercise, plasma glucose steadily declined in both trials but remained significantly higher in the CHO vs CON trial. Plasma insulin levels in the CHO trial significantly increased during the initial 90 min of exercise as compared to CON (P < 0.05; Fig. 2f). Thereafter, plasma insulin steadily declined in both trials. Plasma lactate concentrations increased above baseline levels, with plasma lactate concentrations being greater in the CHO vs CON trial (Fig. 2e). In contrast, during the latter stages of exercise, significantly higher plasma lactate levels were observed in the CON vs CHO trial. Plasma norepinephrine concentrations increased threefold within the first 20 min of exercise and continued to rise throughout the exercise period in both trials, with no significant differences between trials (Fig. 3). Plasma epinephrine levels increased gradually in both trials during the first 2 h of exercise, after which, levels increased substantially (P < 0.001; Fig. 3). However, in the CHO trial, there was an attenuated increase in epinephrine, resulting in significantly (P < 0.05) lower concentrations vs CON. Fig. 2Plasma metabolite concentrations during 3 h of cycling at 50% Wmax in CON and CHO trials. Values are means ± SD, n = 10. Crosses, significantly different than CON trial (P < 0.05)Fig. 3Plasma catecholamine concentrations during 3 h of cycling at 50% Wmax in CON and CHO trials. Values are means ± SD, n = 10. Crosses, significantly different from the CON trial (P < 0.05) Plasma tracer kinetics As plasma FFA and glucose concentrations varied over time in both trials (Fig. 2a,d), nonsteady-state Steele [32] equations were applied to calculate tracer kinetics. At rest, tracer kinetics did not differ between trials as carbohydrate supplementation did not start until the onset of exercise. At rest, plasma glucose Ra and Rd averaged 14.9 ± 1.6 and 15.0 ± 1.8 μmol kg−1 min−1, respectively. Resting palmitate Ra, Rd, and Rox averaged 1.92 ± 0.43, 1.89 ± 0.55, and 0.74 ± 0.22 μmol kg−1 min−1, respectively, with 38 ± 4% of palmitate Ra being oxidized. The onset of exercise significantly elevated plasma glucose and palmitate Ra, Rd, and/or Rox (Table 2). Plasma glucose Ra and Rd, and palmitate Ra, Rd, and Rox increased continuously during the exercise in both trials (Table 2). During the entire exercise period, average glucose Ra and Rd was 49 ± 9% greater in the CHO compared with the CON trial (P < 0.05; Table 2). In contrast, plasma palmitate Ra, Rd, and Rox was 41 ± 12% lower during exercise in the CHO vs CON trial (P < 0.05; Table 2). Acetate label recovery maintained a constant value during exercise and averaged 86 ± 4% of the acetate infusion rate. In a pilot experiment, we confirmed that carbohydrate supplementation does not affect acetate label recovery during exercise, which was in accordance to previous findings [44]. Table 2Tracer kinetics and respiratory measures during exercise First hourSecond hourThird hourExercise averageCONCHOCONCHOCONCHOCONCHOGlucose Ra (μmol kg−1 min−1)21.1 ± 2.746.8 ± 9.6a28.8 ± 4.855.8 ± 6.8a38.1 ± 7.572.9 ± 8.2a30.4 ± 4.7 59.9 ± 7.2a Rd (μmol kg−1 min−1)21.9 ± 3.148.3 ± 12.3a30.3 ± 7.960.0 ± 11.2a39.5 ± 7.272.9 ± 8.5a31.7 ± 4.7 61.9 ± 7.3aPalmitate Ra (μmol kg−1 min−1)2.49 ± 0.802.03 ± 0.40a3.55 ± 0.781.97 ± 0.57a6.43 ± 0.893.60 ± 1.92a4.36 ± 0.712.59 ± 0.70a Rd (μmol kg−1 min−1)2.48 ± 0.792.06 ± 0.40a3.50 ± 0.791.96 ± 0.56a6.36 ± 0.883.57 ± 1.34a4.32 ± 0.702.58 ± 0.69a Rox (μmol kg−1 min−1)2.28 ± 0.731.84 ± 0.42a3.49 ± 0.721.91 ± 0.68a6.35 ± 0.733.62 ± 1.38a4.26 ± 0.592.53 ± 0.77a %Ra ox91.5 ± 5.990.9 ± 8.698.7 ± 5.8395.6 ± 8.899.4 ± 7.899.8 ± 7.097.2 ± 6.296.0 ± 6.8 Ac recovery81.7 ± 5.7–86.5 ± 4.7–86.2 ± 4.1–85.8 ± 3.9–Respiratory measures VO2 (ml)2,666 ± 912,690 ± 902,713 ± 882,709 ± 982,773 ± 932,772 ± 1092,717 ± 892,724 ± 97 VCO2 (ml)2,361 ± 812,412 ± 762,336 ± 762,424 ± 84a2,327 ± 882,453 ± 94a2,342 ± 792,430 ± 83a RER0.89 ± 0.010.90 ± 0.010.86 ± 0.010.90 ± 0.01a0.84 ± 0.010.89 ± 0.01a0.86 ± 0.010.89 ± 0.01aValues are expressed as means ± SD, n = 10. Tracer kinetics during each hour of exercise and averaged during 3 h of cycling at 50% Wmax in the CON and CHO trial.Ra Rate of appearance; Rd rate of disappearance; Rox rate of oxidation; %Raox %Ra palmitate oxidized; Ac recovery fraction 1,2-13C acetate label recovery in expired CO2aSignificantly different compared with the CON trial (P < 0.05) Substrate source utilization at rest There were no differences in total resting energy expenditure (5.37 ± 0.52 vs 5.32 ± 0.53 kJ min−1 in the CON and CHO trial, respectively) or substrate source utilization rates between trials. Total fat oxidation averaged 0.08 ± 0.01 g min−1, contributing 58 ± 6% to total energy expenditure. Plasma FFA oxidation rates averaged 0.061 ± 0.011 g min−1, contributing 53 ± 7 to total energy expenditure at rest. The use of muscle- and/or lipoprotein-derived TG sources averaged 0.016 ± 0.008 g min−1 and contributed 7 ± 20% to total energy expenditure. Total carbohydrate oxidation rates averaged 0.13 ± 0.03 g min−1, contributing 42 ± 6% to total energy expenditure. Substrate source utilization during exercise The applied 50% Wmax workload resulted in subjects cycling at 199 ± 21 W, corresponding with a relative workload intensity of 63 ± 1% VO2max From rest to exercise, energy expenditure increased tenfold. No differences in energy expenditure were observed over time or between trials (57.3 ± 5.9 and 57.8 ± 6.4 kJ min−1 in the CON and CHO trial, respectively; Fig. 5). Total carbohydrate oxidation rates were significantly higher in the CHO vs the CON trial (2.35 ± 0.37 vs 1.97 ± 0.33 g min−1, respectively; P < 0.001). Concomitantly, total fat oxidation rates were higher in the CON vs CHO trial (0.62 ± 0.12 vs 0.48 ± 0.16 g min−1, respectively; P < 0.001). In the CHO trial, carbohydrate and fat oxidation contributed 66 ± 6 and 34 ± 5% to total energy, respectively. In the CON trial, carbohydrate and fat oxidation contributed 56 ± 5 and 44 ± 7% to total energy expenditure during exercise. The use of specific substrate sources over time during exercise is illustrated in Fig. 4. The calculations for substrate source utilization in Figs. 4 and 5 commences at 40 min because of the nonsteady-state transition from rest to exercise. Plasma FFA and glucose oxidation rates increased over time in both trials (P < 0.01; Fig. 4a,c). In contrast, the respective use of muscle- (and lipoprotein) derived TG and glycogen declined over time (P < 0.01; Fig. 4b,d). Plasma glucose oxidation was significantly greater in the CHO vs CON trial and averaged 13.2 ± 2.1 vs 6.7 ± 1.1 kJ min−1, respectively (P < 0.001: Fig. 4c). Plasma FFA oxidation rates were substantially greater in the CON vs CHO trial and averaged 13.8 ± 2.9 vs 9.1 ± 2.8 kJ min−1, respectively (P < 0.001; Fig. 4a). No significant differences were observed in the use of muscle (and lipoprotein-derived) TG and glycogen over time between trials. Fig. 4a Plasma FFA, b other fat sources, c plasma glucose, and d muscle glycogen rate of oxidation (kJ min−1) during 3 h of cycling at 50% Wmax in CON and CHO trials. Values are means ± SD, n = 10. Crosses, significantly different from CON trial (P < 0.05)Fig. 5Whole-body substrate source utilization (kJ min−1) during the first, second, and third hour of cycling and averaged over the entire 3 h, at 50% Wmax in the CON and CHO trial. Crosses, plasma FFA, other fat sources, total carbohydrate, and/or plasma glucose and/or glycogen use significantly different from the CON trial (P < 0.05) Figure 5 illustrates average substrate source utilization rates calculated over each consecutive hour of exercise. During the first hour of exercise, there were no differences in endogenous fat source utilization between trials. In contrast, plasma glucose oxidation rates were higher in the CHO trial, resulting in a 10 ± 6% lower glycogen use compared with the CON trial (P < 0.05; Fig. 5). During the latter stages of exercise, plasma glucose oxidation rates remained higher in the CHO vs CON trial, with no differences in muscle glycogen use between trials (Fig. 5). In the first hour, fat contributed nearly the same amount of energy in the CON and CHO trial, 36 ± 6 and 33 ± 7%, respectively. In contrast, during the second and third hour of exercise, total fat oxidation rates increased substantially, with greater fat use in the CON vs CHO trial. By the third hour, fat oxidation contributed 51 ± 8% to total energy expenditure in the CON trial, whereas fat oxidation contributed only 36 ± 9% in the CHO trial (P < 0.01; Fig. 5). In the CHO trial, the attenuated increase in fat oxidation rate during exercise was offset by a significant increase in total carbohydrate oxidation rate. Total carbohydrate oxidation rates during exercise averaged 1.97 ± 0.33 and 2.35 ± 0.37 g min−1 in the CON and CHO trial, respectively (P < 0.01). As there were no differences in the average rate of glycogen utilization between treatments during the second and third hour of exercise (Figs. 4d and 5), the increased carbohydrate use in the CHO trial was fully attributed to increases in plasma glucose oxidation over the last 2 h of cycling (Figs. 4c and 5). Muscle (and lipoprotein-derived) TG and glycogen use declined over time but did not differ between trials during the latter stages of exercise (Fig. 5). Skeletal muscle lipid content Preexercise fiber-type-specific muscle IMTG was not different between conditions and averaged 0.067 ± 0.025 and 0.018 ± 0.010 arbitrary units (AU) in type I and II muscle fibers, respectively. There was a significantly greater net decline in intramyocellular lipid content in the type I vs type II muscle fibers after exercise (Fig. 6a). No differences in the net decline in myocellular lipid content were observed between trials. Exercise resulted in a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively (P < 0.01). Type II muscle-fiber lipid content was not significantly reduced after exercise and showed a net decline of 0.003 ± 0.014 and 0.008 ± 0.0012 AU in the CHO and CON trials, respectively (P = NS). The observed decrease in type I muscle intramyocellular lipid content was attributed to significant decreases in both lipid-droplet size (preexercise, 0.560 ± 0.150; vs postexercise, 0.359 ± 0.152 μm2) and lipid-droplet density (preexercise, 0.119 ± 0.031; vs postexercise, 0.041 ± 0.022 droplets μm−2). Fig. 6Fiber-type-specific net intramyocellular lipid and glycogen utilization (expressed as mean arbitrary units ± SD, n = 10) during 3 h of cycling at 50% Wmax in CON and CHO trials. Asterisk, type II significantly different than type I (P < 0.05); Crosses, significantly different from the CON trial (P < 0.05) Skeletal muscle glycogen content At rest, glycogen contents did not differ between trials (0.087 ± 0.018 vs 0.103 ± 0.021 AU in the CHO vs CON trial, respectively; P > 0.05) or between fiber types (0.095 ± 0.019 vs 0.094 ± 0.021 AU in the type I vs type II muscle fibers, respectively; P > 0.05). Exercise resulted in a significantly greater net reduction in muscle glycogen content in the type I vs the type II muscle fibers (P < 0.05; Fig. 6b). The net decline in muscle glycogen content after exercise was significantly greater in the CON vs the CHO trial (38 ± 19 and 57 ± 22% greater in the type I and II muscle fibers, respectively; P < 0.01) Discussion The aim of this study was to examine substrate source utilization with (CHO) or without (CON) carbohydrate supplementation on subsequent glycogen and IMTG use during prolonged cycling exercise in endurance-trained male cyclists. Carbohydrate ingestion increased total carbohydrate oxidation, which was entirely attributed to a greater plasma glucose appearance (Ra) and disappearance (Rd) rate. The greater glucose availability in the CHO trial lowered muscle glycogen use during the first hour of exercise, resulting in significant muscle glycogen sparing in both type I and II muscle fibers after 3 h of exercise. Carbohydrate ingestion inhibited adipose tissue lipolysis, resulting in lower plasma FFA Ra, Rd, and Rox. The lower total fat oxidation rate was entirely attributed to reduced plasma FFA oxidation, as muscle IMTG use was not affected by carbohydrate supplementation. IMTG use during prolonged exercise The importance of the IMTG pool as a substrate source during exercise has been disputed. Many of the inconsistencies in the literature regarding IMTG use seem to be explained by the methodological limitations associated with the biochemical mixed-muscle TG extraction analyses [46]. More recent studies using either stable isotope tracers, 1H-magnetic-resonance spectroscopy, electron microscopy, and/or immunofluorescence microscopy on ORO stained muscle cross-sections have now established that IMTG is indeed a viable substrate source during exercise in healthy humans [37]. In accordance, substantial net reductions in type I muscle-fiber lipid content as assessed by immunohistochemical oil red O staining of muscle cross-sections have been observed after both endurance and resistance type exercise [11, 20, 41, 44]. Furthermore, our laboratory has found a significant correlation between IMTG contents assessed by biochemical TG extraction analyses and immunofluorescence microscopy on ORO stained muscle cross-sections (unpublished observations). In the present study, we observed a significant 20 ± 3 and 19 ± 2% contribution from muscle- and/or lipoprotein-derived TG sources use during exercise in the CHO and CON trial, respectively (Fig. 5). Concomitantly, muscle biopsy analyses revealed a 76 ± 21 and 78 ± 22% net decline in type I muscle-fiber IMTG content in the CHO and CON trial, respectively (Fig. 6a). This translated in a 67 ± 19 and 70 ± 20% decline in mixed-muscle intramycellular lipid content in the CHO and CON trial, respectively. IMTG utilization during prolonged exercise with carbohydrate supplementation Earlier estimations [28] and more recent findings [41, 45] show that the progressive increase in plasma FFA delivery during the latter stages of prolonged exercise suppresses IMTG mobilization and/or oxidation. In agreement, pharmacological inhibition of adipose tissue lipolysis has been shown to reduce plasma FFA availability and augments IMTG use during exercise [42, 44, 47]. This possibly suggests that during situations of decreased FFA availability there is a compensatory increase in IMTG utilization in vivo in humans. Carbohydrate supplementation during exercise also inhibits adipose tissue lipolysis and reduces plasma FFA availability [9, 10]. Therefore, it could be speculated that carbohydrate supplementation could augment IMTG use during the latter stages of prolonged exercise. In the present study, carbohydrate supplementation resulted in a 23 ± 17% reduction in whole-body fat oxidation because of the concerted reductions in adipose tissue lipolysis with a concomitant 41 ± 12% lower plasma palmitate Ra, Rd, and Rox when compared with the CON trial. Despite the attenuated increase in plasma FFA availability in the CHO trial, no differences in estimated IMTG use were observed between trials (Figs. 4b and 5). In agreement, after exercise, type I muscle-fiber lipid content was similarly reduced by 76 ± 21 and 78 ± 22% in the CHO and CON trial, respectively (Fig. 6a). In the present study, we did not observe any differences in IMTG use after carbohydrate ingestion. This might be because of the metabolic effects of increased glucose availability in the CHO trial, as we observed a 49 ± 9% greater glucose Ra (Table 2) and a subsequent 48 ± 9% higher plasma glucose oxidation rate throughout exercise (Fig. 4c). Furthermore, carbohydrate ingestion elevated plasma insulin concentrations by nearly 60% in the first hour of exercise vs CON (Fig. 2f). Elevated plasma insulin is a potent inhibitor of adipose tissue lipolysis [6], resulting in a substantially lower FFA availability in the CHO vs CON trial (Table 2). Although reduced plasma FFA availability could stimulate IMTG hydrolysis by reducing the allosteric inhibition of hormone-sensitive lipase (HSL) [44], we did not observe greater IMTG use in the CHO trial (Figs. 4b and 6a). However, this elevated plasma insulin could also serve to inhibit HSL activity, as a recent study showed blunted HSL activity during exercise when glucose is ingested [48]. Watt et al. [48] suggested that the decreased HSLa was most likely mediated via increased insulin and decreased epinephrine concentrations, and that even in situations when local intramuscular mediators of HSL are altered (i.e., muscle contraction, substrates/products), the concurrent hormonal milieu may play a greater role in the activation of HSL. This previous study also found no effect of glucose ingestion on estimated IMTG oxidation, despite a decrease in plasma FFA availability and HSL activity [48]. Our present findings are not in line with de Bock et al.[11], who reported attenuated IMTG use after carbohydrate supplementation during a more intense exercise session in less-well-trained subjects. As such, we conclude that the proposed stimulating effects of reduced plasma FFA availability on IMTG mobilization and/or oxidation are offset by the inhibitory effects of carbohydrate supplementation on IMTG hydrolysis. The latter likely includes the greater glycolytic flux (Table 2), concomitantly higher circulating insulin (Fig. 2f), and decreased epinephrine levels (Fig. 3), all of which have been associated with reduced HSL activity in muscle tissue [48]. Effects of carbohydrate supplementation on muscle glycogen use It has been established that prolonged moderate-intensity exercise results in greater glycogen depletion in type I vs type II muscle fibers [8, 14]. In accordance, we observed a 73 ± 25 and 42 ± 18% greater net utilization of type I vs type II muscle-fiber glycogen content in the CHO and CON trial, respectively (Fig. 6b). This is the first study to show glycogen sparing during prolonged cycling exercise in both type I and II muscle fibers (Fig. 6b). This finding seems to be in contrast to most previous studies that failed to observed muscle glycogen sparing after carbohydrate supplementation during cycling exercise [5, 9, 13, 17, 24, 25]. In line with our findings, some studies have also reported muscle glycogen sparing after carbohydrate supplementation during running exercise [34, 35]. However, these studies reported muscle glycogen sparing to be restricted to the type I muscle fibers only [34, 35]. The apparent discrepancy with our findings is likely explained by the differences in the type (running vs cycling) and duration (1–2 vs 3 h) of the exercise that was implemented, as with increasing exercise duration, muscle-fiber-type recruitment shifts to include more type II fiber activation as the type I fibers become glycogen depleted [15]. Our findings of muscle glycogen sparing with carbohydrate supplementation during prolonged constant intensity cycling are in line with some [4, 12], but definitely not with the majority of studies [5, 9, 13, 17, 24, 25]. The apparent discrepancy between the current study and prior work that failed to observe muscle glycogen sparing with carbohydrate supplementation during cycling might be explained by differences in training status of the subjects, the intensity and duration of the exercise, the amount and timing of the ingested carbohydrate, the absence of the time-dependent assessment of muscle glycogen use, and the lack of muscle-fiber-type-specific glycogen analyses. Glycogen sparing occurs early in exercise with carbohydrate supplementation In agreement with our findings of skeletal muscle glycogen sparing, whole-body muscle glycogen use was lower during the initial hour of exercise in the CHO vs the CON trial (Fig. 5). In accordance to the hypothesis set forth by Coggan and Coyle [7], we observed a greater dependence on muscle glycogen as a substrate source during the early stages of exercise in the CON trial (Fig. 5). As exercise duration lengthened, the contribution of muscle glycogen decreased over time as muscle glycogen stores became depleted, while blood glucose uptake and oxidation rates increased progressively (Fig. 4). In line with this hypothesis, we observed lower muscle glycogen use during the initial hour of exercise in the CHO trial. At the onset of exercise, carbohydrate administration caused a 49 ± 9% greater plasma glucose Ra and Rd in the CHO compared with the CON trial (Table 2), which paralleled a 60% increase in plasma insulin concentrations as compared to CON during the first hour of exercise (Fig. 2). Concomitantly, these changes because of the augmented glucose availability at exercise onset decreased the reliance of muscle glycogen as a fuel source during the first hour of exercise. The latter is in accordance with a previous work reporting significant glycogen sparing after carbohydrate supplementation after 60 min of running exercise [34]. We conclude that the majority of glycogen sparing because of carbohydrate supplementation occurs during the initial stages (<1 h) of prolonged exercise, resulting in lowered glycogen utilization in type I and II muscle fibers. As our findings show that muscle glycogen sparing after carbohydrate supplementation occurs early in exercise, endurance athletes should be recommended to initiate sports-drink consumption immediately at the onset of exercise to maximize the potential for glycogen sparing, along with maintaining euglycemia and high blood glucose oxidation rates late in exercise. Conclusions This study provides compelling evidence that carbohydrate supplementation spares muscle glycogen utilization in a time-dependent manner during prolonged exercise in endurance-trained cyclists. Carbohydrate supplementation augments plasma glucose uptake and oxidation rate, while reducing muscle glycogen use during the early stages of exercise, thereby sparing type I and II muscle fiber glycogen stores. Carbohydrate supplementation during exercise lowers plasma FFA rate of appearance, uptake, and oxidation, but does not modulate the use of IMTG as a substrate source during cycling exercise.

Mol_Neurobiol-3-1-2039847

Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer’s Disease

The pathologic lesions of Alzheimer’s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-β (Aβ) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. “Professional chaperones”, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. “Amateur” chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of Aβ, and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood–brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention. Introduction Pathological lesions consisting of intra- and/or extracellular accumulations of misfolded proteins are characteristic for neurodegenerative diseases such as Alzheimer’s disease (AD). AD is characterized by three distinct pathological lesions: senile plaques (SPs), neurofibrillary tangles (NFTs), and cerebrovascular amyloid angiopathy (CAA) [1]. Both SPs and CAA are formed by extracellular deposition of aggregated amyloid-beta protein (Aβ), whereas NFTs consist of intracellular aggregates of hyperphosphorylated tau protein in the cytoplasm of neurons [2, 3]. The Aβ protein is a 4-kDa proteolytic cleavage product [2] of the transmembrane amyloid-β precursor protein (APP). The two major forms of Aβ in human brain are Aβ1-40 and Aβ1-42, differing from each other only by two amino acids. Cerebral production of Aβ is balanced by clearance from the brain either via active transport across the blood–brain barrier (BBB) or via uptake and degradation of Aβ by microglial cells and astrocytes [4–6]. Active transport of Aβ is mediated by Aβ receptors that are capable of transporting Aβ, or Aβ in complex with other proteins, across the BBB [7]. In contrast to normal brain, the cerebral Aβ balance is disturbed in AD brains, resulting in accumulation and aggregation of Aβ. Aβ aggregation includes the formation of Aβ oligomers, protofibrils, and eventually, mature fibrils. Both Aβ oligomers and protofibrils are considered the most toxic forms of Aβ that initiate degeneration of neurons and of cells within the vasculature, such as smooth muscle cells and pericytes [8, 9]. Aβ aggregates do not clear from the brain as efficiently as soluble Aβ, and thus, directly lead to increased levels of Aβ in the brain [10]. Furthermore, deposition of Aβ in SPs is accompanied by attraction and activation of both microglial cells and astrocytes [11–13]. Activation of these cell types results in increased secretion of pro-inflammatory cytokines as part of a neuro-inflammatory reaction. Chaperones can be defined as proteins that: (1) have a role in the intracellular handling of misfolded proteins, (2) induce conformational changes of proteins, (3) act as transporter of proteins. “Professional” chaperones, such as the heat shock protein family (Hsp), are defined as proteins that have a specific function in facilitating normal folding of proteins and intracellular handling of misfolded proteins. Members of the Hsp family recognize misfolded proteins and transport them to the proteasome for degradation. Therefore, this protein family acts as the first line of defense against toxicity induced by misfolded proteins such as Aβ and tau. In contrast to professional chaperones, “amateur” chaperones can be defined as proteins that bind to other proteins and induce conformational changes or, alternatively, serve as transporter proteins. Examples of putative amateur chaperones are apolipoprotein E (ApoE), heparan sulfate proteoglycans (HSPGs), and complement factors such as C1q. They have, in contrast to the professional chaperones, primarly an extracellular function. In this paper, we will review the role of both amateur and professional chaperones in the pathogenesis of AD. Aβ-Binding Proteins in Extracellular Interaction with Aβ Apolipoproteins The apolipoprotein family consists of proteins that conjugate with lipids to form different classes of lipoprotein particles. In human brain, several members of this protein family are expressed, such as apolipoprotein E (ApoE), apolipoprotein J (ApoJ), and apolipoprotein D (apoD). ApoE is a major determinant of lipid transport and metabolism and is expressed in brain by astrocytes, microglia, pericytes, and smooth muscle cells [14–18]. In human, three common isoforms are expressed: apoE2, apoE3, and apoE4 which are all products of alleles at a single gene locus [19, 20]. The ɛ4 allele of ApoE is the major genetic risk factor for AD, whereas the ɛ2 allele appears to be protective against AD [21–24]. As ApoE immunoreactivity was found in extracellular amyloid deposits in subjects with AD, it has been suggested that it affects amyloidogenesis [25, 26]. In vitro studies provided evidence for a direct interaction of ApoE with Aβ and the formation of stable complexes [27, 28]. Binding of ApoE to Aβ is, however, ApoE isoform-dependent (ɛ2>ɛ3>>ɛ4) [29, 30] and depends on the degree of lipidation [29]. Lipidation of ApoE also seems a major factor in its effect on Aβ-mediated cellular toxicity [18]. In addition, ApoE4 promotes the conversion of soluble Aβ into β-sheet-rich amyloid more than ApoE3 [31–33]. In contrast to its effect on Aβ in vitro where a consistent accelerating effect of ApoE on Aβ aggregation is observed, the effect of ApoE on Aβ deposition in transgenic (Tg) mice studies is less equivocal. In early studies, both Aβ immunoreactivity and amyloid formation were reduced in ApoE knockout mice [31, 32]. In addition, CAA and associated microhemorrhages were also suppressed in ApoE knockout mice [34]. This effect might be due to the absence of ApoE/Aβ complexes [35]. In contrast to the effects of murine ApoE in the early studies, both human ApoE3 and ApoE4 suppressed Aβ deposition in Tg mice [36]. In addition, when these mice aged, ApoE4 induced a tenfold higher deposition of fibrillar Aβ than ApoE3 [37]. Consistent with these latter results, human ApoE4 accelerated Aβ deposition in APPSwe Tg mice relative to human ApoE3 [38]. In addition, when human ApoE3 or ApoE4 were knocked in in Tg mice, Aβ deposition was reduced compared to mice carrying endogenous ApoE at 9 months, and at 15 months, substantial CAA was observed in mice with human ApoE4, but not with human ApoE3, and, in either case, parenchymal Aβ was sparse [39]. Thus isoform- and species-specific differences in ApoE direct the aggregation or clearance of Aβ. Furthermore, it is suggested that the presence of ApoE facilitates internalization and degradation of Aβ from brain parenchyma by astrocytes [40] and human ApoE may reduce Aβ deposition in mouse brain by facilitating Aβ transport across the BBB [36]. Although these Tg mice studies seem contradictional, ApoE clearly affects conformational changes of Aβ and functions as an Aβ-transporter protein. Besides colocalization of ApoE with Aβ in AD brains, ApoE is also found within neurons containing NFTs [25] where it is able to interact directly with tau protein [41]. Furthermore, ApoE has an isoform-dependent effect on tau phosphorylation, as ApoE3 binds to tau in vitro, whereas ApoE4 fails to bind tau [42]. In addition, an ApoE4-dependent increase in phosphorylated tau has been observed [43–45]. Neuroinflammation in AD comprises both activation of microglial cells and astrocytes and activation of the complement system. Aβ deposits in brain are associated with activated microglia and astrocytes, but also with elevated levels of complement [5, 6, 46]. ApoE may have an anti-inflammatory effect by suppressing microglial and astrocytic activation [47–50]. ApoE-deficient mice demonstrate increased levels of IL-6 and TNFα after LPS stimulation, suggesting a role of ApoE in inflammatory gene regulation [51]. In addition, ApoE isoform-dependent (ɛ2<ɛ3<ɛ4) differences in nitric oxide (NO) levels have been observed in microglia cells [52]. Transgenic mice expressing the ApoE4 protein isoform show a greater NO production than mice expressing the ApoE3 protein isoform. These data indicate that ApoE4 has a less efficient anti-inflammatory affect, and thus, may accelerate the development of AD. Apolipoprotein J, also known as clusterin or SP-40/40, is a highly conserved heterodimeric secreted glycoprotein expressed in brain by epithelial and neuronal cells [53]. ApoJ colocalizes with fibrillar Aβ deposits, and it is suggested that it prevents misfolding and aggregation of soluble Aβ [54–56]. ApoD is a glycoprotein associated with high-density lipoproteins in human plasma and also has a high expression level in human brain [57], but neither its physiological role nor its ligand has been identified. ApoD levels are increased in the hippocampus of AD patients and in ApoE-deficient mice [58, 59]. In conclusion, ApoE and ApoJ can be regarded as amateur chaperones that regulate Aβ aggregation in vitro. By accelerating the Aβ aggregation process towards mature fibril formation, (human) ApoE prevents formation of toxic Aβ intermediates such as oligomers and protofibrils, and thus, may have a protective function towards development of AD. Moreover, ApoE protects against the development of AD by suppressing the inflammatory reactions associated with AD lesions. Besides its role in inducing conformational changes in Aβ, ApoE facilitates Aβ clearance from brain by serving as a transporter molecule of Aβ, which will be discussed in paragraph 4. Heparan Sulfate Proteoglycans Proteoglycans are members of a large family of macromolecules with a wide variety of functions ranging from simple physical support to various effects on cell adhesion, motility, proliferation, differentiation, and even tissue morphogenesis. They are composed of linear sulfated polysaccharides (glycosaminoglycans, GAGs), consisting of disaccharide units, covalently bound to a core protein. One of the members of this superfamily is the heparan sulfate proteoglycan (HSPG) family characterized by polymers of repeating disaccharides, N-acetylglucosamine and glucuronic acid, which can be subsequently modified by sulfatation [60, 61]. HSPGs can be subdivided into a family of extracellular matrix proteins, including perlecan, agrin, and collagen XVIII, and a family of cell surface proteins, including syndecans and glypicans [60, 62]. Ever since GAGs were demonstrated in amyloid deposits, the proteoglycans became of interest in amyloidogenesis. The presence of HSPGs in SPs, CAA, and NFTs in AD brains was already demonstrated in the late 1980s [63–65]. Only when antibodies became available that could identify the various individual HSPG species was it described that perlecan colocalized with all three lesions characteristic of AD brains [65–67]. However, we were not able to confirm these findings [68, 69]. Furthermore, it was shown that in both diffuse and classic SPs, several other HSPGs were found, such as agrin, glypican 1, and syndecan 1-3, whereas collagen XVIII is only present in classic SPs and CAA [69–72]. These data suggest that HSPGs interact with Aβ, thereby contributing to development or persistence of SPs or CAA. HSPGs isolated from Engelbreth–Holm–Swarm tumor prevented proteolytic breakdown of aggregated Aβ [73]. In addition, both agrin and perlecan directly interacted with Aβ and promoted conversion of non-fibrillar Aβ into fibrillar Aβ [70, 74–76]. Although the interaction between HSPGs and Aβ is likely mediated predominantly by the sulfate moieties of the GAGs, a role for the protein backbone in Aβ aggregation could not be excluded [77, 78]. As sulfated GAGs were also demonstrated in NFTs in AD brains [79], these macromolecules may also play a role in tangle development. Indeed, sulfated GAGs may induce the formation of paired helical filaments by stimulating tau phosphorylation [80]. As heparan sulfates bind to Aβ and interfere with its fibrillogenesis, they are interesting candidates for therapeutic intervention [81]. GAG mimetics are able to inhibit this binding and may block the formation of β-pleated sheets and adherence of Aβ to the cell surface [82]. The use of GAG mimetics has already been explored in mouse models where they reduced progression of inflammation-associated amyloidosis [83]. The efficacy of one of these compounds is currently being tested in a human phase III trial. As exemplified by ApoE, Aβ-binding proteins may play a role in the inflammatory reactions in AD brains. Recently, it was demonstrated that the semi-synthetic proteoglycan analogue dextran sulfate blocks activation of the complement cascade [84]. In addition, chondroitin sulfate proteoglycans are also known to bind to C1q and prevent the formation of the C1 complex in vitro [85]. By doing so, chondroitin sulfate proteoglycans inhibit normal complement function. Furthermore, heparin has long been regarded as a potential complement inhibitor [86]. In conclusion, HSPGs do not only colocalize with Aβ and tau, but they also contribute to the development of these lesions. The role of HSPGs in Aβ aggregation might even be a protective one. HSPGs prevent the persistence of toxic Aβ forms, e.g., oligomers or protofibrils, and transform them into more harmless aggregates, i.e., the classic senile plaques containing mature Aβ fibrils that are less toxic than the intermediate aggregates. In addition, HSPGs might play a role in the development of AD lesions by inhibiting complement activation. According to the definitions, HSPGs can therefore be regarded as amateur chaperones. Their ability to recognize a variety of proteins may originate from the heterogeneous structure of the heparan sulfate chains. The negatively charged HS chains are structurally heterogeneous and bind a diverse repertoire of proteins, such as amyloid A, protease-resistant prion protein, α-synuclein, and tau, providing HSPGs with the ability to interact with a wide range of intracellular and extracellular amyloidogenic proteins [61]. Complement Factors The complement system is an ancient host defense mechanism which is involved in boosting antibody activity. The system consists of a group of soluble serum proteins C1–C9 and is activated either by immunoglobulin M or G bound to a foreign particle or directly by microorganisms. Proteins such as Hageman factor, C4 binding protein, CDS46, CD59, and C1 inhibitor regulate the complement system. In AD, the complement system is overexpressed and activated [46]. The Aβ protein itself activates this system, and complement factor concentrations are increased in AD brains [87–89]. Aβ induces C3 and C4 in AD, and elevated levels of the membrane attack complex (MAC) composed of C5–C9 have been observed [90, 91]. In addition, factors such as Hageman factor, C1q, C3, and C5–9 are commonly found in SPs and NFTs [87, 92, 93]. C1q is associated with Aβ deposits and directly binds fibrillar Aβ which activates the complement cascade [94]. In contrast, the complement inhibitor (C1 INH) is downregulated in AD [95, 96]. Thus, an activated complement system is a general feature observed in AD. However, the contribution of complement to the pathogenesis of AD is controversial. On the one hand, it is suggested that complement activation protects against Aβ-induced toxicity and even contributes to reducing the accumulation of Aβ in SPs [97]. Transgenic mice expressing complement inhibitors develop increased AD-pathology, whereas increased complement C3 production was associated with a reduction of Aβ deposition [97]. Thus, the complement activation in the brain may be beneficial in AD and possibly also other neurodegenerative diseases [98–100]. However, complement activation may lead to accelerated neurodegeneration as well. Activation of complement in an antibody-independent fashion is achieved by binding of aggregated, but not soluble, Aβ to C1q [12, 90, 101, 102]. This latter finding suggests that in AD, aggregated Aβ induces chronic complement activation. Thus, C1 binding to fibrillar Aβ deposits may precede microglial activation. Both Aβ and pro-inflammatory stimuli are able to activate microglia, which results in increased Aβ and cytokine production [103]. Furthermore, cultured human microglial cells show an increase in cytokine production after co-stimulation of Aβ with C1q and serum amyloid P (SAP) [104]. This suggests that microglia may get triggered by both Aβ- and SP-associated factors such as C1q, which results in the secretion of pro-inflammatory cytokines and Aβ, both accelerating neurodegeneration. Although none of the complement factors directly regulate conformational changes of Aβ, complement activation as a whole plays a role in the Aβ aggregation in vivo. Therefore, complement factors might act as amateur chaperones, although their exact role in Aβ aggregation remains to be elucidated. Professional Chaperones Heat shock proteins (Hsp) are professional chaperones. They are highly conserved proteins constitutively expressed in most cells under normal conditions where they play a role in cellular metabolism and help normal folding processes [105]. In addition, during cell stress, they bind unfolded proteins to keep them in their native state [105]. Heat shock proteins can be divided into two different families based on size and function: classic Hsps such as Hsp100, Hsp90, Hsp70, Hsp60, and the small heat shock proteins (sHsps). Hsps with a molecular weight of 60 kD or more possess an ATP-binding site and are actively involved in the process of refolding of misfolded proteins [106]. Small Hsps, with a molecular weight of 40 kD or less, lack this ATP-binding site and assist the Hsps in their refolding function [107]. The role of Hsps in misfolded protein recognition and refolding is illustrated in Fig. 1. Fig. 1The role of heat shock proteins (Hsp) and small heat shock proteins (sHsps) in recognition and refolding of unfolded and misfolded proteins. Unfolded or misfolded proteins are recognized by Hsps and sHsps. Together with these unfolded or misfolded proteins, Hsps and sHsps form a complex. In addition, Hsps recover unfolded or misfolded proteins back to their native form using ATP. If unfolded or misfolded protein are not recognized by the Hsp/sHsps, these unfolded or misfolded proteins are capable of forming aggregates Small Heat Shock Proteins Small Hsps function as molecular chaperones that can prevent proteins from adopting an incorrect conformation [108]. The sHsp family is characterized by the presence of an α-crystallin domain, a stretch of 80–100 amino acids in the C terminal half of the proteins [109]. So far, the sHsp family comprises ten sHsps, including αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2/B3 [110]. Although sHsps are predominantly expressed in muscle cells, several family members are also found in human brain. In AD, αB-crystallin and Hsp27 are upregulated and expressed by astrocytes surrounding SPs and NFTs [111–114], whereas Hsp20, HspB2, and HspB8 colocalize with Aβ in SPs and CAA [115, 116]. Although αB-crystallin or Hsp27 do not colocalize with Aβ in SPs, direct interaction between Aβ and these sHsps in addition to Hsp20 and HspB8 has been demonstrated [111, 117–119]. In addition, high-affinity binding of αB-crystallin and Aβ has been observed in eye lenses from AD patients [120]. Furthermore, αB-crystallin is able to prevent mature Aβ fibril formation, retaining it in a non-fibrillar, but likely a protofibrillar state, which is highly toxic to neurons [121]. Recently, we demonstrated that αB-crystallin, Hsp20, and HspB8 inhibit Aβ-mediated toxicity towards cerebrovascular cells probably by preventing aggregation of Aβ at the cell surface [116, 117]. Others showed that Hsp27 directly binds to hyperphosphorylated tau, thereby protecting against cell death [122]. Hsps are involved in the formation and persistence of misfolded protein aggregates. They are upregulated in several neurodegenerative diseases, such as AD, Creutzfeldt–Jakob disease, and Parkinson’s disease probably as a reaction to the formation of misfolded proteins [113, 123–126]. However, despite of increased intracellular levels, they are unable to prevent accumulation of Aβ in AD possibly because of decreased chaperone activity. In aged rats, this was illustrated by a significant decrease of Hsp90 function [127], resulting in diminished hepatic chaperone capacity. Furthermore, the increasing amount of damaged or misfolded proteins as a result of defects in protein degradation might lead to a total decrease in chaperone activity in aged cells [128]. Thus, the state of misfolded protein recognition and repair systems, such as the (s)Hsp system, might be of great importance in the development of neurodegenerative diseases. Miscellaneous Proteins Apart from the above-described proteins, several other molecules are also associated with the pathological lesions of AD, and some of these can be regarded as amateur chaperones. Acute phase proteins, such as α1-antichymotrypsin (ACT), α2-macroglobulin (α2M), and SAP, are all associated with Aβ deposition [129–132]. ACT is a serine protease inhibitor of the serpin family, and in AD, ACT levels are upregulated, and binding of ACT with Aβ induces Aβ fibrillogenesis [133–135]. Furthermore, when ACT is overexpressed in transgenic mice, an increased plaque load in the brains of these mice and impaired spatial learning is observed [134, 135]. α2M also binds Aβ, although in contrast to ACT, this binding prevents Aβ fibril formation and fibril-associated neurotoxicity [136, 137]. α2M promotes the protease-mediated degradation of α2M/Aβ complexes and contributes to clearance of Aβ from the brain (discussed in paragraph 4) [138, 139]. The glycoprotein SAP belongs to the pentraxin family and is a common component of all known types of amyloid fibrils. SAP is upregulated in AD and protects amyloid fibrils from proteolysis in vitro [140, 141]. SAP not only colocalizes with SPs and interacts with aggregated Aβ; SAP oligomers also bind and activate C1 [142]. Both C1 and SAP may bind to fibrillar Aβ deposits in vivo and induce microglial activation, as cultured human microglial cells show an increase in cytokine production after co-stimulation of Aβ with C1q and SAP [104]. These observations further strengthen the above-noted suggestion that not only Aβ, but also several Aβ-binding proteins, are capable of activating the complement system, and thus, contribute to neuroinflammation in AD. In addition, both α2M and ACT, in contrast to SAP, can be regarded as amateur chaperones, as they regulate conformational changes of Aβ. Tissue-type plasminogen activator (tPA) regulates activation of plasminogen into plasmin and is expressed in various regions of the brain especially in the hippocampus [143]. Several reports suggested an important role for tPA in AD, as the tPA system is involved in Aβ turnover [144, 145]. Fibrillar forms of Aβ stimulate tPA activity in vitro, whereas in AD patients, a reduction of tPA activity is observed in the affected areas [144, 146]. Although tPA has no effect on conformational changes of Aβ, it might play a role in the clearance of Aβ from the brain (paragraph 4]. The actin-regulatory protein gelsolin is found both intracellularly and in plasma [147, 148]. Plasma gelsolin can be considered an amateur chaperone, as it binds Aβ and not only inhibits its Aβ fibrillization but is also capable of degrading preformed Aβ fibrils [149, 150]. Furthermore, gelsolin inhibits Aβ-mediated neurotoxicity [151]. One of the major gangliosides in the brain is GM1. Soluble Aβ binds GM1 and the formed complexes accelerate Aβ fibrillogenesis by acting as a seed for Aβ [152]. In the presence of GM1, Aβ is more neurotoxic than Aβ alone, and cholesterol-rich membranes demonstrate accelerated Aβ binding due to the formation of GM1 clusters [153, 154]. As GM1 is a major component of lipid rafts and recent studies suggest that Aβ accumulation in these lipid rafts is an early event in AD development, GM1 might play an important role in the early steps in AD pathogenesis [155, 156]. In summary, several proteins are associated with Aβ aggregates in the AD brain and contribute to the aggregation of Aβ and should, therefore, be considered as amateur chaperones. In addition, they might play a role in triggering inflammation. Aβ-Binding Proteins and Intracellular Interactions with Aβ Intracellular accumulation of Aβ already starts in the ER or in the Golgi apparatus of the cell [157–159]. Intracellular Aβ is associated with neuronal damage [160, 161], and intraneuronal accumulation of Aβ in transgenic mice was correlated with impairments in synaptic plasticity [162]. Intraneuronal accumulation of Aβ in those brain areas affected earliest in AD suggests a possible relation between intracellular Aβ and development of AD [160]. A few proteins that interact with intracellular Aβ and affect its intracellular fate have been identified. The endoplasmic reticulum amyloid beta-peptide-binding protein binds intracellular Aβ and mediates neurotoxicity in neuronal cells by forming an intracellular target for Aβ [163]. In addition, the mitochondrial enzyme amyloid-β alcohol dehydrogenase also binds Aβ inside neurons, resulting in the production of free radicals [164]. However, whether these intracellular Aβ-binding proteins affect aggregation of Aβ within the cells remains unknown. Therefore, both these proteins cannot, for the time being, be defined as amateur chaperones of Aβ. The first lines of defense against misfolded and aggregated proteins are the professional chaperones, which counteract these processes and are able to stimulate clearance of misfolded proteins by proteosomal degradation. Newly synthesized proteins are folded by several other proteins, such as immunoglobulin-binding protein (BiP)/glucose-regulated protein (GRP78), and calnexin. GRP78 is a member of the Hsp70 protein family and interacts with intracellular APP. GRP78 regulates APP and Aβ secretion by intervening between APP and β-/γ-secretases within the cell [165]. It is not surprising that in AD, where misfolded protein molecules accumulate, both Hsp90 and Hsp70 synthesis is increased. Several members of the Hsp family directly interact with intracellular Aβ, but only recently, Hsp70 was identified as a protector against intracellular Aβ accumulation [166, 167]. Besides, immunoreactivity of both Hsp90, 70, and Hsp60 is found in SPs [132], which suggests that these professional chaperones may not only interact with misfolded protein in the cell interior [168–171]. In addition, it has also been postulated that up-regulation of Hsp90 and Hsp70 may suppress the formation of NFTs by partitioning tau into a productive folding pathway and thereby preventing its aggregation [172]. Recently, it was demonstrated that the chaperone CHIP–Hsc70 complex conjugates ubiquitin to hyperphosphorylated tau, which enhances cell survival by elimination of soluble hyperphosphorylated tau [173]. These data suggest that the cell increases production of the Hsps to cope with the presence of misfolded proteins such as hyperphosphorylated tau and accumulating Aβ. At some point, this protective mechanism seems to fail, however. In line with this hypothesis, it was shown that the actin and tubulin specific chaperone Hsp60 is decreased in AD, resulting in a decrease of cytoskeletal proteins in AD-affected neurons [174]. Thus, both production and function of Hsps seems to be disturbed in AD, which might result in the accumulation of misfolded proteins. The role of other Hsps in regulating intracellular Aβ or tau folding remains to be investigated (Table 1). Table 1Summary of the expression of chaperones in AD brains and their interaction and effects on Aβ and tau SP/CAANFTDirect interactionEffects on Aβ or tau in generalApolipoproteins ApoE++Aβ/tau↑ Fibrillar Aβ /↓ hyperph. Tau ApoJ+?Aβ↓ Aβ aggregationHSPGs Perlecan±±AβHSPGs: Agrin+−Aβ↓ Proteolytic breakdown Aβ Glypican 1+−?↑ Non-fibrillar → fibrillar Aβ Syndecan 1–3+−?↑ Phosphorylation tau Collagen XVIII+−? GAGs++Aβ/tauComplement factors Hageman Factor++?Aβ activates complement in AD C1q++AβC3 ↓ Aβ deposition C3/C4++Aβ C5-9++?Heat shock proteins Hsp90+?Tau↓ Tau aggregation Hsp70+?Aβ/tau↓ Tau aggregationSmall Hsps αB-crystallin−−Aβ↓ Aβ fibril formation Hsp27−±Aβ/tau↓ Aβ fibril formation Hsp20+−Aβ↓ Aβ fibril formation HspB2/B3++–No effect HspB8+−Aβ↓ Aβ fibril formationAcute phase proteins α1-antichymotrypsin+−Aβ↑ Aβ fibrillization α2-macroglobulin+−Aβ serum amyloid P++Aβ↑ Aβ fibrillizationMiscellaneous compounds tPA−−Aβ↓ Aβ fibril formation Gelsolin−−Aβ↑ Aβ fibrillizationExpression of chaperones in a specific lesion is illustrated as follows: present (+), by conflicting reports (±), absence (−), and unknown (?); ↓ = inhibition or down-regulation, ↑ = induction or up-regulationSP Senile plaques, CAA cerebral amyloid angiopathy, NFT neurofibrillary tangles, HSPGs heparan sulphate proteoglycans, Aβ amyloid-beta, Hsp heat shock proteins, Apo apolipoproteins, SAP serum amyloid P, tPA tissue-type plasminogen activator, GAGs glycosaminoglycans, LDLR low-density lipoprotein receptor, LRP-1 LDL receptor protein-1, BBB blood–brain barrier Aβ-Binding Proteins and Aβ Clearance Aβ-binding proteins, amateur chaperones, play a role in the clearance of Aβ from brain by functioning as a transporter molecule. Two major pathways govern Aβ clearance. By the first pathway, Aβ is removed from brain to blood via active transport across the BBB. This active transport is performed by specialized transporters, so-called “Aβ-receptors”, expressed by endothelial cells. Second, Aβ is removed from brain via phagocytosis by both microglial cells and astrocytes. In both pathways, interaction of Aβ with cell surface Aβ-receptors is crucial; therefore, the expression levels of Aβ-binding proteins might contribute to Aβ clearance by regulating its binding with Aβ receptors. The low-density lipoprotein receptor-related protein-1 (LRP-1) binds Aβ in a complex with ApoE at the abluminal side of the endothelium and internalizes these ApoE/Aβ complexes followed by degradation in lysosomes or transport into the plasma [4, 175]. However, LRP-1 also mediates transport of free Aβ across the BBB [10]. In contrast to LRP-1, the receptor for advanced glycation end products (RAGE) transports Aβ from the circulation into the central nervous system [176]. Similar to RAGE, the Aβ receptor megalin is also involved in the transport of Aβ from blood to brain, although megalin probably plays only a minor role in Aβ transport. Furthermore, megalin binds Aβ/ApoE complexes rather than free Aβ [177, 178]. Clearance of Aβ/ApoE complexes from brain might be ApoE isoform-dependent. ApoE4 forms less stable complexes with Aβ than ApoE3 or ApoE2; therefore, ApoE4 reduces Aβ transport efficiency across the BBB. Additionally, as described above (paragraph 2), ApoE4 enhances Aβ aggregation more efficiently than ApoE3, which also inhibits clearance. On the other hand, the LDL receptor shows a marked preference for the ApoE3 and ApoE4 isoforms and binds the ApoE2 isoform poorly [179]. Given the similarity between the LDL receptor family, other LDL receptors, such as the LRP-1 receptor, might display similar specificities towards the ApoE isoforms, but this has not been reported yet. Moreover, lipidation of ApoE also affects clearance of ApoE and ApoE/Aβ complexes from brain, as LRP preferentially binds lipid-rich forms of ApoE [179]. These data indicate that Aβ-binding proteins, especially ApoE, and possibly, ApoJ, play an important role in transport of Aβ across the BBB and that both the ApoE isoform and the ApoE lipidation state affect Aβ clearance. In addition to ApoE and ApoJ, the Aβ-binding protein α2M also forms complexes with Aβ. As α2M is a ligand for LRP-1, these α2M/Aβ complexes might undergo LRP-1-mediated endocytosis and degradation or translocation into the plasma [7, 139]. Stimulation of the transport of Aβ across the BBB demonstrated to be an effective therapeutic approach in AD, as several studies demonstrated elevated levels of Aβ in the plasma of mice after passive immunization with anti-Aβ antibodies or Fab fragments [180–182], and decline in cognitive performance was arrested in patients that received vaccination [183]. However, the occurrence of severe meningoencephalitis in human patients after active immunization with Aβ hampered widespread application of this type of therapy. Administration of Aβ-binding proteins that demonstrate similar positive effects, but possibly, without the severe immune reactions associated with antibody therapy, might provide an alternative strategy. An interesting example of such an Aβ-binding protein is gelsolin. This protein has high affinity for Aβ and reduces Aβ levels in a transgenic mouse model of AD [184]. Furthermore, administration of gelsolin or GM1 in PS/APP mice resulted in decreased Aβ aggregation in the brains [184]. Both gelsolin and GM1 act as a “peripheral sink” for Aβ. Although both compounds did not enter the brain, they lowered soluble Aβ concentrations in the blood, shifted the balance between blood and cerebral Aβ concentrations, and accordingly, stimulated Aβ transport over the BBB. Therefore, other Aβ-binding proteins administered in the circulation might also act as “peripheral sinks” [181, 184]. Both activated microglial cells and activated astrocytes are associated with Aβ deposition and may internalize Aβ fragments via phagocytosis [185–187]. Activation of the complement system is, among others, achieved by Hsps such as Hsp60 and Hsp70, which are able to induce phagocytosis by microglia, and thus, clearance of Aβ [132, 188, 189]. In addition, the absence of ApoE reduces internalization and degradation of Aβ by astrocytes in the brain, demonstrating that ApoE is directly involved in the clearance of Aβ from brain via phagocytosis by microglial cells and astrocytes [40]. tPA might also contribute to clearance of Aβ, as it accelerates Aβ clearance from transgenic mouse brains [146]. Thus, as Aβ-chaperones contribute to activation of the complement system or activation of microglial cells and astrocytes, these proteins might contribute to the clearance of Aβ from the brain via phagocytosis. Concluding Remarks Professional chaperones, such as the heat shock protein family, and amateur chaperones, such as apolipoproteins and HSPGs and several other proteins, have a role in the intracellular handling of misfolded proteins, induce conformational changes of proteins, or act as transporter of proteins (Fig. 2). This suggests that these chaperones form interesting therapeutic targets in the prevention and treatment of neurodegenerative diseases. Fig. 2The putative role of chaperones in amyloid-β (Aβ) fibril formation, proteolytic breakdown, and clearance from the brain. In Alzheimer’s disease, soluble Aβ, predominantly produced in neurons, is converted into β-sheet rich protofibrils and eventually forms mature Aβ fibrils. The conversion from soluble Aβ to protofibrils and fibrils, which accumulate in senile plaques and cerebral amyloid angiopathy, is enhanced by chaperones as apolipoprotein E (ApoE), Gelsolin, α1-antichymotrypsin (ACT) and several heparan sulphate proteoglycans (HSPGs), which function as catalysts. In contrast, the heat shock protein family, tissue-type plasminogen activator (tPA) and complement factors prevent the transition of soluble Aβ into protofibrils and mature fibrils. Furthermore, heat shock proteins and tPA stimulate the proteolytic breakdown of (proto)fibrils, whereas HSPGs prevent this breakdown. Finally, the clearance of Aβ from the brain across the blood–brain barrier is stimulated by ApoE, ApoJ, and α2-macroglobulin (α2M), whereas complement factors stimulate phagocytosis-mediated clearance of Aβ by activated microglia and astrocytes In the process of clearance of Aβ from the brain, Aβ-binding partners might play important roles by acting as Aβ transporter proteins in both the receptor-mediated clearance of Aβ across the BBB but also as a “peripheral sink” for Aβ. Both ApoE isotype and local concentrations in the brain might regulate Aβ transport across the BBB, but as this transport is receptor-mediated, other Aβ-binding proteins might also fulfill such a role. In addition, transport of aggregated Aβ across the BBB is less efficient than soluble Aβ. Thus, by preventing self-aggregation of Aβ, Aβ-binding proteins contribute to the clearance of Aβ from the brain. As a therapeutic strategy, Aβ-binding proteins serving as a “peripheral sink”, such as gelsolin, seem promising [184]. Overexpression of professional chaperones, such as the Hsps, to prevent aggregation of misfolded proteins will have to be evaluated carefully, as they also interact with other chaperones and are dependent on this interaction to fulfill some of their functions. This strategy may therefore result in instability of the cell-stress mechanism, which may cause the system to collapse. A solution may be found in the overexpression of several chaperones, which may be required to achieve an impact on the progression of the disease. Another pitfall in the use of professional chaperones as therapeutic agents is their ability to bind misfolded proteins and keep them in an intermediate conformation. This type of conformation might even be more toxic than the aggregated state. As an example, co-incubations of αB-cystallin with Aβ are more toxic to neurons than Aβ alone [121]. Furthermore, Hsps are most likely to be involved in early development of neurodegenerative diseases, given their natural function. Yet, the role of this protein family in maturation of the neurodegenerative lesions remains to be elucidated. In conclusion, studying the role of chaperones, both professional and amateur, in the pathophysiology of AD will provide us with a better understanding of the mechanisms underlying the formation and accumulation of toxic aggregates in AD, which, eventually, will lead to the design of more effective therapeutic strategies.

Eur_J_Clin_Pharmacol-3-1-2071966

Evaluation of patients’ experiences with antidepressants reported by means of a medicine reporting system

Objective To assess experiences related to antidepressant use reported to an internet-based medicine reporting system and to compare the nature of the side effects reported by patients with those reported by health care professionals (HCPs). Introduction Non-adherence to prescribed medication regimens is a persistent problem in the treatment of depression with antidepressants. Premature discontinuation, dosing lapses and partial non-adherence often occur [1–3]. The illness itself as well as physician-, patient- and treatment-related factors have been suggested to contribute to antidepressant non-adherence, but the ability of health care professionals (HCP) to predict who discontinues medication remains poor [4–6]. Earlier studies provide scant information on how patients in clinical practice perceive their treatment with antidepressants, thus insight and an understanding of the patients’ perspectives on non-adherence and discontinuation are limited. Side effects and ineffectiveness are the major reasons cited for the discontinuation of antidepressant therapy [7, 8]. However, the information available on side effects is mostly based on controlled clinical trials, which standardly do not evaluate how bothersome side effects are to the individual patient. Moreover, both the side effects and the effectiveness of the antidepressant therapy observed in clinical trials may not reflect the experiences of the patients in real world settings. Many interventions have been developed to improve adherence to antidepressants, but to date these have failed to demonstrate a clear benefit [9, 10]. Treatment non-adherence remains one of the least understood health-related behaviours. Information received directly from the patient may improve our understanding of the relative importance of antidepressant treatment issues and, consequently, help to explain patients’ behaviour towards antidepressant use. Reporting systems have shown to be a useful tool in collecting experiences and identifying issues related to the daily use of medicines [11]. In 2004, an internet-based medicine reporting system was established in The Netherlands where users of medicines are able to report all types of experiences with medicines – side effects as well as experiences with effectiveness and practical and reimbursement issues. We extracted data from this medicine reporting system in order to (1) assess the type of experiences related to antidepressant use reported by patients, (2) assess the relevance of these experiences and (3) compare the nature of the suspected side effects reported by patients with those reported by HCPs. Methods Setting Reports were obtained from an internet-based medicine reporting system launched on 11 May 2004 in The Netherlands. This reporting system was initiated by DGV, The Dutch Institute for the Proper Use of Medicine, the Science Shop for Medicines and patient and consumer organisations. Individuals are able to report their experiences with medicines anonymously by completing a report form downloaded by accessing a website (http://www.meldpuntmedicijnen.nl). Users of the medicine themselves as well as their relatives, acquaintances or (professional) carers, can report an experience. All reports submitted between 11 May 2004 and 13 May 2005 related to the use of antidepressants were reviewed for the clarity and completeness of the description of the experience and checked for multiple reports from the same IP-address. One duplicate report had to be excluded. All other reports were included in the study. The data were stored in an Access database. Report form The report form contained items which requested information on age, gender, type of medicine used, nature of the experience, a description of the experience and the relevance of the experience. Individuals filling in the report had to indicate whether the experience was related to either effectiveness, a side effect, a practical issue (e.g. difficulties with swallowing tablets) or a reimbursement issue. More than one experience related to the same drug could be submitted. Experiences related to effectiveness had to be further specified by the individual by selecting one of the following categories: ineffectiveness, a positive effect or a different type of effect (e.g. drug interactions). Experiences related to side effects had to be further specified by selecting one of the following categories: negative side effects, positive side effects (e.g. less side effects as compared with previous medication) or the absence of side effects. Relevance of the experience was assessed by asking the individual filling in the report to indicate both the impact of the experience on a 5-point scale (ranging from very negative to very positive) and whether the experience caused a change in antidepressant use. To specify the change in antidepressant use, those reporting had to select one of the following categories: discontinuation, switching to other treatment, adjustment of dosage or administration, any other action taken or no action taken. In the case of discontinuation of the antidepressant, the report also asked whether the HCP has been informed. Side effects All reported side effects (including experiences on ineffectiveness) were coded by one of the authors (SvdW) and checked by a second author (EvG), using preferred terms of the World Health Organisation Adverse Reaction Terminology (WHO-ART) [12]. Preferred terms were combined into groups of similar side effects. Spontaneous reports of adverse drug reactions on antidepressants received from HCPs from May 2004 to May 2005 were included for comparison. These reports were received by The Netherlands Pharmacovigilance Centre Lareb either on paper forms or electronically via http://www.lareb.nl. Coding and assessment were carried out by qualified assessors from Lareb [13]. Data analysis The relevance of the side effects was assessed by comparing the impact of the (grouped) side effects as a proportion of the side effects that were perceived as “very negative”. Relevance was also assessed by comparing the proportion of the side effects that caused discontinuation of initial antidepressant use, including the categories “discontinuation” and “switching to other treatment”. The impact and discontinuation proportions of the different groups of side effects were compared using the chi-square test. For the comparison between patients and HCPs, the groups of side effects were compared and expressed as an Odds Ratio (OR) and 95% confidence interval (CI). The 15 most frequently reported (groups of) side effects by patients as well as the 15 most frequently reported (groups of) side effects by HCPs were included in the analysis. Results Characteristics In total 2232 individuals submitted a report to the medicine reporting system during the study period, of whom 258 (12%) submitted a report on antidepressants. Of these 258 individuals, 248 (96%) reported for themselves and ten reported for a relative or acquaintance. The mean (±SD) age of the of the antidepressant users was 42.8 (±13.5) years and 72% were female. The majority of those reporting on antidepressants (63%) were reporting on a serotonin-reuptake inhibitor (SSRI); 12% were reporting on a tricyclic antidepressant (TCA) and 25% on another type of antidepressant. The antidepressants most frequently reported on were paroxetine (35% of total), venlafaxine (15%), citalopram (10%) and mirtazapine (8%). The use of a benzodiazepine as a concomitant drug was reported by 19% of those reporting on antidepressant use. Nature of experiences The 258 individuals reporting on antidepressant use described 327 experiences. Table 1 shows the nature of the experiences described. Ninety-two individuals (36%) described an experience with effectiveness, of whom 40 (16%) described an experience with ineffectiveness. Four individuals claimed generic substitution as the reason for ineffectiveness, and 45 (17%) reported a positive experience with the effectiveness of an antidepressant. “Other type of effects” in terms of effectiveness that were submitted by seven (3%) individuals included experiences on suspected drug interactions. Table 1Nature of the reported experiences related to antidepressant use reported to a medicine reporting system (http://www.meldpuntmedicijnen.nl)aIssueProportion of total number of individuals (n = 258) reporting on antidepressant useNature of issueProportion individuals reporting on a specific issueEffectiveness36% (n = 92)Ineffectiveness16% (n = 40)Positive effect17% (n = 45)Other3% (n = 7)Side effects84% (n = 217)Negative side effect78% (n = 202)Positive side effect1% (n = 3)Absence of side effect5% (n = 12)Practical issues5% (n = 14)Reimbursement and availability issues2% (n = 4)aA total of 258 reporters submitted 327 experiences. The reported experiences are expressed as a percentage of the total number of individuals submitting reports In total, 217 of the 258 individuals (84%) reporting on antidepressant use submitted a report on side effects. Of these, 202 (78%) described a total of 630 side effects that were experienced as negative. The number of reported side effects ranged from 1 to 11 per reporter experiencing a negative side effect. Twelve individuals (5%) reported that they had not experienced any side effect at all, and three (1%) submitted a positive experience with side effects, reporting that their current antidepressant therapy caused fewer side effects than their previous medication. Fourteen individuals (5%) reported a practical issue (such as problems with swallowing tablets or bad taste), and four (2%) reported a reimbursement or availability issue (receiving treatment for a relative short duration). Box 1 shows a number of the reported experiences. Relevance of side effects Table 2 shows the relevance, impact and discontinuation rates of the most frequently reported side effects. Of all side effects, 48% resulted in discontinuation of the initial antidepressant therapy. The proportion of side effects that caused discontinuation of the initial antidepressant did not differ significantly between the different side effects. Of those individuals who simultaneously reported an experience on side effects and one on ineffectiveness, 59% (17/29) reported that their experience resulted in discontinuation of the initial antidepressant therapy. Of all individuals who reported discontinuation of therapy, 29% did not inform their HCP. Table 2Relevance of the most frequently reported side effects related to antidepressant useSide effectNumber of reported experiences (n)Impact (% perceived as “very negative”)Discontinuation of antidepressant use (%)Sleep disorder684750 Somnolence, drowsiness, fatigue395159 Insomnia, sleeplessness294138Weight increase484656Sexual problems434433Discontinuation symptoms404350Ineffectiveness403553Apathy284654Excessive sweating234339Nausea, gagging233930Dizziness, fainting236535Headache226850Dry mouth185061Suicidal attempt, thought or tendencya97878aSuicidal attempts, thoughts or tendency is also included, although the number of reported experiences was less than 15. Of all side effects, 52% were perceived as “very negative”. The impact of the side effects differed almost significantly between the groups of side effects (p = 0.052), with headache, dizziness and fainting perceived as most negative. Side effects compared between patients and HCPs Table 3 presents those side effects most frequently reported by patients and HCPs. Patients and HCPs differed in the nature of reported side effects. Compared with HCPs, patients reported significantly more events such as apathy, excessive sweating, ineffectiveness, discontinuation symptoms, somnolence, insomnia, sexual problems and weight increase. HCPs reported significantly more rash, pruritis, laboratory abnormalities, muscle and joint complaints, congenital disorders, eye disorders, extrapyramidal disorders and menstrual disorders than patients. Table 3Reported side effects on antidepressants by patients compared with reported side effects on antidepressants by health care professionals (HCPs)Side effectNumber of reported side effects by patientsPercentage of total number of side effects (n = 670)Number of reported side effects by HCPsPercentage of total number of side effects (n = 471)Odds ratio (95% CI)aTop 15 most frequently reported side effects by patients  Weight increase487.261.35.98 (2.54–14.09)  Sexual problems436.461.35.31 (2.24–12.59)  Discontinuation symptoms406.030.614.14 (4.35–45.93)  Ineffectiveness406.030.614.14 (4.35–45.93)  Somnolence, drowsiness, fatigue395.881.73.58 (1.66–7.73)  Insomnia, sleeplessness294.340.85.28 (1.84–15.13)  Apathy284.200–  Excessive sweating233.400–  Nausea, gaggingb233.481.72.05 (0.91–4.64)  Dizziness, faintingb233.481.72.05 (0.91–4.64)  Headacheb223.3112.31.42 (0.68–2.96)  Dry mouth182.730.64.31 (1.26–14.71)  Abdominal pain142.130.63.33 (0.95–11.65)  Anxiety131.920.44.64 (1.04–20.66)  Depressed mood111.630.62.60 (0.72–9.39)Top 15 most frequently reported side effects by HCPs  Rash, urticaria and pruritis30.4234.90.09 (0.03–0.29)  Laboratory abnormalities00224.7–  Muscle and joint complaints30.4204.20.10 (0.03–0.34)  Congenital disorders00153.2–  Eye and vision disorders60.9153.20.27 (0.11–0.71)  Paraesthesia81.2122.50.46 (0.19–1.14)  Headacheb223.3112.31.42 (0.68–2.96)  Extrapyramidal disorders, Parkinsonism10.1102.10.07 (0.01–0.54)  Menstrual disorders, vaginal bleedings30.4102.10.21 (0.06–0.76)   Heart rhythm problems71.0102.10.49 (0.18–1.29)  Convulsions, epilepsy0091.9–  Drug substitution problems71.081.70.61 (0.22–1.70)  Serotonin syndrome0081.7–  Nausea, gaggingb233.481.72.06 (0.91–4.64)  Dizziness, faintingb233.481.72.06 (0.91–4.64)95% CI, 95% confidence intervalaOR > 1: Patients were more likely to report the side effect than the HCPs; OR < 1: HCPs were more likely to report the side effect than the patientsbSide effects that appear in both the patients’ and HCPs’ list Discussion Both side effects and a lack of effectiveness appear to be important treatment issues for patients who reported on antidepressants in our study. Most of the individuals reporting on antidepressant use described one or more side effects, of which the most frequent were weight increase, sexual problems, somnolence, insomnia and apathy. Ineffectiveness was also reported by a considerable number of individuals. Ineffectiveness is not often reported in literature as an adverse effect of treatment [14]. However, our study reveals that it is a relevant issue for patients during antidepressant therapy. The fact that side effects tend to occur before the therapeutic effect of the antidepressant is perceived may play an important part in explaining early discontinuation of the therapy. Ineffectiveness should therefore receive attention from HCPs in order to prevent early discontinuation of antidepressants. One of the unique features of this medicine reporting system is the possibility to gather information on the relevance of the experiences – that is the impact of the experience and the change in initial treatment. Overall, half of the experiences were perceived as very negative. Experience of one or more bothersome side effects means an individual is threefold more likely to stop taking antidepressants [7]. Our results show that one half of the side effects resulted in discontinuation of the initial antidepressant therapy. Moreover, of all those individuals who reported discontinuation of therapy, 29% did not even inform their HCP. This supports the concept that this medicine reporting system provides data of which HCPs are often not aware, but which are of crucial importance to any understanding of patients’ behaviour related to the use of antidepressants. Patients and HCPs differed in the nature of the side effects reported. Patients were found to report more frequently those events which may be less tangible and visible to HCPs, such as sleeping problems and apathy. In addition, HCPs may consider symptoms reported by patients with psychiatric disorders as a symptom of the disease rather than as one related to the medication. Other notable differences between patients and HCPs in terms of the frequency of reported side effects were those of weight increase, sexual problems, discontinuation symptoms and excessive sweating. The differences in the nature of the reported side effects show that patients and HCPs differ in which type of side effects can be considered to be bothersome and/or relevant to report. The burden of side effects is clearly underestimated by HCPs [15]. Although HCPs are knowledgeable on the side effects related to SSRI use, they underestimate the frequency of these side effects and how bothersome they are to patients [16]. In addition, HCPs may be reticent in reporting a side effect to a reporting system. More than patients, HCPs evaluate the side effect reported by the patient according to perceived relevance and causality related to the medication. Side effects which are considered by the HCP as well-known or not related to the medication consequently get lost to the health care system [17]. Patient reporting is not yet widely accepted, and the number of systems collecting experiences from patients is still limited. In the UK, the Prescription-Event Monitoring (PEM) system seeks to identify adverse events recorded following the use of newly marketed drugs selected for monitoring on the first 20,000–50,000 patients given the new drug [18]. Since the middle of 2003, the Danish Medicines Agency and the Netherlands Pharmacovigilance Centre Lareb also accept reports on adverse drug reactions from patients [19]. In Sweden, the KILEN Consumer Institute for Medicines and Health started a consumer database in 1997 that collects experiences related mainly to dependence and side effects of benzodiazepine and antidepressant use [19]. However, these systems focus only on adverse events, while other aspects of medicine use, including experiences on ineffectiveness, practical and reimbursement issues, have also been shown to be relevant to patients. A limitation of this internet-based reporting system is that the reporters are anonymous and, therefore, further contact and feedback are not possible. Consequently, a thorough assessment of causality between the side effect and the antidepressant was not always possible. The Netherlands Pharmacovigilance Centre Lareb, on the other hand, whose primary aim is the early detection of new adverse drug reactions, has the facilities to request further medical information by contacting the patient and/or his/her HCP [20]. However, we believe this does not detract from our conclusion. The individuals reporting through the internet-based reporting system proved to be capable of providing clear descriptions of their experiences and of balancing the benefits and burden of treatment. The patient experiences provide important information on how patients in clinical practice perceive treatment with antidepressants. Such patients experience sleep disorders, weight increase, sexual problems, apathy and ineffectiveness as events which have a negative impact and which frequently lead to discontinuation of the antidepressant therapy. Because adherence decisions are mostly a rational balance of perceived benefits versus burden [21], an assessment of patient experiences may improve the understanding of patients’ behaviour towards antidepressant use. This information can be used in the development of more targeted adherence-enhancing strategies that may lead to optimisation of antidepressant treatment from the perspective of both HCPs and patients.

Int_J_Hematol-4-1-2330060

Differential expression of HOX genes upon activation of leukocyte sub-populations

The HOX genes are key determinants of cellular identity both in early development and in the renewal and differentiation of adult blood cells. Although a number of studies have examined the expression of individual HOX genes in defined blood cell lineages, we have undertaken a comprehensive analysis of HOX gene expression in resting and activated lymphocytic and monocytic subpopulations. This has revealed distinct patterns of expression between different cell types and resting and activated states. (Main category A: Erythrocytes, Leukocytes and Hematopoiesis, subcategory: 8: Lymphocytes). Introduction The HOX genes are a family of homeodomain-containing transcription factors that define specific positional identities, both in vertebrate and invertebrate embryos [4]. They are also involved in the regulation of haemopoiesis (reviewed by [13]), and blocking HOX gene function by the use of antisense oligos or gene knock out perturbs a number of different haemopoietic events. Thus for example myeloid, erythroid and lymphoid haemopoiesis are all defective in HoxA9 knock out mice [8], and the antisense ablation of HoxB5, HoxB6, HoxB7 [15], or HoxA5 [5] blocks erythroid differentiation. Forced expression of HOXA10 in CD34+ progenitor cells purified from cord blood results in a significant reduction in the number of B cells and natural killer cells but a marked increase in monocytes [14]. There are also examples of specific regulatory functions for HOX genes, including the modulation of globin transcription by HOXB6 [12]. In addition, HOX genes can also act to regulate the proliferation of haemopoietic stem cells (HSCs). Hence HOXB4 is a strong, positive regulator of HSC self renewal (reviewed by [10]), and an estrogen-dependant version of HOXB8 can be used to drive the production of neutrophils and macrophages from their respective progenitor cells [16]. Despite these studies, relatively little is still known about the expression of HOX genes in the mature blood lineages (reviewed by [6]). In order to address this we purified different cell populations from human peripheral blood on the basis of their expression of the surface markers CD4, CD8, CD14 and CD19 that are present on T-lymphocytes (CD4 and CD8), monocytes, and B-lymphocytes respectively. HOX expression was compared in ‘resting’ cells and cells that had been activated by challenging with specific antigenic stimuli—antibodies against CD2, CD3 and CD28 for CD4+ and CD8+ cells, Lipopolysaccharide for CD14+ cells and anti-IgG for CD19+ cells. Each population of cells underwent proliferation in response to this treatment, as measured by [3H]thymidine incorporation (Fig. 1). RNA was extracted from each cell type and the expression of each of the 39 HOX genes was assed by quantitative PCR (QPCR), relative to the amount of Beta-actin transcript present (Fig. 2). Fig. 1Proliferation of activated lymphocyte populations. Proliferation was measured by [3H]thymidine incorporation 4 h after activation. Values shown are the fold increase in incorporation compared to resting cells. Error bars: SEM from triplicate experimentsFig. 2HOX gene expression in resting (a) and activated (b) after 6 h, (c) after 24 h) lymphocytic and monocytic populations. Expression was assessed by quantitative PCR and is shown as a relative value to the number of beta-actin transcripts (‘Relative expression’). HOX genes are abbreviated to show only their family and paralogue position, hence HOXA2 is shown as ‘A2’. HOX genes for which no expression was detected are not shown in the figure. Each value shown is the mean from three experiments. Significant changes in expression levels (p < 0.05) 6 and 24 h after activation are marked (*). Error bars: SEM The highest level of expression is generally shown by the HOXA and HOXC genes, with HOXD genes generally exhibiting a tenfold lower expression level and HOXB genes a 100 fold lower expression level, a feature that is also true for the other cell populations examined. For all of the HOX genes expressed in CD4+ cells there is a significant reduction in HOX expression upon activation. This is particularly striking for the most posterior genes of the HOXA, HOXC and HOXD clusters. A similar pattern is also observed in CD8+ cells, although there is a general reduction in HOX expression with every HOX gene except HOXC10 being present at lower levels in these cells. There are a number of notable differences between the HOX expression profile of CD14+ cells and the CD4+/CD8+ populations. HOXC4, HOXC6 and HOXC8 are not expressed. The most striking difference though is the change of HOX expression upon activation—in contrast to CD4+/CD8+ cells, CD14+ cells show a large increase in HOXA2, HOXB13, HOXC10, HOXC13, HOXD1 and HOXD9. Other HOX genes such as HOXB4 and HOXA10 show no significant change in expression upon activation, which again contrasts with the response of CD4+/CD8+ cells. CD19+ cells show a broadly similar pattern of HOX expression to CD14+ cells prior to activation, but have a distinct response to activation. Like CD4+/CD8+ cells, CD19+ cells exhibit a large reduction in expression of most HOX genes with the exception of HOXD10, HOXD11 and HOXD12 that show a very strong increase in expression upon activation. To our knowledge this is the first comprehensive analysis of HOX gene expression in these leukocyte populations. Taking the data as a whole, each population shows a unique pattern of HOX expression that may define cellular identity. Generally the expression levels are in agreement with those previously reported for individual HOX genes in other studies [6, 7, 9]. However the changes of HOX gene expression upon cellular activation have not previously been reported. The magnitude of these changes is striking, with the activated T cell subsets exhibiting an almost complete loss of expression of many HOX genes especially the more posterior members of the groups (HOXA10, HOXC13, and HOXD12). Conversely, activated monocytic and B-lymphocyte cells show a large increase in expression of the same genes. Given the key regulatory roles of HOX genes it is likely that these changes in expression facilitate the cellular changes associated with activation. One of these is increased proliferation, a response that is known to be mediated by HOX genes in a number of hematopoietic lineages (reviewed by [1]). Indeed the antisense mediated ablation of HOXB2 or HOXB4 in T-lymphocytes was previously shown to block the proliferation of activated cells (Care et al. 1995). Furthermore HOXA10 deficiency causes a severe immunological disturbance in uterine tissues characterised by rapid T cell proliferation and a failure of progesterone-mediated immunosuppression [17]. The HOX genes are also likely to regulate lineage-specific transcription upon activation, and in this context the contrasting expression levels of these genes presumably reflect the dramatically different response of each cell type. Materials and methods CD4+, CD8+, CD14+ and CD19+ cells were isolated using the Dynabead system (Invitrogen) and cultured as previously described [2]. Quantitative PCR was performed as previously described [11]. [3H]thymidine incorporation was evaluated by a standard procedure. Briefly, 2 μCi of [3H]thymidine (20 Ci/mmol) was added for 4 h to each well. Cells were then recovered, washed, and processed for the determination of TCA precipitable radioactivity. Each evaluation was performed in triplicate. T cell activation was achieved using anti-CD2, anti-CD3 and anti-CD28 antibodies as supplied in the T cell activation/expansion kit (Miltenyi Biotec).

Childs_Nerv_Syst-3-1-2092440

Deliberate termination of life of newborns with spina bifida, a critical reappraisal

Objects Deliberate termination of life of newborns (involuntary euthanasia) with meningomyelocele (MMC) is practiced openly only in the Netherlands. ‘Unbearable and hopeless suffering’ is the single most cited criterion for this termination, together with the notion that ‘there are no other proper medical means to alleviate this suffering’. In this paper, both (and other) statements are questioned, also by putting them in a broader perspective. “An infant with spina bifida cannot ‘suffer unbearably’. Infants might be able to experience unbearable pain, but spina bifida does not cause it.” (Chervenak 2006) Introduction and historical overview of the treatment of newborns with MMC Before the 1960s, most newborns with meningomyelocele (MMC) were not treated given the consideration that it was in the best interests of the child and family, in most cases resulting in the death of the child. They died from sepsis, meningitis, hydrocephalus, or renal failure. Contraindications to operation included “hydrocephalus, irreparable deformities, paralysis of the sphincters, complete paraplegia or any ulcerative process in the region of the spina bifida” [22]. The influence of the surgical approaches since the 1960s allowed gradually effective early treatment of the complicating hydrocephalus, resulting in many patients surviving into childhood. Early treatment of the spinal lesion and hydrocephalus resulted in decreased mortality and morbidity [66]. ‘Wait and see’ changed into active treatment. Lorber (1971) published the first large series of patients treated between 1959 and 1969 [44]. He found that only 7% of the survivors had less than “crippling disability”, “most had a quality of life inconsistent with self-respect, learning capacity, happiness, and even marriage”. To “spare children and families prolonged suffering” he proposed the well-known ‘Lorber selection criteria’. These four major adverse criteria are: severe paraplegia, gross enlargement of the head, severe kyphosis or scoliosis and associated gross congenital anomalies (such as heart disease) or major birth injuries. No active treatment was advised for children who had one or any combination of these criteria. The result of this proposed selection was to treat only those with lesions on lumbar and sacral level without any other major complication. Others would, according to Lorber, “die quickly”. Since the Lorber era there have been tremendous successes in the treatment of patients with MMC [48, 50]. Over the last 30 years, continual progress has not only been made in the neurosurgical (closure of the back defect, shunting, or endoscopic techniques for the complicating hydrocephalus, treatment of tethered spinal cord and Chiari II malformation), orthopedic (treatment of scoliosis, correction of club feet), urological (preserving normal renal function, securing social continence), and psychosocial treatment of patients with MMC, but also in support for independence by modern rehabilitation programs. Nevertheless, early decision making (for restriction) and selective treatment of newborns with MMC is still propagated [27, 69], resulting in approvals but also in critiques [7, 23, 41, 42, 49, 79]. What is considered ethical at one time, or in one country, can be considered unethical or unacceptable at/in another. The ethical debate on treating children with MMC is unique in the sense that these children will/can have a life of substantial and sometimes severe physical disability but often with normal mentation, this in contrast to severely psycho-motor handicapped children. Children and adults with MMC can be aware of their deficits. Lorber (1971) stated that untreated children will die quickly [44]. However, he delivered the children anticonvulsants to prevent seizures, opioids and chloral hydrate to prevent pain [25]. One pediatrician has said that these untreated children received the same care and attention as others, being fed and loved, but they also received 60 mg/kg body weight of chloral hydrate, four times a day. This was being administered for pain, but the children who were operated on did not receive this regimen. Another physician just said: we don’t feed them [79]. Consequently, the duration of life seems to depend on the attitude of the responsible doctor. In the United States, untreated children were not given morphine or anticonvulsant drugs and were fed every 4 h, resulting in many ‘survivors’ with severe handicaps. On the contrary, in the United Kingdom the children died quickly. However, the question remains if these children were really in pain or suffered from seizures; perhaps not. Not all physicians did follow the ‘Lorber criteria’, but treated all children unselectively, regardless of the level of the lesion. This approach resulted in multidisciplinary MMC clinics and MMC teams. The nonselective treatment of newborns with MMC started in the early 1970s and gives us a picture of long-term outcome of newborns with MMC [4, 9, 32, 48, 52] and also of the quality of life and health status of adults with spina bifida [58, 63]. Some physicians became bitter after treating children with MMC for years [2]. In the early 1980s, the ‘Baby Jane Doe (‘Stony Brook baby’) case resulted in the United States in an ethical debate on the prospect physicians make about the future life of handicapped children born with MMC. This girl was born with MMC, complicated with hydrocephalus (although diagnosed by the attending physician as microcephalic). The team of physicians told the parents that the child would spend her life “lying in bed, bottle fed....She would experience no joy, sadness or any such emotion except response to pain and would develop no cognitive skills. She might survive for 20 years” [51]. Based on the given information, the parents agreed not to treat their child. However, the physicians turned out to be wrong. Although the child developed meningitis, sepsis, and hydrocephalus (which were only treated to some extent), she survived all these complications, was taken home, became ambulating and communicating on an acceptable level of intelligence, all this despite the partial treatment [24, 25]. After the introduction of alpha feto-protein (AFP) screening in the late 1980s (followed by amniocentesis) and ultrasound diagnosis, many fetus with MMC can now be detected early in pregnancy. Nowadays, after being counseled, most pregnant couples decide to abort a fetus with MMC (in many Western countries resulting in an abortion rate of 95%, resulting in fewer children with MMC born [13, 14, 53]), seen by many as ‘prevention’ of MMC. But what advice should neonatologists, neurologists, or neurosurgeons give the parents when a full-term child with MMC is born? Is the advice not to treat, or to terminate the life of the newborn deliberately (involuntary euthanasia) an ethically defendable one? Deliberate and active termination of life in newborns with MMC is only discussed and practiced in an open way in the Netherlands [35, 38_40, 71_76, 78]. The judgment “unbearable and hopeless suffering” of the newborn and “the prospect of unbearable and hopeless suffering” in the future, together with the notion that there is “no other proper medical means to alleviate the unbearable suffering” form the basis for this decision [73]. Because these judgments are questioned, and to quantify the level of discomfort in these neonates, the “Rotterdam Prospective Study on Discomfort in Newborns with Spina Bifida” was started in 2005 in the Erasmus Medical Center (EMC)–Sophia Children’s Hospital (Rotterdam, the Netherlands). Although in due time the results of this study will be published, some important aspects will already be addressed in this paper by elaborating on the case of a newborn with a very severe form of MMC and hydrocephalus. Deliberate termination of life in newborns with MMC in The Netherlands The Dutch neurosurgeon De Lange analyzed the selection criteria for treatment of newborns with MMC in the Netherlands already in the 1970s [41, 42]. It became clear from an enquiry among Dutch neurosurgeons that a number of them advised against treatment on the basis of physical handicaps alone. Others regarded the expected mental abilities as an overruling factor. When basic communication skills could be expected, most children were treated. At the time, deliberate termination of life was never practiced. De Lange (1970) also concluded “If we were to decide that in our opinion patients with spina bifida who are below a certain level of physical or mental ability should not be kept alive, it should be illogical not to apply the same rules to other categories of handicap.” [41] In the 1980s, the Dutch ethicist Hertogh (1988) published his paper “Ethical considerations on the dilemmas caused by the selective treatment of newborns with MMC” in the leading Dutch medical Journal [30]. He elaborated on the philosophical and ethical aspects of the Lorber selection criteria: “The objective medical fact of a physical shortcoming is something completely different than the subjective reality of ‘being-handicapped’ itself. Studies have shown that living as ‘being-handicapped’ does not correlate directly to the severity of physical shortcomings. The question as to what can be considered as an acceptable handicap transgresses, in other words, the scope of what can be ascertained in a medical-scientific way and this, therefore, refers inevitably to the subjective experience. It has to be questioned whether the quantification of qualitative aspects of medical treatment perhaps offers nothing more than pretence-rationality, caused by the used decisive terminology. In conclusion: up to date the criterion ‘quality of life’ appears to be more deceptive than clarifying in the medical decision about the possible continued existence of a newborn with spina bifida.” [30] He also referred to the ethics of the philosopher Levinas: “Ethos cannot be founded in scientific facts; it is founded in the human relationship itself. Mutual dependency, which is characteristic for ‘human being’, makes us responsible just by itself and therefore defines ‘the disposition of ethos’ as a rational fact. This relationship always precedes facts themselves.”[30] In other words: human beings, just because of the fact that they are human beings, have an intrinsic and mutual responsibility for the well-being of others, this responsibility itself preceding physical qualities. This thoughtful paper, however, was not given much attention in the Dutch medical press. In 1993, baby Rianne was born in a small town in the Netherlands with a MMC at level of the first and second lumbar vertebrae complicated by hydrocephalus. The lower limbs were deformed and paralyzed. Computed tomography (CT) scan of the head confirmed the diagnosis of severe hydrocephalus. On the first day of her life, the decision was made by the pediatrician and the gynecologist in consultation with a neurologist, neurosurgeon, and pastor not to operate the back lesion and the hydrocephalus. On the fourth day of her life, at the request of the parents, the gynecologist Prins terminated the life of the baby by a lethal injection. In an interview, Prins stated [37]: “Closing the defect and fighting infections with maximal treatment could enable the child to survive for years. No one knows how long”. On the question of what an operation could have accomplished, Prins answered: “The defect would have been closed. The possibility of secondary infection, which was an immediate threat to her life, would have been diminished. Her brain pressure would rise, however, and it would have been necessary to implant a shunt”. On the question of how he would calculate the benefit of treatment, he answered: “It (the operation) would have prolonged her life without treating the symptoms and consequences of the defect because the operation would not have alleviated them. The operation would only have minimized the possibility of infection, but it would have created the necessity for repeated operations involving the shunt and the urological system because there were obstructions in the passage between the kidney and the bladder. Her brain damage was so extensive and the possibility of movement so limited. She would never have been able to walk or sit unsupported; she would have been incontinent in her bladder and bowel. There was sensory damage as well; she could not feel sensation in her lower body. She would have started life with repeated operations, need for persistent medical attention, no possibility for self-support, and very low communicative ability” [37]. As a definition of a severe handicap he stated: “This can relate to the estimated possibility of becoming happy” [37], and he further referred to the Dutch Association of Paediatrics who developed criteria, including the lack of capacity to communicate, lack of capacity for self-maintenance, persistent need for medical assistance, and the existence of pain. All these criteria grouped together gave a picture of the baby’s quality of life. Because most of the criteria were satisfied, the baby was considered to be severely handicapped, resulting in a very poor expected quality of life; the child was not operated on because of the severity of the expected handicaps. On the necessity of life-termination, Prins (1997) explained: “The baby was in a great deal of pain and the normal analgesics did not work well. So it would have been necessary to use narcotic analgesics—morphine-like drugs. And that would have caused depression of consciousness. It is clear to me that the use of such drugs would have depressed normal functioning—behavior, expressions, and sensibility. Using morphine-like drugs would have caused a situation of floating between heaven and earth. It would have been dishonest and should be considered as unethical and unjust. There is no moral difference between killing and withholding useless medical treatment. There is an emotional difference. And there should not be a legal difference” [37]. Although elements of the offense of murder were proven, the physician was acquitted based on the unavoidable conflict of duties between his duty to prolong life and his duty to alleviate unbearable suffering [26]. In 1996, three Dutch child neurologists and one neurosurgeon questioned in the leading Dutch medical journal the active termination of life of newborns with MMC [59]. They defended the opinion that (severe) handicapped life could also be meaningful. Deliberate termination of life was not considered appropriate; sedation and the delivery of opioids were mandatory in the case of suffering. Not treating a child with MMC should not result in a more severe handicapped life; the decision to withhold treatment was only made in the case of severe pulmonary or cardiac complications with the prospect of early death. The possible suffering of the parents should not be an indication for deliberate termination of the life of a newborn with MMC. They referred to the study of Steinbok et al. (1992) [68] for their argument that the life of children and adults with (severe) MMC can be meaningful [59]. In 2003, the Dutch physician/medical ethicist Van de Vathorst stated that whenever it is decided not to treat a defective newborn, deliberate termination of the life of this newborn should be regarded as a moral demand, based on the principle of ‘mercy’ [72]. She illustrated her arguments with a case of a newborn with a thoracic MMC. Interestingly enough, the life of this child was not terminated. It died by itself, a few days old, because of pulmonary insufficiency. Although she received a national award for this paper, it resulted in several disapproving letters to the editor. In January 2005, two pediatricians, one physician, and one pediatric neurologist reported on the deliberate termination of life of 22 newborns in the Netherlands between 1997 and 2004 [73]. Explaining the fact that all 22 cases were newborns with MMC and hydrocephalus, Verhagen et.al. referred to the ‘baby Rianne case’, stating that “her life was dominated by unbearable suffering” and “since this case it is established that newborns with a severe form of spina bifida do suffer severely” [73]. According to the authors, the deliberate termination of life was necessary because of the presence of “unbearable and hopeless suffering, acutely and long-term, with no other proper medical means of alleviating the suffering” [73]. All parents consented to the proposal of termination of life; in four cases, they explicitly requested for it. For the public prosecutor, the termination of life was acceptable if four requirements were properly fulfilled: (1) the presence of hopeless and unbearable suffering, (2) consent of the parents to termination of life, (3) consultation of another physician or of a multidisciplinary MMC team, and (4) a proper and careful execution of the termination. These four criteria eventually became the cornerstone of the so-called ‘Groningen Protocol’, a protocol meant to insure that the decision for life termination, the executing itself, together with reporting to the legal authorities, are all conducted in a proper way. This protocol was approved and adopted by the Dutch Association of Paediatrics in 2005. The motivation for the judgement of ‘unbearable and hopeless suffering’ in the 22 cases of spina bifida was [73]: ‘suffering’ (acute and chronic pain, physical pain) (100% of the 22 cases);the ‘lack on ability to live or do things independently’ as the result of ‘severely disturbed sensomotoric development’ (100% of the 22 cases);the ‘lack of possibility to verbal and non-verbal communication’ (82% of the 22 cases);‘the prospect to dependency to the medical circuit as the result of frequent hospital admissions and operations’ (77% of the 22 cases)the ‘life expectation’ (‘the burden of severe suffering increases by a longer life span’) (in 59% of the 22 cases). None of the cases led to prosecution; all cases were found to be in accordance with good medical practice. This article of Verhagen et al. (2005a) gave rise to only a few reactions in the Netherlands. Kompanje et al. (2005) [38] questioned the validity of ‘unbearable suffering’ as a useful criterion. They also stated that with the use of modern palliative care, ‘suffering’ always can be dealt with in an adequate way, as did Laane (2005) [39, 40]. De Jong (2006) [35] made a plea for ‘letting them die’ instead of ‘making them die’ in the case of an untreated newborn with MMC. On request of the editor of the New England Journal of Medicine, Verhagen and Sauer (2005) published another article concerning the Groningen protocol and the 22 cases [75] and a similar article appeared in Pediatrics [76]. The reactions in the international medical press were mostly negative. In his article “Control of suffering on the slippery slope of care” in the Lancet, Feudtner (2005) also addressed the poor quality and the insufficiency of the offered palliative care and questioned whether legalization of active termination of life might not lead to the abuse of it [21]. Saugstad (2005) stated in the Acta Paediatrica: “Neonatologists must be extremely careful not to start on the slippery slope ending in the Dutch practice of euthanasia of newborns. I would strongly warn against this attitude, which I find non-compatible with Western humanistic traditions” [61]. Jotkowitz and Glick (2006), in the Journal of Medical Ethics, were worried: “We feel an important line has been crossed if the international medical community consents to the active euthanasia of severely ill infants and are concerned about the extension of the policy to other at risk groups” [36]. Bondi et. al. (2006), in Pediatrics, made a plea for an open discussion: “Rather than performing an in-depth examination of the philosophical, ethical, and moral implications of this protocol, the authors focus on its technical details and acceptability within the European medical community. The Groningen Protocol centers not on patient self-determination, but instead on the perception of the patient’s current and future suffering. Although the authors note that an open discussion of these issues is ‘extremely important,” they fail to initiate it. We hope that thoughtful ethicists and legislators will initiate the open discussion that the authors advocate” [8]. In their article ‘Why the Groningen Protocol should be rejected’, Chervenak et al. (2006) concluded that: “The justification they offer is woefully inadequate. It relies on appeals to the concepts of “hopeless and unbearable suffering,” the “best interests of the patient,” and “medical-ethical values,” but it never explains these concepts, and it gives no ethical argument for their clinical application. In fact, not only is no argument for the Protocol given, none can hope to succeed. The Groningen Protocol should therefore be rejected as the basis for obstetric and neonatal practice in The Netherlands and throughout the world.” (11) Manninen (2006a), finally, made an important contribution with her article “A case for justified non-voluntary active euthanasia: exploring the ethics of the Groningen Protocol” in the Journal of Medical Ethics [45]. She made a thoughtful plea for the ethics of the Groningen Protocol: “What the Groningen Protocol proposes to do is both ethical and also the most human alternative for these suffering and dying infants.” Newborns eligible for this protocol “do face no future at all—that is, the infants must be terminally ill”, “will die soon no matter what actions doctors undertake, modern medicine can do nothing to save them”, “have no prospects, have crossed the threshold into a comparatively valueless state of existence”, “will spend the remainder of their lives in a chronic state of nothing but pain and suffering” [45]. Interestingly enough, on November 23 an additional letter of herself, “Regarding the Groningen Protocol” [46], was published in the same journal. Some key passages of this letter: “It has recently been brought to my attention that the physicians of the Groningen Hospital who originally proposed the Groningen Protocol have proposed to violate the first two conditions of the original Groningen Protocol: (1) The suffering must be so severe that the infant has no prospects for a future; (2) There is no possibility that the infant can be cured or alleviated of her affliction with medication or surgery. For example, Dr. Verhagen has maintained that the Protocol could apply to infants that are not necessarily terminally ill, but nevertheless may face a compromising future. In essence, the Groningen Protocol has begun to make quality of life judgments. If the Groningen physicians have commenced making quality of life judgments, they have strayed away from the original purpose of the Protocol, and this, I believe, may have concerning ethical implications. As I write in my paper: “...the Groningen Protocol does not make quality of life judgments, and it is because of this very important requirement that the Protocol strikes me as humane and morally permissible, for it seems utterly vicious to extend the life of a suffering infant with no prospects for a future. If the infant did have a possible future ahead of her, I would be more hesitant to condone a legal practice that begins to make quality of life judgments, for such judgments can be, and have been, subject to error.” [46] In her opinion, in these end-of-life decisions ‘quality of life judgments’ should not be made and, in a way, with this letter she also highlights the inconsistencies in the reports by Verhagen et al. Being of crucial importance, the subject of ‘quality of life judgments’ will be discussed more extensively in paragraphs 4 and 5. Finally, in 2006, in The Netherlands a national multidisciplinary expert review committee has been appointed. In the case of active termination of the life of a newborn, the responsible physician is supposed to inform this committee, which then has to determine whether the criteria for life termination have been met. Subsequently this committee has to advise the Public Persecutor’s Office (OM) whether the physician should be prosecuted or not. Although such a committee seems to guarantee more certainty, it can be doubted, however, if this ‘checking on afterhand’ is of much value for the newborn in question. It seems to be more sensible to change this procedure to a ‘checking on beforehand’. Legally, this might perhaps be difficult to establish, but it seems not justifiable that legal procedures themselves do dominate the question of appropriateness of life termination; errors in judgment being unacceptable (cf. the level of certainty required for a death penalty in crime in, e.g., the USA). Analysis of the mentioned criteria for non-treatment and deliberate termination of life Suffering The actual presence of acute unbearable and hopeless suffering of the newborn and the prospect of future/chronic unbearable and hopeless suffering as the result of handicaps, pain, and discomfort is the most given argument for deliberate termination of life in neonates with MMC. In the recent Dutch series, this suffering was stated to be present in 100% of the 22 cases [73, 75, 76]. Acute pain Not any convincing evidence can be found in the literature that newborns with MMC actually do suffer from untreatable pain in the days after birth caused by their back lesion or by other complications. A pediatric surgeon who devoted much of his professional life of more than 30 years to the treatment of those who have been born with spina bifida stated: “I personally have seen little evidence that the babies have pain in the newborn period, nor have I found them unable to sleep.” [79] Delight and Goodall (1988; 1990) [17, 18] studied the experiences of parents of 44 children born with MMC, all managed without any surgery. All 44 children died before their first birthday. Only a quarter of them did require some medication such as analgesics and anticonvulsants; half of them were given drugs in the dying period. Analgesics were given in a low dose simply to enhance their quality of life [17, 18]. Chronic pain The literature on chronic pain in MMC is scarce. The first systematic examinations of the nature and prevalence of pain in children with MMC was published in 2005 [12]. Headache is not uncommon in adults with MMC and is associated with hydrocephalus. In this series, 56% of 68 patients reported experiencing pain once a week or more frequent. Of the shunted children, 88% reported headaches compared to 79% of the children without a shunt [12]. Children in this study reported frequent and sometimes intense pain at multiple locations associated with the physiological sequelae, equipment usage, and medical management of the disability. The authors concluded that children with spina bifida frequently report clinically significant, under-recognized and untreated pain, and they propagated proper care and adequate medical management of these children. In another series, 55% of 42 patients underwent investigations for one or more episodes of chronic headache due to shunt malfunction, ventriculostomy failure, or symptomatic Arnold–Chiari malformation [20]. Although all these problems sometimes can be fairly serious, the notion of ‘unbearable and hopeless suffering’ is never mentioned. The ‘lack of ability to live or do things independently’ as the result of ‘severely disturbed senso-motoric development’ The second argument for unbearable and hopeless suffering is the prospect of lack of ability to live or do things independently as the result of severely disturbed senso-motoric development experienced by the patient in childhood, puberty, and adult life [73]. McLone reported in 1982 the results of the unselective treatment of 100 consecutive newborns with MMC [48]. After a follow-up period of 3.5 to 7 years there were 86 survivors. He concluded that the total percentage of children who could be independent and competitive is approaching 70–80%. Hunt and Oakeshott (2003) [32] and Oakeshott and Hunt (2003) [52] studied the outcome in 117 unselected and actively treated patients with MMC at age 35. The patients were treated between 1963 and 1971 without any attempt at selection. Forty-two patients had a severe lesion at a sensory level above Th11, 30 of whom died, most before their first birthday. They died mostly of cardio-respiratory or renal failure, hydrocephalus, or CNS infection. Two of the 12 survivors live independently, drive cars, and have an open employment; six have an IQ >80. In another study [62], 29% out of 60 adolescents with spina bifida had the lesion at a high lumbar or thoracic level. The authors studied the quality of life in these patients. They concluded that there were no significant relationships between the level of the lesion or spina bifida severity and the overall quality of life, which appeared to be 70 to 80 on a 100-point scale; both patients and their parents view the overall quality of life positively [62]. Besides these results, criteria such as ‘living independently’ or ‘doing things independently’ can be confusing when deciding about the future life of vital newborns. Most people who do live a meaningful life are dependent on others or interdependent on each other. In fact, interdependency and especially the willingness to care for other (perhaps dependent) people can be considered as a criterion of a truly civilized society, certainly so when this means to offer ‘something for nothing’ (this being real mercy). The ‘lack of possibility of verbal and non-verbal communication’ In 18 of the 22 studied Dutch cases, it was predicted “(future) communication of and with the child would not be possible, neither verbally nor nonverbally” [73]. In other words, these babies were predicted to be or to become in a state of deep coma or in a persistent vegetative state. Neither of these states of very severe disturbed consciousness, however, is applicable to a newborn with MMC and hydrocephalus. Furthermore, several publications concerning the level of mental functioning of adults with MMC have been published [3, 4, 9, 32, 33, 47, 67, 68, 70]. Intellectual development varies between normal IQ (this being the case in 60% [9, 68] to 70% [32] of the studied population) and severe mental disability, but the predicted complete absence of the possibility to communicate is never mentioned. On the contrary: “Virtually all infants with spina bifida are capable of meaningful human relationships, independent of the level of the lesion. Indeed, most are of normal intelligence” [23]. Aside from this incorrect medical description there are some other important aspects that need to be highlighted. When all communication is claimed to be impossible, one wonders by what methods at all (the amount of) suffering can be established. Furthermore, when a person is in such a state of deep coma or in a persistent vegetative state, there simply is no cognitive experience whatsoever, also not of (unbearable) suffering. This was confirmed in a recent conviction of the Dutch Medical Disciplinary Committee against a general practitioner who euthanized a comatose patient; the ground of the verdict being “because of the comatose state there was no longer any question of unbearable suffering nor any question of euthanasia” [15]. ‘The prospect of dependency to the medical circuit as the result of frequent hospital admissions and operations’ It seems hardly possible to quantify this criterion in such a way that it can be used to justify life termination of vital newborns. When compared to persons with other congenital malformations or congenital diseases or some acquired diseases, patients with MMC do not appear to need substantially more medical care than, e.g., patients with intracranial tumors or with chronic diseases such as severe pulmonary, cardiac, or neurological disorders. Furthermore, the claim of Verhagen (2004) that “this child would have to undergo at least 60 operations in the course of a year to temporarily alleviate its problems” [71] again cannot be substantiated: a retrospective survey of treated newborns with MMC over 2 years in the Sophia Children Hospital showed that three to four operations were needed in their first year of life. ‘Life expectation’ (‘the burden of severe suffering increases by a longer life span’) It is clear that the longer a patient lives, the more medical care will be needed in a quantitative way. Whether this means that this will affect the experienced quality of life in a negative way remains totally unclear and cannot be predicted. Furthermore, patients with MMC do require relatively much of their medical care in their first two decades, this steadily decreasing when they grow older and when disabilities and coping with these disabilities are gradually stabilized. Can ‘suffering’ be applied as a reasonable criterion in newborns? Suffering, especially the ‘unbearable and hopeless’ aspects of it, is a complex psychosocial phenomenon and by definition strictly individualized: only the individual itself can experience the existence, the intensity, and hopelessness of it. Others (parents, physicians, nurses) can only to some degree make an estimation of it. It is therefore tentative whether ‘suffering’ can be applied to newborns at all. What is possible, however, is to establish quite precisely the level of ‘(dis)comfort’ of a newborn by making use of standardized score charts such as the Comfort score and the Visual Analogue Scale (VAS) (see below). The Dutch physician Beijk(1998) stated it as follows: “suffering will be interpreted by every patient, by every doctor, and by every other involved person in a different way and therefore can lead to completely different conclusions. The bearableness is especially dependent on personal and environmental factors; it is all about the subjective experience of the individual itself who is suffering, about the durableness of the suffering, and about the wish to die” [6]. In 2005, in his Ph.D. thesis, also Hamburg (physician and jurist) concluded “for the criterion ‘suffering’ there are no objective or scientifically validated criteria available, with the consequence of decisions of life-termination being dependent of the subjective judgment of the doctor, leading to inconsistent decisions” [29]. Therefore, according to Hamburg, “in a time of evidence-based medicine ‘suffering’ is not acceptable anymore” [29], as also was suggested by Kompanje et al. (2005) [38]. Although Beijk and Hamburg are referring to the practice of euthanasia (which in the Netherlands is strictly referring to competent patients wishing to die), their statements are the more applicable to newborns, as in these cases predictions are being made about future suffering of others. According to Chervenak et al. (2006): “An infant with spina bifida cannot have and therefore cannot have lost the ability to realize intentions, desires, and hopes for the future; it can feel pain but cannot ‘suffer’ as a psychosocial phenomenon” [11]. Laane (2005a) came to the same conclusion: “having no biographical consciousness, ‘suffering’ cannot be applied to newborns [39]. To date, it is unclear why this improper use of ‘suffering’ still continues in this debate. Can physicians predict quality of life? Counseling the parents In a time in which most fetuses with MMC will be detected by AFP and amniocentesis or by ultrasound diagnosis (in most cases being followed by iatrogenic abortion), the birth of a newborn with MMC is increasingly becoming a rare phenomenon [14]. The decision to terminate a pregnancy in the case of a fetus with MMC is usually made shortly after the diagnosis. It can be questioned whether there is enough time for proper counseling about the prospect of living with spina bifida, as a patient and also as parents. Freeman (1998) stated: “Decisions are made by the parents without knowledge of the child’s future; without knowing whether the child will have minimal deficit and walk with only short leg braces or walk with no braces at all, and without awareness of whether this child will have a thoracic lesion with high paraplegia and spend his or her life in a wheelchair. Few parents at that stage in the pregnancy know much about spina bifida or about what life will be like for that child and for that family. In other words, these rarely are informed decisions” [25]. He points in the same direction concerning physician experience: “Today, few younger physicians have experience with the decision-making process for spina bifida either before or after birth. Thus, physician advisors will lack experience with the joys of caring for a child, even a handicapped child, as well as experience with the pains and problems. They will have little experience with the feelings of the handicapped children themselves. With the lack of experience, how should the physician counsel? On what basis can the inexperienced physician advise uninformed parents? What decision should the parents make?” [25] In their article “Tell the truth about spina bifida,” Bruner and Tulipan(2004) also showed their worries about the quality of counseling: “Unfortunately, many healthcare professionals are equally ignorant of the current prognosis of children with spina bifida who have ready access to comprehensive care in a modern multidisciplinary clinic. As a result, much of the information initially provided to couples with a newly diagnosed fetus is biased and misleading. As medical ethicists Bliton and Zaner at Vanderbilt University Medical Center observed, “To date we have met and held intense conversations with more than 150 pregnant women and their partners. Many times, couples admitted, the initial counseling they received from their obstetrician was slanted—both against disability and toward termination of pregnancy. What they remembered was how the initial obstetric consultation portrayed as grim a picture as possible about their future child’s prognosis.” [10] The statements of Verhagen et al. (2005) mentioned earlier, together with other incorrect predictions (“these newborns have no prospect of a future and no chance to survive at all; are barely able to breathe”, “these children face a life of agonizing pain that cannot be alleviated by any proper medical means”, “the child would suffer such unbearable pain that it has to be constantly anaesthetized” [71], that “they all have spina bifida of the most severe form at level of the neck and all have non-functioning kidneys” [78]), unfortunately seem to confirm these worries, also in counseling the parents of a newborn with MMC. To date, it is not clear why such incorrect information is presented to parents, colleagues, and society as a whole. When parents are told that communication with and of their child never will be possible, that their child will face a life of agonizing pain and will suffer unbearably, that this suffering cannot be alleviated by any means, that annually at least 60 operations will be needed to offer some (but only temporary) relief, then these parents are left without any hope and their consent/decision to non-treatment, or to active termination of life, almost can be predicted. However, in such situations the fundamental concept of ‘informed decisions’ is obviously violated. Does the level of the lesion matter? Does the level of the lesion determine the amount and severity of disturbances? In general, the higher up the spine the lesion is, the more likely are severe neurological disabilities to be found. Cervical lesions often do not contain neural tissue and are simple meningoceles. At lower levels (thoracic and especially lumbar), the converse is true: the infant nearly always being (partially) paralyzed below the level of the lesion, the cord showing the classical features of dysplasia [66]. However, the extent of handicaps that a person with MMC experiences is determined not by the site of the lesion, but almost entirely by the neurological deficit. Verhoef (2005) concluded that young adults with a mild form of spina bifida (and subsequently with few disabilities), did experience their disabilities more often as problematic than contemporaries with a serious form of spina bifida [77]. Likewise, in the study of Seller (1990) [65], eight patients ended up with minimal handicaps, having bony lesions in the thoracic (one patient), lumbar (two patients), and lumbosacral (five patients) spine. Three others with lumbosacral bony lesions were among the most gravely handicapped [65]. According to Hetherington (2005), low lesions do not necessarily indicate a mild course; physical well-being was not related to the level of the spinal lesion [31]. Although persons with sacral lesions showed less impairment compared to persons with higher spinal lesions, Padua et al. (2002) did not find a correlation between quality of life (both physical and mental) and the site of the lesion [54]. According to Pit-ten Cate et al. (2002), lesion level, type of spina bifida, and gender were not significantly related to the quality of life in children with spina bifida [55]. This is in agreement with the results of the study of Sawin et al. (2002) [62]: no significant relationship with lesion level, spina bifida severity or shunt status and quality of life was found in 60 adolescents. Schoenmakers (2003) [64] concluded that being independent in mobility contributes more to quality of life than other functional abilities such as whether or not being wheelchair-dependent. And, although children with the most severe forms of MMC have significantly more and severe disabilities than children with milder forms of MMC, scores about self-esteem did not differ significantly between the two groups [64]. In conclusion, the level of the lesion does not correlate with the actual experience of the handicaps themselves. Is the diagnosis ‘extensive brain damage’ easy to make? There is little doubt that surgical procedures are unjustified in hydrocephalic newborns with little or no potential for independent survival, the decision as to what constitutes as irreversible and severe brain damage may however be difficult to make. Ventricular size alone cannot be used as a conclusive predictive criterion. It has been repeatedly demonstrated that there is no consistent correlation between the thickness of the cortex and the eventual psychomotor development. Barf et. al. (2003) [3] concluded that cognitive status was especially negatively influenced by multiple shunt revisions and by other pathologies associated with hydrocephalus. Amacher and Wellington (1984) [1] treated 170 children with hydrocephalus in 636 operative procedures and concluded that “there is no difference in results based upon measurement of the initial thickness of the cerebral mantle.” They found normal intelligence in 63% of the 5-year survivors. In a recent study also, Beeker et al. (2006) described the difficulty in making a reliable prognosis of the intellectual development based on ventricular size [5]. ‘Quality of life’, to conclude It seems impossible to predict with certainty the future quality of life of newborns with MMC. One also has to be aware of the fact that the self-reported quality of life of children with handicaps does not differ from that of children without disabilities, and health care professionals are known to underestimate disabled persons’ quality of life as compared to self-reports [11]. Generally speaking, prognostic judgments about quality of life are conceptually plausible; their failing is simply that, given the available evidence, they do not appear to be reliable [11]. Finally, and perhaps the most important, quality of life judgements are considered as being ethically unacceptable in end-of-life decisions [45, 46]. Non-discrimination principle In his Ph.D. thesis, Dorscheidt (2006) raises the issue as to whether the deliberate termination of the life of disabled newborns is compatible with the ‘non-discrimination principle’, in particular the legal prohibition of discrimination on the ground of disability [19]. Deliberate termination of the life of newborns with spina bifida is always preceded by a non-treatment decision (as in the baby Doe case and the baby Rianne case) and according to Dorscheidt, the decision of non-treatment should be questioned with regard to the non-discrimination principle as well. He elaborates on two fundamental rights of the child, being the child’s right to life and its right to health(care), mentioned in international human rights instruments such as the Universal Declaration of Human Rights (UDHR), The United Nations Convention on the Rights of the Child, and the European Convention of Human Rights, and on the authoritative views of international human rights bodies such as the United Nations Children’s Rights Committee (UNCRC) and the Committee on Economic, Social and Cultural Rights (CESCR). Some examples: Article 25, UDHR: “Everyone has the right to a standard of living adequate for the health and well-being of himself, including medical care and the right to security in the event of disability. All children shall enjoy the same social protection”. CESCR, par. 26: According to standard Rules: ”States should ensure that persons with disabilities, particularly infants and children, are provided with the same level of medical care within the same system as other members of society.” UNCRC: “In its examination of States parties reports, the Committee should commit itself to highlighting the situation of disabled children and the need for concrete measures to ensure recognition of their rights, in particular the right to live, survival and development”. UN, General Counsel: “We will take all measures to ensure the full and equal enjoyment of all human rights and fundamental freedoms, including equal access to health, education and recreational services, by children with disabilities and children with special needs, to ensure the recognition of their dignity, to promote their self-reliance and to facilitate their active participation in the community”. Proposal text of the American delegacy for the UN Convention to Promote and Protect the Rights and Dignity of Persons with Disabilities: ”States Parties reaffirm the inherent right to life of all persons with disabilities, shall take all necessary measures to ensure its effective enjoyment by them, and shall ensure that disability, or perceived quality of life, shall not serve as a basis for infringement of the right to life.” [19] These international political statements and agreements are also reflected in the resolution of the International Federation for Spina Bifida and Hydrocephalus (IF) as formulated at the 12th International Conference (“The Right to be Different”) for Hydrocephalus and Spina Bifida in Toulouse [34]: People with spina bifida and hydrocephalus can live a full life with equal value to that of any other citizen and they should not be seen as a medical condition. Their views should be sought and heard by governments and health professionals, who should acknowledge the right of people with spina bifida and hydrocephalus to speak for themselves.People with disabilities have the right to aim for lives as rewarding as those of their peers who have what is regarded as a normal life. Supportive systems must be in place when they are needed.Experience over the past 30 years has improved considerably the medical outcome and the quality of life of people with spina bifida and hydrocephalus.Most adults with spina bifida underline that their quality of life is not automatically—and should not be given as—a reason for abortion [34]. As a recognition of the importance of non-discrimination on the basis of disability (that is to say, present as well as expected disability) or (expected) quality of life in deciding whether or not a newborn child with, e.g., spina bifida should be treated or whether or not it is justified to deliberately terminate such a child’s life, Dorscheidt (2006) recommends to involve the legal concept of ‘objective justification’ in the medical-decision making process. By using a particular questionnaire, the physician in charge may take advantage of this concept when considering (and deliberating on) how to act properly and professionally. Through this, the physician can ensure the disabled newborn child’s rightful claim to equal protection of its right to life and its right to health(care). Doing so would offer possibilities to replace the usual doctor-centered approach of decision making by a more patient-centered approach as well. To summarize from another perspective: “Prominent theories of justice emphasize equality of opportunity for human experience and development; if society has an obligation to provide citizens this very broad equality of opportunity, then it has an obligation to shoulder the burden of care associated with citizens who have spina bifida” [11]. Palliative care for untreated newborns with MMC When a newborn with spina bifida is not treated, discomfort can be expected in the nearby future caused by the lesion itself (e.g., by becoming infected), by a subsequent meningitis/ventriculitis, or by raised intracranial pressure caused by an untreated hydrocephalus. Therefore, the decision not to treat should always be accompanied by offering proper palliative care. It is becoming increasingly evident that, taken modern palliative techniques in newborns seriously, pain and discomfort can always be dealt with in an adequate way [43, 57]. In their article “Strong opioids in pediatric palliative medicine” Hain et al. (2005) also stated that “clinical evidence is accumulating that strong opioids can be used safely and effectively; they should be used as part of a rational approach to the management of pain” [28]. This use of strong pain killers is also highlighted in the recent Oxford Textbook of Palliative Care for Children [16]. In the highly unlikely event of insufficient effect of modern pain medication, terminal palliative sedation should be the next step in the palliative approach [16]. With palliative sedation, the possible experience itself of discomfort can always be dealt with adequately. In the “Consensus guidelines on sedation and analgesia in critically ill children” by Playfor et al. (2006) in Intensive Care Medicine, some 20 guidelines are mentioned by which pain and discomfort in critically ill children can be treated in an efficient way; life termination not being mentioned at all [56]. When it is decided that surgical treatment is not a feasible option in a newborn with spina bifida, it is regarded by some to be an act of ‘mercy’ to terminate the life of the newborn [72]. This notion, however, is a misconception: they are not terminally ill because of the MMC and/or hydrocephalus per se; they are ‘terminally ill’ because of the decision not to treat their congenital malformations. The needs of these untreated newborns, however, are still the same as the needs of all newborns: proper care, feeding, comfort, respect and love; till death (and apparently not death itself). The ‘need’ for life termination in these patients can only be regarded as an indication of insufficient palliative care. Life termination as an act of mercy can only be applied to hopeless situations that can occur, e.g., on battlegrounds or in mountaineering, when there are no proper means indeed to alleviate real unbearable suffering. This is obviously not the case in a modern equipped hospital (at least, it should not be) with experience in palliative care and pain management. This is all confirmed in the paper of Sauer in 2001, “Ethical dilemmas in neonatology: recommendations of the Ethics Working Group of the CESP (Confederation of European Specialists in Pediatrics)” in the European Journal of Pediatrics [60]. In this paper, some ethical principles, applicable to every newborn, are being formulated, like: “decisions to withhold or withdraw treatment should always be accompanied by optimal palliative therapy and dignified and comforting care” and “every form of intentional killing should be rejected in paediatrics” [60]. This is followed by: “However, giving medication to relieve suffering in hopeless situations which may, as a side effect, accelerate death, can be justified”. To date, it is not clear why these recommendations should not hold for cases of untreated spina bifida and hydrocephalus in the Netherlands and why the Dutch Association of Paediatrics does not adopt these recommendations. Illustrative case After an uneventful pregnancy, a full-term female child was delivered spontaneously, Apgar scores being 4 and 5. Physical examination revealed a huge MMC at high thoracic level (Fig. 1), a very large head with a large but soft fontanel, a marked deformation of the thorax with scoliosis, and clubfeet. After counseling the parents, they persisted in their wish to treat the child, if this probably could save her life. The MMC team decided to respect the parents will and on day 3 the back lesion was closed by the neurosurgeon in collaboration with the plastic surgeon, making use of extensive rotational flaps together with free skin grafting (Fig. 2). Shunt placement could be postponed till day 9; a medium pressure valve being used. Because postoperatively the fontanel did not soften adequately, 2 weeks later the medium pressure valve was changed in a low-pressure valve, now with satisfying clinical results. Fig. 1Photograph of the newborn, showing the extensive MMC, extending from +/− the 4th thoracic vertebra till the midlumbar regionFig. 2Photograph at the end of the surgical procedure. The MMC was closed by making use of extensive rotational flaps together with free skin grafting All period long, from birth till 1 week after the shunt revision, the well-being of the baby was estimated by making use of the Comfort score and the VAS and also medication used for treatment of pain or discomfort (paracetamol and morphine) was recorded (Fig. 3a,b). Fig. 3a Comfort scores during the first month of life (min = 5: no discomfort; max = 35: extreme discomfort). Green bars: only paracetamol (intermittently) being used. Green/black bars: low-dose morphine together with paracetamol being used. b The same as a, but now with the VAS (min = 0: no pain; max = 10: most severe pain) Note: The combination of a Comfort score of at least 17 and a VAS of at least 4 indicates that the well-being of the newborn is in jeopardy. In such a situation, the attending nurse is expected to determine the cause of the discomfort and to take adequate measures (changing a full nappy, feeding a hungry child, emptying a full bladder, giving adequate painkillers if appropriate, etc.) to secure the child’s well-being Studying these data and particularly the amount of medication needed, several conclusions can be made: When untreated (the first 3 days), the child was not discomfortable.Closing of the back lesion, in this case certainly a major procedure, did not contribute significantly (and only temporarily) to more discomfort. In other words, closing of the open spine can be qualified as a proportional treatment modality.The same holds true for placing a ventricular shunt.Treating an active hydrocephalus adequately contributes to the well-being of the patient.In 1 month’s time, this active treatment resulted in a stable situation of overall well-being in this patient.Only paracetamol and low-dose morphine were necessary in the treatment of actual discomfort. In summary, this case not only demonstrates that there is no such thing as ‘suffering’ in these newborns, but also the incorrectness of the statement of Verhagen et al. (2005a) [73] that “the suffering of these newborns cannot be alleviated by any means in a proper medical way” (Fig. 4). The overall impression of this case is in accordance with the preliminary results of the Rotterdam Prospective Study on Discomfort in Newborns with MMC: there is no “acutely unbearable suffering” in these newborns (Figs. 5a,b) Fig. 4The same child at 7 months old; fixating and following with the eyes, vocalizing, normal movements of the arms, moving both legs vigorously (perhaps nonvoluntary), still being partially fed by a nasogastric tubeFig. 5a and b Preliminary results of the Rotterdam Prospective Study on Discomfort in Newborns with MMC (n = 13) Although in an interview Verhagen himself reported that “all 22 cases had a lesion of the most severe form, at level of the neck” [78], in their published articles Verhagen et al. [73_76] do not quantify the level of the lesion. However, it seems very unlikely that his statement is correct or that the levels of lesion in these 22 cases are of the same level and/or magnitude as in the case described. Synthesis and conclusion There is no evidence that newborns with MMC and hydrocephalus do either ‘suffer’ unbearably or hopelessly and certainly not without the prospect to relieve this suffering by standard care. ‘Suffering’ itself is a nonconclusive, and in newborns, inapplicable denominator that should not be used anymore in this debate. Although they will in their future life be confronted with handicaps, sometimes very severe, their future prospects and their actual experienced quality of life cannot be predicted with such certainty at birth that their lives can be regarded as hopeless or meaningless (‘quality of life judgments’ as such being unacceptable in this decision making). Possible discomfort in these newborns can easily be treated in a straightforward way by active treatment (closure of the defect and shunting the hydrocephalus) and, when necessary, by the use of a professional pain/symptom protocol. The decision not to treat such a newborn, when based on expected handicaps, possibly violates the ‘non-discrimination’ principle (Dorscheidt, 2006). When not being treated, they are not terminally ill because of the MMC and/or hydrocephalus per se; they are ‘terminally ill’ because of this nontreatment decision. Not being terminally ill, it is not ‘humane’ or ‘merciful’ to terminate their life, this also being not in accordance with international legislation and international medical recommendations. When untreated and when it is the intention to alleviate actual discomfort (this question of intent being crucial to a moral and legal analysis of end-of-life decisions including active termination of life), this can always be achieved in an effective way by using one of the widely accepted palliative protocols. Such a child can and should be cared for in a respectful and dignified way, providing all its actual needs (which apparently is not death itself). This being the case, there is no indication whatsoever for the deliberate termination of the life of children born with MMC. While Verhagen et al. (2005, 2006) must be credited for bringing the discussion about deliberate termination of newborns in the open, to date, there does not seem to be much in their qualifications that can be quantified properly, especially not in cases of MMC, which is prone to quality of life judgments (and thereby neglecting the needs and rights of the newborn). To date, the Groningen protocol, therefore, cannot be regarded as very useful. Being a tool, it seems to have been changed in a means in itself. To gain more usefulness, at least it has to be extended in a way as proposed by Dorscheidt (2006), by adding charts such as the Comfort Score and the VAS and by adding palliative medication charts. Thus, it should be changed from a doctor-centered protocol to a patient-centered protocol. To throw more light on these end-of-life decisions, Verhagen et al. should consider to extend their retrospective study, focusing on the medical aspects of the 22 cases. Thus, it must be possible to get a basic understanding of the extent of the malformations themselves, of possible co-morbidities, whether or not quality of life judgments were made, how ‘unbearable suffering’ was ascertained, how palliative care was offered, and why this failed. Such a study would contribute significantly to the open discussion they propagate. Verhagen is undoubtedly correct indeed in his appeal: “It’s time to be honest about the unbearable suffering endured by newborns with no hope of a future”. [71] Finally, to date it remains unclear on what grounds the Dutch Association of Paediatrics has adopted the Groningen protocol and why international legislation, international human rights instruments, and international medical recommendations appear not to hold for the Netherlands, especially not in newborns with MMC.

Acta_Neuropathol-4-1-2270355

Applicability of current staging/categorization of α-synuclein pathology and their clinical relevance

In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) α-synuclein (αS) pathology is seen that displays a predictable topographic distribution. There are two staging/categorization systems, i.e. Braak’s and McKeith’s, currently in use for the assessment of αS pathology. The aim of these diagnostic strategies in pathology is, in addition to assess the stage/severity of pathology, to assess the probabilities of the related clinical symptomatology i.e. dementia and extrapyramidal symptoms (EPS). Herein, we assessed the applicability of these two staging/categorization systems and the frequency of dementia and EPS in a cohort of 226 αS-positive-subjects. These subject were selected from a large autopsy sample (n = 1,720), irrespective of the clinical presentation, based on the detection of αS-immunoreactivity (IR) in one of the most vulnerable nuclei; in the dorsal motor nucleus of vagus, substantia nigra and basal forebrain. The frequency of αS-IR lesions in this large cohort was 14% (248 out of 1,720). If applicable, each of the 226 subjects with all required material available was assigned a neuropathological stage/category of PD/DLB and finally the neuropathological data was analyzed in relation to dementia and EPS. 83% of subjects showed a distribution pattern of αS-IR that was compatible with the current staging/categorization systems. Around 55% of subjects with widespread αS pathology (Braak’s PD stages 5–6) lacked clinical signs of dementia or EPS. Similarly, in respect to those subjects that fulfilled the McKeith criteria for diffuse neocortical category and displaying only mild concomitant Alzheimer’s disease-related pathology, only 48% were demented and 54% displayed EPS. It is noteworthy that some subjects (17%) deviated from the suggested caudo-rostral propagation suggesting alternative routes of progression, perhaps due to concomitant diseases and genetic predisposition. In conclusion, our results do indeed confirm that current staging/categorization systems can readily be applied to most of the subjects with αS pathology. However, finding that around half of the subjects with abundant αS pathology remain neurologically intact is intriguing and raises the question whether we do assess the actual disease process. Introduction There are two staging/categorization systems commonly in use for the assessment of the progressive regional distribution of the pathology seen in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Both of these staging/categorization systems are based on the assessment of misfolded α-synuclein (αS) protein within selectively vulnerable neuronal populations which is considered to be either directly responsible or at least intimately linked to the neuronal dysfunction seen in PD and DLB. In this respect, αS-immunoreactive (IR)-inclusions in the brainstem have been claimed to be responsible for the extrapyramidal symptoms (EPS), whereas dementia has been attributed to the limbic and neocortical spread of these lesions. Thus, PD and DLB are thought to form a clinico-pathologic continuum wherein the clinical manifestation of EPS and/or dementia depends on the anatomical distribution and the load of αS pathology [4, 17, 19, 25, 28, 30]. In 2003, Braak and colleagues reported that the αS pathology begins in clearly defined induction sites and advances, not in a random, but in a predictable sequence with increasing severity throughout the brain [4, 11]. Based on the analysis of the regional distribution of αS-IR inclusions in a cohort including both neurologically unimpaired subjects and patients with PD, a staging system was devised whereby αS pathology was divided into six successive stages. In the central nervous system, the proposed sequence begins in the dorsal motor nucleus of vagus (dmV), and then proceeds with an upward progression via locus coeruleus (LC) (stage 2) to the substantia nigra (SN) (stage 3), and then to the basal foreberain (BFB) and transentorhinal region (stage 4) until it finally reaches the neocortex (stages 5–6). Already in 1996, the consortium on DLB international workshop proposed their consensus guidelines for the clinical and pathologic diagnosis of DLB [27] that later, in 2005 were somewhat revised [28]. These consensus criteria of DLB subdivide subjects into three different neuropathological categories; brainstem predominant, limbic/transitional and diffuse neocortical depending on the anatomical distribution of the αS-IR structures [28]. These criteria also include the semiquantitative grading of lesion density, although the pattern of regional involvement has been assumed to be more important than the actual count of inclusions. It is noteworthy that in the revised recommendations by McKeith et al from 2005 [28], it is emphasized that the concomitant pathologies should be taken into account when assessing the causative relationships between pathologies and symptoms. Thus, the most common pathology seen in aged demented individuals, i.e. Alzheimer’s disease (AD)-related pathology, should be evaluated while assessing the likelihood of causation, i.e. that the αS pathology is associated with a DLB clinical syndrome. Herein, we assess the applicability of these two current staging/categorization systems of synucleinopathies in a large autopsy material collected, not on the basis of clinical presentation, but by αS immunoreactivity in some of the most vulnerable nuclei; dmV, SN and BFB. Thus, the selection of material was entirely based on the presence of αS pathology irrespective of clinical phenotype. All subjects, if applicable, were assigned a stage following in detail Braak staging recommendations and a McKeith neuropathological category following in detail recommendations by the consortium on DLB international workshop [4, 28]. The frequency of dementia and EPS was assessed in each stage and the likelihood that dementia was due to AD-related pathology or αS pathology was also examined. Materials and methods Selection of subjects and the clinical assessment The flowchart delineates the logistics of this study (Fig. 1). A total of 1,720 elderly individuals (age at death >40 years) that had undergone an autopsy together with an examination of the brain during the years 1996–2005 in the Kuopio University Hospital were included in this study. From this large autopsy sample, we selected 248 (14%) subjects that displayed αS-IR structures in the SN and/or BFB nuclei: nucleus basalis of Meynert (nbM) and amygdaloid complex (AC). Screening of dmV was also carried out in 1996–2000 and revealed that in 24 subjects out of 904 (3%) αS-IR-structures were restricted to the lower brain stem nuclei (dmV and/or LC) [33]. Five subjects were excluded from the analysis because they had received a pathological diagnosis of MSA. Seventeen subjects were excluded due to lack of clinical information or inadequate amount of brain material required for the classification as recommended by Mc Keith and Braak [4, 28], and thus ultimately, this study examined 226 subjects. Fig. 1The flowchart delineating the logistics of this study. a The αS-immunoreactive inclusions were screened in substantia nigra, amygdaloid complex and dorsal motor nucleus of vagus. b Schematic presentation of both Braak staging and McKeith categorization [4, 28] and c schematic presentation of assessment of likelihood that αS-immunoreactive inclusion are associated with dementia with Lewy bodies [28] The diagnosis of dementia was based on the DSM-III criteria and the criteria of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) [29]. The diagnosis of PD followed the criteria established by the United Kingdom Parkinson’s Disease Society Brain Bank whereby PD was considered present if the patient had at least two of the four cardinal symptoms (resting tremor, rigidity, hypokinesia and postural instability) and exhibited a positive response to levodopa [9]. The initial screening for both dementia and EPS had taken place in a primary health care centre. With respects to dementia, all patients scoring 26 or less in minimental State Examination (MMSE) had been referred to a neurologist for further evaluation and all patients scoring >26 in MMSE but displaying signs of memory impairment had been referred to a CERAD test. Consequently, many subjects had been examined by a neurologist and all had at least visited a general physician (within a maximum of 1 year before death). It is noteworthy that many of the subjects included in this study had been under continuous clinical follow-up due to some chronic disease. Admittedly, some subjects may have developed subtle extrapyramidal signs or mild cognitive impairment between the last clinical examination and death, but it is most unlikely that the presence of moderate or full-blown parkinsonian syndrome/dementia would have been overlooked. Neuropathological assessment According to the dissection protocol used in the Kuopio University Hospital, the brains were weighed, evaluated for grossly detectable lesions and vessel abnormalities, perfused with and immersed in 10% buffered formalin for at least one week and cut in coronal slices of 1 cm thickness. Brain specimens were embedded in paraffin and cut into 7 μm-thick sections. Immohistochemical (IHC) methodology was used to visualize the expression of αS and hyperphosphorylated τ (HPτ). The sections were deparaffinized and rehydrated according to a routine procedure. For αS IHC, the sections were autoclaved (120°C) in citrate buffer for 10 min and pretreated with 80% formic acid at room temperature for 5 min. Monoclonal antibodies to human αS1–140 (Novocastra, Newcastle upon Tyne, UK) at a dilution of 1:1,000 and to human HPτ at a dilution of 1:500 (Innogenetics, Ghent, Belgium) were applied. For detection, Histostain SP kit (Zymed, San Francisco, CA) was used with Romulin AEC chromogen (Biocare Medical, Walnut Creek, CA). The expression of αS was assessed in 10 brain regions: (1) medulla with dmV; (2) pons with LC and raphe nucleus (RN); (3) midbrain with SN; (4) BFB including nbM, AC, and transentorhinal cortex; (5) posterior hippocampus at the level of geniculate nucleus including the CA2 region of the hippocampus and temporo-occipital gyrus; (6) insular cortex; (7) anterior cingulate gyrus; (8) superior temporal gyrus (Broadman area 22); (9) frontal cortex (Broadman area and (10) parietal cortex (Broadman areas 39, 40). The selection of regions was based on the currently used staging systems [4, 28] (See Fig. 1). HPτ IHC was carried out on sections from hippocampus, temporal and occipital cortices and the regional distribution of AD-related neurofibrillary pathology was subdivided into 6 stages (I-VI) as has been described previously [6]. The number of αS-IR inclusions was counted within the microscopic field at ×200 magnification (diameter of 1 mm) and assessed semiquantitatively in all brain areas examined. The total thickness of the cortical grey matter and AC were assessed according to the established pathological guidelines [28] and rated as follows: 1 = mild (sparse inclusions at ×100); 2 = moderate (>1 inclusion in low power field at ×200 magnification); 3 = severe (≥4 inclusions in low power field at ×200 magnification); 4 = very severe (numerous inclusions). In the nbM and all subcortical regions, αS-IR inclusions were counted unilaterally within entire nuclei and assessed following an arbitrary grading system: in nbM and SN, + =mild (<25 inclusions); ++ = moderate (25–50 inclusions); +++ = severe (>50 inclusions), in LC and dmV, + = mild (1–2 inclusions); ++ = moderate (2–10 inclusion), +++ = severe (>10 inclusions). Several inclusions within one neuron were counted as a single inclusion. The αS-IR structures were designated as being present (+) or not (0) in raphe nucleus and CA2 region of the hippocampus. If no αS-IR inclusions were identified in the sections of medulla, pons, midbrain or BFB, the result was verified in at least 3–4 consecutive sections i.e. the subsequent 5th, 10th, 15th and 20th sections were stained. This was done in order to increase the likelihood of capturing incipient neurons with αS-IR inclusions. Results Assessment of clinical data The 226 αS-positive subjects examined included 114 (50%) patients with a clinical diagnosis of a neurodegenerative disorder, 15 (7%) patients with other neurological disorders and 97 (43%) individuals in whom the clinical information indicated that they had no neurological impairment. The mean age at death was 77 ± 0.6 years, ranging from 44 to 98 years and the gender was rather evenly distributed (108 females/118 males). Frequency of αS-immunoreactivity in the most vulnerable anatomical regions The most frequently affected regions in the 226 αS-positive subjects were dmV (197/223) and SN (197/225) where the αS-IR was seen in 88% of the analyzed subjects. The LC was affected in 81% (181/223) of subjects, making this area the second most vulnerable nuclei. The large neurons in the nbM were αS-immunopositive in 78% of cases (171/219), whereas the involvement of AC, particularly the cortico-medial nuclear group, was seen in 73% (162/223) of subjects. Staging of Parkinson disease-related αS pathology according to Braak and the frequency of dementia and extrapyramidal symptoms Out of the 226 αS-positive subjects, 187 (83%) displayed a distribution pattern of αS-IR that was compatible with the current staging systems of PD/DLB-related synucleinopathies [4, 28]. Braak stage 1–2 was applicable for 22 subjects, 3–4 for 38 subjects and 5–6 for 127 subjects (Table 1). When all subjects with severe AD-related neurofibrillary pathology, i.e. Braak’s AD stage V-VI, were excluded, 168 cases of the 187 subjects remained. Notably, only 25–30% of the subjects with PD-related Braak stage 5 and 50% of subjects with PD-related Braak stage 6 were demented and/or had EPS. It is noteworthy that when only demented subjects were included in the analysis (53 out of 168), 91% (48/53) were assigned to PD-related Braak stages5–6. Similarly, when only subjects with EPS were included in the analysis (52 out of 168), 94% (49/52) were in the PD-related Braak stages 5–6.Table 1Applicability of Braak staging and the incidence of dementia and extrapyramidal signs (EPS) in each stage Categorization of the distribution of αS pathology following the recommendations by the consortium on DLB international workshop, i.e. McKeith’s categorization, and the frequency of dementia and extrapyramidal symptoms Sixty-six subjects fulfilled Mc Keith’s criteria for the brainstem predominant form, 30 for the limbic (transitional) and 91 for the diffuse neocortical form of DLB (Table 2). Table 2Applicability of neuropathological categorization as recommended by the consortium on DLB international workshop, i.e. McKeiths categorization and the incidence of dementia and extrapyramidal signs (EPS) in each category Fifty-seven percent of subjects with the diffuse neocortical form of DLB were demented and 47% displayed EPS (Table 2). When AD-related pathology was taken into account as suggested by McKeith and colleagues [28] (Table 3), the percentage of demented in the high likelihood category varied from 24 to 60%. Irrespective of the αS pathology, in Braak’s AD stage V-VI, all subjects were demented. Notably, when only demented subjects were included in the analysis and the subject with severe AD-related pathology (Braak’s AD stage V-VI) were excluded, 85% (45/53) were assigned to a high likelihood category of DLB. Table 3Assessment of likelihood that the pathological findings are associated with a DLB clinical syndromeCategory of Lewy body type pathologyAlzheimer type pathologyBraak stage 0-IIBraak stage III-IVBraak stage V-VIBrainstem predominant n = 66Low n = 58 5 demented (9%) 5 with EPS (9%)Low n = 7 2 demented (29%) 0 with EPS (0%)Low n = 1 1 demented (100%) 0 with EPS (0%)Limbic transitional n = 30High n = 25 6 demented (24%) 9 with EPS (36%)Intermediate n = 1 1 demented (100%) 0 with EPS (0%)Low n = 4 4 demented (100%) 1 with EPS (25%)Diffuse neocortical n = 91High n = 63 30 demented (48%) 34 with EPS (54%)High n = 15 9 demented (60%) 4 with EPS (29%)Intermediate n = 13 13 demented (100%) 5 with EPS (36%) Atypical cases i.e. cases that could not be classified as recommended Thirty-nine cases were not classifiable as recommended and thus were assigned as being atypical i.e. neither Braak staging nor McKeith categorization systems could be applied. The deviating topographical distribution of αS-IR inclusions of these subjects is shown in Table 4. In four subjects, the BFB nuclei (AC predominant) were severely affected (cases 1–4) together with some cortical inclusions, whereas the brainstem had been preserved. Five subjects displayed isolated minor involvement of SN without any αS-IR inclusions in the lower brainstem nuclei (cases 5–9). In six subjects (cases 10–15) minor involvement of both SN and BFB nuclei was detected (cases 10–15). In addition to these two areas, three cases exhibited severe αS pathology in AC (cases 16–18, AC predominant). In seven subjects, some inclusions were seen in the BFB, SN and LC, but not in the dmV (cases 19–25). In the final 14 subjects (cases 26–39), inclusions were seen in the dmV and SN, but not in the LC, together with variable affection of BFB and other cortical regions. In only minority of these cases EPS symptoms were seen (13%), whereas dementia was more common (54%). In 14 cases dementia was due to AD-related neurofibrillary pathology (Braak stage V-VI), in six cases numerous multifocal microscopic infarcts and concomitant severe white matter rarefaction was considered as the causative agent regarding dementia and in one cases neuropathological lesions consistent with frontotemporal dementia with ubiquitin only lesions were seen. Table 4Topographic distribution of αS immunoreactive lesions in the 39 atypical cases, i.e. not classifiable following current recommendationsIDAgeSexEPSDemAD stageTopographical distribution of αS-immunoreactive structuresdmVRNLCSNnbMACTrEnt CxCA2Temp-OccIns CxCGTCxFCxPCx179FNoYes400000410000000262FNoYes600000420100000374FNoYes20000+420201100479FYesYes60000+300011100586FNoNo4000+0000000000644MNoNo0000+0000000000768MNoNo1000+0000000000883MNoNo2000+0000000100983MNoNo10+0+00000000001075FNoNo0000++0000000001176MNoNo1000++0000002001270MNoNo0000++0000000001377FNoNo2000++2100000001473MYesYes2000+++0000001001571FNoYes6000++++0001111001679FNoYes5000+03300100001787FNoYes2000++4nana0000001874FNoYes6000+na4na+1000001962MYesYes000++00000000002079MNoYes000+++0000000002184FNoYes200++++0000000002285FNoNo000+++11nana000002382FNoYes300++++na200100002458MNoNo000++++++0000000002584MNoNo300+++++3201020002676MNoNo1+00+00000000002787FNoNo1+00++0000na00002878MNoNo1+00++2200000002984FNoYes5+00+01na+1000003075FYesYes5++00+01000000003160FNoNo0+++00+00000000003280MNoYes6+00++00000010003369MNoNo1+++0++++1100010003480MYesYes5++00+++32+1110003586FNoYes6++0+044+3001003664FNoYes6+00+na4na+na001003774FNoYes6+00++440na012003878MNoYes5+00++33+1211003989MNoNo2+00++220001110EPS extrapyramidal signs, Dem clinical signs of dementia, AD stage the stage of Alzheimer’s disease related neurofibrillary pathology stages as recommended by Braak from 0 to VI., dmV dorsal motor nucleus of vagus, RN raphe nucleus, LC locus coeruleus, SN substantia nigra, nbM nucleus basalis of Meynert, AC amygdaloid complex, TrEntCx transentorhinal cortex, CA2 CA2 region of the Cornu Ammonis of the hippocampus, Temp-Occ temporo-occipital gyrus, Ins Cx insular cortex, CG cingulate gyrus, TCx temporal cortex, FCx frontal cortex, PCx parietal cortex; na not available. The αS-immunoreactive inclusions were examined in the total thickness of the cortical grey matter and AC and rated as follows: 1 mild (sparse inclusions in ×100), 2 moderate (>1 inclusion in ×200 magnification), 3 severe (≥4 inclusions in ×200 magnification), 4 very severe (numerous inclusions), in the nbM and subcortical regions, αS- immunoreactive inclusions were counted unilaterally within entire nuclei and assessed following an arbitrary grading system: in nbM and SN, + mild (<25 inclusions), ++ moderate (25–50 inclusions), +++ severe (>50 inclusions), and in LC and dmV, + mild (1–2 inclusions), ++ moderate (2–10 inclusion), +++ severe (>10 inclusions). In Raphe nucleus and CA2 region of the hippocampus, αS-immunoreactive structures (inclusion and/or neurites) were marked to be present (+) or not (0) Discussion Most of our (83%) αS-positive cases could be assigned to one of the six PD stages as described by Braak and also into the brainstem, limbic or diffuse neocortical neuropathological category as recommended by McKeith and colleagues [4, 28]. Braak and colleagues depicted the topographical distribution of αS-IR structures by assessing 110 αS-positive subjects (69 incidental and 41 symptomatic PD patients) [4]. The initial intent of Braak and colleagues was not to correlate the designated neuropathological stages with clinical symptoms, however this was later contemplated [5]. Stages 1 and 2, i.e. stages where αS pathology is confined to the dmV and/or LC, are considered to be presymptomatic, whereas EPS appear and the cognitive decline increases with each stage. In stage 3, when SN is affected, EPS appear and subsequently in stage 4 when amygdaloid complex, transentorhinal region and temporo-occipital gyrus become involved, moderate cognitive impairment is observed (MMSE scores 21–24) and finally in stage 5 and 6 when the neocortex succumbs to the pathology, severe cognitive impairment is evident (MMSE scores 11–20 and 0–10, respectively) [5]. Our results also suggest that the risk of EPS increases with disease progression though not to the same extent as earlier reported. In our study, we found one subject with EPS already in stage 2, whereas none of our cases in stage 4 displayed EPS, and more importantly no EPS had been reported in 55% of subjects who exhibited widespread pathology (Braak stages 5–6), i.e. this being compared with the 14% previously reported by Braak and colleagues [4]. The initial decline in cognition was postulated to occur already during stages 3 and 4 i.e. around the same time when the initial manifestation of somatosensor dysfunction start to appear. When assessing 88 subjects, Braak and colleagues reported, that 36% of their subjects in stage 3, 67% in stage 4, 94% in stage 5 and 100% in stage 6 were demented [5]. This is clearly in odds with our results where the percentage of demented increased from none to 50% between stages 3–6. It is noteworthy that when only demented subjects were included, 91% were assigned to PD-related Braak stages 5–6 and when only subjects with EPS were included, 94% were in the PD-related Braak stages 5–6. Thus the key difference between our study when compared to most other clinico-pathological correlation studies that have reported good correlation between risk of disease and progression of pathology is the study design [5]. Consequently, when we only included subjects with clinical signs in our analysis the correlation between stage/severity of αS pathology and EPS/dementia was excellent in line with previous reports. PD and DLB are distinguished as separate clinical entities and in 1996 the consortium of the DLB international workshop subdivided the neuropathological features of DLB into three categories: brainstem predominant, limbic and diffuse neocortical type [27]. The foundation of these three categories is also based on the progressive propagation of αS pathology along a caudo-striatal axis. Similarly to the Braak staging [4], when applying this categorization it is presumed that 100% of subjects with widespread αS pathology, i.e. in the diffuse neocortical stage all will be demented and display EPS. When we followed the classification strategy proposed by McKeith and colleagues [28], our results also differed from those expected, i.e. only a subset of our subjects who were classified to be in the diffuse neocortical category displayed dementia and/or EPS (57 and 47%, respectively). In line with the above, when only demented subjects were included in the analysis the correlation between αS pathology and dementia was close to excellent (85%). The clinical relevance of cortical αS pathology in relation to dementia is a matter of intense debate. Some authors have emphasized their key causative role [1, 18, 21, 26], whereas others have reported that there may be abundant cortical pathology in non-demented PD patients [8] as well as in neurologically unimpaired subjects [10, 24, 32]. It is noteworthy that the current study differs significantly from most other studies since it is based on neuropathologiacal findings rather than on clinical presentation. Our results emphasize that abundant pathology may be detected in many subjects without notable signs of dementia (MMSE >26) (43%), if it is sought. This has one unexpected consequence, i.e. a detailed regional assessment of αS pathology cannot reliably predict the clinical status observed premortem [33]. There has been much discussion concerning the significance and influence of concomitant AD-related pathology, particularly as this is quite frequently seen in aged subjects. Therefore, the revised consensus criteria have recommended taking AD-related pathology into account while assessing subjects with suspected DLB [28]. It was presumed that this would increase the diagnostic specificity since it was believed that the pathological substrate behind DLB was indeed αS pathology. When we assessed our unique material, we found, that within the neuropathological high likelihood categories of DLB, i.e. those cases where limbic/diffuse neocortical αS pathology is combined with mild/moderate AD-related changes, 56% of subjects remained cognitively intact. However, when we examined only demented subjects without severe AD-related pathology (Braak’s AD stage V-VI), 85% were assigned to a high likelihood category of DLB. This shows that when αS pathology is examined in clinically demented subjects, the correlation received between particular pathologic change and dementia is good. It is noteworthy that with respect of AD-related neurofibrillary pathology, all cases in the neocortical stage (Braak stage V-VI) were indeed demented. One important issue with respect to the pathogenesis of synucleinopathies is not only to understand the molecular mechanisms behind the intracytoplasmic aggregation of αS, but also to appreciate where this process first appears and how it may progress through the brain. Thus, many recent studies have attempted to localize the most vulnerable neuronal populations. In this study, dmV and SN were found to be equally susceptible nuclei, but even earlier affected structures have been reported to appear in the spinal cord, dmV, olfactory bulb and AC [2, 4, 11, 16, 22, 35]. Thus, mapping out the “trigger site” for αS pathology appears to depend on the screening process i.e. if one screens medulla alone, those cases where lesions are restricted to other areas (e.g. SN) are not found and vice versa. At variance to the studies of Del Tredici and Braak [4, 11] we identified a number of subjects where dmV and/or LC were not affected but αS-IR inclusions were found in the SN, BFB and or other cortical regions, and thus, the distribution of αS pathology did not strictly follow the caudo-rostral propagation pattern described by Braak and colleagues [4]. Thus, the proposed ascending pathway is not the only possible route and our results indicate that pathology can emerge simultaneously in subcortical and cortical regions. Jellinger has also reported subjects with multiple αS-IR inclusions but with preservation of medullary nuclei [20]. Furthermore, in some subjects we found the AC to be devoid of pathology although αS-IR structures were detected elsewhere in the neocortex. This refutes the proposal that in order to have neocortical involvement then the subcortical lesions have to expand through the basal forebrain nuclei. In addition, according to Braak and colleagues [4], the αS pathology in previously involved regions should become exacerbated with disease progression. It is difficult to evaluate this proposal when one takes into account the increasing neuronal loss. If these two pathological hallmarks are linked in a causative chain, the load of αS-IR structures should show an inverted u-shape distribution over time where the number of inclusions would increases with the progression of the disease until the neurons start to die [12]. We observed some cases with severe αS pathology in AC and in that situation this structure was either only involved region or was affected together with BFB and SN. All these subjects were demented and exhibited coexistent severe AD-related pathology (Braak AD stage V-VI [6]). In one case, the dementia was considered to be vascular in origin. This is in line with the results of Uchikado and colleagues who have reported the AC predominant form to be common finding among patients with AD [35]. In conclusion, our results confirm that the current staging/categorization systems can readily be applied to most of the subjects when assessing regional distribution of αS-pathology. It is noteworthy, however, that outliers do exist and in these cases the presumed distribution may have been modified by other coexisting pathologies or genetic factors [23, 35]. It is intriguing that around every second subject displaying abundant pathology did remain neurologically intact. It is noteworthy that these results were seen when a rather unselected sample of cases was investigated. It has been suggested that the key lesions begin to develop a considerable time prior to the appearance of clinical symptoms [13], but based on our results there do seem to be some subjects who can tolerate substantial amounts of pathology. As only 50% of subjects with widespread αS pathology were demented (MMSE < 26) and displayed EPS, the clinical relevance of αS-IR inclusions as such still remains to be resolved. Hitherto, the aggregation of αS has been thought to lead to neuronal death but recent evidence has suggested that the formation of large inclusions may actually represent a protective process [3, 14, 34]. Many biophysical studies have suggested that it is a protofibrillar form of αS rather than the “mature” fibrils that are responsible for the cell death [7, 15, 36], and moreover, the fibrillar form that is typically observed at autopsy may actually be a sign of a well functioning neuroprotection [31, 34]. Thus, when we are assessing regional distribution of αS pathology, the question arises if we are really evaluating a stage of degeneration or conversely monitoring the level of functional neuroprotection.

Anal_Bioanal_Chem-3-1-1802726

Vibrational microscopy and imaging of skin: from single cells to intact tissue

Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications, ranging from studies of isolated single cells (corneocytes) to characterization of endogenous components in intact tissue. Two applications are described to illustrate the power of these techniques for skin research. First, the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated. Two solvents, DMSO and chloroform/methanol, commonly used in dermatological research, are shown to induce large reversible alterations (α-helix to β-sheet) in the secondary structure of keratin in isolated corneocytes. Second, factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 μm in intact pigskin, demonstrates the delineation of specific skin regions. Two particular components that are difficult to identify by other means were observed in the epidermis. One small region was formed from a conformationally ordered lipid phase containing cholesterol. In addition, the presence of nucleated cells in the tissue (most likely keratinocytes) was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA. Introduction Technological developments in infrared and Raman microscopic imaging have permitted several new types of biomedical applications. As demonstrated elsewhere in this volume, the diagnosis of pathological states in a wide range of tissues is now feasible, and is being pursued vigorously in laboratories around the world. The vast volume of spectral data generated from imaging experiments permits sophisticated multivariate statistical analysis of data and enables the experimentalist to discern tissue regions where spectral signatures from pathological states differ from those of healthy tissue. An important attribute of vibrational spectroscopy is the availability of spectra–structure correlations from many tissue components. This extensive background information can provide a useful supplement for biomedical diagnostics. By way of example, Boskey and her collaborators have characterized a new form of brittle bone disease (osteoporosis) based on structural changes in the collagen component of mineralized tissue [1]. This pathological state differs from the more widely known forms of osteoporosis, which are characterized by altered hydroxyapatite levels/structure. The origin of the spectral changes in the collagen component of pathological tissue biopsies was traced to increased levels of collagen cross links and alterations in their spatial distribution. Evidently, the availability of such information facilitates the development of strategies for tracing the origin (i.e., genetic predisposition, external factors, etc.) of the pathology. Skin is another tissue where the availability of spectra–structure correlations as well as the ability to detect spectral signals from exogenous constituents opens up new possibilities for vibrational microscopic imaging experiments relevant to pharmacology, physiology, and biochemistry [2–4]. In this article, we provide two examples depicting the interplay of vibrational microscopic imaging with spectra/structure correlations. The first example uses both IR and Raman microscopy to evaluate structural changes in the keratin of single cells (corneocytes) induced by treatment with dimethyl sulfoxide (DMSO) or chloroform/methanol (C/M). These solvents have been widely used in dermatology for studies of permeation enhancement and for extracting lipid from the stratum corneum (SC), respectively. The characterization of DMSO-induced structural changes in keratin will aid in the determination of DMSO permeation pathways and in the evaluation of SC barrier recovery. Assessing C/M’s effect on keratin in isolated corneocytes including the reversibility of potential structural changes, will facilitate the delineation of solvent treatment effects from that of lipid removal. In the second example, we demonstrate that confocal Raman microscopy along with multivariate (factor) analysis provides the detection of particular skin regions (stratum corneum, epidermis, dermis) as well as endogenous sub-areas (keratinocytes, lipid inclusions) in the epidermis. The availability of spectra–structure correlations permits molecular structure information to be deduced from either the spectra or from the factor loadings. Materials and methods Preparation of corneocytes The Rutgers University Internal Review Board has approved all protocols. Corneocytes were collected by tape stripping (Sellotape, 3M Scotchguard) human forearm skin. The forearm was flushed with water for several minutes prior to tape stripping. The first two strips were discarded and corneocytes from the third tape strip were isolated as follows. Corneocytes were flushed from the tape with HPLC-grade hexane. The hexane/corneocyte suspension was sonicated for approximately 5 min to break up desmosomes and lipid cohesion between individual corneocytes. Corneocytes were then isolated using nitrocellulose membrane filters with a pore size of 0.22 μm (GE Osmonics Labstore, Minnetonka, MN) and resuspended with fresh hexane. This procedure was repeated three times. A small aliquot of this suspension was separately deposited on either CaF2 windows for transmission IR or gold-coated silicon substrates for Raman measurements. Samples were dried overnight under house vacuum prior to spectroscopic measurements. The remaining corneocytes were dried under N2 gas prior to treatment with DMSO or C/M. Corneocytes were placed on substrates that were directly dipped in pure DMSO for about 1 min. Separate aliquots of harvested corneocytes were transferred to glass vials using 5 mL C/M (2:1 v/v). Non-covalently bound lipids were extracted for 68 h while stirring at room temperature in sealed vials. Corneocytes, collected by centrifugation, were further washed 3 times using fresh C/M before the final suspension was placed on the appropriate substrate. Samples were dried overnight under vacuum prior to spectroscopic measurements. No vibrational bands from residual solvent were observed in corneocyte spectra. Subsequently, corneocytes from both DMSO and C/M treatment were rehydrated on the solid substrates by exposure to 100% relative humidity overnight before a final set of spectra were acquired. Intact porcine skin preparation Skin biopsies from Yucatan white, hairless pigs were purchased from Sinclair Research Center, Inc (Columbia, MO). The stratum corneum of the biopsies was washed with water and samples were cut to a size of approximately 1 × 1 cm2, 0.5-cm-thick sections. Sections were placed, SC side up, into a milled brass cell, covered, and sealed with a microscope coverslip for confocal Raman measurements. IR microspectroscopy of corneocytes IR spectra were acquired with the PerkinElmer Spotlight system described elsewhere in detail [4]. The instrument has imaging capabilities, but in this study the point mode was used. Since the typical diameter of corneocytes is about 40–60 μm, an aperture size of 40 × 40 μm2 was chosen to optimize the spectral quality. A total of 256 scans was acquired for each spectrum using 4 cm−1 spectral resolution. One level of zero-filling yielded data encoded every 2 cm−1. Approximately 50 corneocytes were examined from each sample preparation. Raman microspectroscopy Raman spectra were acquired with a Kaiser Optical Systems Raman Microprobe. The instrument has been described in detail elsewhere [5, 6]. A 785-nm diode laser generates approximately 7–12 mW of single mode laser power at the sample with a spot size of about 2 μm using a ×100 objective. The backscattered radiation illuminates a near-IR CCD (ANDOR Technology, Model DU 401-BR-DD). Spectral coverage is from 100 to 3,450 cm−1 at spectral resolution of 4 cm−1. Following linearization, data are encoded every 0.3 cm−1. Spectra were acquired from 10–15 corneocytes from each sample preparation using an 80-s exposure time, 4 accumulations, and cosmic ray correction. Optical images of corneocytes were acquired with a Leica microscope (Model DMLP) coupled to the Kaiser system with a ×100 objective. Confocal Raman spectra of intact pigskin were acquired using a 60-s exposure time, 2 accumulations, and cosmic ray correction. A ×100 oil immersion objective was used for the intact pigskin samples. To evaluate axial characteristics of the optical set-up used in these experiments, the following observations were made with the oil immersion lens using protocols previously published [5]. The test sample consisted of five layers of a polymer laminate which incorporated two chemically identical layers (out of the five), each with a thickness of 7.5 μm separated by an intermediate layer of thickness 15 μm. This provides a center-to-center distance of 22.5 μm between the identical layers. Raman intensity profiles show a baseline-resolved width for each of the chemically identical layers of approximately 12–13 μm, a half-width of the intensity profile for each peak of about 8 μm, and a peak-to-peak distance of about 25 μm, in good agreement with the known test sample physical characteristics. The actual axial spatial resolution in the skin samples cannot be directly ascertained. Data analysis Grams/32 AI software version 6.0 (Thermo Galactic, Salem, NH) was used for processing individual Raman and IR spectra. Raman spectra were Fourier smoothed (80–90%) and 2-point baselines were applied to spectral regions of interest. This degree of Fourier smoothing could be applied without inducing spectral lineshape distortions. Confocal Raman spectra were analyzed (e.g., factor analysis) and Raman image planes were generated using ISys software version 3.1 (Spectral Dimensions, Inc. Olney, MD). Factor analysis was performed using the ISys score segregation routine. The analysis seeks to detect simple patterns in the relationships between observed variables in order to reduce the dimensionality of the data. Score segregation begins by normalizing PCA scores which may then be sharpened by raising them to a power specified by the acceleration parameter. The default value of 10 was used. Factor loadings are then calculated according to (S′S)−1*S′*X, unless S′S is not invertible, in which case iteration stops, and a factor is deleted before continuing. Results and discussion Solvent-induced conformational changes in the keratin of single cells Although its mechanism of action is not well understood, DMSO has often been utilized as an efficient permeation enhancer for a wide variety of drugs, including antiviral agents, steroids, and antibiotics [7]. It has been suggested to alter protein conformation in the stratum corneum (SC), the outermost layer of skin and the main barrier to permeability. Anigbogu et al. [8] monitored the protein (keratin) α-helix to β-sheet transition in hydrated SC. In addition, Caspers et al. [3] monitored protein structure following DMSO penetration into human SC in vivo by confocal Raman spectroscopy. A broadening of the Amide I contour was noted, although major new spectral features were not resolved. It is noted that DMSO concentrations differed in the two experiments. IR (1,200–3,600 cm−1) and Raman (800–1,720 cm−1) spectra, shown in Fig. 1a and b, respectively, demonstrate the quality of data obtained from untreated human corneocytes. Several strong bands characteristic of the protein and lipid constituents are observed in each spectrum. The Amide I and Amide II bands observed at approximately 1,650 and 1,550 cm−1 in the IR spectra, respectively, are of particular interest. The Amide I band is also one of the strongest bands in the Raman spectrum. In both types of spectra, the measured Amide I frequency reflects the predominantly helical secondary structure found in epidermal keratin. Absorbance in the Amide I region due to ceramides and other components, some containing amide groups and others C=C, is expected to be low compared to keratin absorbance in corneocytes isolated from the topmost layers of the stratum corneum [6]. Raman spectral quality is very good considering the thickness of an individual corneocyte (0.5–0.8 μm) and is comparable to spectra obtained from much thicker stratum corneum samples and intact skin [2, 5, 8]. It is likely that the observed signal has been enhanced due to a surface-enhanced Raman scattering (SERS) effect between the corneocytes and gold-coated substrate. Raman band assignments pertinent to stratum corneum samples have been listed previously [5, 9, 10]. Fig. 1Vibrational microscopy of human corneocytes. a IR point mode spectrum (1,150–3,600 cm−1 region) acquired using a 40 μm2 aperture of a single corneocyte isolated from the third sequential tape strip applied to human forearm skin. The inset shows the optical image of the corneocyte (bar = 10 μm). b Raman spectrum (800–1,720 cm−1 region) of a similarly isolated corneocyte displaying bands characteristic of the lipid and protein (keratin) components IR and Raman spectra of isolated corneocytes prior to and following immersion in DMSO, and after an overnight rehydration period (top to bottom trace) are displayed in Fig. 2a and b, respectively. A similar series of IR and Raman spectra acquired from corneocytes treated with C/M are shown in Fig. 2c and d. Both solvents induce several alterations in the spectra. The most significant change in the IR spectra is the appearance of an intense low frequency Amide I component observed for the DMSO-treated sample at 1,626 cm−1 in conjunction with a weak high frequency shoulder at approximately 1,695 cm−1. This pattern is diagnostic for formation of antiparallel β-sheet structure [11]. A redistribution of the maximum intensity in the IR Amide II band from 1,550 to 1,517 cm−1 for the same two spectra is also consistent with β-sheet formation [11]. Fig. 2Solvent-induced conformational changes in the keratin of single corneocytes. a and b IR and Raman spectra, respectively, of an isolated corneocyte prior to and following immersion in DMSO, and after an overnight rehydration period (top to bottom, respectively). c and d IR and Raman spectra, respectively, of a corneocyte prior to and following C/M treatment, and after an overnight rehydration period (top to bottom, respectively). In the IR spectra (a and c), α-helical and β-sheet components of the Amide I (ca. 1,650 cm−1) and II (ca. 1,500–1,550 cm−1) bands are noted. In the Raman spectra (b and d), α-helical and β-sheet components of the Amide I are marked along with disordered constituents at 1,685 cm−1. One component of the Amide III band is marked at 1,240 cm−1 Helix:sheet transitions are well documented for different types of keratin, and have been detected by IR spectroscopy. In particular, Oertel [12] used IR to observe similar DMSO-induced changes in human SC. The current work extends observation of this conformational transition to isolated human corneocytes. It is also noted that, based on the IR Amide I and II band contours, a fair amount of helical structure remains subsequent to DMSO treatment. The Raman spectrum (Fig. 2b, middle trace) corroborates the β-sheet assignment with the appearance of a high frequency Amide I component at about 1,669 cm−1 and a strong Amide III component at approximately 1,240 cm−1. Both features are within frequency ranges assigned to antiparallel β-sheets [13, 14]. The observed changes in the Amide I region of the Raman spectrum are consistent with a previous report detailing the effects of a series of aqueous DMSO solutions on human SC [8]. Comparisons between individual cells and the SC in the Amide III region are difficult due to overlapped bands from lipid components in the SC sample. In the current work, a second slightly weaker band is also observed for the DMSO-treated cells at approximately 1,270 cm−1. Bands in this region are usually assigned to α-helical structure [13, 14]. Generally, caution is required in associating characteristic frequency ranges with conformation for the Amide III mode given that the NH in-plane bend internal coordinate contributes to a number of modes in the 1,200–1,400 cm−1 region [15] and secondary-structure-dependent coupling occurs between the Amide III and the Cα–H bending vibration [16, 17]. This complexity, in addition to the inherent heterogeneity of corneocytes and variable lipid content of the samples examined herein, precludes an in-depth analysis of the Amide III contour in the current context. The majority of the DMSO-induced secondary structure change is reversed upon rehydration of the corneocytes as demonstrated by comparison of the top and bottom spectra shown in Fig. 2a and b. Some residual broadening remains on the low frequency side of the Amide I band in the IR spectrum. The average full width at half height for the Amide I band in the IR spectra increased from 44 cm−1 for untreated corneocytes to 52 cm−1 following DMSO treatment and rehydration, indicating that some residual sheet structure may be present. An accurate measure of the Amide I bandwidth in the Raman spectra is precluded by the presence of a weak side chain vibration at approximately 1,608 cm−1. The general reversibility of the conformational change upon hydration was also observed by Oertel [12] for isolated SC. C/M solutions are commonly used to extract lipids from isolated SC and intact skin samples, but solvent effects on the structure of the remaining constituents have not been detailed. The utility of vibrational spectroscopy for evaluation of keratin secondary structure after lipid removal is somewhat hampered by the native ceramide contribution to the Amide I and II band prior to solvent exposure. In SC samples it is therefore difficult to accurately access the contribution ceramides make to Amide I and II band intensities and position. In the current work, this concern is mostly overcome by acquiring spectra of isolated corneocytes in which the ceramide contribution to the Amide modes is substantially reduced. Several changes are apparent in the IR and Raman spectra of corneocytes upon exposure to C/M (Fig. 2c and d, compare top to middle traces). Some of the solvent-induced alterations are similar to those just discussed for the DMSO-treated cells, e.g., the low (high) frequency Amide I component in the IR (Raman) spectrum at 1,626 cm−1 (1,670 cm−1) and the appearance of an Amide III band at 1,240 cm−1 in the Raman spectrum. These features provide strong evidence that C/M also induces antiparallel β-sheet formation. Differences are also evident when spectra from the two treatments are compared. Broadening on the high frequency side of the Amide I band in the Raman spectra is much more intense after C/M treatment vs. DMSO (compare the middle trace in Fig. 2d to b). Amide I Raman intensity in this region (ca. 1,685 cm−1) has been assigned to non-hydrogen-bonded disordered structures [13]. The broadening in the Raman Amide I band is not completely reversible upon rehydration of the sample that had been treated with C/M, even though the intensity of the β-sheet component at about 1,670 cm−1 has diminished (Fig. 2d, bottom trace). In addition, upon rehydration, intensity remains in the Amide III band at approximately 1,240 cm−1, although this band also broadens. Finally, measurements of the Amide I bandwidth in IR spectra upon rehydration of several C/M-treated corneocytes are essentially the same (±2 cm−1) as widths measured for untreated cells. The close proximity of Amide I bands arising from helical and random coil structures makes it difficult to differentiate between these two forms. Considering both the Raman and IR spectral data after rehydration, it seems safe to conclude that the majority of keratin exists in a helical form with some disordered structure remaining. The extent of the C/M-induced protein structural changes has been observed to vary among corneocytes. IR spectra of some cells with significant lipid bands (i.e., those characterized by relatively intense methylene stretching modes near 2,850 and 2,918 cm−1) still exhibit notable protein conformation changes (data not shown), while some cells from which the lipid has been essentially completely extracted do not show significant changes in protein secondary structure. It thus appears that the extent of lipid extraction is not directly related to C/M-induced protein conformational changes. Similar results have been noted in IR studies of DMSO-induced protein conformation changes in SC samples [12]. Additional experiments under conditions of controlled hydration are needed to further explore possible origins of these conformational changes. One explanation for the α-helix to β-sheet structure change in keratin is that DMSO displaces bound water necessary for maintaining secondary structure [12]. DMSO is known to form an association complex with water stronger than that formed between water molecules alone [8]. It may be that C/M destabilizes keratin’s native secondary structure in a manner similar to DMSO, since the majority of the native structure is restored after both solvent treatments when the corneocytes are exposed to high relative humidity. The importance of the current experiments lies in the fact, that in studies of supposedly native protein structures in the SC, C/M is often used to remove lipid components. As shown in the current work, this procedure is not innocuous, but can induce irreversible changes in protein structure in corneocytes. Confocal Raman microscopy: delineation of skin regions from factor analysis As demonstrated above, Raman microscopy complements the information available from IR. Although Raman scattering is inherently weaker than IR absorbance, the technique offers two major advantages for tissue studies. First, the spatial resolution of the method is inherently better than IR. For an excitation wavelength of 785 nm, Rayleigh’s rule for spatial resolution (ΔZ = 1.22λ/2(NA)) predicts a spatial resolution of approximately 0.5 μm for a wavelength (λ) of 785 nm and a numerical aperture (NA) of the ×100 objective of 0.95. In practice, as has been discussed in Materials and methods and elsewhere, the apparent depth and related measured optical properties are degraded by refractive index variations in the tissue [5, 18, 19]. Second, and equally important, spectra may be acquired in a confocal manner. Thus, physical sectioning of samples is not required to obtain molecular structure information from within the tissue. This evidently opens up the possibility for in vivo examination of skin, especially at the low laser powers (ca. 10 mW) used in these experiments. Puppels’ group has demonstrated the utility of confocal Raman determination of water levels in vivo in skin [20]. The benefit of using both a multivariate approach and existing spectra–structure correlations to characterize the microanatomy of skin is demonstrated in the next few figures. An optical micrograph of a 5-μm-thick pigskin section, shown in Fig. 3a, allows delineation of different skin regions. The outermost layered stratum corneum (SC) region appears to be ca. 15-μm thick with the underlying epidermis (ca. 50-μm thick) and dermis discernible. The physical sampling used in the confocal Raman measurement precludes the acquisition of a meaningful micrograph from the same sample; however to allow for comparison, confocal Raman spectra were acquired from a similar pigskin biopsy using approximately the same dimensions. Spectra were acquired from an untreated piece of pigskin, in steps of 2.5 μm in the z direction and 5 μm in the x (or y) direction. The area sampled was 75×40 μm so that 240 spectra (30 × 8) were acquired during the experiment. These were analyzed with factor analysis as described in Materials and methods and as applied to liposome-treated pigskin in a previous report [21]. In the current work, five significant factors were determined as shown in Fig. 3b, while score images for each factor are depicted in Fig. 3c. Three of the score images and corresponding factor loadings in Fig. 3 may be directly correlated with the known regions of skin as follows: Factor 1 corresponds to the SC, Factor 4 to the viable epidermis, and Factor 5 to the dermis. Thus, delineation of the skin regions based on the factor score images is wholly consistent with the optical image. The two remaining factor score images (Factors 2 and 3 ) highlight particular features within the viable epidermis. Examination of the factor loadings or averaged spectra corresponding to those pixels where the respective scores are high provides an indication of the molecular origin of the components. In general, factor plots tend to offer somewhat higher S/N ratios, since they represent a substantial condensation of data. However, it is important to remember that factors include all sources of variance in the data. For example, baseline distortions may appear as “features” in the factor loadings, which however are not related to vibrational modes in the sample. Thus, it is useful to routinely compare factors with appropriately selected spectra. In Fig. 4 an average spectrum of the stratum corneum is overlaid with the average localized to the small spatial region highlighted in Fig. 3c, labeled Factor 2. The average spectrum labeled “ordered lipid” has strong bands near 1,058, 1,130, 1,296, 2,880, and 2,850 cm−1 (Fig. 4b and c). These bands are readily assigned [9, 22] to lipid C–C stretching vibrations (1,058, 1,130 cm−1) of a conformationally ordered (all-trans) chain, lipid CH2 twist (1,296 cm−1) and lipid CH2 stretch (2,850 and 2,880 cm−1). In addition, the bands observed at 605 and 700 cm−1 (Fig. 4a) are indicative of cholesterol [23]. Thus, the small pocket in Fig. 3c (Factor 2) likely contains an ordered lipid phase. The fact that the same bands are present in the average stratum corneum spectrum (Fig. 4, top), albeit at lower intensity, strengthens the assignment. Highly ordered lipid phases (ceramides, fatty acids, and cholesterol) comprise the inter-corneocyte matrix of the stratum corneum. In addition, infrared spectroscopic evidence exists (D.J. Moore, unpublished results) for ordered lipid regions with relatively low protein content in appendages (i.e., pores and hair follicles) well below the stratum corneum. It is possible that the confocal Raman image planes shown in Fig. 3c cut through such an appendage. Fig. 3a An optical micrograph of a microtomed 5-μm-thick pigskin section acquired using the PerkinElmer Spotlight (bar = 10 μm). The results of factor analysis in the 800–1,150 cm−1 region conducted on a confocal Raman map acquired from intact pigskin. b The five distinct factor loadings generated by the ISys score segregation algorithm (see Materials and methods) are offset and labeled Factor 1–5. c The spatial distribution of factor scores for each of the loadings as marked. Dark blue indicates the lowest score with green, yellow, orange, and red indicative of progressively higher scores. Factor loadings and score images have been assigned to different micro regions in skin as described in the textFig. 4Averaged Raman spectra from within the stratum corneum (top) and lipid (bottom) regions as noted in Fig. 3b and c as Factor 1 and 2, respectively. a The 590–790 cm−1 region with bands assigned to cholesterol marked at 605 and 700 cm−1. b The 800–1,380 cm−1 region displays several bands (1,058, 1,130, and 1,296 cm−1) characteristic for ordered (all-trans) lipid acyl chains. c The CH stretching region (2,800–3,030 cm−1) with methylene symmetric (2,850 cm−1) and asymmetric (2,880 cm−1) stretching modes noted. The frequencies and relative intensity of the bands are consistent with well-ordered lipid acyl chains Finally, Factor 3 (Fig. 3b), whose score image reveals several small-localized pockets in the epidermis, contains one broad spectral feature centered at approximately 1,090 cm−1 that is not present in the other factor loadings. Based on both the position of this particular band and the spatial distribution of the factor score, the band is tentatively assigned to the phosphodiester mode of DNA [24, 25]. Since the identification of DNA is of interest, as it would reveal the location of nuclei within keratinocytes or other cells in the tissue, an additional strategy was employed to acquire better spectra from the epidermis. The experiment, whose results are depicted in Fig. 5, was carried out as follows. The top ≈20 μm of a pigskin section, including most of the SC, were removed by tape stripping prior to confocal Raman imaging. This allows for the acquisition of higher quality spectra in deeper regions of the viable epidermis. The Raman experiment was also carried out with more data points/unit area (2-μm step size) providing a closer examination of the tissue compared to the sample presented in Fig. 3. The presence of cell nuclei was revealed by factor analysis conducted over two wavenumber regions, each separate analysis restricted to compute only two factor loadings. An additional DNA band assigned to cytosine is detected in the factor loadings shown in Fig. 5b, which also strengthens the previously identified phosphodiester DNA band assignment at about 1,090 cm−1 [24, 25]. As expected, in the tape stripped sample, the phosphodiester band is again observed (Fig. 5c). The score image shown in Fig. 5a is derived from the factor with the strong cytosine band and is essentially the same as the score image generated from the phosphodiester factor (not shown). Thus, the image shown in Fig. 5a, depicting keratinocyte or other epidermal cell (e.g., melanocyte) nuclei, demonstrate the ability of confocal Raman to detect DNA in intact skin tissue. Fig. 5The results of factor analysis conducted in two separate spectral regions for a confocal Raman map of pigskin after stratum corneum removal by tape stripping. Both analyses were performed allowing the generation of only two factors. In each case, one factor contained a spectral feature specific to DNA and the other mapped in a fairly uniform way throughout the epidermal region. a Factor score image for the factor-labeled nuclei shown in (b) with red corresponding to the highest score and dark blue to the lowest. b Factor loadings for the 600–820 cm−1 region with a distinctive vibrational band at approximately 785 cm−1 assigned to cytosine. c Factor loadings for the 800–1,150 −1 region with a second characteristic DNA vibrational band due to the phosphodiester backbone stretching mode, at about 1,090 cm−1 noted Conclusions The current experiments highlight some unique advantages of vibrational microscopic imaging. An understanding of the spectroscopy of the various tissue components permits insight into the molecular origins of the images generated from factor analysis. Thus, identification of the lipid pocket in Fig. 3c (Factor 2) and of the cell nuclei (as detected from the DNA spectral signatures) in Fig. 3c (Factor 3) and Fig. 5a were greatly facilitated by the availability of Raman spectra of lipids and of DNA, respectively. In addition, the availability of correlations between lipid spectra and chain conformation, permitted assignment of the lipid inclusions as having arisen from ordered lipid phases. The biological relevance of this observation remains to be determined. From a pharmacological perspective, the detection of cell nuclei (most likely keratinocytes) within intact skin permits co-localization experiments to begin to be designed. For example, for classes of drugs targeted to these cells, confocal Raman measurements will permit determination of whether the putative therapeutic agent reaches its intended target. In addition, although all microscopic methods can obviously readily detect single cells, the ability of IR- or Raman-based methods to detect conformational changes within a particular component of a single isolated cell is unique. The example we have chosen in Fig. 2 is of practical importance in skin research, since solvents such as DMSO or chloroform/methanol are used for permeation enhancement and lipid extraction, respectively. In each case these solvents were found to be far from innocuous. Each induced large conformational changes in the cellular keratin. This fact had been known previously from spectra of intact SC, but two aspects of the current measurements are novel. The observation of IR data from single cells provides a sharper spectral assignment of the bands. We note for example that ceramides, a major component of the SC, have strong bands arising from the amide bond located in these lipids. These modes contribute significantly to the spectra of the intact SC and overlap protein Amide I and II vibrations; however, they are much reduced in relative intensity in spectra of cells. Thus, studies of the reversibility of the solvent-induced conformational changes benefit from the reduced intensity of ceramide interference. Finally, it is feasible to begin to examine biochemical heterogeneity at a single cellular level. Issues such as the relationship of protein structure (either native or solvent-modified) to hydration levels or to the levels of natural moisturizing factors, in single cells may be profitably probed. In addition, changes in the spectra of cells from pathological states may be examined.

Apoptosis-4-1-2217618

RGD-avidin–biotin pretargeting to αvβ3 integrin enhances the proapoptotic activity of TNFα related apoptosis inducing ligand (TRAIL)

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL’s potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin–biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity. Introduction TNF ligands have strong potential as anti-tumor agents, acting in different ways on either tumor cells or tumor vasculature, or by promoting the induction of tumor-directed immune responses [1]. The broad clinical use of TNF ligands is prohibited however due to lack of tumor selectivity, causing various side effects. A recently identified TNF family member is TNF related apoptosis-inducing ligand (TRAIL) [2]. Many tumor types are sensitive to TRAIL or become sensitive after treatment with chemotherapeutic drugs [3]. Soluble TRAIL variants showed no cytotoxic effects on most primary human cells and were well tolerated in mice and chimpanzees [4, 5]. However, the selectivity of TRAIL for tumor cells over normal cells is lost when TRAIL forms multimeric complexes. For example, it has been observed that cross-linked TRAIL induces apoptosis more efficiently than non-aggregated trimeric TRAIL [6], but such cross-linked TRAIL also caused cell death in normal hepatic cells [7]. It seems therefore attractive to design clustered TRAIL variants with enhanced specificity for tumor tissue. The increased tissue retention of cellular targeted TRAIL variants may counterbalance or prevent side effects elsewhere in the body. In the present paper, we investigated the development of TRAIL complexes equipped with targeting ligands directed to the tumor vasculature. Enhanced retention of TRAIL complexes at the tumor vasculature will enhance their accumulation in the tumor tissue, thereby favoring the desired therapeutic activity and reducing potential toxicity. Several recent papers described the development of recombinant TNF family members with enhanced target specificity. One approach is to modify the intrinsic specificity of TRAIL for its target Death-receptor 5 specific by substituting amino-acids located in the receptor binding interface of TRAIL [8]. Development of chimaeras is another successful approach. Linkage of TRAIL to scFv antibody fragments yielded constructs which bind to additional surface-exposed receptors on tumor cell types [9–11]. Similarly, other TNF family members like Fas-ligand and TNFα (have been conjugated to tumor-cell directed targeting ligands, yielding chimeric proteins with full or even enhanced therapeutic activity [11–15]. Although the recombinant approach has been applied successfully for the production of chimeric proteins, low production yields may hamper the feasibility of such a strategy. Especially when multiple targeting ligands are introduced into a protein subunit, which is preferred for high avidity binding, aberrant folding of chimeric proteins can occur. In the current study we therefore pursued a chemical approach, in which TRAIL was biotinylated and complexed to avidin equipped with multiple targeting ligands. Avidin–biotin complexation is a widely accepted approach in diagnostic applications that employ immunodetection steps, and is also considered an attractive strategy for the preparation of tumor targeted complexes, especially in so-called pretargeting approaches in which the complexes are assembled in vivo at the cell surface of tumor cells. Commonly, such strategies employ biotinylated tumor-specific antibodies and a therapeutic or diagnostic cargo coupled to avidin [16, 17]. The approach presented in this paper differs to the above-discussed strategies in several aspects. First, we now equipped TRAIL with targeting ligands directed to angiogenic endothelium rather than to tumor cells, allowing an enhanced binding to the tumor blood vessels. The vascular wall is in direct contact with the systemic circulation, which makes it an easily attainable target for therapeutic proteins. Furthermore, intratumoral pressure, which hampers the penetration of macromolecules into tumor tissue will not affect the homing to of RGD-equipped complexes to the angiogenic endothelium. Second, we have prepared a different type of targeted avidin that can interact directly via covalently attached RGD-groups with tumor endothelial cells. Thus, the developed avidin–biotin-TRAIL complexes will home to angiogenic endothelium without the need of complexation with a biotinylated antibody, thereby leaving its biotin-binding sites available for complexation with the biotinylated TRAIL. The functionalization of avidin with RGD-homing ligands was carried out using a cyclic RGD-peptide with high affinity and specificity for αvβ3 integrin on angiogenic endothelium [18]. We furthermore applied a polyethylenglycol (PEG) linker for coupling of the RGD-ligand, which brings several advantages, such as a reduced non-specific binding of the avidin and reduced immunogenicity [19]. The now developed RGDPEG-avidin conjugates contain the RGD homing ligand attached to the distal end of the PEG groups. In order to minimize loss of functional TRAIL activity, we have compared two different biotinylation strategies that react at different functional groups in the protein. We used a conventional Biotin-NHS reagent that binds to primary amino groups in the protein, and we employed the Biotin-ULS (ULS: universal linkage system) reagent, which reacts at methionine residues. Figure 1 shows a schematic representation of the developed complexes and their interaction with target cells. The products were evaluated for their association with αvβ3 integrin expressing endothelial cells and for their antitumor activity in both endothelium and cultured tumor cells. Our results demonstrate that recombinant TRAIL can be complexed with endothelial specific targeting ligands in such a way that it maintains its extensive cell killing activity either in solution or when displayed on the surface of endothelial cells. Fig. 1Schematic representation of RGD-avidin:TRAIL complexes. Soluble recombinant human TRAIL was modified with biotin groups via two different reagents that either react at lysine or methionine residues. Biotinylated TRAIL was complexed to an avidin-based targeting device equipped with approximately 5 RGD-peptide ligands per avidin molecule. A hydrophilic PEG-linker was used to append the RGD-peptides to avidin. The assembled complexes can bind to tumor blood vessels via RGD//αvβ3-integrin interactions, while apoptosis can be induced via TRAIL/DR ligation Materials and methods Preparation and characterization of RGDPEG-avidin Avidin was modified with heterobifunctional PEG groups that can react with primary amino groups in avidin, after which RGD-peptide groups can be conjugated to their distal ends. The hydrophilic PEG groups of 3.4 kDa will furthermore reduce non-specific protein interactions. Briefly, avidin (2 mg, 32 nmol, Sigma) was dissolved in PBS at 10 mg/ml and treated with a 50-fold molar excess of the heterobifunctional vinylsulfone-polyethylenglycol-N-hydroxysuccinimide ester (1 mg, 292 nmol, VNS-PEG-NHS; Nektar, Alabama, USA), which had been dissolved in water at a concentration of 20 mg/ml. The PEG linker was added drop wise. The reaction mixture was protected from light with tin foil and incubated for 1 h at room temperature on a spiramix rollerbank. Meanwhile, the RGD peptide c(RGDf(ε-S-acetylthioacetyl)K) (Ansynth Service, Roosendaal, The Netherlands) was dissolved at 10 mg/ml in a 1:4 acetonitrile/water mixture. The peptide was added drop wise to the reaction mixture at a molar ratio of 55:1, after which freshly prepared hydroxylamine was added to a final concentration of 50 mM. The reaction was carried out overnight at room temperature while protected from light. Remaining VNS groups were quenched by addition of cysteine (55:1 molar excess over the amount of avidin), after which the product was purified by size exclusion chromatography on a Highload superdex 200 column (Amersham Biosciences), pre-equilibrated in PBS. The final product RGDPEG-avidin was stored in PBS at −20°C. RGDPEG-avidin conjugates were characterized for protein content by BCA protein assay (Pierce). Coupling of RGDPEG groups to avidin was demonstrated by SDS-PAGE performed on a mini-Protean II system (Bio-Rad) using 12% Ready Gels (Bio-Rad, The Netherlands). Duplicate gels were either stained for protein (Coomassie Brilliant Blue staining) or blotted on a PVDF membrane (Roche, Mannheim, Germany). The membrane was blocked with BSA and subsequently incubated with an in-house raised anti-RGD polyclonal rabbit antiserum specific for the conjugated RGD-peptide [20]. Blots were further developed with goat-anti-rabbit peroxidase (GARPO)/DAB detection. The grade of RGD-pegylation was furthermore determined by MALDI-TOF analysis as described previously [21]. Expression and purification of recombinant human His-tagged TRAIL A cDNA corresponding to human soluble TRAIL (amino acids 114–281) was cloned downstream of the hexahistidine (His) tag sequence in pET15B (Novagen) using NdeI and BamHI restriction sites. Escherichia coli BL21 (DE3) (Invitrogen) was subsequently transformed with the His-TRAIL construct. The transformed host was grown in shake flasks at four liter scale as described previously [22]. The culture was centrifuged at 2,500 rpm and the supernatant was discarded. The isolated pellet was resuspended in three volumes of extraction buffer (20 mM phosphate buffer pH 8 containing 200 mM NaCl, 10% (v/v) glycerol and 7 mM β-mercaptoethanol). Cells were disrupted using sonication and extracts were clarified by centrifugation at 40,000 g. Subsequently, the supernatant was loaded on a cobalt-charged IMAC TALON column (Clontech) and His-TRAIL was eluted with a step gradient of extraction buffer containing 0.5 M imidazole. Trimeric His-TRAIL was purified by size-exclusion chromatography on a Hiload Superdex 75 column (Amersham Biosciences), to remove impurities. The final product was flash frozen and stored at −80°C in storage buffer (phosphate buffer pH 7.4 containing 150 mM NaCl, 3.5 mM DTT and 20 μM ZnSO4). Purified protein was >95% pure as determined by colloidal Coomassie Brilliant Blue-stained SDS–PAGE gel. Preparation and characterization of bio-NHS-TRAIL and bio-ULS-TRAIL We studied two different linkage strategies to introduce biotin into His-TRAIL, by applying either a conventional biotinylation reagent that reacts with primary amino groups in the protein (biotin-NHS), or one that reacts to methionine residues (biotin-ULS™, ULS = universal linkage system). Such an alternative strategy may prove less harmful to the therapeutic activity of TRAIL, depending on the extent to which side chain residues in the active domains are modified. Biotin-ULS was provided by Kreatech Biotechnology (Amsterdam, The Netherlands). All syntheses were performed in a 10:1 molar ratio of biotinylation reagent over TRAIL. His-TRAIL in storage buffer was extensively dialyzed against PBS containing 10% glycerol at 4°C using Slide-A-Lyzer dialysis cassettes (10,000 MWCO, Pierce) to remove DTT and other components of the storage buffer that can interfere in the reactions. Typically, the purified His-TRAIL (500 ug, 7.6 nmol) at 0.7 mg/ml was mixed either with Sulfo-NHS biotin (Pierce) (10 mg/ml in DMF) or with biotin-ULS (10 mg/ml in 20 mM NaCl), after which the mixtures were incubated for 4 h at 37°C. Unreacted biotinylation reagent was removed by dialysis against PBS at 4°C. The products were sterilized by filtration via disposable 0.2 μm filters and stored at −20°C. Biotin-TRAIL conjugates were characterized for protein content using BCA protein assay (Pierce). The relative number of coupled biotin molecules was determined by anti-biotin ELISA. Wells were coated with serial dilutions of the biotinylated proteins for 1 h at room temperature, washed with PBS containing 0.05% Tween20 and incubated for 1 h at room temperature with streptavidin-peroxidase complex (Dako, 1:2500 in PBS), followed by a standard incubation with OPD. The protein concentration at which 50% of the maximum absorbance was measured (EC50) was calculated by nonlinear regression (Graphpad Prism), and used to calculate relative biotin:protein ratios in each conjugate. The grade of biotinylation was furthermore assayed by MALDI-TOF analysis as described above for RGDPEG-avidin. Cells Jurkat T cells were cultured in RPMI 1640 culture medium (containing 25 mM HEPES and l-glutamine), supplemented with 10% heat-inactivated FCS (Bodinco, Alkmaar, The Netherlands), 1 mM sodium pyruvate, 2 mM l-glutamine, 0.5 mM 2-ME, and 0.1 mg/ml gentamycin sulfate obtained from BioWhittaker, and 0.02 μg/ml fungizone. (Bristol-Meyers, Woerden, The Netherlands) HUVEC were isolated by the UMCG Endothelial Cell Facility by previously described methods [23]. Primary isolates were cultured in 1% gelatin-coated tissue culture flasks (Corning, Costar, The Netherlands) at 37°C under 5% CO2/95% air. The culture medium, hereafter referred to as EC medium, consisted of RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 20% heat inactivated fetal calf serum (Integro, Zaandam, The Netherlands), 2 mM l-glutamine (Invitrogen, Breda, The Netherlands), 5 U/ml heparin (Leo Pharmaceutical Products, Weesp, The Netherlands), 100 U/ml penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands, 100 μg/ml streptomycin (Radiumfarma-Fisiopharma, Italy), and 50 μg/ml endothelial cell growth factor supplement extracted from bovine brain. After attaining confluence, cells were detached from the surface by trypsin EDTA (0.5/0.2 mg/ml in PBS) treatment and split in 1:3 ratio. Cells were used up to passage four. Binding of RGDPEG-avidin to HUVEC Binding affinity of RGDPEG-avidin to αvβ3-integrin expressed on the surface of HUVEC was determined by competitive displacement of 125I-labeled Echistatin, which is a known ligand for αvβ3 [24]. Confluent monolayers of HUVEC in 24-well plates (Costar) were incubated with 100,000 cpm 125I-Echistatin either in the presence of excess of RGDPEG-avidin, free RGD-peptide or avidin as competitors (competitors all in a concentration of 10 μM), or a serial dilution of RGDPEG-avidin and free RGD-peptide. Competition experiments were carried out with divalent cation containing binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 1% BSA) at 4°C for 4 h. Cells were washed three times with binding buffer and lysed with 1 M NaOH. Radioactivity was counted in a Packard RIASTAR multiwell gamma counter (GMI, Minnesota, USA). Data were analyzed by non-linear regression using the GraphPad Prism program. Complexation of biotinylated TRAIL with RGDPEG-avidin We studied the complexation of biotinylated TRAIL with RGDPEG-avidin in an ELISA-like experimental setup. Wells were coated with 50 ng of avidin or RGDPEG-avidin overnight at room temperature, blocked with BSA (1% in PBS) and incubated with 10 ng of biotinylated TRAIL for 2 h at room temperature. After the wells were washed with PBS/0.05% Tween20, the amount of TRAIL associated with the avidin was detected by anti-TRAIL immunodetection (anti-TRAIL antibody 2E5, Alexis, Breda, The Netherlands) for 1 h, followed by standard detection with GARPO/OPD. Apoptotic activity of biotinylated TRAIL The apoptosis inducing activity of the biotinylated products was evaluated on Jurkat T cells and on endothelial cells, using a cell viability assay and a caspase activity assay. Experiments were conducted with the single cell types, Jurkat tumor cells or HUVEC, or with a combination of tumor and endothelial cells. Assessment of TRAIL activity by MTS cell viability assay Briefly, cells were seeded in flat-bottom 96-well plates at a density of 3 × 104cells/well (Jurkat) or 1 × 104cells/well (HUVEC) in 100 μl medium supplemented with the indicated compounds. Concentrations of TRAIL, bio-NHS-TRAIL and Bio-ULS-TRAIL ranging from 1 μg/ml to 1 ng/ml were tested on Jurkat. Only one concentration (100 ng/ml) was tested on HUVEC. The MTS cell viability assay (Promega) was performed after 48 h of incubation, according to the manufacturer’s recommendations. Experiments were performed in triplicate. Assessment of TRAIL activity by caspase 3/7 assay We investigated the induction of apoptosis in Jurkat T cells using a caspase 3/7 activity assay (Promega) employing luminescence detection. Different experimental settings were evaluated either reflecting direct incubation of the products with tumor cells, or settings in which biotinylated TRAIL was complexed to RGDPEG-avidin and/or anchored onto endothelial cells. First, we determined pro-apoptotic effects of non-complexed TRAIL. About 5 × 103 Jurkat cells were incubated with biotinylated TRAIL at a concentration of 100 ng/ml. Experiments were carried in 96-well plates in 50 μl medium supplemented with the indicated compounds for 4 h, after which 50 μl of caspase 3/7 substrate was added to the cells, followed by luminescence detection with a microplate luminometer according to the provider’s instructions. Next, we determined proapoptotic effects of RGDPEG-avidin:TRAIL complexes in Jurkat cells. These experiments were performed in the presence of endothelial cells, to allow adherence of the RGD-equipped complexes to αvβ3 integrin expressing cell layers. HUVEC were preincubated with 10 μg/ml RGDPEG-avidin for 2 h at room temperature after which the excess of non-cell associated RGDPEG-avidin was removed by washing with PBS. Control experiments included RGDPEG-avidin treated HUVEC that were not washed and cells to which no RGDPEG-avidin was added. All wells were supplemented with biotinylated TRAIL (100 ng/ml in culture medium), after which Jurkat cells (5 × 103 cells in 50 μl of medium) were added. Cells were incubated for 4 h at 37°C, after which caspase activity was assayed as described above. Experiments were conducted in triplicate. Statistical analysis Statistical analysis was performed using Student’s two-tailed t-test, assuming equal variances. Difference were considered to be significant when P < 0.05 unless otherwise stated. Results Preparation and characterization of RGDPEG-avidin We modified avidin with PEG-linked RGD-peptides to obtain an avidin molecule which could bind to angiogenic endothelial cells. Successful synthesis of RGDPEG-avidin was proven by several analytical techniques. SDS-PAGE analysis (Fig. 2a) showed a single band for avidin of approximately 15 kDa, corresponding to the weight of one of its four subunits. Different protein bands were observed for RGDPEG-avidin with molecular weights corresponding to the unmodified subunit and to subunits modified with one or multiple RGDPEG moieties. Second, Western blot of the product showed positive staining of these bands by an anti-RGD-peptide antiserum. Due to the size heterogeneity of the PEG polymer, the RGD-band stained in a broad streak rather than a discrete band (Fig. 2b). Of note, the tetrameric band of avidin was not observed due to the denaturating conditions of SDS-PAGE. Third, we analyzed the product by MALDI-TOF mass spectrometry (Fig. 2c), which showed peaks corresponding to avidin subunits modified with RGDPEG to a different degree. From the average molecular weights (Mn) we calculated a relative derivatization ratio of 1.4 RGDPEG groups per avidin subunit, or a total of 5.6 RGDPEG groups per tetrameric avidin. Fig. 2Characterization of RGDPEG-avidin. Panel A and B: SDS-PAGE separation followed by CBB protein staining (a) or Western blotting and anti-RGD detection (b). lane 1: MW marker, lane 2: avidin, lane 3: RGDPEG-avidin. Unmodified avidin was separated into its 15 kDa subunits, while RGDPEG-avidin was disassembled into different bands corresponding to subunits with 0, 1, 2 or 3 attached RGDPEG groups. The intrinsic heterogeneity of PEG polymers can be observed in the broadly stained band of the RGDPEG-avidin Western blot. Panel C: MALDI-TOF analysis of RGDPEG-avidin. Upper line: RGDPEG-avidin, bottom line: avidin. During ionization, the tetrameric proteins have disassembled into subunits, yielding a single peak for avidin and multiple peaks for RGDPEG-avidin corresponding to modification degree 0, 1, 2, 3, 4 Next, we investigated the interaction of RGDPEG-avidin with HUVEC, which express αvβ3-integrin when being at a subconfluent state. To determine binding affinity of RGDPEG-avidin for αvβ3-integrin, we studied its capability to compete with the αvβ3-integrin ligand 125I-echistatin. While RGDPEG-avidin completely displaced 125I-echistatin from the cells, similar to the free RGD-peptide, avidin did not affect binding of the radiolabeled peptide (Fig. 3a). The sigmoid displacement curve with RGDPEG-avidin (Fig. 3b) demonstrated a 2.9-fold increase in affinity (IC50 = 134 nM) as compared to the single RGD-peptide (IC50 = 383 nM). Fig. 3Association of RGDPEG-avidin with endothelial cells. Subconfluent HUVEC were incubated with radiolabeled echistatin, which is a known ligand for αvβ3 integrin, and RGDPEG-avidin, free RGDpeptide or unmodified avidin. Panel A: Binding specificity. Echistatin was displaced by excess of RGDPEG-avidin or free RGD-peptide (both at 10 μM) but not by excess of avidin. *P < 0.05. Panel B: Affinity of binding. Echistatin binding was displaced by different concentrations of the added competing ligands, showing the increased affinity of RGPEG-avidin over the single RGDpeptide ligand Preparation and characterization of biotinylated TRAIL Since the modification of side-chain residues may diminish the activity of a therapeutic protein, we studied two different biotinylation strategies that target chemical modifications at different amino acid residues, i.e., at lysine groups (biotin-NHS) or methionine residues (biotin-ULS). Such different labeling positions at the surface of the protein may correlate to differences in pharmacological activity of the biotinylated proteins. Anti-biotin ELISA (Fig. 4a) demonstrated a higher biotin/TRAIL modification of biotin-NHS over biotin-ULS, which is in line with the lower abundance of methionine residues as compared to the number of lysine residues. We tried to confirm these data by analysis of the biotinylation extent by MALDI-TOF mass spectrometry, which provided reliable data on the average number of incorporated RGDPEG units in avidin. Although this approach succeeded for biotin-NHS-TRAIL (Fig. 4b), demonstrating the incorporation of about 1 biotin group per subunit of TRAIL, MALDI-TOF analysis failed to detect biotin-ULS modification of TRAIL. Most likely, the mass ionization step induced the dissociation of biotin-ULS from the protein. From the collective results, we concluded that both strategies had yielded biotinylated TRAIL with approximately 1–3 biotin groups per protein (summarized in Table 1). Fig. 4Characterization of biotinylated TRAILs. Panel A: Anti-biotin ELISA with streptavidin-HRP. Signals were corrected for background and expressed relative to the maximum detected intensity. Panel B: MALDI-TOF analysis of TRAIL and biotin-NHS-TRAILTable 1Characteristics of biotinylated TRAIL variantsRelative biotin content (Biotin/TRAIL)aActivity in cell viability assay (IC50 in ng/ml)bActivity in caspase 3/7 assayc (%)TRAIL–40100Bio-NHS-TRAIL1.612335Bio-ULS-TRAIL0.860126aExpressed as mole biotin bound per mole of trimeric TRAIL. Relative biotin content was determined by MALDI-TOF analysis (bio-NHS-TRAIL) or ELISA (BIO-ULS-TRAIL)bAs determined after 48 h of incubation with Jurkat cellscAs determined after 4 h of incubation at 10 ng/ml with Jurkat cells We studied complexation of biotinylated TRAIL with RGDPEG-avidin that had been coated in 96-well plates. Highest binding was observed for Bio-NHS-TRAIL, probably due to its higher biotin content, while bio-ULS-TRAIL showed approximately 40% less binding (Fig. 5). Unmodified His-TRAIL showed non-specific binding to RGDPEG-avidin as well, despite the absence of biotin groups in this product. Non-specific binding of TRAIL to RGDPEG-avidin was however far lower than binding to unmodified avidin, most likely due to the PEGylation (data not shown). Fig. 5Association of biotinylated TRAIL with RGDPEG-avidin. 96-well plates were coated overnight with 50 ng RGDPEG-avidin and incubated with indicated compounds for 2 h. TRAIL binding was assessed by anti-TRAIL immunodetection and expressed relative to the highest signal obtained with biotin-NHS-TRAIL. Experiments were performed in triplicate Cytotoxic and proapoptotic activity of TRAIL complexes To test whether our products were functionally active, i.e., capable of inducing cell death and apoptosis, we assessed their activity on Jurkat T leukemic cells and HUVEC endothelial cells. Figure 6a shows the dose-dependent cytotoxic effects at Jurkat cells after treatment for 48 h. Both His-TRAIL, Bio-NHS-TRAIL and Bio-ULS-TRAIL effectively killed Jurkat cells with IC50 values between 40 ng/ml and 123 ng/ml (Table 1). While the Bio-NHS modification resulted in a considerable reduction in activity compared to the unmodified protein, Bio-ULS-TRAIL displayed almost similar activity as unmodified TRAIL. The pro-apoptotic activity of His-TRAIL and biotinylated TRAIL derivatives in Jurkat cells was furthermore determined by analyzing caspase 3/7 activity, as shown in Fig. 6b. These experiments confirmed the relative higher activity of bio-ULS-TRAIL versus bio-NHS-TRAIL, and the absence of caspase induction by RGDPEG-avidin (data not shown). In contrast to Jurkat cells, no cytotoxic effects were observed when HUVEC were incubated with the products (Fig. 6c). Fig. 6TRAIL activity assays. Panel A: Cell viability of Jurkat T cells after 48 h of incubation with indicated compounds. Cell viability was assessed by MTS assay. Experiments were performed in triplicate. Panel B: Caspase 3/7 activity in Jurkat T cells after 4 h of incubation with 100 ng/ml of the indicated TRAIL derivatives. RLU: relative light units. Panel C: Cell viability (MTS assay) of HUVEC after 48 h of incubation with 100 ng/ml of indicated compounds Finally, we investigated the proapoptotic effects of RGDPEG-avidin:TRAIL complexes in Jurkat cells. These experiments were performed in the presence of endothelial cells, to allow adherence of the RGD-equipped complexes to αvβ3 integrin expressing cell layers. First, we determined the effects of avidin:TRAIL complexes formed in the presence of an excess of RGDPEG-avidin. As can be observed in Fig. 7, such complexes with both types of biotinylated TRAIL showed similar activity to the non-complexed biotinylated TRAIL derivatives (striped bars versus white bars, respectively). Furthermore, in agreement with Fig. 6b, Bio-NHS-TRAIL showed a reduced activity as compared to unmodified TRAIL, while Bio-ULS-TRAIL retained its activity. Fig. 7Proapoptotic activity of RGDPEG-avidin:TRAIL complexes. Assays were performed in 96-well plates seeded with HUVEC that were subsequently incubated at room temperature with RGDPEG-avidin (10 μg/ml) and biotinylated TRAIL (100 ng/ml), to allow assembly of the complexes. Next, Jurkat T cells were added followed by 4 h of incubation at 37°C, after which the activation of caspase 3/7 was determined by a luminescence based assay. Experiments were carried out in the absence of RGDPEG-avidin (control experiment, white bars), in the presence of excess of RGDPEG-avidin (striped bars, 100-fold molar excess over biotinylated TRAIL) or in wells incubated with RGDPEG-avidin that had been washed before addition of biotin-TRAIL and Jurkat cells (black bars). *P < 0.05 versus TRAIL; #P < 0.05 versus biotin-TRAIL in the absence of RGDPEG-avidin A striking result was observed when the excess of RGDPEG-avidin was removed before addition of TRAIL and Jurkat cells, leaving only endothelial-associated RGDPEG-avidin available for complexation with biotinylated TRAIL. This procedure significantly potentiated the proapoptotic effect for both Bio-NHS-TRAIL and Bio-ULS-TRAIL, the latter even beyond the activity of unmodified TRAIL. Discussion In the present study we describe the development and functional evaluation of RGD-avidin:TRAIL complexes. These targeted complexes of TRAIL showed enhanced binding to endothelial cells and maintained their pro-apoptotic activity. As a result of the binding to angiogenic endothelial cells of these complexes, an improved tumor accumulation and enhanced residence within tumor tissue can be expected. Such an improvement in the pharmacokinetic profile of TRAIL may further enhance its therapeutic efficacy and enable a reduction in dosing frequency. αvβ3 integrin is a validated target for drug delivery and imaging approaches directed to angiogenic endothelium. One of the most popular ligands for αvβ3 integrin is the cyclic RGD-peptide cyclo(RGDfK). Due to the special constrained conformation of the cyclic RGD-peptide and the d-phenylalanine amino acid, its binding motif adopts a high affinity conformation for αv-integrins [25]. The cyclo(RGDfK) peptide has been used successfully for the delivery of a wide range of drug or gene carrying systems (e.g., liposomes, polymers, adenoviruses) [18]. Since the cyclopeptide is an unnatural peptide, it can not be encoded by a DNA sequence and requires chemical synthesis and hence chemical coupling techniques to prepare multivalent targeting devices. The present paper describes a versatile approach in which multivalent RGD-avidin is complexed with biotinylated TRAIL. The RGD-peptides are linked to avidin via bifunctional PEG linkers, thereby surrounding the avidin by a hydrophilic PEG shell tethered with targeting ligands. PEGylation is a widely accepted methodology for improvement of therapeutic proteins, and affects both the immunological properties as well as the renal filtration of proteins [26]. The functionalization of avidin with RGD-PEG groups thus will combine several advantageous properties. RGDPEG-avidin displayed nanomolar affinity for αvβ3 integrin expressing endothelial cells, which is in good agreement with the data obtained with other RGD-equipped proteins prepared in our group [27]. We have compared two different biotinylation procedures, which react at different amino acid side chain residues. While biotin-NHS couples at lysine residues, biotin-ULS binds at methionines residues in TRAIL. The methionines in TRAIL are not solvent exposed, as can be derived from the crystal structure of TRAIL [28]. Therefore, the most likely binding site for biotin-ULS is the methionine in the HIS-tag of the recombinant protein. In contrast, most lysine residues are readily accessible, including ones at positions that will interface with the Death receptor. This could explain why Biotin-ULS-TRAIL better maintained its proapoptotic activity than biotin-NHS derivatized TRAIL. Whether the greater loss in activity of biotin-NHS-TRAIL is due to the relative higher extent of derivatization or to modifications of lysine residues in the activity domains of protein can not be concluded from our data. Upon complexation of biotinylated TRAIL with RGDPEG-avidin, a bulky structure has been introduced onto the surface of the therapeutic protein. The assembled complexes, however, fully maintained their TRAIL activity indicating that steric hindrance did not interfere in the TRAIL-death receptor ligation. Figure 7 showed similar proapoptotic activities for either biotinylated TRAIL or the complexes formed in the presence of excess of RGDPEG-avidin. Remarkably, we observed an enhancement of caspase activity when the excess of RGDPEG-avidin was removed, leaving only surface-exposed RGDPEG-avidin at the endothelial cell layer. One of the explanations that may account for this is the assembly of multiple TRAIL molecules onto a single avidin, thereby affording a ‘mega’TRAIL complex with enhanced death-inducing activity, as was also observed for multimeric forms of Fas-ligand [29]. Such types of complexes would facilitate the close assembly of multiple death receptors, thereby potentiating the induction of death signaling. The presence of an excess of RGDPEG-avidin would avoid the formation of such complexes, as both components were added in a 100:1 molar ratio of avidin:TRAIL. Gently washing of the endothelial cells after RGDPEG-avidin incubation removed the excess of unbound RGDPEG-avidin, leaving only cell-associated avidin after incubation at room termperature. These conditions therefore provided an excess of biotinylated TRAIL over avidin, now favoring the formation of multimeric TRAIL complexes. Alternatively, the exposure of TRAIL via RGD-PEG-avidin onto the surface of endothelial cells may explain its increased activity, as has been observed for chimeric TRAIL variants directed to tumor receptors [10]. HUVEC were not killed by TRAIL or its derivatives, as has been described by others [30]. On the other hand, although TRAIL does not induce apoptosis in endothelial cells, it activates other processes such as production of NO and reactive oxygen species which may alter the tumor responsiveness to TRAIL [31]. This can not explain the results shown in Fig. 7, since endothelial cells were preincubated with RGDPEG-avidin only and preincubations were performed at room temperature rather than at 37°C. Such effects of RGD-equipped TRAIL may occur however in vivo. In addition, tumor derived growth factors might sensitize endothelial cells to TRAIL’s apoptosis inducing capacity, similar to the findings reported for TNF-sensitivity [32]. The complexation of biotinylated TRAIL with RGDPEG-avidin and its association with angiogenic endothelial cells may afford an increased accumulation into the tumor blood vessels, similar to the enhanced tumor accumulation of other RGD-modified proteins [18]. An unresolved issue is whether RGD-avidin/TRAIL complexes bound to the tumor vasculature can interact with tumor cells behind the endothelial lining in vivo. To interact with tumor cells and induce tumor apoptosis, TRAIL complexes bound to the tumor vasculature should dissociate and penetrate into the tumor tissue. Several considerations may be important in this respect, such as the expression of αvβ3 integrin on both luminal and abluminal side of the endothelium and the fact that many tumor cells express αvβ3 integrin [18]. Both of these will favor the interaction of RGD-avidin complexed TRAIL with tumor cells. Furthermore, RGD-avidin binding to endothelial cells is less tight than the typical antibody binding. This may favor redistribution of the complexes upon their initial binding to αvβ3 integrin. Homing to the tumor vasculature will provide elevated concentrations within the tumor microenvironment, which may subsequently provide elevated intratumoral concentrations. Together with the observed enhancement of TRAIL activity, this may favor a more efficient induction of apoptosis in tumor cells. To summarize, we have prepared a novel type of RGD-targeted TRAIL with binding specificity for angiogenic endothelium and maintenance of strong cytotoxic activity. Whether in vivo application of this product will lead to an enhanced tumor accumulation still needs to be investigated. We now succeeded in coupling the well known homing device RGD to the TRAIL molecule, without losing the apoptosis-inducing activity of TRAIL or the receptor-binding properties of RDG peptides. An enhanced activity could even be demonstrated in vitro. This may, in combination with an improved pharmacokinetic profile induced by the PEG and RGD moieties, lead to improved effects of TRAIL in vivo. Lastly, the followed synthetic approach offers opportunities for the complexation with alternative targeting ligands that bind directly to tumor cells, such as tumor-specific antibodies or peptide ligands.

Matern_Child_Health_J-2-2-1592161

Promising Practices in Preconception Care for Women at Risk for Poor Health and Pregnancy Outcomes

Objectives: Two programs targeting urban African-American women are presented as promising models for preconception care, which includes interconception care. Methods: The Grady Memorial Hospital Interpregnancy Care (IPC) Program in Atlanta, Georgia, and the Magnolia Project in Jacksonville, Florida, are described. The IPC program aims to investigate whether IPC can improve the health status, pregnancy planning and child spacing of women at risk of recurrent very low birthweight (VLBW). The Magnolia Project aims to reduce key risks in women of childbearing age, such as lack of family planning and repeat sexually transmitted diseases (STDs), through its case management activities. Results: Seven out of 21 women in the IPC were identified as having a previously unrecognized or poorly managed chronic disease. 21/21 women developed a reproductive plan for themselves, and none of the 21 women became pregnant within nine months following the birth of their VLBW baby. The Magnolia Project had a success rate of greater than 70% in resolving the key risks (lack of family planning, repeat STDs) among case management participants. The black to white infant mortality (IM) ratio was better for the babies born to women managed in the Magnolia Project compared to the same ration for the United States. Conclusions: Preconception care targeted to African-American women at risk for poor birth outcomes appears to be effective when specific risk factors are identified and interventions are appropriate. Outreach to women at risk and case management can be effective in optimizing the woman's health and subsequent reproductive health outcomes. Introduction Current national level statistics demonstrate the potential for preconception care to improve the health of women and impact their reproductive health outcomes in terms of achieving optimally spaced, planned pregnancies and averting adverse birth outcomes. Each year in the United States, approximately 6 million pregnancies result in over 4 million live births. Almost half of all pregnancies are unintended, despite the fact that 62% of the 62 million women of reproductive age use a contraceptive method [1]. Chronic diseases and adverse health behavior known to affect pregnancy outcomes are prevalent among women of reproductive age (15–44 years). In 2002, 9.3% of women of reproductive age had diabetes, and 11.4% of pregnant women smoked tobacco and 10.1% of pregnant women consumed alcohol [2]. The U.S. Public Health Service has designated preconception care as a critical component of prenatal care [3]. Initiating interventions to address a woman's health risks upon her entry into prenatal care is often too late to impact the outcome of pregnancy. For example, if a woman has her first prenatal visit after the first month of pregnancy, it is too late to prescribe folic acid supplementation to help prevent neural tube defects as the neural tube closes by the 28th day of gestation. Likewise, women who are advised to discontinue their use of potentially teratogenic medications at the first prenatal visit may have already sustained damage to the fetus, as most organogenesis is complete by day 56 following conception [4]. Preconception care is the care of women of reproductive age before a first pregnancy or between pregnancies (commonly known as interconception care) to ensure that conditions and behaviors which may pose a risk to mothers and infants are identified and managed. A specific element of interconception care is the identification and reduction of risks indicated by a prior adverse pregnancy outcome. The major components of preconception care are risk assessment (identification of conditions that may be amenable to intervention that adversely affect reproductive health outcomes), education and health promotion, and medical and psychosocial interventions. There are varying concepts of preconception care ranging from a pre-pregnancy planning visit in the months prior to conception to a component of each health encounter for women. Ideally, preconception care would be an integral component of primary and preventive care for women of reproductive age. Presently, the status of preconception care in the United States is far from ideal. Approximately one of six obstetrician/gynecologists or family physicians had provided preconception care to the majority of the women for whom they provided prenatal care [5]. Given the diversity of women of reproductive age, the numerous health and psychosocial issues that impact reproductive health outcomes and the varied settings for delivery of health care, a single ‘best’ model of preconception care for all women is unlikely. Rather, tailoring the delivery of preconception care interventions to women with specific circumstances and in specific care settings will be necessary. This paper describes two programs for delivering preconception care for women at-risk for poor health and pregnancy outcomes. One targets women whose risk status is defined by African-American ethnicity and delivery of a very low birth weight (VLBW; <1500 g) infant and, thus, specifically involves the provision of an interconception intervention. The other program targets women whose risk status is defined by African-American ethnicity and residence in Jacksonville-Duvall County, Florida, and involves the delivery of an intervention with preconception and interconception components. The grady memorial hospital Interpregnancy Care (IPC) program Background Georgia ranks among the 10 states with the highest feto-infant mortality rate. The largest contributor to Georgia's comparatively high feto-infant mortality is the delivery of VLBW infants. VLBW delivery comprises only 2% of births in Georgia, yet accounts for approximately 50% of feto-infant mortality. African-American women in Georgia have 3–4 times the rate of VLBW delivery compared to whites. Approximately 66% of the observed racial disparity in feto-infant mortality rates between African-Americans and whites can be attributed to the higher rate of VLBW delivery among African-American women [6]. There is a close link between the poor health status of women and VLBW delivery [7–17]. The poor health status of women in Georgia is estimated to account for approximately 60% of Georgia's excessive infant mortality [18]. With each subsequent VLBW delivery, there is an increased chance that the woman's next pregnancy will result in recurrence of VLBW delivery [19]. In fact, the best predictor of whether a woman will have a VLBW delivery is her history of a previous VLBW delivery, with the rates of recurrence after a single VLBW delivery being approximately 1.5 times higher for African-American compared to white women (13.4% vs. 8.2%, respectively, for non-teen mothers; 26.8% vs. 16.4%, respectively, for teen mothers). The reason for recurrence of VLBW delivery is likely that aspects of the woman's pre-existing health status—including untreated or poorly managed medical problems and unaddressed nutritional, social, and behavioral risk factors—that may have contributed to delivery of the VLBW delivery persist after delivery and in subsequent pregnancies. In 1998 the Georgia Task Force on Perinatal Care was convened to make recommendations for reducing Georgia's overall feto-infant mortality rate and racial disparities in feto-infant mortality. From its findings as outlined above, the Task Force specifically recommended that interpregnancy care be initiated and evaluated for women at risk for having recurrence of VLBW delivery. The Grady Memorial Hospital Interpregnancy Care (IPC) Program was initiated in response to the recommendation of the Task Force. Target population African-American women residing in Fulton or DeKalb counties, Georgia who qualify for county-supported indigent care services and who deliver liveborn or stillborn VLBW infants at Grady Memorial Hospital in Atlanta are considered eligible for the IPC program. Program description The Grady Memorial Hospital IPC program provides 24 months of integrated primary health care and dental services through enhanced nurse case management and community outreach via a Resource Mother. The Resource Mother is a layperson who is trained by the Fulton County Health Department using a specific set of objectives to facilitate life skills and health education acquisition to support high-risk women. The nurse case manager offers all women who deliver a VLBW (stillborn or liveborn) infant at Grady Memorial Hospital enrollment in the program during or soon after their delivery admission. Women who choose to enroll in the IPC program have their initial home visit with the Resource Mother within 1–2 weeks of discharge from the delivery admission; they are scheduled for their initial IPC clinical evaluation at a clinic within Grady Memorial Hospital at 4–6 weeks postpartum. At the initial IPC clinical evaluation, the IPC program's family physician or nurse- midwife performs a comprehensive survey of medical, obstetrical, nutritional, psychological, and social issues (using standardized assessment tools); a thorough physical examination including pelvic exam; and laboratory evaluations to screen for anemia, nutritional deficiencies, sexually transmitted diseases (STDs) and reproductive tract infections. Standard postpartum care issues are addressed in the initial IPC clinical evaluation, thus eliminating a separate postpartum appointment. As part of the evaluation, the participant and provider explicitly discuss a care plan for the 24-month period of the program. For each woman, the care plan addresses the following seven areas epidemiologically linked to LBW delivery: 1) Pregnancy intendedness and child-spacing through the provision of health education concerning the importance of achieving at least a 9-month (and preferably an 18-month) interpregnancy interval, assisting the woman to articulate her own reproductive plan and select a corresponding contraceptive method; 2) Management of chronic disease (if present) through the promotion of self-care and adherence to scheduled appointments in Grady Health System that are facilitated by nurse case management; 3) Screening and treatment for nutritional deficiencies; 4) Prevention, screening, and treatment for STDs and reproductive tract infections; 5) Treatment and referral for substance abuse (if present) including linkage with rehabilitation programs for illicit substance abuse, and support in and linkage with existing programs for tobacco and alcohol abuse; 6) Screening and treatment or support for depression, psychosocial stressors, and domestic violence; 7) Prevention, screening and treatment for periodontal disease. Subsequent visits to the IPC clinic are offered every 1–3 months, depending upon the extent and severity of the woman's health and social issues. Elements of the care plan are addressed at each subsequent visit to the IPC clinic. Peer group learning experiences are integrated with IPC health care visits and are modeled on the Centering Pregnancy concept of group prenatal care [20]. Resource Mother support services are focused on identification and management of psychosocial stressors, and life skills enhancement, including parenthood preparedness, safe housing, skills training, employment acquisition, and relationship issues. Resource Mother support is offered at least twice monthly in the form of home visits and telephone contact. Participants are able to contact the Resource Mother via her cell phone to request support, if needed. Providers Primary care and outreach services are delivered by a multidisciplinary team comprised of a family physician, an advanced practice nurse trained as both a nurse midwife and a family nurse practitioner, a periodontist, a nurse case manager, and a Resource Mother. Social workers affiliated with Grady Health System also support the IPC program and interface with the IPC team. Outcome objectives The purpose of the Grady Memorial Hospital IPC program is to investigate whether interpregnancy care can improve the health status, pregnancy planning and child spacing of women at risk of recurrent VLBW delivery. Findings will contribute to the field of primary health care of reproductive age women in several important ways: (a) the content of a successful IPC package for improving the health of high-risk women will be explored; (b) the concept of IPC will be tested as a means of improving attainment of desirable interpregnancy intervals and decreasing the occurrence of subsequent adverse pregnancy outcomes for high-risk women; (c) the cost of providing IPC to high-risk women will be studied. Funding The health care services rendered to IPC participants were provided through the services of the Grady Health System. Funding for the research and evaluation components of the IPC Program, including Vasser-Wooley Foundation, Healthcare Georgia Foundation, Centers for Disease Control and Prevention (CDC), Rockdale Foundation, and March of Dimes. Results from the first year of the IPC program Enrollment During November 2003 through March 2004, the feasibility phase of the IPC program was initiated by enrolling the first sequential 29 eligible women who gave their informed consent for participation. During the enrollment period, a total of 47 women delivered VLBW infants at Grady Memorial Hospital. Of these 47 women, nine were not African-American or did not reside in Fulton or DeKalb counties, four declined enrollment, three who wished to be contacted afterdischarge from the hospital were unable to be contacted, and two were discharged from the hospital within 24 h and before being offered enrollment. Participation Twenty-one of 29 enrolled women (72.4%) successfully completed the initial 12 months of the planned 24 months of follow-up. During the first 12 months, eight women became disenrolled from the program two moved out of state (and informed us of their move), three electively disenrolled (two before prior to the initial home visit and the initial IPC clinical evaluation; one after a single IPC clinic visit), and three became lost to follow-up (two before the initial IPC clinical evaluation; one after a single IPC clinic visit and four home visits) all of whom who had noted problems with cocaine abuse in their medical records during their pregnancy. Outcomes A detailed program description and final results for all evaluated outcomes for the full 24 months of follow-up will be published when all follow-up and outcome ascertainment is complete (anticipated June 2006). Important findings related to the health status of women in the IPC intervention cohort who had at least two visits during the first 12 months of IPC are given below:Chronic diseases that were previously unrecognized or poorly managed were identified for 7/21 participants, and include valvular heart disease (1/21), sickle cell anemia (1/21), hypertension (2/21), diabetes (1/21), asthma (1/21), systemic lupus erythematosus (1/21), prolactinoma (1/21), panic attacks (1/21), and generalized anxiety disorder (1/21);Reproductive tract infections were diagnosed and treated for 15/21 participants;Iron-deficiency anemia was diagnosed and treated for 5/21 participants;Concerns about finances, employment, and needs of the child are almost universal stressors;Average annual outpatient charges per participant for IPC are $1,801 (average 4.6 visits, $389 per visit). Important findings related to the reproductive outcomes during the first 12 months of IPC are as follows:With extensive case management and patient education, 21/21 women who had at least two visits during their first 12 months of IPC, stated a reproductive plan for themselves and initiated a contraceptive plan in accordance with their stated reproductive plan. Despite a stated desire to either avoid or delay a pregnancy (21/21 women), a significant number of barriers to effective contraception existed and were dealt with, including misinformation about contraceptive methods and reproduction, concerns about side effects of contraceptive methods, and perceptions of partners’ desires regarding child bearing and contraception.All of the women who had at least two visits during their first 12 months of IPC (21/21) achieved at least a nine-month interpregnancy interval (i.e., none have become pregnancy within nine months of the index VLBW delivery). For the 21 actively participating women, pregnancy ascertainment involved ongoing contact with the women and reviewing the Grady Healthy System medical record system for any evidence of health care visits or laboratory tests in which a pregnancy was diagnosed, incidentally noted, or treated. No pregnancies are known to have occurred within nine months of the index VLBW delivery for the eight women who became disenrolled, for which pregnancy ascertainment involved telephone follow-up, where possible (two women), and review of the Grady Health System medical record system. It is possible that those women with whom contact was lost have had a pregnancy for which no care was sought or care was sought outside of Grady Health System. Barriers In the United States, a major barrier to studying the impact of interpregnancy care on the health status and reproductive outcomes of high-risk women has been the lack of financial coverage for the health care component of the IPC intervention. For the IPC program in Atlanta, the Grady Health System has provided the two years of primary health care and treatment of all identified diseases for all enrollees. Specific barriers exist for retaining participation in the IPC program. Women with substance abuse problems who do not enroll in formal substance abuse treatment programs are difficult to maintain in the IPC program. These women seem to have poor insight into the effects that substance abuse has on their repeated poor pregnancy outcomes. For some enrolled women, receiving health care services is less of a priority than securing employment, which negatively influences their health care seeking behaviors. Next steps Upon conclusion of the planned 24 months of follow-up, the final results for all evaluated outcome objectives from the feasibility phase will be reported. The IPC program is currently seeking grant support to conduct a randomized clinical trial to test the hypothesis of whether IPC can decrease the occurrence of subsequent adverse pregnancy outcomes for pregnancies conceived within 18 months of a VLBW delivery at Grady Memorial Hospital. The project plans to enhance participant retention by offering more opportunities for life skills enhancement, job training, and delivery of health care services via the community-based Grady Neighborhood Health Centers. The magnolia project Background In 1998, infant mortality rates in Jacksonville-Duval County, Florida were increasing while statewide rates were declining. The Northeast Florida Healthy Start Coalition, Duval County Health Department and other community partners undertook a community assessment and identified racial disparities as a major factor contributing to the city's high infant mortality. The Coalition utilized the Perinatal Periods of Risk (PPOR) and findings from its Fetal & Infant Mortality Review (FIMR) project to analyze linked birth and death data. The results of this analysis indicated the need for interventions that focused on the health of women, particularly African-American women, before conception. The Coalition used this information to apply for federal Healthy Start funding under a 1999 initiative to address racial disparities in birth outcomes. This funding was used to establish The Magnolia Project, which adapts selected Healthy Start program models to at-risk African-American women of childbearing age who are not pregnant, but sexually active and likely to become pregnant. The project is implemented in a five-zipcode area of Jacksonville-Duval County that accounts for over half of all African-American infant mortality and about 30% of African-American births. Target population The Magnolia Project targets African-American women ages 15–44 living in a socioeconomic high-risk area of Jacksonville-Duval County. The project focuses on women of childbearing age who are likely to become pregnant and have some identified risk factor associated with poor birth outcomes. The project also maintains a small caseload of pregnant women (<10% of patients). Program description The Magnolia Project provides outreach, case management, risk reduction, support, well-woman prenatal care, health education, and community development. The project engages high-risk women through an empowerment model that promotes improved wellness and health, rather than future childbearing. Clinical services and health education are available to all eligible women in the target area. The project offers intensive case management to a subset of women who have specific risk factors identified through FIMR, including previous fetal or infant death or delivery of a LBW infant; repeated STDs; lack of family planning; substance abuse; first pregnancy before age 15; and lack of access to health care. The project's intensive case management is a collaborative team approach that supports participants based on a comprehensive assessment and a client care and goal plan. The plan is developed in collaboration with each participant and reflects her choices, preferences, and goals for coordination of services and activities that address identified risk factors associated with infant mortality. Case management services include:A comprehensive assessment of each participant's abilities and needs at program entry. This assessment includes completion of a problem checklist; screening for depression and stress; and evaluation of self-esteem. Participants are re-assessed periodically based on need and progress to determine resolution or recurrence of problems and risks, achievement of personal goals, and improvements in coping skills and self-esteem.Participant care and goal plan. This plan addresses the needs and preferences of each participant. The written plan is a collaborative process involving the participant and the case manager. It addresses health and social risks and outlines specific steps that the participant must follow to improve her health and well-being and to meet her goals for social, economic, and personal development.Ongoing monitoring and service coordination. The case management team monitors services to ensure that quality care is being provided, participants are addressing their care plans, and participants who are referred to another agency follow through and receive appropriate services. The risk profile (problem checklist) and other measures (stress, self-esteem) are updated at regular intervals (at 6 and 9 months).Anticipatory guidance, health education and advocacy. Case management staff addresses participant risk factors, ensures compliance with services, and promotes achievement of personal goals. Educational support groups are organized monthly. Women receive an incentive ($50 gift certificate) for each six months that they remain in the program, provided they have made progress in addressing their goals. Providers The clinical component of the program is staffed by a nurse-midwife and related nursing, lab and support staff. The program has a full-time health educator who provides a brief counseling intervention on key issues (e.g., use of multivitamins, nutrition, douching, and safe sex) to every woman who comes into the clinic. The clinic serves approximately 800 women annually. A team that includes a nurse, a social worker and two specially trained paraprofessionals provides intensive case management to about 75 women a year. Community development and outreach staff, as well as members of the Magnolia Project Community Council, who are indigenous neighborhood leaders, conduct outreach and community awareness activities. The project uses a collaborative, multi-agency staffing model. The Northeast Florida Healthy Start Coalition is the grantee and project administrator. Subcontractors include the Duval County Health Department (clinical care and case management) and Shands Jacksonville, a tertiary care and teaching hospital (case management and outreach). Staff is co-located at a storefront community site within walking distance of six public housing complexes. The University of North Florida Center for Community Initiatives serves as project evaluator. Outcome objectives The project addresses all the outcome objectives required by the Maternal and Child Health Bureau of the Health Resources and Services Administration (HRSA) regarding birth outcomes (LBW, VLBW, infant mortality, adequacy of prenatal care, etc.). However, because the project primarily focuses on women who are not pregnant, it has developed specific outcome objectives that examine its success in reducing key risks associated with poor outcomes through its case management activities. These include lack of family planning and STDs. Specific outcome objectives have been developed to measure the program's success in addressing participant risks identified at program entry and closure. Funding The Magnolia Project is primarily funded through a grant from the federal Healthy Start program ($925,000). Additional funding is provided through Medicaid and other third- party reimbursement for prenatal, family planning and STD services; state categorical funding through the health department (about $250,000 a year); and smaller community grants for special projects. Total 2004–2005 budget was just over $1 million. Results to date From 2001 to 2005, Magnolia Project clients were predominantly African-American (83%) and single (82%). The average age of participants was 25 years old. More than half (55%) had less than a high school education. Based on patient history and assessment, case management participants had an average of 7.9 risk factors or problems (current or history of) that could potentially impact a pregnancy. Seventy-two percent of all current problems were social problems (alcohol use, drug abuse, depression, family planning, housing, stress, and domestic violence). The remainder were medical problems (anemia, diabetes, hypertension, poor nutrition, previous poor outcome, STDs). The most common risk factors addressed in case management were family planning issues, education/training, bacterial vaginosis, repeated STDs, stress, and poor nutrition. Table 1 identifies frequency of participant risks/problems.Table 1Frequency of risks/problems, case management participants, The Magnolia Project, July 2001–May 2005 Risk factor/problemPercentage with history (n=1214)Percentage with current (n=1727)Social problems Family planning issues2643.5 Job placement8.233.0 Education/training11.327.3 Stress8.721.7 Housing4.115.7 Domestic violence7.211.3 Depression8.211.1 No source of care6.78.7 Lack of exercise7.08.7 Injury prevention/safety2.88.0 Drug abuse10.06.7 Transportation4.45.9 Sexual abuse7.04.4 Alcohol abuse6.44.1Medical problems Bacterial vaginosis12.930.2 Poor nutrition17.819.3 Repeated STDs12.416.5 Tobacco use13.215.5 Breast health3.911.3 Douching8.510.8 Abnormal pap9.59.5 Overweight11.69.5Source: Magnolia Project database; compiled by UNF Center for Community Initiatives, August 2005. Two-hundred-forty-seven participants received at least three months of case management and were closed to care because they completed their care plans, voluntarily withdrew or were lost to service. For key risk factors, case management participants were most successful in resolving or managing issues related to domestic violence (68%) and poor nutrition (63%). The project was least successful in addressing substance abuse by participants (31%). In October 2004, the HRSA Office of Performance Review conducted a site visit and assessment of Magnolia Project outcomes and activities. Its report noted that participants receiving case management services from 2001 to 2003 successfully addressed two priority risks: 86% of participants with family planning issues were consistently using a method at closure; 74% of participants with repeated STDs had no recurrent STDs at closure. Barriers Insufficient funding has limited expansion of the Magnolia Project. Existing funding streams focus primarily on pregnancy, not women's health. The Magnolia Project has successfully cobbled together categorical funding and Medicaid reimbursement for most clinical services, however, in Duval county, the delivery of most categorically funded services remains siloed (e.g., STDs, primary care, prenatal care). Additionally, case management/risk assessment is not universally accepted or valued in the medical model of health care, even though many of the risk factors associated with poor outcomes are social, rather than medical, in nature. More longitudinal evaluations are needed to determine the impact of preconception intervention on pregnancy outcomes. Longitudinal evaluations are not easily incorporated into service delivery projects that rely on short-term outcomes for continued funding. Finally, although outcomes have improved in the target area since the implementation of the Magnolia Project, infant mortality rates in the overall community remain high. The project is unlikely to affect these rates because of its comparatively small reach. Next steps The Magnolia Project is aggressively seeking funding for a longitudinal evaluation. Current program evaluation is limited to an assessment of how well the project is achieving short-term objectives and objectives set by the funding agency (primarily focusing on birth outcomes for the small number of pregnant women served by the project). The CDC recently provided support to the Duval County Health Department for the initial design and piloting of tools for a longitudinal assessment of case management participants served by the project. Additionally, the project is working to interest local providers in replicating the intervention in other at-risk areas in Jacksonville-Duval County. Funding for replication is a significant challenge. Discussion The Grady IPC program and the Magnolia project use a classic public health model of care. They identify women at known risk for an adverse outcome and attempt to alter the woman's risk status to improve outcomes. These programs deliver aspects of preconception care to women at risk for poor health and pregnancy outcomes. The programs do, however, target different categories of high-risk African-American women and utilize different sites for contacting and interfacing with them. The IPC program identified women at risk based on race/ethnicity, qualification for charity care (based on financial status and geographic residence in two counties of metropolitan Atlanta) through Grady Memorial Hospital, and a prior poor birth outcome (VLBW delivery). The IPC program enrolled women soon after their VLBW delivery and provided interconception care in the clinical setting of the hospital (with community outreach). The Magnolia project serves women at risk based on race/ethnicity and residence in Jacksonville-Duval County, Florida. It provides preconception care in a community-based storefront setting with enhanced interconception care for women with a previous LBW delivery or a previous fetal or infant death. Access to the Magnolia project is enhanced by offering evening clinics and walk-in Wednesdays. Both programs deliver services via a multidisciplinary team approach, though there are differences in the exact composition of the team. Inherent in the team approach of each program is attention to participants’ physical and psychological health and social well-being. The programs emphasize several comparable intervention strategies, including community outreach via lay persons; psychosocial support; group education and health promotion modeled on the Centering Pregnancy philosophy of care; and provision of health services. Both programs also emphasize the provision of family planning services for helping women achieve intendedness of any subsequently conceived pregnancy. Each program offers unique strategies as part of the interconception intervention. Because of the link between periodontal disease and preterm delivery (almost all VLBW deliveries are preterm), the IPC program incorporates the services of a periodontist to screen and treat women. To specifically enhance particpants’ life skills, the IPC program utilizes a layperson as a Resource Mother who has undergone a tailored training program through the local department of health. A unique feature of the Magnolia Project's approach is its reliance on indigenous neighborhood leaders (e.g., Magnolia Project Community Council) and community-based organizations to provide outreach, community awareness, and education. The Magnolia Project also has established a partnership with the Ryan White III program for STD/HIV/AIDS screening and treatment. Both programs have had success in accessing and providing services to the specific target group of women. Women who choose not to avail themselves of the services seem to have greater socioeconomic issues, such as substance abuse and lack of housing, jobs, and childcare. Due to the magnitude of some of the problems, maintaining contact is challenging and care often is not continuous. Existing literature supports the strategy of addressing family planning to improve pregnancy outcomes. Preterm delivery, the underlying factor responsible for most LBW and VLBW deliveries, is the leading cause of neonatal mortality and among the leading causes of infant mortality. Haas and colleagues examined whether a woman's health status and risk factors before pregnancy are associated with her risk of preterm birth, independent of risk factors that occur during pregnancy. They found that, after adjusting for sociodemographic characteristics and prepregnancy and pregnancy risk factors, women who reported poor physical function during the month before conception were nearly twice as likely to deliver preterm. The authors concluded that a broader focus on the health of women before pregnancy might improve rates of preterm birth [21]. In a related editorial, Goldenberg suggests that reducing smoking, depression, and stress; treating periodontal disease and genital tract infections; and reaching a normal weight might all be necessary to substantially reduce the risk of preterm birth [22]. Goldenberg advocates for a Medicaid mandate that women who have had an adverse pregnancy outcome be eligible for continued medical coverage in the postpartum period to reduce risk factors and improve health status before the next pregnancy. Data from the 1999 PRAMS survey indicate that the prevalence of unintended pregnancies in the United States ranges from 34 to 52% with 27 to 36% of these pregnancies being mistimed, and 6 to14% being unwanted [23]. In the United States, a clear causal association between unintended pregnancy and poor pregnancy outcomes has not been established. However, women with unwanted pregnancies are at greater risk for poor pregnancy outcomes than women with wanted pregnancies. Hogue asserts that one of the key approaches to prevention of high-risk pregnancy is assuring that the pregnancy is consciously desired, with adequate attention to preconception care [24]. Both programs demonstrate several components essential to any successful preconception care program: identification of risk factors amenable to change by the target population; tailored interventions for the target population; integration of preconception care into existing services; incorporation of family planning counseling and clinical services, health education, and community outreach. Preconception care aims to promote the health of women of reproductive age before conception and thereby improve pregnancy-related outcomes. Both the IPC program and the Magnolia Project show promise in achieving this goal for their target audiences.

J_Membr_Biol-2-2-1764598

Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years. Introduction Hearing relies on faithful synaptic transmission at the ribbon synapse of auditory hair cells. Figure 1 shows an electron micrograph of a typical hair cell ribbon synapse. The synapse is characterized by the presence of a large presynaptic organelle, the synaptic ribbon or dense body, that tethers synaptic vesicles and is itself anchored to the active zone. The performance of this synapse is remarkable in several aspects. Figure 1Hair cell ribbon synapse. Electron micrograph of an inner hair cell afferent synapse of an 8-week-old mouse. The ribbon (rib) is anchored to the presynaptic plasma membrane and faces the postsynaptic density (PSD) of the auditory nerve fibers. The arrowhead and the star indicate a ribbon-associated synaptic vesicle and a free cytoplasmic coated vesicle, respectively. PHASIC RELEASE PROPERTIES The synapse’s precision to code the temporal fine structure of acoustic stimuli is unparalleled. For example, our capacity to locate sound sources in space depends on interaural time differences in the arrival of sound of only tens to hundreds of microseconds. Unlike a conventional synapse that is driven by action potentials, the hair cell afferent synapse responds to graded changes in membrane potential. While the action potential results in a strong all-or-none stimulus-secretion coupling, the hair cell afferent synapse is faced with the challenge of encoding sounds of extremely different intensities and, hence, must rely on mechanisms that allow for variable stimulus-secretion coupling. At the same time, the hair cell afferent synapse must also maintain temporal fidelity. For example, the firing of an auditory nerve fiber can follow at a constant phase relationship the periodicity of tonal stimuli into the low kHz range of frequencies, and we infer that the hair cell neurotransmitter release must comply with this extraordinary timing. Such temporal precision in synaptic transmission is even observed with sub-threshold stimuli, where the temporal pattern of spike firing regularizes already before there is an overall increase in the discharge rate. TONIC RELEASE PROPERTIES As we all can tell from our own experience, the cochlear hair cell afferent synapse must be able to encode prolonged, ever-present sounds. For example, despite adaptation, the constant background noise of the room’s air handling system is faithfully reported by the peripheral auditory system. To meet this requirement of audition, sustained neurotransmitter release must be maintained and implicates the need for extensive synaptic vesicle cycling (both, vesicle exo- and endocytosis). The hair cell afferent synapse’s capacity for sustained signal has often been attributed to the presence of the synaptic ribbon, and apparent depot of synaptic vesicles. However, recent morphological and physiological studies suggest additional/different roles for the hair cell synaptic ribbon, and have fuelled the structure-function debate at the ribbon synapse. Molecular Anatomy and Physiology of the Hair Cell Synapse We are still far from a comprehensive understanding of the molecular composition of the presynaptic active zone and the postsynaptic density of the hair cell afferent synapse. Homologies between retinal and inner ear sensory ribbon-type active zones are evident (Safieddine & Wenthold, 1999). Given the larger number of receptor cells in the retina (millions of photoreceptors vs. thousands of coding hair cells) and relative accessibility of the visual end organ, a more advanced molecular description is available presently for retinal ribbon synapses (e.g., tom Dieck et al., 2005) and promises to guide our understanding of auditory and vestibular ribbon synapses. For example, ribbons in both organs contain RIBEYE (Schmitz, Konigstorfer & Sudhof, 2000; Zenisek et al., 2003; Khimich et al., 2005), Bassoon and Piccolo (Khimich et al., 2005; tom Dieck et al., 2005). RIBEYE is thought to be a structural component of the ribbon but may also have enzymatic activity at the synapse (Schmitz et al., 2000). The photoreceptor ribbon also contains the close RIBEYE homologue and transcriptional co-repressor C-terminal binding protein 1 (tom Dieck et al., 2005). A putative role for this enzyme’s lysophosphatidic acid acyl-CoA transferase activity in membrane fission has been proposed (Valente et al., 2005, but see for an opposing view Gallop, Butler & McMahon, 2005). Some hint for a ribbon function in membrane fission may be seen in the accumulation of cisternal membrane profiles in ribbon-deficient inner hair cells (IHCs) (Khimich et al., 2005). However, any hypothesized enzymatic role for this ribbon constituent in synaptic transmission will require further investigation. Bassoon, like Piccolo, is a large cytomatrix molecule found at the active zone, and most likely contributes to the anchoring of the ribbon at the active zone (Dick et al., 2001; Dick et al., 2003; Khimich et al., 2005). Deletion of a major part of the Bassoon gene (Altrock et al., 2003) results in the lack of synapse-anchored ribbons at most active zones of mouse IHCs. Moreover, the amplitude of a readily releasable pool of IHC synaptic vesicles is decreased, and both synchronous afferent synaptic transmission and hearing are impaired (Khimich et al., 2005). In addition, a minor reduction in the IHC Ca2+ current is observed, potentially reflecting impaired insertion of Ca2+ channels at the active zone due to the Bassoon and/or ribbon deficiency. Although less severe than the complete synaptic dysfunction resulting from the lack of CaV1.3 channels (Brandt, Striessnig & Moser, 2003), the loss of ribbons causes a synaptopathic hearing impairment with the previously described audiological signature of an auditory neuropathy (preserved cochlear amplification but impaired auditory evoked potentials). Interestingly, hearing was found intact in a Piccolo mutant (Sendin and Moser, unpublished data), but some immumoactivity was observed in the Synapses of the mutants. Perhaps the failure of the Piccolo mutation to impair hearing reflects a sufficient abundance of residual functional Piccolo protein in the mutant mouse or the action of some unknown compensatory mechanism. Hair cells express the SNARE proteins syntaxin 1, SNAP-25 and synaptobrevin 1 (Safieddine & Wenthold, 1999). These proteins are thought to build the key exocytic machinery, as their cleavage by clostridial neurotoxins shuts off exocytosis in other systems (e.g., Xu et al., 1998; Sakaba et al., 2005). The effects of clostridial neurotoxin action on hair cell afferent synaptic transmission remain to be investigated. Hair cell afferent synapses, like photoreceptor synapses, lack synapsins (Favre et al., 1986; Safieddine & Wenthold, 1999), a class of proteins that are involved in synaptic vesicle mobilization. Moreover, synaptotagmins 1 and 2, which are considered the calcium sensors of vesicle fusion in the central nervous system and neuroendocrine cells (Sudhof, 2004), have not been observed in hair cells (Safieddine & Wenthold, 1999). Genetic ablation of Synaptotagmin 7 in mice, which is expressed in IHCs (Safieddine & Wenthold, 1999) and involved in exocytosis of hippocampal neurons (Virmani et al., 2003), did not result in a hearing impairment (Nouvian and Moser, unpublished data). Thus, taken together, these findings suggest that either other synaptotagmin isoforms or other proteins altogether may act as the calcium sensor at the hair cell afferent synapse. Despite some homologies, IHC ribbon synapses differ molecularly from retinal ribbon synapses. Most prominently, IHC transmitter release and sound coding are controlled by CaV1.3 Ca2+ channels (Platzer et al., 2000; Brandt et al., 2003; Brandt, Khimich & Moser, 2005), while visual signalling relies on stimulus-secretion coupling by retinal CaV1.4 channels (Bech-Hansen et al., 1998; Strom et al., 1998; Mansergh et al., 2005). Some vestibular hair cells, in addition, may involve non-L-type Ca2+ channels (Rodriguez-Contreras & Yamoah, 2001). Evidence obtained by various techniques in various species all suggests that CaV1.3 channels cluster at the ribbon-type active zones of the hair cell. These include loose-patch recordings (Roberts, Jacobs & Hudspeth, 1990), cell-attached recordings (Rodriguez-Contreras & Yamoah, 2001), Ca2+ imaging (e.g., Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003) and immunohistochemistry (Sidi et al., 2004; Brandt et al., 2005). Estimates of the total number of Ca2+ channels per active zone have been obtained in frog saccular hair cells (∼90 channels, Roberts et al., 1990; but see alsoRodriguez-Contreras & Yamoah, 2001) and mouse cochlear IHCs (∼80, Brandt et al., 2005) by ensemble fluctuation analysis. This similarity in channel number is unexpected given the size differences between the active zones of the two species (see below and Table 1) and highlights how much more we have to learn about this intriguing synapse. Table 1Size and kinetics of release components related to the synaptic ribbon and to morphological vessel pools associated from different species Classical work by Robert’s and Fettiplace’s groups on intracellular calcium dynamics at the plasmalemma face of hair cells advocated the view that calcium domains were micromolar in concentration and micrometer in dimension. This work was based largely on computational models and studies of calcium-activated potassium channel gating, but was assumed to be also applicable to hair cell afferent synapse exocytosis. However, more recent experiments on the stimulus-secretion coupling in mouse IHCs suggested that exocytic fusion of a given readily releasable vesicle is controlled only by few nearby Ca2+ channels (Brandt et al., 2005). These authors argued that such a “nanodomain” control would support the temporal precision of coding also for weak sounds. Additional experiments using complimentary techniques are needed to further test this provocative hypothesis. Rim binding protein, interacting with the active zone protein RIM and the CaV1.3 channel, is currently the best characterized candidate for a molecular link of channel and release site of a given vesicle (Hibino et al., 2002). However, functional evidence is still lacking and it remains possible that other proteins, e.g., scaffolding proteins of the cytomatrix of active zones like Bassoon or Piccolo, contribute. It is also possible that the synaptic ribbon itself is responsible for positioning of the CaV1.3 channel and readily releasable vesicles (Khimich et al., 2005). Clearly, molecular work, physiological analysis of mutants and particular immunoelectronmicroscopy are required to characterize the molecular interactions and the ultrastructural topography at the active zone. Figure 2 illustrates the subcellular localization of a few synaptic proteins. Figure 2Molecular composition of the hair cell synapse. (A) Montage of a Nomarski image (note the hair cell bundles) and a confocal reconstruction of the mouse organ of Corti with immunolabeled ribbons (stained for RIBEYE/CtBP2, red) and postsynaptic transmitter receptor clusters (GluR2/3 glutamate receptor subtypes, green). Presumptive hair cell border (white) and postsynaptic fibers (orange) contacting an IHC were drawn illustration. (B) Previously identified molecular determinants of the hair cell ribbon synapse. 1Safieddine & Wenthold, 1999; 2Furness & Lawton, 2003; 3Eybalin et al., 2002; 4Schmitz et al., 2006; 5Khimich et al., 2005; 6Platzer et al., 2000; 7Brandt, Striessnig & Moser, 2003; 8Brandt, Khimich & Moser, 2005; 9Matsubara et al., 1996; 10Eybalin et al., 2004. Besides a molecular coupling, other mechanisms contribute to localized Ca2+ signalling. The presence of the mobile, proteinaceous Ca2+ buffers calretinin, calbindin and parvalbumin has now been documented in a variety of cochlear and vestibular hair cells (e.g., Edmonds et al., 2000; Heller et al., 2002; Hackney et al., 2005). Their relevance for presynaptic Ca2+ signalling has been inferred from recordings of Ca2+-activated large-conductance K+ channels (Fettiplace, 1992; Roberts, 1993; Tucker & Fettiplace, 1996; Edmonds et al., 2000), simulations (Roberts, 1994) and capacitance measurements (Moser & Beutner, 2000; Spassova et al., 2004). Most likely they spatiotemporally restrict the presynaptic Ca2+ domains and, hence, improve the timing of synaptic transmission. However, discrepancies regarding both the amount and kinetic properties of buffers in different hair cells (Roberts, 1993; Edmonds et al., 2000 vs Moser & Beutner, 2000) suggest that our understanding of calcium buffers at the hair cell active zone synapse is far from complete. Examination of hair cells from mutant mice lacking the major proteinaceous Ca2+ buffers promises further insight into this important question. In addition to synchronous synaptic transmission, the molecular machinery needs to be suited for long-lasting transmitter release. By yet unknown mechanisms CaV1.3 currents in hair cells are much less inactivating than in heterologous expression systems (Koschak et al., 2001). Most likely this is due to interactions with a special combination of auxiliary subunits and proteins, such as the class of Ca2+ binding proteins (Haeseleer et al., 2004). None of these auxiliary determinants of hair cell Ca2+ current are known so far. Once the neurotransmitter is released into the synaptic cleft, it activates an AMPA-like receptor residing within a receptor cluster at the postsynaptic density of afferent auditory nerve fibers. In a classical study, Ottersen and his colleagues showed the abundance of the glutamate receptor subtypes (GluR) 2, 3 and 4 by immunogold labelling (Matsubara et al., 1996). Interestingly, the distribution of the glutamate receptor was not homogenous in the postsynaptic density (PSD): the receptor numbers increase at the border of the PSD. Using patch-clamp recordings from the postsynaptic endings contacting rat IHCs, Glowatzki & Fuchs (2002) showed that excitatory postsynaptic currents of spiral ganglion neuron afferent fibres are mediated by AMPA receptors. They failed to detect NMDA or Kainate currents. Recently, Eybalin and colleagues studied the developmental expression changes of postsynaptic glutamate receptors using western blotting, RT-PCR and immunohistochemistry (Eybalin et al., 2004). They showed that GluR2 becomes expressed only around the onset of hearing, while GluR3 and 4 are present before. Little is known about the other molecular determinants of the PSD. Davies et al. (2001) report the presence of PSD-95 and PSD-93 in the PSD of auditory nerve fibres. To avoid saturation and excitotoxicity due to an excess of glutamate in the synaptic cleft, glutamate is taken up by glutamate transporters (EAAT) into supporting cell transporters (Furness & Lehre, 1997; Furness & Lawton, 2003; Rebillard et al., 2003). Interestingly, glutamate-aspartate transporters (GLA ST) are more abundant on the pillar IHC side, where high spontaneous rate fibers are located, compared to the modiolar cell side, where low spontaneous rate fibres are located (Liberman, 1982; Furness & Lawton, 2003). Relating Structural and Functional Vesicle Populations MORPHOLOGY Synaptic Ribbons The active zone of the hair cell is characterized by an electron dense structure called synaptic body or ribbon. Most synaptic ribbons found in mature hair cells are anchored to the plasma membrane, one ribbon per active zone, and oppose a postsynaptic terminal. A small number of “floating” ribbons (<5 %) are observed, and probably reflect the turnover of these subcellular organelles (Zenisek et al., 2004; Khimich et al., 2005). Shape, size and number of the ribbons differ (see Table 1) between species as well as along the tonotopic axis, during development (Shnerson, Devigne & Pujol, 1981; Sobkowicz et al., 1982; Khimich et al., 2005) and even among different active zones within a single hair cell (Merchan-Perez & Liberman, 1996). For example, frog saccular hair cells display about 20 large, synaptic bodies irrespective of the cells’ location in the end organ (Roberts et al., 1990; Lenzi et al., 1999, 2002). Whereas in the chick cochlea, the number of synaptic bodies is constant in tall hair cells (functional homologue of the mammalian IHCs), the synaptic body diameter increases with increasing characteristic frequency (Martinez-Dunst, Michaels & Fuchs, 1997). Conversely, the number of ribbons varies along the tonotopic axis in many other species including turtle (plus small size differences: Sneary, 1988; Schnee et al., 2005), cat (Liberman, Dodds & Pierce, 1990), gerbil (Slepecky et al., 2000) and guinea pig (Hashimoto, Kimura & Takasaka, 1990). Both the number and shape of the synaptic ribbons can also change with the onset of hearing. Mouse IHCs contain a large number (>20 in the apical cochlea, Nemzou et al., unpublished data) of small synaptic bodies (Shnerson et al., 1981; Sobkowicz et al., 1982) before the onset of hearing. After the onset of hearing only fewer but larger ellipsoid ribbons are observed (Shnerson et al., 1981; Sobkowicz et al., 1982; Francis et al., 2004; Khimich et al., 2005). An interesting feature described for cat IHCs is the spatial segregation of synapses with fibres of different spontaneous rates and thresholds. Thus, low-spontaneous-rate, high-threshold fibres contact the IHCs on their modiolar side, whereas high-spontaneous-rate, low-threshold fibres synapse on the pillar IHC side. EM analysis showed that the ribbon size and accordingly the number of ribbon-associated synaptic vesicles tend to increase with decreasing spontaneous rate of the contacted fibres (Liberman, 1980; Liberman et al., 1990; Merchan-Perez & Liberman, 1996). Synaptic vesicles Ribbons tether a monolayer of synaptic vesicles at a packing density that is ∼55% of maximum by 20 nm long filaments (Lenzi et al., 1999). These ribbon-associated vesicles represent only a tiny fraction of the huge number of synaptic vesicles contained in a hair cell. At least three vesicle populations can be morphologically distinguished at the hair cell ribbon synapse as illustrated by Figure 3:Docked vesicles (no discernible space between vesicle and plasma membranes)Ribbon-associated vesiclesFree cytosolic synaptic vesicles. These vesicle populations have been quantified for hair cells of various species (Table 1). A variety of approaches have been used to assess the vesicle populations and accordingly, varying degrees of certainty surround these vesicle counts. The different approaches have included: precise three-dimensional EM counts based on few synapses (EM tomography: Fig 3, Lenzi et al., 1999; Lenzi et al., 2002; reconstructions based on ultrathin serial sections: Schnee et al., 2005); extrapolations from random ultrathin EM sections of tens of synapses (Khimich et al., 2005) and approximations based on ribbon surface areas and vesicle packing density estimates (Spassova et al., 2004; Khimich et al., 2005: more than 200 ribbons analyzed). Figure 3Morphology of synaptic ribbons. (a, b and c) Electron micrographs of inner hair cell ribbon synapses of 8-week-old mouse. Ribbons are attached to the plasma membrane and surrounded by a monolayer of synaptic vesicles. Colored dots in (b) illustrate the morphologically defined classes of synaptic vesicles: red dots indicate the docked vesicle associated with the ribbon; yellow marks ribbon- associated SV which are not docked and green indicates outlying cytoplasmic vesicles. (d) Slice through an osmium-stained frog saccular hair cell synapse, reconstructed by electron tomography. Note the round shape of the synaptic body in contrast to the elipsoid one of the mouse. (e) Three-dimensional structure of presynaptic organelles from the same reconstruction shown in (d). The synaptic body (blue) is open and lies adjacent to the hair cell’s plasma membrane in red. Synaptic body-associated vesicles (yellow) surround the SB; outlying vesicles (green) lie further out in the cytoplasm. Also visible are coated vesicles (gold) and cisterns (purple). Docked vesicles at a conventional synapse are viewed as a homogeneous anatomical pool. However, at the hair cell ribbon synapse docked vesicles are found in a variety of anatomical states; this includes underneath the ribbon (docked and ribbon-associated), at the active zone without contact to the ribbon, and outside the active zone. Lenzi et al., (1999) termed these last two classes of docked vesicles as “outlying”. The functional implications of the different classes of docked vesicles are poorly understood. Obviously, proximity to a calcium channel increases the likelihood that a docked vesicle is of functional significance. Accordingly, Lenzi et al., (1999) attempted to correlate the positions of docked vesicles and Ca2+ channels at the active zone. While the “outlying” vesicles are substantial in number, they are located further from the presumed calcium channel containing presynaptic densities than are other docked vesicles. “Outliers” has been alternatively used in evanescent wave microscopic studies (Zenisek, Steyer & Almers, 2000) to describe the “docking” and fusion of synaptic vesicles outside of functionally defined active zones. PHYSIOLOGY One fruitful way to investigate the compact afferent presynaptic terminal of the inner ear is to monitor exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (Cm) using the patch-clamp technique (for review seeNeher, 1998). During the fusion of the synaptic vesicle to the plasma membrane the cell surface and consequently Cm increase. This then is usually followed by slower fission accompanying Cm decrease. This technique has become increasingly popular to study the presynaptic function of hair cells in various species (Table 1). Upon depolarization, calcium influx, mainly through CaV1.3 L-type Ca2+ channels, triggers exocytosis of readily releasable synaptic vesicles. Depending on the stimulus intensity and duration, the resulting Cm increase may report exocytosis occurring at all active zones as well as the fusion of synaptic vesicles outside of active zones or even the fusion of non-synaptic membrane organelles (Coorssen, Schmitt & Almers, 1996). This lack of specificity associated with capacitance measurements can complicate its interpretation. At least two kinetic components of exocytosis are commonly discriminated in the recordings of hair cell membrane capacitance following stimuli thought to trigger exocytosis. Such findings from a variety of species are also summarized in Table 1. The first kinetic component of these membrane capacitance increases mediates exocytosis at extremely high rates but saturates within a few milliseconds. Hence, it is generally agreed that this component represents exocytosis of a small, finite pool of vesicles. Because it is the fastest discernible and exhaustible component of exocytosis several authors denominated it the readily releasable vesicle pool (RRP, Moser & Beutner, 2000; Spassova et al., 2004; Rutherford & Roberts, 2006) following the classical terminology of functional vesicle pools (Liley & North, 1953; Birks & MacIntosh 1961; Elmqvist & Quastel, 1965). Three major findings indicate that the first kinetic component is, indeed, involved in synaptic sound-coding by hair cells:Correlation between the RRP size and the number of afferent synapses made by a hair cell as it varies along the tonotopic axis of the cochlea (Schnee et al., 2005).Correlation between the RRP size and the number of synapse-anchored ribbons (Khimich et al., 2005).Correlation between the RRP size and the amplitude of the spiral ganglion compound action potential (reflecting synchronous activation of spiral ganglion neurons) (Khimich et al., 2005). Several other lines of evidence support the notion that the first kinetic component of exocytosis represents a synaptic relative pool of vesicles. Resistance of the readily releasable pool to inhibition by exogenous slow calcium buffers infers a co-localization of this functional pool with Ca2+ channels (Moser & Beutner, 2000; Spassova et al., 2004). Calcium channels are widely believed to be concentrated at hair cell active zones (e.g., Roberts et al., 1990; Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003; Sidi et al., 2004; Brandt et al., 2005). Nanodomain control of synaptic vesicle exocytosis (Brandt et al., 2005) requires an intimate spatial relationship between the readily releasable pool and calcium channels. There is also good correspondence between the quantal content predicted by measurement of action potential-driven exocytic Cm changes in immature IHCs (Beutner & Moser, 2001; Johnson, Marcotti & Kros, 2005) and direct measures of postsynaptic currents from the afferent fiber (Glowatzki & Fuchs, 2002). Finally, additional supportive evidence comes from comparison of the timing of peripheral auditory adaptation and the kinetics of RRP depletion and recovery. Adaptation is a common property of sensory systems. It reduces encoding redundancy by decreasing the sensitivity to constant stimulation, is proposed to optimize a sensory system’s operating range (Koutalos & Yau, 1996) and maximize its information transfer (Brenner, Bialek & de Ruyter van Steveninck, 2000). In the peripheral auditory system, adaptation is likely localized to the cochlear hair cell afferent synapse because hair cell receptor potentials do not adapt to maintained acoustic stimulation (Holton & Weiss, 1983; Russell & Sellick, 1978). Previous work (Furukawa & Matsuura, 1978; Moser & Beutner, 2000) suggested that the exhaustion and replenishment of the readily releasable pool of vesicles in the hair cell contribute to a fast form of adaptation and recovery from it in the auditory nerve (Westerman & Smith, 1984; Yates, Robertson & Johnstone, 1985). Parsons and colleagues (Spassova et al., 2004) employed both in vitro patch-clamp recordings of hair cell synaptic vesicle fusion and in vivo single-unit recording of cochlear nerve activity at the same synapse to further examine this hypothesis and probe the possible auditory significance of the hair cell ribbon synapse structure-function relationship. Similar to other hair cell preparations, exocytosis of the chick cochlear tall cell’s readily releasable pool is fast, saturating in < 50 ms, and its recovery is also rapid, regaining 95% of its initial amplitude following a 200 ms period of repolarization. Interestingly, sound-evoked afferent synaptic activity also adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. This work supports the view that the fast exocytic component is of synaptic relevance by providing evidence that exhaustion and replenishment of the readily releasable pool mediates behaviorally relevant phenomena — namely short-term auditory nerve adaptation and its recovery from adaptation. In an elegant two-photon imaging study Ashmore and colleagues utilized the turnover of the membrane dye FM1-43 to track the exocytosis of synaptic vesicles at hair cell active zones. Their findings on the readily releasable pool of the adult guinea pig IHCs were similar to descriptions provided in other species by capacitance measurements. The significance of these findings is that the imaging experiments, unlike the capacitance measurements, are spatially resolved and lend credence to previous interpretations of the first exocytic component. Optically recorded exocytosis was extremely fast at the onset of a depolarization (initial rate of 3,000 vesicles per second) and saturated with a time constant of 30 ms in mature guinea pig hair cells (Griesinger, Richards & Ashmore, 2005). This intial rate is comparable to the initial rate of exocytosis evoked at one ribbon synapse of mature mouse IHCs by maximal stimulation amounting to ∼6,000 vesicles per second (after Cm conversion into vesicle numbers using 28 aF/vesicle). Moreover, estimates of the time course of recovery from depletion obtained by the two methods were quite similar, too (Table 1). Both the amplitude and kinetics of the first kinetic component in mature mouse and guinea-pig hair cells were found to depend on the stimulus strength (Brandt et al., 2005; Griesinger et al., 2005). This is in contrast to a study on frog saccular hair cell exocytosis where the kinetics, but not amplitude of exocytosis, varied with stimulus amplitude (Edmonds, Gregory & Schweizer, 2004). The reasons for this discrepancy among the different experiments are currently unclear. Moser and colleagues have suggested the Ca2+ nanodomain control of RRP exocytosis in mouse IHCs (Brandt et al., 2005). This hypothesis predicts that an increase in intensity of stimuli would recruit more calcium channel-release site units, presumably increasing the amplitude of the RRP without major changes of release kinetics. Classical quantal analysis at the goldfish hair cell synapse (Furukawa, Kuno & Matsuura, 1982) also revealed intensity-dependent increases in the number of available vesicles (as opposed to changes in the release probability). While the synaptic relevance of the first kinetic component is indisputable, clearly a detailed mechanistic understanding of it awaits further experimentation. Our current knowledge about the further kinetic component (s) is much more limited. This is mainly due to the lack of spatial resolution of Cm recordings of exocytosis, which report fusion to the plasma membrane no matter whether it happens at the synapse or elsewhere. The slower kinetic components are typically triggered by prolonged, strong depolarizing steps (tens of ms to s in duration) and have the potential to drive calcium levels within the cell to levels that could not be achieved with more physiological time-varying, smaller-amplitude stimuli. The relative resistance of the CaV1.3 calcium channel to inactivation allows for the influx of large numbers of calcium ions during prolonged depolarizations that may be sufficient to saturate cellular calcium buffers. Clearly, aphysiologic calcium gradients within the hair cell promise to further compound the limitations of membrane capacitance measurements and cloud the interpretation of secondary, slower exocytic components. Since the first Cm measurements of hair cell exocytosis (Parsons et al., 1994), we have been astonished and puzzled by the huge amount of membrane turnover during prolonged depolarization. During the first second of depolarization thousands of vesicles are released (see Table 1). A membrane turnover of more than one equivalent of the initial hair cell’s surface has been reported for maximal stimulation over several seconds (Schnee et al., 2005). In this respect, the hair cell’s exocytic capacity exceeds that of retinal photoreceptors and bipolar neurons. This difference between retinal cells and hair cells is most dramatic when examining the fast exocytosis evoked by Ca2+ uncaging. A uniform step increase in calcium concentration through the cell’s cytoplasm triggers the exocytosis of all fusion-competent vesicles. Flash photolysis of caged calcium in the goldfish bipolar nerve terminal results in an average capacitance increase of ∼150 fF (Heidelberger et al., 1994), whereas a similar stimulus in the mouse IHC drives a 10-fold larger increase in membrane capacitance (Beutner et al., 2001). This discrepancy in the amount of exocytosis is even more remarkable given that a hair cell contains nearly 4 times fewer, but similar-sized and vesicle-populated synaptic ribbon active zones as the retinal bipolar terminal (von Gersdorff et al., 1996; Khimich et al., 2005). Hence we have to either assume that the turnover at a hair cell ribbon synapse is 40 times faster than that of a bipolar neuron or to conclude that hair cells contain an extremely large pool of fusion-competent vesicles that reside outside of the active zone. Spatially-resolved exocytic measurements, such as imaging or conventional post-synaptic recordings, will be required to ascertain the contribution of the hair cell’s large pool of fusigenic vesicles to synaptic relevant exocytosis. Is it Possible at all to Relate Anatomically and Physiologically Defined Pools of Synaptic Vesicles? It would be a significant breakthrough in our understanding of the hair cell ribbon synapse if we managed to arrive at a joint anatomical and physiological definition of synaptic vesicle populations. This goal requires relating physiologically derived capacitance or fluorescence changes to anatomically defined synaptic vesicle numbers. Unfortunately, several technical issues limit the accuracy and certainty of this pursuit. Estimations of single-vesicle capacitance are required to determine the equivalent number of vesicles associated with a given Cm increase. Surface calculations based on the vesicle diameter in fixed tissue (possibly correcting for a potential shrinkage bias) and an assumption of the specific membrane capacitance (usually 10 μF/μm2, Breckenridge & Almers, 1987) are commonly employed. Both are prone to errors complicating this structure-function approach. The inherent problems with the fixation artefacts are highlighted by the factor of 2 discrepancy between “in vitro” single-vesicle capacitance values derived from anatomical studies (37 aF, Lenzi et al., 1999 and 28 aF, Khimich et al., 2005) and the more direct “in vivo” estimates obtained by electrophysiology in other systems (∼50 aF, Klyachko & Jackson, 2002 and 65 aF, Sun, Wu & Wu, 2002). The physiological approach to pool definition also faces technical limitations. The rate of the first or fastest kinetic component is limited by the bandwidth and signal-to-noise ratio of the recording equipment. Much confusion in the retinal bipolar terminal literature stems from the initial definition of a readily releasable pool with a time constant of ∼100 ms (von Gersdorff & Matthews, 1994) prior to the subsequent discovery of a 5-fold smaller, but nearly 50-fold faster kinetic component of exocytosis (Mennerick & Matthews, 1996; Neves & Lagnado, 1999). At the chick hair cell synapse, which features some of the smallest-numbered and -sized synaptic ribbons, the total number of docked vesicles is ∼100. Exocytosis of these vesicles would generate a capacitance increase perhaps as small as 3 fF, at or below the resolution of whole-cell capacitance measures and likely obscuring the observation of this pool that is readily measured in cells with large ribbon structures. What Is the Anatomical Substrate of the Fast Kinetic Component in Hair Cell Exocytosis? Several caveats described above constrain our ability to answer this question. However, recognizing these limitations and uncertainties, it remains an important and interesting question underlying our understanding of the hair cell afferent synapse. The recent literature provides two alternative hypotheses: it may preferentially draw on readily releasable vesicles docked at the plasma membrane of the active zone (Moser & Beutner, 2000; Khimich et al., 2005; Schnee et al., 2005; Rutherford & Roberts, 2006) or involve release of all ribbon-associated vesicles (Edmonds et al., 2004; Spassova et al., 2004). Table 1 highlights these views. The first hypothesis is consistent with independent fusion of the individual vesicles, similar to what is expected for docked vesicles at the active zone of conventional synapses. The second hypothesis requires the rapid recruitment and fusion of non-docked ribbon-associated vesicles. As active vesicle transport seems too slow (see discussion in Edmonds et al., 2004), different modes of exocytosis, such as compound and “piggy back” (cumulative) fusion, have been considered (Parsons & Sterling, 2003; Edmonds et al., 2004; Spassova et al., 2004). Functional evidence for multivesicular release at the hair cell ribbon synapse was first obtained by Glowatzki and Fuchs (2002). Using direct post-synaptic recordings at rat afferent synapses, they observed large variations in the amplitude of kinetically quite homogeneous postsynaptic currents, as well as clear composite events characterized by multiple quanta (Glowatzki & Fuchs, 2002). Clearly, the exact presynaptic release mechanism of vesicle release is likely to have great impact on the transfer characteristics of the hair cell synapse and remains an extremely important problem for continued study. This review cannot solve the debate over the anatomical basis of the hair cell fast exocytic component. However, we aim to fuel the on-going discussion with some selected comparisons of data complied from various studies and summarized in Table 1. Any considerations of structure-function comparisons are complicated by the fact that the physiological pool size estimates obtained by different groups can vary substantially even for hair cells of the same species (Edmonds et al., 2004; Rutherford & Roberts, 2006, see discussion in Rutherford’ paper). Moreover, the morphological vesicle counts or estimations vary dramatically in their degree of precision (from direct counts in electron microscopical 3-D reconstructions to approximations based on ribbon size, see Morphology section above and Table 1). In addition, we need to remember that our capacitance-based approximations probably overestimate the number of vesicles due to underestimation of single-vesicle capacitances (due to the shrinkage during chemical fixation, see above). With these caveats in mind, studies on hair cells of the frog (work from the Roberts lab) and the mouse (work from the Moser lab) provide the most compelling structure-function correlations, in part because both the anatomy and physiology were carried out in the same laboratory. In each case, their observations support the hypothesis that for these particular hair cells RRP is defined by the fusion of docked vesicles (Table 1). Correcting the conversion factor for shrinkage would tend to still improve the match between RRP size and docked vesicle number. The question still lingers as to whether non-docked vesicles can be released rapidly at ribbon synapses. Zenisek et al. (2000), using evanescent wave microscopy of single FM1-43 stained vesicles in retinal bipolar nerve terminals, provide some of the most direct evidence addressing this question. They reported that the first component of exocytosis was dominated by “resident” (probably docked) vesicles at the hot-spots of exocytosis, whereas arrivals or “newcomers” contributed only marginally within the first milliseconds. This argues against a major contribution of non-docked, ribbon-associated vesicles. On the other hand, Griesinger et al. (2005) concluded from two-photon imaging of basolateral hot-spots of FM1-43 fluorescence that rapid exocytosis of non-docked vesicles does, indeed, occur at hair cell ribbon synapses. A certain degree of caution, however, is required in interpreting either of these studies, as the best resolution of an optical microscope (200 nm) is comparable to the size of most synaptic ribbons. The apparent differences between the behavior of ribbon-tethered vesicles in these two types of ribbon synapses highlight the fact that it could be an oversimplification to assume that a common anatomical substrate underlies the first/fastest recorded kinetic component of hair cell exocytosis. The physical dimensions of hair cell active zones, shapes of synaptic ribbons, best stimulation frequency, and possibly endogenous calcium buffering vary dramatically across end organ, species and in some cases even within the same end organ of a given species. Thus, subsequent studies may reveal that different hair cells use different anatomical substrates for different functional pools of synaptic vesicles to meet their respective synaptic demands. What about the Anatomical Substrate of Subsequent, Slower Kinetic Components of Exocytosis? Things are still more complicated for the further kinetic component(s) of exocytosis reported by capacitance measurements. Most studies concur that replenishment of readily releasable vesicles occurs at high rates, exceeding those of conventional synapses and even retinal ribbon synapses (Moser & Beutner, 2000; Spassova et al., 2004; Edmonds et al., 2004; Griesinger et al., 2005). However, whether the slow kinetic components of exocytosis simply represent the serial refilling of the readily releasable pool and or parallel exocytosis at “ectopic” fusion sites is debatable. Schnee et al. (2005) proposed a purely serial and synaptic model to explain the three kinetic components of the depolarization-induced exocytic Cm changes in turtle hair cells. They inferred that the second component reflected exocytic turnover of the ribbon-associated vesicle population, while vesicle recruitment to the ribbon would become rate-limiting thereafter. Not surprisingly, a similar model was appropriate to describe destaining of FM1-43 hotspots and recovery of staining in the study of Griesinger and colleagues. However, these optical studies differ from the capacitance recordings in the turtle hair cell (Schnee et al., 2005), as they are spatially resolved and focus on membrane turnover at individual active zones. Findings that argue for a significant contribution of ribbon-independent and at least in part parallel extrasynaptic exocytosis to sustained exocytic Cm changes include:Robust sustained exocytosis in hair cells that lack 90% of their synapse-anchored ribbons (Khimich et al., 2005)Presence of docked synaptic vesicles at extrasynaptic stretches of the plasmamembrane (Lenzi et al., 1999, 2002)The extensive fast exocytosis during flash photollysis, which by far exceeds fusion of the readily releasable pool (Beutner et al., 2001), potentially arguing for the presence of many fusion-competent vesicles outside the active zone. In addition, exocytosis of such “outlying” vesicles has been described in retinal bipolar nerve terminals by evanescent microscopy (Zenisek et al., 2000). Hence, taken together, present evidence supports the interpretation that sustained hair cell exocytosis reported by Cm tracking represents both the serial re-supply of vesicles to the active zones and the parallel extrasynaptic turnover of synaptic vesicles. However, the quantitative contribution of the slow exocytic components to synaptically relevant neurotransmitter release remains to be determined. Conclusion and Outlook The multidisciplinary approach to the hair cell ribbon synapse has for the first time quantitatively described important aspects of the synapse’s structure and function. Most importantly, despite all limiting uncertainty, we begin to relate molecules, structure and function. Although still debated, the concept of a readily releasable pool has been substantiated for the ribbon synapse of several hair cells. We have gained a few insights into the mechanisms underlying the incredible temporal precision of synapses that participate in the coding of sound. Moreover, in addition to learning about the molecular physiology, the availability of mouse mutants with defined synaptic lesions provided the possibility to study the properties of synaptopathic hearing impairment in greater detail. However, we are far from a comprehensive molecularly defined model of ribbon structure and function. The molecular dissection of the hair cell synapse is technically challenging due to the low amount of tissue and will require much more time and effort. Ear-specific genetic deletion also will be helpful to investigate synaptic protein function in hair cell sound coding. A more precise and direct biophysical analysis of single hair cell synapses will require combined pre- and postsynaptic recordings as well as optical measurements, such as evanescent wave microscopy and confocal techniques. The optical approach will be strongly facilitated by the generation of genetically targeted fluorescent vesicle tags.

Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764203

Characteristics associated with involuntary versus voluntary legal status at admission and discharge among psychiatric inpatients

Background The objective of this analysis was to determine the ways in which patients’ legal statuses at hospital admission and discharge are associated with select sociodemographic and clinical variables. This study specifically investigated differences between patients who were voluntary during both admission and discharge, patients who were involuntary on admission but voluntary on discharge (having converted to voluntary status during hospitalization), and patients who were involuntary during both admission and discharge. Introduction In the United States, criteria for involuntary psychiatric hospitalization generally require imminent dangerousness to oneself or others, or complete inability to care for oneself [1, 2], though specific criteria vary between states [3]. An important rationale for involuntary psychiatric hospitalization is that patients admitted for evaluation often have severe deficits in their capacities to make treatment decisions [4, 5]. Patients often revise their beliefs about the need for treatment during or after hospitalization. A study conducted by Gardner et al. [6] found that slightly more than half of patients who initially stated (upon admission) that they did not need hospitalization later admitted they indeed had needed treatment when re-interviewed several weeks after discharge. It has been suggested that involuntary treatment may reduce the likelihood that patients will voluntarily seek care in the future [7, 8]. Subsequent aversion to psychiatric services may be due to a distrust of clinicians arising from coercive tactics or a lack of procedural justice (e.g., the patient is treated unfairly, disrespected, ignored, or excluded from the decision-making process) encountered during hospitalization [1, 8, 9]. On the other hand, involuntary hospitalization may provide patients with needed treatment, even if involuntarily, that results in improved outcomes and treatment adherence [10]. Some research indicates that the presence or absence of coercion is accurately reflected by the patient’s legal status, but other studies demonstrate that coercive pressures and perceived coercion do not necessarily correlate well with legal status in all settings [8, 11]. Evidence associating legal status with sociodemographic and clinical variables is sparse and inconsistent. One study found that higher re-admission rates and more unstable discharge living arrangements were associated with involuntary legal status on hospital admission [12]. A meta-analytic review conducted using studies from the 1950s through the early 1980s found that the only significant outcome measure correlated with admission legal status was length of stay [13]. Studies have reached opposing conclusions regarding whether or not male gender is significantly associated with involuntary legal status, especially when controlling for severity of illness [7, 12, 14]. With regard to clinical characteristics, psychotic disorder diagnoses, greater illness severity, and more frequent preadmission assaultive behaviors may be associated with involuntary admission [13]. Even fewer studies have examined factors associated with converting from involuntary to voluntary legal status during hospitalization [14–16]. Cuffel [14] identified greater clinical improvement, less severe diagnoses, and non-minority ethnicity as factors predictive of converting to voluntary status early in the hospitalization. Later during hospitalization, the only factor associated with legal status conversion was having discharge living arrangements involving family and friends. Studies that find significant associations between legal status and outcomes may influence policies relating to resource allocation during hospitalization, discharge planning, and outpatient follow-up [12, 15]. For instance, research characterizing those at risk for very short involuntary hospitalization (i.e., discharge upon expiration of short-term commitment) addresses the need for more thorough and engaged evaluation and treatment while hospitalized. One may be able to enhance treatment adherence with a more focused treatment and evaluation plan, in conjunction with closer follow-up after discharge [12]. Though legal status is often overlooked in studies in which it would be particularly relevant, it is clearly an important variable for further research. The purpose of the present study was to compare three groups of hospitalized psychiatric patients: (1) patients admitted and discharged voluntarily (VOL-VOL), (2) patients admitted involuntarily who later signed-in voluntarily and were discharged voluntarily (INV-VOL), and (3) patients admitted and discharged on an involuntary legal status (INV-INV). As such, this trichotomous criterion variable addressed the change (or lack thereof) in patients’ legal status during hospitalization, allowing an examination of whether or not certain sociodemographic and clinical factors are associated with differences in admission-to-discharge legal status. Treating legal status as a trichotomous variable (rather than solely considering either admission or discharge legal status) may yield meaningful results that add to the current body of research on correlates of patients’ legal status. Methods Subjects and setting This analysis was part of a study aimed at determining predictors of non-adherence with the first scheduled community mental health outpatient appointment following hospitalization [17]. Inclusion in the study was restricted to consecutively discharged patients with a scheduled follow-up appointment at any of 12 participating community mental health settings in a single urban county. Patients re-hospitalized during the course of data collection were not included a second time, in order to maintain independence of observations. This analysis included data that were collected on 227 patients from December 2003 to July 2004. All patients were admitted to the 22-bed inpatient unit or the 8-bed crisis stabilization unit of a large, public-sector hospital in the southeastern United States. Both the longer-stay inpatient unit and the crisis stabilization unit admit patients primarily for evaluation and treatment of first episodes of illness or exacerbations of severe and persistent mental illnesses (primarily schizophrenia and other psychotic disorders and severe affective disorders). This county hospital serves a predominantly low-income, urban, African American population. In the state of Georgia, commitment for involuntary evaluation can be initiated by a licensed physician, psychologist, clinical social worker, or psychiatric clinical nurse specialist. Taking custody for transportation to an emergency evaluation facility also can be carried out by a law enforcement officer. In either case, this initial commitment allows for the individual to be taken to and held at a locked evaluation facility. Commitment criteria include the fact that the person appears to have a mental illness, and either (1) presents a substantial risk of imminent harm to self or others as manifested by recent overt acts or threats of violence presenting a probability of physical injury, or (2) is so unable to care for his/her own physical health and safety as to create an imminently life-endangering crisis [3]. Within 48 h of admission, if the individual continues to meet commitment criteria, a physician may sign for commitment for further evaluation and treatment of up to five business days in duration. During this period of time, some patients will sign-in as voluntary patients. If the patient does not sign-in voluntarily, then he or she is typically discharged after the 5 days, unless commitment criteria clearly continue to be present. In that case, a court hearing is scheduled to determine whether or not the legal standard is met for longer-term involuntary hospitalization. However, in this setting extended commitment is used relatively rarely, and only in circumstances in which the threat to self or others is very apparent. Procedures The majority of data collected were abstracted from patients’ medical charts. Subjectively rated variables (e.g., treatment plan adherence during hospitalization, the treatment team’s opinion of likelihood of follow-up) were obtained by consulting with the social worker, the psychiatry resident or psychology intern, and the attending psychiatrist caring for the patient during hospitalization. As described in two recently published reports, basic demographic, socioeconomic, psychosocial, and clinical data were recorded in a systematic fashion using a standardized data collection instrument [17, 18]. The criterion variable of interest in this analysis was a three-level variable representing the patient’s legal status at admission and at discharge: VOL-VOL, INV-VOL, and INV-INV. The research was approved by the university’s institutional review board and the hospital’s research oversight committee. Analysis Bivariate analyses were performed using a variety of independent variables in relation to the criterion variable. Continuous variables were analyzed using one-way analysis of variance and categorical variables were analyzed using chi-square tests of association. These bivariate analyses were conducted to inform the selection of variables for inclusion in the subsequent multivariate regression analysis. Polytomous logistic regression modeling was required due to the trichotomous nature of the admission/discharge legal status criterion variable. Those patients admitted and discharged voluntarily (VOL-VOL) were designated as the referent group in the regression model. Entering variables into the logistic model was based on significant bivariate test results (P < 0.05). A thorough assessment of confounding was conducted, and relevant confounding variables were controlled for in the model. The backward elimination method was used to identify variables that were independently significantly associated (P < 0.05) with legal status. Appropriate modeling diagnostics were conducted to ensure the fit of the model to the data. Adjusted odds ratios and 95% confidence intervals were computed to determine effect estimates for independent variables that were significantly associated with legal status, controlling for all other covariates in the model. The SAS version 9.1 software (SAS Inc., Cary, North Carolina, USA) was used for all statistical analyses. Results Basic sociodemographic and clinical descriptive statistics for the overall sample are shown in Table 1. Slightly over half (55.5%) of patients were female. The racial composition of the sample is consistent with that of the population served by the two inpatient units, and by the hospital in general. Specifically, 84.9% of patients were Black/African American. At the time of admission, 94.2% of patients were unemployed, and 25.6% reported being homeless. The majority of patients were single/never married (67.8%). The majority of patients had been previously hospitalized for psychiatric treatment (75.1%). An even greater percentage of patients were reported as having psychotic symptoms at the time of admission (85.8%). The mean age was 38.7 ± 12.4 years, and the mean educational level was 12.1 ± 2.3 years. The mean Global Assessment of Functioning (GAF) scale score on admission was 30.3 ± 8.2, ranging from 10 to 55. Table 1Basic sociodemographic and clinical descriptive statistics for the overall sample (n = 227)VariableFrequencyGender, female126 (55.5%)Race    Black/African American191 (84.9%)    Hispanic/Latino4 (1.8%)    White/Caucasian/European American 28 (12.4%)    Other2 (0.9%)Employment status, unemployed213 (94.2%)Homeless on admission 58 (25.6%)Marital status    Single/never married154 (67.8%)    Married/living as married 19 (8.4%)    Separated/divorced/widowed 54 (23.8%)Prior psychiatric hospitalization169 (75.1%)Psychotic symptoms present on admission194 (85.8%)VariableMean ± SDMedianRangeAge 38.7 ± 12.44017–76Years of education 12.1 ± 2.312 4–18GAF score on admission 30.3 ± 8.23010–55 Among the 227 patients, 56 (24.7%) were in the VOL-VOL group, 81 (35.7%) were in the INV-VOL group, and 90 (39.6%) were in the INV-INV group. As shown in Table 2, select demographic and social variables were examined across the three groups. The difference in percentage of patients documented as having Axis IV psychosocial problems in the area of housing was statistically significant (χ2 = 6.43, df = 2, P = 0.04), with patients admitted and discharged voluntarily most likely to have housing problems (60.7%). The percentage of patients who were receiving disability payments—Supplemental Security Income (SSI) or Social Security Disability Insurance (SSDI)—over the past 3 months also differed significantly across the three legal status groups (χ2 = 6.82, df = 2, P = 0.03), with patients admitted and discharged involuntarily most likely to be receiving disability payments (61.1%). Table 2Sociodemographic characteristics of 227 hospitalized patientsVariableLegal status on admission and dischargeaTest statistic (df)P-valueVOL-VOL (n = 56)INV-VOL (n = 81)INV-INV (n = 90)Gender, female32 (57.1%)48 (59.3%)46 (51.1%)1.23 (2)0.54Race, Black/African American45 (81.8%)66 (81.5%)80 (89.9%)2.87 (2)0.24Employed part-time or full-time2 (3.64%)7 (8.64%)4 (4.44%)1.99 (2)0.37Homeless on admission17 (30.4%)23 (28.4%)18 (20.0%)2.48 (2)0.29Current marital    Single/never married32 (57.1%)56 (69.1%)66 (73.3%)4.32 (4)0.36    Married/living as married6 (10.7%)7 (8.6%)6 (6.7%)    Separated/divorced/widowed18 (32.1%)18 (22.2%)18 (20.0%)Age on admission (Mean ± SD)41.0 ± 11.537.0 ± 13.238.8 ± 12.11.73 (222)0.18Axis IV housing problems34 (60.7%)35 (43.2%)36 (40.0%)6.43 (2)0.04Axis IV economic problems32 (57.1%)51 (63.0%)41 (45.6%)5.40 (2)0.07Receiving disability payments over past 3 months (SSI or SSDI)22 (39.3%)40 (49.4%)55 (61.1%)6.82 (2)0.03aLegal status on admission and discharge: VOL-VOL, Patients having voluntary legal status on both admission and discharge; INV-VOL, Patients having involuntary status on admission and voluntary status on discharge; INV-INV, Patients having involuntary legal status on both admission and discharge Table 3 shows select clinical and diagnostic characteristics. The three legal status groups differed significantly on 13 of these 20 variables. The INV-INV group was most likely to have had previous psychiatric hospitalizations (84.3%), followed by 71.6% among the INV-VOL group, and 65.5% among the VOL-VOL group (χ2 = 7.27, df = 2, P = 0.03). Compared to the VOL-VOL group, patients who were admitted and discharged involuntarily were more likely to have a primary diagnosis of a psychotic disorder, to have a change in GAF score from admission to discharge of <20 points, and to have psychotic symptoms on discharge. Patients admitted and discharged involuntarily were less likely to have documentation of medical problems requiring medications at discharge, to be taking >2 psychiatric medications at discharge, and to have good treatment plan adherence during hospitalization compared to the VOL-VOL group. Not unexpectedly, length of stay in days (F = 20.45, df = 224, P < 0.0001) and the inpatient unit to which patients were admitted (χ2 = 32.84, df = 2, P < 0.0001) were highly significantly different across the groups. Table 3Clinical and diagnostic characteristics of 227 hospitalized patientsVariableLegal status on admission and dischargeaTest statistic (df)P-valueVOL-VOL (n = 56)INV-VOL (n = 81)INV-INV (n = 90)Prior psychiatric hospitalization36 (65.5%)58 (71.6%)75 (84.3%)7.27 (2)0.03Psychotic symptoms on admission43 (76.8%)71 (87.7%)80 (89.9%)5.20 (2)0.07Primary diagnosis, schizophrenia or other psychotic disorder26 (47.3%)56 (69.1%)63 (70.0%)9.03 (2)0.01Comorbid substance use disorder diagnosis26 (46.4%)34 (42.5%)40 (44.4%)0.21 (2)0.90Comorbid personality disorder diagnosis10 (17.9%)13 (16.1%)12 (13.3%)0.58 (2)0.75Change of <20 in GAF score (Discharge–Admission)23 (42.6%)26 (32.9%)47 (53.4%)7.14 (2)0.03Depressive symptoms at discharge23 (41.1%)23 (28.4%)22 (24.4%)4.69 (2)0.10Anxious symptoms at discharge23 (41.1%)43 (51.1%)28 (31.1%)8.49 (2)0.01Psychotic symptoms at discharge27 (48.2%)51 (63.0%)67 (74.4%)10.34 (2)<0.01Documented medical problems requiring medication at discharge35 (62.5%)41 (50.6%)32 (35.6%)10.52 (2)<0.01>2 Psychiatric medications at discharge20 (36.4%)13 (16.1%)14 (15.9%)10.40 (2)<0.01Experiencing side effects at discharge7 (12.5%)23 (28.4%)13 (14.4%)7.41 (2)0.02Required seclusion5 (8.9%)11 (13.6%)18 (20.0%)3.52 (2)0.17Required restraints4 (7.1%)8 (9.9%)8 (8.9%)0.31 (2)0.86Required PRN medicine14 (25.0%)30 (37.0%)38 (42.2%)4.48 (2)0.11Patient has an established outpatient clinician15 (26.8%)20 (25.0%)38 (42.2%)6.78 (2)0.03Good treatment plan adherence during week prior to discharge41 (73.2%)60 (74.1%)50 (55.6%)8.06 (2)0.02Treatment team’s opinion of likelihood of follow-up    Poor15 (26.8%)16 (20.0%)35 (38.9%)15.02 (4)<0.01    Fair19 (34.0%)29 (36.3%)38 (42.2%)    Good22 (39.3%)35 (43.8%)17 (18.9%)Length of stay in days (Mean ± SD)11.3 ± 7.515.5 ± 8.48.4 ± 6.020.45 (224)<0.0001Unit, longer-stay inpatient unit36 (64.3%)67 (83.8%)37 (41.1%)32.84 (2)<0.0001a Legal status on admission and discharge: VOL-VOL, Patients having voluntary legal status on both admission and discharge; INV-VOL, Patients having involuntary status on admission and voluntary status on discharge; INV-INV, Patients having involuntary legal status on both admission and discharge The average lengths of hospital stay (in days) across the three groups of patients are displayed in Fig. 1. Patients in the INV-INV group had an average length of hospitalization of 8.4 days, whereas patients in the VOL-VOL group had an average length of stay of 11.3 days. Patients in the INV-VOL group had the longest average length of hospital stay, at 15.5 days. Based on post-hoc tests using the Tukey approach, the difference between mean lengths of stay was significant for the INV-VOL group compared to the VOL-VOL group and for the INV-VOL group compared to the INV-INV group. Fig. 1Average lengths of hospital stay (days) in the three groups of patients Figure 2 illustrates the differences among the three groups in terms of the percentage of patients adhering with the first scheduled community mental health appointment following hospitalization. The average time interval between hospital discharge and the first scheduled community mental health appointment was 14 days and ranged from 0 (i.e., the appointment was scheduled to occur on same day as discharge) to 59 days. (Follow-up was assessed by a single phone call to the community mental health center where the discharged patient had been scheduled for the first outpatient appointment. The follow-up phone call was made a few days after the appointment, and further outpatient adherence was not tracked [17].) Patients in the INV-VOL group were the most likely to adhere with the first follow-up appointment (52.0%), whereas patients in the VOL-VOL group (30.9%) and the INV-INV group (27.1%) were far less likely to adhere (χ2 = 11.75, df = 2, P < 0.01). Fig. 2Percentage of patients adhering with the first scheduled community mental health appointment following hospitalization Based on the significant bivariate test results and a thorough consideration of relevant confounding variables, 18 factors were entered into the polytomous logistic regression model. In this model, three of these factors were independently associated with legal status: psychotic symptoms at discharge, documented medical problems requiring medication, and number of psychiatric medications. Psychiatric inpatient unit, length of hospital stay, prior psychiatric hospitalization, and receiving disability payments (SSI/SSDI) were deemed confounding variables and were, therefore, controlled for in the final model. Because of sparse missing values, modeling results were generated using data from 221 individuals. Table 4 shows model coefficients, standard errors, adjusted odds ratios, and 95% confidence intervals for each of the three significant variables, by legal status category. Table 4Independent variables significantly associated with admission and discharge legal status among 221 hospitalized patientsVariableLegal status categoryaBSEaOR95% CIPsychotic symptoms at dischargeINV-VOL−0.020.440.980.42, 2.32INV-INV1.490.464.421.80, 10.86Documented medical problems requiring medication at dischargeINV-VOL−0.480.400.620.28, 1.35INV-INV−1.510.430.220.10, 0.51Number of psychiatric medications at discharge (>2)INV-VOL−1.800.520.170.06, 0.46INV-INV−0.880.510.410.15, 1.12The model controls for psychiatric hospital unit, length of hospital stay, prior psychiatric hospitalization, and receiving disability payments (SSI/SSDI)a VOL-VOL (voluntary legal status on both admission and discharge) is the referent group for all comparisons; INV-VOL, involuntary legal status on admission, voluntary status on discharge; INV-INV, involuntary legal status on both admission and dischargeB, Coefficient value from the final regression model; SE, standard error; aOR, adjusted odds ratio; CI, confidence interval Patients who were involuntary on admission and discharge (INV-INV) were approximately 4.4 times more likely to have psychotic symptoms at discharge, relative to the VOL-VOL group. Patients in the INV-INV group were also about 4.5 times less likely (or equivalently, 0.22 times as likely) to have documented medical problems requiring medications at discharge, relative to those in the VOL-VOL group. Patients who were involuntary on admission and voluntary on discharge (INV-VOL) were approximately 6 times less likely (0.17 times as likely) to be prescribed more than two psychiatric medications at discharge. Discussion Multivariate logistic regression modeling yielded three clinical variables that were independently significantly associated with admission/discharge legal status, even after controlling for psychiatric unit, length of hospital stay, prior psychiatric hospitalization, and whether or not the patient received disability payments. As described below, the associations between legal status and each of these three significant variables potentially may be explained by the lower overall treatment engagement common among involuntary patients. First, patients in the INV-INV group were much more likely to be experiencing psychotic symptoms at discharge, compared to the VOL-VOL group. The most plausible explanation for this finding relates to a difference in the level of acceptance of treatment between the two groups. Involuntary legal status is generally an indicator of a lack of treatment engagement, resistance to treatment, and/or impaired insight. Bivariate test results for “treatment plan adherence during the week prior to discharge” support the idea that involuntary patients generally had poorer overall treatment adherence. When patients refuse to sign-in voluntarily and no longer meet dangerousness criteria, they may have to leave the hospital before resolution of psychotic symptoms. The abbreviated hospitalization may not have allowed for sufficient time for stabilization and adjustments to psychiatric medications. Due to the laws governing involuntary inpatient commitment in this state, individuals who have been hospitalized for the duration of the short-term commitment and who have refused to convert to voluntary status, must be discharged unless they clearly meet further commitment criteria [3]. On the other hand, patients with voluntary legal status are generally discharged based on clinical, rather than legal, criteria—they are released from the hospital when symptoms have sufficiently resolved and the patient is stable enough to return to the community. A significant difference in psychotic symptoms at discharge was not found between the INV-VOL group and the VOL-VOL group. This is not surprising given the fact that these two groups were virtually equally as likely to be rated as having good treatment adherence during the week prior to discharge. Furthermore, these patients were discharged after sufficient resolution of symptoms rather than legal expiration. From a clinical vantage point, it appears that INV-INV patients are being discharged “too soon” due to legal constraints. An alternate explanation for the difference in psychotic symptoms at discharge among the three groups is that INV-INV patients were more likely to have psychotic symptoms at the time of admission compared to the other two groups. However, the bivariate test revealed no statistically significant difference between the three groups in terms of psychotic symptoms present on admission. Second, patients admitted and discharged involuntarily (INV-INV) were much less likely to have documented medical problems requiring medications at the time of discharge, relative to the VOL-VOL group. Because length of stay was controlled for in the model, the most likely reason for the difference in medical problems at discharge is a generalized lack of treatment engagement among involuntary patients. That is, poor treatment engagement is associated with a decreased likelihood of medical problems being reported and diagnosed. Involuntary patients are more likely to refuse medical history taking, physical examination, vital sign checks, diagnostic blood work, and other diagnostic procedures during hospitalization. Thus, clinicians may not have sufficient opportunities or information to detect common medical problems, such as hypertension, diabetes mellitus, and anemia. It is notable that a significant difference in documented medical problems requiring medication was not found between the INV-VOL and VOL-VOL groups. As noted earlier, these two groups had similar treatment adherence during the week prior to discharge. These results support the idea that converting to voluntary status is associated with better treatment engagement, affording more opportunities for medical problems to be detected and medication to be prescribed. Future research is needed to determine whether a general lack of treatment engagement alone, or some additional factors in combination with poor treatment engagement, accounts for the difference in documented medical problems requiring medication. Some alternate explanations as to why the INV-INV patients had fewer medical problems requiring medication at discharge merit consideration. For example, it is conceivable that even when clinicians detect a medical problem, they may be biased toward not prescribing medication to involuntary patients who are highly unlikely to agree to take it. However, in this particular study setting, when medical problems are detected in involuntary patients, clinicians typically prescribe the indicated medication, regardless of the likelihood that the patient will comply with taking it. Another possible explanation is that patients in the INV-INV group are generally healthier than the other two groups. However, because the three legal status groups were very similar with respect to age, gender, and race, it is highly unlikely that one group of patients would have been healthier than the other two. Third, though the INV-VOL and VOL-VOL groups did not differ with respect to psychotic symptoms and medical problems requiring medication at discharge, these two groups did differ in the number of psychiatric medications at time of discharge. Those patients in the INV-VOL group were much less likely to have been prescribed more than two psychiatric medications at discharge, relative to the VOL-VOL group. The difference in number of psychiatric medications between the INV-INV and VOL-VOL groups was not significant, though the effect for the INV-INV group was in the same direction as with the INV-VOL group. The most plausible explanation for this finding may be related to both a lower overall treatment engagement among involuntary patients and differences in clinician prescribing behavior for involuntarily admitted patients compared to voluntary patients. That is, perhaps clinicians err on the side of caution with patients recently converting to voluntary status by limiting the number of psychiatric medications used to treat symptoms. Clinicians may limit the number of prescribed medications, especially if psychiatric symptoms are resolving, in order to simplify the medication regimen and sustain good treatment adherence. Interestingly, a much higher percentage of patients in the INV-VOL group adhered with the first community mental health appointment compared to the VOL-VOL group and the INV-INV group. It is conceivable that a simpler treatment regimen (one or two psychiatric medications) may contribute to higher rates of adherence with initial community mental health follow-up. Several methodological limitations of this study merit consideration. First, the fact that patients are admitted voluntarily or involuntarily is a legal distinction, which may not accurately reflect the patient’s level of treatment engagement [11, 12, 19, 20]. Patients admitted voluntarily do not necessarily always have the motivation and adherence behaviors of individuals truly pursuing treatment for mental illness of their own accord [11–13]. For example, a voluntary patient who is pressured or coerced by family members to seek inpatient treatment may have motivation, adherence, and insight more similar to patients admitted involuntarily than to those voluntarily seeking treatment [11, 12]. Likewise, some involuntary patients may be more similar to voluntary patients than to other involuntary patients. This might occur when patients who would willingly have signed-in voluntarily are admitted involuntarily in order to overcome obstacles associated with voluntary admission procedures in certain mental health systems [11]. Because legal status may not be an appropriate surrogate for measuring perceived coercion or treatment engagement [5, 8, 11], future research should consider utilizing MacArthur’s Perceived Coercion Scale [21] and a continuous scale to assess treatment engagement. A second methodological limitation is that commitment statutes “censor” the number of patients that convert to voluntary status, since non-converters must be discharged upon expiration of the commitment. Therefore, the true difference between the INV-VOL and INV-INV groups is somewhat obscured because of the limited timeframe in which patients have to convert to voluntary status. One cannot assume that any given INV-INV patient would never have converted to voluntary status; rather one can only infer that he or she had not done so by time of discharge. In other words, caution must be exercised in assigning ontological status to the INV-INV group. A third limitation is that patients re-admitted during the data collection period were excluded from the analysis. Future research might consider focusing on INV-INV patients with multiple hospitalizations. Another limitation with regard to data collection was that clinical and diagnostic variables (some of which were subjectively rated) were based on clinicians’ reports or information in the hospital chart, without using rating scales administered directly to patients. However, given the goals of this study, the researchers deemed patient assessments, beyond that done as part of routine evaluation and treatment, unnecessary. Also, involuntary patients would have been less likely to participate in formal research evaluations compared to voluntary patients, thus restricting the ability of the analyses to examine the issues of interest. Lastly, the findings from this study may have limited generalizability, given the specific sociodemographic and diagnostic characteristics of the sample. However, the population of interest to the researchers was that of minority individuals with severe psychiatric illnesses being treated in a large, urban, public-sector hospital. Despite the methodological limitations inherent in the study design, there were also several strengths in the methodology and data analysis process. First, an extremely thorough assessment of confounding was conducted. The final model controlled for four variables that were considered likely confounders. Second, alternate modeling techniques were conducted, all of which resulted in the same findings. Lastly, a concerted effort was made to minimize missing values during data collection, and to prevent biases, the few variables with significant missing values were excluded from the analysis. Conclusions Research on associations between patients’ legal status and sociodemographic and clinical variables can directly and indirectly affect psychiatric treatment during and after hospitalization. For instance, patients in the INV-INV group have the shortest average length of hospitalization, which is a direct result of the commitment law dictating that patients cannot be involuntarily hospitalized for more than 5 days unless they continue to meet dangerousness criteria [3], even if symptoms have not yet resolved. In addition to being more likely to have psychotic symptoms at discharge, patients in the INV-INV group are less likely to have documented medical problems requiring medication at discharge. Thus, involuntary patients frequently may be discharged from the hospital with unresolved psychotic symptoms and undetected, untreated medical problems. The poor outpatient treatment follow-up rate among INV-INV patients suggests that this is a particularly vulnerable group needing focused attention by the mental health system. During hospitalization, clinicians should be attentive to the manner in which involuntary patients are treated because this can greatly influence patients’ perceptions of coercion. It has been suggested that a concerted effort to reduce the level of coercive practices used during the admission process could mitigate some of the negative effects of coercion, such as future non-adherence with treatment and outpatient follow-up [8]. After discharge from the hospital, involuntary outpatient commitment can be employed to stabilize patients in the community, though this form of coercive treatment is also controversial. Recent research has suggested efficacy of involuntary outpatient commitment in enhancing treatment compliance and reducing re-admission rates among persons with severe psychiatric illnesses [22, 23]. Increased resource allocation for involuntary patients during the admission process and throughout hospitalization, discharge planning, and outpatient follow-up may have the downstream effect of preventing future hospital admissions and worsening of medical comorbidity.

J_Gen_Intern_Med-3-1-2150633

Performance of a Web-Based Clinical Diagnosis Support System for Internists

BACKGROUND Clinical decision support systems can improve medical diagnosis and reduce diagnostic errors. Older systems, however, were cumbersome to use and had limited success in identifying the correct diagnosis in complicated cases. INTRODUCTION The best clinicians excel in their ability to discern the correct diagnosis in perplexing cases. This skill requires an extensive knowledge base, keen interviewing and examination skills, and the ability to synthesize coherently all of the available information. Unfortunately, the level of expertise varies among clinicians, and even the most expert can sometimes fail. There is also a growing appreciation that diagnostic errors can be made just as easily in simple cases as in the most complex. Given this dilemma and the fact that diagnostic error rates are not trivial, clinicians are well-advised to explore tools that can help them establish correct diagnoses. Clinical diagnosis support systems (CDSS) can direct physicians to the correct diagnosis and have the potential to reduce the rate of diagnostic errors in medicine.1,2 The first-generation computer-based products (e.g., QMR—First Databank, Inc, CA; Iliad—University of Utah; DXplain—Massachusetts General Hospital, Boston, MA) used precompiled knowledge bases of syndromes and diseases with their characteristic symptoms, signs, and laboratory findings. The user would enter findings from their own patients selected from a menu of choices, and the programs would use Bayesian logic or pattern-matching algorithms to suggest diagnostic possibilities. Typically, the suggestions were judged to be helpful in clinical settings, even when used by expert clinicians.3 These diagnosis support systems were also useful in teaching clinical reasoning.4,5 Surprisingly and despite their demonstrated utility in experimental settings, none of these earlier systems gained widespread acceptance for clinical use, apparently related to the considerable time needed to input clinical data and their somewhat limited sensitivity and specificity.3,6 A study of Iliad and QMR in an emergency department setting, for example, found that the final impression of the attending physician was found among the suggested diagnoses only 72% and 52% of the time, respectively, and data input required 20 to 40 minutes for each case.7 In this study, we evaluated the clinical performance of “Isabel” (Isabel Healthcare Inc, USA), a new, second generation, web-based CDSS that accepts either key findings or whole-text entry and uses a novel search strategy to identify candidate diagnoses from the clinical findings. The clinician first enters the key findings from the case using free-text entry (see Fig 1). There is no limit on the number of terms entered, although excellent results are typically obtained with entering just a few key findings. The program includes a thesaurus that facilitates recognition of terms. The program then uses natural language processing and search algorithms to compare these terms to those used in a selected reference library. For Internal Medicine cases, the library includes 6 key textbooks anchored around the Oxford Textbooks of Medicine, 4th Edition (2003) and the Oxford Textbook of Geriatric Medicine and 46 major journals in general and subspecialty medicine and toxicology. The search domain and results are filtered to take into account the patient’s age, sex, geographic location, pregnancy status, and other clinical parameters that are either preselected by the clinician or automatically entered if the system is integrated with the clinician’s electronic medical record. The system then displays a total of 30 suggested diagnoses, with 10 diagnoses presented on each web page (see Fig. 2). The order of listing reflects an indication of the matching between the findings selected and the reference materials searched but is not meant to suggest a ranked order of clinical probabilities. As in the first generation systems, more detailed information on each diagnosis can be obtained by links to authoritative texts. Figure 1The data-entry screen of the Isabel diagnosis support software. The clinician manually enters the age, gender, locality, and specialty. The query terms can be entered manually from the key findings or findings can be pasted from an existing write-up. This patient was ultimately found to have a B-cell lymphoma secreting a cryo-paraproteinFigure 2The first page of results of the Isabel diagnosis support software. Additional diagnoses are presented by selecting the ‘more diagnoses’ box The Isabel CDSS was originally developed for use in pediatrics. In an initial evaluation, 13 clinicians (trainees and staff) at St Mary’s Hospital, London submitted a total of 99 case scenarios of hypothetical case presentations for different diagnoses, and Isabel displayed the expected diagnosis in 91% of these cases.8 Out of 100 real case scenarios gathered from 4 major teaching hospitals in the UK, Isabel suggested the correct diagnosis in 83 of 87 cases (95%). In a separate evaluation of 24 case scenarios in which the gold standard differential diagnosis was established by two senior academic pediatricians, Isabel decreased the chance of clinicians (a mix of trainees and staff clinicians) omitting a key diagnosis by suggesting a highly relevant new diagnosis in 1 of every 8 cases. In this study, the time to enter data and obtain diagnostic suggestions averaged less than 1 minute.9 The Isabel clinical diagnosis support system has now been adapted for adult medicine. The goal of this study was to evaluate the speed and accuracy of this product in suggesting the correct diagnosis in a series of complex cases in Internal Medicine. METHOD We considered 61 consecutive “Case Records of Massachusetts General Hospital” (New England Journal of Medicine, vol. 350:166–176, 2004 and 353:189–198, 2005). Each case had an anatomical or final diagnosis, which was considered to be correct by the discussants. We excluded 11 cases (patients under the age of 10 and cases that focused solely on management issues). The 50 remaining case histories were copied and pasted into the Isabel data-entry field. The pasted material typically included the history, physical examination findings, and laboratory test results, but data from tables and figures were not submitted. Beyond entering the patient’s age, sex, and nationality, the investigators did not attempt to otherwise tailor the search strategy. Findings were compared to the recommended (but slightly more time consuming) strategy of entering discrete key findings, as compiled by a senior internist (MLG). Because the correct diagnosis is presented at the end of each case, data entry was not blinded. RESULTS Using the recommended method of manually entering key findings, the list of diagnoses suggested by Isabel contained the correct diagnosis in 48 of the 50 cases (96%). Typically 3–6 key findings from each case were used. The 2 diagnoses that were not suggested (progressive multifocal encephalopathy and nephrogenic fibrosing dermopathy) were not included in the Isabel database at the time of the study; thus, these 2 cases would never have been suggested, even with different keywords. Using the copy/paste method for entering the whole text, the list of diagnoses suggested by Isabel contained the correct diagnosis in 37 of the 50 cases (76%). Isabel presented 10 diagnoses on the first web page and 10 additional diagnoses on subsequent pages up to a total of 30 diagnoses. Because users may tend to disregard suggestions not shown on later web pages, we tracked this parameter for the copy/paste method of data entry: The correct diagnosis was presented on the first page in 19 of the 37 cases (51%) or first two pages in 28 of the 37 cases (77%). Similar data were not collected for manual data entry because the order of presentation depended on which key findings were entered. Both data entry approaches were fast: Manually entering data and obtaining diagnostic suggestions typically required less than 1 minute per case, and the copy/paste method typically required less than 5 seconds. DISCUSSION Diagnostic errors are an underappreciated cause of medical error,10 and any intervention that has the potential to produce correct and timely medical diagnosis is worthy of serious consideration. Our recent analysis of diagnostic errors in Internal Medicine found that clinicians often stop thinking after arriving at a preliminary diagnosis that explains all the key findings, leading to context errors and ‘premature closure’, where further possibilities are not considered.11 These and other errors contribute to diagnoses that are wrong or delayed, causing substantial harm in the patients affected. Systems that help clinicians explore a more complete range of diagnostic possibilities could conceivably reduce these types of error. Many different CDSSs have been developed over the years, and these typically matched the manually entered features of the case in question to a database of key findings abstracted from experts or the clinical literature. The sensitivity of these systems was in the range of 50%–60%, and the time needed to access and query the database was often several minutes.3 More recently, the possibility of using Google to search for clinical diagnoses has been suggested. However, a formal evaluation of this approach on a subset of the same “Case Records” cases used in our study found a sensitivity of 58%,12 in the range of the first-generation CDSSs and unacceptably low for clinical use. The findings of our study indicate that CDSS products have evolved substantially. Using the Isabel CDSS, we found that data entry takes under 1 minute, and the sensitivity in a series of highly complex cases approached 100% using entry of key findings. Entry of entire case histories using copy/paste functionality allowed even faster data entry but reduced sensitivity. The loss of sensitivity seemed primarily related to negative findings included in the pasted history and physical (e.g., “the patient denies chest pain”), which are treated as positive findings (chest pain) by the search algorithm. There are several relevant limitations of this study that make it difficult to predict how Isabel might perform as a diagnostic aid in clinical practice. First, the results obtained here reflect the theoretical upper limit of performance, given that an investigator who was aware of the correct diagnosis entered the key findings. Further, clinicians in real life seldom have the wealth of reliable and organized information that is presented in the Case Records or the time needed to use a CDSS in every case. To the extent that Isabel functions as a ‘learned intermediary’,13 success in using the program will also clearly depend on the clinical expertise of the user and their facility in working with Isabel. A serious existential concern is whether presenting a clinician with dozens of diagnostic suggestions might be a distraction or lead to unnecessary testing. We have previously identified these trade-offs as an unavoidable cost of improving patient safety: The price of improving the odds of reaching a correct diagnosis is the extra time and resources consumed in using the CDSS and considering alternative diagnoses that might turn out to be irrelevant.14 In summary, the Isabel CDSS performed quickly and accurately in suggesting correct diagnoses for complex adult medicine cases. However, the test setting was artificial, and the CDSS should be evaluated in more natural environments for its potential to support clinical diagnosis and reduce the rate of diagnostic error in medicine.

Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764202

Help-seeking pathways in early psychosis

Introduction Understanding the help-seeking pathways of patients with a putative risk of developing psychosis helps improving development of specialised care services. This study aimed at obtaining information about: type of health professionals contacted by patients at putative risk for psychosis on their help-seeking pathways; number of contacts; type of symptoms leading to contacts with health professionals; interval between initial contact and referral to a specialised outpatient service. Introduction In recent years, the aim of intervening in the early phases of psychosis has drawn great interest. Early intervention may shorten duration of untreated psychosis or even prevent onset of psychosis. Exact knowledge about the help-seeking pathways of patients is pivotal in order to provide early intervention and, thus, supply specialised and focussed health care. One study has investigated the patterns of referral in patients known to be at risk for developing a psychotic illness [1]. Further studies investigating these trajectories in first-episode patients [2–5] were able to show the important role of GPs. GPs were reported to be commonly contacted by patients who later develop psychosis. In particular, GPs were often reported to be contacted early along the help-seeking pathways and were shown to commonly be the first contacted professional group [3]. It can thus be hypothesised that a considerable part of GPs are contacted by patients who may still be presenting more insidious features such as functional disability or social withdrawal, given that these insidious features have been found to be highly prevalent in the early course of psychosis [6]. It must therefore be expected that studies of help-seeking pathways which, in addition to first-episode patients, further include patients in presumed at-risk states for psychosis would be able to corroborate the importance of GPs as early contacted professional groups. Up to date, only one other such study has been reported on patients who are considered to be at-risk for psychosis, but who have not yet developed psychosis [1]. This study is the first to compare the help-seeking behaviour of at-risk patients, patients with first psychotic episodes and of patients neither meeting clinical at-risk nor first episode criteria, but who are concerned about their mental health and thus were assessed in a prodromal service. To the best of our knowledge, it is also the first study to assess symptoms in patients at risk for psychosis at their first presentation to a prodromal service. We expected GPs to be highly represented among the early help-seeking contacts of at-risk patients. Method Recruitment strategy In August 2002, a specialised, low-threshold outpatient service for the assessment of patients considered at-risk for psychosis was established in a semi-urban catchment area of North Western Switzerland (population = 300.000). This “prodromal clinic” is part of the only general psychiatric outpatient clinic of the catchment area and is associated with a public psychiatric hospital. As is the case all over Switzerland, patients can refer themselves directly to any public or private psychiatric facility and do not require referral to mental health systems via gate-keeping GPs. Patients can also be referred by any other source such as GPs, school counsellors, paediatricians or social workers. Between August and December 2002, the majority of GPs (n = 240), private psychiatrists and psychologists (n = 130) of the catchment area were educated either individually or in small groups about the early warning signs of emerging psychosis and manifest psychotic features. Education strategies were based on the findings from a large, nation-wide survey among 1089 GPs [7] that showed that GPs often underidentified the insidious features of beginning psychosis, such as functional decline. In keeping with these findings, education strategies primarily focussed on the importance of the insidious features of beginning psychosis such as sustained functional decline and social withdrawal as important early warning signs. With the inclusion of the adolescent psychiatric service into our prodromal clinic in January 2004, all private youth psychiatrists as well as paediatricians were equally enrolled in this campaign so as to ensure additional referrals of patients aged 14–17 years, as symptom onset in this young age group has been associated with less favourable outcomes [8]. In our study, sensitised primary and secondary health care providers were encouraged to contact the outpatient clinic whenever one of their patients showed sustained decline in social functioning in a still relatively asymptomatic state; if they showed attenuated or brief intermittent positive psychotic symptoms; or if they suspected that “something odd had been going on” for some time in their patients. The research protocol was approved by the “Ethikkommission beider Basel”. It was designed to obtain data on help-seeking pathways and to collect longitudinal information about progression of symptoms and social as well as neuropsychological functioning, and was not a treatment trial. Patient sample As of January 1st 2003, patients could be referred to our prodromal clinic. Consenting patients were included in the study. Each individual was fully informed about the research protocol. Written informed consent was obtained from all patients, and additionally from the parents if under 18. Patients were considered ineligible for further assessment within the study if they presented a history of a past psychotic episode, traumatic brain injury, epilepsy or other known neurological disorder; other significant medical conditions considered to affect cognitive performance and self-perception; an IQ of below 70; or patients below the age of 18 in the first year and below the age of 14 years in the second year of the present study. Symptom ratings We assessed the entire range of potential symptoms from early prodrome to first psychotic episode using the following instruments: potential early prodromes were assessed using the Schizophrenia Prediction Instrument—Adult Version (SPI-A) [9], potential late prodromes using the Scale of Prodromal Symptoms (SOPS) with its companion interview manual (Structured Interview for Prodromal Symptoms, SIPS) [10, 11]. For the assessment of potentially manifest psychosis, we used the Positive and Negative Syndrome Scale (PANSS; 12). Ratings were performed by an experienced consultant research psychiatrist (A.S.) or by a trained masters-level psychologist (D.D.). Interrater reliability was established by extensive training by one of the authors of the SPI-A and by repeated training sessions involving all raters. However, formal assessment of interrater reliability was not conducted. The SPI-A has been developed from a hierarchical cluster analysis of the BSABS [13] and includes those 10 basic symptoms for which a good positive predictive value for later schizophrenia has previously been reported [14, 15]. These basic symptoms include: thought interferences; thought perseveration; thought pressure; thought blockages; disturbances of receptive language; decreased ability to discriminate between ideas and perception, phantasy and true memory; unstable ideas of reference; derealisation; visual and acoustic perception disturbances. The structure of the SIPS manual and the SOPS rating scale as well as its high validity and reliability have been reported elsewhere [10, 11]. In summary, these authors classified patients with either attenuated positive psychotic symptoms, brief limited intermittent positive psychotic symptoms or with a combined functional decline and genetic high-risk as ultra high-risk (UHR). Group assignment Depending on symptom severity, patients were assigned to one of the three following groups: (1) the First Episode (FE) group was constituted by patients scoring 6 on any of the five SOPS positive symptom items for more than 1 week, which is identical with a minimum score of 4 on any of the PANSS positive items for the same time period. (2) The at-risk (AR) group included: patients meeting severity, but not duration criteria of the FE group; patients meeting scores of 3–5 on any SOPS positive psychotic symptom item at least once a week over the past month; patients with a first degree relative with any psychotic disorder and/or patient meeting DSM-IV Schizotypal Personality Disorder criteria and a 30% or greater drop in the GAF [16] score during the last month compared to 12 months ago; patients scoring a minimum of 3 on any of the 10 predictive basic symptoms according to the SPI-A. (3) Help-seeking patients who were referred to the prodromal clinic for risk assessment, but who did neither meet FE nor AR criteria, were assigned to the Patient Control (PCo) group. Assessment of help-seeking pathways and of symptoms at prior contacts After symptom assessment, patients were asked which other professional groups they had previously contacted for similar problems. Further, timing and number of contacts as well as type of symptom leading to each single prior contact were recorded and, whenever possible, corroborated with information from family members. These data were assessed using a semi-structured interview that was designed for this study. However, no information was collected whether referrals to mental health services were voluntary or involuntary. Analysis Data on patients who were enrolled between January 2003 and December 2004 were analysed using the computer package SPSS Version 11.04. We compared number of contacts and professionals contacted along the help-seeking pathways between groups. Similarly, symptoms that patients had presented during their previous help-seeking contacts and the interval between initial contact and referral to our prodromal service were compared between groups. Continuous variables were compared with t-tests or ANOVA, categorical variables with χ2 tests or, if ranked, with Kruskal–Wallis and Mann–Whitney-U tests. An alpha level of 0.05 was considered significant. Results Sample characteristics Of the 104 patients included into this study, 28 were female and 76 male. The mean age was 23.2 years (SD: ±5.43; range: 16–38) in the first year of the study, and 20.0 years (SD: ±6.08; range: 14–40) in the second year. 28 patients met criteria for the FE group, 50 for the AR group, and 26 patients were assigned to the PCo group. A summary of the socio-demographic characteristics across these study groups is shown in Table 1. Table 1Socio-demographic characteristicsFirst episodeAt-riskControlsTotalSignificance valuesNumber of patients285026104Mean age23.42122.122χ² = 3.744, df = 2, P = 0.153Gender    m (%)22 (79)31 (62)23 (88)76 (73)χ² = 6.676, df = 2, P = 0.036    f (%)6 (21)19 (38)3 (12)28 (27)Marriage status    Unmarried (%)26 (93)45 (90)25 (96)96 (92)χ² = 0.929, df = 2, P = 0.629Children    No children (%)28 (100)48 (96)25 (96)101 (97)χ² = 1.140, df = 2, P = 0.566Living situation    Alone (%)5 (18)7 (14)6 (23)18 (17)χ² = 8.412, df = 4, P = 0.078    With partner or friends (%)5 (18)5 (10)8 (31)18 (17)    With parents or relatives (%)18 (64)36 (72)10 (39)64 (62)City size    >200,000 (%)5 (18)5 (10)4 (15)14 (14)χ² = 3.337, df = 6, P = 0.766    10,000–200,000 (%)7 (25)12 (24)6 (23)25 (24)    <10,000 (%)16 (57)33 (66)16 (62)59 (62)School education    None completed (%)1 (4)1 (2)2 (8)4 (4)χ² = 4.688, df = 6, P = 0.584    Obligatory school (%)19 (68)36 (72)18 (69)73 (70)    Maturity (%)5 (18)4 (8)4 (15)13 (13)    Still in education (%)3 (11)9 (18)2 (8)14 (13)Professional training    None (%)9 (32)22 (44)8 (31)39 (38)χ² = 10.429, df = 6, P = 0.108    Apprenticeship (%)12 (42)18 (36)12 (46)42 (40)    Academic education (%)5 (18)1 (2)1 (4)7 (7)    Still in training (%)2 (7)9 (18)5 (19)16 (15)Work situation    No occupation (%)12 (43)19 (38)10 (39)41 (39)χ² = 4.097, df = 6, P = 0.664    At work (%)10 (36)11 (22)6 (22)27 (26)    In training (%)6 (21)20 (40)10 (39)36 (35) Number of contacts and professionals contacted The mean number of contacts over all three patient groups was 2.38 (SD: ±1.42; median: 3.0; range: 1–8) with no significant between-group differences (χ2 = 208.375, df = 2, P = 0.605). The overall patient sample reported a total of 247 contacts prior to their referral to our prodromal clinic. Table 2 shows the type of professional groups contacted by the patients across the three study groups along their help-seeking trajectories. FE patients more often presented themselves to mental health professionals (psychiatrists, psychologists as well as psychiatric out- and in-patient services) than AR and PCo patients (71.2% vs. 57.6% vs. 53.5% of contacts); however, this comparison did not reach significance (χ2 = 4.724, df = 2, P = 0.094). When comparing FE patients with the combined non-psychotic AR and PCo patients, FE patients significantly more often presented themselves to mental health professionals (χ2 = 4.461, df = 1, P = 0.024). Furthermore, when comparing the numbers of contacts with mental health professionals (149/247 = 60.3%) with those to non-mental health professionals (98/247 = 39.7%), FE patients more often contacted in-patient services, whereas PCo and AR patients more often attended GPs and “other” professionals such as alternative medical practitioners, non-medical counselling services and non-specified professionals (χ2 = 20.189, df = 8, P = 0.010). Of the overall sample, 51.0% (53/104) contacted a GP at some point in time, whereas 83.7% had at least once visited a mental health professional. Table 2Distribution of professionals contactedFEARPCoTotal(n) % of total(66) 26.7(125) 50.6(56) 22.7(247) 100General practitioners21.2% (14)21.6% (27)37.5% (21)25.1% (62)Private Psychiatrists/Psychologists21.2% (14)24.8% (31)21.4% (12)23.1% (57)Psychiatric out-patient services30.3% (20)25.6% (32)16.1% (9)24.7% (61)Psychiatric in-patient services19.7% (13)7.2% (9)16.1% (9)12.6% (31)Other professionals7.6% (5)20.8% (26)8.9% (5)14.6% (36)Percentages (and absolute numbers) of contacts made with professional groups at any stage of the help-seeking pathway However, as shown in Fig. 1, there was a trend for progression from non-psychiatric towards psychiatric services. Contacts with GPs were often at an earlier stage on the help-seeking pathways. Moreover, GPs were the most often first-contacted professional groups, whereas psychiatric in-patient services were most commonly the last contacted professional group (χ2 = 15.816, df = 4, P = 0.003). However, differences among study groups did not reach significance (first contacts: χ2 = 13.102, df = 8, P = 0.108; last contacts: χ2 = 9.699, df = 8, P = 0.287). Fig. 1Progression to specialized services. Distribution of first (n = 104) and last contacts (n = 104) in percent made to different helper groups. Contacts representing the only help-seeking attempt by a subject appear in both first and last contacts Symptoms at prior presentations The complete set of signs and symptoms which led patients or their relatives to seek help at each of the recorded contacts was obtained from 100 of the total patient sample (96.2%). The frequencies of the most commonly presented symptoms are shown in Table 3. In the AR and PCo groups, the trias depression, social decline and social withdrawal were presented most frequently. In the FE group, unusual thought content was the most frequent symptom, followed by the above-mentioned triad. Table 3Frequency of presented symptomsFEARPCoTotalNumber of contacts6612556247Positive symptoms  Ideas of reference (%)15 (23.1)2 (<5.0)1 (<5.0)18 (7.7)  Unusual contents of thought (%)36 (55.4)18 (14.8)1 (<5.0)55 (23.5)  Hallucinations (%)13 (20.0)11 (9.0)2 (<5.0)26 (11.1)  Perceptual disturbances (%)5 (7.7)21 (17.2)026 (11.1)  Alienation or derealisation (%)16 (24.6)29 (23.8)045 (19.2)Negative symptoms  Deterioration of social functioning (%)32 (49.2)54 (44.3)16 (34.0)102 (43.6)  Social withdrawal (%)32 (49.2)42 (34.4)14 (29.8)88 (37.6)  Avolition (%)9 (13.8)26 (21.3)12 (25.5)47 (20.1)Cognitive symptoms  Impaired concentration (%)13 (20.0)29 (23.8)1 (<5.0)43 (18.4)  Impaired attention (%)7 (10.8)12 (9.8)1 (<5.0)20 (8.5)  Impaired memory (%)10 (15.4)5 (<5)2 (<5.0)17 (7.3)  Formal thought disorders (%)17 (26.2)25 (20.5)042 (17.9)Other symptoms  Depression (%)24 (36.9)74 (60.7)38 (80.9)136 (58.1)  Anxiety (%)14 (21.5)27 (22.1)9 (19.1)50 (21.4)  Lack of impulse-control (%)2 (<5.0)14 (<5.0)11 (23.4)27 (11.5)Absolute numbers (percentages) of presented symptoms. Percentage values do not add to 100% due to multiple symptoms recorded for most contacts FE patients had presented at least one positive symptom in 80.3% of their prior contacts, AR patients in 56.6% and PCo patients in 5.9% of their prior contacts (χ2 = 65.680, df = 2, P < 0.001). Similarly, when comparing numbers of patients rather than numbers of contacts, 89% FE, 65% AR and 8% PCo patients had reported positive symptoms at any stage along their help-seeking pathway (χ2 = 35.483, df = 2, P < 0.001). Of patients presenting positive symptoms along their help-seeking pathways, 81.2% already did so during their first contact. Positive symptoms were significantly more often reported in first help-seeking contacts with psychiatric out-patient (63.6%) and in-patient (100%) services as compared to GPs (37.1%) and private psychiatrists/psychologists (31.8%) (χ2 = 13.425, df = 4, P = 0.009). Table 4 shows the relationship between positive and non-positive symptoms and the professional groups to whom they were presented. Positive symptoms were significantly more often reported in contacts with both psychiatric out- and in-patient services (67.2%, 70.0%) as compared to GPs, who were contacted for positive symptoms only in 37.3% recorded contacts. Consequently, GPs were contacted with more unspecific symptoms (62.7%) such as depression and negative symptoms (χ2 = 18.046, df = 4, P = 0.01). Table 4Positive vs. non-positive symptoms presented to professional groupsGeneral practitionersPsychiatrists psychologistsOut-patient servicesIn-patient servicesOther xprofessionalsTotalNumber of contacts5955613034239Contacts made with positive symptoms37.3% (22)40% (22)67.2% (41)70% (21)55.9% (19)(125)Contacts made with non-positive symptoms62.7% (37)60% (33)32.8% (20)30.0% (9)44.1% (15)(114)Percentages (absolute numbers) of symptoms presented to professional groups. Percentage values do not add to 100% due to multiple symptoms recorded for most contactsPositive symptoms included items according to Table 3, non-positive symptoms included items according to “negative”, “cognitive” or “other” symptoms in Table 3 Duration from initial contact to referral The median interval from initial contact to time of referral to our prodromal clinic was 36 weeks (range: 1 day to 7.6 years; mean: 124.0 weeks; ±SD 217.1). About a third (33.3%) of the patients whose initial contact was over 18 months before referral to the prodromal clinic accounted for the relatively long mean help-seeking duration. Two-thirds (66.6%), however, were referred within 18 months, with one-third (35.4%) being referred within 2 months. The median help-seeking duration of the FE group (12.5 weeks) was shorter than for the AR (42.5 weeks) and PCo groups (46.5 weeks), however, between-group comparisons dropped just below level of significance (z = −1.879, P = 0.06). More significantly, patients presenting with positive symptoms at some stage along their help-seeking trajectories (n = 56) showed shorter pathway durations (median = 24.5 weeks) than patients presenting only with negative, affective or “unspecific” symptoms, i.e. patients never presenting positive symptoms (n = 44; median = 46.0 weeks) (z = −2.072, P = 0.038). We further calculated the median intervals from any of the prior contacts to the time of referral and compared these “delays to referral” for the different professional groups that were contacted. Median “referral delay” across all three-study groups was 28 weeks with no between-group differences (z = −1.260, P = 0.208). Median “delays” were 15 weeks for contacts with GPs, 68.5 weeks for private psychiatrists and psychologists, each 13.0 weeks for psychiatric in- and out-patient services, and 52.2 weeks for contacts made to “others” (z = −3.554, P < 0.001). For 165 of the 247 recorded contacts, we were able to obtain information on whether a continuous treatment of three or more consecutive sessions was provided. The significantly longer “delays to referral” following contacts with private psychiatrists and psychologists was associated with the finding that in 58% of all contacts made with these groups, three or more sessions were administered. In comparison, in the vast majority of the cases (83.7%) contacts with GPs were single consultations. Discussion To the best of our knowledge, this is the first study to investigate the help-seeking pathways of a patient cohort, which was referred to a prodromal clinic for a suspected at-risk state for psychosis. We were able to confirm the important role of GPs along the help-seeking pathways of patients with emerging psychosis. Furthermore, GPs were contacted in particular by those patients who presented insidious features. It was these patients that showed the longest delays in referral to our specialised outpatient service. The importance of GPs along the early pathways This study was able to confirm recent reports from Australia [1] and from Germany [17, 18] that a substantial number of contacts made along the help-seeking pathways were with mental health care professionals. As the gatekeeping model applies for Australia, but not for Germany or Switzerland, mental health care professionals seem to play an important role independent of a particular health system. At the same time, our study also confirms earlier reports [2–5] about the pivotal role of GPs in the early pathways to care of help-seeking individuals that were referred to our prodromal clinic for an assessment of a potential psychotic at-risk state. While they were solicited in half of all contacts, GPs even constituted the most frequently first-contacted professional group (34.6%). It is noteworthy that in Switzerland patients can refer themselves to GPs without a “gate-keeping” GP. It can be thus assumed that in health systems with “gate-keeping” GPs [19], the present results may be even more representative. The challenge of detecting the insidious symptoms In addition, our results revealed that patients in less symptomatic states more commonly seek help with their GPs. Patients with manifest psychotic symptoms, however, more often contacted specialists. These results are paralleled by the finding that a shift from contacts made with primary carers to contacts with more specialised professional groups is taking place along the pathways (as shown in Fig. 1). Similar findings were reported by Lincoln et al. [3]. We assume that this process may also be an expression of the symptom progression along the course of early psychosis [20, 21]. As we thus had expected, GPs are faced with the difficult task to detect potential at-risk states in patients that do not yet present psychotic, but the unspecific insidious features. This finding is of particular relevance as a recent comprehensive survey among 1089 Swiss GPs was able to show that GPs commonly under-identified the insidious features of emerging psychosis [7]. Preliminary results of an international replication study (IGPS) of the Swiss survey across 10 countries were able to confirm these findings [22]. Although features such as functional disability or social isolation may not necessarily lead to overt psychosis and may either be expression of another psychiatrically relevant process or remit after time, it is the detection of the earliest signs and symptoms of emerging psychosis that has become the aim of preventive efforts. Similarly, depression has been shown to be highly prevalent in emerging psychosis [6]. While this triad—functional disability, social isolation and depression—was found to be highly prevalent across all groups in the present study, it is also a characteristic of the deficit syndrome of schizophrenia [23]. If such symptoms are true-positive precursors of later psychosis, the non-detection of such patients contributes to a substantial delay on their way to specialised services and adequate treatment, which in turn has been shown to negatively affect the outcome of patients with first-episode psychosis [24]. Accordingly, our study confirmed earlier reports that patients with more insidious features showed longer pathway durations than patients with predominant positive symptoms [25]. Deficit syndrome patients, however, may show lower adherence to treatment and are per se characterised by worse outcomes [23]. Delays in referral Interestingly, when compared to specialists and other professional groups, GPs referred patients more rapidly to other professional groups once they were contacted. In contrast, more contacts per patient were found with private psychiatrists and psychologists before final referral to the prodromal clinic was established. Partially, this finding may be explained by our large and repetitious sensitisation of GPs about the insidious features of early psychosis. However, some of the sampled patients had contacted a GP before the sensitisation had taken place, suggesting that GPs tend to refer such patients more rapidly. This would be in line with the findings from both the Swiss survey [7] and its international replication study [22], in which GPs indicated that they wished to rapidly refer patients in whom they suspected a beginning psychosis to specialised outpatient services. Given their degree of specialty, private psychiatrists and psychologists, in contrast, seem not to engage into rapid referral of these patients. Given the considerable potential for recall bias, our study did not assess type of treatment that was provided to patients at each of their contacts. Thus, no conclusions can be drawn about the adequacy of applied treatment strategies. It may be the psychiatrists and psychologists that are seeing the more symptomatic and “difficult-to-treat” patients who require a stable and continuous treatment setting. However, all patients included into this study were finally referred to our prodromal clinic, which may have either taken place for diagnostic assessment or for optimising treatment. It may thus be suggested that referrals of patients could have ideally been initiated already at an earlier stage along their help-seeking pathways. Interestingly, in two German studies on the pathways to care of patients with first-episode psychosis, contacts with private psychiatrists and psychologist were associated with longer duration of untreated psychosis as compared to GPs and general casualty services [17, 18]. Taken together, these findings suggest that education may not only need to include primary carers such as GPs, but also other professional groups as well as secondary carers such as private psychiatrists and psychologists. Number of contacts and duration of help-seeking pathways Finally, the comparatively low mean number of contacts (2.38) on the pathways to care over all patient groups may express the effect of the large sensitisation of professional groups that was conducted when our prodromal clinic was established. This finding is identical with the Phillips et al. study [1] who reported a mean number of contacts of 2.36 in their at-risk sample. Additionally, the median duration from initial contact to referral to our prodromal service was very similar to the one reported by Phillips et al. [1] (42.5 vs. 41.4 weeks). In those studies assessing help-seeking pathways of first-episode cohorts, only one other study reported lower mean contacts (1.7; range 1–4) and also was conducted as part of an early psychosis service [5]. In comparison, number of contacts of patient samples not treated in specialised services were reported to be higher. A mean number of contact of 4.9 was reported by Johnstone et al. [2], a finding which was identically reported in a study that included patients treated before and after the establishment of an early psychosis service [3]. These findings point to the importance of specialised early psychosis services. Weaknesses of this paper Finally, a few weaknesses of this survey should be acknowledged. First, this study, like any retrospective research, is limited by an inherent potential for recall bias. Given the considerable length of pathway duration in the case of some patients, information on the exact number and on timing of contacts may be subject to errors. Ideally, all contacted professional groups would need to be contacted and interviewed in order to validate the information obtained by patients and their relatives. Second, pathways were studied on a patient sample, which was mainly constituted by subjects without manifest psychosis. Contacted professional groups may therefore not have been alarmed to refer their patients, and referral to our prodromal clinic may have only occurred in order to exclude an at-risk state for psychosis once the prodromal clinic was established. It can, however, be suggested that referrals would only take place if there is particular need for further assessment. Moreover, referrals may also have been initiated by patients themselves, although we did not control for that aspect in our study. Third, we cannot exclude that pathways may differ in patients that are never referred to our prodromal clinic. It may well be possible that an unknown number of patients in presumed at-risk states are treated by private specialists and undergo complete remission. It may therefore be a per se selection of more impaired patients that will be referred to a prodromal clinic. Although the at-risk criteria applied in the current study are now well-established in the early psychosis research community [26], it may well be possible that not all true at-risk patients meet these criteria. Thus, our study may have only captured a fraction of the individuals developing psychosis [27]. Fourth, we did not control for patient-related factors that may in a large part contribute to referral delays. These factors may include lack of insight, poor social adjustment, paranoid thoughts or avolition. Finally, the training and role of GPs in the health care system may vary across countries; thus, not all of the findings of our study may be generalisable to other settings. However, our findings emerge from a health system where specialists may be contacted without the referral of the “gate-keeping” GPs. Thus, in health systems with gate-keeping models our findings that GPs are more commonly contacted by individuals with insidious features may warrant appropriate education efforts even stronger. Conclusions Our study confirms earlier reports of the GPs’ important role on the help-seeking pathways of psychotic patients. Furthermore, our study revealed that GPs are often contacted by patients with suspected at-risk states for psychosis early on their pathways to care. The insidious nature of the symptoms presented by these patients in the early stages of their pathways imposes an important challenge for GPs, as insidious features of early psychotic stages are difficult to detect. Our results thus support the need for appropriate education of GPs on these insidious features and for rapidly accessible specialised outpatient services. In a few countries, such specialized outpatient services have been established in recent years [28]. For example, in England, under the National Health Service Plan, 50 early intervention teams have been established at a cost of £70 million [29]. It can thus be expected that help-seeking pathways of patients in early phases of psychosis may be reduced, thus maybe improving the overall outcome of patients.

Health_Qual_Life_Outcomes-2-_-395842

A new instrument to describe indicators of well-being in old-old patients with severe dementia – The Vienna List

Background In patients with very severe dementia self-rating of quality of life usually is not possible and appropriate instruments for proxy-ratings are not available. The aim of this project is to develop an instrument of clinical proxy-ratings for this population. Background In industrial societies the proportions of old people and of people suffering from dementia are steadily increasing. Consequently, the number of people depending on different types of institutional care is growing. The care is provided in general hospitals, geriatric hospitals, nursing homes, private homes, and senior residences and within the families. Obviously, there are large differences in costs and efficacy of these different types of care. In general, quality of life data have been increasingly appreciated as the key outcome measure for the assessment of therapeutic interventions and for the usefulness of various treatment facilities. Quality of life is defined by WHO as "an individual's perception of their position in life, in the context of the culture and value systems in which they live, and in relation to their goals, expectations, standards, and concerns. It is a broad ranging concept, effected in a complex way by the person's physical health, psychological state, level of independence, social relationships and their relationships to salient features of their environment" [1]. As concerns people with dementia, many of the instruments in use represent brief global measures, which cannot be applied to patients with severe dementia. The most important reason for this slow growth of empirical data in this area probably relates to the obvious methodological problems of obtaining reliable subjective accounts of individuals with severe dementia who have compromised cognitive abilities, frequently with concurrent impaired communicative skills. Due to these impairments in this group of patients most of the available instruments are not applicable for assessing quality of life related issues by means of traditional measures such as questionnaires or interviews which require a highly complex procedure of introspection and evaluation, involving several components of cognition including implicit and explicit memory [2]. Facing these problems, attempts have been made to develop direct observational methods [3] measuring positive affects such as pleasure, interest, and contentment as well as negative affects such as sadness, anxiety/fear, and anger according to operationalized criteria during a series of 10-minutes direct observation. An alternative approach was chosen by the Bradford Research Group in the UK [4] with the Dementia Care Mapping (DCM) based on the psychosocial model of "person-centred care" which provides detailed observational ratings covering aspects of articulation, feeding, social withdrawal, passive engagement, walking and a number of indicators of well-being. Literature searches (-2003) of Medline, Embase, Psyclit, Cinahl using the keywords dementia and well-being and quality of life were conducted. Lawton et al. [5] developed the Minimum Data Set (MDS) comprising cognition, activities of daily living, time use, depression, and problem behaviours. He proposed to include observations of demented patients' emotional behaviours [6]. Most of the measurements are derived from existing instruments and are confined to few of the following dimensions: affect, activity, enjoyment, self-esteem, and social interactions [7]. Ready and Ott concluded from their review [7] that the psychometric properties of most of the available instruments have to be regarded as preliminary. As mentioned above, there were no quality of life assessment tools for patients with very severe dementia as representative of our population. Unfortunately, the instruments developed for demented patients were not found applicable to our patients who were in much more advanced stages of dementia (e.g. the Mini Mental State Examination could not be completed by any of our patients). For this reason a project was launched in 1998 by the department of Palliative Geriatrics (Geriatriezentrum am Wienerwald), to develop a new instrument based on observations made by the staff (physicians, nurses and physiotherapists) completely independent on the patients' cooperation. Methods Patients In the present study 217 consecutive patients (44 males, 173 females) were included. The average age was 84 years (range 61–105 years). The majority of patients suffered from severe dementia according to ICD-10 (34,5 % F00 – Dementia in Alzheimer's Disease; 61,5 % F01 – Vascular Dementia; 4,0 & F02 – other). It appeared that more women (38 %) had been diagnosed as suffering from Alzheimer's disease compared with men (23 % – χ2 (df = 2) = 6,05; p = 0,049). As concerns age, the patients with Alzheimer's disease (87,3 ± 5,7 years) and vascular dementia (86,3 ± 7,0 years) were significantly older than those with other diagnoses (78,5 ± 15,6 years – t = 3,27; p = 0,002 and t = 2,74; p = 0,007 respectively). Development of the item-pool Thirteen staff members comprising doctor, nurses, and therapists from the department of Palliative Geriatrics at the Geriatriezentrum am Wienerwald in Vienna had observed severely demented patients during a one year period (May 1998 to April 1999). The patient's behaviour was documented at one of the wards (32 beds). Based on this documentation, 65 items for the description of behaviour in demented inpatients were derived and allocated to categories, supposedly reflecting relevant aspects of their well-being such as voice, language, mood, eye contact, acceptance of body contact, gait, muscular tension, hand movement, sleep, activities, communication, independence concerning food intake. This approach is different from prevailing approaches, which mainly are based on the use of items from existing instruments measuring specific aspects. Subsequently, by means of this item-list, 771 assessments of 217 in-patients in various situations such as eating, dressing, grooming had been obtained between June 1999 and September 2000 by physicians and nurses. Each of the original 65 items was scored on a 5-point Likert scale from 0 = never to 4 = always. Further assessments Patients were diagnosed according to ICD-10. They were rated by means of the Brief Cognitive Rating Scale (BCRS) [8], the Global Deterioration Scale (GDS) [9], the Barthel-Index [10], and the Spitzer Index [11]. The BCRS describes the severity of cognitive impairment providing five main axes (concentration, short term memory, long term memory, orientation, and self-care ability) and five co-axes (language, psychomotoric, mood and behaviour, drawing skills, calculating skills) each rated on a 7-step scale. The GDS is a proxy rating scale to assess the severity of dementia in elderly people on a seven point Likert-type scale (1 = no impairment; 7 = most severe impairment). The Barthel-Index was used to assess the activity of daily living in 10 areas (feeding, transfers bed to chair and back, grooming, toilet use, bathing, mobility, climbing stairs, dressing, stool control, bladder control). The Spitzer Index is a global quality of life measure covering five areas (activity, daily life, health, social relations, future) with a maximum score of 10 points. The BCRS, GDS, and Spitzer Index had been rated by physicians and the Barthel index by nurses. All ratings including the 65 items list were made on the same occasion. Data collection was carried out using electronic questionnaires implemented through the Quality-of-Life-Recorder technology [12]. Statistical analysis Descriptive statistics were generated for demographic data and diagnostic categories and for the BCRS and GDS scales, for the Barthel and Spitzer Indices, as well as for the newly developed instrument. A factor analysis (principle component analysis, oblimin with Kaiser normalisation as rotation method), based on these 771 assessments was performed. The number of interpretable factors was determined by interpretation of a Scree plot. The consistency of the factors was tested by Cronbach's alpha coefficients. To improve the consistency of the scales, items have been deleted based on the criteria of changes in magnitude of the Cronbach's alpha coefficients and on the fit of the item with the content of the core items of the factors. To test the stability of the factor structure, we conducted separate analyses for doctors and nurses. For testing construct validity, we used the two external criteria, Brief Cognitive Rating Scale and the Barthel-Index. To test for inter-rater reliability Spearman rank correlation coefficients were calculated. We included only data in which the electronic recording confirmed that it was obtained at exactly the same time. Results Most patients suffered from severe dementia as indicated by the results of the BCRS (mean ± SD: 57 ± 8.8) and GDS (mean ± SD: 5.7 ± 1.1) and the low level of activity of daily living (mean ± SD of Barthel Index: 26.8 ± 29.7) in the present sample also reflected by the distribution of diagnoses according to ICD-10. Of the 771 assessments 386 had been performed by nurses and 385 assessments by physicians. By means of the electronic recordings we identified 22 pairs of assessments made at the same time by a nurse and a physician. A planned feasibility analysis after 120 assessments resulted in the exclusion of the Spitzer Index because of a general floor effect (mean score <3). The factor analysis suggested five factors based on the interpretation of the Scree plot. The results of the five-factor solution are given in Table 1 with high Eigenvalues and an explained variance of more than 60 per cent. Table 1 Eigenvalues and explained amounts of variance for the 5-factor solution Eigenvalues % of Variance Cumulative % Factor nurse physician nurse physician nurse physician 1 10.0 10.7 24.4 26.0 24.4 26.0 2 5.7 6.1 13.8 15.0 38.2 41.0 3 4.9 4.8 11.9 11.8 50.0 52.1 4 2.5 2.1 6.1 5.0 56.1 58.0 5 2.0 2.5 4.9 6.1 61.0 63.9 Extraction Method: Principal Component Analysis. To improve the consistency of the five factors, 18 of the original 65 items were deleted based on the criteria of changes in magnitude of the Cronbach alpha coefficients and on the fit of the item with the content of the core items of the factors. As a next step, eight further items were excluded due to different factor-loadings between raters and of content considerations resulting in the following five factors: communication (15 items), negative affect (10 items), bodily contact (5 items), aggression (4 items) and mobility (6 items). The factor structure matrix including the single items for physicians and nurses is shown in table 2 and demonstrates a high congruency of the factor structure between both groups of raters on the item level. Table 2 Structure Matrix Factor 1 2 3 4 5 Nurse Physician Nurse Physician Nurse Physician Nurse Physician Nurse Physician Communication ITEM 59 0.82 0.83 -0.19 -0.14 0.04 -0.32 0.29 0.05 0.06 0.25 ITEM 62 0.81 0.64 0.02 0.16 0.08 -0.39 0.28 0.22 -0.09 0.34 ITEM 6 0.79 0.78 -0.26 0.13 -0.07 -0.29 0.18 0.08 0.10 0.17 ITEM 61 0.79 0.67 -0.23 -0.13 0.10 -0.56 0.22 0.09 0.08 0.25 ITEM 8 0.78 0.81 -0.20 0.08 -0.16 -0.25 0.14 0.12 0.12 0.18 ITEM 65 0.73 0.77 -0.27 -0.13 -0.05 -0.05 0.52 0.10 0.01 0.48 ITEM 56 0.70 0.69 -0.08 0.08 -0.15 0.07 0.07 -0.06 0.05 0.10 ITEM 14 0.70 0.85 -0.11 -0.11 0.02 -0.10 0.44 -0.06 0.15 0.26 ITEM 32 0.66 0.64 -0.14 -0.06 0.35 -0.48 0.03 -0.11 0.15 0.22 ITEM 31 0.65 0.65 -0.12 0.01 0.32 -0.54 0.08 0.00 0.15 0.19 ITEM 13 0.64 0.76 -0.24 -0.04 0.06 -0.42 0.11 0.13 0.12 0.20 ITEM 50 0.64 0.77 -0.40 -0.19 -0.04 -0.17 0.46 -0.03 0.19 0.35 ITEM 64 0.62 0.61 0.05 0.13 0.06 -0.17 0.49 -0.21 0.03 0.40 ITEM 60 0.59 0.69 0.12 0.17 -0.27 0.03 0.01 0.28 -0.11 0.07 ITEM 15 0.45 0.73 0.03 -0.13 -0.08 0.19 0.34 -0.19 0.12 0.06 Negative Affect ITEM 18 -0.08 0.04 0.83 0.87 -0.03 -0.14 -0.08 0.11 -0.28 -0.02 ITEM 22 -0.04 0.09 0.80 0.74 0.11 -0.34 -0.10 0.08 -0.20 0.00 ITEM 3 -0.07 0.02 0.75 0.82 0.16 -0.18 -0.13 0.17 -0.11 -0.06 ITEM 24 -0.09 -0.01 0.74 0.76 -0.05 -0.18 -0.05 0.12 -0.37 0.08 ITEM 27 0.04 0.02 0.66 0.77 -0.13 -0.10 -0.17 0.45 -0.45 -0.05 ITEM 17 -0.28 -0.17 0.66 0.71 -0.08 0.14 -0.16 0.38 -0.58 -0.12 ITEM 25 -0.30 0.17 0.65 0.63 0.03 -0.02 -0.29 -0.02 -0.08 -0.07 ITEM 54 -0.20 -0.20 0.62 0.58 -0.09 0.10 0.19 0.28 -0.34 0.05 ITEM 53 -0.10 -0.01 0.60 0.50 -0.07 0.23 0.14 0.17 -0.28 0.01 ITEM 47 -0.28 -0.26 0.53 0.61 0.00 0.11 -0.26 0.37 -0.45 -0.11 Bodily contact ITEM 35 0.00 0.17 0.02 -0.03 0.93 -0.76 -0.08 -0.13 0.11 -0.11 ITEM 34 0.01 0.27 0.00 0.07 0.93 -0.81 -0.09 -0.12 0.12 -0.09 ITEM 33 0.03 0.37 0.00 0.14 0.90 -0.77 -0.09 -0.06 0.12 0.00 ITEM 36 -0.06 -0.21 0.01 0.10 0.89 -0.81 -0.13 -0.15 0.16 -0.08 ITEM 37 -0.06 -0.27 0.01 0.15 0.86 -0.76 -0.12 -0.16 0.17 -0.06 Aggression ITEM 4 -0.14 0.01 0.25 0.27 -0.19 0.08 0.13 0.91 -0.88 0.08 ITEM 19 -0.24 -0.05 0.31 0.18 -0.22 0.16 0.13 0.84 -0.83 0.09 ITEM 1 0.05 0.20 0.28 0.20 -0.07 0.12 0.01 0.71 -0.78 0.08 ITEM 29 0.22 0.11 0.20 0.21 -0.10 0.05 0.03 0.84 -0.77 0.15 Mobility ITEM 40 0.42 0.56 -0.31 -0.23 -0.20 0.08 0.79 -0.14 0.22 0.80 ITEM 41 0.49 0.64 -0.28 -0.27 -0.23 0.07 0.71 -0.19 0.23 0.63 ITEM 57 0.01 0.01 0.01 0.08 -0.05 0.00 0.68 0.14 -0.18 0.70 ITEM 42 0.04 0.07 0.07 0.06 -0.02 0.05 0.68 0.20 -0.18 0.80 ITEM 43 0.43 0.63 -0.43 -0.15 -0.08 -0.09 0.66 0.00 0.22 0.64 ITEM 55 0.41 0.46 -0.14 -0.07 -0.10 0.04 0.42 -0.23 0.03 0.34 Extraction Method: Principal Component Analysis. Rotation Method: Oblimin with Kaiser Normalization. In addition, it appeared that the factors were generally unrelated to each other except for significant correlations between the factors 'communication' and 'bodily contact' (r = 0.25; p < 0.001 each) and of 'mobility' with 'negative affect' (physicians: r = 0.22; p = 0.001; nurses: r = -0.33; p < 0.01). The Cronbach alpha coefficients as a measure of internal consistency were high for both nurses and physicians (table 3). The congruence of nurses' and physician's ratings is further demonstrated by similar item severity (relative ratings) and selectivity of the single factors (table 4). Table 3 Cronbach alpha coefficients Factor Number of Items Nurse Physician 1 – Communication 15 0.93 0.94 2 – Negative Affect 10 0.88 0.89 3 – Bodily Contact 5 0.90 0.90 4 – Aggression 4 0.86 0.87 5 – Mobility 6 0.81 0.82 Table 4 Item severity and Item selectivity Item severity Item selectivity Communication Nurse Physician Nurse Physician 59 Responding to distant calls 0.57 0.63 0.82 0.85 62 Looking for contact 0.53 0.56 0.81 0.71 06 Speaks comprehensibly 0.64 0.69 0.79 0.81 61 Contact possible 0.83 0.90 0.79 0.74 08 Speaks meaningful groups of words 0.59 0.68 0.77 0.82 65 Eats and drinks by him-/herself 0.66 0.68 0.79 0.78 56 Reads newspaper 0.24 0.19 0.69 0.64 14 Carries out simple orders 0.39 0.64 0.73 0.84 32 Maintains visual contact 0.72 0.74 0.65 0.71 31 Visual contact possible 0.80 0.85 0.63 0.72 13 Comprehends single words 0.89 0.88 0.63 0.81 50 Uses both hands intentionally 0.62 0.67 0.70 0.79 64 Worries about others 0.25 0.23 0.65 0.63 60 Rings the bell 0.29 0.31 0.58 0.67 15 Carries out complicated orders 0.08 0.24 0.47 0.64 Negative Affect 18 Full of despair 0.40 0.43 0.82 0.86 22 Sad/crying 0.32 0.36 0.77 0.75 03 Whining voice 0.33 0.32 0.72 0.83 24 Nervous/anxious 0.37 0.40 0.76 0.75 27 Wailing 0.30 0.31 0.69 0.80 17 Tensed 0.36 0.43 0.73 0.72 25 Resignated 0.27 0.38 0.65 0.63 54 Restless/confused 0.25 0.31 0.62 0.59 53 Problems to fall asleep 0.25 0.36 0.59 0.49 47 Muscular tension 0.37 0.43 0.60 0.62 Bodily Contact 35 Bodily contact possible at shoulders 0.77 0.87 0.92 0.78 34 Bodily contact possible at arms 0.81 0.91 0.90 0.80 33 Bodily contact possible at hands 0.82 0.94 0.88 0.73 36 Bodily contact possible at the head 0.63 0.63 0.91 0.88 37 Bodily contact possible in the face 0.56 0.55 0.88 0.84 Aggression 04 Aggressive voice 0.27 0.27 0.89 0.90 19 Aggressive acts 0.21 0.19 0.85 0.84 01 Loud voice 0.32 0.33 0.80 0.80 29 Insulting others 0.23 0.22 0.81 0.88 Mobility 40 Walking upright 0.37 0.40 0.92 0.95 41 Walking straightup to 0.33 0.36 0.87 0.87 57 Departs from ward 0.07 0.05 0.53 0.46 42 Wanders around 0.16 0.17 0.53 0.62 43 Sitting upright 0.57 0.57 0.80 0.81 55 Ready to help on ward 0.13 0.09 0.55 0.54 For testing construct validity, we used two external criteria, the Brief Cognitive Rating Scale (BCRS) used by physicians and the Barthel-Index used by nurses (table 5). The correlation coefficients between the various areas of the BCRS and the two relevant scales of the new instrument (communication and mobility) point to a satisfactory validity. Concerning the second criteria, the Barthel-Index (a measure of activities of daily living), it was significantly correlated with the scales "communication" and "mobility" of our instrument. Furthermore, the latter was correlated with the scale "negative affect" and "acceptance of body contact" in the expected direction. Table 5 Correlations with BCRS scores and Barthel Index Communication Negative Affect Bodily contact Aggression Mobility Physicians BCRS 1 – concentration -0.71* 0.05 0.07 -0.02 -0.45* BCRS 2 – short time memory -0.67* 0.02 0.11 -0.01 -0.42* BCRS 3 – long time memory -0.68* 0.14 0.09 0.08 -0.46* BCRS 4 – orientation -0.65* 0.12 0.12 0.11 -0.40* BCRS 5 – everyday life competency -0.47* -0.04 -0.04 -0.10 -0.44* BCRS 6 – language -0.71* -0.02 0.02 -0.09 -0.37* BCRS 7 – psychomotorics -0.41* -0.01 0.06 -0.10 -0.59* BCRS 8 – mood and behaviour -0.60* 0.10 -0.02 0.03 -0.34* BCRS 9 – constructive skills -0.55* 0.03 0.02 -0.06 -0.34* BCRS 10 – calculation skills -0.59* 0.17* 0.09 0.09 -0.35* Main axis -0.73* 0.07 0.09 0.02 -0.49* Co-axis -0.71* 0.07 0.04 -0.03 -0.49* BCRS total score -0.74* 0.07 0.06 -0.01 -0.50* Nurses Barthel Item 1 – feeding 0.70* -0.21* -0.10 0.02 0.63* Barthel Item 2 – transfer 0.46* -0.27* -0.17* -0.08 0.83* Barthel Item 3 – personal care 0.41* -0.17* -0.02 -0.12 0.36* Barthel Item 4 – toilet use 0.47* -0.25* -0.20* -0.12 0.67* Barthel Item 5 – bathing 0.08 -0.07 0.04 -0.06 0.09 Barthel Item 6 – moving 0.43* -0.28* -0.18* -0.09 0.83* Barthel Item 7 – stairs 0.32* -0.23* -0.14 -0.03 0.72* Barthel Item 8 – dressing 0.51* -0.23* -0.22* -0.09 0.67* Barthel Item 9 – bowel 0.48* -0.20* -0.16* -0.15* 0.57* Barthel Item 10 – bladder control 0.44* -0.20* -0.22* 0.14 0.56* Barthel Index 0.56* -0.28* -0.20* -0.10 0.83* * p < 0.001 When testing for gender differences concerning the factors, we found significant differences for all but one factor (table 6). Table 6 Factor Scores of Observations by Gender of the Patients Factor Observations of Males (x ± SD) N = 123 Observations of Females (x ± SD) N = 648 t-score p value 1 – Communication 34.8 ± 13.2 33.8 ± 11.8 0.82 0.415 2 – Negative Affect 9.5 ± 6.7 14.8 ± 6.7 -8.37 <0.001 3 – Bodily Contact 14.9 ± 4.3 16.7 ± 4.9 -4.18 <0.001 4 – Aggression 3.1 ± 3.0 4.3 ± 3.5 -3.21 0.001 5 – Mobility 8.1 ± 5.5 6.3 ± 5.5 3.38 0.001 The interrater-reliability between sub-samples of physicians and nurses proved to be satisfactory (table 7). Table 7 Paired sample test and Spearman Rank correlation coefficients between nurses and physicians related to the same patient at the same day (22 pairs) Factor Nurse (x ± SD) Physician (x ± SD) t-score/p R/p 1 – Communication 25.8 ± 10.5 26.4 ± 8.9 -0.35/0.727 0.71/<0.001 2 – Negative Affect 11.9 ± 7.6 8.6 ± 5.0 2.46/0.023 0.57/0.006 3 – Bodily contact 15.9 ± 5.7 18.4 ± 2.9 -2.22/0.038 0.53/0.011 4 – Aggression 4.3 ± 3.0 2.2 ± 2.1 3.69/0.001 0.35/0.112 5 – Mobility 6.5 ± 5.7 5.1 ± 5.8 2.03/0.056 0.81/<0.001 Discussion The special problem in the assessment of well-being in patients with severe dementia is their lack of competence which is compromising the reliability of their reports. Consequently, observer ratings are the only alternative for such self-ratings. However, observer ratings inherit the potential risk of overrating the well-being of patients if the provider and rater of health care services are identical. We have controlled for this risk by semi-quantitatively describing the frequency of distinct behaviour patterns in demented patients. The results of this study demonstrate that the behaviour of old-old patients with severe dementia can be described by five factors of the Vienna List. By explaining more than 60% of the total variance these five factors obviously cover a considerable part of the possible spectrum of behaviour in these patients. Since nurses and physicians have different intensity of contact and corresponding different perspectives, it was surprising that their assessments were highly correlated in three of the five factors. The two factors, aggression and mobility, yielded higher scores among the nurses as compared to doctors. As concerns aggression, there are mainly two explanations for this difference. Firstly, nurses spend more time and have closer contact with the patients and consequently have a higher risk to induce aggressive behaviour in the patients. In addition, the extended period of contact increases the chance to experience an episode of aggressive behaviour. Secondly, patients normally behave differently towards nurses and doctors due to differences in role expectation and familiarity related to the frequency of contact. However, we consider this later explanation as unlikely in these patients due to their cognitive impairment. Regarding mobility it is plausible that the doctors report lower scores for mobility of the patients as the doctor mainly sees the patient under certain circumstances, i.e. during the rounds where the ward routines limit the mobility of the patient. Since these five factors encompass most of the behavioural repertoire of demented old-old patients we assume that these factors can be regarded as a useful approach to describe the well-being in these patients.

Acta_Neuropathol_(Berl)-3-1-2039821

Detection of amyloid beta aggregates in the brain of BALB/c mice after Chlamydia pneumoniae infection

Neuroinflammation, initiated by cerebral infection, is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer’s disease (AD). We investigated whether Chlamydia pneumoniae (Cpn) infection results in extracellular aggregation of amyloid beta (Aβ) in BALB/c mice. At 1 week post intranasal infection (p.i.), Cpn DNA was detected predominantly in the olfactory bulbs by PCR, whereas brains at 1 and 3 months p.i. were Cpn negative. At 1 and 3 months p.i., extracellular Aβ immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected. However, these extracellular Aβ aggregates showed morphological differences compared to extracellular Aβ aggregates detected in the brain of transgenic APP751SL/PS1M146L mice. These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like Aβ plaques in the brain of BALB/c mice, as suggested before. However, future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis. Introduction The most common cause of dementia is Alzheimer’s disease (AD), which manifests in a familial and a sporadic form. The neuropathology in both conditions is characterized by neuritic or senile plaques, consisting mainly of amyloid beta protein (Aβ) and neurofibrillary tangles with hyperphosphorylated tau protein as its key components. Mutations in the amyloid precursor protein (APP), presenilin-1 (PS-1) or -2 (PS-2) genes have been associated with the familial form of AD [27]. However, the most common type of this disease, sporadic AD, is not due to these mutations, and although risk factors have been identified, the initiating trigger is still unknown [18]. The potential importance of infections in the development of sporadic AD has been recently recognized [1, 21, 31]. Specifically, not only various viruses like the human immunodeficiency virus, herpes simplex virus and cytomegalovirus but also bacteria like Borrelia burgdorferi, have been linked to the process of neurodegeneration [6, 12, 13, 17, 20]. Some data also suggest that Chlamydia pneumoniae (Cpn) should be added to the list of pathogens possibly involved in the pathogenesis of AD. Cpn is a widespread, obligatory intracellular pathogen, often causing acute respiratory infections including pneumonia, sinusitis and bronchitis [10, 11, 15]. This microorganism has also been implicated in a variety of severe, chronic diseases including some disorders of the central nervous system [7, 14, 30, 32]. In an initial study, Balin et al. demonstrated the presence of Cpn in brains of 17 out of 19 patients with sporadic AD, using a variety of techniques, including PCR, electron microscopy and immunohistochemistry, while the pathogen could only be demonstrated in 1 out of 19 controls [2]. The presence of Cpn was demonstrated in various cell types like microglia, astrocytes, perivascular macrophages and monocytes, and predominantly in brain areas linked with AD pathology. Recently, these observations were confirmed [8]. On the contrary, others were unable to detect Cpn in AD patient tissues [9, 22, 23, 28]. The only animal study so far supporting a role for this pathogen in AD demonstrated an AD-like pathology in the brain of BALB/c mice post Cpn infection [19]. Three-month-old mice were inoculated intranasally with Cpn and the authors claimed that this resulted in the deposition of Aβ in the brain up to 3 months post infection with increasing pathology as the infection progressed. Based on these results, it was concluded that in naïve BALB/c mice, AD-like pathology could be induced by Cpn infection. In spite of these results, doubts remain concerning the amyloid-like plaques as being only unrelated elevations in amyloid [24]. Because of the possible implications of this study and the need for confirmation, it was our aim to repeat these experiments under quite corresponding conditions. Moreover, we compared our findings with the pathology found in a transgenic mouse model of AD. Materials and methods Chlamydia pneumoniae The Cpn strain, TWAR 2043 (ATCC) was cultured and continuously propagated on Hep2 cells as previously described [25]. Bacterial titres were determined by titration in Hep2 cells [7] and staining with a monoclonal Chlamydia LPS-specific antibody followed by a FITC-conjugated secondary antibody (RR-402; DAKO, Glostrup, Denmark). Titres were expressed as the number of inclusion forming units (IFU) per ml. Animals and infection protocol A total of 43 female, specific pathogen-free inbred BALB/c mice were obtained from Charles River (Someren, The Netherlands). Animals were kept under standard housing and care conditions at the Central Animal Facilities of Maastricht University (Maastricht, The Netherlands). All experimental procedures were approved by the Ethical Committee for the Use of Experimental Animals of Maastricht University. At 3 months of age n = 24 mice were inoculated intranasally with 107 IFU Cpn in 10 μl (5 μl per nostril), and were analyzed at 1 week, 1 month or 3 months p.i., respectively, as shown in Table 1. Pilot experiments revealed that this dosage ensures both survival of the mice and optimal dissemination throughout the body. Another n = 15 mice were mock infected with a sucrose-phosphate-glucose solution (SPG, solution for storage of Cpn) at 3 months of age and were also analyzed at 1 week, 1 month or 3 months after mock infection, as shown in Table 1. In addition, two mice, which did not receive any treatment, were investigated at 4 months of age, as well as two mice at 6 months of age (Table 1). Table 1Numbers of animals per group investigated in the present studyMouse strain; analysisInfection with CpnMock infectionNo treatment1w1m3m1w1m3mM4M6M17BALB/c; PCR444111–––BALB/c; IHC–66–6622–APP751SL/PS1M146L––––––––1CpnChlamydia pneumoniae, IHC immunohistochemistry, 1w/1m/3m, analysis 1 week (or 1 month or 3 months, respectively) after Cpn infection or mock infection at 3 months of age. M4/M6/M17, 4, 6 or 17 months of age, respectively In addition, a 17-month-old transgenic mouse expressing human mutant APP751 (carrying the Swedish and London mutations KM670/671NL and V717I, under the Thy1 promoter) and human mutant presenilin-1 (PS-1 M146L, under the HMG promoter) was used (APP751SL/PS1M146L mouse; for a detailed description see refs [4] and [24]) (Table 1). This APP751SL/PS1M146L mouse was bred on a CBA (12.5%) × C57Bl6 (87.5%) background. Detection of Cpn DNA in brain tissue with real-time PCR As shown in Table 1, 15 mice were used to detect Cpn DNA in brain tissue with real-time PCR at different time points after infection with Cpn or mock infection. To this end the animals received a weight-adjusted dose of pentobarbital (Nembutal; Sanofi Sante B.V., Maassluis, The Netherlands) and were then perfused with sterile phosphate buffered saline. Brains were removed quickly and dissected into five different regions, i.e., olfactory bulbs, cerebellum, hippocampus, subventricular zone and neocortex. The isolated regions were snap-frozen in liquid nitrogen and stored at −80°C until further processing. DNA was isolated from brain material using methods previously described [5]. The Nanodrop® ND-1000, a full-spectrum spectrophotometer (220–750 nm) was used to calculate the DNA content of each sample. Finally, the Cpn DNA copy number/μg DNA in the collected brains was determined by real-time PCR (ABI Prism 7000; Applied Biosystems, Foster City, CA, USA) using the following primers and probe; forward (5′TCCGCATTGCTCAGCC3′), reverse (5′AAACAATTTGCATGAAGTCTGAGAA3′) and probe (5′-FAM-TAAACTTAACTGCATGGAACCCTTCTTTACTAGG3′-TAMRA) as described previously [5, 29]. All PCR runs were performed in duplo. Mean Cpn DNA copy numbers/μg brain DNA of three independent experiments (±SEM) was calculated. Then, comparisons between all groups of mice, analyzed for the presence of Cpn DNA in brain tissue, were performed with three-way ANOVA with Treatment (Cpn infection or mock infection), Time-point of analysis (1 week, 1 month or 3 months p.i.) and Brain region (olfactory bulbs, cerebellum, hippocampus, subventricular zone or neocortex) as fixed factors. P values smaller than 0.05 were considered statistically significant. Calculations were performed with SPSS v. 12.0.1 for Windows (SPSS, Chicago, IL, USA). Detection of Aß aggregates in brain tissue with immunohistochemistry As also shown in Table 1, n = 29 mice were used to detect Aβ aggregates in brain tissue with immunohistochemistry at different time points after infection with Cpn, mock infection or without any treatment, respectively. To this end, the animals received a weight-adjusted dose of pentobarbital (Nembutal; Sanofi Sante B.V., Maassluis, The Netherlands) and were then subjected to intracardial perfusion fixation, as previously described [26]. In case of the APP751SL/PS1M146L mouse, anaesthesia was performed with chloral hydrate (10% aqueous solution, 0.005 ml/g body weight, i.p.). Brains were removed rapidly and post-fixed for 2 h at 4°C in the same fixative, omitting the glutaraldehyde. In case of the APP751SL/PS1M146L mouse, the brains were halved in the midsagittal line. Brain tissues were then cryoprotected by immersion in 30% sucrose in Tris-buffered saline at 4°C overnight. Afterwards, brains (or brain halves, respectively) were quickly frozen and stored at −80°C until further processing. For immunohistochemical analysis, the entire brains (or the right brain half in case of the APP751SL/PS1M146L mouse) were cut into entire series of 30-μm-thick frontal sections on a cryostat (Leica CM 3050 S; Leica, Nussloch, Germany). After cutting, sections were again stored at −80°C until further processing. Immunohistochemical detection of Aβ (as well as of glial fibrillary acidic protein) was performed with standard immunofluorescence procedures using a first layer, consisting of a combination of mouse anti-human amyloid 17–24 (4G8 clone; 1:1,200 in TBS-T; Sigma-Aldrich, St Louis, USA) [3, 16, 33] and polyclonal rabbit anti-mouse GFAP (1:1,600 in TBS-T; DAKO, Glostrup, Denmark). The corresponding secondary antibodies were donkey anti-mouse Alexa 488 (1:100 in TBS-T; Molecular Probes) and donkey anti-rabbit Alexa 594 (1:100 in TBS-T; Molecular Probes). Finally, counterstaining was performed with Hoechst (1:500 in TBS; Sigma, St Louis, MO, USA) and sections were mounted on gelatine-coated glasses. As a negative control, sections were only incubated with the secondary antibodies. Detection of extracellular Aß aggregates in brain tissue with thioflavin-S For the detection of extracellular, fibrillary Aβ, a standard thioflavin-S (Sigma) staining was performed. Finally, the sections were counterstained with Hoechst and mounted as described above. Microscopy and photography Microscopic inspection of sections was performed with a MBF Bioscience Stereo Investigator Confocal Spinning Disk (SI-SD) system (MBF Bioscience; Williston, VT, USA), consisting of a modified Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) with UPlanSApo objectives 10 × (N.A. = 0.4), 40 × (N.A = 0.9) and 100 × (oil; N.A = 1.4); Olympus, customized spinning disk unit (DSU; Olympus), computer-controlled excitation and emission filter wheels (Olympus), three-axis high-accuracy computer-controlled stepping motor specimen stage (4 × 4 Grid Encoded Stage; Ludl Electronic Products, Hawthorne, NY, USA), linear z-axis position encoder (Ludl), ultra-high sensitivity monochrome electron multiplier CCD camera (1,000 × 1,000 pixels, C9100-02; Hamamatsu Photonics, Hamamatsu City, Japan) and controlling software (MBF Bioscience). In case of sections processed for the detection of Aß and GFAP, digital RGB image stacks showing the same microscopic field in 50 consecutive focal planes with a distance of 0.5 μm between the focal planes were generated with the 40× objective. In the case of sections stained with thioflavin-S, digital image stacks showing the same microscopic field at 150 consecutive RGB focal planes with a distance of 0.2 μm between the focal planes were generated with the 100× objective. From these image stacks three-dimensional reconstructions (maximum intensity projections) were produced with Imaris software (Version 4.0; Bitplane, Zurich, Switzerland). In addition, low-power photomicrographs (10×) were taken from sections stained with thioflavin-S. Final figures were constructed using Corel Photo-Paint v.11 and Corel Draw v.11 (Corel, Ottawa, Canada). Only minor adjustments of contrast and brightness were made, without altering the appearance of the original materials. No deconvolution was performed on images obtained by confocal microscopy. Results Presence of Cpn in the brain of BALB/c mice infected with Cpn At 1 week p.i., the mean Cpn DNA copy number per μg DNA was 310.97 ± 61.6 (mean ± SEM) in the olfactory bulbs, 2.6 ± 0.8 in the cerebellum and 7.9 ± 0.8 in the hippocampus, while no Cpn DNA was detected in the subventricular zone and the neocortex (Table 2). No Cpn DNA was detected in any brain region of the Cpn infected mice at 1 and 3 months p.i.. Furthermore, Cpn DNA was not detected in the brain of mock-infected BALB/c mice at any time point. Statistical analysis revealed significant differences among the groups of mice with respect to Treatment (T) (P = 0.024), Time point of analysis (Tpoa) (P = 0.008) and Brain region (Br) (P = 0.002), as well as with respect to the combinations T–Tpoa (P = 0.008), T–Br (P = 0.002), Tpoa–Br (P < 0.001) and T–Tpoa–Br (P < 0.001). Table 2Detection of Cpn DNA in various brain regions (olfactory bulbs, cerebellum, hippocampus, subventricular zone and neocortex) with real-time PCR at 1 week, 1 month and 3 months post infection1 week p.i.1 month p.i.3 months p.i.Olfactory bulbs310.97 ± 61.600Cerebellum2.6 ± 0.800Hippocampus7.9 ± 0.800Subventricular zone000Neocortex000Data were expressed as mean values of three independent experiments ±SEM Detection of Aβ and GFAP in the brain of BALB/c and APP751SL/PS1M146L mice Amyloid beta immunoreactivity was found in the brains of all mice, i.e. in Cpn-infected BALB/c mice at both 1 and 3 months p.i. (Fig. 1a), mock-infected BALB/c mice at both 1 and 3 months after mock infection (Fig. 1b), not inoculated BALB/c mice (Fig. 1c) and in APP751SL/PS1M146L mice (Fig. 1d). However, Aβ-immunopositive aggregates were found only occasionally in the brains of the BALB/c mice (on average one to two aggregates per section without a preference for a certain brain region), whereas they were found abundantly in hippocampus and neocortex of APP751SL/PS1M146L mice (see also Fig. 2H in [24]). Furthermore, there were several qualitative differences between the APP751SL/PS1M146L and the BALB/c mice. First, in the brain of APP751SL/PS1M146L mice, the materials immunopositive for Aβ formed a dense core, devoid of Hoechst-positive structures (i.e. cell nuclei). In contrast, the materials immunopositive for Aβ in the brain of BALB/c mice did not present with a dense core, and Hoechst-positive structures were scattered within these materials. Second, the astrocytic reaction in the brain of APP751SL/PS1M146L mice, surrounding the Aβ-immunopositive materials, was much stronger than in the brain of BALB/c mice (arrowheads in Fig. 1a–d). Third, in the brain of APP751SL/PS1M146L mice, cells with intracellular deposits of Aβ were found (asterisk in Fig. 1d), which was not observed in the brain of BALB/c mice, irrespective of treatment. Fig. 1Immunohistochemical detection of Aβ (green) and GFAP (red) in 30 μm-thick cryostat sections counterstained with Hoechst (blue) from the brain of a 6-month-old Cpn-infected BALB/c mouse 3 months post infection (a), a 6-month-old mock infected BALB/c mouse 3 months after mock infection (b), a 6-month-old BALB/c mouse neither infected with Cpn nor mock infected (c) and a 17-month-old APP751SL/PS1M146L transgenic mouse (d). Note that material immunopositive for Aβ was found in the brain of all mice (arrows). However, there were differences between the APP751SL/PS1M146L mouse and the BALB/c mice: (1) in the brain of the APP751SL/PS1M146L mouse the material immunopositive for Aβ formed a dense core devoid of Hoechst-positive structures (i.e. cell nuclei). In contrast, the material immunopositive for Aβ in the brain of the BALB/c mice did not present with a dense core, and Hoechst-positive structures were scattered within it. (2) The astrocytic reaction in the brain of the APP751SL/PS1M146L mouse surrounding the material immunopositive for Aβ was much stronger than in the brain of the BALB/c mice (arrowheads). (3) In the brain of the APP751SL/PS1M146L mouse cells with intracellular deposits of material immunopositive for Aß were found (asterisk in d). This was not observed in the brain of the BALB/c mice. Furthermore, extracellular thioflavin-S-positive structures in the brain of the 17-month-old APP751SL/PS1M146L mouse are shown [e (arrowheads) and f, resembling fibrillary Aβ deposits. Scale bar = 25 μm in a–d, 100 μm in e and 10 μm in f Detection of thioflavin-S-positive structures in the brain of APP751SL/PS1M146L but not in the brain of BALB/c mice In the brain of APP751SL/PS1M146L mice, extracellular thioflavin-S-positive structures were found (Fig. 1e, f) as also previously reported [4, 26], resembling fibrillary Aβ deposits. Such extracellular thioflavin-S-positive structures were not found in the brain of BALB/c mice. Discussion The role of inflammatory factors, predominantly caused by infections, in the initiation or acceleration of neurodegenerative diseases is increasingly recognized. Next to several viruses [6, 12, 13, 17], the obligatory intracellular bacterium Cpn has also been linked to a variety of neurodegenerative diseases such as AD. In 1998, the presence of this pathogen was described in postmortem brains from patients with AD [2]. That Cpn may contribute to neurodegenerative processes was recently supported by data from our laboratory, as we demonstrated that Cpn is able to infect neurons, ultimately resulting in neuronal cell death in vitro [5]. However, to the best of our knowledge only one experimental in vivo study has been published so far supporting a role for Cpn in AD [19]. In this study, naïve BALB/c mice were inoculated intranasally with Cpn at an age of 3 months. Brains were recovered at 1–3 months p.i., revealing the presence of amyloid-like plaques and astrocyte reactivity. Due to the possible impact of this study, it was our goal to confirm these data by repeating these experiments. Focusing on our results of Cpn-inoculated mice at 3 months p.i., material immunopositive for Aβ was indeed detected in the brain. Accordingly, at first glance, our results are in line with the study of Little et al. [19]. Comparing the morphological appearance of this material immunopositive for Aβ with corresponding material in the brain of APP751SL/PS1M146L mice, however, some significant and important differences were observed. First, one characteristic of neuritic (AD) plaques is the presence of filamentous, star-shaped aggregates of Aβ fibrils with a thioflavin-S-positive dense core. However, neither such star-shaped aggregates of Aβ fibrils nor extracellular thioflavin-S-positive structures were found in any of the brains of Cpn infected or mock infected BALB/c mice, whereas both lesions were abundant in the brain of APP751SL/PS1M146L mice. Second, no damage was found in the tissue surrounding the material immunopositive for Aβ. Rather Hoechst-positive structures (i.e. cell nuclei) could be detected within the material immunopositive for Aβ, a feature that was not seen in the brain of APP751SL/PS1M146L mice. Finally, although astrocytes were found in the vicinity of the material immunopositive for Aβ in the brain of Cpn infected or mock infected BALB/c mice, they were not intimately associated with the Aβ immunoreactivity, as seen in the brain of APP751SL/PS1M146L mice. In summary, except that Aβ plaques were detected in most brains, our data suggest that at 3 months after Cpn infection most characteristic features of AD pathology could not be detected in 6-month-old naïve, non-transgenic BALB/c mice, whereas these were prominent in the brain of the “control” APP751SL/PS1M146L mice. It may still be argued that the material immunopositive for Aβ in the brain of the Cpn-infected mice represented early precursors of neuritic plaques. To our surprise, however, nearly identical patterns of Aβ immunoreactivity were detected in mock-infected BALB/c mice as well as in BALB/c mice, neither Cpn infected nor mock infected. This suggests that the observed immunoreactivity for Aβ in the brain of BALB/c mice does not directly result from Cpn infection but might represent unrelated elevations in amyloid. Positive staining for Aβ in the brain of mock-infected mice was also observed by Little et al. [19], who found that the amyloid deposition load and size were significantly enhanced in Cpn-infected mice compared to mock infected ones. As outlined above, this was not found in the present study. Furthermore, Little et al. also demonstrated the presence of a small subset of thioflavin-S positive plaques, which were not found in our mice. These discrepancies between the study by Little et al. [19] and the present study could be due to the fact that Little et al. [19] used a Cpn strain (C. pneumoniae, 96-41, isolated from the postmortem brain of an AD patient) that was rather different from the Cpn strain used in the present study (TWAR 2043, ATCC). Notably, Little et al. [19], were able to detect Cpn in olfactory tissues of infected mice up to 3 months p.i., by both light and electron microscopy. In the present study, however, we were only able to detect Cpn DNA in the olfactory bulb of infected BALB/c mice at 1 week p.i. but not at 1 or 3 months p.i., despite the use of a more sensitive detection method (real-time PCR). As it is well established that the clinical isolates of pathogens are often more infectious than their laboratory counterparts, this could partially explain the discrepancy between both studies. Furthermore, it cannot be excluded that differences in solutions used for mock infection (Hanks balanced salt solution in the study by Little et al. [19] vs. SPG solution in the present study) may have affected the final outcome. Yet, in the present study, the immunosignal in mice that were neither Cpn infected nor mock infected appeared somewhat weaker than in the brain of the BALB/c mice that were either Cpn- or mock-infected (a detailed quantitative analysis was not performed). This suggests that intranasal application of Cpn or other infectious agents may trigger the production and deposition of Aβ in the brain of BALB/c mice. For the time being, however, the impact of this remains unclear. In conclusion, our data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD like Aβ plaques in the brain of BALB/c mice, as suggested by Little et al. [19]. Accordingly, future studies are required to investigate whether Cpn infections may aggravate AD pathology by stimulating the formation of Aβ plaques as has been suggested in atherosclerosis, a disease that is also characterized by pronounced local inflammation.

J_Gastrointest_Surg-3-1-1852383

Liver Transplantation Across Rh Blood Group Barriers Increases the Risk of Biliary Complications

Background Cold ischemia time and the presence of postoperative hepatic arterial thrombosis have been associated with biliary complications (BC) after liver transplantation. An ABO-incompatible blood group has also been suggested as a factor for predisposal towards BC. However, the influence of Rh nonidentity has not been studied previously. Introduction Biliary complications (BC) after liver transplantation (LT) are still a significant cause of morbidity and mortality.1,2 Hepatic arterial thrombosis, long ischemia time, inadequate exposure of the biliary epithelium to the preservation solution, and chronic rejection have been associated with BC.3–5 However, complications may also occur in the absence of these pathogenic factors. The expression of donor ABH antigen in the vascular and biliary epithelium of hepatic allografts has been linked to higher risk of biliary and arterial complications, especially in ABO-incompatible grafts.6 However, the influence of Rh nonidentity in LT has not been analyzed until now. The aim of this study is to analyze the incidence of BC after LT, to define their etiological risk factors, and to study the influence of Rh nonidentity on presentation of BC after LT. Materials Three hundred and fifty-six (n = 356) liver transplants were performed at our hospital between January 1995 and November 2000. Eleven were excluded from the study because important data were missing (n = 345). The study was closed on May 2001 with a 6-month-minimum graft follow-up. The procurement procedure was based on the rapid flush technique.7 After reperfusion of the allograft, cholecystectomy was performed, the donor bile duct being divided just above the cystic duct junction in most cases. Biliary anastomosis was performed with interrupted stitches of 6-0 Polydioxanone suture Ethicon® (Johnson & Johnson, Brussels, Belgium). The biliary reconstruction technique was end-to-end anastomosis in the majority of LTs (315 patients, 91%). T-tubes were used in four patients due to diameter discrepancy between the biliary ducts of the donor and the recipient. If the primary disease affected the biliary tract, or if technical factors made end-to-end anastomosis difficult, Roux-en-Y-hepaticojejunostomy was performed (25 out of 345, 7%). The outcome was assessed in terms of biliary and arterial complications and patient status (alive, retransplanted, or dead). BCs were studied routinely with abdominal ultrasonography at day 1 post LT, weekly before discharge from hospital and monthly thereafter or when there was clinical or biochemical suspicion of BC, and this was confirmed with magnetic resonance cholangiography, percutaneous transhepatic cholangiography, or endoscopic retrograde cholangio-pancreatography when necessary. The immunosuppressive regimen was based on quadruple sequential therapy with antilymphocyte globulin, Sandimmune Neoral® (Novartis, Basel, Switzerland), steroids, and azathioprine in most patients, as reported elsewhere.8 Cellular rejection was diagnosed according to histological criteria.9 Statistical analysis Chi-squared analysis was used to compare dichotomous variables and the presence of BC. Variables that were statistically significant in the univariate analysis were introduced in a multivariate logistic regression analysis. Other known risk factors for developing BC were introduced into the model as covariates: donor age, acute rejection, and chronic rejection. Variables with P > 0.05 were excluded from the final equation. Kaplan–Meier estimates of the rate of BC for both groups and the results were compared with a log-rank test. Results Perioperative data and surgical details The preservation solutions used were the University of Wisconsin solution in 332 (97%), Celsior solution in 5 (1%), and histidine-tryptophan-ketoglutarate solution in 8 (2%). Twenty grafts (6%) were shipped by another team. The study of donor risk factors demonstrated that there was no hemodynamic instability (systolic pressure lower than 60 mmHg for more than 1 h) in 265 cases (77%), and that the main cause of death was traumatic head injury (129/36%). Donor Rh blood group was positive in 301 (87%) and negative in 44 cases (13%). Recipient Rh blood group was positive in 284 (82%) patients, and negative in 61 (18%). Rh blood groups of donor and recipient were positive to positive in 255 cases (74%), positive to negative in 46 (13%), negative to positive in 30 (9%), and negative to negative in 14 (4%). Donors and recipients had identical Rh in 269 cases (78%) and nonidentical in 76 (22%). The donor ABO blood group was A in 157 (46%), B in 29 (8%), AB in 19 (5%), and O in 140 (41%), while recipients were A in 161 cases (46%), B in 34 (10%), AB in 20 (6%) and O in 130 (38%). The ABO groups of transplanted grafts and hosts were identical in 97% of cases (335 patients) and compatible in 10 cases. There were no cases of incompatible grafts.During postoperative development, 25 LTs presented arterial thrombosis (7%). Eight grafts presented primary nonfunction and were retransplantated (2.2%), and 43 presented initial poor function that recovered spontaneously (12%).3 Biopsy-proven acute rejection was diagnosed in 66 grafts (19%) after transplantation, with development to chronic ductopenic rejection in 8 (2%). BCs and Rh mismatch BCs appeared in 70 of the 345 patients (20%). Cross-tabs were built to analyze differences between the grafts that suffered BCs and the rest. Both groups were similar in terms of donor preoperative evaluation and support, recipient descriptive data, and surgical terms. The incidence of BC in nonidentical Rh graft–host cases (23/76; 30%) was significantly higher than in cases of identical Rh graft–host (47/269; 17%, P = 0.01). Cases of arterial thrombosis after LTs and ischemia time longer than 430 min were also associated with a higher incidence of BC (Table 1). Table 1Demographics and Major Complications Occurring in Both Groups Studied, Chi-square Test BCs (n = 70)No BCs (n = 275)Chi-squareDonor dataABO blood group0.2 A25/157 (16%)132/157 (84%) B6/29 (21%)23/29(79%) AB6/19 (32%)13/19(68%) O33/140 (24%)107/140 (76%)Rh blood group0.4 Positive59/301 (19%)242/301 (81%) Negative11/44 (25%)33/44 (75%)Sex0.4 Male51/233 (22%)182/233 (75%) Female19/110 (17%)91/110 (83%)Donor age0.4 ≤70 years66/320 (20%)254/320 (80%) >70 years4/25 (16%)21/25 (84%)Recipient dataRh blood group0.2 Positive54/284 (19%)230/284 (81%) Negative16/61 (26%)45/61 (74%)Rh D-R crossing0.1 Positive–positive45/255 (18%)210/255 (82%) Positive–negative14/46 (30%)32/46 (70%) Negative–positive9/30 (30%)21/30 (70%) Negative–negative2/14 (14%)12/14 (86%)Rh D-R identity0.01 Identical47/269 (17%)222/269 (83%) Nonidentical23/76 (30%)53/76 (70%)ABO blood group0.4 A27/161 (17%)134/161 (83%) B7/34 (20%)27/34(80%) O30/130 (23%)100/130(77%) AB6/20 (30%)14/20 (70%)ABO D-R identity0.4 Identical67/335 (20%)268/335 (80%) Nonidentical3/10 (30%)7/10 (70%)Sex of recipient0.4 Male48/214(22%)166/214 (78%) Female22/130(17%)108/130(83%)Recipient age0.2 <60 years41/221 (18%)180/221 (82%) ≥60 years29/124 (24%)95/124 (76%)Diagnosis0.3 Choleostasis2/14(15%)12/14(85%) Cirrhosis32/162 (20%)130/162(80%) Hepatocarcinoma27/103(26%)76/103(74%) Other etiology1/18(5%)17/18(95%) Re-OLT7/39(18%)32/39 (89%) Other tumors1/9(11%)8/9 (89%)Surgical dataCold ischemic time0.01 ≤430 min26/178 (15%)148/178 (85%) >430 min44/171 (26%)127/171 (74%)Type of anastomosis0.6 Termino-terminal64/315(20%)251/315(80%)Graft evolution dataArterial thrombosis0.03 Yes9/25 (36%)16/25(64%) No60/319 (19%)259/319(81%)Initial poor function0.6 Yes10/43 (23%)33/43 (77%) No60/302 (20%)242/302 (80%)Primary nonfunction0.1 Yes08/8 (100%) No70/336 (20%)266/336 (80%)Acute rejection0.4 Yes11/66(17%)55/66(83%) No59/279(21%)220/279(79%)Chronic rejection0.5 Yes1/8(12%)7/8(88%) No69/337 (20%)268/337(80%)OLT orthotopic liver transplantation Multivariate Analysis of Risk Factors for BCs In multivariate analysis, arterial thrombosis presented an adjusted relative risk (aRR) = 2.6, CI 95% = (1.1–6.4) (P = 0.02), cold ischemia time aRR = 1.8 (CI 95%  = 1–3.2) (P = 0.02), and Rh graft–host nonidentity aRR = 2 (CI 95% = 1.1–3) (P = 0.02) were confirmed as independent risk factors for BC. Other variables included in the initial analysis were nonsignificant (Table 2). Kaplan–Meier estimator and log-rank test confirmed these findings (P = 0.01, Fig. 1). To discard the possible association between the Rh match and the two other risk factors, we demonstrated that arterial thrombosis had similar incidence in Rh-nonidentical grafts (3/75, 4%) and in Rh-identical grafts (22/269, 8%; P = 0.1). Moreover, grafts with long ischemia times (>430 min) had similar incidence in the Rh-nonidentical (42/76, 55%) group and in Rh-identical patients (129/269, 48%; P = 0.1). Table 2Biliary Complications Univariate logistic regressionMultivariate logistic regressionDonor age >70 years0.50.7Rh D-R identity identical nonidentical0.01; 2(1.1–3.6)0.02; 2 (1.1–3.6)Cold ischemic time >430 min0.01; 1.9(1.1–3.3)0.02; 1.8(1–3.2)Arterial thrombosis (yes)0.04; 2.4(1–5.7)0.02; 2.6 (1.1–6.4)Acute rejection (yes)0.40.4Chronic rejection (yes)0.50.5Univariate and multivariate logistic regression.Figure 1Kaplan–Meier estimates for the onset of BCs for nonidentical Rh graft–host (P = 0.01). Type and Management of BCs BCs were diagnosed in 70 patients. Biliary duct anastomosis stricture was the main complication, presented clinically, with (7/10%) or without postoperative leak (21/30%). Solitary leaks (17/24%), ischemic-type BCs (ITBC) with arterial thrombosis (6/9%), ITBC without arterial thrombosis (10/14%), and lithiasis (9/13%) were also related complications. The therapeutic approach was surgical in 23 patients (33%), endoscopic in 20 (28%), retransplantation in 11 (16%), and conservative treatment in 16 (23%). Chi-square test was performed to analyze differences between the type of BC and the three risk factors found. As would be expected, arterial thrombosis was identified in all the cases of ITBC with thrombosis, in a higher percentage than the other BCs (P < 0.001). However, the different types of BCs were not associated with long ischemia time (P = 0.2) or Rh-identity (P = 0.4). Finally, chi-square test was also performed to analyze differences among the three BC risk factors and medical management. The therapeutic approach regarding Rh-mismatch was similar in both groups (P = 0.3). Retransplantation was a frequent approach in arterial thrombosis management (4/9, 40%; P = 0.04). Interestingly, when the BC was presented in grafts with short ischemic time (<430 min), management by surgery (10/26, 38%) or an endoscopic approach (10/26, 38%) were sufficient, and it was not necessary to retransplant. However, when the BC arose in a graft with a long ischemic time (>430 min) the management was more aggressive, with 29% of patients (13/44) needing surgery and 25% (11/44) needing retransplantation. Discussion Etiopathogenesis of BC Currently, orthotopic liver transplants are performed with good results at several centers without taking the donor–recipient Rh relationship into account. In fact, no prior work has shown greater morbidity or mortality after the usage of Rh-incompatible liver grafts. However, it was in studying the causes of BC in our grafts in prior studies that we began to suspect the existence of a possible relationship between BC and Rh incompatibility. First, we observed a greater rate of BC in the presence of preservation lesions in postreperfusion biopsies.10 Thus, we found that the biliary epithelium is very sensitive to changes during preservation. Second, the description of lesions like ITBC,11 to which surgical technique does not contribute as a primary cause, led us to suspect possible immunological pathogenesis of the BC. Lastly, it is paradoxical to find that, while surgical technique is improving and satisfactory results are achieved in liver transplants, BCs are still a problem, leading us to suspect that there are unidentified factors that cause them. The arterial thrombosis and long cold ischemia time were independent risk factors for developing BC, as was expected and reported by others.11,12 In our study we have demonstrated that even though BC in grafts with short ischemia can be resolved with surgery or endoscopy, the prognosis of BC was worse in cases of long ischemic grafts requiring retransplantation. Relationship Between ABO and Rh in Liver Transplants As for the donor–recipient ABO relationship, the usage of ABO-incompatible grafts has been discouraged due to the high rate of BC and poor graft survival.6,13,14 In the Sanchez-Urdazpal study,6 82% of the 18 ABO-incompatible grafts presented BC in comparison with 6% of the control group, while Farges,14 published slightly better results several years later with BC of 54%. Therefore, these grafts are used in extremely urgent cases when there is no other possible alternative. Immunological phenomena, such as rejection,3 may also lead to biliary strictures. In the same way, the ABO system was shown to cause more BC and worse graft survival in LT.6,13–15 However, Rh nonidentity seems to have better tolerance and is not a cause of graft refusal when a donor appears. Surprisingly, our study demonstrated a higher incidence of BC in the Rh-nonidentical group. Some authors16,17 reported low rates of alloimmunization in Rh-negative recipients of Rh-incompatible transfusion after LT. It was suspected that immunosuppressant drugs modified the immunosuppressive response.16 However, debate still exists as to whether the D barrier can be crossed in LT. Previous studies of ABO barrier and BC suggested the hypothesis of an immunological injury to the bile duct epithelium, and the expression of ABH antigens in the donor 150 days after transplantation.6 However, the D antigen is only expressed in erythrocytes. The nonidentical Rh group has two mismatch possibilities: positive donor to a negative recipient or negative donor to positive recipient. In the first case (positive to negative) the immunologic mechanism is easy to understand because the humoral anti-D (Rh) response may be responsible for the graft injury. The other subgroup (negative to positive) may have a different pathogenic explanation. Bryan et al.17 hypothesized that two mechanisms could be involved in the same process in kidney transplantation: other Rh antigenic loci (C and E) and histocompatibility antigenic crossings. A negative liver graft in a positive recipient with lymphatic cells and tissues predisposes to cellular response against it. Finally, the biliary tract can probably suffer immunological damage, and thus further BC. Therefore, while the results of the study lead to suspicion of an immunological pathogenesis, the mechanism is still unclear. In conclusion, Rh-nonidentical LT involves a higher rate of BCs. Future studies should examine the influence of Rh donor and blood group on graft development. Finally, our results suggest that there is a summation effect of BC risk factors. In our opinion, Rh-nonidentical liver grafts should not undergo a very long ischemia time.

Appl_Microbiol_Biotechnol-3-1-1914237

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains. Introduction The sequencing and annotation of the Pseudomonas putida KT2440 genome (Nelson et al. 2002) has greatly catalyzed research on this strain and other academically and biotechnologically relevant but non-sequenced P. putida strains. A number of key scientific advances (both pure and applied) have been made via proteome and transcriptome analysis of P. putida strains (Dominguez-Cuevas et al. 2006; Hallsworth et al. 2003; Morales et al. 2006; Reva et al. 2006; Santos et al. 2004; Segura et al. 2005; Volkers et al. 2006; Yuste et al. 2006). In our laboratories, we have studied both P. putida KT2440 (for recent publications: Hallsworth et al. 2003; Martins dos Santos et al. 2004; Nelson et al. 2002; Timmis 2002) and the solvent-tolerant P. putida S12 (De Bont 1998; Hartmans et al. 1990; Wery and De Bont 2004). Unlike most pseudomonads, solvent-tolerant strains possess an extraordinary tolerance to a broad variety of toxic solvents (for reviews: De Bont 1998; Kieboom and De Bont 2000; Ramos et al. 2002). A large number of high-added value bioconversions involve toxic, generally apolar, products (aromatics, aliphatic alcohols, epoxides, etc.). Especially for bioprocesses involving such products, the use of solvent-tolerant P. putida strains renders advantages in terms of productivity and the application of multiphase media for product recovery (Ramos-Gonzalez et al. 2003; Rojas et al. 2004; Wery and De Bont 2004; Wery et al. 2000; Wierckx et al. 2005). Despite their biotechnological potential, the genomes of S12 and other Pseudomonas strains have not been sequenced. So, recent studies of their metabolic potential have been limited to comparative proteomics (Segura et al. 2005; Volkers et al. 2006) or transcriptomics based on the database information for P. putida KT2440. The genus Pseudomonas is very heterogeneous (Anzai et al. 2000); even strains of one species tend to vary greatly in terms of both phenotypic (Grimont et al. 1996) and genotypic characteristics (Brosch et al. 1996). Strains belonging to the species P. putida can be categorized into biovar A and B: the former grouping (A) is the more heterogeneous (Brosch et al. 1996) and the phylogenetic and metabolic diversity of biovar A strains has yet to be fully characterized and industrially exploited. Comparative transcriptomics-based approaches have played a pivotal role in recent investigations of complex cellular responses of P. putida strains (Dominguez-Cuevas et al. 2006; Duque et al. 2007; Yuste et al. 2006). As transcriptome profiling is based on the highly-sensitive detection of DNA–cDNA hybridization, DNA sequence similarity determines the validity of these analyses. The inherent heterogeneity of the P. putida grouping provides additional cause for concern that microarray platforms derived from strain KT2440 may provide a valid framework for the study of nonsequenced P. putida strains. We therefore carried out this study to assess the utility of KT2440-based high-density DNA microarrays for transcriptomics studies of DSM 6125 (subculture of KT2440), DSM 3931 (subculture of mt-2), DSM 291T (the P. putida type strain), the solvent-tolerant S12, and other nonsequenced Pseudomonas strains. In addition, the use of such microarrays to establish highly discriminative phylogenetic relationships between these strains was demonstrated. Materials and methods Strains, cultivation conditions, and DNA extraction Single colonies from each Pseudomonas strain studied (see Table 1) grown on Pseudomonas Isolation Agar (Difco) were used to inoculate Luria–Bertani broth (LB) (Sambrook et al. 1982) in duplicate. After cultures were grown overnight at 30°C, genomic DNAs were prepared from 5 ml of culture (approximately 3 × 108 cells/ml) using the Genomic DNA 100/G Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA concentrations were determined at 260 nm using ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and purity was confirmed by agarose (1% w/v) gel electrophoresis. Table 1Source and designations of Pseudomonas strains used in this studySpecies and strain numberaSource or referencebOther designationscP. putida DSM 6125DSMZKT2440, ATCC 47054; NCIMB 11950P. putida DSM 3931DSMZmt-2, ATCC 23973; ATCC 33015; JCM 6156; NCIMB 12182P. putida S12Hartmans et al. 1990ATCC 700801P. putida DSM 291TDSMZDSM 50202T; ATCC 12633T; ICPB 2963T; NCTC 10936T; CCUG 12690T; LMG 2257TP. putida DSM 50198DSMZATCC 17453; ICPB 2563-77; JCM 6157; NCIMB 10007P. putida DSM 50208DSMZATCC 17485; ICPB 2789-111; JCM 6158; NCIMB 12092P. monteilii DSM 14164DSMZATCC 700476; CCUG 38736; LMG 21609; CIP 104883P. fluorescens DSM 50090TDSMZATCC 13525T; ICPB 3200T; NCIMB 9046T; NCTC 10038T; CCUG 1253T; LMG 1794TaStrain designations used in this studybDSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, GermanycSubcultures of the used strains available in other culture collections. ATCC American Type Culture Collection, ICPB International Collection of Phytopathogenic Bacteria, USA, NCIMB National Collections of Industrial and Marine Bacteria, UK, NCTC National Collection of Type Cultures, UK, JCM Japan Collection of Microorganisms, Japan, CIP Collection bacteriènne de l’Institut Pasteur, France, CCUG Culture Collection University Göteborg, Sweden, LMG BCCM/LMG Bacteria Collection, Belgium. Preparation of biotin-labelled fragmented genomic DNAs Approximately 17 μg of heat-denatured genomic DNA was fragmented using 0.3 U DNaseI (Pharmacia)/μg DNA at 37°C for 20 min (Wolfgang et al. 2003). DNaseI was inactivated by immediate heat treatment at 95°C for 10 min, followed by cooling on ice. An aliquot of fragmented DNA was analyzed by agarose (4% w / v in TAE buffer) gel electrophoresis to confirm the purity and establish the mean size of DNA fragments (20–50 bp). Approximately 7 μg of fragmented DNA was labelled with biotin according to the Affymetrix “Expression Analysis Technical Manual for Prokaryotic Samples”. Design of high-density oligonucleotide microarrays and hybridization with genomic DNAs High-density oligonucleotide microarrays based on the annotated genome of P. putida KT2440 (NC 002947.3) were constructed according to Affymetrix specifications (http://www.affymetrix.com/support/technical/other/custom_design manual. pdf ) with the pair-wise configuration of 13 perfect match (PM) and mismatch (MM) 25-mer oligonucleotides per gene (probe set). The microarrays included 7,781 probe sets: 5,338 representing 5,350 annotated genes or open reading frames (orfs), and 2,443 targeting intergenic regions. Hybridization and washing of the microarrays were performed on a GeneChip® Node system (Hybridization temperature 49°C; Hybridization oven 640, Fluidics station 450; Affymetrix, Santa Clara, CA, USA) following the supplier’s instructions. Scanning was carried out by ServiceXS (Leiden, The Netherlands) on a high resolution Gene Chip® Scanner 3000 7G system with autoloader (Affymetrix, Santa Clara, CA, USA) using the default analysis settings of the manufacturer (filter: 570 nm; pixel size: 2.5 μm). Hybridization intensity data were extracted from the scanned array images, and the designations for presence or absence were calculated with Affymetrix Microarray Suite (MAS) 5.0 software. A one-sided Wilcoxon’s Signed Rank was used to calculate p-values reflecting the statistical significance of differences between PM and MM of a probe set. The significance levels for detection calls of probe sets were p-value < 0.05 for present and p-value ≥ 0.05 for absent. Differences between PM and MM were considered insignificant and removed from further consideration by comparisons of the discrimination score [(PM−MM)/(PM+MM)] with the defined discrimination threshold τ (discrimination score<0.015). Comparisons of present/absent designations for genes in the different strains were made with GeneSpring version 7.2 (Agilent). Replicate assays of biological duplicates were performed for P. putida DSM 6125 and S12. The deviation caused by present/absent designations of genes in only one of both replicates was 0.8% for DSM 6,125 and 4.2% for S12 regarding all probe sets for putative genes and intergenic regions (Table 2). Table 2Similarity indices based on designations of presence or absence (stated in brackets) from hybridization signals of genomic DNA fragments from different Pseudomonas members on P. putida KT2440-based microarraysaStrainPresent (absent) in percent (%)Putative genes and intergenic regions (7,781 probe sets)Putative genes (5,338 probe sets)Putative genes with assigned function (3,670 probe sets)P. putida DSM 6125b97.6 (1.4)99.6 (0.0)99.8 (0.0)P. putida DSM 393198.6 (1.4)99.9 (0.1)100.0 (0.0)P. putida S12b78.0 (16.9)81.8 (13.9)86.9 (9.7)P. putida DSM 291T60.5 (39.5)64.2 (35.8)69.3 (30.7)P. putida DSM 5019856.2 (43.8)60.7 (39.3)66.5 (33.5)P. putida DSM 5020858.8 (41.2)62.6 (37.4)67.8 (32.2)P. monteilii DSM 1416457.7 (42.3)61.7 (38.3)66.8 (33.2)P. fluorescens DSM 50090T22.0 (78.0)27.2 (72.8)30.7 (69.3)aPresent/absent designations (present: p < 0.05; absent: p ≥ 0.05; τ = 0.015) derived from a decision matrix in Affymetrix MAS 5.0bReplicate assays were performed for P. putida DSM 6125 and S12 and the values listed include only genes that were designated present or absent in both replicates. Calculation of dendrograms Simple matching similarity matrix based on the present/absent detection calls in microarray analysis of different pseudomonads (SM = m/n; m, number of matching probe sets; n, total number probe sets), and the microarray genotyping dendrogram were calculated using UPGMA parameters (unweighted pair group method, arithmetic average) and agglomerative hierarchical clustering with XLSTAT version 7.5.3 (Addinsoft, Paris, France). AFLP analysis In this paper, we used amplified fragment length polymorphism (AFLP) (Janssen et al. 1996; Savelkoul et al. 1999; Vos et al. 1995) to analyze different strains of P. putida a P. monteilii and a Pseudomonas fluorescens. AFLP is based on selective amplification of restriction fragments from totally digested genomic DNA. AFLP fingerprints were performed by KeyGene (Wageningen, The Netherlands). Genomic DNA of the different pseudomonads was digested using restriction enzyme combination NlaIII (Westburg, Leusden, The Netherlands) and Csp6I (Fermentas, St. Leon-Rot, Germany) according to manufacturers instructions. Each restriction enzyme was combined with the ligation of specific linker oligonucleotide pairs (NlaIII: 5-GACGATGAGTCCTGAG-3/5-TGTACGCAGTCTAC-3; Csp6I: 5-GACGAT GAGTCCTGAG-3/5-TACTCAGGACTCAT-3). For each of these linker combinations, AFLP was performed using nine N/C AFLP primer combinations, which were selected using KT2440 genome sequence as reference and software package REcomb (Keygene) for prediction analysis. These primers were extended with a 3′ terminal dinucleotide (+ 2) and the extensions were CA/AC, CA/CA, CA/CC, CA/GG, CA/TT, CT/TT, CT/CA, CT/CT, and CT/TC. For further detail, please refer to Van den Braak et al. (2004). PCRs were performed in the presence of radioactive nucleotides, and the amplimers obtained were separated on 50 × 20 cm polyacrylamide slabgels. Using phosphor-imaging, the individual presence/absence in a total of 757 markers (DNA bands) per strain was analyzed. The total marker score data table (presence/absence of individual DNA bands) was subjected to genetic distance analysis using simple matching similarity matrix (SM = m/n; m, the number of matched scores; n the total sample size), consisting of similarity indices for all combinations of AFLP-banding patterns. Simple matching coefficients were calculated using NTSYSpc-software version 2.2 (Exeter Software, Setauket, NY, USA). To visualize the relationship between the strains, a dendrogram was generated using Sequential Agglomerative Hierarchical Nested (SAHN) cluster analysis with the use of UPGMA parameters. Standard PCR and sequencing Diagnostic PCR of selected putative orfs (PP1265, PP5224) that were called absent in microarray analyses of P. putida DSM 3931 genomic DNA was performed using proofreading enzyme mixture High Fidelity PCR master (Roche Diagnostics, The Netherlands) and specific primers (PP1265: forward: 5′-CTGCTGCACCAGGCCTAT-3′, reversed: 5′-TTGGTCACATAGCCGTCAAC-3′; PP5224: forward: 5′-CAACGGCTAAACCTTTGCAT -3′, reversed: 5′-AGGATCGAGACCTTGCCTTC-3′). Yielded PCR-amplicons of expected sizes, 1,108 and 1,062 bp, respectively, were sent to Baseclear (Leiden, The Netherlands) for sequencing according to Sanger et al. (1977) using nested sequencing primers (PP1265: 5′-CCAGGCAATCCGTGTCAT-3′; PP5224: 5′-GGTGTCCTGACCGTCAAGTT-3′), and the resulting sequences were used for sequence alignments. Biological function-derived phylogenetic analysis The concept of Clusters of Orthologous Genes (COG; see Tatusov et al. 1997, 2003) was used during analysis of the genomic content of nonsequenced Pseudomonas strains by linkage to primary biological function (Table 3). A COG consists of individual proteins or groups of paralogs from at least three lineages and thus corresponds to an ancient conserved domain (Tatusov et al. 1997, 2003). In the NCBI-COG database, 4,497 proteins of 5,350 putative orfs in P. putida KT2440 were identified as COGs and were arranged in functional categories (see http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=266 ). Because of the limited number of COGs in the functional categories (A) RNA processing and modification, (B) chromatin structure/dynamics, and (W) extracellular structures (1, 2, and 3, respectively), these COGs were collectively grouped together with general function prediction COGs (Category R) under the designation R′(see Table 3). Table 3Primary functional designationa of genes identified in P. putida S12 genomic DNACodebDescriptionCOGs in KT2440cCOGs KT2440 in S12FNucleotide transport and metabolism9387 (93.5%)JTranslation194178 (91.8%)HCoenzyme transport and metabolism183166 (90.7%)TSignal transduction mechanisms427387 (90.6%)ILipid transport and metabolism194174 (89.7%)NCell motility130116 (89.2%)GCarbohydrate transport and metabolism264232 (87.9%)PInorganic ion transport and metabolism368322 (87.5%)UIntracellular trafficking and secretion119104 (87.4%)EAmino acid transport and metabolism630550 (87.3%)OPosttranslational modification, protein turnover, chaperones194169 (87.1%)CEnergy production and conversion321276 (86.0%)QSecondary metabolites biosynthesis, transport and catabolism161138 (85.7%)KTranscription499426 (85.4%)VDefense mechanisms6656 (84.8%)R′General function prediction only733620 (84.6%)SFunction unknown442365 (82.6%)MCell membrane biogenesis288227 (78.8%)DCell cycle control5641 (73.2%)–Not in COGs853544 (63.8%)LReplication, recombination and repair269158 (58.7%)aBased on the Clusters of Orthologous Groups (COG) system, also see Materials and methods section)bCodes of functional categories of COGcProteins, 4,497 of 5,350 putative orfs in P. putida KT2440 can be found in COG database (http://www.ncbi.nlm.nih.gov/ COG/) Results Genomic DNA hybridizations with P. putida KT2440-based high-density DNA microarrays Total genomic DNA from P. putida strains, and other nonsequenced Pseudomonas members (see Table 1), was hybridized to custom KT2440-based high-density oligonucleotide microarrays. Presence or absence designations for each probe set (designed for specific genes and intergenic regions) were calculated by the Affymetrix MAS 5.0 algorithm from the significant difference (see Materials and methods section) in hybridization intensities between the corresponding perfect match and mismatch oligonucleotides (Table 2). Absence designation is a synonym for the absence of significant signal values and stands for divergent DNA still coding a similar biological function or for the complete absence of the specific DNA. Replicate array hybridizations were performed for DSM 6125 DNA and S12 DNA only. For these DNAs the values given in Table 2 represent only probe sets designated present or absent in both replicates. As expected, the DSM 6125 DNA yielded an almost perfect score: 97.6% for probe sets corresponding to all genetic elements (including the intergenic regions) and 99.8% for probe sets designed for genes with an assigned function. Strain DSM 3931 (subculture of P. putida mt-2: Teruko, 2002) is a TOL plasmid (pWW0)-containing variant of DSM 6125 and was used as an additional control for the accuracy of the microarray experiments. As expected, both strains were virtually indistinguishable in the array hybridization study (Table 2). Nonetheless four orfs were indicated absent in DSM 3931. These orfs were found to be called absent in only one of the DSM 6125 replicates. We therefore used diagnostic PCR to investigate the presence of two of them (PP1265, PP5224) in DSM 3931; the other two appeared less important due to their limited size (~90 bp). In both cases, PCR products of the expected sizes (1,108 and 1,062 bp) were obtained that, after sequencing, proved to be identical to the KT2440 homologs. Apart from DSM 3931, the solvent-tolerant S12 showed the highest genomic similarity to KT2440. Nearly 3,188 of 3,670 (86.9%) genes with assigned functions in the KT2440 genome were identified in the genomic DNA of S12 (Table 2). Approximately 71% of 1,668 (putative) genes without an assigned function, and 70% of the intergenic regions were found to be present in the S12 (data not shown). Microarray-based genomotyping A dendrogram was constructed (Fig. 1a) based on genomic similarity of all 7,781 genetic elements of KT2440 in the tested genomic DNAs except for the control strain DSM 3931 (Table 2). DSM 6125, DSM 3931 (not shown), and S12 clustered in a group separate from the other P. putida strains (Fig. 1a). The nonsequenced P. fluorescens (DSM 50090T), that was included as an out-group, did not cluster with any other strain. By contrast, Pseudomonas monteilii DSM 3931 that was included as a non-putida member clustered with other P. putida strains suggesting a closer relationship with the P. putida taxonomic grouping (Fig. 1). To assess the validity of these microarray-derived phylogenetic relationships, AFLP DNA fingerprinting was used to obtain an independent phylogenetic classification of strains (Janssen et al. 1996; Savelkoul et al. 1999; Vos et al. 1995). The phylogenetic tree constructed following AFLP analyses showed an identical pattern in terms of strain grouping. (Fig. 1b). Fig. 1Genome similarity between different Pseudomonas strains. a High-density microarray genotyping tree based on absent/present designations generated by Affymetrix MAS 5.0 algorithm (Table 2) using simple matching similarity coefficient, UPGMA, and agglomerative hierarchical clustering. b AFLP-genotyping. Tree is based on the score of 757 AFLP markers using the simple matching similarity coefficient and Sahn cluster analysis An inventory was made of genes encoding proteins belonging to COGs (Tatusov et al. 1997, 2003) that were identified in the different genomic DNAs (Fig. 2). Of 5,350 orfs in the KT2440 genomic DNA sequence, 4,497 encode proteins that have been categorized into classes of primary biological function based on the COG system (http://www.ncbi.nlm.nih.gov/ COG/). It was found that, after DSM 3931, P. putida S12 showed the highest present score in all functional classes (Fig. 2). The “present” designations for S12 genes encoding COG members ranged from 58.7 to 93.5%, depending on their primary biological function (Table 3, Fig. 2). The unequal distribution of the present designations over the different functional classes was also characteristic of other Pseudomonas strains (Fig. 2). Strikingly, all pseudomonads other than DSM 6125, DSM 3931, S12 and the out-group DSM50090T showed a comparable distribution pattern of identified genes over the different COGs. Fig. 2Distribution of genes encoding COG proteins over different functional categories as identified in Pseudomonas strains. Functional categories were adopted from the COG database for P. putida KT2440 (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=266 ). The categories of biological function corresponding to COG codes are given in Table 3. P. putida strains are well-known for their broad metabolic potential regarding aromatic compounds (Jimenez et al. 2002; Wackett 2003, for reviews). Several pseudomonads, including P. putida KT2440, have been reported to degrade and/or transform a variety of aromatics. Among these are compounds of industrial importance, and there is an interest in studying these metabolic pathways on the level of gene expression and regulation thereof. The utility of KT2440-based microarrays in transcriptomics studies of aromatics metabolism of the pseudomonads under investigation was appraised. The presence of 70 genes of central and peripheral metabolic routes of aromatics were chosen based on reports by Jimenez et al. (2002) and Wackett (2003) (Table 4). All genes (100%) were detected in DSM 6125, S12 and in DSM 3931. Higher divergence was found for P. putida DSM 291T (82.9% detectable), DSM 50198 and DSM 50208 (both 75.7% detectable), and DSM 14164 (74.3% detectable). DSM 50090T again behaved as an out-group with only 25.7% of the 70 genes detectable. In the P. putida strains used in the present study and DSM 14164, almost all genes for degradation of benzoate (ben), homogentisate (hmg, mai), phenylalanine/tyrosine (phh, tyrB), and catAB were detected. In DSM 291, 50198, 50208 and 14164 putative regulatory genes of different pathways (e.g. catR, pcaQ, pobR, phaNM) and some isoenzymes (e.g., catA2) were not found (Table 4). Interestingly, in all these strains, the muconolactone isomerase (catC), and in the catabolism of phenylpropenoid compounds, vanillin dehydrogenase (vdh), putative conifer aldehyde dehydrogenase (calB; except DSM 50198), and feruloyl-CoA synthetase (fcs; except for DSM 50208) were not identified and can thus not be studied in KT2440-based microarray-based transcriptome analysis. Table 4Comparison of the presence/absence designations of genes encoding metabolic pathways of aromatic compounds in different pseudomonads derived from Affymetrix decision matrix MAS 5.0StrainPresent (%)aGenes of aromatic pathways not identified in hybridizations with genomic DNAP. putida DSM 6125100.0P. putida DSM 3931100.0P. putida S12100.0P. putida DSM 291T82.9catC; fadAxB2x; fcs; pcaQ; phaDHMN; vdh; calB; catA2P. putida DSM 5019875.7benE-1; catCR; ech; fadAxB2x; fcs; pcaDQ; phaGHMN; pobAR; vdh; catA2P. putida DSM 5020875.7catCR; ech; fadAxB2x; pcaBCQ; phaDHJMN; phhR; pobR; vdh; calB; catA2P. monteilii DSM 1416474.3benE-1; catCR; ech; fadB2x; fcs; pcaBDQ; phaEGHMN; pobAR; vdh; calB; catA2P. fluorescens DSM 50090T25.7aat; acdA; benACDE-1E-2FKR; catABCR; fadAxBB1xB2xDx; fcs; hpd; pcaCDHIJKQT; pcm; phaAB-EG-ILMN; phhBR; pobAR; tyrB-2; vanAB; vdh; calB; ferR; catA2aSelected 70 genes (100%) involved in catabolism of aromatic compounds annotated in P. putida DSM 6125: protocatechuate (pcaBCDFGHIJKQRT), catechol branches β-ketoadipate pathway (catABCRA2), homogentisate pathway (hmgA, mai), central meta-cleavage (pcm), p-hydroxybenzoate (pobAR), benzoate (benABCDE-1E-2FKR), phenylacetate pathway (phaABCDEFGHIJKLMNJ1), n-phenylalkanoic acids (fadABDHAxB1xB2xD2Dx), phenylpropenoid compounds (vanAB, vdh, calB, ferR, fcs, ech, aat, acdA), and phenylalanine/tyrosine (phhABR, tyrB-1B-2, hpd, pcm). Discussion A major advantage of microarray-based comparisons of species is the ability to pinpoint differences in individual genes and intergenic regions. Through these comparisons, detailed insight was gained in the utility of P. putida KT2440-based microarrays in transcriptomics studies of different pseudomonads at the level of specific categories of biological function. It was found that genes involved in, e.g., “translation” and “nucleotide transport and metabolism” could be identified at a high frequency (>80%) in all P. putida strains tested, in contrast to other functional groups where the frequency of gene identification dropped below 60%. Whereas P. fluorescens DSM 50090T behaved as a typical out-group in these functional studies, it was clear that, depending on the functional category, up to 45% of the genes of this strain could still be identified. There is a biotechnological requirement for P. putida biocatalysts that can function at high solvent concentrations, such as strain S12, and that can be swiftly optimized for different bio-based production processes. In our group, studies have focused on the construction of strains that are able to convert renewable substrates, such as sugars, into aromatics of interest via central metabolites (Nijkamp et al. 2005, 2006; Wierckx et al. 2005). These conversions take place via multiple metabolic pathways each consisting of several enzymatic steps with regulatory mechanisms that are being investigated using S12 as a model system. A comparative transcriptomics approach is invaluable to gain detailed insights into the complex cellular systems of the metabolically versatile pseudomonads. The employment of the high-density KT2440 microarrays would enable highly sensitive and reproducible transcriptome analyses that are compatible with those used for model species such as P. aeruginosa (Ochsner et al. 2002; Wagner et al. 2003; Whiteley et al. 2001) and Escherichia coli (Woo et al. 2004). In the present study, we showed that the use of KT2440-based microarrays would enable reliable transcriptomics analysis of P. putida S12. Significantly, we found that of all pseudomonads tested, the genomic content of P. putida S12 showed the highest similarity to that of P. putida DSM 6125 (KT2440). The vast majority of KT2440 genes coding for proteins involved in primary metabolism, including biosynthesis of important intermediates such as amino acids, and the genes involved in the conversion of aromatic compounds were shown to be sufficiently similar to those of S12. The high resolution achieved by comparative genomotyping enabled the identification of minute genotypic differences between tested strains, making a meaningful analysis of phylogenetic relationships feasible. For example, the genomic DNA of P. putida DSM 6125 was shown to be virtually identical to that of the control strain DSM 3931, and this is consistent with the origin of strain KT2440 as a derivative of strain mt-2 (Regenhardt et al. 2002). The relationship between the P. putida DSM 291T and KT2440 has been an issue of controversy. Based on 16S rRNA gene comparisons (99% identity), both strains appeared closely related; however, a DNA–DNA hybridization experiment indicated only 50.5% genome relatedness between both strains (Regenhardt et al. 2002). The results lead to doubts about the classification of both strains as part of the same Pseudomonas species, given the widely accepted recommendation that strains of the same species shall have genome similarities higher than 70% (Stackebrandt and Goebel 1994). In the same study, an appreciable distance between DSM 291T and KT2440 was established by REP-PCR genomic fingerprint patterns and Biolog GN metabolic profiling. In our genomotyping approach, 69.3% of the genes with an assigned function, 60.5% of all genetic elements (including intergenic regions), and 52.2% of the intergenic regions (not shown) were identified in the genomic DNA of P. putida DSM 291T. These differences, which are supported by the AFLP analysis, indicate a considerable phylogenetic distance between DSM 291T and KT2440. The diversity within the species P. putida was previously reflected in studies concerning genomic DNA ribotyping (Brosch et al. 1996), whole cell protein electrophoretic fingerprinting (Vacanneyt et al. 1996) and Biolog/Biotype-100 experiments (Grimont et al. 1996). In our genomotyping studies, strains of P. monteilii and P. fluorescens were included as out-groups. P. fluorescens DSM 50090T was shown to be distantly related to the other Pseudomonas members tested, which supports its classification as a separate species. However, P. monteilii DSM 14164 clustered well with P. putida DSM 50198 and to a lesser extent with DSM 291T and P. putida DSM 50208. AFLP analysis showed a comparable result and confirmed the close relation between P. putida and P. monteilii. The present study thus suggests that DSM 14164 should more accurately be classified as a P. putida. Other studies based on classification of P. monteilii by classical, well established taxonomic methods do not support our findings. DNA–DNA hybridizations among P. monteilii CFML 90-60T, DSM 291T, and DSM 50208 generated relative bindings of genomic DNA of 40 and 48%, with ΔTm values of 9.2 and 7.9, respectively (Elomari et al. 1997). Pyoverdine typing (siderotyping) analyzing the excreted siderophores during iron starvation of P. monteilii CFML 90-60T and DSM 291T produced different patterns for both strains (Dabboussi et al. 2002). Phenotypically, however, P. monteilii was previously shown to be highly similar to P. putida and could only be differentiated by assimilation experiments of the substrates inositol, α-aminobutyrate, and o-/m-hydroxybenzoate (Dabboussi et al. 2002; Elomari et al. 1997). In conclusion, the genomotyping of different pseudomonads using KT2440-based DNA microarrays yielded novel insights in their phylogenetic relationships and the underlying identification of genes and their distribution over different primary and secondary biological functions. This revealed the utility of KT2440-based microarrays in transcriptomics and classification studies of these strains.

Diabetologia-4-1-2292424

Prediction of outcome in individuals with diabetic foot ulcers: focus on the differences between individuals with and without peripheral arterial disease. The EURODIALE Study

Aims/hypothesis Outcome data on individuals with diabetic foot ulcers are scarce, especially in those with peripheral arterial disease (PAD). We therefore examined the clinical characteristics that best predict poor outcome in a large population of diabetic foot ulcer patients and examined whether such predictors differ between patients with and without PAD. Introduction Diabetic foot ulcers are a common and much feared complication of diabetes, with recent studies suggesting that the lifetime risk of developing a foot ulcer in diabetic patients may be as high as 25% [1]. Foot ulceration requires long and intensive treatment, has important effects on quality of life of both patients and care-givers [2] and is associated with major healthcare costs [3–5]. Although in recent years much effort has been put into the development of international guidelines in order to stimulate the delivery of uniform and structured care [6], prospective data on outcomes and predictors of outcome in patients with diabetic foot ulcers are limited. The population of diabetic patients who present with foot ulceration is heterogeneous: although most patients have peripheral polyneuropathy, there are several other characteristics that may vary among patients, such as the presence of peripheral arterial disease (PAD), infection and co-morbidities. PAD is present in approximately one-half of all patients with foot ulcers [7] and is considered an important predictor of outcome [8, 9]. Therefore, outcome data on this important subgroup of patients with diabetic foot disease are needed. Such a requirement is underlined by the fact that although diabetic foot ulcers are usually reported and analysed as one clinical entity, marked differences in patient, foot and ulcer characteristics can exist between patients with and without PAD [7]. These observations raise the question of whether predictors of outcome in patients with and without PAD may differ. The aim of the present study was therefore: (1) to obtain prospective data on outcome of individuals presenting with a new diabetic foot ulcer, including patients both with and without PAD; (2) to assess clinical characteristics that best predict poor outcome (i.e. non-healing of the foot ulcer) from this large set of patients; and (3) to examine whether such predictors differ between patients with and without PAD. Methods Study design and population The EURODIALE consortium is an international collaborative network that was created to stimulate further research in the field of diabetic foot disease. Its main objective was to assess outcome and the major predictors of clinical outcome in a large sample of European patients with diabetic foot ulcers. The design and rationale of this study have been described in detail elsewhere [10]. Briefly, between 1 September 2003 and 1 October 2004, 1,232 patients with a new foot ulcer were included in 14 diabetic foot centres in ten European countries. The mean (range) number of included patients per centre was 88 (40–125). All participating centres have a longstanding expertise in the field of diabetic foot disease. Patients included were those presenting for the first time with a new foot ulcer within a period of 12 months, either at the outpatient or inpatient clinics of participating centres. Excluded patients were those who had been treated at the participating centres for an ulcer on the ipsilateral foot during the previous 12 months and those with a life expectancy of less than 1 year. Participants attended follow-up visits on a monthly basis. At baseline and during all follow-up visits, data were collected and recorded on standardised case record forms. This was done by dedicated investigators in each centre who were trained during plenary meetings and on-site visits. Recorded data included demographics, data on co-morbidities and foot and ulcer characteristics, as well as management. The local ethics committees of the 14 hospitals approved the study protocol and all patients gave written informed consent. Management of diabetic foot ulcer All patients were treated according to protocols based on the International Consensus on the Diabetic Foot [11], which include off-loading, diagnosis and treatment of infection, assessment of vascular status, treatment of PAD and regular wound debridement. Potential predictive factors Potential determinants of healing were chosen on the basis of (1) current literature; (2) expert opinion after extensive discussions during EURODIALE meetings; and (3) suitability for use in daily clinical practice. In addition to sex, age at baseline and duration of diabetes, several disease-specific characteristics and co-morbidities were investigated [10]. Ulcer characteristics All patients underwent a standardised examination according to the PEDIS system. This was developed by the International Consensus on the Diabetic Foot to enable classification of patients for clinical research purposes [11, 12] and classifies foot ulcers according to five categories: perfusion, extent, depth, infection and sensation.Perfusion assessment included evaluation of the presence of pedal pulses and measurement of the ankle–brachial pressure index (ABPI) using a handheld Doppler device; PAD was considered to be present if ABPI was <0.9 and/or two foot pulses were absent.Extent (i.e. size) was determined by multiplying the largest by the second largest diameter perpendicular to the first and divided into three categories: <1 cm2, 1–5 cm2 and >5 cm2.Depth was described as either deep or superficial if a full thickness lesion of the skin was or was not extending through the subcutis, respectively.Infection was diagnosed if two or more of the following signs were present: frank purulence, local warmth, erythema, lymphangitis, oedema, pain, fever and foul smell. The term infection covers both soft tissue infection and bone infection.Evaluation of sensation (peripheral neuropathy [PNP]) included pressure sensation (10 g monofilament on plantar aspect of hallux, metatarsophalangeal joints 1 and 5), tactile sensation (cotton wisp on dorsum of foot), vibration sensation (128 Hz tuning fork on dorsum of the hallux) and blunt/sharp discrimination (dorsum of foot). PNP was diagnosed if the results of two or more of the aforementioned tests were abnormal.In addition, the location of the ulcer was divided into plantar (on the plantar toes, plantar mid- or forefoot and plantar hind foot) and non-plantar (on the dorsal or interdigital part of the toes, on the dorsal or lateral aspect of the foot and heel ulcers). Ulcer duration was divided into three categories: <1 week, between 1 week and 3 months, and >3 months. Co-morbidities The following disabling co-morbidities were assessed: presence of severe visual impairment (defined as the inability to read a newspaper after correction), end-stage renal disease (ESRD) (defined as dependency on haemodialysis or peritoneal dialysis or a previous renal transplant procedure), heart failure (New York Heart Association [NYHA] classification III or IV), any neurological disorder (excluding diabetic polyneuropathy) resulting in loss of motor or sensory function (e.g. stroke) and inability to stand or walk without help. Study main outcome Main outcome was complete healing (with or without minor amputation) of the foot, within the maximum follow-up period of 1 year. Healing was defined as healing (intact skin) of the whole foot at two consecutive visits. If more than one ulcer was present, the foot was defined as healed once all ulcers were healed. Outcome information was not obtained in 144 patients (11.7% of the patients included) who dropped out of the study and were therefore excluded from the analyses. Reasons for dropout were non-compliance (n = 24), inability to follow the patient (lack of transportation, no social support, too sick to attend; n = 25) or if care had been taken over by other specialists (n = 29); in 66 patients the reason for dropout could not be discovered. At baseline these participants were slightly older and had a higher incidence of heart failure, deeper ulcers and ulcers of longer duration than those included in the analyses (n = 1,088; Table 1). Table 1Baseline characteristics of participants included and those excluded (dropouts) from the present studyVariableIncluded (n = 1,088)Dropouts (n = 144)p valueAge (years)64.7 ± 12.568.0 ± 11.60.003Male sex, n (%)a703 (64.6)85 (59.0)0.189Duration of diabetes, n (%)a0.418<5 years148 (14.1)19 (13.5)5–10 years169 (16.1)17 (12.1)>10 years731 (69.8)105 (74.5)Deep ulcer, n (%)a476 (43.8)80 (55.6)0.007Size of ulcer, n (%)a0.843<1 cm2403 (37.2)50 (35.0)1–5 cm2563 (52.0)76 (53.1)>5 cm2117 (10.8)17 (11.9)Duration of ulcer, n (%)a<0.001<1 week184 (17.0)10 (7.0)1 week–3 months627 (58.1)68 (47.6)>3 months269 (24.9)65 (45.5)Plantar location, n (%)a493 (48.2)62 (46.3)0.675Pretibial oedema, n (%)a197 (18.2)29 (20.3)0.538Heart failure NYHA III–IV, n (%)a117 (10.9)23 (16.1)0.065Neurological disorder, n (%)a70 (6.5)9 (6.3)0.918Inability to stand or walk without help, n (%)a107 (9.9)15 (10.4)0.843Visual impairment, n (%)a164 (15.3)19 (13.2)0.507ESRD, n (%)a63 (5.8)7 (4.9)0.639Polyneuropathy, n (%)a826 (78.5)105 (76.1)0.515Infection, n (%)a591 (57.2)82 (61.2)0.380PAD, n (%)a505 (47.5)78 (56.1)0.056Unless otherwise stated, data are mean values ± SD.aPercentages may not sum to 100 due to missing information Statistical analyses All statistical analyses were carried using the STATA software package version 9.2 (STATA, College Station, TX, USA). Comparisons between groups’ characteristics were made with χ2 tests (frequency data) or Student’s t test (continuous data). Multiple imputation of missing values of predictor variables Values for one (n = 188), two (n = 35) or three (n = 13) predictor variables were not available for 236 participants; the number of missing values per predictor ranged from 0 to 6%. In order to decrease bias and increase power of the analyses [13], we used multiple imputation chained equations (procedure ‘ICE’ in STATA) to impute those missing values (1.7% of all required values) rather than performing complete case analyses [14, 15]. With ICE the imputation model of a single variable uses all the other variables as predictors by appropriate regression models (i.e. linear, logistic or multinomial if imputed variable is continuous, dichotomous or categorical). We generated five imputed datasets that were used to fit the regression models of interest (in each dataset and in the final, i.e. the combined dataset). Parameter estimates and standard errors were combined across the five replicates according to the procedure described by Rubin [16] and Carlin et al. [17] (procedure ‘micombine’ in STATA). Development of predictive models First, univariable logistic regression analyses were performed for all potential predictor variables with the outcome of interest (non-healing), with values presented as univariable odds ratios (ORs) along with the respective 95% CI. Second, all potential predictors were entered simultaneously in a multivariable logistic regression model that was reduced to a most parsimonious model using a backward selection method based on Akaike’s Information Criterion. These models yielded a set of variables that best predict (and can be regarded as independent predictors of) outcome. Results Clinical outcome Within the 1 year follow-up, 77% of the 1,088 patients healed, 12% were still undergoing treatment, 5% underwent a major (i.e. above the ankle level) amputation and 6% died (before healing of the foot ulcer). Among the patients who healed, 17% underwent a minor amputation; this rate was similar to that in those patients who did not heal (20%, p = 0.425). When stratifying patients according to the presence or absence of PAD, significantly (p < 0.001) worse healing rates were observed in patients with than in those without PAD (69% vs 84%, respectively). Major amputation and mortality rates were also higher in patients with (8% and 9%, respectively) than in patients without PAD (2% and 3% respectively; p < 0.001). Baseline characteristics of patients with PAD compared with those without PAD are provided in Table 2. Table 2Patients’ baseline characteristics according to their PAD statusVariablePatients with PAD (n = 505)Patients without PAD (n = 558)p valueAge (years)69.1 ± 11.260.5 ± 12.3<0.001Male sex, n (%)a321 (65.6)366 (63.6)0.490Duration of diabetes, n (%)a0.265<5 years63 (12.9)80 (14.9)5–10 years72 (14.7)93 (17.4)>10 years354 (72.4)363 (67.7)Deep ulcer, n (%)a266 (52.7)200 (35.8)<0.001Size of ulcer, n (%)a0.002<1 cm2173 (34.4)219 (39.5)1–5 cm2259 (51.5)294 (53.0)>5 cm271 (14.2)42 (7.5)Duration of ulcer, n (%)a<0.001<1 week58 (11.5)120 (21.7)1 week–3 months296 (58.0)318 (57.5)>3 months148 (29.5)115 (20.8)Plantar location, n (%)a197 (40.9)284 (55.0)<0.001Pretibial oedema, n (%)a111 (22.0)83 (14.9)0.002Heart failure NYHA III–IV, n (%)a64 (12.7)47 (8.5)0.027Neurological disorder, n (%)a40 (8.0)27 (4.9)0.039Inability to stand or walk without help, n (%)a65 (12.9)36 (6.5)<0.001Visual impairment, n (%)a89 (17.9)66 (12.0)0.007ESRD, n (%)a35 (7.0)25 (4.5)0.082Polyneuropathy, n (%)a383 (77.2)424 (79.3)0.429Infection, n (%)a293 (60.9)282 (53.4)0.016Unless otherwise stated, data are mean values ± SDaPercentages may not sum to 100 due to missing information Predictors of healing Table 3 shows the univariable associations of the potential predictors of non-healing in the overall population and Table 4 presents the variables retained in the predictive models after backward selection in the combined imputed datasets. The estimates were similar to those obtained in the complete cases dataset (n = 854) indicating that missing values were non-selective (data not shown). These include the following eight characteristics, all of which predict lower probabilities of healing: older age, male sex, larger ulcer size, heart failure, inability to stand or walk without help, ESRD, PNP and PAD. These variables were consistently identified in all five imputed datasets. Table 3Association of each potential predictor with non-healing in the overall population (n = 1,088)Predictor variablesOutcome: healingOR95% CIp valueAge, per 10 year increase1.321.17–1.49<0.001Sex, men vs women1.501.07–1.970.018Duration of diabetes0.712 5–10 vs <5 yearsa0.960.56–1.65 >10 vs <5 yearsa1.050.69–1.60Depth of ulcer, deep vs superficial1.661.25–2.20<0.001Size of ulcer<0.001 1–5 vs <1 cm2a2.251.60–3.17 >5 vs <1 cm2a4.222.64–6.72Duration of ulcer<0.001 1 week to 3 months vs <1 weeka1.811.15–2.85 >3 months vs <1 weeka2.611.60–4.27Location, plantar vs non-plantar0.730.55–0.980.035Pretibial oedema, yes vs no1.791.27–2.510.001Heart failure (NYHA III–IV), yes vs no2.031.35–3.050.001Neurological disorder, yes vs no1.440.85–2.460.176Inability to stand or walk without help, yes vs no2.501.62–3.79<0.001Visual impairment, yes vs no1.360.94–1.980.105ESRD, yes vs no2.201.30–3.730.004Polyneuropathy, yes vs no1.410.98–2.040.065Infection, yes vs no1.471.09–2.000.012PAD, yes vs no2.311.72–3.10<0.001aReference categoryTable 4Multivariable models with independent predictors of non-healing in the whole study population and in patients with and without PADVariableAll patientsPatients with PADPatients without PADOR95% CIp valueOR95% CIp valueOR95% CIp valueAge, per 10 year increase1.281.11–1.470.0011.420.17–1.73<0.0011.550.91–2.630.105Sex, men vs women1.721.23–2.400.0021.971.25–3.110.003–––Size of ulcer<0.001<0.0010.008 1–5 vs <1 cm2a2.261.58–3.223.221.95–5.321.250.74–2.12 >5 vs <1 cm2a3.882.37–6.343.841.97–7.483.481.62–7.46Duration of ulcer––0.086 1 week to 3 months vs <1 weeka––––2.141.05–4.36 >3 months vs <1 weeka–– ––2.180.98–4.84Heart failure (NYHA III–IV), yes vs no1.550.99–2.430.0541.540.87–2.740.141–––Inability to stand or walk without help, yes vs no2.001.27–3.140.0032.361.34–4.170.0031.910.86–4.240.112ESRD, yes vs no2.511.41–4.480.0023.041.38–6.700.0062.000.76–5.250.161Polyneuropathy, yes vs no1.420.96–2.080.078–––1.700.89–3.250.108Infection, yes vs no–––1.631.03–2.580.036–––PAD, yes vs no1.711.23–2.370.001N/AN/AaReference categoryN/A, not applicable Since we hypothesised that, from an aetiological point of view, predictors of non-healing would differ between patients with and those without PAD, predictive models were also fitted for these two groups separately (Table 4). In patients with PAD almost all of the predictors identified in the whole study population, with the exception of PNP, were again found to be independent predictors of healing. In addition, the presence of infection emerged as an additional independent predictor of non-healing. In patients without PAD, older age, larger ulcer size, inability to stand or walk without help, ESRD, PNP and, in addition, longer ulcer duration were independent predictors of poorer healing. The observed interaction between infection and PAD status partly supports the classification of foot ulcer disease into four stages as suggested by Armstrong et al. (University of Texas classification system) [9]. Accordingly, upon analysis of the odds of non-healing per PAD × infection status, it was only in those patients with both PAD and infection that the odds of non-healing were markedly increased compared with those without PAD or infection: OR 2.82, CI 1.88–4.22, p < 0.001 in unadjusted analyses (Fig. 1) vs OR 1.87, CI 1.20–2.91, p < 0.001 after adjustments for the other variables included in the predictive model. Fig. 1ORs of healing per PAD and infection (Inf) status Discussion The EURODIALE study is one of the few large prospective, international studies on outcome and determinants of outcome in diabetic foot disease. Despite the severity of the underlying disease and the important co-morbidity [7], clinical outcome of this population within a 1 year follow-up can be considered favourable. In our cohort, 77% of the patients healed (with or without a minor amputation), 5% underwent a major amputation and 6% died. However, healing rates in patients with PAD were considerably worse. In addition, predictors of healing also differed between the groups with and without PAD. The presence of infection, which is generally regarded as an important predictor of healing, was only predictive in individuals with PAD. With regard to the overall outcome in our cohort, two recent studies found relatively comparable outcomes. Jeffcoate et al. [18] reported healing (excluding minor amputations) rates of 66% and an amputation rate of 5% with a similar prevalence of PAD. In a German cohort, Beckert et al. [19] found healing rates between 57% and 93% and major amputation rates of 3%, although the data as presented in that report cannot be easily compared because of their unique classification system. Oyibo et al. [20] also found similar rates of major amputation in their cohort (5%). Our study shows that the combination of PAD and infection has a major impact on healing rates (Fig. 1); this significant interaction between PAD and infection is, in our opinion, one of the major findings of this study. In the patients without PAD we did not observe an association between infection and non-healing, which suggests that in these patients current antibiotic regimens and surgical techniques seem adequate to save a limb with adequate perfusion. However, within the total population of individuals with diabetic foot disease in developed countries, the group of infected and ischaemic ulcers accounted for almost one-third of all patients in our earlier report [7]. In a recent study, a significant relation between PAD, infection and poor outcome was also observed: in that study’s large cohort of outpatients with type 2 diabetes, PAD was an independent predictor of infection-related mortality [21]. Unfortunately, there is very little insight into the pathophysiology and treatment of infection in individuals with PAD. Currently it is not clear why infection is more prevalent and more difficult to treat in individuals with PAD. Remarkably, very few patients with PAD were included in most of the randomised trials on antibiotic therapy in diabetic foot infections [22, 23]. It has previously been demonstrated that lower limb tissue levels of antibiotics can be markedly decreased as a result of impaired perfusion in PAD [24]. It is open to speculation whether aggressive revascularisation will improve control of infection in these patients. Although some earlier studies have examined the impact of co-morbidities on ulcer healing [25], no studies, to our knowledge, have systematically assessed in a multivariable analysis the effects on ulcer healing of patient characteristics including co-morbidities, as well as foot and ulcer characteristics at baseline. In our study, older age, ulcer size and several co-morbidities were independent predictors of non-healing in patients with and without PAD. Recently a number of larger studies reported data on determinants of outcome in diabetic foot disease such as the single-centre study by Beckert in Germany and the UK multi-centre study initiated by Jeffcoate [19, 26]. The former focused on wound-based characteristics and also found that the presence of PAD (defined as absence of pedal pulses) was an independent predictor of outcome (healing), while infection was not. In the recent UK multi-centre study, ulcer area was a strong predictor of outcome, as was the presence of PAD; co-morbidities were not taken into account in the regression analyses. Surprisingly, depth of the ulcer was not associated with outcome in our multivariable model, a finding also shown by Ince et al. [26]. In a retrospective study, Miyajima et al. [27] reported on patient characteristics that determined major lower extremity amputation and found that haemodialysis was an independent predictor of major amputation; in this study wound characteristics were not part of the regression analyses. The poor prognosis of foot ulcers for individuals in our study with ESRD is in line with earlier reports, in which amputation rates of 57% in individuals on haemodialysis were observed [25]. Although in our study ESRD was a predictor of non-healing in patients with and without PAD, it seemed to have a particularly negative effect in the latter patient group. PAD is frequently diagnosed and associated with adverse outcomes in haemodialysis patients [28]. PAD in ESRD patients is more severe and is accompanied by diffuse vascular calcifications, involvement of both distal infrapopliteal and foot arteries, and by impaired microcirculatory perfusion [29–31]. The severity of PAD in ESRD may explain the importance of ESRD in our healing models; additional mechanisms are probably impaired host defences in chronic renal failure and uraemia, or the presence of more resistant micro-organisms [32, 33]. The outcome of patients without PAD in our study was relatively favourable: 84% of the patients healed with or without minor amputation, 2% underwent a major amputation and 3% died. In our multivariate models, loss of sensation was associated with a poorer outcome in these patients, suggesting that loss of protective sensation is not only a key factor in the development of an ulcer, but also affects its outcome. This may be related to the preserved mechanism of off-loading the ulcer in individuals with intact protective sensation. However, neuropathy may also have direct effects on wound healing. Although data on the effect of neuropathy on wound healing in humans are scarce, some animal studies suggest that denervation may contribute to impaired wound healing in diabetes [34, 35]. A large dataset on individuals with neuropathic ulcers comes from retrospective database analyses in which healing rates of 47% were observed [36]. Although this study reported on healing at 20 weeks (whereas the current one examined healing rates at 1 year), the different results compared with our study are striking and may be related to an increased awareness of the importance of adequate off-loading, as a result of publication of international guidelines and reports on casting techniques [37–39]. There are several limitations to our study. Individuals who were lost to follow-up were excluded from the analyses as healing status could not be obtained; these individuals were slightly older and had a greater incidence of heart failure, deep ulcers or ulcers with a longer duration and PAD at baseline. In addition, we excluded patients who had had a previous ulcer within 12 months prior to presentation (i.e. we probably excluded patients with recurrent ulcers). Also we excluded patients with a life expectancy shorter than 1 year because of anticipated problems with follow-up. The estimates obtained in our models may therefore have underestimated the probability of non-healing in patients with a recurrent foot ulcer, although in one earlier study healing rate of neuropathic foot ulcers did not decrease in patients with multiple recurrences [40]. Since our study was embedded in daily clinical practice, limitations had to be set with regard to the number and type of data collected. It was therefore not possible to record more characteristics of these patients such as medication and extensive documentation of all complications. Moreover, to facilitate data collection, some continuous data (e.g. ulcer size) had to be transformed into a limited set of categories. Nevertheless, the set of potential predictors used in the present study do cover relevant patient and disease-specific aspects that can be easily assessed and used for patient risk estimation in clinical practice. Finally, our predictive model is based on outcomes that can be obtained in developed countries with access to the necessary resources such as antibiotic treatment and revascularisation; our results, therefore, are most relevant for diabetic foot ulcer patients in developed countries. In conclusion, the results of this study have several implications. Both ulcer characteristics and several patient-related characteristics affected the outcome of diabetic foot ulcers. Therefore, a holistic approach by healthcare professionals who are familiar with the treatment of complicated diabetic patients is essential in order to identify the high-risk patient and start appropriate treatment. We found that patients with and without PAD differ in clinical characteristics, outcome and predictors of outcome. Taking into account these findings and the different pathophysiology and treatment of PAD and non-PAD ulcers, we feel that that diabetic foot ulcer with and without PAD should be defined as two separate disease states. The prevalent combination of PAD and infection is a unique entity; an important challenge lies in the development of evidence-based strategies to improve the poor outcome of these patients. Both studies comparing different antibiotic regimens in PAD, and studies evaluating the effects of early revascularisation on control of infection are urgently needed.

Naunyn_Schmiedebergs_Arch_Pharmacol-4-1-2323035

Dipeptidyl peptidase IV inhibitors in diabetes: more than inhibition of glucagon-like peptide-1 metabolism?

Inhibitors of the protease dipeptidyl peptidase IV (DPP-IV) are promising new drugs for the treatment of type 2 diabetes. They are thought to act by inhibiting the breakdown of glucagon-like peptide-1 and, thereby, selectively enhancing insulin release under conditions when it is physiologically required. These drugs are selective for DPP-IV, but the enzyme itself has a broad range of substrates other than glucagon-like peptide-1. Other high affinity substrates of DPP-IV including peptide YY may also play a role in the regulation of energy homeostasis. Moreover, DPP-IV is also known as CD26 and considered to be a moonlighting protein because it has a wide range of other functions unrelated to energy homeostasis, e.g. in immunity. The potential role of DPP-IV inhibition on substrates other than glucagon-like peptide-1 in diabetes patients remains to be elucidated. Glucagon-like peptide-1 (GLP-1) is a hormone which is released following meals and stimulates insulin release from the pancreas. Its effects are terminated by breakdown by the enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, inhibition of DPP-IV increases GLP-1 levels in the circulation and, hence, insulin release under conditions when it is needed, i.e. after a meal but not during fasting. Consequently, inhibition of GLP-1 inactivation is an insulinotropic principle which is unlikely to cause hypoglycaemia between meals. The lower risk for hypoglycaemic events as compared with other insulinotropic or insulin-sensitising agents makes DPP-IV inhibitors very promising candidates for a more physiological treatment of type 2 diabetes (Combettes and Kargar 2008). In recent years, a number of selective DPP-IV inhibitors such as vildagliptin and sitagliptin have been evaluated in clinical trials (Hermansen et al. 2007; Utzschneider et al. 2008) and may have a future role in the treatment of type 2 diabetes (Combettes and Kargar 2008). In this issue of the journal, a novel DPP-IV inhibitor, ASP8497, is being introduced, which is highly selective for DPP-IV as compared to other peptidases (Someya et al. 2008). However, DPP-IV itself is not selective for GLP-1 but has a wide range of other natural substrates (Boonacker and Van Noorden 2003). Therefore, we wish to highlight potential implications of this lack of selectivity of DPP-IV for the use of DPP-IV inhibitors in diabetes treatment. Another high affinity substrate of DPP-IV is peptide YY (PYY; Mentlein et al. 1993). Whereas cleavage of GLP-1 by DPP-IV causes inactivation, cleavage of PYY yields the long C-terminal fragment PYY3–36, which is inactive at some but active at other subtypes of PYY receptors. Specifically, it converts the non-subtype-selective agonist PYY into a selective agonist at Y2 and Y5 receptors (Michel et al. 1998). Thus, DPP-IV does not inactivate PYY but, rather, qualitatively alters its biological activity. Indeed it has been reported that peripherally administered PYY3–36 inhibits food intake in rats, whereas PYY is a potent central stimulator of food intake (Batterham et al. 2002). Given the role of obesity in type 2 diabetes, prevention of the formation of such an endogenous food intake inhibitor by a DPP-IV inhibitor may be undesirable. It has been proposed that the effects of peripherally administered PYY3–36 on central nervous functions such as food intake may be mediated by excitation of afferent vagal fibres (Koda et al. 2005). However, the majority of subsequent rodent studies did not confirm inhibition of food intake by PYY3–36, particularly not following chronic administration (Boggiano et al. 2005). On the other hand, recent studies in non-rodents such as pigs (Ito et al. 2006) or humans (Degen et al. 2005; Sloth et al. 2007a; Sloth et al. 2007b) have reported reduced food intake upon peripheral administration of PYY3–36 but typically, these effects were found only at relatively high concentrations. Moreover, the effect of PYY3–36 on food intake was biphasic, depending on the duration of its administration (Parkinson et al. 2008). Two additional findings deserve consideration. Firstly, PYY3–36 was reported to promote fat oxidation and ameliorate insulin resistance in mice even under conditions of chronic administration where it did not reduce food intake (van den Hoek et al. 2006). Secondly, PYY3–36 was reported to lower plasma glucose levels even in the absence of alterations in circulating insulin levels (Bischoff and Michel 1998). Taken together, the presently available data on PYY3–36 on food intake and metabolic parameters are not yet conclusive. However, it is clear that PYY3–36 is largely formed by DPP-IV, raising the possibility that selective DPP-IV inhibitors may exert part of their effects by modulating the PYY/PYY3–36 ratios. Therefore, it remains to be explored how possible effects on PYY cleavage contribute to metabolic effects of DPP-IV inhibitors in diabetic patients. Moreover, DPP-IV is not only a protease for substrates relevant to energy homeostasis, but it also has a range of additional functions (Boonacker and Van Noorden 2003). Therefore, it is considered to be a moonlighting protein. As a protease, it has several other substrates, and it also acts as a receptor and costimulatory protein in the immune system. In this regard, CD26 is considered to be an important regulator of T-cell function (Reinhold et al. 2008). These pleiotropic effects of DPP-IV or CD26 lead to numerous potential uses of its inhibitors other than type 2 diabetes including inflammatory diseases (Ohnuma et al. 2006; Reinhold et al. 2007; Thielitz et al. 2008; Thompson et al. 2007) and, perhaps, certain types of cancers (Kikkawa et al. 2005; Thompson et al. 2007). Some of these effects may manifest as useful secondary actions when being used for the treatment of diabetic patients whereas others may manifest as adverse events. Most of these potential additional effects may not yet have manifested in the currently published diabetes literature, but it appears prudent to keep an eye on them.

Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2413123

Refractive properties of the healthy human eye during acute hyperglycemia

Purpose To measure the refractive properties of the healthy human eye during acute hyperglycemia by means of Scheimpflug imaging and Hartmann-Shack aberrometry. Transient refractive changes, due to a variation in blood glucose levels, are well-known complications of diabetes mellitus (DM). Both myopic shifts [2, 6, 10, 23, 31, 36] and hyperopic shifts [9, 13, 18, 26, 32, 37] have been reported in patients with DM after several days or weeks of hyperglycemia. It has been suggested that the predominant cause of the refractive changes is a change in the thickness of the lens [16, 17, 19, 25, 29] or shape of the lens [21], and/or a change in its refractive index [24, 27, 29, 33]. Two studies have been conducted in which hyperglycemia was induced under controlled circumstances to investigate refractive changes during hyperglycemia. Firstly, Gwinup et al. [15] administered glucose intravenously to ten patients with DM. A myopic shift of maximal -0.75 D was measured with autorefractometry. Secondly, Furushima et al. [11] induced hyperglycemia in seven healthy, young subjects through somatostatin injections and an oral glucose load. By means of autorefractometry and ultrasound biometry a large change in ocular refraction (-2.0 diopters (D)) and thickness of the lens (1.0 mm) was measured within 150 minutes after administration of the glucose load (maximal hyperglycemia: 13.9 mmol/l). The results of these two studies indicated that induced hyperglycemia can cause changes in refraction and that these changes appeared to be larger in healthy subjects. In the study of Furushima et al. [11], only the thickness of the lens was measured, and there is no information about the change in the shape and the refractive index of the lens due to acute hyperglycemia. Therefore, the aim of our study was to induce hyperglycemia in healthy subjects under controlled circumstances, and accurately measure ocular geometry and refraction by means of corrected Scheimpflug imaging and Hartmann-Shack aberrometry, in order to investigate the mechanism underlying refractive changes during hyperglycemia. From the ocular geometry and refraction, in combination with the measurement of the axial length of the eye, it is also possible to calculate the equivalent refractive index of the lens [4, 5]. Subjects and methods Five healthy Caucasian subjects (two males and three females) were recruited for this study. Mean age was 24.8 years (SD ±4.6 and range 21.2–32.6) and mean body mass index (BMI) was 24.2 kg/m2 (SD ±3.2 and range 21.4 - 29.7). The Medical Ethics Committee of the VU University Medical Center in Amsterdam approved the study protocol, and written informed consent was obtained from all subjects after the nature of the study had been explained. Subjects with a history of diabetes mellitus (or a fasting plasma glucose >5.5 mmol/l), a BMI of >30 kg/m2, visual acuity of <0.5 (Snellen), or a history of ocular pathology were excluded from the study. Procedure to induce hyperglycemia After a 10-hour overnight fast, the subjects received a subcutaneous injection of 100 µg synthetic somatostatin (Sandostatin, Novartis, Basel, Switzerland) in order to suppress the endogenous insulin secretion during glucose loading. Each subject had an oral glucose tolerance test (OGTT) (glucose 75 g) 30 minutes after the somatostatin injection. Blood glucose levels were measured with a blood glucose analyzer (HemoCue Diagnostics BV, Oisterwijk, the Netherlands). Endogenous insulin levels were determined with immunometric assays (Luminescence, Bayer Diagnostics, Mijdrecht, the Netherlands) in the Laboratory of Endocrinology of the Department of Clinical Chemistry in the VU University Medical Center. The subjects remained in a fasting state during the entire procedure. Measurement of ocular parameters Before and 120 minutes after the OGTT, 1.0% cyclopentolate and 5.0% phenylephrine eye-drops were administered to the right eye of the subjects. Hartmann-Shack aberrometry was performed with an IRX3 aberrometer (Imagine Eye Optics, Paris, France) and Scheimpflug imaging was performed with a Topcon SL-45 Scheimpflug camera, in which the film was replaced by a charge-coupled device (CCD) camera (St-9XE, SBIG astronomical instruments) with a range of 16 bits of grey values (512 × 512 pixels, pixel size 20 × 20 µm, magnification: 1×). Before and every 30 minutes after the OGTT, three measurements were made with each apparatus. To obtain accurate measurements of the shape of the lens, ray-tracing was performed to correct the Scheimpflug images for the distortion that is inherent to this technique [4, 5]. By combining the measurements of the corneal thickness (d1), the depth of the anterior chamber (ACD), the anterior (R1) and posterior (R2) radius of the cornea, the lens thickness (d3) and the anterior (R3) and posterior (R4) radius of the lens, the axial length of the eye, and ocular refraction, it was possible to calculate the equivalent refractive index of the lens (n lens) by means of an iterative process [5]. In these calculations it was assumed that the refractive indices of the cornea, aqueous humor, and vitreous do not change, because in vitro experiments have shown that large changes in glucose levels induce negligible changes in the refractive indices of the ocular media [8, 22]. The axial length of the eye was measured with an IOL-master (Carl Zeiss Inc., North America). The equivalent refractive error (ERE) was calculated as: ERE = sphere + (cylinder/2). Furthermore, the higher order aberrations (HOA) of each eye were analyzed for a pupil size of 5.7 mm and they were summarized in root mean square errors (RMS), including the third up to the eighth Zernike orders [34]. Furthermore, the HOA, including the spherical aberrations, were analyzed separately. Measurements at baseline and during hyperglycemia were compared in the whole group by means of Wilcoxon signed rank sum tests. A refractive change of more than 0.2 D and a change in HOA of more than 0.025 µm were considered to be meaningful, according to the precision (defined as 95% confidence interval) for measuring the ERE and HOA of the aberrometer [3, 30]. Error analysis indicated that a change in R1, R2, d1, ACD, d3, R3, R4, and n lens of more than 0.05 mm, 0.05 mm, 0.02 mm, 0.14 mm, 0.15 mm, 0.30 mm and 0.40 mm, and 0.007 respectively, could be considered as significant, according to the precision of corrected Scheimpflug imaging [4]. In each subject individually, the significance of a change was determined from the precision of the measurements and the difference in the ocular parameters at baseline and during hyperglycemia. P values ≤0.05 were considered statistically significant. Results Figure 1 shows the changes in blood glucose after the OGTT. In all subjects the mean blood glucose levels rose from 4.0 mmol/l (range 3.4–4.5 mmol/l) to 18.4 mmol/l (range 16.1–22.0 mmol/l) in 126 minutes (range 90–210 minutes) after the OGTT. Subject 1 had a delayed elevation of blood glucose level, and therefore this subject received a second 75 g oral glucose load at time = 30 minutes. Furthermore, venous blood samples of subject 1 were taken from the antecubital vein, which was kept open with 0.9% saline (100 cl). Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1 pmol/l (range 0.4–4.5 pmol/l), and remained below basal secretion level (<110.0 pmol/l) for 147 minutes (range 75–270 minutes). Fig. 1Changes in blood glucose (BG) levels in the five subjects after the administration of somatostatin and glucose; the oral glucose load (75 g) was administered at time = 0 minutes No significant change in ERE was found in the whole group or in four of the subjects individually. In subject 1, a hyperopic shift in ERE of 0.4 D and an increase in the spherical aberration (±SE) from 0.03 ± 0.01 µm at T = 0 to 0.11 ± 0.01 µm during hyperglycemia was measured (p < 0.001). The other HOA did not change in subject 1. Furthermore, there was no significant change in the HOA separately or the RMS values in the group or in any of the other subjects individually. No changes in corneal thickness (d1), corneal shape (R1 and R2), anterior chamber depth (ACD), lens thickness (d3), or posterior lens shape (R4) were found in the group or in any of the subjects individually. In the whole group and in four subjects, no changes were found in the anterior radius of the lens (R3) or refractive index (n lens). However, in subject 01, the R3 and n lens changed significantly during hyperglycemia, compared to normal conditions; R3 decreased from 11.65 to 9.69 mm (mean decrease R3 = 1.96 mm; p < 0.001) and n lens decreased from 1.436 to 1.422 (mean decrease n lens = 0.014; p = 0.003). Figure 2 presents graphs of the normalized ERE, ACD, d3, R3, R4 and n lens of the five subjects. Figure 3 shows two corrected Scheimpflug images and a schematic drawing of the lens of subject 1, in order to illustrate the differences in lens geometry during hyperglycemia compared to baseline. In all subjects, both hematological and ocular parameters normalized within 6 hours after the OGTT. Fig. 2Graphs of the normalized equivalent refractive error (ERE), lens thickness (d3), anterior chamber depth (ACD), anterior (R3) and posterior (R4) radius of the lens, and the refractive index of the lens (n lens) of the five subjects. Data are normalized by subtracting the value at baseline from the measured value in each subject. In subject 01 a hyperopic shift of 0.4 D, an increase in R3 and decrease in n lens were found during hyperglycemia at T = 240 minutes (p < 0.01). The oral glucose load was administered at T = 0Fig. 3Corrected Scheimpflug images of the right eye of subject 1. a At baseline time = 0 minutes, the shape of the cornea and lens are indicated with a solid line. b During hyperglycemia at time = 240 minutes after the first oral glucose load, the shape of the cornea and lens are indicated with a line of dashes. c A drawing of the changes in the shape of the lens in hyperglycemic condition (line of dashes) compared to the normal condition (solid line) Discussion Refractive changes occur frequently in patients with DM. The underlying mechanism is still unclear, and therefore the aim of our study was to measure ocular refraction and geometry during hyperglycemia, in an attempt to identify a possible explanation for these refractive changes. The effect of reproducible hyperglycemia was studied in healthy subjects without the systemic effects of DM. An OGTT in combination with a somatostatin injection was used to induce hyperglycemia. In an earlier study, this was shown to induce large changes in the refractive error and lens thickness [11]. Somatostatin inhibits insulin secretion [7], and to our knowledge there are no reports of refractive errors or changes in the lens due to this agent. In general, and in four of the five subjects individually, no changes in ocular refraction or geometry were found during hyperglycemia. It could be that a more prolonged and severe hyperglycemia is needed to induce changes in refractive error or geometry of the eye. Glucose within the lens is metabolized via the sorbitol pathway, which consists of two enzymes that catalyze the conversion of glucose into its sugar alcohol sorbitol and the further conversion of sorbitol to fructose. These sugar alcohols tend to accumulate within the lens, because they penetrate cell membranes poorly. This accumulation of sugar alcohols causes the lens to swell [12]. This process might have taken longer than the observation period of the present study. Furthermore, the subjects measured in our study were young, and it could be that their lenses, which still have a fast metabolic reaction capacity, tolerate short hyperglycemic stress without swelling. The results of our study are not in accordance with the findings of Furushima et al. [11], who observed a large myopic shift (-2 D) and a large increase in the thickness of the lens (1 mm) during hyperglycemia in healthy subjects. It must be noted that in our study the mean increase in the blood glucose level was even higher than that of the study of Furushima et al.. One main difference between our study and the study by Furushima et al. is the methods that were used to measure ocular refraction (aberrometry versus autorefractometry) and ocular geometry (corrected Scheimpflug imaging versus ultrasound biometry). However, the precision of aberrometry and autorefractometry are comparable for the measurement of defocus, astigmatism and, consequently, the equivalent refractive error [3, 30, 35]. Furthermore, the precision of corrected Scheimpflug imaging and ultrasound biometry are comparable as well. It could be that the difference in ethnicity (Caucasian subjects in the present study versus Asian subjects in the study by Furushima et al.) caused the inconsistency between the two studies, since Asian people generally have a more myopic ocular refraction than Caucasian subjects [14, 38]. In our study, small but significant changes in ocular refraction and lens geometry were found in one subject. A hyperopic shift of 0.4 D was found, in combination with an increase in anterior convexity of the lens. A combination of hyperopia and an increase in lens thickness during hyperglycemia has been described by Kluxen et al. [21], who found a 6 D hyperopic shift and a 0.4 mm increase in lens thickness in a diabetic patient with hyperglycemia. Saito et al. [29] reported hyperopic shifts (1.1 to 4.9 D) and an increase of approximately 0.2 mm in the thickness of the lens in five diabetic subjects during hyperglycemia. Therefore, it has been suggested that hyperglycemia causes a change in the refractive index of the lens [21, 24, 27, 29, 33]. The results of our study support this hypothesis; in subject 1, a decrease in the equivalent refractive index of the lens was calculated during hyperglycemia. It could be suggested that if the change in the shape of the lens is small, hyperopia will predominate during hyperglycemia due to a decrease in the refractive index of the lens. Alternatively, if the change in the shape of the lens is large in comparison to the decrease in the refractive index of the lens, the overall refractive error will result in myopia. The controversy in the literature with regard to refractive changes during hyperglycemia could be explained by this underlying mechanism of a balance between changes in the shape or the refractive index of the lens, which eventually determine the overall refractive outcome [20, 23, 28, 37]. It is surprising that a change in refraction and ocular parameters could be determined in only one subject. It must be noted that the procedures for inducing hyperglycemia and monitoring blood glucose were, to some extent, different for subject 1 compared to the other subjects. Because of a delayed elevation in blood glucose level, a second oral glucose load (150 g instead of 75 g glucose) was administered. Nevertheless, the maximum blood glucose value of subject 1 did not exceed that of the other subjects, and the endogenous insulin level was adequately suppressed during the glucose loading. Furthermore, in order to obtain sufficient blood samples, 0.9% saline had to be administered to keep the antecubital vein open. Therefore, it could not be excluded that the administration of saline contributed to the refractive change and alterations in the lens of subject 1. However, no studies have yet reported any refractive change due to saline administration. The change in refractive error in patients with hyperglycemia could also be caused by a change in the shape of the cornea. However, previous research has shown that hyperglycemia has no influence on the shape of the anterior corneal surface [10, 24, 29, 32]. The results of our study also agree with these findings. No change in the anterior or posterior corneal radius was found during hyperglycemia. Therefore, the cornea does not seem to play a role in the explanation of refractive changes during hyperglycemia. This also applies for the higher order aberrations. Applegate et al. [1] reported that an increase in the higher order aberrations could cause a decrease in visual acuity. However, no changes in the higher order aberrations were found in any of the subjects in our study. Therefore, it can be assumed that blurred vision in hyperglycemia cannot be explained by changes in the higher order aberrations of the eye. In sum, the results of this study show that induced hyperglycemia generally does not cause changes in the refractive properties of the healthy human eye. However, there were interindividual variations, as illustrated by subject 1, who had a hyperopic shift of refraction and a change in shape and equivalent refractive index of the lens during hyperglycemia. This could provide an explanation for the mechanism underlying the refractive changes often experienced by patients with DM and hyperglycemia.

Histochem_Cell_Biol-4-1-2413109

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance. Introduction In multicellular organisms, cell–cell adhesion is involved in most developmental processes. It is necessary for example for the assembly of coherent sheets of barrier-forming epithelial and endothelial cells which line the inner and outer surfaces of the organism like those of the intestine, the skin or the blood vessels. However, also in adult tissues cell–cell contacts are far from being static structures which maintain the barriers by simply holding cells together. During the turnover of growing tissues such as the intestine or the skin, they are constantly remodeled to allow the extrusion of “old” cells and the incorporation of “young” cells derived from stem cells without a concomitant loss of the barrier function (Fuchs et al. 2004). Similarly, during leukocyte extravasation in secondary lymphoid organs or at sites of an ongoing immune response, the homotypic interactions between endothelial cells must be altered to allow the passage of the leukocytes without affecting the barrier properties of the endothelium (Ley et al. 2007). Finally, during wound healing and tissue repair, cells undergo a coordinated movement, proliferate and establish new cell–cell contacts once they encounter cells from the opposing site of the wound (Perez-Moreno and Fuchs 2006). These different demands in different cell types and different physiological situations require a sophisticated regulatory network which enables a partial dismantling and re-establishment of cell–cell contacts while simultaneously preventing the loss of an epithelial phenotype which in adult tissues frequently correlates with tumor progression and metastasis (Thiery 2002). Not surprisingly, the organization of cell–cell contacts of epithelial has attracted a great deal of attention. Due to the easy accessibility of cultured epithelial cells many discoveries have been made in epithelial cells. Epithelial cell–cell contacts contain three major adhesive structures which can be identified at the ultrastructural level, the tight junctions (TJs), the adherens junctions (AJs), and the desmosomes. In polarized epithelial cells of certain tissues like the intestine, TJs and AJs are asymmetrically distributed at the apical region of the lateral cell contact forming the apical junctional complex (AJC) which encircles the apex of the cells and demarkates the border between the apical and the basolateral membrane domains (Nelson 2003). Common to all three types of structures is the presence of several adhesion molecules that link the neighbouring cells through homophilic and heterophilic adhesive interactions, and the presence of cytoplasmic scaffolding proteins that organize signaling complexes and which provide a mechanical link to the actin cytoskeleton (or intermediate filaments in the case of desmosomes). The scaffolding proteins might also link different protein complexes—at least temporarily—and thus organize supramolecular protein complexes. It should be noted that these protein complexes are dynamic, and that their composition is subject to regulation depending on junctional maturation and integrity. During the last few years, a rapid progress has been made in identifying new proteins at cell–cell contacts and in particular in deciphering the molecular composition of the TJs. Among the most exciting findings was probably the discovery of protein complexes at TJs which are highly conserved through evolution and which regulate cellular polarization in different organisms and various cell types. Here, I will review the major multiprotein complexes present at cell–cell contacts of vertebrate epithelial cells and highlight the most recent advances in the understanding of their role in the organization and functions of epithelial cell–cell contacts in vertebrates. Adherens junctions The function of adherens junctions A main function of AJs is to connect cells to regulate tissue formation and morphogenesis during development as well as the maintenance of solid tissues in the adult organism (Gumbiner 1996). The major cell adhesion molecules at AJs, the classical cadherins, connect adjacent cells through homophilic interactions and are linked to the cytoskeleton through proteins associated with their cytoplasmic tail, the catenins. This link generates a transcellular network of actin filaments running through the entire sheet of cells with the cadherin–catenin complexes serving as connectors of the actin filaments bundles at the intercellular space. During morphogenetic events, for example during neural tube formation, mechanical forces can thus be applied to the whole cellular sheet. In adult tissues, cadherin-mediated cell adhesion is absolutely required for cell–cell adhesion to be maintained as a loss of cadherin adhesion by Ca2+-depletion results in a loss of cell–cell interaction and rounding up of the cells despite a number of other adhesion molecules (which are not Ca2+-dependent) at cell–cell contacts (Takeichi 1977). Cadherins and catenins Since the cadherin–catenin complex has been the subject of a number of reviews, I will only summarize the central interactions and refer the reader to recent comprehensive reviews (Gumbiner 2005; Halbleib and Nelson 2006; Perez-Moreno and Fuchs 2006; Perez-Moreno et al. 2003; Pokutta and Weis 2007). As mentioned above, the cytoplasmic tail of classical cadherins like E-cadherin forms a multiprotein complex with β-catenin, γ-catenin and p120 catenin (p120ctn), members of the armadillo repeat domain-containing family of proteins. In this complex, β-catenin and p120ctn are directly associated with the membrane-distal and membrane-proximal region, respectively (Fig. 1). Both regions are highly conserved among classical cadherins underscoring the importance of these interactions. Under conditions where β-catenin is limiting, its binding site in E-cadherin can be occupied by the β-catenin-related plakoglobin/γ-catenin which otherwise associates preferentially with desmosomal cadherins (Zhurinsky et al. 2000). The interaction with β-catenin is required for the transport of E-cadherin from the ER to the basolateral cell surface (Chen et al. 1999). It is also required for the adhesive function of E-cadherin as post-translational modifications of β-catenin which alter its affinity towards E-cadherin alter the strength of E-cadherin adhesive activity (Perez-Moreno et al. 2003; Pokutta and Weis 2007). The adhesive activity is thus dependent on its association with β-catenin. Conversely, this association is also necessary to stabilize β-catenin which is otherwise rapidly degraded by the ubiquitin-proteasome pathway (Nelson and Nusse 2004). Fig. 1Major protein complexes at adherens junctions. Two major protein complexes exist at AJs of epithelial cells. The cadherin–catenin complex consist of the Ca2+-dependent adhesion molecule E-cadherin and the armadillo repeat proteins p120ctn and β/γ-catenin which directly bind to the cytoplasmic domain of E-cadherin. α-catenin directly associates with β-catenin but not simultaneously with F-actin. The nectin–afadin complex consists of the Ca2+-independent adhesion molecule nectin and the PDZ protein afadin. Afadin contains a F-actin-binding domain and thus can link the nectin–afadin system to the actin cytoskeleton. The two adhesion complexes can be linked through several molecular interactions. Afadin can directly interact with α-catenin. It also interacts with ponsin/SH3P12 which can interact with the F-actin-binding protein vinculin, and it interacts with LMO7 and ADIP which both can interact with the F-actin binding protein α-actinin. The nature of the link between the cadherin–catenin complex and the actin cytoskeleton is still unclear. Double arrows indicate direct interactions, the question mark symbolizes the missing link The association of E-cadherin with p120ctn is subject to a similar reciprocal regulation of cell–cell contact localization: The cadherin molecule is necessary to localize p120ctn at the cell contact, and p120ctn is required for the stable localization of the cadherin molecule at AJs (Reynolds and Roczniak-Ferguson 2004). In contrast to β-catenin, p120ctn stabilizes cadherin which is constitutively endocytosed (Bryant and Stow 2004) by regulating its turnover rate at the surface (Davis et al. 2003). Besides its additional role in regulating transcription by interacting with transcription factors (van Roy and McCrea 2005), p120ctn has a also a function in regulating the activity of Rho small GTPases. The formation of early cell–cell contacts correlates with the activation of Cdc42 and Rac1 and the inhibition of RhoA (Noren et al. 2001), and these changes in activities of the small GTPases are at least in part regulated by p120ctn which interacts with the Rac activator Vav2 (Noren et al. 2000) and the RhoA inhibitor p190RhoGAP (Wildenberg et al. 2006). The activation of Cdc42/Rac1 and the downregulation of RhoA activity serves to facilitate new cell adhesion by increasing the cell surface interacting with membranes of neighbouring cells through lamellipodia and filopodia formation and simultaneously inhibiting cell migration by inhibiting stress fiber formation, respectively (Perez-Moreno and Fuchs 2006). The role of α-catenin is less clear. For a long time considered to bridge the AJs to the actin cytoskeleton through direct interactions with both β-catenin and F-actin, this role has been challenged by the observations that its binding to F-actin (occurs only as homodimer) and its heterodimeric association with β-catenin are mutually exclusive (Drees et al. 2005; Yamada et al. 2005). Therefore, the physical bridge between the cadherin–catenin complex and the actin cytoskeleton remains to be identified. It is possible that α-catenin present in the cadherin-associated heterodimeric β-catenin–α-catenin complex is able to bind to other proteins which associate with F-actin. The putative linker is required to fulfill two critera: to bind directly to α-catenin and simultaneously–either indirectly or directly—to F-actin. From several proteins which fulfill these requirements, vinculin and α-actinin turned out not to mediate actin binding to the cadherin–catenin complex excluding them from the list of possible candidates (Yamada et al. 2005). Other candidate proteins including AF-6/afadin (Pokutta et al. 2002), ZO-1 (Itoh et al. 1997), formin (Kobielak et al. 2004), spectrin (Pradhan et al. 2001) or the LIM protein ajuba (Marie et al. 2003) remain to be tested. Nectins and afadin The second major adhesive protein complex at AJs consists of members of the nectin family of adhesion molecules and a scaffolding protein that is directly associated with the cytoplasmic domain of nectins named AF-6/afadin (Takai and Nakanishi 2003). Nectins are immunoglobulin-like proteins and comprise a family consisting of four members (nectin-1 to -4), which are localized at AJs of epithelial cells (Reymond et al. 2001; Sakisaka and Takai 2004). Unlike classical cadherins which undergo only homophilic interactions in trans, nectins undergo both trans-homophilic and trans-heterophilic interactions. The major heterophilic binding partners are other members of the nectin family as well as members of nectin-related adhesion molecules Nectin-like (Necl)-1 to -5 (Sakisaka et al. 2007). Nectins are “true” adhesion molecules as they support cell aggregation when ectopically expressed in cells (Aoki et al. 1997; Lopez et al. 1998; Satoh-Horikawa et al. 2000; Takahashi et al. 1999). They also seem to influence the E-cadherin-mediated adhesion (Martinez-Rico et al. 2005; Sato et al. 2006) suggesting that they are contributing to the overall strength of cell–cell adhesion. Like E-cadherin, nectin-2 appears very early at cell–cell contacts during junction formation and is present at so-called primordial, spot-like AJs (pAJs) or puncta (Asakura et al. 1999), which are formed at the tips of protrusions of two contacting cells (Perez-Moreno and Fuchs 2006). An important function of nectins which is similar to that of cadherins is their ability to activate Cdc42 and Rac1 small GTPases. Trans-interaction of nectins results in the recruitment and activation of c-Src, followed by the activation of the two guanine-nucleotide exchange factors “FGD-1-related Cdc42 GEF” (FRG) and Vav2, which are specific for Cdc42 and Rac1, respectively (Fukuhara et al. 2004; Kawakatsu et al. 2002, 2005). As pointed out above, the activation of these small GTPases is probably required to facilitate junction formation suggesting that nectins cooperate with cadherins in the regulation of the actin cytoskeleton at sites of cell adhesion. However, in addition they might also help to regulate the formation of tight junctions and their physical separation from AJs during junctional maturation (see below). All nectins directly associate with afadin (Reymond et al. 2001; Takahashi et al. 1999). L-Afadin, the longer version of two afadin isoforms with a F-actin-binding domain, is a scaffolding protein which interacts with both nectins and F-actin through independent domains suggesting that it directly links nectin-based adhesion sites to the actin cytoskeleton (Mandai et al. 1997) (Fig. 1). However, through additional protein interactions, it might also establish an indirect link to the actin cytoskeleton as well as to the cadherin–catenin complex by its interaction with “classical” AJ-associated proteins. Afadin directly associates with α-catenin (Pokutta et al. 2002; Tachibana et al. 2000), with vinculin through its association with ponsin/SH3P12 (Mandai et al. 1999), and with α-actinin through its association with “afadin Dil domain-interacting protein” (ADIP) and “Lim domain only 7” (LMO7) (Asada et al. 2003; Ooshio et al. 2004) (Fig. 1). It should be mentioned that some of these protein interactions might occur specifically in certain tissues, and that the molecular mechanisms underlying these interactions are not revealed in detail. Nevertheless, it is likely that through these multiple interactions the two major protein complexes at AJs are physically linked and that they influence each other in their localization and activity (Sakisaka et al. 2007). Tight junctions The function of TJs In polarized epithelial cells, the TJs forms a belt-like structure at the apical region of the cellular junction and represent a boundary between the apical and the basolateral membrane domains which differ in the composition of lipids and proteins (Tsukita et al. 2001). At the TJ area, two structures can be distinguished by ultrathin electron microscopy: sites where the intercellular space is basically obliterated and where the outer leaflets of the adjacent membranes appear to be in direct contact, and regions where the membranes of the two adjacent cells are separated by intercellular space. By freeze-fracture electron microscopy, the TJ area appears as a branched network of strands where the strands reflect the sites of direct membrane contacts (Tsukita et al. 2001). Two major functions are attributed to TJs: First, the regulation of the paracellular permeability of the epithelial sheet for ions and small solutes, which is an organ-specific function and which varies for different epithelia depending on the specific requirements of the organ (Furuse and Tsukitas 2006; Van Itallie and Anderson 2006); second, the formation of a physical barrier to prevent intramembrane diffusion of lipids and proteins, a rather cell-autonomous function which is necessary for a cell to maintain an asymmetric distribution of membrane components and to develop membrane polarity (Tsukita et al. 2001). In the recent years it has become clear that the molecular basis of the TJ strands are claudins (Fig. 2), a family of integral membrane proteins at TJs which are constituents of the TJ strands and which induce the formation of TJ strands upon ectopic expression in fibroblasts (Furuse et al. 1998). Claudins do not just create TJ strands, but within the strands form a selective permeability barrier by forming size- and charge-selective aqueous pores (Tsukita and Furuse 2000; Van Itallie and Anderson 2006). Surprisingly, the TJ strands do not seem to be the basis for the second major function of TJs, the diffusion barrier for intramembrane particles (Umeda et al. 2006). It has also become clear that the TJs harbor peripheral membrane proteins which regulate cell polarity and membrane asymmetry suggesting that these proteins are involved in the role of TJs in regulating apico-basal polarity. Fig. 2Integral membrane proteins at tight junctions of epithelial and endothelial can be grouped into three classes based on their overall organizations. The first class is characterized by two extracellular loops, four transmembrane regions, and two cytoplasmic tails (occludin, claudins, tricellulin). The second class consists of Ig-SF members which all contain two Ig-like domains. The third class (contains only one member, CRB3) is characterized by a short extracellular domain (36 AA), a single transmembrane domain and a short cytoplasmic tail. In contrast to the other integral membrane proteins, the function of the extracellular domain of CRB3 is not clear Integral membrane proteins at TJs Three different classes of integral membrane proteins have been identified at TJs (Fig. 2). One class comprises occludin, claudins and tricellulin, which all contain four transmembrane domains, two extracellular loops, and the N-terminal and C-terminal ends are localized in the cytoplasm. Twenty-four claudins have been identified in humans. The major role of claudins is to form paired strands through homophilic and heterophilic cis and trans interactions. The large number of claudins, their ability to undergo heterophilic interactions and their ion selectivity allows for the formation of TJ strands with specific permeability properties depending on the needs of a specific tissue (Furuse and Tsukita 2006). Occludin is incorporated into TJ strands but its ectopic expression does not induce strand formation (Furuse et al. 1998) suggesting that it has rather an accessory role in TJ strand formation (Yu et al. 2005). Tricellulin differs from claudins and occludin in its specific enrichment at tricellular contact sites (Ikenouchi et al. 2005). The second class of integral membrane proteins comprises members of the CTX subfamily of the immunoglobulin superfamily (Fig. 2) which is characterized by one V-type and one C2-type Ig-like domain (Williams and Barclay 1988). Based on sequence homology, the length of their cytoplasmic tails and the type of the C-terminal PDZ domain motifs, the Ig-SF proteins at TJs can be further subdivided into a group consisting of JAM-A, JAM-B and JAM-C, and in a second group consisting of CAR, CLMP, ESAM, and JAM4 (Ebnet et al. 2004). Except for JAM-A, there is only little information on the role of these proteins in the development or the function of tight junctions. All but CLMP interact with TJ-associated scaffolding proteins like ZO-1 (CAR, JAM-A, -B, -C (Cohen et al. 2001; Ebnet et al. 2000, 2003), PAR-3 (JAM-A, -B, -C (Ebnet et al. 2001, 2003) or MAGI-1 (ESAM, JAM4; (Hirabayashi et al. 2003; Wegmann et al. 2004) (Fig. 3), and in some cases, their ectopic expression increases the transepithelial electrical resistance (TER) or decreases the paracellular permeability suggesting a regulatory role in TJ formation (Cohen et al. 2001; Hirabayashi et al. 2003; Mandicourt et al. 2007; Raschperger et al. 2003). For JAM-A, there is good evidence that it regulates TJ formation through its direct association with the scaffolding protein PAR-3 (Ebnet et al. 2001; Itoh et al. 2001). JAM-A localizes very early at sites of cell adhesion during cell–cell contact formation (Ebnet et al. 2001; Suzuki et al. 2002), and this localization probably serves to recruit PAR-3 to these site to initiate the polarization of the lateral membrane resulting in TJ formation (see below for details). Both RNA interference-mediated downregulation of JAM-A and ectopic expression of a JAM-A dominant-negative mutant that mislocalizes PAR-3 result in decreased TER, increased paracellular permeability and a defect in the development of membrane asymmetry (Mandell et al. 2005; Rehder et al. 2006) pointing to a general defect in the formation of functional TJs. The third class of integral membrane proteins comprises only one protein, Crumbs3 (CRB3), a homologue of the Drosophila Crumbs protein with a very short extracellular domain of only 36 AA and a short cytoplasmic domain of 41 AA (Makarova et al. 2003; Medina et al. 2002). CRB3 directly associates with two peripheral membrane proteins, Pals1 (Roh et al. 2002b) and PAR-6 (Lemmers et al. 2004), which are components of the two major cell polarity protein complexes localized at TJs, i.e. the Pals1–PATJ complex and the PAR-3–aPKC–PAR-6 complex (Fig. 3, see also below). Its overexpression delays TJ formation (Lemmers et al. 2004; Roh et al. 2003), and its ectopic expression in a cell line that expresses only little endogenous CRB3 results in the development of functional TJs (Fogg et al. 2005). These effects can most likely be attributed to its association with cell polarity proteins and the regulation of their subcellular localization (see below). Fig. 3Major protein complexes and functional classes of molecules at tight junctions. The TJs contain three major multi-protein complexes consisting largely of scaffolding proteins, the ZO protein complex, the CRB3–Pals1–PATJ complex and the PAR-3–aPKC–PAR-6 complex. Besides these three protein complexes which seem to be constitutively associated at TJs, a number of proteins with different functions has been identified at TJs. These include additional scaffolding proteins like MUPP1 and MAGI-1, adaptor proteins, transcription regulators and RNA processing factors, regulatory proteins like small GTPases and G-proteins, kinases and phosphatases, and heat shock proteins. Double arrows indicate direct interactions. Not all direct interactions that have been identified are depicted Multiprotein complexes at TJs Since the discovery of ZO-1 as the first protein at TJs and its molecular cloning (Itoh et al. 1993; Stevenson et al. 1986), the number of proteins that are localized at TJs has steadily increased. These proteins comprise scaffolding and adapter proteins, regulatory proteins like small GTPases, G-proteins, kinases and phosphatases, as well as transcription factors or factors regulating RNA processing (Fig. 3). The large number and the functional diversity of these proteins suggest that TJs are a focus of incoming and outgoing signals and that their composition is dynamic. In accordance with this, many proteins identified at TJs are found at other compartments of the cell as well including the nucleus or the cytoskeleton and are actively shuttling between these compartments and the TJs (Matter and Balda 2007). The organization of these networks is regulated by proteins containing multiple protein–protein interaction domains such as PDZ domains, GuK domains, SH2 or SH3 domains (Pawson and Nash 2003). At the TJs three major protein complexes exist which involve one or several scaffolding proteins, the ZO protein complex, the Pals1–PATJ complex, and the PAR-3–aPKC–PAR-6 complex (Fig. 3). The ZO protein complex ZO-1 is a classical scaffolding protein of the MAGUK family with three PDZ domains, one SH3 domain and one GuK domain (Funke et al. 2005). It can directly associate with several integral membrane proteins at TJs including occludin, claudins, JAMs and CAR through independent domains (Fig. 4), and it probably serves to cluster these proteins at the TJs. It also interacts with other cytoplasmic proteins including its homologues ZO-2 and ZO-3, and in addition with the actin cytoskeleton (Fanning et al. 1998; Fanning et al. 2002; Wittchen et al. 1999). The exact composition of the ZO complex is not completely understood, yet. ZO-1 forms independent complexes with ZO-2 and ZO-3 (Wittchen et al. 1999), and both ZO-2 and ZO-3 can also interact with F-actin and share with ZO-1 some of the integral membrane proteins at TJs like occludin and claudins (Itoh et al. 1999; Wittchen et al. 1999). Alltogether, the ZO complex provides the major link to the actin cytoskeleton at the TJs (Fig. 4). However, regarding the role of the individual ZO proteins, some redundancy might exist. In agreement with this, the absence of ZO-1 results in a slight delay in TJ formation but does not impair the formation of functional TJs in two epithelial cell lines (McNeil et al. 2006; Umeda et al. 2004). Only when all three ZO proteins are absent the formation of TJs is blocked as indicated by the absence of TJ strands, the lack of other TJ proteins like occludin, claudin-3 and JAM-A, and the complete loss of the barrier function (Umeda et al. 2006). Together, these findings indicate a critical role for the ZO proteins for the development of TJ strands, probably by forming the physical scaffold for the strand-forming proteins like claudins and occludin. As will be discussed below, ZO proteins also form a platform for signaling proteins to regulate epithelial proliferation and morphogenesis. Fig. 4Organization of the tight junctional plaque. The major protein complexes at TJs interact with specific transmembrane proteins. CRB3 recruits the Pals1–PATJ complex to TJs. CRB3 interacts with the PDZ domain of Pals1, Pals1 interacts with PATJ through a heterodimeric L27 domain interaction. CRB3 is also localized at the apical membrane domain of epithelial cells (Makarova et al. 2003). The CRB3–Pals1–PATJ complex regulates TJ formation but the mechanism is largely unknown. The ZO complex is associated with the membrane through multiple interactions of ZO-1 with various integral membrane proteins including occludin, claudins, JAM-A and CAR. ZO-2 and ZO-3 can interact with both ZO-1 and also with claudins. The ZO protein complex probably serves to link TJs to the cytoskeleton as all three ZO proteins directly interact with F-actin. The PAR-3–aPKC–PAR-6 complex is associated with the membrane through the interaction of PAR-3 with JAM-A. PAR-3 interacts with aPKC through its aPKC-interacting domain, PAR-6 interacts with aPKC through a PB1–PB1 domain interaction. A direct interaction between the PDZ domain of PAR-6 and PDZ domain 1 of PAR-3 has also been described. The PAR complex regulates the formation of TJs and apico-basal polarity. The JAM-A-related Ig-SF member JAM4 interacts directly with MAGI-1; the role of this protein complex is not clear. It should be noted that this drawing is incomplete as it does not depict interactions among the various protein complexes which have been described (e.g. PAR-6 can also associate with CRB3 and Pals1, ZO-3 can associate with PATJ). Also, the multiple PDZ domain protein MUPP1 (not depicted in this Figure, see Fig. 3 for a schematic representation) associates with claudins, JAM-A and CAR, as well as with angiomotin family members (Coyne et al. 2004; Hamazaki et al. 2002; Sugihara-Mizuno et al. 2007). Double arrows with solid lines indicate direct protein–protein interactions, double arrows with broken lines indicate interactions with F-actin The CRB3–Pals1–PATJ complex The CRB3–Pals1–PATJ complex has originally been described in Drosophila as a protein complex (the Crumbs–Stardust–Discs lost complex; this complex is now called Crumbs–Stardust (Sdt)–dPATJ complex (Pielage et al. 2003)) involved in the regulation of apico-basal polarization (Tepass et al. 2001). In Drosophila epithelial cells, this complex localizes to the apical region of the lateral membrane domain, called subapical region (SAR) or marginal zone (Knust and Bossinger 2002), that is positionally analogous to the TJs in vertebrate epithelial cells. The localization of the CRB3–Pals1–PATJ complex at the TJ is mediated through a direct and PDZ domain-dependent interaction of Pals1 with the C-terminal PDZ domain motif in CRB3, and a direct interaction between Pals1 and PATJ involving the L27N domain of Pals1 and the L27 domain present at the NH2-terminal region of PATJ (Fig. 3) (Roh et al. 2002b). RNA interference-mediated knockdown of Pals1 leads to defects in the formation of TJs as well as in the development of lumen-containing epithelial cysts (an assay system for apico-basal polarity development (O’Brien et al. 2002)), and is accompagnied by a loss of PATJ protein expression (Straight et al. 2004). Conversely, knockdown of PATJ impairs the barrier function of TJs and results in a loss of Pals1 at TJs, an internalization of CRB3, and a redistribution of other TJ components like occludin and ZO-3 (Michel et al. 2005; Shin et al. 2005). These observations strongly suggest that the CRB3–Pals1–PATJ complex is important for the development of functional TJs in vertebrate epithelial cells. The PAR-3–aPKC–PAR-6 complex In evolutionary terms, the PAR-3–aPKC–PAR-6 complex is the most ancient among the three major protein complexes at TJs. As opposed to the ZO protein complex and the CRB3–Pals1–PATJ complex, all three proteins of this complex exist in C.elegans where they cooperate to regulate the development of membrane asymmetry in the zygote (Kemphues 2000). The acronym Par stands for partitioning-defective and reflects the lack of partition of cytoplasmic P granules in C.elegans mutant embryos in response to sperm entry (Kemphues et al. 1988). The initial screen identified six par genes, and their molecular characterization revealed that they encoded proteins of different structures and functions which include scaffolding/adapter proteins with several protein–protein interaction domains (PAR-3, PAR-6), serine/threonine kinases (PAR-1, PAR-4), a protein containing a RING finger domain typical for E3 ubiquitin ligases (PAR-2) and a member of the 14-3-3 family of signaling proteins (PAR-5) (reviewed in (Goldstein and Macara 2007; Suzuki and Ohno 2006)). With the exception of PAR-2, all PAR proteins exist in Drosophila and vertebrates. Two PAR proteins, PAR-3 and PAR-6, form a functional unit with aPKC, the PAR-3–aPKC–PAR-6 complex (Ohno 2001). In this complex, PAR-3 and PAR-6 undergo direct interactions with aPKC (Fig. 3). The interaction of PAR-6 with aPKC is mediated by a heterotypic PB1–PB1 domain interaction, the interaction of PAR-3 with aPKC by the CR3 domain of PAR-3 and the kinase domain of aPKC (Ohno 2001). The interactions of the two scaffolding proteins PAR-3 and PAR-6 with aPKC are assumed to regulate the localization and the activity of aPKC, respectively. Among all integral membrane proteins tested, PAR-3 binds specifically to JAM-A, -B, and -C (Ebnet et al. 2001, 2003), and the interaction with JAM-A might serve to anchor the PAR–aPKC complex to TJs. The interaction with PAR-6 is assumed to regulate the activity of aPKC (Lin et al. 2000). In the absence of small GTPases like Cdc42 or Rac1 aPKC is inactive. The binding of active Cdc42 or Rac1 to the CRIB domain of PAR-6 activates aPKC, probably by inducing a conformational change of PAR-6 which allows aPKC to become active (Yamanaka et al. 2001). What is the function of the PAR–aPKC complex in TJ physiology? A large body of evidence indicates a critical role of the PAR complex in TJ formation rather than in TJ maintenance (Chen and Macara 2005, 2006; Gao et al. 2002; Hirose et al. 2002; Joberty et al. 2000; Mizuno et al. 2003; Nagai-Tamai et al. 2002; Ooshio et al. 2007; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). Many of these studies applied dominant-negative mutants of either PAR-3 or PAR-6 or aPKC. The negative effects on TJ formation were only observed when these mutants were expressed during the process of cell–cell contact formation but not when expressed in fully polarized epithelial cells where TJ formation had already been completed (Gao et al. 2002; Nagai-Tamai et al. 2002; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). This strongly suggests that the PAR–aPKC complex develops its polarizing activity at an early stage of cell–cell contact formation and that it is critical for the formation of TJs rather than for their maintenance. Regulation of membrane asymmetry and TJ formation by the PAR-3–aPKC–PAR-6 complex The formation of cell–cell contacts and the development of intercellular junctions with distinct structures like AJs and TJs is a step-wise process (Fig. 5). In the absence of cell–cell contacts, cells form thin protrusions filled by axial actin filaments which upon encountering protrusions of other cells form multiple transient contacts which are subsequently stabilized (McNeill et al. 1993). The first sites of cell–cell contact formation are called “primordial, spot-like AJs” (pAJs) or “puncta” (Adams et al. 1996; Yonemura et al. 1995). The formation of multiple pAJs between the protrusions of adjacent cells results in a zipper-like appearance of the early cell contact sites (McNeill et al. 1993) (Fig. 5). During maturation of cell–cell contacts, the pAJs gradually fuse to form a linear contact region, the cells start to polarize and eventually develop cell junctions with AJs and TJs. Fig. 5A step-wise recruitment of proteins to cell–cell contacts. The earliest sites of stable physical interaction during cell–cell contact formation are primordial, spot-like AJs (pAJs) or puncta at the tips of cellular protrusions. During junctional maturation, the cellular protrusions of adjacent cells interdigitate, and multiple puncta are formed along the sides of protrusions. These puncta gradually fuse to form linear arrangements of cell–cell contacts sites thus generating a zipper-like appearance. During further maturation, cell–cell contacts are formed along the entire lateral cell surface, and the zipper-like cell–cell structure disappears. Cell contact-associated proteins are recruited in a step-wise manner. The pAJs/puncta are positive for integral membrane proteins (E-cadherin, JAM-A, nectin-2), but also peripheral membrane proteins (ZO-1, α-catenin, afadin) and contain proteins associated with AJs as well as TJs in polarized cells. During the formation of zipper-like cell contacts, occludin is recruited, probably through its interaction with ZO-1. Thereafter, PAR-3 is recruited by JAM-A and/or nectin-2, and claudins are recruited, probably through interaction with ZO-1. Indirect evidence suggests that aPKC and PAR-6 appear slightly later than PAR-3. The vertical bar reflects the increase in the contacting membrane area during cell–cell contact formation The pAJs are positive for typical AJ proteins like E-cadherin, α-catenin, β-catenin, nectin-2, AF-6/afadin and ponsin but also for typical TJ proteins like ZO-1 and JAM-A (Adams et al. 1996; Asakura et al. 1999; Ebnet et al. 2001; Suzuki et al. 2002; Yonemura et al. 1995). During maturation, occludin is recruited to these sites, and during further maturation, claudin-1, PAR-3 and aPKC appear (Suzuki et al. 2002) (Fig. 5). Although direct comparison has not been performed yet, it is likely that aPKC together with PAR-6 appear slightly later than PAR-3 (Suzuki et al. 2002). The formation of cadherin-based pAJs marks the early sites of cell–cell adhesion and probably serves as a “landmark” or “positional cues” for membrane growth and for the recruitment of other integral and peripheral membrane proteins (Yeaman et al. 1999). After the localization of the first set of proteins at pAJs other proteins can be recruited through direct physical interactions with those already present. For example, α-catenin-associated ZO-1 could serve to recruit occludin and claudins, afadin could serve to recruit JAM-A and nectin-2 (or vice versa), and JAM-A or nectin-2 could recruit PAR-3 which serves as scaffold to assemble the PAR-3–aPKC–PAR-6 complex. Once the PAR-3–aPKC–PAR-6 complex has been recruited to nascent cell–cell contacts aPKC has to be activated as suggested by the observation that ectopic expression of a kinase-dead, dominant-negative mutant of aPKC prevents the maturation of pAJs into belt-like AJs and TJs (Suzuki et al. 2001, 2002). The activation occurs most likely by the Rho GTPases Cdc42 and Rac1 which bind to the Crib domain of PAR-6 thereby inducing a conformational change which leads to the activation of PAR-6-associated aPKC (Garrard et al. 2003; Ohno 2001; Peterson et al. 2004; Yamanaka et al. 2001). Both E-cadherin and nectin-2 could be responsible for the activation of Cdc42 and Rac1. Whereas E-cadherin seems to activate Rac1 but not Cdc42 (Betson et al. 2002; Kovacs et al. 2002; Nakagawa et al. 2001; Noren et al. 2001; Yamada and Nelson 2007), nectin-2 induces the activation of both Cdc42 and Rac1 after ectopic expression in cultured epithelial cells (Fukuhara et al. 2003, 2004; Fukuyama et al. 2005; Kawakatsu et al. 2002, 2005). The association of the Rac1 GEF Tiam1 with PAR-3 (Chen and Macara 2005; Mertens et al. 2005) could regulate a locally restricted activation of Rac1 specifically at those sites where cell–cell adhesion has occured and where the activity of aPKC is required to promote the maturation of cell–cell contacts and the development of TJs from pAJs. The exact mechanism how the maturation of cell–cell contacts is regulated by aPKC is not clear. One could imagine that aPKC phosphorylates components of TJs and thereby regulates their specific localization or their specific functions at the TJs. Phosphorylation of occludin, claudin-1 and ZO-1 by aPKCζ has been found in vitro (Nunbhakdi-Craig et al. 2002). However, a physiological relevance of these phosphorylations has not been demonstrated, yet. Alternatively, aPKC could regulate TJ formation indirectly by regulating the development of membrane asymmetry along the lateral cell–cell contacts. The following example might serve to illustrate an example for this activity. In polarized epithelial cells, aPKC and PAR-1, another Ser/Thr kinase, are separately localized along the apico-basal axis: aPKC localizes to TJs whereas PAR-1 localizes to the basolateral membrane domain (Bohm et al. 1997). PAR-1 is a substrate for aPKC, and aPKC-mediated phosphorylation of PAR-1 leads to its dissociation from the membrane into the cytoplasm (Hurov et al. 2004; Suzuki et al. 2004). As a result, PAR-1 is absent from aPKC-containing membrane domains. A reciprocal mechanism has been described in Drosphila epithelial cells (Benton and St Johnston 2003). Drosphila PAR-1 phosphorylates PAR-3/Bazooka thereby inhibiting its dimerization and blocking its ability to assemble a functional PAR-3–aPKC–PAR-6 complex. As a result, the PAR-3–aPKC–PAR-6 complex is absent from PAR-1-containing membrane domains. Through these reciprocal inhibitory interactions two distinct membrane domains are generated characterized by the mutual exclusion of aPKC and PAR-1. Thus, by regulating the formation of a specific membrane domain from which certain proteins are actively excluded, aPKC could indirectly promote the formation of TJs. It is not clear, yet, if the abilities of the PAR-3–aPKC–PAR-6 complex to regulate TJ formation and to regulate membrane asymmetry are mechanistically linked. Regulation of TJ maintenance by the Rich1–Amot complex Recently, a new protein complex has been identified which has been suggested to regulate the maintenance rather than the formation of TJs. The functional core of this complex is a set of two proteins which regulate the activity of Cdc42, the Rich1 and angiomotin (Amot) proteins (Wells et al. 2006) (Fig. 6). Rich1 is a Rho GTPase activating protein (RhoGAP) for Cdc42 and Rac1 with a Cdc42-selective activity in epithelial cells; it contains a BAR domain and a RhoGAP domain (Richnau and Aspenstrom 2001; Richnau et al. 2004; Wells et al. 2006). Amot is a scaffolding/adapator protein with a coiled-coil domain region and a C-terminal PDZ domain-binding motif (Bratt et al. 2002). The Rich1–Amot complex is targeted to TJs through a PDZ domain-dependent interaction of Amot with PATJ (Fig. 6). Both overexpression of Amot and partial downregulation of Rich1 by RNA interference affect the barrier function of TJs. More importantly, the loss of the barrier function in response to Ca2+-removal is accelerated after partial Rich1 downregulation (Wells et al. 2006) suggesting that the Rich1–Amot complex is important for the maintenance of functional TJs. This function of the Rich1–Amot complex probably resides in regulating the cycling of Cdc42 and maintaining the pool of active Cdc42 at TJs at a low level. Fig. 6The Rich1–Amot complex at TJs. Rich1 is a RhoGAP with specificity for Cdc42 in epithelial cells. Rich1 interacts with Amot through a reciprocal BAR domain-dependent interaction, and Amot binding regulates Rich1 activity. Rich1 associates with Cdc42 through its GAP domain. Amot directly interacts with one (or several) of the PDZ domains 3–10 of PATJ that in turn associates with CRB3–Pals1. Surprisingly, Rich1 is directly or indirectly associated with PAR-3 and aPKC, and this PAR complex is distinct form the PAR-3–aPKC–PAR-6 complex because it does not contain PAR-6 and because aPKC cannot be associated with the 100 kDa isoform of PAR-3 which lacks the aPKC-binding domain. All three Amot-like proteins (Amot, Amotl1, Amotl2) form Rich1-independent protein complexes with MUPP1 and PATJ. Double arrows with solid lines indicate direct protein–protein interactions, arrows with broken lines indicate the presence of the two proteins in the same complex but the nature of the interaction has not been characterized in detail, yet Besides Amot, PATJ and Pals1, the Rich1 immunoprecipitates contain PAR-3 and aPKC (Wells et al. 2006). Surprisingly, the Rich1 immunoprecipitates do not contain PAR-6, and they contain the 100 kDa isoform of PAR-3 which lacks the aPKC-interacting domain and thus cannot directly associate with aPKC (Lin et al. 2000). This suggests that PAR-3 and aPKC can undergo interactions with the Rich1–Amot complex which are independent of the PAR-3–aPKC–PAR-6 interaction with the Pals1–PATJ complex (Hurd et al. 2003) (Fig. 6). The functional relevance of this interaction is not clear. Two Amot-like (Amotl) proteins—Amotl1/JEAP and Amotl2/MASCOT—which have been described earlier as TJ components (Nishimura et al. 2002; Patrie 2005) are present in Amot but not Rich1-containing protein complexes. All three Amot proteins directly interact with the scaffolding protein MUPP1 and its paralogue PATJ (Sugihara-Mizuno et al. 2007) (Fig. 6). These findings suggest that additional Amot protein-containing complexes exist with functions different from regulating Cdc42 activity. Signaling from TJs In addition to the relatively stable protein complexes described so far (Fig. 4), many protein complexes at TJs assemble only transiently. In addition, some proteins are not exclusively associated with TJ but shuttle between the TJ and other compartments in the cell. The identification of such proteins has revealed that TJ proteins are engaged in receiving signals but also in delivering signals to the cell interiour and thereby regulate epithelial proliferation and differentiation (Matter and Balda 2003). The mechanism by which TJ proteins influence for example gene expression is most likely indirect through binding and sequestration of regulatory molecules at the TJs as exemplified by ZO-1. ZO-1 associates with ZONAB/DbpA, a transcription factor which promotes proliferation of epithelial cells, in part by interacting with the cell division kinase CDK4 and also by regulating the expression of genes involved in proliferation like cyclin D1 and PCNA (Balda et al. 2003; Balda and Matter 2000; Sourisseau et al. 2006). In proliferating cells, which have little ZO-1, ZONAB/DbpA expression is high and ZONAB/DbpA protein is localized in the nucleus. When cells reach confluence and develop intercellular junctions, ZO-1 is accumulating at cell–cell contacts and recruits ZONAB/DbpA to the junctions thus sequestering it away from the nucleus (Balda and Matter 2000). Interestingly, during cellular stress ZONAB/DbpA associated with ZO-1 can be re-activated. The heat shock protein Apg-2 that is distributed in the cytoplasm under normal conditions is recruited to cell–cell contacts in response to heat shock where it binds directly to ZO-1 using the same binding interface like ZONAB/DbpA, i.e. the SH3 domain of ZO-1 (Tsapara et al. 2006). This leads to a loss of ZONAB/DbpA from cell junctions and in activation of the transcriptional activity of ZONAB/DbpA (Tsapara et al. 2006). Thus, ZO-1 influences gene expression and cell cycle progression in a cell density-dependent manner, and this function can be regulated during cellular stress. ZO-2 is another scaffolding proteins at TJs which is involved in signaling. In contrast to ZO-1, however, ZO-2 seems to actively shuttle between TJs and the nucleus. ZO-2 contains functional nuclear localization and nuclear export signals (Gonzalez-Mariscal et al. 2006; Jaramillo et al. 2004) and interacts with various proteins that have nuclear functions including the transcription factors AP-1 and C/EBP (Betanzos et al. 2004), the DNA-binding protein SAF-B (Traweger et al. 2003), and the p120ctn family member ARVCF (Kausalya et al. 2004). ZO-2 probably inhibits the activity of the transcription factors AP-1 and C/EBP by regulating their export from the nucleus which is consistent with the predominant nuclear localization of ZO-2 in sparse cells and the localization of ZO-2 as well as AP-1 and C/EBP at TJs in confluent cells (Betanzos et al. 2004). In the case of ARVCF, ZO-2 regulates its nuclear import (Kausalya et al. 2004) where it might regulate transcription similar to other p120ctn family members (Hatzfeld 2005). In summary, the identification of protein complexes formed by typical TJ proteins and typical nuclear proteins involved in the regulation of transcription indicates that the TJs participate in the regulation of proliferation and differentiation. A protein complex at the lateral membrane which regulates TJ formation: the Scribble–Discs Large–Lethal Giant Larvae complex The Scribble complex comprises the proteins Scribble (Scrib), Discs Large (Dlg), and Lethal Giant Larvae (Lgl) (Fig. 7) which have originally been identified in Drosophila as tumor suppressor proteins (Bilder 2004). Mutations in the scrib, dlg or lgl genes result in overgrowth of certain tissues ultimately leading to a “giant larvae” phenotype and also in a disruption of apico-basal polarity (Bilder 2004). All three proteins are membrane-associated in Drosophila epithelial cells. Scrib and Dlg localize to the Septate Junctions (SJ), a structure that is a functional homologue of vertebrate TJs but which is localized basally of the AJs; Lgl localizes along the lateral membrane domain and is excluded from the SAR (Tepass et al. 2001). Importantly, the Scrib complex genetically interacts with the PAR-3–aPKC–PAR-6 and the Crumbs–Sdt–PATJ complexes to regulate apico-basal polarity (Bilder et al. 2003; Tanentzapf and Tepass, 2003). All three proteins are conserved and exist in vertebrates. In mammals, one homologue of Drosophila Scrib, four homologues of Dlg (Dlg1–Dlg4) and two homologues of Lgl (Lgl1, Lgl2) exist, and the proteins localize to the basolateral membrane domain of epithelial cells (Dow and Humbert 2007; Humbert et al. 2003). As opposed to the PAR-3–aPKC–PAR-6 and the CRB3–Pals1–PATJ complexes, Scrib, Dlg and Lgl do not seem to form a ternary complex through direct interactions. Recent evidence indicates that Scrib exists in a complex with Lgl2 (Kallay et al. 2006), but it is not clear if this interaction is direct or indirect as described in Drosophila (Mathew et al. 2002). The role of the Srib protein complex in regulating apico-basal polarity and cell–cell contact formation seems to be conserved in vertebrates. Scrib knockdown results in a delayed TJ formation (Qin et al. 2005), and knockdown of Dlg1 disturbs TJ formation after Ca2+-switch-induced new cell–cell contact formation (Stucke et al. 2007). Furthermore, Lgl has been described to regulate cell–cell contact and apico-basal polarity formation by forming a complex with PAR-6 and aPKC (Plant et al. 2003; Yamanaka et al. 2003) (Fig. 7). The interaction of Lgl with PAR-6 and aPKC precludes the binding of PAR-3 to PAR-6 and aPKC. Also, the Lgl–aPKC–PAR-6 complex counteracts the activity of the PAR-3–aPKC–PAR-6 complex by suppressing not only its formation but also its activation by Cdc42 (Yamanaka et al. 2006). According to the current model (Fig. 7), Lgl forms a complex with aPKC and PAR-6 early during cell–cell contact formation and blocks the formation of the PAR-3–aPKC–PAR-6 complex at this stage. Upon phosphorylation by aPKC, Lgl dissociates from the complex thus allowing for the formation and activation of the PAR-3–aPKC–PAR-6 complex and the development of apical TJs harboring the PAR–aPKC complex and a basolateral domain harboring Lgl (Yamanaka et al. 2003, 2006). Fig. 7The Scribble (Scrb), Discs large (Dlg) and Lethal giant larvae (Lgl) proteins localize to the basolateral membrane domain in polarized epithelial cells. Scrb and Lgl exist in a complex but it is not clear if the interaction is direct. Inset: During the process of cell–cell contact formation, Lgl forms a transient complex with aPKC and PAR-6 from which PAR-3 is excluded. After aPKC-induced dissociation of Lgl from the complex, PAR-3 associates with PAR-6 and aPKC which promotes TJ formation and the development of apical and basolateral membrane domains. Double arrows with solid lines indicate direct protein–protein interactions, double arrows with broken lines indicate the presence of the two proteins in the same complex but the nature of the interaction has not been characterized in detail, yet Protein complexes at cell junctions and cancer Given the important role of protein complexes localized at cell junctions of epithelial cells in regulating cell–cell contact formation, cell polarity and cell proliferation, it is not surprising that altering the compositions of these protein complexes will result in changes in cell–cell adhesion, a loss in cell–cell contact integrity and eventually in uncontrolled proliferation and cancer. A loss of cell polarity is frequently associated with cancer (Bissell and Radisky 2001; Wodarz and Nathke 2007). As pointed out above, many tumor suppressor genes identified in Drosophila including dlg, scrib and lgl turned out to encode proteins which regulate epithelial cell polarity (Bilder 2004), and loss of their expression correlates with more invasive and aggressive cancers in mammalian cells (Dow and Humbert 2007). Recent evidence identified the PAR-3–aPKC–PAR-6 complex at TJs as a target for the oncogenic receptor tyrosine kinase ErbB2. When cultured on reconstituted basement membrane, MCF-10A mammary epithelial cells form three-dimensional spheroids that resemble glandular structures (acini) with a single-layered, polarized epithelium surrounding a luminal space (Debnath and Brugge 2005). ErbB2 signaling leads to multiacinar structures as a result of hyperproliferation and to filling of the luminal space in individual acini due to a block of apoptosis of the inner cells. The latter of these two effects turned out to be regulated by the PAR-3–aPKC–PAR-6 complex (Aranda et al. 2006). Although the molecular mechanism has not been revealed in detail, the findings indicate that in response to ErbB2 activation ErbB2 physically interacts with the PAR-3–aPKC–PAR-6 complex which leads to a removal of PAR-3 and the formation of a ErbB2–PAR-6–aPKC complex (Fig. 8). The newly formed ErbB2–PAR-6–aPKC complex disrupts apico-basal polarity and blocks apoptosis of inner acinar cells through aPKC activity (Aranda et al. 2006). A role of PAR-6–aPKC in regulating apoptosis of inner cells has been observed in MDCK cysts as well (Kim et al. 2007). Thus, the ErbB2 oncogene exploits the PAR–aPKC system to regulate survival of the ErbB2-transformed cells. Fig. 8The ErbB2 oncogene targets the PAR complex. In normal cells, PAR-3, aPKC and PAR-6 form a stable complex at TJs of epithelial cells (left panel). This complex is required for the fomation of TJs and the development of apico-basal polarity. ErbB2 activation triggers the association of the ErbB2 homodimer with PAR-6 and aPKC thereby disrupting the PAR-3–aPKC–PAR-6 complex. As a consequence, the development of apico-basal polarity is inhibited, and inner cells do not undergo apoptosis (right panel) Besides increased proliferation and reduced apoptosis, epithelial-mesenchymal transition (EMT) is another hallmark of tumor progression (Thiery 2002). During EMT, polarized epithelial cells adopt a mesenchymal or fibroblastoid, highly motile phenotype, and this is required during phases of embryonic development when epithelial cells leave a primitive epithelium to migrate to a distinct site in the embryo in order to induce new organ formation. Not surprisingly, typical characteristics of EMT are transcriptional repression of E-cadherin expression, profound changes in the cytoskeleton concomitant with a loss of apico-basal polarity (Thiery 2002). When this developmentally regulated programme is re-activated in the adult organism, it easily contributes to tumor progression by facilitating invasion and metastasis. Among the various physiological inducers of EMT, TGFβ signaling turned to be critically involved in EMT through cooperation with receptor tyrosine kinases (RTKs) and oncogenic Ras (Huber et al. 2005). Recent evidence indicates that TGFβ signaling affects not only the integrity of AJs through its known effect on E-cadherin expression but also the integrity of TJs by targeting the PAR-3–aPKC–PAR-6 complex. TGFβ signaling is mediated by two TGFβ receptors, the Ser/Thr kinases TGFβ-receptor I (TβRI) and TβRII. In polarized NMuMG cells, TGFβ induces EMT by recruiting TβRII to TJs which results in TJ dissolution. TβRI localizes to TJ through a direct interaction with occludin (Barrios-Rodiles et al. 2005). Interestingly, TβRI interacts also directly with PAR-6 (Ozdamar et al. 2005) (Fig. 9). A TGFβ signal triggers heterodimeric complex formation between the two TGFβ receptors bringing TβRII in close vicinity of the PAR complex. PAR-6 is then phosphorylated by TβRII, and this leads to the recruitement of the ubiquitin ligase Smurf1 which in turn mediates ubiquitination of RhoA. RhoA, however, is critical for junctional integrity (Sahai and Marshall 2002), and its localized degradation might thus lead to TJ dissolution. TGFβ does not only induce RhoA degradation via PAR-6 and Smurf1 but also induces the downregulation of PAR-3 (Wang et al. 2007) (Fig. 9). Although the fate of aPKC during this process has not been clarified, these findings suggest that the PAR-3–aPKC–PAR-6 complex is a major target of TGFβ signaling at TJs during TGFβ-induced EMT. Fig. 9TGFβ signaling targets the PAR complex at TJs. Left panel: Under normal conditions, TGFβ receptor I (TβRI) localizes to TJ through direct interactions with occludin and PAR-6. The cells maintain a polarized morrphology. Middle panel: TGFβ induces heterodimer formation of the two TGFβ receptors TβRI and TβR2 leading to activation of TβRII folllowed by TβRII-mediated phosphorylation of PAR-6 at Ser345. Right panel: Ser345-phosphorylated PAR-6 recruits Smurf1 leading to ubiquitination and degradation of the local pool of RhoA. As a consequence, the integrity of TJs is disturbed, the polarized morphology can not be maintained and the development of a fibroblastoid morphology is facilitated. In addition to PAR-6 phosphorylation, TGFβ signaling also induces downregulation of PAR-3. By targeting the PAR-3–aPKC–PAR-6 complex at TJs, TGFβ impairs the ability of cells to maintain a polarized morphology Protein complexes as targets for pathogens Many pathogens need to overcome epithelial and endothelial barriers to invade the host and establish infection. For this purpose, various strategies have evolved to disrupt the barrier and allow the pathogen the passage into tissues. These strategies include the release of proteolytic enzymes that cleave adhesion molecules like occludin, E-cadherin or desmoglein (Hanakawa et al. 2004; Pentecost et al. 2006; Wu et al. 1998, 2000), or the release of toxins that act via cell surface receptors to induce intracellular changes (e.g. of the actin cytoskeleton) which eventually lead to alterations of the barrier (Hopkins et al. 2003; Nusrat et al. 2001). More “advanced” strategies involve the delivery of pathogen-derived proteins via secretion systems into the host cell cytoplasm where these proteins directly associate with host cell proteins to influence their function. One example is the Helicobacter pylori (H.pylori) effector protein “Cytotoxin-associated gene A antigen” (CagA). H.pylori induces morphological changes of epithelial cells, alterations of the composition of the apical junctional complex as well as a breakdown of the epithelial barrier function (Amieva et al. 2003; Bagnoli et al. 2005), and H.pylori infections can result in mucosal damage (ulceration), inflammation (gastritis) and cancer (gastric carcinoma) (Peek and Blaser 2002). The CagA protein is involved in many of these processes through multiple interactions with a number of host proteins. After the translocation into the host cell, CagA is phosphorylated by src-family kinases and recruits the phosphotyrosine phosphatase SHP-2 (Higashi et al. 2002). In addition, CagA associates with several proteins involved in the regulation of TJs and in the formation of apico-basal polarity. First, it recruits and thereby mislocalizes the TJ proteins ZO-1 and JAM-A to the site of bacterial attachment (Amieva et al. 2003). Second, it directly interacts with the serine/threonine kinase PAR-1 (Saadat et al. 2007). Under normal conditions, PAR-1 cooperates with the PAR-3–aPKC–PAR-6 complex through reciprocal phosphorylations to regulate the formation of distinct membrane domains (Hurov et al. 2004; Suzuki et al. 2004) (see also above). The binding of CagA to PAR-1 blocks the kinase activity of PAR-1 thus preventing the phosphorylation of PAR-3; at the same time, it prevents the phosphorylation of itself by aPKC. As a result, the integrity of cell–cell contacts is disturbed and cells are extruded from the monolayer (Saadat et al. 2007). Thus, through its multiple interactions with signaling molecules, scaffolding proteins and cell polarity proteins, CagA disregulates critical cellular functions to enter the sub-epithelial tissues which also leads to inflammation and eventually to cancer (Hatakeyama 2004). Conclusions and perspectives The last decade has witnessed a steady increase in the number of new proteins localized at cell–cell contacts of epithelial cells. The identification of claudins at TJs has strongly increased the understanding of the molecular basis of TJ function. The identification of cell polarity protein complexes like the PAR-3–aPKC–PAR-6 complex and the CRB3–Pals1–PATJ complex at TJs has added new aspects on the mechanisms underlying the development of TJs. The identification of the nectin–afadin system provided evidence for a second major adhesive system besides the cadherin–catenin system at AJs. It also became evident that TJs and AJs are signaling centers which are actively engaged in regulating proliferation and differentiation through feed-back mechanisms with the cytoskeleton and the nucleus. Meanwhile, many of the proteins that regulate formation and maintenance of cell–cell contacts in epithelial cells have been found in other cellular systems as well suggesting a general function in cell–cell contact regulation. Claudins, nectins, and JAMs are used by cells of the male reproductive system to mediate homotypic Sertoli–Sertoli cell as well as heterotypic Sertoli cell–spermatid interactions (Gliki et al. 2004; Gow et al. 1999; Takai and Nakanishi 2003), and similar functions are performed by these molecules as those proposed in epithelial cells, i.e. formation of TJ strands, regulation of the actin cytoskeleton and regulation of cellular polarization, respectively (Gliki et al. 2004; Gow et al. 1999; Ozaki-Kuroda et al. 2002). In the peripheral nervous system (PNS), a large number of proteins typically associated with TJs or AJs was found to mediate autotypic Schwann cell interactions within the myelin sheath as well as heterotypic Schwann cell–axon interactions. For example, different claudins, JAM-C, Necl-1, Necl-2, Necl-4, and various scaffolding proteins like MUPP1, PATJ, ZO-1, ZO-2, PAR-3 and MAGI-2 are localized at areas of non-compact myelin including Schmidt-Lanterman-incisures, paranodal loops, and mesaxons, and some proteins preferentially localize to some but not other areas (Maurel et al. 2007; Poliak et al. 2002; Spiegel et al. 2007) (Fig. 10). The absence of claudin-19 or JAM-C in mice results in defective nerve conduction indicating that both are critically important for the proper functioning of the PNS (Miyamoto et al. 2005; Scheiermann et al. 2007). Necl-1 and Necl-4 mediate heterotypic interactions between axons and Schwann cells during myelination (Maurel et al. 2007; Spiegel et al. 2007). And PAR-3 has recently been identified at the interface between pre-myelinating Schwann cells and axons along the internodal region (Chan et al. 2006). PAR-3 is expressed by the Schwann cells, not by the axon, and recruits the p75 neurotrophin receptor to the glial–axon junction to regulate myelination of the axon. Fig. 10Proteins at autotypic and heterotypic cell–cell contacts in the peripheral nervous system (PNS). A multitude of proteins localized at TJs of polarized epithelial cells localizes to autotypic glial–glial cell contacts and heteroytpic glial cell–axon contacts. Abbreviations: JXP juxtaparanodal region, MV microvilli, PNL paranodal loops, PNJ paranodal junction, SLI Schmidt-Lanterman incisure It is not clear if these proteins form the same complexes like in epithelial cells, and it is likely that differences exist in the composition of protein complexes to regulate the specific requirements in the context of the given cell or tissue. As one example, JAM-C deficiency in mice leads to a mislocalization of PAR-6, aPKC and PATJ bot not PAR-3 in spermatids (Gliki et al. 2004) suggesting that PAR-3 is not part of the JAM-C-associated polarity complex in spermatids despite its ability to interact with PAR-6, aPKC and also directly with JAM-C (Ebnet et al. 2003; Suzuki and Ohno 2006). As another example, in endothelial cells two PAR protein complexes have been identified, a “conventional” PAR-3–aPKC–PAR-6 complex and a second PAR complex in which PAR-3 and PAR-6 are independently associated with VE-cadherin and which lacks aPKC (Iden et al. 2006). Nevertheless, the use of a set of conserved proteins by morphologically diverse cell types to regulate cell–cell contact formation highlights both the importance of the proteins for cellular function as well as their versatility that allows the regulation of similar aspects in different cell types. Many open questions remain. For example, what is the molecular nature of the intramembrane diffusion barrier (fence function) of the TJs? The absence of TJ strands in cells lacking all three ZO proteins results in a complete loss of the barrier function of the epithelial sheet but, unexpectedly, the fence function which has been attributed to the presence of TJ strands is retained (Umeda et al. 2006). Membrane diffusion barriers exist also in other cells even in the absence of a physical cell–cell contact, for example at the axonal hillock of neurons to separate somatodendritic and axonal membrane domains (Winckler et al. 1999). It has been suggested that in these cells the accumulation of integral membrane proteins that are anchored to the submembranous cytoskeleton function as rows of pickets which prevent the free diffusion of even small molecules (Nakada et al. 2003). A second unresolved issue is if the activity of the PAR-3–aPKC–PAR-6 complex to regulate membrane asymmetry is mechanistically linked to its role in TJ formation. Finally, the functional interrelationship of the three major polarity complexes at TJs (depicted in Figs 3, 4) has not been resolved, yet. Members of individual complexes interact with each other. For example, CRB3 can also interact with PAR-6 (Lemmers et al. 2004), PAR-6 can also interact with Pals1 (Hurd et al. 2003), and ZO-3 can interact with PATJ (Roh et al. 2002a). Genetic evidence in Drosophila suggests a functional hierarchy among the protein complexes (Johnson and Wodarz 2003) but it is unclear if a similar hierarchy exists in vertebrate epithelial cells and which aspects of cell–cell contact formation are regulated by these interactions. Most likely, many of these interactions are dynamically regulated and occur in a temporally and spatially restricted manner. The large number of scaffolding and signaling molecules identified at cell–cell contacts and the multitude of physical interactions described so far among these proteins indicates that cell–cell contact formation, the development of TJs from pAJs and the aquisition of membrane polarity is a highly dynamic process the complexity of which is still far from being completely understood.

Matern_Child_Health_J-2-2-1592140

Ensuring the Safe and Effective Use of Medications During Pregnancy: Planning and Prevention Through Preconception Care

Introduction Whether to use a medication during pregnancy can be a concern for both women and health care providers. It has been estimated that up to 10% of congenital anomalies may be caused by environmental exposures–that is, exposures to medications, alcohol, or other exogenous factors that have adverse effects on the developing embryo or fetus [1]. Avoiding all potentially adverse exposures can prevent these adverse effects. However, it is not always possible to avoid taking medications during pregnancy. Women who are pregnant continue to experience short-term and long-term health conditions that must be managed. Discontinuing treatment of a serious condition when a woman becomes pregnant can have profound, long-term implications both for her health and that of her baby. In a retrospective study from eight health maintenance organizations, researchers estimated that approximately 59% of pregnant women were prescribed a medication other than a vitamin or mineral supplement at some time during pregnancy [2]. Use of over-the-counter medications during pregnancy may be even higher, and many women take a dietary or herbal supplement other than multivitamins or folic acid while pregnant [3, 4]. Preconception care provides the opportunity to optimize a woman’s use of medications in preparation for pregnancy. Such care includes identifying patterns of medication use before pregnancy occurs; adjusting those patterns to avoid the use of nonessential medications; minimizing exposure to medications known to be harmful to the embryo or fetus; and adjusting the dose, route of administration, and timing of essential treatments to optimize maternal health at each stage of pregnancy while safeguarding the embryo, fetus, and infant. In this paper, we summarize the basic principles of teratology and the current state of knowledge about the effects of medication use during pregnancy; outline basic components of preconception care that can help minimize the risk of birth defects; and provide examples of approaches to planning for the safe and effective use of medications during pregnancy through preconception care. Principles of teratology The term “teratogen” is sometimes used to describe an agent that can produce structural or functional abnormalities in a developing embryo or fetus. Although this implies that an agent is inherently either teratogenic or not, teratogenicity is actually a property of the exposure taken as whole—not only the physical and chemical nature of the agent, but also the dose, route of administration, and timing in gestation, as well as concurrent exposure to other agents and biological susceptibility of the mother and embryo or fetus. The developmental stage of the embryo or fetus at the time of exposure is critical. During conception and for about 2 weeks thereafter, most cells of the conceptus are not yet committed to a specific developmental program. One damaged cell can be replaced by another, and normal development will usually ensue, although the embryo will not survive if too many cells are damaged or killed. This is known as the “all-or-nothing” period, but even during this period some exposures can adversely affect the development of surviving embryos [5]. The subsequent period of organogenesis, from 18 to 60 days after conception (about 4.5–11 weeks after the start of the last normal menstrual period) is the time of greatest sensitivity to most teratogenic exposures. Fetal exposure later in gestation usually does not produce gross structural abnormalities, although there are exceptions. Adverse exposures during the fetal period more often result in growth restriction or functional disorders of the central nervous system, kidneys, or other organs. Dose is a critical feature of any teratogenic exposure. Teratogenic effects occur only when the dose of an agent exceeds a certain threshold [1]. Medications that are generally considered safe during pregnancy can have adverse effects on the embryo or fetus if the mother takes them in doses that are so high that they cause maternal toxicity. Chronic exposure is usually of more concern than a single exposure, if the doses are similar. The route of exposure is also important. For example, risk is unlikely from the use of dermal agents that lack substantial systemic absorption. The teratogenicity of an exposure is also influenced by both the maternal and fetal genotypes, which can result in differences in cell sensitivity, placental transport, metabolism, receptor binding, or drug distribution. Some medications are metabolized extensively by the mother; their teratogenicity depends upon whether a toxic form reaches the embryo or fetus in sufficient quantities to produce adverse effects. Current state of knowledge about the effects of medication use during pregnancy Maternal effects Many women begin pregnancy with medical conditions that require ongoing or episodic treatment. Examples include asthma, epilepsy, and hypertension. In addition, other medical problems, such as migraine headache or auto-immune disorders, may be exacerbated by pregnancy. When planning for the management of maternal conditions during pregnancy, it is important to distinguish conditions for which withholding treatment could be harmful to the mother, embryo, or fetus, from those for which cessation of treatment is unlikely to pose significant risk. For example, women with major depression who discontinue antidepressant medication before conception are at high risk of relapse and consequent self-injurious or even suicidal behavior [6]. In contrast, cessation of treatment for moderate hypercholesterolemia with a statin drug while a woman is pregnant is unlikely to increase her cardiovascular morbidity or mortality significantly. Physiologic changes occur during pregnancy that can alter the effective dose of medications a woman is taking. Some changes occur abruptly, while others evolve slowly. Most begin during the first trimester and peak during the second trimester of pregnancy [7]. It may be necessary to adjust the dose and/or frequency of medication use repeatedly during pregnancy. Physiologic changes that can affect the pharmacokinetics and/or pharmacodynamics of medications during pregnancy include:Changes in total body weight and body fat composition.Delayed gastric emptying, prolonged gastrointestinal transit time, and decreased gastric acid secretion, all of which can affect the bioavailability of drugs [8–10].Expanded plasma volume and significantly increased extracellular fluid space and total body water content. These vary with the patient’s weight and can affect the volume of distribution of drugs [11].Increased cardiac output, stroke volume, heart rate, and blood flow to the uterus, kidneys, skin, and mammary glands. The percentage of cardiac output attributed to hepatic blood flow is lower during pregnancy [12].Decreased concentration of plasma albumin, which can reduce the protein binding of some drugs [13].Increased glomerular filtration rate early in pregnancy, with a continued rise throughout pregnancy [14].Changes in the activity of hepatic enzymes, including the cytochrome P450 enzymes, xanthine oxidase, and N-acetyltransferase [15, 16]. Unfortunately, there are relatively few studies of drug pharmacokinetics during pregnancy. The dose of medications usually prescribed during pregnancy is the same used in nonpregnant adults, but this may result in substantial under- or over-dosage during pregnancy. When blood or serum concentrations of medications can be measured and the most effective level is known, they should be monitored throughout pregnancy and appropriate dosage adjustments made as needed. Further well-designed and well-conducted pharmacokinetic and pharmacodynamic studies of medications during pregnancy are needed. Fetal effects Maternal treatment with conventional doses of some medications during a susceptible period of pregnancy is known to be harmful to the developing embryo. Thalidomide and isotretinoin are the most notable examples, but there are others [17]. In contrast, taking some other medications or dietary supplements such as folic acid helps to prevent adverse pregnancy outcomes [18]. Because pregnant women are traditionally excluded from clinical trials for ethical reasons and because premarketing animal studies do not necessarily predict the effects of treatment in human pregnancy, little information about the teratogenic risks or safety of most drugs is available at the time they are marketed. Animal teratology studies are not routinely conducted for non-prescription drugs, vitamins, and dietary and herbal supplements, although these substances may produce pharmacological or toxic effects in the fetus. Moreover, there is no standard requirement for studies of adverse effects among children of women who took a drug during pregnancy after it has been approved by the FDA. In a review conducted in 2001, researchers found that there was not enough information to assess the teratogenic risk or safety during human pregnancy of more than 90% of prescription medications approved by the FDA in the previous 20 years [19]. Even when available, studies addressing fetal effects of maternal medication use during pregnancy may provide conflicting results or insufficient information to assess all potential outcomes or levels of risk. The concept of safety implies the absence of risk, which is impossible to demonstrate conclusively with any kind of study. Thus, it can be difficult for women and health care providers to decide whether to use a medication during pregnancy. The balance of risk, benefit, and efficacy of treatment for both mother and fetus is not always clear and must be individualized for different women under different circumstances. Basic components of preconception care that can minimize the risk of birth defects Serious congenital anomalies, including chromosome abnormalities and Mendelian disorders, can be identified in about 2% of infants at birth [52]. However, some anomalies do not become apparent until later in life [20]. While most birth defects are not preventable, some can be avoided through appropriate planning and medical interventions. The following components of preconception care can help minimize the risk of birth defects:Optimize health before conception occurs. This includes counseling women to avoid smoking, use of excessive alcohol and illicit drugs, and exposure to potentially toxic environmental or occupational hazards before they are pregnant.Establish effective treatment for chronic conditions before conception occurs.Carefully manage all chronic conditions and intercurrent illnesses throughout pregnancy.Counsel women to avoid the use of nonessential medications, including prescription and over-the-counter medications and dietary or herbal supplements.Avoid the use of medications with high teratogenic risk when equally effective treatments with lower risks are available.Limit the use of essential medications to the smallest number of drugs possible that will effectively treat maternal disease without compromising the health of the woman or her fetus.Limit each essential medication to the smallest dose that can be used to effectively treat maternal disease without compromising the health of the woman or her fetus.Recommend that all women who are capable of becoming pregnant take a vitamin supplement or eat fortified foods to assure consumption of 0.4 mg (400 micrograms) of folic acid per day. Effective pregnancy management in women with chronic conditions requires careful planning, close medical supervision before and during pregnancy, and continuous communication between the pregnant woman and her health care providers. Examples of approaches to preconception planning for the use of medications during pregnancy in different clinical settings Exactly how the components of preconception planning for the use of medications during pregnancy are implemented depends on the nature of the condition requiring treatment, the known risks and safety of use of the specific drugs during pregnancy, and the woman’s individual circumstances, among other factors. In this section, we provide three examples of approaches to planning for the safe and effective use of medications in clinical settings where these factors vary Avoiding teratogenic treatments for non life-threatening maternal conditions—isotretinoin Isotretinoin is indicated for the treatment of severe nodular cystic acne unresponsive to other therapy but is also used to treat non-nodular, but scarring, acne. A single course of therapy typically lasts 15–20 weeks and can result in complete and prolonged remission of the acne in many patients. However, isotretinoin treatment in the first trimester of pregnancy is teratogenic. Exposed infants can have craniofacial, cardiac, thymic, and central nervous system malformations [21]. Research has also shown a high incidence of developmental delay in children whose mothers used isotretinoin early in the first trimester, regardless of whether the children had structural malformations [22]. Isotretinoin is indicated for use only in men and nonpregnant women. It should never be used during pregnancy. However, because approximately half of pregnancies in the United States are unintended, some women use isotretinoin in the early weeks of gestation before realizing they are pregnant [23]. Teratogenic outcomes have been reported after only one dose of isotretinoin during pregnancy [24]. The half-life of isotretinoin is approximately 24 h, but about 2 weeks are required to eliminate 99% of the drug from the body after cessation of use. Several risk management strategies have been implemented to prevent the use of isotretinoin during pregnancy, but pregnancy exposures continue to occur [25]. An enhanced risk management program called iPLEDGE became fully operational in March 2006 [26, 27]. iPLEDGE is a single, mandatory program for all marketed isotretinoin products. It requires that wholesalers, pharmacies, doctors, and patients register with the program in order to obtain the drug. Female patients who are capable of having children must obtain counseling about the risks of isotretinoin treatment and the requirements for its safe use; complete an informed consent form; have two negative pregnancy tests documented before starting isotretinoin therapy; use two different forms of contraception simultaneously or agree not to engage in heterosexual intercourse for one month prior to starting isotretinoin, throughout the course of treatment, and for one month after completing treatment; and have a negative pregnancy test documented every month during treatment and one month after completing treatment. More information about the iPLEDGE program can be found at www.ipledgeprogram.com. Preconception care provides a unique and critical opportunity to prevent exposure to teratogens such as isotretinoin. Any woman who is considering pregnancy should be asked whether she is taking any medications including any preparation containing isotretinoin. If so, her health care provider should emphasize the risks of isotretinoin use during pregnancy, reevaluate the need for and duration of treatment, assess thoroughly the use and effectiveness of contraceptive measures, and reinforce the key elements of the iPLEDGE program. Women should be informed that conception cannot be attempted without risk of teratogenicity until one full month after the last dose of isotretinoin was taken. Educational information and materials from the iPLEDGE program can also be helpful in the setting of preconception care. Managing maternal conditions that require continuous treatment—epilepsy At least one in every 250 pregnant women, or about 0.4%, takes an anticonvulsant drug [28]. Approximately half take the drugs to prevent seizures, but anticonvulsants are also used to manage mood disorders, migraine headaches, and chronic pain. Anticonvulsant drugs have several different modes of action, such as targeting a specific receptor or enzyme [29]. As a result, anticonvulsant medications vary in their effectiveness for specific types of epilepsy. Individuals with epilepsy may respond differently to particular medications, reflecting genetic differences such as polymorphisms in the cytochrome P450 enzymes. It is not yet possible to screen for pharmacogenetic differences that would help select the appropriate anticonvulsant for individual use. Physiologic changes during pregnancy also can affect the disposition of anticonvulsant drugs and the dose needed to prevent seizures [30]. While not all anticonvulsant drugs have been studied in pregnancy, a number of adverse effects have been identified in infants and children of women treated with these medications during pregnancy. Major malformations, midface and digit hypoplasia, microcephaly, growth restriction, and deficits in IQ are sometimes seen, although the pattern of abnormalities and specific effects vary for individual drugs [28, 31–33]. For example, spina bifida occurs in approximately 1% of fetuses exposed to carbamazepine and in 2 to 5% of fetuses exposed to valproic acid [34, 35]; microcephaly and growth restriction are observed more frequently in infants whose mothers took two or more anticonvulsant drugs simultaneously during pregnancy [28]; and subtle effects on intelligence have been identified in some studies of children exposed to carbamazepine, phenytoin, or phenobarbital in utero, although developmental delay and deficits in cognitive function are much more frequent with exposure to valproic acid [36–38]. While some infants exposed to an anticonvulsant drug in utero have abnormalities, others do not. Genetic differences in the fetal response to medications probably play a role. It has been hypothesized that maternal epilepsy might cause fetal abnormalities independent of any drug effect, but several studies suggest that anticonvulsant treatments are usually responsible [39]. However, it seems likely that repeated or prolonged maternal seizures, such as occur in status epilepticus, can be devastating to the fetus [40]. Discontinuation or inadequate treatment of maternal epilepsy may be more dangerous to the fetus than effective anticonvulsant therapy. Preconception care provides an opportunity to choose a plan of anticonvulsant treatment that will pose the least risk to the fetus while appropriately managing maternal symptoms. New data about the effects on the fetus of anticonvulsant medications are emerging steadily, and the latest information should always be sought. The treatment plan must be individualized for each woman in collaboration with her neurologist, psychiatrist, or other specialist. Primary considerations include assessing whether taking an anticonvulsant drug is essential to the mother’s health; using the fewest number of anticonvulsant drugs possible; using the lowest dose for each drug that will effectively treat maternal symptoms [41]; establishing the most effective blood level of each drug before conception; and monitoring drug levels throughout pregnancy. For carbamazepine, phenobarbital, phenytoin, primidone, and valproic acid, levels of the non-protein bound, or “free,” concentration should be measured. Because abrupt cessation of a medication may result in increased seizures or exacerbation of other symptoms, the number and dose of medications should be adjusted, and the woman’s response stabilized, over a period of time before attempting conception. In addition, preconception care offers the opportunity to consider whether additional measures, such as the use of a higher daily dose of folic acid, might be beneficial. Only limited and somewhat conflicting information is available about whether periconceptional supplementation with folic acid at levels higher than 0.4 mg per day decreases the risk for neural tube defects associated with anticonvulsant drug exposure in utero [33, 42]. Currently, many women who require anticonvulsant drug therapy during pregnancy take 4 or 5 mg of folic acid daily. Managing maternal conditions with intermittent symptoms—asthma Asthma is a chronic condition with intermittent symptoms, for which treatment during pregnancy is essential to safeguard the health and well-being of both the mother and fetus. Abrupt cessation or undertreatment of asthma during pregnancy can endanger both. Studies suggest that maternal asthma during pregnancy can increase the risk for perinatal mortality, preeclampsia, preterm delivery, and low birth weight [43, 44]. Maternal asthma can lead to alkalosis with decreased blood flow to the uterus, decreased venous return, and a leftward shift of the oxyhemoglobin dissociation curve, all of which may contribute to fetal hypoxia. In the extreme, maternal hypoxia can result in decreased umbilical blood flow, increased systemic and pulmonary vascular resistance in the fetus, and decreased fetal cardiac output. Fetal compromise may occur well before maternal symptoms become severe [45]. Additional pathophysiologic mechanisms that could contribute to these adverse outcomes include hyperactivity of uterine and bronchial smooth muscle and the release of bioactive mediators during symptomatic asthma [43]. For these reasons, it is considered safer for pregnant women to be treated with asthma medications than to experience asthma symptoms and exacerbations [46]. A variety of medications are available to treat acute and chronic asthma. They include beta2-agonoists, corticosteroids, cromolyn, leukotriene modifiers, theophylline, and anticholinergics. These drugs have different mechanisms of action and thus potentially different effects on the fetus. Before attempting conception, it is important to maximize asthma control using medications that can also be used to manage asthma symptoms during pregnancy. This will ensure maximum oxygen delivery to the developing embryo and fetus while avoiding unintended medication exposures. A period of time may be needed to wean patients off some medications and to stabilize symptoms using others before attempting conception. In addition, because an estimated one third of pregnant women with asthma experience an increase in the frequency or severity of their symptoms during pregnancy, it is important to develop a plan before conception occurs for regular monitoring of asthma symptoms and pulmonary function during pregnancy and for early recognition and prompt treatment of exacerbations [47, 48]. This plan should include a review of the proper use of medications, including the proper technique for using inhalers; education about self-monitoring and self-management at home; and specific instructions about when to contact a health professional for additional care. The National Asthma Education and Prevention Program Working Group outlines a step-wise approach for managing asthma during pregnancy in its report Managing Asthma During Pregnancy: Recommendations for Pharmaceutical Treatment—Update 2004. This report was developed after a systematic review of the current evidence from safety studies of asthma medications during pregnancy [46]. The approach is centered on the use of beta2-agonoists and corticosteroids, by inhalation when possible to minimize systemic absorption. The dose, frequency, and number of medications are decreased when possible but increased as needed, and pulmonary function is measured regularly (e.g., by monthly spirometry testing) during pregnancy. The approach also emphasizes the need for a treatment plan tailored to each patient’s needs and circumstances, the frequency and severity of her symptoms, and her individual response to treatment. Regardless of the specifics, the benefits and risks of asthma treatment and of uncontrolled asthma during pregnancy for both the mother and fetus should be discussed during preconception planning so that a fully informed plan for asthma management can be agreed upon. Additional measures can be instituted preconceptionally to minimize the severity or frequency of asthma symptoms during pregnancy, and thus decrease reliance on, and fetal exposure to, medications. Identifying, reducing, or eliminating exposure to allergens such as animal dander, house-dust mites, cockroaches, pollen, and indoor mold; to irritants such as perfumes, sprays, and cleaning agents; and to smoke, including primary and secondhand tobacco smoke, wood burning stoves, or fireplaces, may decrease asthma symptoms. Preconception counseling also provides an opportunity to discuss the use of over-the-counter medications for symptoms such as rhinitis, congestion, and esophageal reflux that are often associated with asthma. While over-the-counter medications can be purchased without a prescription, they contain pharmacologically active ingredients that may affect the fetus. For example, studies have suggested that use of pseudoephedrine or aspirin in the first trimester may increase the risk of gastroschisis, a rare abdominal wall defect [49–51]. It has been theorized that gastroschisis could result from vascular disruption. Both aspirin and pseudoephedrine can have vasoactive effects, although the causal pathway has not been established for either drug, and the absolute risk of their use is very small. Most over-the-counter medications have not been adequately studied in pregnancy. Conclusion Although it is always better to avoid unnecessary medical treatment during pregnancy, some women with chronic conditions may not be able to become pregnant without appropriate therapy. In many other cases, proper treatment of a chronic condition during pregnancy may be safer for both the woman and her baby than stopping this treatment. It is important that women who are planning a pregnancy talk with their health care provider before beginning a new medication or making changes in current medications for the management of acute or chronic conditions. Continuous communication between a pregnant woman and her health care providers, careful preconceptional planning, effective management of conditions prior to pregnancy, and close medical supervision during pregnancy can help assure the best possible outcome for every woman and baby. New information about the effects of medication use and the optimal management of maternal conditions during pregnancy continually becomes available. The following are regularly updated sources of such information and advice:The Organization of Teratology Information Specialists (OTIS) provides medical consultation, usually by phone, to individuals and health care providers about the reproductive risks of prenatal exposures.Phone: 866-626-6847Website: http://otispregnancy.orgREPROTOX® is an information source for laboratory scientists, practicing physicians, and government agencies that contains commentaries on the effects of chemicals and physical agents on human pregnancy, reproduction, and development.Website: www.reprotox.orgThe Teratogen Information System (TERIS) is a computerized database of summaries of individual agents designed to assist health care professionals in assessing the risks of exposures in pregnant women. Each summary is based on a review of the published literature and includes a risk assessment derived by consensus of an advisory board of authorities in clinical teratology.Website: http://depts.washington.edu/∼terisweb/teris Information about the results of premarketing studies of individual medications, including animal reproductive studies and human clinical trials, is usually available from the drug manufacturer. A list of many of the existing postmarketing pregnancy registries that seek to monitor exposure to specific medications during pregnancy is maintained by the Office of Women’s Health, U.S. Food and Drug Administration at www.fda.gov/womens/registries. Additional information is available from the March of Dimes at www.marchofdimes.com/pnhec/173_1453.asp.