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Purinergic_Signal-3-4-2072928

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X1,2,4,7 and P2Y1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects. Introduction Microglial cells are the principal intrinsic immune competent cells of the brain. Resting microglial cells with a ramified morphology become activated under almost all pathological conditions, e.g., mechanical or chemical injury or inflammation. They change into ameboid-shaped motile phagocytotic and cytotoxic cells, which invade the site of injury and initiate repair or defense processes by secreting proteases, neurotrophic factors, and inflammatory mediators [1, 2]. This suggests that microglial cells are able to sense signal molecules entering the extracellular space by disturbances of brain homeostasis and promoting their transformation into the active state [3, 4]. Further, microglia express major histocompatibility complex antigen class II and may perform phagocytotic activity to clear cellular debris [4–7]. One of the most prominent compounds released by injury is the purine nucleotide ATP. Extracellularly applied ATP induces the activation of a cation conductance and a subsequent increase in intracellular Ca2+ [8]. ATP was shown to stimulate microglial cells to respond with rapid movements of their processes [9]. Further, ATP also induced the outgrowth of processes and membrane ruffling in cultured systems [10, 11]. Dependent on the concentration of ATP applied to in vitro systems, microglial cells secrete various biologically active substances, such as the cytokines tumor necrosis factor-α (TNF-α) [12], interleukin (IL)-1β [13, 14], or IL-6 [15]. Boucsein and colleagues [16] have shown that in the presence of purinergic ligands, the lipopolysaccharide-induced release of TNF-α, IL-6, IL-12, and macrophage inflammatory protein-1α was attenuated. In patch-clamp investigations with ATP-induced Ca2+ wave recordings of astrocytes, a current response in an adjacent microglial cell was observed, suggesting that microglia can sense the activity of astrocytes [17]. All these data suggest that a variety of ATP-sensitive ionotropic (P2X) and metabotropic (P2Y) receptors is expressed at resting and/or activated microglial cells [18–22]. The functional expression of ATP receptors on cultured microglia is well known [18, 23, 24]. The basic problem in studying this type of glial cells is that any manipulation ex vivo or in vitro will promote their transformation into the active form. We used an in vivo model of a stab wound injury in the rat nucleus accumbens (NAc) to observe the P2 receptor-mediated response of microglial cells to ATP released by injury. This technique enables local administration of receptor-specific purinergic ligands by microinfusion. Furthermore, our investigations focused especially on the P2X7 receptor subtype on microglial cells in vivo and the functional consequences of injury-induced stimulation. The possible involvement of P2X7 receptors in apoptotic and proliferative processes in microglia and astrocytes, respectively, was also investigated. Material and methods Materials Ketamine hydrochloride (Ketanest; Ratiopharm, Ulm, Germany), xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany), and thiopental natrium (Trapanal, Altana Pharma, Konstanz, Germany) were used for anesthesia. For intra-accumbal microinjection, 2-methylthioATP (2MeSATP), α,β-methyleneATP (α,βmeATP, both RBI; Natick, MA, USA); adenosine 5′-(β-thio)diphosphate (ADPβS), 2′,3′-O-(4-benzoyl-benzoyl)ATP (BzATP), brilliant blue G (BBG), and 5-bromo-2′-deoxyuridine (BrdU) (all from Sigma, Deisenhofen, Germany); uridine 5′-O-(γ-thio)triphosphate (UTPγS, Inspire Pharmaceuticals Inc., Durham, NC, USA); pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; Biotrend, Köln, Germany); and artificial cerebrospinal fluid [ACSF (in mM): 126 NaCl; 2.5 KCl; 1.2 NaH2PO4; 1.3 MgCl2, and 2.4 CaCl2; pH 7.4] were used. Antibodies directed against glial fibrillary acidic protein (GFAP; mouse anti-GFAP, Sigma, Deisenhofen, Germany; rabbit anti-cow GFAP, DakoCytomation, Hamburg, Germany); BrdU (mouse anti-BrdU, Clone Bu20a; DakoCytomation), active caspase 3 (rabbit, Promega, Madison, WI, USA); OX 42 (CD 11b, mouse anti-rat, complement receptor type 3; Serotec, Oxford, UK); (a) lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) isolectin B4 (BSI-B4, peroxidase labeled) and (b) lectin from B. simplicifolia (G. simplicifolia) isolectin B4 (GSA-B4, biotin conjugated; Sigma, Deisenhofen, Germany, each); phosphorylated serine/threonine kinases (pAkt1/2/3; rabbit, Ser 473, raised against the short amino acid sequence containing phosphorylated Ser 473 of Akt 1, Akt 2, and Akt 3 of mouse origin, recommended for the detection of Ser 473 phosphorylated Akt 1 and corresponding phosphorylated Akt 2 and Akt 3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and the following P2 receptor antisera were used: rabbit anti-P2X1, anti-P2X2, and anti-P2X4,5,6 (Alomone Labs, Jerusalem, Israel) as well as rabbit anti-P2X7 receptor-subtype (intracellular C-terminus binding, Alomone Labs), anti-P2X3 (guinea pig; Neuromics Inc., Northfield, MN, USA). Furthermore, a goat anti-rat ecto-P2X7 receptor antibody (a kind gift of Dr. M. Kim; [25]) was used for immunofluorescence double-labeling studies. Additionally, anti-P2Y1, anti-P2Y2, anti-P2Y4, anti-P2Y12 (rabbit; Alomone Labs, Jerusalem, Israel), anti-P2Y6 (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-P2Y1 (rabbit, GlaxoSmithKline, Brentford, Middlesex, UK) receptor antibodies were tested. The secondary carbocyanine (Cy)2- and Cy3-conjugated IgGs as well as Cy2- and Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA, USA) were used. For histochemical detection, 3,3′-diaminobenzidine hydrochloride (DAB; Sigma Chemical Co., St. Louis, USA) was applied. Animals Male Wistar rats (280–320 g) were housed under a 12-h light, 12 h-dark cycle and allowed access to lab food and water ad libitum. All procedures using animals were approved by the committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection. Surgery/microinjection Anesthetized rats were fixed in a stereotaxic frame. At the coordinates AP = 1.7 mm (rostral to bregma), L = 1.5 mm (lateral to the sagittal suture), and V = 6.5 mm (below the surface of the hemisphere), the injection cannulae connected to a syringe pump via an FEP tubing was inserted into the NAc [26]. By microinfusion, rats received ACSF, which was used as control as well as vehicle for the following P2 receptor agonists: 2MeSATP (nonselective), α,βmeATP (P2X1,3), ADPβS (P2Y1,11,12,13), UTPγS (P2Y4,6), given as 0.1 nmol each, and BzATP (preferential P2X7) 0.3 nmol. As antagonists, PPADS (30 pmol; nonselective) and BBG (1 pmol, P2X7) were applied. When the effects of the P2 receptor agonists were pharmacologically proven, the microinfusion of the respective antagonists preceded infusion of the agonist and antagonist mixture. As a proliferation marker, BrdU (0.1 nmol) was applied together with the antagonist, the agonist, or ACSF alone. Test substances were injected in a volume of 1 μl at a rate of 12 μl/h. Immunocytochemical studies and double-immunofluorescence studies Immunocytochemical and double-immunofluorescence studies were done as previously described [26, 27]. After a postinjection time of 2 h and 4 days, the rats were transcardially perfused under thiopental sodium anesthesia with paraformaldehyde (2%) in sodium acetate buffer (pH 6.5; solution A) followed by paraformaldehyde (2%)/glutaraldehyde (0.1%) in sodium borate buffer (pH 8.5; solution B). The brains were immediately removed and stored overnight in solution B without glutaraldehyde. Serial coronal sections (50-μm thick) from the NAc were obtained using a vibratome (Leica, Typ VT 1000S, Nussloch, Germany) and collected as free-floating slices (0.1 M Tris; pH 7.6). Immunocytochemical studies BSI immunoreactivity Free-floating sections were rinsed with 0.05 M Tris-buffered saline (TBS, pH 7.6) and were treated with 1% hydrogen peroxide for 30 min to inactivate endogenous peroxide activity. Immunolabeling was performed with lectin from BSI-B4 (1:200) in TBS containing 2% bovine serum albumin overnight at 4°C, followed by washing in 0.05 M Tris buffer (pH 8.0). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) and hydrogen peroxide, which renders a black reaction product. Active caspase 3 immunoreactivity Free-floating sections were rinsed with 0.1 M TBS (pH 7.4) and treated with 1% hydrogen peroxide for 25 min to inactivate endogenous peroxide activity. Immunoreactivity (IR) was studied with rabbit anti-active caspase 3 (1:500) in TBS containing 10% normal horse serum (NHS) and 0.1% Triton X-100 overnight at 4°C, followed by biotinylated horse anti-rabbit IgG (1:100, Vector Labs. Burlingame, CA, USA) and preformed streptavidin/biotin-peroxidase complex (1:125, StreptABComplex; DakoCytomation) for 2 h. DAB (0.05%) served as chromogen. GFAP/BrdU immunoreactivity The procedure was applied to free-floating slices as previously described [26]. Briefly, the GFAP staining procedure was carried out with rabbit anti-cow GFAP antiserum (1:600) and biotinylated protein A (1:400; Calbiochem, La Jolla, CA, USA). To detect the astroglial marker, the streptavidin/biotin technique based on a StreptABComplex (1:125) and DAB (0.05%) as chromogen were used. Mitotic astrocytes were identified by immunostaining of the incorporated BrdU. After DNA denaturation (2 N HCl) and neutralization (borate buffer; 0.15 M; pH 8.5), the slices were incubated with a mouse monoclonal antibody against BrdU (1:75), followed by incubation with horse biotinylated anti-mouse immunoglobulins (1:100; Vector Labs., Burlingame, CA, USA) and with ABC Elite Kit (1:50; Vectastain; Vector). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) plus cobalt chloride (1%) (DAB-Ni/Co) and hydrogen peroxide, which renders a black reaction product. For GFAP/BrdU double-staining experiments to characterize mitogenic changes, slices were first processed for anti-GFAP labeling followed by BrdU immunolabeling. Double-immunofluorescence studies After washing with TBS (0.05 M; pH 7.6) and blocking with normal goat serum (1% NGS) in TBS, slices were incubated in an antibody mixture of mouse anti-OX 42 (CD11b; 1:100) and rabbit anti-P2X receptor antibodies (P2X1 1:400; P2X2 1:400; P2X4 1:400; P2X5 1:400; P2X6 1:400; P2X7 1:1,000) or P2Y receptor antibodies [P2Y1 1:400; P2Y2 1:1,000; P2Y4 1:1,000; P2Y12 1:400 (Alomone Labs); P2Y1: 1:1,500 (GlaxoSmithKline); P2Y6 1:200 (Santa Cruz Biotechnology, Inc.)] with 0.1% Triton X-100 in 1% NGS in TBS for 48 h at 4°C. The secondary antibodies employed for simultaneous localization of the two primary antisera were Cy2-conjugated goat anti-mouse IgG (1:500) and Cy3-conjugated goat anti-rabbit IgG (1:800), respectively. Cy2-conjugated goat anti-guinea pig IgG (1:400) and Cy3-conjugated goat anti-mouse IgG (1:1,000) were used for visualization of guinea pig anti-P2X3 (1:1,000). Sections were washed three times for 5 min each in 1% NGS [or 5% fetal calf serum (FCS) for guinea pig anti-P2X3] in TBS and then incubated for 2 h in a solution containing a mixture of the secondary antibodies with 1% NGS (5% FCS) in TBS. GSA-B4 and P2X or P2Y double labeling was performed in two steps: first the incubation of the slices with GSA-B4 (1:300), followed by treatment with Cy2-conjugated streptavidin (1:350); and second, by incubation with rabbit P2X/Y antibodies followed by Cy3-conjugated goat anti-rabbit IgG (1:800) as described above. The ecto-P2X7 receptor antibody (goat anti-rat ecto-P2X7 receptor, 0.12 μg/ml [25]) was combined with rabbit anti-caspase 3 (1:500), rabbit anti-pAkt (1:600), or rabbit anti-P2X7 (C-terminal, 1:1,000) and subsequent detection with Cy2-conjugated donkey anti-goat IgG (1:1,000) and Cy3-donkey anti-rabbit IgG (1:1,000). Incubation of slices with goat anti-rat ecto-P2X7 (1:600), rabbit anti-active caspase 3 (1:500), or rabbit anti-pAkt (1:600) and GSA-B4 (1:300) was followed by treatment with Cy3-conjugated donkey anti-goat (1:1,000) or Cy3-conjugated goat anti-rabbit IgG (1:400) and Cy2-conjugated streptavidin (1:350). Control experiments were carried out without the primary P2 receptor antibody or by preadsorption of the antibody with the peptides used for their generation. When slices were incubated with PBS instead of the primary antibody or with primary antibody serum, which had been preabsorbed with peptide antigen for 1 h before use, no immunofluorescence with either of the control procedures was observed. After intensive washing and mounting on glass slides, all stained sections were dehydrated in a series of graded ethanol, processed through n-butylacetate, coverslipped with entellan (Merck, Darmstadt, Germany), and analyzed by light microscopy or confocal laser scanning microscopy. Confocal microscopy Double immunofluorescence was investigated by a scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with an argon laser emitting at 488 nm (yellow-green Cy2-immunofluorescence) and a helium/neon laser emitting at 543 nm (red Cy3-immunofluorescence). Quantification and statistical analysis Changes in BSI-B4-IR were determined by densitometry (in regions 1 and 2, Fig. 1a) using a digital image analyzer (Diana II) in combination with an advanced image data analyzer (AIDA 2.0). Results are expressed as percentage of the BSI-B4-IR of the ACSF-treated sides in the same regions. Fig. 1a A rat brain slice including needle tracts and regions of interest within the nucleus accumbens (NAc) in which immunoreactivity (IR) and cells were evaluated (1 core 1; 2 core 2; according to [29]). b BSI-B4-marked microglial cells after stab-wound injury (overview). c Quantification of the number of activated microglial cells after injury and microinjection of P2-receptor-agonists 2MeSATP, ADPβS, α,βmeATP (0.1 nmol each), and BzATP (0.3 nmol) after a postinjection time of 4 days. Values are expressed as percentage of ACSF-treated controls and represent mean ± SEM of six animals per group. d–f BSI-B4-IR of microglial cells in the NAc of rats, illustrating changes in the glial morphology: d A great number of resting (process-bearing) microglial cells (arrows) under control conditions; e, f changes in the number of resting microglial cells (arrow) and activated microglial cells (arrowhead) after different treatment conditions (scale bar = 200 μm). g Quantification of the effects of agonists 2MeSATP, ADPβS, α,βmeATP (0.1 nmol each), and BzATP (0.3 nmol) alone and in combination with PPADS (0.1 nmol/0.03 nmol each) or BBG (1 pmol) on BSI-B4-IR of microglial cells in brain slices of the NAc of the rat after a postinjection time of 4 days. Data are expressed as percentage of the ACSF-treated side and represent the mean ± SEM of six animals per group (*p < 0.05, vs. ACSF group; +p < 0.05, agonist vs. antagonist/agonist group) Additionally, the number of BSI-B4-labeled ramified and activated microglial cells as well as GFAP and GFAP/BrdU double-labeled cells were counted under a light microscope (Axioskop; Zeiss) with a 20x objective within a square (0.5 × 0.5 mm) in region 1 (Fig. 1a). Each value represents five replications for each condition. The results were expressed as a percentage of ACSF-treated sides of the same regions. The individual groups were compared with one-way analysis of variance (ANOVA) using Bonferroni t test. A probability level of 0.05 or less was considered to be statistically significant. Results Immunocytochemistry After mechanical injury (Fig. 1a,b), a characteristic BSI-B4 immunolabeling around the stab wound was observed in comparison with untreated controls, decreasing in relation to the distance from the lesion. In untreated rats, a great number of resting (process-bearing) microglial cells was present (Fig. 1d, arrows), whereas after stab-wound injury, changes in the number of resting microglial cells with retracted processes (Fig. 1e, arrow) and activated microglial cells (Fig. 1f, arrowhead) were observed. Quantification of BSI-B4-immunolabeling in the NAc (region 1; Fig. 1c) of rats 4 days after stab-wound injury indicated a variable influence of P2 receptor agonists on the number of activated cells. Whereas α,βmeATP had no effect, 2MeSATP was more potent in tendency than was ADPβS in increasing the number of activated microglial cells. BzATP, which was injected into the NAc in a higher dose than were the previous ligands [28], also induced a marked enhancement of activated microglial cells (Fig. 1c). UTPγS was without effect, both on the number of activated microglia and on resting microglia (not shown). Quantification of BSI-B4-IR in the NAc (region 1 plus 2) by densitometry showed a significant increase for all agonists used with the exception of UTPγS (not shown) when compared with ACSF-treated animals. The effects of α,βmeATP, ADPβS, and 2MeSATP could be inhibited by pretreatment with PPADS and those of BzATP by pretreatment with BBG (Fig. 1g). PPADS and BBG given alone inhibited and failed to alter BSI-B4-IR, respectively. Double-immunofluorescence studies at P2X and P2Y receptor subtypes Multiple immunofluorescence labeling in combination with laser scanning microscopy was used to characterize the P2X/Y receptor subtype expression after injury in comparison with untreated controls. Under control conditions, no P2X or P2Y (with the exception of P2Y1) receptor-IR was coexpressed on single GSA-B4-positive cells. The mechanical injury associated with the introduction of the injection cannula resulted in the expression of P2X1,2,4,7 and P2Y1,2,4,6,12 receptors in the lesioned area, observed earliest 2 h until 4 days after injury. An up-regulation of immunolabeling of P2X1,2,4,7 receptor subtypes was unequivocal; examples are shown in Fig. 2 (a–i). In the affected area, P2Y1,2 and P2Y12 fluorescence labeling was observed on GSA-B4-positive cells with low intensity. P2Y4 labeling appeared preferentially on process-bearing microglial cells (not shown). P2X2-IR (Fig. 2c,d), P2X4 (Fig. 2e,f), and P2Y6-IR (Fig. 2l,m) were found on both resting and especially on activated microglial cells, the latter being identified by the lack of processes. No immunofluorescence was observed for P2X3,5,6 receptor subtypes on either type of microglial cells before and after stab wounding. Fig. 2Confocal images of examples of double immunofluorescence labeling to characterize the colocalization of P2X1,2,4,7 and P2Y1,6,12 receptor subtypes on a–f OX42- or g–o GSA-B4-labeled microglial cells in the NAc of rats 4 days after stab-wound injury (thin arrow process-bearing microglial cell; thick arrow activated microglial cell) (scale bars: a, b = 20 μm; c, d = 100 μm; e–i = 20 μm; j, k = 100 μm; l, m = 20 μm; n, o = 50 μm) Injury-induced prominent expression of the P2X7 receptor was observed on microglial cells by double labeling with GSA-B4 (Fig. 2g–i) and also on astrocytes, as described earlier [29]. The two different P2X7 receptor antibodies, one directed against the extracellular domain of the rat P2X7 receptor [25] and the other against its C terminus (C-terminal P2X7) (Alomone Labs) labeled the same cells (Fig. 3a,b; thin arrow) but also different cells (Fig. 3c,d; thick arrow). Additionally, the ecto-P2X7 antibody labeled GSA-positive cells (data not shown). Fig. 3Apoptosis or proliferation after P2X7 receptor stimulation after stab-wound injury in the NAc of rats 4 days after injury. a–d Colocalization of P2X7-IR detected by antibodies directed against the C-terminus (C-term) (b, d) and the ectodomain (a–d) of the rat P2X7. e–j Confocal images of double-labeling studies: Coexpression of active caspase 3 on single GSA-B4-positive cells (e, f) and active caspase 3 and the ecto-P2X7 receptor subtype (g, h). No double labeling of pAkT on GSA-B4-labeled microglial cells 4 days after injury (i, j) (scale bars: b–g = 20 μm). k Quantification of the number of active caspase 3-positive cells in region 1 (see Fig. 1a). Values are expressed as percentage of controls and represent the mean ± SEM of six animals per group. (*p < 0.05, vs. ACSF group; +p < 0.05,agonist vs. antagonist/agonist group; #p < 0.05, antagonist vs. antagonist/agonist group) Possible role of the P2X7 receptor after injury in vivo To find out whether the P2X7 receptor is related to apoptotic and/or proliferative processes after injury, we used the early apoptotic marker active caspase 3 and the proliferation marker BrdU. After BzATP microinjection, a significant increase in the number of active caspase-3-positive cells was found in comparison with ACSF-treated animals (Fig. 3k). This effect was reduced by pretreatment with the P2X7 receptor antagonist BBG. Further, BBG given alone decreased the number of active caspase 3-immunopositive cells in comparison with ACSF-treated rats (Fig. 3k). Double immunofluorescence-labeling showed colocalization of active caspase 3 at single GSA-B4-positive cells in the NAc of rats (Fig. 3e,f). Furthermore, colocalization of the ecto-P2X7 receptor subtype on caspase-3-positive cells was also documented (Fig. 3g,h). Additionally, a possible coexpression of the anti-apoptotic and possibly proliferation-promoting pAkt on GSA-B4-positive cells was studied 4 days after injury. However, no colocalization of pAkt with GSA-B4-positive cells (Fig. 3i,j) was found by immunofluorescence labeling. Eventually, no colocalization of P2X7 and pAkt was observed to this time point (data not shown). A possible influence of P2X7 receptor stimulation on the increase in cell number of activated and ramified microglial cells after BzATP stimulation was also studied. Changes in the number of activated microglia are shown in Fig. 1c. By quantification of ramified (resting) microglial cells in region 1 (Fig. 1a), an increase to 123.8 ± 5.3% in comparison with ACSF-treated rats was found, similar to that after treatment with 2MeSATP (124.0 ± 7.8%). For comparison, no characteristic changes were found after microinfusion of ADPβS (108.3 ± 11,6%) and α,βmeATP (101.7 ± 6.9%). Besides the effects of BzATP on the number of active and resting microglial cells, no influence of BzATP treatment on the number of GFAP (specific marker for fibrous astrocytes)-positive cells (BzATP 1.7 ± 7.3%; BBG 0.4 ± 3.0%; BzATP/BBG 0.96 ± 4.8%) was found in comparison with ACSF-treated rats. There was also no effect on the number of GFAP/BrdU double-labeled cells (proliferating cells) (BzATP 14.8 ± 4.5%; BBG 8.8 ± 11.2%; BzATP/BBG 15.9 ± 5.9%) in comparison with ACSF-treated rats. Discussion The results presented here demonstrate the involvement of various P2 receptors in microglial response to the placement of cannulas and microinjection, both resulting in tissue damage and thereby in high extracellular ATP concentrations [30]. Microglial cells stimulated by ATP may participate both in neurodegeneration (apoptosis and/or necrosis) and neuroprotection. Cultured microglial cells from mouse or rat brain were shown to respond to ATP with the activation of a cationic conductance, accompanied by an increase in cytosolic Ca2+ [8], followed by changes of microglial morphology [31, 32]. The exposition of cultured microglial cells to ADP or ATP further induced membrane ruffling and markedly enhanced chemokinesis, a prerequisite to perform tissue surveillance in the brain [33]. In vivo, microglial cells are homogenously distributed within the NAc characterized by rod-shaped somata with thin and highly ramified processes extended in all directions. Injury by stab wound caused microglial cell activation around the lesion track, characterized by swollen somata and thicker processes. The additional administration of P2X and P2Y receptor agonists enhanced the occurrence of activated microglial cells detected by their BSI-IR. The observed changes suggest that the stimulation of P2X1,3 receptors has only minor relevance in the process of microglia activation, whereas in agreement with the subsequent pharmacological investigations, involvement of P2X7 and P2Y1,2,4,6,12 receptors in this process is highly probable. Whereas P2X1–7 receptor-IR was absent in the nonaffected NAc, the expression of P2X1,2,3,4,7 receptors but not of P2X5,6 receptors was detected after the stab-wound lesion. P2X2,4,6 receptors are widely expressed in central and peripheral neurones as well as other excitable cells [22, 34, 35], and the P2X7 receptor is expressed in macrophage-type cells such as microglia but also mediates purinergic responses in astrocytes and neurons [36]. Changes in microglial expression of P2X receptors (P2X1–7) during postnatal development of the rat have been described [37]. Obviously, microglial cells are involved in long-term trophic events such as cell proliferation, differentiation, movement, and death during development [37]. Upregulation of P2X1-IR at microglial cells, as found in this study, was described at activated astrocytes after stab-wound injury in the adult rat NAc [29]. Further, the P2X2 receptor was found to be situated at activated microglia in our study and previously at astroglial cells [29]. After ischemia in vivo, P2X2 and P2X4-receptor-IR in the hippocampus became up-regulated in different cellular phenotypes [38]. P2X2 was expressed on neuronal cell bodies and fibers in hippocampal pyramidal cells, whereas intensive P2X4-IR was localized at microglial cells. Confocal microscopic analysis with OX 42 [39] at organotypic cultures showed that P2X4 and P2X7 are expressed on microglia, whereas P2X1 and P2Y1,2,12, although present in the slices, do not colocalize at microglia, and the P2X6 receptor is absent. P2X4 receptors seem to be functionally relevant for microglial chemotaxis, which was suppressed by the knockdown of the P2X4 receptor in cultured microglia by ribonucleic acid (RNA) interference through the lentivirus vector system [33]. After spinal-nerve injury, P2X4 receptor expression strikingly increased at the ipsilateral site in hyperactive microglia but not in neurons or astrocytes [40]. Intraspinal administration of P2X4 receptor antisense oligodeoxynucleotide decreased induction of P2X4 receptors and suppressed tactile allodynia after nerve injury. The major P2X receptor expressed at microglial cells is the P2X7 receptor, characterized in several reports in vitro [13, 16, 41]. This receptor subtype appeared to be strongly coexpressed with the microglial marker GSA-B4 after the mechanical lesion used in our study, suggesting an outstanding role of this receptor in injury-induced microglial responses. It is thought to be closely related to the immune functions of microglia, such as production of inflammatory cytokines, superoxide, and nitric oxide [42]. The P2X7 receptor is reported to be little susceptible to desensitization in the presence of ATP, which makes it quite different from other purinergic receptors, including the P2X4 receptor, which can be desensitized within a few minutes [23, 43]. Up-regulation of microglial P2X7 receptors has been observed in several pathological models, such as in vivo ischemia [41, 44, 45], as well as multiple sclerosis and amyotrophic lateral sclerosis of the human spinal cord [46]. The P2X7 receptors were also specifically up-regulated around β-amyloid plaques in a mouse model of Alzheimer’s disease (Tg2576) [47] and in human proliferative vitreoretinopathy on Müller glial cells [48]. Within the P2Y receptor family, in our study, the P2Y1 receptor was markedly expressed on microglial cells of untreated rats and was up-regulated in concert with P2Y2,4,6,12 receptors after injury. In the rabbit retina, microglial cells express functional P2Y1 receptors [49]. Activation of these receptors stimulates phenotypic alterations characteristically for microglial activation. Our data also demonstrate a strong colocalization of the P2Y6 receptor with GSA-IR. In addition to the microglial function to build a barrier between injured and healthy tissue, these cells are able to clear dead cells or dangerous debris from brain tissue. This phagocytotic process was shown to be triggered by activation of P2Y6 receptors by the endogenous agonist UDP [50]. Several authors report on ATP-induced migration of microglial cells to the affected sites of the brain [9, 10, 51–53]. Expression of the P2Y12 receptor on microglial cells after injury seems to be strongly related to the motility of this cell type. In primary cultured microglial cells, exposition to ADP or ATP induced membrane ruffling and markedly enhanced chemokinesis, which could be blocked by the P2Y12 receptor antagonist cangrelor (AR-C69931MX), suggesting that Gi/o-coupled P2Y receptors are involved [10]. The high dynamic of intact microglial processes as well as their chemotactic response to injury or local injection of ATP was shown in mouse cortex using time-lapse, two-photon laser imaging [9]. Investigations in P2Y12 receptor-deficient mice gave further support that P2Y12 receptors are required for the nucleotide-evoked chemotaxis [33, 54]. Eventually, expression of P2Y12 receptors was observed in native and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy, considered to be a crucial component in the regeneration cascades of motor neurons [55]. The published data confirm that not all known P2 receptors can be detected by their immunoreactivity. However, the mRNA of the P2X1–7 [29] and P2Y1,2,4,6,12 subtypes [27] was detectable in the NAc of untreated rats. The P2 receptors present on microglia may be differently involved in the processes of their activation, as shown by the inhibition of BSI-B4-IR using PPADS and BBG. It can be suggested by pharmacological studies that P2Y1 and P2X7 receptors may play a predominant role in the response of microglia to the consequences of a stab wound in the NAc. Retinal detachment and application of ADPßS onto control retinas induced phenotype alterations of microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density [49]. In the stab-wound model, stimulation of P2 receptors with agonists preferring P2Y1 and P2X7 receptor subtypes promoted the morphological changes of resting to activated microglial cells. P2X7 receptors are ATP-gated ion channels; their sustained activation leads to cytolysis in an apoptotic fashion in the microglia [56]. P2X7 receptor-mediated apoptosis involves activation of the proteolytic pathway of caspase activation, which leads to nuclear DNA damage [57]. In our study, immunocytochemistry showed expression of P2X7 and active caspase 3 at GSA-positive cells after injury without agonist application. The active caspase 3 has been defined as the main executioner of programmed cell death and as an early apoptotic marker [58]. The microinfusion of BzATP increased the number of caspase-3-positive cells, suggesting an involvement of P2X7 receptors in apoptotic changes after traumatic events. The BzATP-induced increase in ramified and activated microglia (and proliferating astrocytes) was only marginal. The phosphoinositide-3-kinase/serine-threonine-kinase Akt (PI3-K/Akt) pathway is associated with cell proliferation, promoting cell survival and inhibiting apoptosis [59]. Because in the present study performed at 4 days after injury no coexpression of pAkt with GSA and P2X7 was found, the evidence for a preferential apoptotic pathway triggered by P2X7 receptors is unlikely. Up-regulation of P2 receptors after mechanical injury might be both the cause and the result of the induction of apoptotic/necrotic cell death activating microglial cells with harmful or beneficial effects. The present results from in vivo investigations implicate that ATP via both P2X and P2Y receptor-stimulation acts as a signaling molecule in microglial cells in vivo. An up-regulation of P2X7-IR after injury suggests involvement of this receptor subtype in apoptotic rather than in proliferative events.

J_Mol_Med-3-1-2121654

Modulation of muscle contraction by a cell-permeable peptide

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells. Introduction Therapeutical delivery of protein or peptides into living primary cells is hampered by the lack of a suitable and efficient method for the introduction of macromolecules. Most commonly used transfections methods work in immortalized cells but mostly fail in primary cells or require specialized and time-consuming protocols, i.e., production of viral vectors. Moreover, the limited life span of primary cells often does not provide sufficient time for expression of gene products and subsequent analysis of their effects. Physical methods such as microinjection and bead loading are highly invasive procedures and are not tolerated by several cell types. It has been known for quite some time that basic proteins like histones or poly-ornithine as well as stretches of basic amino acids within proteins aid the uptake of proteins into mammalian cells [14, 17, 20, 30]. In the 1990s, transducible peptides like penetratin-1 from the homeodomain of Antennapedia [11] and amino acids 48–57 from the transactivator of transcription of human immunodeficiency virus (HIV)-1 [14] were identified and exploited to introduce drugs or biological macromolecules into mammalian cells. Together with other native and synthetic transducing peptides, they are referred to as cell-penetrating peptides (CPPs) [12]. However, it became evident that the invasive methods used to analyze the transduction mediated by CPPs or effects of interconnected cargoes had led to overestimated transduction results and overrated effects of cargoes fused to CPPs [27]. Nevertheless, CPPs mediate the introduction of fused cargoes into living cells, with cargo-dependent mechanistical differences. One uptake mechanism can be described as a slow adsorptive endocytosis and is preferentially used by globular proteins, whereas small compounds like peptides linked to CPPs favor a rapid membrane potential-dependent uptake with overall intracellular availability of the transduced species [33]. CPP-mediated peptide transduction has recently been used to deliver cardioprotective peptides derived from protein kinase C [4, 7–9, 24], the antiapoptotic BH4 peptide derived from the apoptosis regulator protein Bcl-xL [26, 28] and a Nox2-derived peptide [34] into isolated cardiomyocytes or whole hearts. We have tested the feasibility of this approach to study muscle function in living adult primary cardiomyocytes. Therefore, we targeted the interaction between actin and the essential myosin light chain (MLC-1) of cardiac type II myosin by a peptide competition approach. Transgenic overexpression of the N terminus of the human ventricular MLC-1 (residues 1–15; hereafter termed VLC-1), which binds to actin and targets actin/MLC-1 interaction, significantly increased the magnitude and kinetics of the contraction of isolated perfused hearts [21]. We fused the same human VLC-1 peptide to the TAT CPP (VLC-1-TAT) and investigated its uptake, intracellular distribution, and functional consequences in primary living adult rat cardiomyocytes. Materials and methods Isolation of adult rat cardiomyocytes Primary cultures of cardiomyocytes from male WKY rats aged 3 months were performed as described previously [1]. Peptides The peptides consisted of either l-amino acids (capital letters) or of d-amino acids (lower case letters). In case of d-amino acid peptides, functional motifs were synthesized as retro-all d-variants (underlined) to maintain their functionality. Peptides were either labeled with the fluorophore 5,6-carboxy-tetramethylrhodamine (TAMRA) or fluorescein isothiocyanate (FITC). TAT (TAMRA- or FITC-rrrqrrkkrg), PTD4 [23, 33] (TAMRA-araqraaaray), and TAT-p21 (TAMRA-rrrqrrkkrgaaAGRKRRQTSMTDFYHSKRRLIFSa-amide) [33] were labeled directly at their N termini with the indicated fluorophores (Peptide Speciality Laboratories, Heidelberg). In the case of VLC-1 (MAPKKPEPKKSSAKA-C-TAMRA-ME-amide) and VLC-1-TAT (MAPKKPEPKDDAKAPAGRKKRRQRRR-C-TAMRA-ME-amide; Biosyntan, Berlin) the TAMRA fluorophore was coupled via an additional cysteine at the C terminus leaving the N terminus free for the interaction with actin. Uptake of transducible peptides and controls To analyze the transduction ability of different compounds into living adult ventricular cardiomyocytes, the primary cells were plated into a laminin-coated μ-slide eight-well ibiTreat (ibidi, Martinsried) or four-well labtek (Nunc, Wiesbaden) microscopy observation chamber. The respective peptides and controls were added directly to Hank’s balanced salts solution buffered with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 7.4 (HBSS) or medium and gently shaken to yield a final concentration of 10 μM in case of the peptides and 5,6-TAMRA-fluorophore. Trypan blue (Sigma-Aldrich) was added to a final concentration of 0.5% (v/v) to the HBSS buffer. For the experiments summarized in Fig. 1 confocal images were acquired 1 h after addition of the respective compounds to living cardiomyocytes. To study the intracellular localization of transducible peptides in Fig. 3, after 15 min of peptide incubation, the medium was removed, and the cells were washed twice with the HBSS buffer. For subsequent confocal image collection, the cells were kept in medium or buffer. To study the effect of the peptide on muscle contraction and intracellular Ca2+ (Fig. 4), cardiomyocytes were electrically stimulated (see below) to permit the access of the peptides to all actin molecules with potential binding sites. During confocal image collection, the electric stimulation was temporarily switched off. Fig. 1Peptide uptake and intracellular distribution in living adult cardiomyocytes. To study the uptake capacity of different compounds into primary rat cardiomyocytes, different fluorescent compounds were directly added for 1 h to the medium or buffer of freshly isolated cardiomyocytes. After the incubation period, confocal images of the cells were taken without exchange of the medium and after removal of the fluorescent solution and exchange against the fresh medium. Internalization into healthy adult cardiomyocytes occurred after application of 10 μM of the transducible peptides FITC-TAT shown at low (a) and high magnification (b) and TAMRA-TAT-p21 shown at low (c) and high magnification (d) but not with application of 10 μM of a basic control peptide PTD4 (e). Moreover, 0.5% of the vital dye trypan blue (f) or 10 μM of the TAMRA-fluorophore (g) itself were not able to enter healthy cardiomyocytes (rod-shaped cells in culture). Scale bar, 10 μm. Stars indicate the nuclei and arrowheads the nucleoli (N) in the higher magnification images. PC Phase contrast, NP nucleoplasm Microscopy, image acquisition, and analysis Confocal images were acquired with a Zeiss laser scanning microscope LSM510 Meta mounted on an Axiovert 200M inverted microscope using a 63× phase-contrast oil immersion plan-apochromat objective NA1.4 or a 100× phase-contrast oil immersion plan-neofluar objective NA1.3. For all settings, the main beam splitter was HFT UV/488/543/633, and the specific parameters for the single fluorophores were: FITC, excited at 488 nm light, detected with a 500–530-nm bandpass filter; TAMRA or rhodamine excited at 543 nm, detected with 565–615 bandpass filter; and trypan blue, excited with 633 nm, detected with 650 longpass filter. Phase-contrast images were recorded with excitation at 488 nm and detection in the transmission channel. Laser power for observation was typically 1–5% (488 nm, 25 mW) and 50–60% (543 nm, 1 mW) unless otherwise indicated. Settings were adjusted in a way that image pixels were not over- or underexposed with the range indicator function in the Zeiss LSM image acquisition and examiner software version 3.2. Measurement of cardiomyocyte shortening and Ca2+ transients Attached cardiomyocytes were washed with HBSS. Cells were loaded with Fura-2-AM for 30 min at room temperature in the dark. The dye solution was removed, and cells were left on HBSS for another 15 min. Only cardiomyocytes of optically intact rod-shaped morphology with clear cross-striation were analyzed. Cardiomyocytes were electrically stimulated until a stable steady-state contraction and Fura-2 signal could be monitored. Cell shortening and Fura-2 signals were simultaneously measured at 30°C on an Ionoptix Contractility and Fluorescence System (Ionoptix). Cardiomyocytes were electrically stimulated with bipolar pulses of 5 ms duration at 1 Hz. Cell shortening, expressed as percentage of resting cell length, was measured using the video-edge technique at a sampling rate of 240 per second. Ca2+ transients were monitored as ratio of fluorescence emission at 510 nm was obtained by alternate excitation at 340 and 380 nm (340/380 ratio). Data files from 15 consecutive beats recorded at intervals were averaged for analysis. Subsequently, the cardiomyocytes were incubated with the peptides for 15 min without electrical stimulation. The peptide was then removed by replacing the peptide-containing HBSS buffer with normal HBSS buffer without the peptide. The cardiomyocytes were subsequently electrically stimulated, and both the shortening and Fura-2 signals were simultaneously recorded. The effect of the peptide on shortening amplitude and the Fura-2 signal was expressed in percent change compared to the steady-state signals obtained in the preincubation period. Results and discussion We recently demonstrated by live-cell confocal microscopy that the TAT CPP was able to shuttle fused peptides into primary blood cells and subsequently modulated their functional properties [10]. In this study, we tested whether TAT CPP could penetrate and if a peptide derived from the N terminus of VLC-1 fused to TAT is able to modulate the contractile function of primary adult cardiomyocytes. Different basic peptides were incubated with freshly prepared cultures of cardiomyocytes, and peptide internalization was analyzed live by laser-scanning confocal microscopy. The fluorescently labeled minimal transduction domain TAT CPP of the transactivator of transcription from HIV-1 was readily taken up into cardiomyocytes and displayed a strong affinity to the nuclear compartment (Fig. 1a). It could also be visualized in a punctate pattern throughout the entire cardiomyocyte (Fig. 1a). To determine whether the TAT CPP is able to shuttle an attached peptide into cardiomyocytes, we used the TAMRA-labeled TAT-p21WAF/CIP (TAT-p21) fusion peptide [33], which in addition to the transducing moiety contains 26 amino acids derived from the C terminus of human p21WAF/CIP protein. The uptake of this cargo peptide (assessed by appearance in nucleoli) occurred on a timescale of 5 to 10 min after peptide application. Its pattern of localization inside the cardiomyocytes was similar than that seen for TAT alone (Fig. 1c). Basic amino acid residues are a prerequisite for the transduction ability of CPPs [13, 25, 35], and peptides containing a minimum of six basic amino acids have been shown to traverse biological membranes [5, 18]. As a nontransducing control peptide, we chose the TAMRA-labeled peptide PTD4, which contains three arginines in total [23, 33]. More than 1 h after application of this peptide to the cardiomyocyte culture, none of the healthy intact cardiomyocytes took up the fluorescent peptide (Fig. 1e). Similar results were obtained for the vital dye trypan blue (Fig. 1f) and the TAMRA fluorophore (Fig. 1g). Approximately 5% of cardiomyocytes in the cultures were dead or harmed (visible in the phase-contrast images as not rod shaped and with no striations). These cells were flooded with both the CPPs as well as any other control dyes (Fig. 1c,e–g). The cell-permeable TAT-derived peptides displayed a similar enrichment in the nuclear compartment with strong accumulation inside nucleoli (arrowheads) as can be seen in the higher magnification images (Fig. 1b,d). This intranuclear distribution is basically determined by the TAT moiety, which includes a nuclear localization sequence [22, 29, 33]. Having shown that CPP TAT was able to shuttle fused peptides into living cardiomyocytes, we designed a transducible peptide comprising the actin-binding element of the ventricular isoform of human MLC-1 (VLC-1), i.e., the N-terminal residues 1–15 (VLC-1 peptide) connected to TAT, termed VLC-1-TAT (Fig. 2). The hearts of transgenic animals harboring minigenes encoding for the VLC-1 peptide revealed enhanced myosin motor activity and positive inotropic effects [21]. A possible molecular mechanism for the increase in myosin motor activity and contractility is illustrated in the scheme in Fig. 2. MLC-1 isoforms of vertebrate striated muscle play a regulatory role in myosin motor function: The N-terminal part of MLC-1 contains a sticky element (SE), which consists of several positively charged amino acids that bind to the C terminus of actin [3, 6, 16, 31]. The 46 N-terminal amino acids of MLC-1 have previously been modeled on the basis of contemporary crystallographic data [2]. Interactions between the sticky MLC-1 element and actin serve as a negative modulator and decrease the activity of the myosin motor domain. Intracellular delivery of a synthetic sticky MLC-1 element by TAT-mediated transduction therefore would antagonize MLC-1/actin interaction. We predict this would increase myosin motor activity and enhance the shortening amplitude without change of the systolic free Ca2+ activation level of intact cardiomyocytes. Fig. 2Molecular hypothesis for the enhancement of cardiomyocyte contractility by the cell-permeable VLC-1 peptide. a Primary sequence of VLC-1-TAT peptide color-coded orange for the VLC-1- and yellow for the TAT-moiety. b Schematic sliding filament model displaying actin in violet and the MHC in red. The higher magnification shows the catalytic domain (CD) and the neck domain (ND) of one molecule of the MHC. MLC proteins are colored green with the N-terminal SE inside the essential MLC-1 protein highlighted in red. Upon intramolecular conformational changes in the CD of MHC, the ND swings out, exposing the SE of MLC-1 to an actin molecule in direction to the Z-line. Treatment of cardiomyocytes with the transducible TAT peptide (yellow ball) fused to the SE-peptide (orange) blocks potential binding sites of the native MLC-1 N terminus. After the MHC rearrangement, the MLC-1 protein can no longer interact with actin and downregulate the myosin motor activity. The scheme is based on the pre- and postpower stroke model [19] developed with respect to crystallographic data on the Dicytostelium myosin motor domain [15, 32] To first test whether the VLC-1 peptide itself might be taken up by isolated cardiomyocytes because of its basic charge, VLC-1 and VLC-1-TAT were applied for 15 min to freshly isolated cardiomyocytes under resting conditions or, in another set of experiments, constant electric stimulation (1 Hz, 20 V). After the fluorescent peptides were washed away, the cells were analyzed live by confocal laser-scanning microscopy (Fig. 3a). VLC-1 alone was not internalized into healthy cardiomyocytes and stained only compromised cells (upper panel, Fig. 3b). To ensure that low concentrations of intracellular VLC-1 were not missed, the laser intensity was increased to its maximum, and still no fluorescent signals were measured (lower panel, Fig. 3b). In contrast, VLC-1-TAT reached the intracellular compartments of more than 95% of adult cardiomyocytes (Fig. 3c). Fig. 3Transduction and intracellular localization of VLC-1-TAT in isolated adult cardiomyocytes. a Scheme for the application of the VLC-1-derived peptides and confocal imaging. Peptides were applied at a concentration of 1 μM, incubated for 15 min to freshly isolated cardiomyocytes. After removal of the peptide solution, the cells were washed twice and kept in buffer or medium for direct live-cell confocal imaging microscopy. Subsequently, the transduced cardiomyocytes were electrically stimulated with 1 Hz for maximally 1 h. b The negative control peptide TAMRA-labeled VLC-1 stained only dead cardiomyocytes (low magnification, upper panel) but failed to get internalized into healthy cardiomyocytes even when 100% laser power was used to detect potentially weak signals (lower panel). c Application of TAT-VLC-1 to living cardiomyocytes led to rapid internalization of the peptide (upper panel). The lower panel shows transduced VLC-1-TAT at higher magnification in an overlay of the phase contrast and the TAMRA-fluorescence. The arrow indicates the direction of the intensity profile of gray and red intensities in the line scan for the localization analysis of VLC-1-TAT, which was present at sarcomeric structures with enrichment at the actin-containing I-bands (light bands in phase-contrast image) Unlike the localization of the TAT and TAT-p21 peptides (Fig. 1), the VLC-1-TAT was not detected in the cell nucleus. Detailed analysis of the intracellular distribution of transduced VLC-1-TAT and correlation of the fluorescent signals with the phase contrast images (merge image, lower panel, Fig. 3c) demonstrated that the peptide associates with sarcomeric structures. It showed strong enrichment at the I-bands representing the nonoverlapping zones of the thin actin filaments with the thick myosin filaments (linescan, lower panel, Fig. 3c). After transduction, the VLC-1-TAT peptide remained inside the sarcomeric compartments of the cardiomyocytes for at least 1 h after transferring the loaded cardiomyocytes into the peptide-free buffer solution. Using the same incubation protocol, we studied the effects of the cell-permeable VLC-1 peptide on muscle function by simultaneously monitoring the shortening amplitude and the systolic and diastolic cytoplasmic Ca2+ fluctuations (measured by Fura-2 fluorescence) of paced adult rat cardiomyocytes (Fig. 4a). As shown in Figs. 4b and c, 1 μM, but not 0.5 μM, of VLC-1-TAT significantly increased the shortening amplitude of adult cardiomyocyte, while the cytosolic Ca2+ signal remained unchanged. The same concentrations of control TAT peptide alone did not change the contractile state or the Fura-2 signals if compared to the basal state during the preincubation period. Higher concentrations of both control and test peptides induced deleterious effects on the cardiomyocyte contraction. It is interesting to note that not only the maximal amplitude of shortening rose but also the maximal rate of shortening and the maximal relaxation velocity increased significantly. Because the Fura-2 signal remained unchanged, the selective effect of the VLC-1-TAT peptide on the contraction parameters suggests a selective and direct effect on the function of the myosin cross-bridges. The Ca2+-sensitizing effect of the VLC-1-TAT peptide could be predicted from the mechanism of action, as the inhibition of the MLC-1/actin interaction may rise selectively myosin motor activity and therefore muscle contraction at a given activating free Ca2+ concentration, i.e., an inotropic effect without additional recruitment of Ca2+. It should be noted that the VLC-1-TAT peptide left the intracellular Ca2+-handling system unchanged, as seen by the fact that the kinetic parameters of the Fura-2 fluorescence signals remained normal (Fig. 4c). Fig. 4Functional effects of cell permeable VLC-1-TAT in living adult cardiomyocytes. a Original registration of cell length (top) and cytosolic Ca2+ signals (Fura-2 signal, expressed as ratio R between 340 and 380 excitation; bottom) of electrically paced adult rat cardiomyocytes before (basal) and after incubation with 1 μM VLC-1-TAT or 1 μM TAT as control. b Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1 μM) on fractional shortening (FS; given in percent of basal contraction obtained before incubation with the peptide), maximal rate of FS (+dFS/dtmax), and maximal relaxation rate (-dFS/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. c Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1 μM) on peak Fura-2 fluorescence signal (expressed as ratio R between 340 and 380 excitation, given in percent of basal R340/380 obtained before incubation with the peptide), maximal rate of R340/380 (+dR340/380/dtmax), and maximal rate of fluorescence decline (-dR340/380/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. Values are means ± SEM, n = 6–9 different cardiomyocytes at each peptide concentration used. Double asterisk, p < 0.01; triple asterisk, p < 0.001 (t test for paired values) In summary, we demonstrate by live-cell microscopy that CPPs and CPPs fused to peptides were taken up by adult cardiomyocytes with high efficiency and localize to their targets inside the cytoplasm and/or nucleus. The cell-permeable peptide VLC-1-TAT accumulated in the actin-containing I-band of the sarcomeres and was able to enhance the contractility of isolated adult cardiomyocytes without changing the myoplasmic Ca2+ levels. Importantly, the fact that VLC-1-TAT did not affect intracellular Ca2+ concentration and that it only has targets in striated muscle cells makes this peptide uniquely suited as a novel potential therapeutic tool to modulate heart function. VLC-1-TAT is a powerful new drug candidate to improve the contractile state of the failing heart.

Eur_J_Appl_Physiol-4-1-2226001

Identification of elderly fallers by muscle strength measures

For efficient prevention of falls among older adults, individuals at a high risk of falling need to be identified. In this study, we searched for muscle strength measures that best identified those individuals who would fall after a gait perturbation and those who recovered their balance. Seventeen healthy older adults performed a range of muscle strength tests. We measured maximum and rate of development of ankle plantar flexion moment, knee extension moment and whole leg push-off force, as well as maximum jump height and hand grip strength. Subsequently, their capacity to regain balance after tripping over an obstacle was determined experimentally. Seven of the participants were classified as fallers based on the tripping outcome. Maximum isometric push-off force in a leg press apparatus was the best measure to identify the fallers, as cross-validation of a discriminant model with this variable resulted in the best classification (86% sensitivity and 90% specificity). Jump height and hand grip strength were strongly correlated to leg press force (r = 0.82 and 0.59, respectively) and can also be used to identify fallers, although with slightly lower specificity. These results indicate that whole leg extension strength is associated with the ability to prevent a fall after a gait perturbation and might be used to identify the elderly at risk of falling. Introduction One-third of those over 65 years of age fall at least once per year. About 50% of them will suffer recurrent falls (Masud and Morris 2001). Even in the large group of relatively fit and healthy elderls, falls are common (Stel et al. 2003a). Consequences of falls are known to contribute substantially to the prevalence of health problems (Lord and Dayhew 2001), health care costs (Stevens et al. 2006), and to lost quality of life (Cumming et al. 2000). Prevention of falls is therefore imperative and should address the people at risk and the physiological causes of falling (Lord et al. 2003). Numerous epidemiological studies have shown that falls are associated with many risk factors (for overviews see e.g., Lord et al. 2001). Reduced muscle strength, especially of the lower limbs, has been indicated as one of the most important risk factors for falls (Lord et al. 2003; de Rekeneire et al. 2003; Moreland et al. 2004; Rao 2005; Rubenstein 2006; Skelton et al. 2002). Experimental studies can provide causal verification and insight into the contribution of muscle strength to fall incidence in standardized situations. For example, tripping over an obstacle, which accounts for up to 60% of falls (Berg et al. 1997; Roudsari et al. 2005), can be induced and measured in older adults in experimental settings (Pavol et al. 1999; Pijnappels et al. 2005c; Schillings et al. 2005). Previous findings provided insight into the (neuro)physical requirements of preventing a fall after tripping over an obstacle (for overviews see van Dieën et al. 2005; Pijnappels et al. 2007). It was shown that older adults were less able to successfully recover balance than younger adults, due to lower maximum ankle moments and lower rates of moment generation around all leg joints during the push-off phase of balance recovery (Pijnappels et al. 2005c). These results suggest that leg strength may be the limiting factor in preventing a fall. Pavol et al. (2002) have investigated the relation between muscle strength and the outcome of an induced trip. In a group of older adults, they compared isolated ankle, knee, and hip flexion and extension strength between those who recovered from a trip, and those who fell. Although some strong adults fell, presumably due to their high walking velocity, they also found that muscle strength was lower in several other fallers. If low muscle strength indeed is a physiological cause of falls, this raises the question whether people with a high risk of falling can be identified by means of relatively simple maximum muscle strength measures. The aim of this study was to find the best predictor of falls after a gait perturbation in a standardized situation, from a range of muscle strength measures in older adults. There are numerous maximum strength capacity measures that have been assessed in aged populations, for example to evaluate training interventions (Ferri et al. 2003; Reeves et al. 2005b; Verschueren et al. 2004) or to compare fallers with non-fallers (Gehlsen and Whaley 1990; Pavol et al. 2002). We focused on the most common measures by use of a dynamometer, isometric ankle and knee extension moment and rate of moment generation. In addition to isolated joint moments, total lower limb push-off force can be measured by a leg press dynamometer (Gehlsen and Whaley 1990). Performance on a maximum vertical jump could be a more functional test used to assess maximum lower limb strength (Izquierdo et al. 1999; Runge et al. 2004). Finally, hand grip strength is thought to reflect general body strength and has been used as predictor of falls in epidemiological studies (Pluijm et al. 2006). Elderly volunteers performed these maximum strength capacity tests and we measured their ability to prevent a fall after being tripped. We hypothesized that maximum muscle strength measures can be used to differentiate fallers from non-fallers. If so, this would not only allow identification of potential fallers, but also indicate specific limiting factors to target with exercise-based interventions. Methods Seventeen healthy older adults participated [10 women: age 71 (SD 4.5) years, mass 75 (SD 9) kg, height 1.68 (SD 0.09) m]. All participants were fit and had no orthopedic, neuromuscular, cardiac or visual problems. The Ethics Committees the VU Medical Center and of the Manchester Metropolitan University approved the procedure and all subjects gave their written, informed consent before participation. Capacity measures Participants performed a series of static and dynamic maximum force generating capacity tests. First, isometric ankle plantar flexion moment and knee extension moment (maximum as well as rate of moment development) were assessed using a dynamometer. In view of constraints on equipment and subject availability, nine of the participants were tested using an isokinetic dynamometer (Cybex Norm, New York, USA) (Reeves et al. 2004) and eight using a custom-built dynamometer (de Ruiter et al. 2004) (Fig. 1a, b). Isometric knee extension moment was measured in both settings with a knee joint angle of 90° (0° full extension) and a hip angle of 85° (0° supine position). Isometric plantar flexion moment was measured using the Cybex with the knee in full extension and the ankle at 0° (anatomical position). In the custom ankle dynamometer, the knee joint was fixed at 90° and to obtain a similar gastrocnemius muscle length compared to that during the tests using the Cybex, the ankle angle was set at 20° dorsiflexion (see equation in Reeves et al. 2005a, p. 2280). Plantar flexion moment of the right leg (push-off leg in the tripping experiments) and knee extension of the left leg (landing leg for tripping) were measured. Measurements resulted in comparable values between settings and were repeated 3 times with 3 min rest between contractions. Subjects first performed a maximum isometric contraction by increasing their effort in a linear ramp fashion, so that maximum moment was reached within 2 s and then maintained for 3–4 s. From these measurements, the maximum moments (anklemax and kneemax) were calculated, relative to body mass. Next, subjects were instructed to increase their moment from 0 to at least 70% of their maximum as quickly as possible, without counter movement. Moment onset (2 N m above baseline) was determined and rate of moment development (anklertd and kneertd) was calculated over the first 100 ms after moment onset and expressed relative to body mass. Fig. 1Pictures of the experimental settings for a right plantar flexion moment measurements, b left knee extension moment measurements, c leg press push-off force measurements, d jumping measurements, and e tripping experiments Maximum leg press push-off force of each lower limb was measured for all subjects using the same leg press fitness instrument (MR Systems, Haarlem, the Netherlands) (Fig. 1c). Subjects performed a one-legged (horizontal) push-off, starting with the ankle joint angle at 0° and the knee joint angle at 30°. This position was comparable to the configuration of the support limb during tripping (Pijnappels et al. 2005a, c). The fore-foot was positioned on a 10 × 10 cm force transducer (AMTI M3-1000, Watertown, USA) that was mounted in the push-off platform. Subjects first performed a maximum isometric contraction with the sled fixed and subsequently a maximum dynamic contraction with the weight stack set equal to body mass, so the only external resistance was the mass and inertia of the body and sled. Both conditions were repeated 3 times for both legs with a 3 min rest between contractions. The maximum horizontal leg press push-off force (legpressmax) was calculated from the isometric contractions and the rate of force development (legpressrfd) was calculated from the dynamic contractions over 100 ms after onset of force generation, averaged over both legs and expressed relative to body mass. For the jumping measurement, subjects performed a maximum two-legged counter movement jump (Fig. 1d). Position data of markers placed bilaterally on the trochanter major were measured by Optotrak cameras (Northern Digital, Waterloo, Canada). From the best of two attempts, maximum jump height (jumph) was calculated as the vertical distance between the highest averaged hip position during jumping and averaged hip height during normal standing. Hand grip strength was measured using a digital strain-gauge dynamometer (Takei TKK 5401, Takei Scientific Instruments, Tokyo, Japan). Subjects performed two maximum force trials with each hand. The maximum values of the right and left hand (in kg) were added together (handf). Tripping measurements and falls The ability to prevent a fall after a gait perturbation was determined by tripping measurements (Pijnappels et al. 2005c). Subjects walked at a self-selected velocity over a 12 m walkway. Fourteen 15 cm high obstacles were hidden at the left side and seven obstacles were hidden at the right side of the walkway, over a total distance of 1.5 m (Fig. 1e). In 2–5 of 40 trials, one of the obstacles suddenly appeared (based on online kinematic data) to catch the subject’s left swing leg at mid-swing, usually causing an elevating strategy for balance recovery. This strategy involves the limb contacting the obstacle being elevated over the obstacle (Eng et al. 1994; Schillings et al. 2000), whilst the contralateral support limb provides push-off. Subjects were aware that they could be tripped on either leg, but did not know whether and where an obstacle would appear in the ongoing trial. They wore a safety harness that moved along a track above the walkway. Subjects were classified as fallers based on visual detection of full body support by the harness (checked by video), in more than 50% of the tripping trials. Statistical analysis Pearson’s coefficients of correlation between the capacity measures were calculated. In case of high correlations between the dependent variables, as we found, the power of a multivariate analysis of variance (MANOVA) decreases when the expected effect sizes are reasonably large and consistent in the same direction (Cole et al. 1994). We therefore focused on separate univariate ANOVAs to determine which capacity measures differed between fallers and non-fallers. The variables that appeared to be determinants of falls from the ANOVAs were used in discriminant analyses, in order to find the variable(s) best discriminating between fallers and non-fallers and to determine sensitivity and specificity. In addition, a cross-validation was performed to assess the generalizability of the predictive models; each case was classified by the functions derived from all other cases (leave-one-out-classification). SPSS statistical software was used and the level of significance was set at p < 0.05. Results Tripping measurements and falls Seven of the participants (all women) were fully supported by the safety harness in more than half of the tripping trials. Six of them fell in all trials; the other fell in three out of five trials. Non-fallers were never fully supported by the safety harness. The averaged walking velocity was 1.38 (SD 0.17) m/s and did not differ between fallers and non-fallers (p = 0.488). Capacity measurement as predictor for falls legpressmax, jumph, and handf were significantly correlated with almost all other capacity measures (Table 1). Leg press push-off force was not significantly different between the left and right leg of the subjects (p = 0.496 for legpressmax and p = 0.149 for legpressrfd), there was no significant interaction with groups (p = 0.133 for legpressmax and p = 0.919 for legpressrfd), and the values were highly correlated between legs (overall, r = 0.813, p < 0.001 for legpressmax and r = 0.829, p < 0.001 for legpressrfd). Table 1Correlation coefficients between capacity measuresanklertdkneemaxkneertdlegpressmaxlegpressrfdjumphhandfanklemax0.63b0.230.320.76b0.370.61b0.40anklertd10.360.70b0.68a0.400.65b0.51akneemax10.72b0.53a−0.020.340.71bkneertd10.57a0.360.55a0.78blegpressmax10.330.82b0.59alegpressrfd10.290.23jumph10.69bSignificant correlations are in bold.ap < 0.05bp < 0.01 All but one of the univariate ANOVAs resulted in significant differences, indicating several measures to be different between fallers and non-fallers (Fig. 2). Ankle and knee capacity measures (both maximum and rate of moment development), maximum leg press force, jump height, and grip strength were significantly lower for fallers compared to non-fallers. Fig. 2Averaged group differences in capacity measures between fallers and non-fallers. Maximum (max) and rate of moment development (RTD) of plantar flexion moment (anklemax and anklertd) and knee extension moment (kneemax and kneertd), maximum and rate of force development (RFD) of leg press push-off force (legpressmax and legpressrfd), jump height (jumph), and hand grip strength (handf). Significant differences between groups are indicated with p-values These seven variables were entered in a stepwise discriminant analysis, which resulted in a significant model (p = 0.001) with legpressmax as the only predictor. This model resulted in 94% correctly classified cases and a cross-validation of 88% (Table 2). For comparison, discriminant analyses with the ankle and knee capacity tests (stepwise) and with jump height and grip strength as single predictors also resulted in significant predictive models, with slightly lower predictive values (Table 2). Figure 3 presents the distribution of individual jumph,, and handf values, both in relation to legpressmax. Table 2Predictive variables resulting from stepwise discriminant analyses and cross-validation on capacity measuresCapacity measuresPredictive variablepDiscriminant analysis (sensitivity/specificity%)Cross-validation (sensitivity/specificity%)legpressmax, anklemax, anklemax, kneemax, kneertd, jumph, handflegpressmax0.00186/10086/90aanklemax, anklertd, kneemax, kneertdanklemax, kneemax0.00771/9071/90jumphjumph0.00286/9086/80handfhandf0.00386/8086/80aBest predictive modelFig. 3Distribution of the individual values of maximum leg press push-off force (legpressmax) with jump height (jumph) and hand grip strength (handf) for fallers and non-fallers. Crosses indicate the incorrect classified cases (x for legpressmax, + for jumph and handf) Discussion It is important to identify individuals most at risk of falling, because they should be considered with priority for receiving targeted exercise interventions aimed at reducing the incidence of falls. This study investigated the possibility to identify fallers from maximum strength measures that could be applied in clinical settings. The results showed that participants who fell after a gait perturbation could indeed be identified based on these measures. Especially, a classification model based on maximum leg press push-off force yielded high sensitivity and specificity in cross-validation. The subjects in this study were relatively fit and healthy older adults. Nevertheless, muscle strength varied greatly between subjects, with coefficients of variation between subjects ranging from 23 to 60% for all strength measures. Pavol et al. (2002) found that not only people with low muscle strength, but also the stronger people had a higher risk of falling after a trip. This was explained by a higher walking velocity in the stronger group, which increases the demands for adequate balance recovery. These subjects mainly used a lowering strategy, which implies that they were tripped at late swing (Eng et al. 1994; Pijnappels et al. 2005b). Since the trip was induced manually, it may be that faster walkers were more likely to be tripped in late swing. It is therefore unclear whether velocity or instant of trip initiation was responsible for a number of stronger subjects falling. In our study, however, walking velocity was not different between fallers and non-fallers and in the range of “fast walkers” of the study of Pavol, even though we did not standardize it and explicitly instructed subjects to walk at their normal and comfortable velocity. Furthermore, we were able to trip all subjects at exactly mid-swing (when the toe height is minimal and a trip is most likely in daily life). This implies that the effect of the trip by impact with the obstacle was equal over subjects and could not affect the outcome of the trip. Yet, this also may suggest that instant of trip initiation and in a more heterogeneous group, differences in preferred walking velocity must be taken into account when attempting to identify fallers. Although we were able to trip our subjects repeatedly, three of the fallers and one non-faller did not complete the whole protocol up to five tripping trials, due to discomfort. Nevertheless, the fallers fell in all trials and the non-faller did not fall in four tripping trials. Hence, the number of tripping trials did not affect the classification of the participants. Hip extension and knee flexion strength were not measured, but might also contribute to fall prediction, as the rate of development of hip extension and knee flexion moments during push-off after tripping were found to be lower in older fallers compared to young adults (Pijnappels et al. 2005c). Since especially hip extension capacity is quite difficult to determine (especially in a clinical setting) and given limitations in the total number of capacity measurements to be performed in a single session, these variables were not included in the present study. During the push-off phase of a tripping reaction, the knee is generating an internal flexion moment while extention (Pijnappels et al. 2005a). Preliminary data suggest that a high extension moment is required in the leg making ground contact at landing (van Dieën et al. 2005) and a limited capacity to generate sufficiently high knee extension moments might thus increase the fall risk. In this study, fallers could not position their recovery foot adequately and were therefore fully supported by the safety harness, mostly even before landing. This underlines that most of the balance recovery takes place during push-off by the support limb. However, as falls can also occur after landing (Pavol et al. 2001), the requirements for landing need further investigation. Fallers and non-fallers were classified in this study based on their ability to prevent a fall after an experimentally induced gait perturbation. In daily life, there are many various ways in which people fall. Furthermore, this experimental study included a small number of participants, which might have influenced the predictive values. A prospective study with a larger cohort is necessary to generalize the results of this study to the prediction of falls in daily life. Overall, sufficient leg extension strength is required for adequate balance recovery after tripping. This might explain why a capacity measure that involves extension of all lower limb joints (i.e., leg press push-off) yielded the best identification of fallers. This one-legged task is highly similar to the actual movement during the push-off phase of tripping (Pijnappels et al. 2005a, c). Jumping also requires rapid moment generation and was therefore expected to be a good and practical measure of whole lower limb strength. Studies have shown that maximum jumping forces are lower in the elderly (Izquierdo et al. 1999; Runge et al. 2004) and related to balance performance (Izquierdo et al. 1999). Jump height indeed resulted in good sensitivity and specificity. This task was less similar to the actual tripping action than the leg press and does not solely reflect strength, as it also requires complex coordination of many joints. One might argue that the older fallers, who were all women, may have been more afraid to jump maximally than their counterparts. However, jump height was strongly correlated to maximum leg press push-off force, which was measured in a secure and comfortable setting. Jumping performance, therefore, more likely reflects lower limb strength than an effect of gender or fear. Fall incidents and the ensuing injury process are multi-factorial. Muscle strength is only one of many factors that associate with falls and that has to be taken into account when identifying one's fall risk. Yet, this particular factor has been indicated as one of the main causal factors for falls and is essential in risk profiles. For example, in the physiological profile approach by Lord et al. (2003) muscle force is tested in lower extremity muscle groups. However, these tests are not based on empirically demonstrated causal relations. Our experimental study allowed investigating the relation between strength measures and gait perturbations in a standardized situation. It can be questioned whether the findings in this study on tripping over an obstacle in mid-swing can be transferred to other fall mechanisms that occur in daily life. Yet, leg extension force has also been indicated to play an important role in other fall mechanics, such as slipping (Chambers and Cham 2007; Tang and Woollacott 1999). The results of this study may therefore contribute to improvement of risk profiles for identification of high-risk fallers, particularly by using relatively simple but specific measures of maximum strength. For clinical use, a maximum vertical jump test might be more practical than the leg press test, as it requires little instrumentation. Rittweger et al. (2004) demonstrated that such a test is feasible also for subjects over the age of 80 and that reliability is high. Hand grip strength is a common clinically used strength capacity measure. It has been shown in epidemiological studies to be related to leg extension strength and to be associated to recurrent falling in daily life (Stel et al. 2003b). Our data indeed showed that grip strength is significantly correlated with lower limb capacities and that it can be used to identify fallers from non-fallers. Although hand grip strength is easy to measure and assumed to be related to whole body strength, one should bear in mind that this measure may be less accurate as a predictor for falls it is not task specific for balance recovery. With respect to fall prevention, the associations found in this study suggest that maximum whole leg extension force is an important target in conducting exercise-based interventions. In a pilot study, we showed that lower limb resistance training has the potential to improve balance recovery, as older trainers improved more in moment generation upon tripping than controls (Pijnappels et al. 2007). It can be questioned whether fall prevention training should focus on strength training alone, as task-specific training of motor skills has been shown to be important (Bieryla et al. 2007; Owings et al. 2001; Troy and Grabiner 2005). A combination of both strength and balance training is most likely to be most beneficial for reduction of fall incidence (Gillespie et al. 2003). Finally, it should be noted that training effects are only valuable if task-specific requirements can be achieved and it can be questioned whether this is the case in older and more frail elderly. Therefore, resistance training may be particularly useful in the group of relatively fit and healthy elderly (Barry and Carson 2004; Hunter et al. 2004; Latham et al. 2004; Macaluso and De Vito 2004), which might prevent these people from becoming recurrent fallers. Conclusion Relatively simple and accessible measures of maximum strength did identify elderly fallers from non-fallers after a standardized gait perturbation. The capacity to generate maximum extension force by the whole leg (e.g., in a leg press apparatus or during jumping) resulted in the best classification of older fallers and non-fallers. Follow up studies on larger cohorts with a wide range of muscle strength and walking velocities are necessary to generalize these results towards a valid prediction of fall risk.

Eur_Spine_J-4-1-2226193

Clinical effect of continuous corrective force delivery in the non-operative treatment of idiopathic scoliosis: a prospective cohort study of the triac-brace

A prospective cohort study of skeletally immature idiopathic scoliotic patients treated with the TriaC brace. To determine if the TriaC brace is effective in preventing curve progression in immature adolescent idiopathic scoliotic patients with a very high risk of curve progression based on reported natural history data. The aim of the newly introduced TriaC brace is to reverse the pathologic transverse force pattern by externally applied and continuously present orthotic forces. In the frontal plane the force system used in the TriaC brace is similar to the force system of the conventional braces. However, in the sagittal plane the force system acts only on the thoracic region. In addition, the brace allows upper trunk flexibility without affecting the corrective forces during body motion. In a preliminary study it is demonstrated that the brace prevents further progression of both the Cobb angle and axial rotation in idiopathic scoliosis. Skeletally immature patients with idiopathic scoliosis with curves between 20 and 40° were studied prospectively. Skeletally immature was defined as a Risser sign 0 or 1 for both boys and girls, or pre-menarche or less than 1-year post-menarche for girls. Curves of less than 30° had to have documented progression before entry. The mean age of the patients at the start of treatment was 11.3 ± 3.1 years. All measurements were collected by a single observer, and all patients were followed up to skeletal maturity. Treatment was complete for all participants when they had reached Risser sign 4 and did not show any further growth at length measurements. This was at a mean age of 15.6 ± 1.1 years, with a mean follow-up of 1.6 years post bracing. In our study a successful outcome was obtained in 76% of patients treated with the TriaC brace. Comparing our data to literature data on natural history of a similar cohort shows that the TriaC brace significantly alters the predicted natural history. The current study demonstrates that treatment with the TriaC brace reduces the scoliosis, and that the achieved correction is maintained in some degree after skeletal maturity is reached and bracing is discontinued. It also prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity is concerned. Introduction The purpose of this paper is to present the results of a prospective study of a new orthotic device for the non-operative treatment of idiopathic scoliosis. Data are provided in a format to facilitate comparison with natural history data. The main concern in patients with idiopathic scoliosis relates to curve progression and the resulting cosmetic deformity. The risk of curve progression is correlated primarily to periods of rapid skeletal growth [3, 5, 6, 11, 14, 41, 42]. Factors related to growth potential, such as patient age at the time of diagnosis, status of menarche, and Risser sign, have been shown to be important predictors of the progression of scoliosis [3, 15, 29, 39]. In addition to future skeletal growth, curve magnitude and curve shape are further predictors of progression of idiopathic scoliosis. Large initial curves, thoracic curves, and double major curves are more likely to progress [3, 29, 39]. Currently bracing is the accepted nonoperative treatment to prevent curve progression in mild to moderate scoliosis during the growth period. A prospective multicenter study, performed by the Scoliosis Research Society, reported that brace treatment has a significant effect on curve progression of idiopathic scoliosis [36]. However, others doubt the effectiveness of braces [12, 16, 19, 32]. Just as for the natural history of untreated curves, Lonstein and Winter [30] found a relationship between the final outcome of brace treatment and curve factors and factors that predict future skeletal growth. In biomechanical theories describing the patho-physiological mechanism of scoliosis, spinal growth is thought to be the main driving force responsible for curve progression [35, 42, 44, 47, 48]. Throughout history, external devices have been used to correct deformities and immobilize the spine. Brace designs have changed periodically over the years, but most modifications have solely focused on improved efficacy and failed to acknowledge the importance, especially to teenagers, of physical appearance. This age group resists acting or looking different from their peers, which obviously occurs when a visible brace is worn. Modern materials, lower profiles, and reduced wearing times have been tried in attempts to reduce resistance to and the emotional difficulties encountered with wearing braces. The aim in the design of a new orthosis was to create a system which is at least as effective as the TLSO’s, with improved comfort for the wearer. The newly introduced brace should reverse the transverse force pattern by externally applied and constantly present orthotic forces without limiting the normal body motions of the patient and an increased comfort level for the patient. Construction and working principle of the TriaC orthosis The name TriaC is based on the three C’s of Comfort, Control, and Cosmetics. Currently, the orthosis is produced by Somas International, St Anthonis, the Netherlands. For this orthosis, the choice has been made to apply continuous correction forces on the chest with the aim to prevent curve progression during the growth period. A basic requirement for such a brace is that the brace force must be able to follow the main body motions of the patient. To achieve this goal required the use of a flexible coupling, connecting the thoracic and lumbar parts of the orthosis. The thoracic part and lumbar sections each generate their own corrective force on the body of the patient. Both parts are connected to each other by a flexible coupling that is placed on the opposite side of the thoracic force pad (Fig. 1). This coupling enables the patient to bend forward, backward and sideways while the correction forces are maintained (Fig. 2). The thoracic force pad is located just below the shoulder blade and applies a corrective force in a lateral–anterior direction. The lumbar force pad is placed between the pelvis and the lower ribs and acts on the lumbar muscles in a lateral direction. A third counter force on the hip region ensures that the orthosis is in equilibrium. Fig. 1The TriaC orthosis, with a thoracic part, a lumbar part and a flexible couplingFig. 2The flexible coupling Placing a flexible coupling between the thoracic and the lumbar part of the orthosis determines the unique force pattern on the patient both with respect to the location of the forces as with respect to the relation between the forces (Fig. 2). Modelling and clinical use have shown that the TriaC is suitable for all curve types, with the exception of curves with an apex at the 12th thoracic and the 1st lumbar vertebra. For these curve type the Lumbar force pad must be placed at the location of the flexible coupling which is not possible in the current construction of the orthosis. The construction as described, makes the orthosis force driven. When the patient moves her or his body in any direction (e.g. flexion, extension, rotation or lateral bending), the force pattern exerted by the orthosis moves along with the patient and thus is maintained constant. For this reason we defined the orthosis as “dynamic” because the orthosis moves dynamically along with the body motions of the patient. In contrast, conventional orthoses are displacement driven. A rigid shell shaped in the desired corrected position of the patient applies a corrective force only when the thorax of the patient presses against the pads of the brace. When the body of the patient moves away from these pads the rigid brace is not able to follow the body motion of the patient and therefore the brace no longer generates forces on the patient. Therefore, we defined these orthosis as “static”. Materials and methods From 1997 to 2005 all consecutive patients who met the inclusion criteria were treated nonoperatively with the TriaC orthosis and studied prospectively. The indication for treatment was a progressive curve with a Cobb angle between 25 and 40°. All curves, except curves already at >30°, had to show documented progression of at least 5°. Patients had to be skeletally immature, defined as a Risser sign 0 or 1 for both boys and girls; in addition, girls are pre-menarche or less than 1-year post-menarche. All the curves had to be flexible as demonstrated by at least a 40% correction on the bending radiograph at the first visit. Patients with the apex of the curve at T12 and L1 were excluded, as were patients with a systemic disease that could possibly influence the outcome of the study (Table 1). Table 1The in and exclusion criteria for the treatment with the TriaC orthosisInclusion criteria Idiopathic scoliosis with a Cobb-angle between 20 and 40° Skeletally immature  Risser 0–1 status   Pre-menarche   Post-menarche <1 year Primary thoracic apex between the 7th and 11th thoracic vertebra Primary lumbar apex between the 2nd and 5th lumbar vertebraFlexible spinal column as evidenced by at least 40% correction on bending filmsExclusion criteria Idiopathic scoliosis <20° and >40° Other types of scoliosis Skeletal age >Risser 1 Rigid curves Thoraco-lumbar curves with an apex at the 12th thoracic and the 1st lumbar vertebra Patients with a systemic disease which could influence the study parameters All patients who met the in- and exclusion criteria were treated with the TriaC orthosis. An informed consent form was obtained from all parents of the subjects in regard of the treatment with a new orthosis. According to the medical ethics committee no informed consent was necessary in case of the follow-up schedule because there is no difference with the patients treated with a Boston brace in our clinic. If during the treatment period the orthosis failed to stop progression and the curve increased significantly, the patient was placed into a Boston Brace. This procedure was chosen to get a first indication whether a Boston brace would be able to prevent curve progression in patients where the TriaC failed. Failure was defined as either a Cobb angle of >45°, i.e. the generally accepted indication for surgery, or if the curve progressed 5° or more compared with the measurement at study entry [16, 24–31]. During the nonoperative treatment patients were checked by an orthotist at the outpatient clinic every 4 months, where the subjective compliance was documented. At every visit a PA and lateral radiographs were made. The patients were allowed to wean of the braces at skeletally maturity, i.e. when the radiographs showed Risser sign 4 or, for girls, 2 years post-menarche and patients did not show any further growth at length measurements. At every visit standing and sitting height of the patient was recorded as an additional measurement, and these had to show no further increase. Because the TriaC is designed as a dynamic orthosis, it possibly preserves the muscle status of the patient. As a result the weaning process could be faster than with rigid Boston type orthoses. In this study, weaning was generally complete within 4 weeks, whereas for rigid braces this process may take up to one year. After terminating the treatment patients were seen once a year with an AP and lateral X-ray. Radiographic analysis All radiographs were obtained in a standing position. The posterior–anterior projection was used to minimize the radiation dose to the breast. All radiographs were made, using a standardized protocol. Anatomical vertebral landmarks are identified and manually marked on a 21 in. high resolution monitor using a pointer [46]. Following the placement of the markers the Cobb angle is calculated, using automatic detection of the apex and end vertebrae [8]. In this article, only the Cobb-angle data are used for comparison with literature data on the natural history. Results Out of a total of 212 patients that were treated at the University Hospital of Groningen during the study period, 63, or 30%, met all the in- and exclusion criteria and form the study group. There were seven patients (11%) with a single curve and 56 (89%) with a double curve. The group included six boys and 57 girls. The mean age of the patients at the start of treatment was 11.3 ± 3.1 years. The treatment was complete when the patients had reached Risser 4 with a mean age of 15.6 ± 1.1 years. The mean Cobb angle of the primary curves before the brace treatment was started was 30.2° ± 7.5°. The secondary curves had a mean Cobb angle of 22.3° ±  6.4°. The distribution of the location of the apex vertebra of the primary curves is shown in Fig. 3. Fig. 3The distribution of the apex level of the primary curves in the study group The patient group was divided into a success group and a failure group whereby failure was defined as a Cobb angle of >45° or curve progression of more than 5° compared to baseline [2, 7, 9, 18, 21, 33, 34, 36, 40]. The X-rays showed a mean initial correction of 22 ± 26% for the primary curves, and a mean correction of and 28 ± 35% for the secondary curves. The large variation was primarily caused by the big difference between the initial corrections seen in patients that were considered a success in comparison to patients that were considered a failure. In the success group the initial correction of the primary curve was 34 ± 17% whereas the initial correction in the failure group was −16 ± 17%. For the entire study group the success rate is 76% (48 successes) for a failure rate of 24% (15 failures). In the single curve group the mean Cobb angle at start of the treatment was 35°± 11. The mean initial correction achieved in this group was 23 ± 9%. There were no failures in this group. Although this difference is remarkable compared to the double curved group the single curve group is too small to draw conclusions. In Table 2 an overview of the success rate is shown, divided per initial Risser sub group (Risser 0 and Risser 1) and divided by the initial Cobb angle at start of the treatment. Table 2Number of successes/total patients in subgroupsCobb angleSuccess rateRisser 0Risser 120–29°76% (16 of 21 patients)30–39°74% (26 of 35 patients)86% (6 of 7 patients)Total75% (42 of 65 patients)86% (6 of 7 patients) The actual number of failures in our patient population was 15 out of 63 patients, or 24%. All patients in whom the brace failed went on to surgery, including the nine patients who were also treated with a Boston style brace after the TriaC brace had failed. A more detailed view of the correction of the primary Cobb angle is presented in Fig. 4. In this figure the mean correction percentage of the Cobb angle is shown as a function of the percentage of the total treatment brace period. The treatment period is considered to be at 0% at the start of the treatment and at 100% at the time the brace is discontinued. Fig. 4Percent correction of the Cobb angle over the entire TriaC treatment period in the success group It is shown that it was not possible to completely maintain the initial correction for the whole treatment period. At the end of treatment, the mean correction of the Cobb angle in the success patients had decreased to 19 ± 13%. This decrease in correction does not appear to follow a linear pattern. When a relation between the time that the patient wears the brace, and the loss of correction in the brace is assumed the correlation is only (very) weak (ρ = −0.33). The mean correction at the first visit after discontinuation of the brace the treatment was 20 ± 15%. All patients in whom the brace treatment was a success were followed with a mean follow-up of 1.6 years and longest follow-up of 6 years post-bracing. Figure 5 shows the mean correction at different follow-up periods postbracing. Fig. 5The mean correction during follow-up after termination of successful brace treatment Because the orthosis does not affect the lateral curvature no significant changes were expected. The analysis of the sagittal curve is displayed in Fig. 6. Fig. 6Lateral curve during treatment Discussion Braces are the oldest recorded method of treatment for spinal injury and deformity. The primary goal in treating patients with scoliosis deformities is to stabilize the curves to prevent further progression of the deformity. Closely related to this is the goal of achieving correction of the spinal deformity, although this is not part of the therapeutic regimen for every patient. Brace treatments do not generally correct the scoliosis, but prevent further progression, i.e. bracing has a “holding effect” [49]. In most published studies, the brace treatment has been considered a failure if the patient needed operative stabilisation or if the curve progressed 5° or more after the initiation of treatment [2, 7, 9, 18, 21, 30, 33, 34, 40]. However, these should not be the only criteria to determine whether brace treatment is successful. The treatment should also improve the patient’s outcome when compared with the expected natural history. The aim of this study was to investigate whether the TriaC-brace affects the natural history of idiopathic scoliosis. Bunnell [3], studied the natural history of idiopathic scoliosis in a group of patients with similar characteristics to our study group with respect to curve magnitude and age at the first visit. He showed that for the group as a whole 68% show a curve progression of 5° or more and only 34% of the patients showed a curve progression of 10° or more. The latter progression rate is in the same range as the failure rate reported in most of the retrospective studies, regarding overall brace effectiveness. However, the progression rates are not equal for every sub group of scoliotic patients. Lonstein and Carlson [29] reported that patients with Risser 0 or 1 maturity and curves larger than 20° are three times more likely to experience curve progression than those with curves smaller than 20°. For those with curves exceeding 20°, patients with Risser 0 or 1 maturity also are three times more likely to experience curve progression than are patients with Risser 2–5 maturity. They classified Risser 0–1 patients with curves between 20 and 29° as high risk (40–70%) and Risser 0–1 patients with curves between 29 and 39 as very high risk (70–90%). Bunnell [3] also found Risser sign and curve size to be good prognostic factors for curve progression in untreated idiopathic scoliosis. In his series patients with a Risser 0 at the time of diagnosis had a 68% risk of progression 10° or more. This risk was decreased to 52% in those who had a Risser sign of 1 or 2 and was further decreased to 18% for those with Risser 3 or 4. Nachemson et al. [36] reported that 66% of skeletally immature female patients with untreated idiopathic curves between 25° and 35° will experience curve progression greater than 5. All patients in our study started at Risser 0–1 maturity and had a curve above 30° or, if they had an initial curve below 30° had to show documented progression of ≥5° before enrolment in this study. Wever et al. [47] showed that there is a strong correlation between the curve progression and spinal growth of the patients. Usually the spinal growth is highest around the beginning of the menarche and therefore we only included female patients when they were before or less than 1 year after menarche. By using these inclusion criteria we aimed to include only patients at the highest risk for curve progression. In order to evaluate whether the TriaC brace alters the natural history of the high-risk patients, we estimated the natural progression rate of our patient group based on the literature as discussed. Our estimates are based on the calculation of a progression factor for all patients in the cohort as suggested by Lonstein and Carlson [29]. According to their algorithm, all our patients individually would have a change of 70–100% of progression of their curves. Based on these data we chose the low end of this range and assumed that in our patient group 70% of the curves would progress without treatment. The actual number of failures in our patient population was 15 out of 63 patients, or 24%, which is a significantly different from the expected number of 44 (P < 0.001). The validity of the sample size was evaluated by calculating the 95% confidence interval for the failure rate, which was between 11 and 36% (7–23 patients). Thus, the difference in outcomes remains statistically significant. These data show that the TriaC brace significantly altered the predicted natural history of curve progression. The comparison of the results of this study and other brace studies reported in the literature is difficult because of differences in sample size, skeletal maturity and curve magnitudes at the initiation of bracing. Furthermore, some reports excluded those patients who were not compliant with their bracing regimen. These large variations render the value of a statistical comparison of the results doubtful [51]. By applying the inclusion criteria used in this study to the literature data, a more uniform group has been created, making the statistical comparison more reliable. Nachemson et al. [36] prospectively compared 111 adolescent idiopathic scoliotic female patients with curves between 25° and 35°, who were treated with an underarm plastic brace, to 129 patients who were not treated. All patients were followed for 4 years. The patient’s thoracic or thoracolumbar curves were during this period evaluated for curve progression of more than 5°. Of the brace treated patients, 23 patients where lost to follow-up. Using survivorship analysis, a successful outcome was estimated in 74% of patients treated with a brace, compared to 34% of those who had no treatment; this difference was significant (P < 0.0001). In a retrospective study Lonstein and Winter [30] evaluated the result of brace treatment in a group of 1,020 patients over 35 years. From these patients a sub group of 177 had a curve between 20 and 29° and a Risser sign of 0–1. Using the natural history data of a similar group of non treated patients from their previous report [29] they where able to show in this sub-group a significant difference (P = 0.0001, chi-square test) of the failure rate between the group of treated patients and not treated patients. The not treated patients had a predicted failure rate of 68% and the failure rate of the braced patients in the sub group was 40%. We used the same report to predict the failure rate in our cohort. Although our results are promising, it is known that 5% of patients with a curve of less than 30° still progress to surgery during mature life [34]. We will follow our patients in whom the TriaC treatment effectively stopped curve progression before the surgical cut-off of 45° during mature life. Besides affecting the lateral curve, most traditional braces reduce lordosis and kyphosis by tilting the pelvis. The purpose of pelvic tilt is to move the lumbar spine closer to the correction pads within the brace. According to Lindh et al. [28] reducing the lumbar lordosis may automatically lead to a reduction of the scoliosis as a result of a coupling mechanism between sagittal and lateral motions of the vertebra [38]. However, reduction of lumbar lordosis will also reduce thoracic kyphosis. The reduction of thoracic kyphosis is an unwanted effect, as reduced thoracic kyphosis already is an integral component of the scoliotic deformity [50]. Schaal et al. therefore, emphasise the need for a system that diminishes the effect of pelvic tilt on the thoracic kyphosis [43]. In the Triac brace the choice has been made to continuously apply corrective forces with the aim of reversing the deforming forces, without affecting the lordosis of the spine [37]. Growth is a continuous process and therefore, the correction forces should be applied continuously as well, even during the normal body motions of the patient. In order to meet this requirement a flexible coupling, connecting the thoracic and lumbar parts of the brace is incorporated in the device. Most importantly, the forces exerted by the brace must be applied in such a way that they are maintained during all body motions of the patient. The new brace incorporates three separate functional elements: frame, springs and pelottes. The springs generate the orthotic forces, which are distributed by the frame and transmitted to the skin by the pelottes. The flexible coupling connects the thoracic and lumbar frame parts resulting in the application of constant forces that cannot be modified by the patients themselves (Fig. 7). The strength of the externally applied forces was chosen on the basis of literature data [10, 20, 27] Fig. 8. Fig. 7TriaC-braceFig. 8Pre-brace Cobb-angle: 30° and 19° apical axial rotation. In the brace Cobb-angle: 17° and 9° apical axial rotation, sagittal curvature unchanged The TriaC-brace provides a good primary correction of idiopathic scoliosis. There was a mean initial correction within the brace of 22 ± 26% for the primary curves and 28 ± 35% for the secondary curves, which is slightly less than reported in the literature [2, 30, 33, 34]. In the beginning of the study, we were not certain how patients would respond to the application of constant forces. Therefore, we started very carefully using relatively low forces. As we found that patients tolerated these forces well we subsequently increased the amount of force which resulted in improved initial corrections. According to the literature, achieved corrections gradually are lost following the termination of the brace treatment [7, 23, 30, 34, 49]. However, being efficacious is not all that is required of a brace; it should also be as comfortable as possible and cosmetically acceptable so that teenagers who require this treatment will actually use it. Non-acceptance of a brace by patients is a real and serious problem. Houghton et al. [24] placed a hidden transducer in their braces and found that actual compliance was considerably less than was reported by the patient; and only 20% of patients wore the brace as prescribed. Modern materials, lower profiles and reduced wearing times have all been tried to improve compliance and reduce the emotional difficulties experienced with brace wear. According to some authors, there is little difference in effect between part-time (12–16 h) and full-time (23 h) wearing of a brace [13, 21]. Kahanowitz [25] reported such findings but only if the pre-brace Cobb-angle were less than 35°; if larger than this more than 50% of curves would progress to such an extent that surgery became necessary. The current study demonstrates that the TriaC brace reduces the scoliosis, and the achieved correction is maintained during brace treatment. It prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity in the coronal plane is concerned. This is expected as the force system of the TriaC brace in the frontal plane is in accordance with the force system in the conventional braces. The new brace offers more comfort to the patient and a better cosmetic appearance. With TriaC brace there are no restrictions regarding daily or sporting activities, and it can be worn with all types of clothing. Summary Compared with published studies on the natural history of idiopathic scoliosis, use of the Triac brace appeared to significantly improve the course of curves between 20 and 40° in skeletally immature individuals. Control or net correction of idiopathic scoliotic curves was achieved in 76% of patients. The new dynamic brace offers more comfort to the patient and a better cosmetic appearance.

Eur_J_Nucl_Med_Mol_Imaging-4-1-2100432

Functional sex differences in human primary auditory cortex

Background We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex (PAC). Regional cerebral blood flow (rCBF) was measured in 10 male and 10 female volunteers while listening to sounds (music or white noise) and during a baseline (no auditory stimulation). Introduction Previously, investigators tried to relate sex differences in behaviour and cognition to differences in brain anatomy, function, or connectivity. A well-documented example of sex differences in the brain is the difference in overall brain size. We now know that the size of the male brain is on average 8–10% larger than the female brain [1–3]. It is also thought that, compared to the female brain, there is more hemispheric asymmetry in the anatomy of the male brain [4–7], which is supported by the finding that in the male brain, functions are represented more unilaterally [8–12]. Sexual dimorphism has also been reported for the anatomy and function of the auditory cortex. The human auditory cortex is located in the superior temporal lobe. The primary auditory cortex (PAC) is situated in the medial two-thirds of the transverse temporal gyrus, also called Heschl’s gyrus (Fig. 1). It is surrounded by secondary and associative auditory regions, which cover the lateral part of the transverse temporal gyrus and extend to the superior temporal plane. The primary auditory region was designated as area 41 by Brodmann [13], the secondary auditory regions are areas 42 and 22 [14]. An example of sexual dimorphism in the anatomy of the auditory system is in the study by Rademacher et al. [15] who reported that both left and right primary auditory cortices are larger in females than in males, although an earlier study found no differences [7]. Anatomical reports also show larger and more symmetrical auditory association cortices in females than in males [7, 16]. However, many studies emphasize that there is intersubject variability regarding the size and location of the primary and secondary auditory cortex [17–21]. Fig. 1Spatial distribution of significant increases in brain activation in men and women when comparing the auditory processing of noise, music, and a baseline. Activations are superimposed on an anatomical MRI template of SPM2. Clusters are significant at p < 0.05 FDR corrected for multiple comparisons. L=left hemisphere, y = −20 and y = −6 means a coronal plane, respectively, 20 mm and 6 mm posterior to the anterior commissure, z = 8 means a horizontal plane 8 mm dorsal to the anterior commissure, z  = −1 means a horizontal plane 1 mm ventral to the anterior commissure. a Contrasting music with noise, women showed activation in the secondary auditory areas only, whereas men showed activation in both PAC and secondary auditory areas. b Comparing music to the baseline, both women and men showed bilateral activation in the PAC and secondary auditory areas. c Comparing noise to the baseline, women showed bilateral activation in the PAC. In men, on the other hand, no significant activation was found. The differences between men and women in a and c are significant (see Table 2) Functional sex differences in auditory processing are widely studied in the light of language. Females depend less on their left hemisphere for language processing than males in some studies [22], whereas in other studies the opposite was found [23, 24] or no sex difference could be detected [25, 26]. Whether a sex difference in language processing can be detected might depend on the nature of the task [10]. Sex-based influence on activation patterns in auditory regions is also found when manipulating the amount of background noise [27], and auditory working memory tasks induce different activation patterns in males and females [28]. These studies suggest that combining men and women in auditory neuroimaging studies may obscure or bias results. However, most of these studies focus mainly on functional sex differences in higher order (associative) brain areas, thereby ignoring possible sex differences in primary auditory cortices. Previously, a sex difference in activation of the PAC during lipreading was found [29]. However, it has not been investigated whether nonspeech sounds can induce different activity in the PAC in both sexes. In the present study, we investigated potential sex differences in the activity of the primary auditory region, using different auditory stimuli. Material and methods Subjects Twenty healthy, right-handed volunteers participated in this study (10 males, 10 females). The mean age for men was 23 years (range 20–25) and for women 22 years (range 19–27). All volunteers gave written informed consent according to the Declaration of Helsinki and the Medical Ethics Committee of the University Medical Center Groningen approved the study. None of the subjects had any history of psychiatric or neurological disorders. Prior to the scanning, subjects were tested for their hearing thresholds using standard audiometric measures. All subjects had normal hearing thresholds (<20 dBHL, 0.25–8 kHz), and only small intersubject variations in hearing thresholds were observed. Data acquisition Regional cerebral blood flow (rCBF) was measured using radioactive water ([15O]-water, half-life 122 s) as a tracer. A Siemens Ecat Exact HR+PET scanner, operated in three-dimensional mode with a 15.5 cm axial field of view, acquired 63 slices simultaneously. Each subject was scanned 12 times to measure the distribution of [15O]-water with a 10 minute interval between two scans to allow for decay. Each scan was performed after an intravenous bolus injection of 500 MBq of [15O]-water per scan. Except for the first scan, scanning started 30 s prior to injection, to account for background activation. Scanning continued for 120 seconds. The activity measured during this period was summed and used as a measure of rCBF. A scan specific calculated attenuation correction was performed to minimize interscan displacement-induced variance [30]. All subjects were scanned at fixed times on fixed weekdays, and male and female subjects were addressed to the scan dates randomly. Experimental design Three conditions were used in this study: baseline (no auditory stimulation), white noise (at an intensity level of 75 dBSL), and music (music of the movie “The Piano” at 75 dBSL). Both music and noise have a wide frequency range, stimulating a large number of haircells in the cochlea and hence a large portion of the cortical auditory areas, but noise has a continuous and uniform frequency spectrum whereas music is a dynamic stimulus. Each condition was presented four times in a random order. For stimulus presentation we used a clinical audiometer (Interacoustics, model AC30), a Tandberg Educational tape recorder, and E.A.RTone 3A insert phones (with E.A.Rlink eartips), which have a flat frequency response between 100 and 4000 Hz, measured in a Zwislocki-coupler. Stimuli were presented binaurally. Ten seconds before injection of radioactive water, the stimulus was started. Because the tracer reaches the brain approximately 10 seconds after injection, subjects were exposed to the stimulus for 20 seconds before the distribution of [15O]-water in the brain starts. Subjects were instructed to close their eyes, not to move during the scans, and to listen to the auditory stimuli. Before each scan we informed the volunteers that the scan was about to start. Immediately after each scan the volunteers were questioned about the scan (did they hear the stimulus? were they uncomfortable or distracted?). During scanning we monitored the subjects with infrared cameras. Data analysis The 2002 version of Statistical Parametric Mapping (SPM2: software from the Wellcome Department of Cognitive Neurology, London, UK) was used for spatial transformations (realignment, transformation into standard stereotactic space, and smoothing with an isotropic Gaussian kernel of 8 mm FWHM), and statistical analysis [31]. An ANOVA estimated the following parameters: two groups (male and female), three conditions (baseline, noise, and music) and the mean perfusion to normalize for global flow differences (multigroup, conditions, and covariates). Each scan was scaled to a mean global activity of 50 ml/100 ml/min. Hypotheses about regionally specific condition effects were tested to compare the estimates by using linear compounds or contrasts. The resulting set of voxel values for these contrasts constituted the associated SPM of the t-statistics. The significance threshold used for the analysis of the two groups separately (male–female) was p < 0.05 (false discovery rate (FDR) corrected for multiple comparisons [32]) with an extent cluster threshold of more than 8 voxels. We used AMIDE software (http://amide.sourceforge.net/) for colour scaling and display of the results on the anatomical MRI template of SPM2. For maximum statistical sensitivity and for testing the significance of the sex related differences, we conducted a region of interest (ROI) analysis in our a priori hypothesized areas, i.e. the left and right PAC, using the SPM anatomy toolbox [33] and the MarsBaR toolbox (MARSeille Boîte À Région d’intérêt [34]). Specifically, we created anatomical ROIs based on the three-dimensional probabilistic cytoarchitectonic maps from the SPM anatomy toolbox brains [18, 20, 33]. To compensate for differences in stereotactic space between SPM and the anatomy toolbox, a linear transformation was applied to the anatomical ROIs. MarsBaR was then used to conduct the statistical analyses on these ROIs. The statistical procedure in MarsBaR is the same as in SPM, but instead of analysing on a voxel-by-voxel basis like SPM does, all voxels in a region are averaged, and hence inferences about the whole region can be made. Also in MarsBaR, contrasts were considered significant at p < 0.05. Results Music versus noise For this contrast, in females the SPM-analysis resulted in significant bilateral activation clusters with a maximal significant voxel in the secondary auditory areas. In men much larger bilateral clusters were found covering not only the secondary auditory areas but also the PAC (Fig. 1a and Table 1). This indicates that males have a much larger activation in the PAC during music than during noise. The voxel-wise analysis of SPM did not reveal any significant activation differences between the two sexes. Table 1Overview of brain areas with statistically significant cerebral blood flow changes  SideRegionTalairach coordinatesNumber of voxelst-valuexyzMusic vs noiseFemalesRightBA 2261−6−11,4597.77LeftBA 22−51−10−13105.02MalesRightBA 41, 42, 22522−33,2347.87LeftBA 41, 42, 22−51−802,1907.87Music vs baselineFemalesRightBA 41, 42, 2257−4−12,7508.85LeftBA 41, 42, 22−48−1731,8667.97MalesRightBA 41, 42, 2251−1022,47510.36LeftBA 41, 42, 22−48−1212,3068.67Noise vs baselineFemalesRightBA 4146−2154466.49LeftBA 41−40−2354636.24MalesNo suprathreshold clustersBaseline vs noiseFemalesNo suprathreshold clustersMalesRightBA 92050299685.20Baseline vs musicMalesNo suprathreshold clustersFemalesNo suprathreshold clustersThe region, covered by the whole cluster and noted in Brodmann areas, and the number of voxels in the cluster are described [14]. BA 41 corresponds to the PAC, BA 22 and 42 correspond to secondary auditory areas. Only the stereotaxic (Talairach) coordinates and t-value of the maximum of the cluster are given. The significance threshold was p  < 0.05 FDR-corrected for multiple comparisons; extent threshold was 8 voxels; voxel size was 2 × 2 × 2 mm. Brain regions were identified using the Talairach atlas and the stereotactic atlas of the human brain of Mai et al. [52, 53] The region of interest analysis in the PAC showed that females do have a larger activation in the PAC during music than during noise (p values 0.005 and 0.001 for the left and right PAC, respectively). But the difference between music and noise is much smaller than in males in both the left and right PAC (p values 0.016 and 0.008, respectively, Table 2, Fig. 2). No significant deactivations were found (i.e. noise versus music). Table 2Region of interest analysis of the left and right primary auditory cortex (PAC) P-valueLeft PACRight PACMusic vs noise females0.005*0.001* Males<0.001*<0.001* Females minus males0.9840.992 Males minus females0.016*0.008*Music vs baseline females<0.001*<0.001* Males<0.001*<0.001* Females minus males0.7210.779 Males minus females0.2790.221Noise vs baseline females<0.001*<0.001* Males0.016*0.005* Females minus males0.042*0.034* Males minus females0.9580.966One-sided statistical significance for various contrasts was tested for men and women separately as well as for the differences between both. *Significant at p < 0.05Fig. 2Regional cerebral blood flow relative to the baseline of each group, based on all voxels in the left and right PAC (with a global mean flow of 50 ml/100 g/min). Error bars indicate the 90% confidence interval of the mean across subjects per condition; the confidence interval of the baseline is also given To determine whether this sex difference can be attributed to the processing of either music or noise, we compared these two stimuli with a baseline without experimental auditory stimulation. Music versus baseline For this contrast, the SPM-analysis showed large comparable activation clusters covering primary and secondary areas in both males and females (Fig. 1b and Table 1). The ROI analysis showed that there were no significant differences in the activation of the PAC between both sexes for this contrast (Table 2, Fig. 2). Again, no significant deactivations were found (baseline versus music). Noise versus baseline Comparing noise to the baseline, the SPM-analysis showed two significant bilateral activation clusters with maxima in the PAC for the female group. In contrast, no significant activations were found in the male brain at a corrected level of p < 0.05 (Fig. 1c and Table 1). Only when the data were analysed at an uncorrected threshold of p < 0.01, a small activation appeared in the PAC of males, primarily on the right side (Fig. 3). The ROI-analysis confirmed in men the involvement of the PAC while processing noise, showing significant bilateral increases of blood flow, but these activations were significantly less than in women in the left (0.042) and right PAC (p = 0.034) (Table 2). Fig. 3Spatial distribution of significant increases in brain activation in men and women for the comparison of noise to the baseline. Clusters are significant at p < 0.01, uncorrected for multiple comparisons. L=left hemisphere, y = −20 means a coronal plane 20 mm posterior to the anterior commissure, z = 8 means a horizontal plane 8 mm dorsal to the anterior commissure. In contrast to Fig. 1c, at an uncorrected level men do show activation in PAC, but it is much smaller than in the female group The voxel-wise analysis of SPM also revealed a significant deactivation in the male group. This deactivation was located in the right dorsolateral part of the prefrontal cortex extending to the posterior part of the middle frontal gyrus, covering primarily BA 9 (Fig. 4 and Table 1). In contrast, no significant deactivation was found in the female group. Fig. 4Spatial distribution of significant decreases in brain activation in men and women for the comparison of noise to the baseline. Clusters are significant at p < 0.05, FDR corrected for multiple comparisons. x = 20 means a sagittal plane 20 mm on the right of the anterior commissure, z = 29 means a horizontal plane 29 mm dorsal to the anterior commissure. Only men showed a significant deactivation in the dorsolateral prefrontal cortex Discussion Our data demonstrate a sex difference in regional cerebral blood flow in the left and right primary auditory cortex (PAC) when comparing auditory processing of music and noise. The PAC was more activated by music than by noise in both men and women. But this difference between the two stimuli was significantly higher in men than in women. To determine whether this sex difference can be attributed to the processing of music, noise, or both, we compared the two stimuli with a baseline of no auditory stimulation. Comparing music with the baseline resulted in extensive activation of the primary and secondary auditory cortex in both sexes, but no significant sex difference was found. On the other hand, a sex difference was detected in the processing of noise, because females activated their PAC significantly more than males. The finding that sex differences in auditory processing can already be detected at the level of the primary cortex is very important, because it is often assumed that sex differences act exclusively upon higher-order cortical areas. There appears to be a mismatch between the SPM and ROI analyses because there is a difference in power between the two analysing methods. The ROI analysis pools the data of all voxels in the PAC and gives a reduced standard error, resulting in a higher t-value and hence more power [34]. Although the SPM analysis showed no activation at all for the male group and a very significant activation for the female group, it did not have enough power to detect a significant difference between the two groups. Because of the enhanced sensitivity of an ROI analysis, this analysis did show a significant sex difference. Of course, caution has to be made when pooling data to improve the sensitivity and is therefore only allowed in a priori defined regions, in this case the PAC. The question is whether the sex difference found is caused by the experimental conditions or by other factors like anatomical differences or methodological errors. The reported differences in activation of the PAC stand or fall by a correct identification of this area in the region of interest analysis. Several authors reported substantial intersubject variability in size and location of the PAC [17–21]. To overcome this problem of variability in size and location of the PAC, we employed linear resizing and shearing as well as nonlinear warping of the brain to normalize the individual brains into a standardized stereotactic frame. In addition, a Gaussian smoothing filter of 8 mm was used to remove residual variance in brain structure that remains after the stereotactic normalization. Furthermore, we used probability maps, based on the cytoarchitecture of 10 subjects, to identify the PAC [18, 20, 33]. Other studies suggested that the volume of the PAC is bilaterally larger in females than in males [15]. It is therefore necessary that the region of interest depicts the PAC of both men and women. This is the case, because the probability maps of the SPM anatomy toolbox are based on the cytoarchitecture of five male and five female brains [18, 20, 33]. For these reasons we believe that possible intersubject or intergroup differences in size and location of the PAC do not bias our results. If the reported sex and contrast dependent differences in rCBF patterns are not caused by underlying anatomical differences, metabolic differences, or methodological errors, they must have been induced by the experimental stimulus. During the baseline condition, subjects had to lie quietly in the scanner and no auditory stimulus was applied. Without auditory stimulation and a specific task, the variance in rCBF might increase, resulting in less statistical power when comparing conditions to the baseline. However, as shown in Fig. 2, the confidence interval for the model parameters was very similar for the three conditions. In addition, the confidence interval was also very similar for the two sexes. This means that the different contrasts tested have similar statistical power. Considering the significant sexual dichotomy in the processing of music versus noise and the results when comparing either the music or noise with the baseline condition, we conclude that males and females differ in the processing of noise. Thus our data demonstrate a sexual dichotomy in auditory processing. But which mechanism could explain this sexual dichotomy? A key to answering this question is the role of the prefrontal cortex, which we found to be deactivated in men when listening to noise. In humans, the prefrontal cortex is engaged in diverse cognitive processes including cognitive control, working memory, and attention [35]. For example, Gisselgård et al. [36, 37] investigated the influence of irrelevant speech on working memory tasks and revealed a functional link between auditory and prefrontal regions. Tzourio et al. demonstrated that prefrontal areas are engaged in auditory tasks that involve sustained or selective auditory attention [38]. In the present study, no explicit (attention) task was implemented. Subjects lay passively in the scanner and were instructed to listen to the auditory stimuli. While listening to an insignificant stimulus like noise, males deactivated the prefrontal attention areas as compared to silence. Females, on the other hand, had no deactivation of the attention areas and had a higher activation in the primary auditory cortex. Deactivation of the prefrontal regions was only seen in the right hemisphere, which is consistent with Tzourio et al. who stated that a right hemisphere dominance exists for attention [38]. The present results suggest a relationship between activation of PAC and prefrontal cortex. From literature, anatomical evidence exists concerning auditory–prefrontal connections. Studies on monkeys have shown that the prefrontal cortex is reciprocally connected with auditory association areas (i.e. belt and parabelt in the monkey brain) [39]. The secondary and primary auditory areas are reciprocally connected (c.f. [40]). More recent studies on monkeys identified two auditory–prefrontal processing streams: dorso and ventrolateral auditory streams [41–43]. Although, one should be cautious when comparing human and nonhuman primate brains, a similar organization of several parts of the human and monkey prefrontal cortex has been reported [44, 45]. To summarize, we know from literature that the auditory and prefrontal regions are anatomically and functionally linked, and our data show a sexual dichotomy in the (de)activation of both regions. Apparently, the male and female brains handle an insignificant stimulus like noise differently, and we speculate that this is done by a different engagement of the auditory–prefrontal attention network. Namely, differences in attention result in a different deactivation of the right prefrontal cortex, which in turn modulates the activation of the PAC and thus explains the sex differences found in the activation of the PAC. This corresponds with previous findings that sex differences exist in the frontal–temporal network, namely, males have higher intrahemispheric functional connectivity of frontal and temporal areas than females [46]. It is also known from ERP studies that sex differences exist in orienting attention to auditory stimuli [47]. It must be noted that even though our data indicate differences in the auditory system, no independent behavioural data regarding the attention levels during scanning are available. Hence, the present experimental design only allows us to speculate about the correlation between the different deactivation of the prefrontal cortex and differences in attention. Further research is needed to fully clarify the role of attention on PAC activation and to determine whether our results can be repeated. Furthermore, other auditory conditions like pure tones, noise bursts, and speech (sense and nonsense) sounds are needed to fully map the influence of sex on central auditory processing and to investigate lateralization differences between the sexes. To our knowledge, this is the first time that a sexual dichotomy in the function of the PAC is demonstrated using nonspeech sounds. Previously, a sex difference in the PAC was demonstrated while lip reading [29, 48]. These studies showed only female activation in the PAC during lip reading because they associate the absent speech sound with the visual lip movements. Males on the other hand, did not display activation in the PAC and focus on the present visual lip image itself. The current study shows that even simple sounds induce different activation patterns, especially in the PAC. Other sexual dichotomies in the central auditory system have been reported. For example, sex differences in auditory feedback loops of the corticofugal network have been found in which men showed more suppression of repeated acoustic stimuli than females. This sex difference in auditory gating is probably the result of differential neuronal inhibition to repeated stimulation [49]. Likewise, the stronger spontaneous otoacoustic emissions (SOAEs) in females are thought to originate from a relatively larger amount of efferent inhibition in males [50]. This efferent innervation would start in the olivary complex and terminate in the outer hair cells of the cochlea [50]. Although the present sexual dichotomy would be consistent with the concept of evolutionary advantages in a hunter–gatherer society (c.f. [51]) where the inhibition of constant irrelevant stimuli in men may facilitate them to focus their attention to a single task, e.g. hunting, this concept remains speculation. In conclusion, a very significant sexual dichotomy was found in the activation of the PAC with different types of acoustic stimuli (noise and music) together with sex differences in deactivation of prefrontal areas. It is known that the auditory and prefrontal regions are anatomically and functionally linked, and the prefrontal cortex is engaged in auditory attention tasks. Hence, we hypothesize that differences in attention might result in a different involvement of the right prefrontal cortex, which in turn modulates the activation of the PAC. This shows that sex influences brain activity already at the level of primary sensory cortex and that in functional imaging studies on primary sensory cortical areas, sex cannot be ignored.

Purinergic_Signal-4-2-2377316

GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal. Introduction Chemorepellents are molecules that inhibit or repel the migration of individual cells. In protozoans, chemorepellents are hypothesized to serve a defense mechanism, helping the organism to avoid predation and environmental hazards. For example, a damaged Tetrahymena thermophila cell may leak cytosolic purines such as adenosine triphosphate (ATP) or guanosine 5'-triphosphate (GTP) into the surrounding environment, causing other cells to avoid the area, thereby avoiding the source of damage to the original cell and increasing the survival rate of the species [1, 2]. It is possible that cytosolic purines may be secreted by these organisms as a means of communication between cells (analogous to paracrine secretion) or in order to trigger second-messenger pathways within the cell doing the secretion (analogous to autocrine secretion). At this time, secretion of purine nucleotide by Tetrahymena has not been documented, although Tetrahymena have been shown to secrete a number of enzymes and peptides. In T. thermophila, extracellular GTP is a known chemorepellent [1, 3, 4]. GTP causes depolarization in the somatic membrane [2], which has been proposed to trigger opening of the voltage-dependent calcium channels in the ciliary membrane [2]. The increase in Ca2+ concentration causes the cilia to reverse their direction [1], resulting in a disruption of normal swimming. This “avoidance reaction” [1] is marked by jerky movements, backward swimming, or spinning. This chemokinetic response involves a series of avoidance reactions, resulting in a “biased random walk,” which orients the organism away from the source of the repellent [3]. Avoidance to chemorepellents may be quantified by observing the response of single cells exposed to repellent using a dissection microscope. T. thermophila avoid micromolar concentrations of GTP [1]. An in vivo binding assay using [32P] GTP showed the KD for GTP binding to intact T. thermophila is 21 ± 2.5 nM. GTP binds to its receptor with high affinity, and binding is saturable as well as reversible [1], all of which are characteristic of membrane receptors. Fluorescent staining with 2′-(or 3′)-O-(2,4,6-trinitrophenyl) (TNP)-GTP has shown GTP binding on the cilia and cell surface of T. thermophila [4]. However, the second messenger pathways involved in GTP signaling have not been previously described. Recently, Iwamoto and Nakaoka [4] found that addition of extracellular GTP to the medium induces cell division in T. thermophila, a result consistent with a tyrosine kinase signaling mechanism, as tyrosine kinases are involved in many major mitotic pathways. Tyrosine phosphorylation has previously been shown to modulate the interaction of calmodulin with target proteins, including protein phosphatase 2B (calcineurin) and neuronal nitric oxide synthase (nNOS) in an in vitro assay [5]. Tyrosine kinase activity has also been shown to activate phospholipase C, NOS (via protein kinase C), and guanylyl cyclase in the rat ileum [6]. Tyrosine kinases are frequently associated with activation of phospholipase C-γ, and with release of calcium from internal stores via the IP3 pathway [7]. In our current study, we examined the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Our data confirm this hypothesis. In addition to tyrosine kinase signaling, we found that phospholipase C, intracellular calcium stores, NOS, and guanylyl cyclase are necessary for behavioral avoidance to occur. All of these second messengers are consistent with the existence of a tyrosine kinase signaling pathway in T. thermophila. Materials and methods Cell cultures T. thermophila, strain B2086, a generous gift from T.M. Hennessey (SUNY-Buffalo) were used for all of the experiments. Cells were grown at 25°C in the axenic medium of Dentler [8] without shaking. Two-day-old cell cultures were used for all assays described below. Chemicals and solutions Behavioral assays were carried out in a buffer of pH 7.0 containing 10 mM Trizma base, 0.5 mM MOPS, and 50 μM CaCl2. All repellents and inhibitors used were dissolved in this buffer. Compounds that were insoluble in aqueous solutions were first dissolved in a small quantity of dimethylsulfoxide (DMSO) and then diluted 1:1,000 or more into the buffer and vortexed vigorously. The chemorepellent guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) was obtained from Alexis Biochemicals (San Diego, CA, USA), along with the G-protein inhibitor pertussis toxin. The tyrosine kinase inhibitor genistein was obtained from Acros Organics (New Jersey, USA). Calphostin C [a protein kinase C (PKC) inhibitor], diadzein a control for genistein inhibition), guanosine-5′-O- (2-thiodiphosphate) (GDP-β-S) (a G-protein inhibitor), Rp-8-Bromoguanosine-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPs) (a competitive inhibitor of PKG), 1-(6-(17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) (a phospholipase C inhibitor), NG-monomethyl-L-arginine (L-NMMA), and N-(3-(Aminomethyl)benzyl)acetamidine (1400W) (NOS inhibitors), 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028) (a PKG inhibitor), and the Quantizyme nitric oxide (NO) assay kit were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). The polyclonal antiphosphotyrosine antibody was purchased from Upstate (Lake Placid, NY, USA); 30X Tris-buffered saline (TBS) and 30X TBS/nonidet P40 (NP40) were obtained from Modern Biology (West Lafayette, IN, USA). The guanosine 3′3;5′monophosphate (cGMP) emzyme immunosorbent assay (EIA) kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Sodium metavanadate (a phosphatase inhibitor) and theophylline (a phosphodiesterase inhibitor) were obtained from Sigma Chemical (St. Louis, MO, USA). All other chemicals were purchased from Sigma as well. Behavioral assays In vivo behavioral assays were carried out as previously described [1, 9, 10]. Briefly, cells were washed in buffer, and 300 μl of cell suspension was transferred to the first well of a three-well spot microtiter plate. Cells were then individually transferred by micropipette to the second well, which contained 300 μl of the test concentration of inhibitor dissolved in buffer. After at least 10–15 min of incubation time, individual cells were transferred to the third well, which contained 300 μl GTP-γ-S and the test concentration of inhibitor. Each cell was briefly observed (1–5 s) for signs of avoidance. Avoidance behavior was characterized by jerky, backward movements; swimming in small, tight circles; sudden reversal of swimming direction; or any deviation from the normal helical swimming pattern of T. thermophila. Western blots Western blots were performed as follows: For each sample, 10 ml of the same 2-day-old culture was washed 3× in behavioral buffer; 100 μM GTP-γ-S (the concentration required to elicit 100% avoidance) was added to the tube of cells marked GTP. The control tube received only buffer; no GTP was added. Both tubes were immediately extracted with 0.1% sodium dodecyl sulphate (SDS) in the presence of 1 mM sodium orthovanadate for 30 min at 4°C. Extracts were spun at 16,000 g at 4°C for 30 min to precipitate insoluble components. Supernatant was collected, and 5× loading buffer was applied in preparation for SDS polyacrylamide gel electrophoresis (PAGE); 100 μl of each sample was applied to a 10% SDS-PAGE. Amount of protein loaded onto the gel was standardized using the BIO-RAD protein assay; in addition, a control, Coomassie-stained gel, was run to show that equal amounts of protein were loaded into each well. Proteins were transferred from the SDS-PAGE to nitrocellulose for 30 min at 150 mA. Blots were then blocked overnight in a 3% solution of powdered milk. Blots were incubated with a 1:1,000 dilution of polyclonal phosphotyrosine antibody in the presence of 3% powdered milk for 1.5 h, washed 3× in TBS, then incubated with a 1:100 dilution of alkaline phosphate conjugated anti-rabbit antibody for 1.5 h in the presence of 3% powdered milk. Blots were then washed 3× with TBS and incubated for 5 min in an alkaline phosphatase buffer containing 100 mM NaCl, 5 mM MgCl2, 100 mM Trizma base, pH 9.5 with hydrogen chloride (HCl). Alkaline phosphatase buffer was then removed, and blots were developed using the Sigma-Fast system until bands were visible. EIA assays NO assays were carried out using 2-day-old cell cultures. Cells were washed twice in buffer and diluted to a final concentration of approximately 720 cells/ml. Cells were exposed to 100 μM GTP-γ-S for approximately 30 s, then spun down in a bench-top microcentrifuge for 30 s. Control cells were spun under identical conditions except that no GTP was added to the solution. Supernatant from both sets of cells was analyzed for NO using a kit from BIOMOL according to the manufacturer’s instructions. cGMP assays were carried out using 2-day-old cell cultures. Cells were concentrated to approximately 7.66 × 106 cells/ml in order to maximize cGMP production. Cells were exposed to 100 μM GTP for approximately 30 s, then immediately lysed by freezing in liquid nitrogen and thawing. Theophylline (1 mM) was added to the lysate to inhibit phosphodiesterases. Lysate was spun at 16,000 g for 30 min. Control cells were treated in identical fashion except for the addition of GTP. Supernatant from both sets of cells was assayed for cGMP using a kit from Cayman Chemicals according to the manufacturer’s instructions. Immunofluorescence Ten milliliters of 2-day-old cells were starved overnight in behavioral buffer then washed 3× in behavioral buffer and concentrated down into 2 ml. Mucocysts were removed by adding Alcian Blue GX to a final concentration of 0.05%, then adding nine volumes of 0.25% proteose peptone [11]. Mucocysts were allowed to precipitate by standing the tube in a rack for 10 min. Supernatant (including cells) was removed from the top of the tube. The cells were then washed 3× in behavioral buffer to remove any remaining mucocysts or Alcian Blue and concentrated down into 3 ml total; 1 ml of cells was set aside as the control cells. Another milliliter of cells was incubated with 100 μM GTP-γ-S. A third aliquot was incubated with 100 μg/ml genistein prior to GTP treatment. Immediately, all three aliquots of cells were fixed in 3.7% formaldehyde (from 37% stock diluted into behavioral buffer) with gentle shaking for 20 min. Cells were washed 3× in 1× TBS/NP40 (Modern Biology) then incubated with a 1:100 dilution of polyclonal antibody in 1× TBS/NP40 for 1.5 h. Cells were again washed 3× in 1× TBS/NP40 then incubated in a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ICN Biomedicals) for 1.5 h. Cells were washed 3× in 1× TBS before being viewed under a Nikon microscope at 400×. Cells were photographed using a SPOT digital camera using a 10-s exposure for each photograph. Results GTP-γ-S is a chemorepellent in Tetrahymena,, with a minimum concentration of 100 μM required for 100% avoidance (Fig. 1). This concentration was used in the inhibition studies that follow as well as in our biochemical studies. We used GTP-γ-S in our experiments rather than GTP to minimize the effects of the ecto-ATPase upon the assays [3]. However, the activity of apyrases and other ectoenzymes may still have degraded some of the GTP-γ-S before it could reach its intended receptor. The concentration of GTP-γ-S required in order to achieve 100% avoidance in this study was 5× higher than that previously published [1]; however, considering that the cell line and growth conditions were different than those published earlier, this finding is not unexpected. The EC50 of GTP-γ-S in this study was approximately 30 μM. Fig. 1Guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) is an effective chemorepellent in Tetrahymena. In vivo behavioral bioassays (see “Materials and methods”) were used to show the concentration dependencies for avoidance reactions to GTP-γ-S. The percentage of cells showing avoidance was determined by observation of a single cell after transfer to the test solution. Each trial consisted of ten cells, which were individually scored as to whether or not avoidance occurred. Each point represents the mean ± standard deviation (SD) of at least three trials. Error bars, representing the standard deviation, are shown for each point. Minimum concentration required to give 100% avoidance was 100 μM for GTP-γ-S. This concentration was used in the inhibition studies that follow. The EC50 of this compound was approximately 30 μM To determine whether a tyrosine kinase activity was involved in GTP avoidance, we used the tyrosine kinase inhibitor, genistein (Fig. 2). Genistein itself did not cause avoidance reactions, nor was it toxic to the cells. Baseline avoidance of ∼ 20% [9] to 100 μM GTP-γ-S was seen at a genistein concentration of 100 μg/ml. The inhibition constant (IC)50 of genistein was approximately 40 μg/ml. Another phytoestrogen, daidzein, served as a control for nonspecific side effects of genistein. As seen in Fig. 2, this compound had no measurable effect on avoidance at the tested concentrations, suggesting that the inhibitory effect of genistein was indeed due to its tyrosine kinase inhibition and not attributable to some side effect of the drug. Fig. 2The tyrosine kinase inhibitor, genistein, blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) in Tetrahymena. The phytoestrogens genistein (closed circles) and daidzein (open circles) were used as inhibitors of avoidance. Baseline avoidance was seen at a genistein concentration of 100 μg/ml. The IC50 of genistein was approximately 40 μg/ml. Daidzein served as a control for nonspecific effects of genistein and had no measurable effect on avoidance at the concentrations tested. Data shown represent the mean +/− standard deviation (SD) for three or more trials As an additional control for specificity, we tested whether genistein had any effect on the transduction of another chemorepellent, adenosine 5′-O-(3-thio)triphosphate (ATP-γ-S). We used 150 μM ATP-γ-S, a concentration that normally causes 100% avoidance in T. thermophila [12]. When cells exposed to ATP-γ-S were first incubated for 10–15 min in 100 μg/ml genistein, no measurable effect on avoidance was seen. Cells continued to avoid 150 μM ATP-γ-S at a rate of 96.6 + 5.8% (n ≥ 3). Genistein also failed to affect avoidance to the chemorepellent pituitary adenylate cyclase-activating polypeptide (PACAP). When testing 0.1 μM PACAP, which normally causes 100% avoidance in T. thermophila [9], we found that prior incubation with 100 μg/ml genistein did not markedly affect avoidance. Cells continued to avoid 0.1 μM PACAP at a rate of 96.6 + 5.8% (n ≥ 3). These data suggest that tyrosine kinase activation is specifically related to GTP exposure rather than being a general response to chemorepellent exposure. In an attempt to localize the phosphotyrosines within the cell, immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody. Three separate trials were done with this antibody, and representative photographs are compiled in Fig. 3a–c. GTP-exposed cells (Fig. 3b) showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells (Fig. 3a,c). Ciliary staining was seen in the GTP-exposed cells, along with punctuate staining of the cytosol. The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern, but ciliary staining was not detected in these control groups, perhaps because the overall fluorescence level of these cells was so low. Fig. 3Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown) To determine which proteins were being phosphorylated by the tyrosine kinase in response to GTP-γ-S, we also performed several Western blots of whole-cell extract obtained from control and GTP-γ-S -treated cells using the polyclonal antiphosphotyrosine antibody as a probe. Protein extracts were standardized so that lanes were loaded equally, and a control, Coomassie-stained gel, was also run to control for lane-loading artifacts (data not shown). A representative Western blot is pictured in Fig. 3d. Both GTP-treated and control cells showed similar staining at 66 kDa. However, increased phosphotyrosine levels were seen in the GTP-γ-S-treated cells at 42, 35, and 21 kDa, consistent with tyrosine kinase activity. These bands were also present in the control cell extract; however, the phosphorylation level in these lanes was so low that they were barely detectable with our staining procedure. Additional bands were also seen in both the GTP-exposed and the control lanes; however, they were also too light to be measurable. A more sensitive assay, such as chemiluminescence based Western blot detection, might help to give a more complete profile of the phosphotyrosine-containing proteins shown in this procedure. In many cells, activation of a tyrosine kinase activates phospholipase C-γ. We used the phospholipase C inhibitor, U73122, to determine whether phospholipase C might be involved in GTP avoidance. GTP avoidance was effectively eliminated at a U73122 concentration of 1 μM. This concentration was ten times lower than that used to inhibit phospholipase C in molluscan ciliary cells [12]. The IC50 of this compound was approximately 0.001 μM. Phospholipase C activates the PKC pathway through the generation of diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3). PKC is then activated by DAG binding in the presence of calcium. To determine whether PKC was involved in GTP avoidance, we used the PKC inhibitor calphostin C, which competes for DAG binding to PKC. In the presence of 10 μM calphostin C, avoidance to 100 μM GTP-γ-S was reduced only to 96.6 +/− 5.8%, compared with 100% avoidance in control cells. This concentration of calphostin C eliminated avoidance to 150 μM ATP-γ-S [13]. Because calphostin C had no measurable effect on GTP avoidance, we concluded that PKC activy is not required for GTP signaling. It is very likely that the two second messengers, IP3 and DAG, are generated by phospholipase C and that PKC is activated, but that PKC targets cellular substrates that are not directly involved in ciliary reversal. Because calcium signaling is often associated with tyrosine kinase pathways and because calcium influx is associated with GTP exposure [2], we decided to eliminate calcium rom cells using the membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester (BAPTA-AM). As seen in Fig. 5a, exposure to this calcium chelator eliminated avoidance in a concentration-dependent manner, with baseline avoidance seen at a BAPTA-AM concentration of 5 μg/ml. This result was certainly not surprising, as all known chemorepellents that have been studied electrophysiologically in Tetrahymena have elicited depolarizations [2, 14, 15] that have been proposed to be linked to calcium. Because our data indicated that phospholipase C was involved in GTP avoidance, we decided to test the hypothesis that internal calcium stores may also contribute to avoidance, consistent with the liberation of IP3 from the plasma membrane. We used an inhibitor of the endoplasmic reticulum (ER) calcium ATPase, thapsigargin, to deplete ER calcium stores to determine whether these stores were necessary for avoidance. Baseline avoidance to GTP was seen at a thapsigargin concentration of 1,000 nM (Fig. 5b). The IC50 of thapsigargin was approximately 1 nM. NO is another second messenger sometimes associated with tyrosine kinases [5, 17] and often associated with calcium signaling [5, 18]. In addition, NO has been found to be a critical second messenger in the Tetrahymena response to both ATP [13] and PACAP-38 [18]. To determine whether NO was involved in GTP signaling, we used the NOS inhibitors 1400W (Fig. 6a) and L-NMMA (Fig. 6b). Both inhibitors blocked avoidance to 100 μM GTP-γ-S in Tetrahymena, with baseline avoidance being reached at 100 nM for 1400W (Fig. 6a) and 50 μM for L-NMMA (Fig. 6b). The IC50 of 1400W was near 0.1 nM, whereas L-NMMA was approximately 3 μM. To confirm the activity of NOS in GTP avoidance, we used the Quantizyme NO assay to determine whether there was a difference in NO metabolism between control and GTP-stimulated cells. Cells stimulated with 8 mg/ml GTP had a 146 +/− 4.7% increase in NO levels over control cells, consistent with the inhibitor studies (n = 3). In many cells, NO activates guanylyl cyclase, which then produces cGMP. Previous studies by Christensen et al. [20] described an NO-linked, possibly soluble guanylyl cyclase activity in T. thermophila. More recent studies, reviewed by Linder and Schultz [20], described a membrane-associated and calcium-dependent guanylyl cyclase in these organisms. To determine whether any guanylyl cyclase isoform was involved in GTP avoidance, we used NS2028, an inhibitor of guanylyl cyclase (Fig. 7a), as well as Rp-8-bromo-cGMPs, a competitive inhibitor of cGMP (Fig. 7b), in behavioral assays. NS2028 and Rp-8-bromo-cGMPs both blocked avoidance to 100 μM GTP-γ-S in Tetrahymena. Baseline avoidance was obtained at 10 nM NS2028 (Fig. 7a) and at 50 μM Rp-8-bromo-cGMPs (Fig. 7b). The IC50 of NS2028 was approximately 0.05 nM and of Rp-8-bromo-cGMPs near 7 μM. To confirm these results biochemically, we performed an EIA assay to determine whether GTP exposure resulted in elevated cGMP levels within Tetrahymena. Exposure to 5 mg/ml GTP resulted in a 1,119 +/− 4.2% increase above control cGMP levels (n ≥ 3), consistent with the hypothesis that GTP avoidance is linked to a guanylyl cyclase. Based on the data we collected, we put together a provisional model of GTP signaling (Fig. 8). In our model, GTP binds to a receptor, which activates tyrosine kinase while interacting with a membrane calcium channel, causing a depolarization. Phospholipase C is activated, generating IP3, which allows calcium efflux from ER stores. The high cytosolic calcium interacts with some (unknown) isoform of NOS, guanylyl cyclase, and other calcium-binding proteins, ultimately resulting in ciliary reversal. Whereas some of the relationships between second messengers are still unknown, the model has been put together to stimulate thought and discussion and to make some order out of a complex pathway. Discussion The data we have compiled here are consistent with the hypothesis that the previously described GTP receptor [1] signals through a tyrosine kinase (Fig. 2, 3). The GTP signaling pathway also involves phospholipase C (Fig. 4), intracellular calcium (Fig. 5), NOS (Fig. 6), and guanylyl cyclase (Fig. 7). The proposed GTP signaling pathway is outlined in Fig. 8. Fig. 4The phospholipase C inhibitor 1-(6-(17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) in Tetrahymena. Baseline avoidance was reached at a U73122 concentration of 1 μM. The inhibition constant IC50 of this compound was near 0.001 μM. Data shown is mean +/− standard deviation (SD) of three or more trialsFig. 5Intracellular calcium is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester (BAPTA-AM) blocks avoidance to 100 μM GTP-γ-S in Tetrahymena. Baseline avoidance was reached at a BAPTA-AM concentration of 5 μg/μl. The inhibition constant IC50 of this compound was near 0.5 μg/μl. Data shown is mean +/− standard deviation (SD) of three trials. b The endothelial reticulum (ER) calcium adenosine triphosphatase (ATPase) inhibitor thapsigargin also blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) in Tetrahymena.. Baseline avoidance was reached at a thapsigargin concentration of 1,000 nM. The IC50 of this compound was near 1 nM. Data shown is mean +/− standard deviation (SD) of three or more trialsFig. 6Nitric oxide synthase (NOS) activity is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The NOS inhibitor 1400W blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) in Tetrahymena. Baseline avoidance was reached at a 1400W concentration of 100 nM. The inhibition constant IC50 of this compound was near 0.1 nM. Data shown is mean +/− standard deviation (SD) of three trials. b The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) also blocks avoidance to 100 μM GTP-γ-S in Tetrahymena. Baseline avoidance was reached at an L-NMMA concentration of 10 μM. The inhibition constant IC50 of this compound was near 3 μM. Data shown is mean +/− standard deviation (SD) of at least three trialsFig. 7Guanylyl cyclase activity is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), an inhibitor of guanylyl cyclase, blocked avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-γ-S) in Tetrahymena. Baseline avoidance was obtained at 10 nM. The inhibition constant IC50 of NS2028 was approximately 0.05 nM. Data shown is mean + standard deviation (SD) of three trials. b Rp-8-Bromoguanosine-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPs), a competitive inhibitor of guanosine 3′3;5′monophosphate (cGMP), also blocked avoidance to 100 μM GTP-γ-S in Tetrahymena. Baseline avoidance was obtained at 50 μM. The IC50 of Rp-8-Br-cGMPs was approximately 7 μM. Data shown is mean + standard deviation (SD) of three or more trialsFig. 8Current model of guanosine 5'-triphosphate (GTP) signaling. Based on data obtained in this experiment, GTP appears to signal through a receptor linked to a tyrosine kinase. Phospholipase C, intracellular calcium release, nitric oxide (NO) production, and guanylyl cyclase are all involved in GTP avoidance. Some of the relationships between pathway components remain unknown. This model is based on the data presented in this paper, along with data obtained from the literature [26, 32, 33] GTP is the first chemorepellent we have studied that does not signal through a G-protein-linked receptor, unlike ATP [13] and PACAP [18, 21]. In contrast to other repellents, GTP signaling is unaffected by either GDP-β-S or pertussis toxin (unpublished data). GTP is also the only one of these three repellents whose signaling is eliminated by genistein (Fig. 2; see “Results”). Previously published cross-adaptation data [2] indicated that GTP and ATP signal through different receptors. These data are consistent with the previously published literature and add the observation that both receptors and second-messenger pathways for these two repellents appear to be different. Multiple target proteins are phosphorylated in response to GTP exposure (Fig. 3d), yet the proteins remain unidentified except for molecular weight. Phosphotyrosine immunolocalization does not help us identify these proteins; however, the ciliary staining (Fig. 3b) opens up the possibility of direct interaction between tyrosine kinase activity and ciliary reversal. This is worthy of note, as Christensen et al. [22] report a similar increase in ciliary phosphotyrosine labeling after exposure to the chemoattractant insulin. However, in their report, the main protein that was phosphorylated was a 66 kDa protein, which they discovered was homologous to the insulin receptor. In the case of GTP exposure, we saw no increase in labeling of the 66 kDa protein (Fig. 3d). Taken together, these data raise the possibility that there are multiple tyrosine kinase pathways that act on cilia to increase forward swim speed in the case of a chemoattractant, or to cause ciliary reversal in the case of a chemorepellent. Becausxe there were multiple phosphotyrosine bands in our Western blot (Fig. 3d) and ciliary as well as cytosolic staining in our immunofluorescence (Fig. 3b), we see that GTP exposure has both ciliary and cytosolic effects. Ciliary reversal is seen immediately upon GTP exposure; however, other effects of GTP exposure, such as mitosis, are seen hours after GTP exposure [4]. Perhaps the cytosolic target proteins are involved in other, longer-term effects of GTP, such as mitosis. Many tyrosine kinase pathways involve phospholipase C. In various Tetrahymena species, evidence for phospholipase C involvement has been seen in stomatin-induced differentiation [24] and in commitment to cell survival or death [25]. Studies by Leondaritis and Galanopoulou [26] suggest that Tetrahymena possess a functional phosphatidylinositol signaling system that is similar to higher eukaryotes. Indeed, in our previous studies of PACAP [10] and ATP [13], phospholipase C involvement is also implicated. In our current study, we found that U73122 effectively eliminated GTP avoidance (Fig. 4), indicating that phospholipase C signaling is involved in behavioral avoidance. However, our calphostin C data (see “Results”) indicate that PKC, a common downstream target of phospholipase C, is not required for GTP avoidance. IP3, a second messenger generated by phospholipase C, is likely involved in GTP signaling. Although we could not test IP3 involvement directly using our behavioral assay, the fact that the ER calcium ATPase inhibitor thapsigargin eliminated GTP avoidance (Fig. 5b) is indirect evidence that IP3 may be binding to the ER and causing release of calcium from internal stores. Calcium is a ubiquitous signaling molecule and has been implicated in ciliary reversal in Tetrahymena. It was no surprise, then, that BAPTA-AM eliminated GTP avoidance (Fig. 5a), as it has previously been shown to eliminate avoidance to PACAP [18] and ATP [13]. Calcium is likely to play multiple roles in signaling. One possible scenario is the direct interaction of calcium with ciliary motor proteins. For example, inner arm dynein 1 (I1) is required for calcium-dependent ciliary reversals in T. thermophila [27]. In this study, a “calcium sensor,” which interacts with I1, was postulated. This calcium sensor protein remains to be isolated. Intracellular calcium may also play other, less direct, roles in GTP avoidance. Indeed, in the protozoan parasite Leishmania donovani, both NOS and guanylyl cyclase are activated by intracellular calcium [28]. Both NOS (Fig. 6) and guanylyl cyclase (Fig. 7) activity are required for GTP avoidance. Previous studies that implicate NOS and guanylyl cyclase in Tetrahymena [13, 18, 19] do not indicate the type of NOS or guanylyl cyclases involved, though Christensen et al. [19] give evidence for a soluble guanylyl cyclase. In L. donovani, there is evidence for a constitutive, neuronal-type NOS that then activates a soluble guanylyl cyclase [28]. Both of these enzymes are activated by intracellular calcium. Our data would be consistent with a similar mechanism in Tetrahymena, as proposed in our model of GTP signaling (Fig. 8). However, neither a soluble guanylyl cyclase nor an nNOS isoform have been purified from Tetrahymena to date. Calcium-binding proteins, such as calmodulin, may also be involved in GTP signaling in Tetrahymena, either by interacting with enzymes that generate second messengers or by interacting with the molecular motors responsible for ciliary reversal. Previous studies [29, 30] indicate that calmodulin activates a ciliary guanylyl cyclase in this organism. Our studies leave open the possibility that this cyclase, activated by calcium/calmodulin, is involved in GTP signaling. In addition to this enzyme, Hirano-Ohnishi and Watanabe [31] reported a calmodulin-dependent phosphorylation of ciliary beta tubulin in Tetrahymena, which they believe to be involved in ciliary reversal. Elongation factor 1α, which functions in protein translation, also interacts with ciliary calmodulin [32]. Whereas this would not necessarily function in ciliary reversal, it may modulate some of the longer-term effects of GTP. Finally, Ueno et al. [33] report that ciliary calmodulin interacts with proteins that are homologues of radial spoke proteins in Chlamydomonas, further implicating calmodulin directly in ciliary reversal. We have added some of these proteins to our model of GTP signaling (Fig. 8), though there are doubtless more proteins to be discovered. In this study, we used behavioral assays and pharmacological inhibitors to explore the intracellular mechanisms employed in GTP signaling in T. thermophila. Because pharmacological inhibitors often have unknown side effects, we used multiple inhibitors when available. Some of these inhibitors had to be used at higher concentrations in our study than have been previously published for mammalian cells. Perhaps this is because of the differences in the structure of the Tetrahymena proteins compared with their mammalian counterparts. Recognizing the potential pitfalls of relying solely upon pharmacological data, we supplemented behavioral assays with other kinds of tests, such as Western blots, immunofluorescence, and EIA when available. In all cases, the additional tests confirmed the data we originally obtained using pharmacological inhibitors. Taken together, our data indicate that a number of enzymes are involved in GTP signaling. However, further experimentation is needed to discern the interrelationships between enzymes in the pathway and to answer the myriad questions that remain. For example, are guanylyl cyclase and NOS truly activated by calcium in Tetrahymena? Would calcium chelation abolish their activity? Is there a soluble guanylyl cyclase in Tetrahymena? What NOS isoforms do Tetrahymena possess? Is tyrosine phosphorylation required for either NOS or guanylyl cyclase activity? Are any additional proteins, particularly ciliary proteins, involved in GTP signaling? Are all of the components of the pathway located in the cilia? These and many other questions remain to be answered. One question we have begun to answer is whether intracellular calcium is required for tyrosine kinase activity. Recent immunofluorescence shows no difference in phosphotyrosine levels between GTP-treated cells and GTP-treated cells that had been pretreated with BAPTA-AM (unpublished data). Similarly, calcineurin (PP2B) does not appear to be involved in GTP signaling, as ascomycin treatment had no effect on GTP avoidance (unpublished data). Endothall, a PP2A inhibitor, did inhibit avoidance (unpublished observation), consistent with observations by Deckman and Pennock [33] that PP2A is involved in ciliary reversal. Further experimentation will help us determine the interrelationship of pathway components and better establish the mechanism by which chemorepellents signal in Tetrahymena.

Ann_Surg_Oncol-3-1-1914276

Pathological and Biological Differences Between Screen-Detected and Interval Ductal Carcinoma in situ of the Breast

Background The incidence of ductal carcinoma in situ (DCIS) has risen dramatically with the introduction of screening mammography. The aim was to evaluate differences in pathological and biological characteristics between patients with screen-detected and interval DCIS. With the introduction of widespread screening mammography, the incidence rates of ductal carcinoma in situ (DCIS) have risen dramatically in Western Europe and North America.1–3 DCIS now accounts for nearly 20% of all screen-detected breast malignancies.4 As a consequence, treating physicians are confronted with a cumulative caseload because it is not known how many women with screen-detected DCIS will develop an invasive carcinoma in their lifetimes. The proportion of untreated cases of DCIS that would progress to invasive malignancy has been difficult to evaluate, because DCIS is usually excised when detected. Because DCIS is a nonobligatory precursor to invasive carcinoma, and, therefore, has a relatively benign nature, screen-detected DCIS has been argued to represent an overdiagnosis.5,6 This argument is supported by autopsy studies in which the median prevalence of DCIS was 8.9%, suggesting some cases do not progress to clinically significant lesions in a patient’s lifetime.7 On the contrary, patients with DCIS treated with biopsy alone in the premammography era had a higher rate of subsequent occurrences (14–50%) of invasive breast cancer than expected.8,9 Large clinical trials, in which patients had been treated with lumpectomy alone, have also indicated that DCIS can recur as invasive ductal carcinoma.10,11 Screen-detected DCIS is more often presented as linear branching microcalcifications on mammography than symptomatic DCIS.12 The screen-detected group in the previously mentioned study had a larger proportion of patients with comedocarcinoma. Therefore, it was suggested that linear branching microcalcifications were related with a more aggressive type of DCIS.12 This is confirmed in other reports that have indicated that linear branching microcalcifications on mammography are associated with high grade DCIS.13,14 We believe that screen-detected DCIS is more often associated with suspicious microcalcifications representing high-grade DCIS, which has been detected before it has had the chance to progress to invasive cancer. Therefore, it is hypothesized that screen-detected DCIS is biologically more aggressive than interval DCIS. To compare screen-detected DCIS with interval DCIS in such a retrospective study, the clinicopathological and biological characteristics of both groups were evaluated for differences. Screen-detected DCIS was classified as DCIS detected by screening mammography, when the examination from two years earlier failed to reveal an abnormality. Interval DCIS was classified as DCIS detected within the two-year interval between two subsequent screening rounds, when the earlier examination failed to reveal an abnormality. Age, tumor size, and pathological grade were studied for their known relation with local recurrence. Finally, the expression of established prognostic biomarkers in breast cancer was studied by immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53, and cyclin D1. PATIENTS AND METHODS Patients and Tumors The Dutch screening program for breast cancer has been gradually implemented in the North Netherlands since 1991. It offered biennial mammography to women 50–69 years old, and since 1999 women 70–74 years old have also been included. Women received mammography in the cranio-caudal and medio-latero-oblique direction for each breast. Two radiologists evaluated the mammograms by a double independent reading. From January 1992 to December 2001, 128 consecutive patients were treated for pure DCIS at our institution. To identify patients for inclusion in the study, all women who had actually attended the screening program at least two subsequent rounds with a two-year interval at the time of diagnosis were considered as attenders. Patients who had skipped one or more screening rounds previous to the diagnosis and patients who had not been attending the program at all were considered nonattenders. Patients’ records were checked to obtain this information, and if there was no information regarding the participation of the screening program at the time of diagnosis the general practitioner was consulted. Out of the 128 consecutive patients, 102 attenders and 26 nonattenders could be identified. For immunohistochemistry, patients were selected on the availability of sufficient paraffin-embedded tissue. Thirteen out of the 26 nonattenders and 74 out of the 102 attenders remained, respectively, for evaluation of Her2/neu overexpression, estrogen receptor (ER) expression, progesterone receptor (PR) expression, p53 expression, and cyclin D1 expression using tissue microarray analysis as part of a project protocol that had been approved by the medical ethics committee. The patients in the study-group (n = 74) were divided into two groups. Patients with DCIS that had been detected by screening mammography were classified as screen-detected patients, when the examination two years earlier failed to reveal an abnormality (n = 54). Patients with DCIS that had been detected within the two-year interval between two subsequent screening rounds were classified as interval patients, when the earlier examination failed to reveal an abnormality (n = 20). Mammography and Pathological Assessment Mammographic and pathological characteristics were derived from mammography and pathological reports, respectively. Data were delivered and evaluated anonymously. If data were missing, mammography and pathological slides were reevaluated. Mammographic appearances were scored as microcalcifications, a mass, a combination of the two, or as occult. Microcalcifications were scored as fine granular, coarse granular, or as linear branching. Pathological size had been estimated, and the grade had been scored according to the European Pathologists Working Group (EPWG15) and according to the Van Nuys classification.16 Tissue Microarray Construction Slides from all blocks were evaluated for representative areas with DCIS, and tissue microarrays were prepared as described earlier.17 In brief, the most representative area of DCIS was marked on the original hematoxylin and eosin (H&E) stained section. With this marked section as an orientation, three 0.6-mm punches were taken from the selected area in the donor blocks and mounted in a recipient block containing approximately 110 biopsies, using a manual tissue microarray device (Beecher Instruments, Silver Springs, MD). The presence of DCIS in the arrayed samples was verified on hematoxylin eosin stained sections. Immunohistochemistry For immunohistochemistry, 3 μm sections of the paraffin-embedded tissue arrays were deparaffinized in 2 changes of xylene for 5 minutes each and gradually rehydrated through changes of graded ethanol from 100% to distilled water. Antigen retrieval methods and antibodies are summarized in Table 1. The endogenous peroxidase reaction was blocked by incubating the sections in 3% perhydrol for 30 minutes. Primary antibodies were diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and incubated at room temperature for 1 hour. Samples were then washed in PBS and incubated with secondary and tertiary antibodies. For visualization of the antibody-antigen complex, the diaminobenzidine tetrahydrochloride/peroxidase reaction was used. After a final wash with distilled water, sections were counterstained with hematoxylin. Sections were dehydrated through rising concentrations of ethanol and mounted. Immunohistochemistry was successful in 81/87 cases for Her2/neu staining, 73/87 cases for ER staining, 71/87 for PR and cyclin D1 staining, and 70/87 for p53 staining. TABLE 1.Antigen retrieval methods and antibodiesAntibodyCloneSupplierDilutionAntigen retrievalSecondary antibodySupplierTertiary antibodySupplierER6F11VentanaaTris/HCL 0.1M (pH 9.5) 30’ 98°C microwaveRAMBIODakoSARBIODakoPR1A6VentanaaTris/HCL 0.1M (pH 9.5) 30’ 98°C microwaveRAMBIODakoSARBIODakoHer-2/NeuCB11VentanaaTris/HCL 0.1M (pH 9.5) 30’ 98°C microwaveRAMBIODakoSARBIODakop53BP-53-12-1Biogenix1:800Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwaveRAMBIODakoSARBIODakoCyclin D1SP4Neomarkers1:50Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwaveRAMBIODakoSARBIODakoa Prediluted by supplier.ER, estrogen receptor; PR, progesterone receptor;; RAMBIO, rabbit anti-mouse biotin; SARBIO, swine anti-rabbit biotin. Evaluation of Immunohistochemical Staining All slides stained for molecular markers were read by two authors (MdR and BvdV). The slides were randomly reviewed by a third author (JW) and in case of disagreement between the other two authors. ER, PR, and p53 were graded based on the percentage of cells showing positive nuclear staining in the ducts with DCIS. ER and PR were considered positive if nuclear staining was present in ≥10% of the cases, and p53 was considered positive in case of a substantial percentage of positively stained nuclei (>30%). Her2/neu expression was graded as recommended by the HercepTestTM scoring guidelines: 0: no staining at all or membrane staining in <10% of tumor cells; 1+: a faint/barely perceptible partial membrane staining in >10% of the tumor cells; 2+: weak to moderate complete membrane staining in >10% of tumor cells; 3+: strong complete membrane staining in >10%. Her-2/neu was considered to be overexpressed if the score was 3+. Cyclin D1 expression was scored using a semiquantitative system as described by Vos et al.18 This system was based on the staining intensity scored as 0 (none), 1 (weak), 2 (moderate), and 3 (strong), and the percentage of positive tumor cell nuclei scored as 0 (0%), 1 (1–25%), 2 (25–50%), 3 (50–75%), and 4 (>75%). The cyclin D1 staining score was calculated as the sum of the intensity and the percentage of positive tumor cells. Statistical Analysis Differences in clinicopathological characteristics between screen-detected and interval patients with DCIS in the study group, and between the study group and the excluded group of patients were analyzed by chi-square analysis. Differences in clinicopathological and biological characteristics between the study group and the nonattenders were also analyzed by chi-square analysis. Differences in age were tested by using the Mann-Whitney U test. Univariate analyses, investigating differences in pathological and biological features, was performed by logistic regression, using screen detection as a dependent variable. Multivariate analyses were performed with a logistic-regression model. The elimination of variables in a stepwise manner identified the statistically significant pathological and biological parameters. A p value of ≤.050 was considered as significant. All calculations were performed with SPSS 12.01 (SPSS inc., Chicago, IL). RESULTS Table 2 shows the clinicopathological characteristics of the patients in the study group (n = 74). Screen-detected DCIS was less often symptomatic than interval DCIS (p < .001). Five patients (25%) in the interval group had no objective signs on presentation; all patients had felt a lump in the breast that could not be verified on clinical examination. On mammography, microcalcifications were more often seen in screen-detected DCIS (p = .002). Screen-detected DCIS was more often presented as linear branching (44.9%) and coarse granular (55.9%) microcalcifications than interval DCIS (p<0.001). High-grade DCIS (Van Nuys, 53.1%, p = .025) was also more often observed in screen-detected DCIS. TABLE 2.Clinicopathological characteristics of the patients in the study group and differences between screen-detected and interval patientsClinicopathological characteristicsScreen-detected n = 54Interval n = 20p-valueaAge (mean), years58.960.7.187Family history of breast cancer.055  Yes8 (14.8)7 (35)  No46 (85.2)13 (65)Signs  Palpable mass5 (9.3)6 (30)<.001  Nipple discharge2 (3.7)9 (45)  Mastodynia1 (1.9)0 (0)  No objective signs46 (85.1)5 (25)Mammography.002  Microcalcifications46 (85.2)10 (50)  Mass2 (3.7)6 (30)  Combination mc’s and mass6 (11.1)4 (20)Microcalcifications <.001  Linear branching22 (44.9)4 (28.6)  Coarse granular27 (55.1)4 (28.6)  Fine granular0 (0)6 (42.9)Mammographic size.183  ≤2 cm21 (42.6)12 (60)  >2 cm33 (57.4)8 (40)  BCS23 (42.6)8 (40).841  Mastectomy31 (57.4)12 (60)Tumor size.787  <16mm19 (35.2)6 (30)  16–40mm17 (31.5)8 (40)>40 mm18 (33.3)6 (30)Grade (EPWG).229  14 (7.5)4 (20)  229 (53.7)11 (55)  321 (38.9)5 (25)Grade (Van Nuys).025  14 (7.4)6 (30)  224 (44.4)9 (45)  326 (53.1)5 (25)Mann-Whitney U test.Values between parentheses are percentages.BCS, breast conserving surgery; EPWG, European Pathologist Working Group.a Chi-square analyses. Differences in clinicopathological characteristics between the study-group (n = 74) and the group of patients that had been excluded (n = 28) because of insufficient paraffin embedded tissue are outlined in Table 3. There was no marked difference in age, microcalcifications, tumor size, and pathological grade according to the EPWG classification between both groups. The excluded group of patients represented a relatively large proportion of low-grade DCIS according to the Van Nuys classification (p < .001) in comparison with the TMA group. TABLE 3.Comparison of clinicopathological characteristics between the study group and the group of patients that were excluded because of insufficient paraffin-embedded tissueClinicopathological characteristicsStudy group n = 74Excluded n = 28p-valueaAge (mean)59.661.5.381Microcalcifications (n = 86).063  Linear branching26 (41.3)5 (25)  Coarse granular31 (49.2)9 (45)  Fine granular6 (9.5)6 (30)Tumor size.458  <16mm25 (33.8)7 (25)  16–40mm25 (33.8)9 (32.1)  >40 mm24 (32.6)12 (42.9)Grade (EPWG).184  18 (10.8)6 (21.4)  240 (54.1)10 (35.7)  326 (35.1)12 (42.9)Grade (Van Nuys)<.001  110 (13.5)15 (53.6)  233 (44.6)6 (21.4)  331 (41.9)7 (25)Mann-Whitney U test.Values between parentheses are percentages.EPWG, European Pathologist Working Group.a Chi-square analyses. There were no differences in clinicopathological and biological characteristics between the study group and the nonattenders (n = 13), except the fact that nonattenders were younger than the patients in the study group (55.9 years versus 59.6 years; p = .042). Table 4 displays the relation among pathological characteristics, biological marker expression, and mode of detection in the study group. Univariate logistic regression analysis indicated that in screen-detected DCIS Her2/neu is more often overexpressed (odds ratio [OR] = 6.5; 95% confidence interval [CI] 1.3–31.0; p = .020). Interval DCIS is more frequently positive for PR staining (OR = 0.3; 95% CI 0.1–1.0; p = .042) and is related to low pathological grade according to the Van Nuys classification (OR = 7.3; 95% CI 1.6–33.3; p = .010). In multivariate logistic regression, including pathological grade according to the EPWG and Van Nuys classification, Her2/neu overexpression and PR expression in the model, Her2/neu overexpression was the only independent indicator for screen-detected DCIS (OR = 12.8; 95% CI 1.6–104.0; p = .018). TABLE 4.Univariate analysis of pathological and biological characteristics in screen-detected versus interval DCIS in the study groupPathological and biological featuresScreen-detected n = 54Interval n = 20OR by screen-detected95% CIp-valueTumor size  <16mm19 (35.2)6 (30)0.80.20.744  16–40mm17 (31.5)8 (40)1.10.20.827  >40 mm18 (33.3)6 (30)1Grade (EPWG)  14 (7.5)4 (20)3.8000.50.377  229 (53.7)11 (55)1.7030.70.124  321 (38.9)5 (25)1Grade (Van Nuys)  14 (7.4)6 (30)7.31.6  224 (44.4)9 (45)1.60.50.416  326 (53.1)5 (25)11.60.010Her2/neu (n = 68)  Positive29 (60.4)2 (10)6.51.3–31.00.020  Negative19 (39.6)18 (90)1ER(n = 62)  Positive33 (75)16 (88.9)0.4  Negative11 (25)2 (11.1)10.1–1.90.236PR (n = 60)  Positive18 (42.9)13 (65)0.3  Negative24 (57.1)5 (35)10.1–1.00.042p53 (n = 60)  Positive10 (24.4)4 (21.1)0.8  Negative31 (75.6)15 (78.9)10.2–3.10.776Cyclin D1 (n = 60)  Positive29 (69.1)13 (72.2)0.90.3–2.90.806  Negative13 (30.9)5 (27.8)1Univariate analysis using logistic regression.Figures in parentheses are percentages.OR, odds ratio; EPWG, European Pathologist Working Group; ER, estrogen receptor; PR, progesterone receptor. 95% CI, 95% confidence interval. DISCUSSION Approximately 1 in every 1300 screening mammography examinations leads to a diagnosis of DCIS.4 Data from a large trial and service screening programs in the United Kingdom, the Netherlands, Australia, and the United States have demonstrated that a woman attending prevalence screen has a 19 times greater chance of having a progressive DCIS or an invasive tumor diagnosed than of having a nonprogressive DCIS diagnosed.19 It is questioned what to do with the high detection rate of screen-detected DCIS. It was hypothesized that screen-detected DCIS is biologically more aggressive than interval DCIS because suspicious microcalcifications, detected by the screening program, will probably more frequently represent high-grade DCIS. Therefore screen-detected DCIS was characterized pathologically and biologically to determine whether screen-detected DCIS differed from interval DCIS. The results of this study indicate that screen-detected DCIS is pathologically (OR = 7.3; 95% CI 1.6–33.3; p = .010) and biologically (OR = 12.8; 95%CI 1.6–104.0; p = .018) more aggressive than interval DCIS. Indeed screen-detected DCIS was related with more suspicious microcalcifications (p < .001). DCIS detected by a prevalence screen was pathologically and biologically comparable to DCIS detected in later rounds (data not shown) suggesting prevalence and incident cases to both be of clinical relevance. The relative incidence of high-grade DCIS in our series of screen-detected patients was 53%, which is comparable to the incidence of high-grade DCIS in a screening population from the Netherlands Cancer Institute (47%).20 Much data point out that poorly differentiated or high-grade DCIS lesions have a greater potential to progress to invasive disease than low-grade DCIS.21 High grade is also an independent risk factor of local recurrence after lumpectomy for DCIS, and approximately 50% of these recurrences are invasive cancers.22,23 There are no studies available comparing screen-detected with interval DCIS in a group of patients that had all been attending the screening program. Reports on screen-detected DCIS regarding histopathological grade vary markedly describing a higher incidence of low grade,24 no difference,25 or a higher incidence of high grade12,26,27 in screen-detected DCIS. In these reports screen-detected DCIS is compared to symptomatic DCIS or to DCIS detected in a period before the screening program was introduced. In the present study, a higher incidence of high-grade lesions, which were classified according Van Nuys (OR = 7.3; 95% CI 1.6–33.3; p = .010) classification, was found in screen-detected DCIS, indicating a higher malignant potential in screen-detected DCIS. These results are consistent with the results from the studies of Evans et al.26 and Kessar et al.27 DCIS lesions from patients in the study group were compared to DCIS lesions from patients who had not attended the screening program. Out of the 26 nonattenders, there were only 13 patients from whom sufficient paraffin-embedded tissue was available. Using chi-square analysis there were no differences in pathological and biological characteristics between the two groups. The difference in age could be explained by the fact that the nonattenders group also contained patients under 50-years of age. From these analyses it seems that DCIS in nonattenders is not pathologically and biologically more aggressive than DCIS in attenders of the screening program. However, because of the very small number of patients in the nonattenders group no hard conclusions can be drawn. Her2/neu overexpression has been found to correlate with various pathologic and biological factors believed to be associated with more aggressive behavior; high grade, presence of necrosis, ER and PR negativity, and overexpression of Ki-67 (indicating an increased proliferation rate) are features that are strongly related with Her-2/neu overexpression.28–30 The report of Walker et al.24 displayed a Her-2/neu expression of 59% in symptomatic DCIS and of 42% in mammographically detected DCIS. Another study by Idvall et al.25 reported no difference in Her-2/neu expression between DCIS before and after introduction of mammographic screening. In this present study Her2/neu overexpression was the only independent feature to be related with screen-detected DCIS in multivariate analysis (OR = 12.8; 95%CI 1.6–104.0; p = .018), which indicates a more aggressive profile of screen-detected DCIS when compared to interval DCIS. Although the statistical methods used were univariate and multivariate analysis, the numbers in both groups are small, which explains the broad 95% CI. The small numbers are due to the selection of patients in this study for study period, attendance of screening rounds, and availability of sufficient paraffin-embedded tissue. Clearly, further studies with larger populations are needed to elucidate the relative significance of the Her2/neu overexpression in women with screen-detected DCIS. Although 28 out of 102 patients were excluded because of lack of sufficient paraffin-embedded tissue, there is no reason to assume that this exclusion results in a significant selection bias. Apart from pathological grade according to Van Nuys, there were no differences in clinicopathological characteristics between the study group and the group of excluded patients. The group of excluded patients displayed a relatively large amount of low-grade DCIS according to Van Nuys (53.6%, p < .001), which is mainly derived from the interval group (n = 10, data not shown). If all patients would have been included the relation of pathological grade and probably Her2/neu expression with screen-detected patients would be even more significant. The inclusion of patients that actually took part in the screening program was necessary to analyze differences between screen-detected and interval DCIS, which, to our knowledge, has not been performed previously. Expression of ER, p53, and cyclin D1 was not related to screen-detected or interval DCIS. There was, however, a relation between screen-detected DCIS and PR negativity in univariate analysis (OR = 0.288; 95%CI 0.087–0.957; p = .042). Other studies could not demonstrate a relation of screen-detected DCIS with the expression of the aforementioned markers.12,24,25 In a review by Boland et al.31 ER and PR positivity are related to low-grade DCIS, whereas p53 and cyclin D1 expression are associated with high grade. The fact that screen-detected DCIS is associated with PR negativity provides indirect evidence for the presence of a more aggressive tumor biology. Obviously, PR expression is related to Her2/neu expression, because PR expression was not significant in multivariate analysis. Ultimately, the question is how we should interpret these findings. The authors think the results from this study represent no evidence to alter patient management and screening recommendations. Instead, they should rather be regarded as support of current clinical practice in DCIS of the breast. They confirm that every DCIS should be treated until we are able to identify DCIS that will progress to invasive cancer if left untreated. In conclusion, since the advent of screening, the increased incidence of DCIS has raised concerns about the possibility of overdiagnosis of DCIS. This study has shown that screen-detected DCIS has a more aggressive tumor profile than interval DCIS. Therefore, screen-detected DCIS should not be regarded as an overdiagnosis per se, and every woman diagnosed with DCIS by mammographic screening should be treated properly according to existing guidelines or standards of care.

Eur_J_Pediatr-4-1-2234442

Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Clinical manifestations include severe airway obstruction, skeletal deformities, cardiomyopathy and, in most patients, neurological decline. Death usually occurs in the second decade of life, although some patients with less severe disease have survived into their fifth or sixth decade. Until recently, there has been no effective therapy for MPS II, and care has been palliative. Enzyme replacement therapy (ERT) with recombinant human iduronate-2-sulphatase (idursulfase), however, has now been introduced. Weekly intravenous infusions of idursulfase have been shown to improve many of the signs and symptoms and overall wellbeing in patients with MPS II. This paper provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides recommendations for the use of ERT. The issue of treating very young patients and those with CNS involvement is also discussed. ERT with idursulfase has the potential to benefit many patients with MPS II, especially if started early in the course of the disease. Introduction First described by Major Charles Hunter in 1917 [15], mucopolysaccharidosis type II (MPS II or Hunter syndrome; OMIM +309900) is an X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). This enzyme cleaves O-linked sulphate moieties from the glycosaminoglycans (GAGs) dermatan sulphate and heparan sulphate as the first step in their degradative pathway. The clinical phenotype of MPS II (Fig. 1) is characterised by progressive pathological lysosomal storage of GAGs in nearly all cell types, tissues and organs. Fig. 1Appearance of a child with mucopolysaccharidosis type II (Hunter syndrome) at (a) 6 years of age and (b) 12 years of age, illustrating the ‘typical’ clinical phenotype at the more severe end of the disease spectrum. The manifestations of the syndrome and the rate of progression, however, may vary widely among individual patients Oropharyngeal and tracheo-bronchial deposition of GAGs leads to severe airway obstruction due to macroglossia, supraglottic narrowing, and tracheomalacia. This obstructive anatomy and physiology leads to sleep apnoea and airway obstruction. As the disease progresses, the respiratory system is further compromised by pulmonary restriction secondary to the effects of the disease on the thoracic skeleton. Deposition of GAGs in the heart, liver and spleen leads to cardiomyopathy, cardiac valve dysplasia and hepatosplenomegaly. Bone and joint involvement results in severe skeletal deformities and limitations of joint mobility. In patients with CNS involvement, severe learning difficulties and progressive neurological decline occur. The clinical manifestations of MPS II generally lead to death in the first or second decade of life, although in the more attenuated form of MPS II, death may occur in early adulthood, and some patients have survived into their fifth and sixth decades of life. Until recently, there has been no effective therapy for MPS II. Care has been palliative, focusing on management of the multiple clinical symptoms. Haematopoietic stem cell therapy (HSCT) has been attempted in some patients [36], but the long-term results have been unsatisfactory. The recent introduction of enzyme replacement therapy (ERT) with recombinant human I2S (idursulfase), however, has raised the possibility that the burden of GAG storage can be reduced and that the progressive tissue and organ damage associated with MPS II can be slowed or even prevented. The availability of ERT thus requires a greater awareness and understanding of the disease amongst a range of medical specialists and primary care physicians, so that early diagnosis can be made and treatment started before organ damage becomes irreversible. This article gives an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides, for the first time, recommendations for the use of ERT. Incidence and inheritance Data from The Netherlands and Germany indicate that the incidence of MPS II is 1.3 per 100,000 male live births [1, 23]. Two-thirds of patients present with CNS involvement, representing the more severe end of the disease phenotype. There have been very few reports of recurrent or common mutations within the gene encoding I2S. As a consequence, genotype–phenotype correlations have been difficult to establish, with the exception of those patients who present with large deletions, which are most often associated with more severe disease [38]. As MPS II is an X-linked recessive condition, it would not be expected to affect females. Despite this, a few girls have been reported with MPS II [35]. Affected females generally have low levels of I2S activity and an attenuated clinical phenotype, although the somatic abnormalities can be severe in some individuals. In affected heterozygous females, skewed inactivation of the X-chromosome prevents expression of the normal allele [35]. Molecular basis of MPS II MPS II is the only known X-linked MPS disorder. The human gene encoding I2S has been mapped to Xq28. It contains nine exons spread over 24 kb. Individuals with major deletions or rearrangements in the gene usually have severe MPS II. An I2S-like pseudogene, comprising copies of exons 2 and 3 and intron 7, is located about 20 kb from the active gene [34]. A recurring rearrangement is due to recombination between the intron region of the gene and a homologous region near exon 3 of the pseudogene, with inversion of the intervening DNA [3]. Very large deletions of the I2S locus may extend to adjoining genes, as the neighbouring DNA is gene rich. A contiguous gene syndrome involving fragile X mental retardation (FMR1 and FMR2) genes could possibly explain the unusual phenotypes seen in some severely affected patients [33]. Over 150 different mutations, resulting in a spectrum of disease phenotypes, have been described. General disease manifestations and diagnosis Treatment of possible disease complications can greatly improve quality of life. Early diagnosis is therefore essential, particularly with the recent introduction of ERT. Table 1 shows the common presenting features of patients with MPS II, together with the range of specialists who are likely to be involved in diagnosis and care. Table 1Presenting features of patients with mucopolysaccharidosis type II (Hunter syndrome) and the specialists likely to be involved in diagnosis and treatmentPresenting featureSpecialistRecurrent upper respiratory tract infections, recurrent otitis media, developmental delay, hepatosplenomegaly, joint restriction, coarse faciesPaediatricianPaediatric neurologistOtolaryngologistRecurrent ear infections, abdominal distension, stiff jointsPrimary care physicianUmbilical and inguinal hernias with recurrenceSurgeonHip dysplasia, arthropathyOrthopaedic specialistUpper airway obstruction, sleep apnoea, recurrent ear infections requiring tube placementOtolaryngologistPapilloedema in the absence of raised intracranial pressureOphthalmologistValvular thickening on echocardiographyCardiologistJoint pain and restrictionRheumatologistPapular pearly rash across the scapulaeDermatologistRestrictive joint range of motionPhysiotherapistCarpal tunnel syndromeHand surgeonCervical myelopathyNeurosurgeon Clinical spectrum of MPS II MPS II is a variable, progressive, multisystem disorder. In most patients, symptoms are severe and death occurs at an early age. In other patients the disease has a more chronic and protracted course. The age of presentation of MPS II is also variable, as are the presenting signs and disease complications. Figure 2 shows the age at onset of the main signs and symptoms of the disease in a cohort of 82 patients enrolled in HOS, the Hunter Outcome Survey. This is a multinational long-term outcomes survey to investigate the natural history of MPS II and the safety and efficacy of ERT with idursulfase [8]. Fig. 2Reported age at onset and prevalence of clinical features in 82 patients with mucopolysaccharidosis type II (Hunter syndrome) enrolled in HOS, the Hunter Outcome Survey The traditional classification of patients into ‘mild’ or ‘severe’ subtypes, on the basis of length of survival and the presence or absence of CNS disease, is a gross simplification. The disorder should rather be regarded as a continuum between two extremes (severe and attenuated). It is important to note that individuals who are diagnosed with an attenuated form of the disease may still have symptoms and complications that lead to significant morbidity and disability, and may present with mild to moderate learning difficulties. Although the clinical course for the more severely affected patients is relatively predictable, there is considerable variability in the clinical phenotype and progression of the more attenuated form of the disease. Individuals with attenuated MPS II are most often diagnosed between the ages of 4 and 8 years. For the majority of these affected patients, the clinical course is very similar to the intermediate form of MPS I (MPS IH/S, Hurler–Scheie disease). Survival to adulthood is common, but death often occurs between the ages of 20 and 30 years from cardiac or respiratory disease (Fig. 3). A few patients are less severely affected. In these, life expectancy can be near normal, and such affected males may have children [9]. Fig. 3Mortality by age of patients with mucopolysaccharidosis type II (Hunter syndrome) in the UK. Data are from the Society for Mucopolysaccharide Diseases (MPS Society) and are based on patients registered with the MPS Society between 1950 and October 2006 Patients with the more severe form of MPS II exhibit a chronic and progressive disease involving multiple organs and tissues. They appear normal at birth, although they tend to be heavy, some have inguinal or umbilical hernias and there is an increased incidence of Mongolian blue spots [25]. The age at diagnosis is usually between 18 and 36 months, compared with 9 months for severe MPS I [6]. Death from a combination of neurological deterioration and cardiorespiratory failure usually occurs in the mid-teenage years. Physical appearance The typical patient with attenuated MPS II is short with some change of facial features. Significant arthropathy and connective tissue involvement lead to joint contractures. A combination of hepatomegaly and lax abdominal muscles leads to abdominal prominence. In the more severely affected patients, the typical facial features become apparent in the first 3 years. They are most likely caused by a combination of storage in the soft tissues of the orofacial region and underlying facial bone dysostosis. Thickening of the alae nasi, lips, ear lobules and tongue becomes progressively more obvious. The patients often develop a plethoric, rosy-cheeked appearance. Thickening of the calvaria results in macrocephaly. Facial and body hypertrichosis is often seen and the scalp hair becomes coarse, straight and thatch-like. Patients also develop specific, pearly, papular skin eruptions, which are usually first seen around the scapulae, but then spread to involve the trunk and upper thighs. These skin eruptions, first noted in Hunter’s original presentation, are said to be pathognomic for MPS II [32]. Development Infants with MPS II appear normal at birth, and early developmental milestones may also be normal, even in the presence of significant somatic disease. Developmental outcomes, however, are highly variable. Significant psychosocial problems occur in affected teenagers and young adults [40]. Even in patients with attenuated disease, cranial magnetic resonance imaging (MRI) scans are often grossly abnormal, with extensive white matter changes as well as dilated perivascular spaces, despite apparently normal intellectual skills [27]. Patients with more severe MPS II also appear normal at birth, and early developmental milestones may be normal. Some patients fail hearing screening tests in the first year, and speech delay is not unusual in more severely affected patients. By 18–24 months, developmental delay is usually apparent. Most patients make very slow progress after this stage, with a developmental plateau beginning between 3 and 5 years of age. Unlike children with severe MPS I, who are usually placid, more severely affected children with MPS II can be hyperactive and aggressive. By the time of death in their second decade, most patients with CNS involvement are severely mentally handicapped and dependant on care providers for all their needs. Biochemical diagnosis Assessing urinary GAGs (heparan and dermatan sulphates) is the usual first screening test for MPS II, and can be quantitative (measurement of total urinary uronic acid) or qualitative (GAG electrophoresis). Although neither test can offer a definitive diagnosis, abnormal measurements indicate the likely presence of an MPS disorder. The sensitivity of these tests, however, is not perfect, particularly when measurements are made on samples of dilute urine. Definitive diagnosis is established by enzyme assay in leukocytes, fibroblasts or plasma, using substrates specific for I2S. Another sulphatase should be measured in conjunction, in order to exclude multiple sulphatase deficiency. This is a much rarer disorder than MPS II and is generally associated with a very poor prognosis. It should be noted that the amount of enzyme activity measured in vitro from cellular extracts does not indicate where an individual falls within the MPS II spectrum of disease severity. Prenatal diagnosis Prenatal testing is available for foetuses at risk of MPS II. This is generally carried out by enzyme assay of I2S in uncultured chorionic villi, allowing early testing and rapid diagnosis of affected foetuses. Very low activities have been measured in some pregnancies with a (heterozygous) female foetus, emphasising the need to combine an enzyme assay with foetal sex determination [7]. Molecular-genetic prenatal testing can be performed if the mutation is known in the family. Carrier testing Previous methods of carrier detection based on an I2S assay in either plasma or hair roots are now known to be unreliable, as there is considerable overlap between the normal and heterozygous ranges. The only definitive test for determining carrier status is based on DNA analysis. In a small number of families, however, the mutation remains elusive, despite full sequencing of the I2S locus. Symptomatic treatment Even with the introduction of ERT, patients with MPS II still require supportive symptomatic treatment from a wide range of specialists. A comprehensive initial assessment of each patient at diagnosis should therefore be undertaken, and should be followed by regular reviews. Supportive management and the anticipation of possible complications can greatly improve the quality of life of affected individuals and their families. Family members should be offered genetic counselling, and contact with other affected families, patients and support groups can be helpful. Skeletal abnormalities Orthopaedic complications, caused by a combination of direct bone involvement and severe arthropathy, can lead to significant disability. The destructive arthropathy, which especially affects the hip joints, is a feature of the skeletal disease in some patients and may be due to secondary events occurring within chondrocytes and/or osteoblasts as a result of storage. Progressive arthropathy may affect all joints and leads to severe restriction of motion. The hip joints appear to be particularly vulnerable and severe erosive hip dysplasia can be especially disabling. Poor hand function, due to the characteristic claw-hand deformity, carpal tunnel syndrome and interphalangeal joint stiffness, is also common. Abnormal joint function is largely a result of both metaphyseal deformities and thickened joint capsules. Secondary erosive joint disease is particularly disabling and is extremely difficult to manage medically and/or surgically. The deformed acetabulum and pelvis makes prosthetic surgery a challenge, and many patients become wheelchair-bound because of hip pain. The role of physical therapy in MPS II is not well studied, but range-of-motion exercises appear to offer some benefit in preserving joint function and should be started at an early age. If significant restriction of joint movement has already occurred, range-of-motion exercises may slow further progression. Cardiovascular system Cardiac abnormalities detected by echocardiography are common in patients with MPS II. Valvular involvement, with thickening and stiffening of the valve leaflets, commonly leads to mitral and aortic regurgitation and/or stenosis [24]. Cardiomyopathy is much less common but may be associated with an increased risk of cardiac arrhythmia [14]. Valve replacement surgery may be necessary, and annual cardiac evaluation with echocardiography is essential. Bacterial endocarditis prophylaxis should be used where appropriate. The prevalence and age at onset of the main cardiovascular signs and symptoms as reported in HOS are shown in Table 2. Table 2Prevalence and reported age at onset (median and 10th–90th percentiles) of the main cardiovascular manifestations of mucopolysaccharidosis type II (Hunter syndrome) in a cohort of 82 patients in HOS, the Hunter Outcome SurveyCardiac manifestationnPrevalence (%)Age at onset (years)Valvular disease34536.2 (2.9–13.8)Murmur32526.4 (3.7–12.3)Cardiomyopathy497.6 (5.2–27.3)Any cardiovascular sign/symptom49726.0 (2.9–13.7) Respiratory disease and upper airway manifestations Chronic recurrent rhinitis and persistent copious nasal discharge without obvious infection are common. Enlargement of the tonsils and adenoids, a narrowed trachea, tracheomalacia, thickened vocal cords, redundant tissue in the upper airway, and an enlarged tongue can contribute to upper airway complications [4, 26, 39]. The upper airway involvement leads to noisy breathing, particularly at night, and is probably a major component of obstructive sleep apnoea, which is a common complication in the later stages of the disease [16]. Because of the airway disease, there is a high risk associated with anaesthesia in these patients [17]. In patients with attenuated MPS II, rhinorrhoea tends to improve with age, but upper airway obstruction and sleep apnoea become more troublesome with increasing age. A progressive restrictive respiratory defect also becomes apparent with increasing age, mainly due to progressive changes in the thoracic skeleton. Sleep studies should form part of the regular assessment schedule, and significant episodes of hypoxia should be managed by continuous or bilevel positive airway pressure devices. Severely affected patients often find such treatment difficult to tolerate. In these patients, supplemental oxygen alone may be an acceptable alternative, but should be used with caution in patients with documented hypercapnia. Tonsillectomy and adenoidectomy are frequently performed to correct Eustachian tube dysfunction and to decrease airway obstruction. Severely affected patients also tend to have frequent ear infections and constant rhinorrhoea. Early placement of ventilating tubes is recommended in severely affected individuals. Gastrointestinal system Protuberance of the abdomen and hernias caused by progressive hepatosplenomegaly are common. Although organ size can become massive, storage of GAGs in the liver and spleen does not lead to either liver or splenic dysfunction. Patients are prone to periodic bouts of watery diarrhoea, which occur without apparent cause and are not associated with malabsorption. Rectal biopsies in affected patients have demonstrated storage within gut neural cells, and an autonomic cause for episodes of diarrhoea has been postulated [10]. With age, loss of muscle strength and physical inactivity lead to constipation. Abdominal hernias should be repaired surgically, though recurrence can occur. Diarrhoea can be controlled by diet and the use of antimotility drugs. Central nervous system Communicating high pressure hydrocephalus, which is common in MPS I, is rare in MPS II. Other CNS complications, such as seizures, are more common. They are usually tonic–clonic in nature and respond to standard anticonvulsant treatment. Progressive compression of the spinal cord with resulting cervical myelopathy due to thickening of the dura (hypertrophic pachymeningitis cervicalis) and hyperplasia of the transverse ligament is common [21]. In addition, MRI scans of the craniocervical junction will often show deposition of GAG around the tip of the odontoid process. Most patients have a well-formed odontoid process and atlanto-axial subluxation is usually not a feature of MPS II. Cervical myelopathy may initially present as reduced activity, exercise intolerance or difficulty in rising from a sitting position. If it remains untreated, irreversible cord damage can occur. Hypertrophic pachymeningitis cervicalis and cervical compression secondary to hyperplasia of the transverse ligaments should be quickly and aggressively treated in patients with attenuated disease. Early and careful cervical decompression performed by an experienced team may prevent severe and devastating consequences. Peripheral nervous system Carpal tunnel syndrome is common in patients with attenuated forms of MPS II and should be promptly investigated and treated. Nerve conduction studies should be conducted to monitor patients, as the typical symptoms of compression (pain, tingling or numbness) do not occur [13]. Surgical decompression of the median nerve at an early stage of involvement results in either partial or complete improvement in most patients. Visual problems Although corneal opacity has been reported [31], it is not generally a feature of MPS II and helps to distinguish the disorder clinically from severe MPS I and MPS VI. In addition glaucoma is rarely present, even in severely affected patients. Retinal degeneration resulting in decreased peripheral vision and poor dark adaptation is common, but often difficult to investigate fully in a severely affected patient. Disc oedema, uveal effusions and epiretinal membranes have all been reported as part of the variable ocular pathology [2, 20, 37]. Electroretinography confirms retinal degeneration in many patients, but a disturbance of vision apart from nyctalopia (‘night blindness’) is rare. Chronic papilloedema without evidence of raised intracranial pressure is common and may be due to deposition of GAGs within the sclera causing pressure on the optic nerve at the interscleral level [2]. Regular, annual, ophthalmological assessment is required and should include a measure of intraocular pressure. Spectacles should be prescribed as appropriate. Hearing Hearing loss is common in patients with severe MPS II and is correlated with the severity of somatic disease. Manifestations leading to hearing loss include frequent middle ear disease secondary to Eustachian tube dysfunction, dysostosis of the ossicles of the middle ear, scarring of the tympanic membrane, and damage to the eighth nerve. Audiological abnormalities usually consist of conductive deafness early in the course of the disease but as the child ages a sensorineural element appears and most patients have combined deafness once the disease is fully established [29]. Moderate to severe hearing loss develops in most adults with attenuated MPS II. In most patients there is a mixture of both conductive and sensorineural deafness [22]. Appropriate regular audiology assessment and provision of the correct auditory aids are required. Education and behaviour Given the intellectual involvement in the more severely affected patients, it is important that infants with MPS II are provided with a stimulating environment to encourage as much learning as possible during the early stages, as some skills may be retained during the later period of general deterioration. For patients with more attenuated forms of the disease, deafness and physical limitations may impact on education. Educational assessments of special needs should be undertaken to ensure that an appropriate educational environment is provided. This may be in a mainstream school with additional help or within a special school system. Many patients with severe MPS II exhibit hyperactivity and aggression. Psychological assessment, behaviour management and the use of medication should be considered in these patients. The hyperactivity generally responds poorly to methylphenidate, and over-sedation is a risk with other forms of medication. Anaesthetic complications All patients with MPS II present major anaesthetic risks, and death can result if appropriate precautions are not taken [12]. Patients should only undergo general anaesthesia in centres staffed with anaesthesiologists experienced in these disorders. The most important complications relate to the following. Dysostosis multiplex leading to rigidity of the neck and spine. Although instability of the spine is rare, significant cervical compression may be present in patients with attenuated disease. This can result in sudden neurological deterioration if excessive neck manipulation is performed during attempts to intubate the patient.Induction may be difficult because of an inability to maintain an adequate airway.Intubation may require smaller-than-anticipated endotracheal tubes and a narrow trachea and thickened vocal cords will impede view. It is recommended that a paediatric pulmonologist or ear, nose and throat specialist should be present during intubation. Fibre-optic laryngotracheoscopy is usually essential.Extubation can also be hazardous for these patients, and there is an increased incidence of post-obstructive pulmonary oedema.Recovery from anaesthesia may be slow, and post-operative airway obstruction is always a risk. Enzyme replacement therapy Recombinant I2S (idursulfase; Elaprase, Shire Human Genetic Therapies, Cambridge, MA, USA) has recently been licensed for treatment of patients with MPS II in the United States, European Union and Switzerland. Idursulfase is produced in a continuous human cell line and is a purified form of the natural lysosomal enzyme I2S. Mannose-6-phosphate (M6P) residues on the oligosaccharide chains of the glycoprotein enzyme allow specific binding of idursulfatase to M6P receptors on the cell surface, leading to cellular internalisation and targeting of the enzyme to lysosomes, and subsequent catabolism of accumulated GAGs. Phase I/II trial Twelve patients were enrolled in a randomized, double-blind, placebo-controlled trial of idursulfase for 24 weeks, followed by an open-label extension study. Urinary GAGs were reduced within 2 weeks of initiating idursulfase treatment and remained low for the 48 weeks of the study (P < 0.0001). Both liver and spleen volumes were decreased at 24 weeks (P < 0.01) and 48 weeks (P < 0.001). The distance that patients could walk in 6 min (6-min walk test) increased by an average of 48 m after 48 weeks (P = 0.013). Six patients developed IgG antibodies that did not appear to influence the clinical response to idursulfase [18]. Phase II/III trial A multinational, randomized, double-blind, placebo-controlled trial was performed to evaluate the safety and efficacy of idursulfase, 0.5 mg/kg administered weekly, compared with placebo. Additionally, the trial evaluated idursulfase, 0.5 mg/kg every other week, compared with placebo. Ninety-six patients were randomized to one of three groups with each patient receiving a total of 52 infusions of either idursulfase, idursulfase alternating weekly with placebo, or placebo. The primary efficacy endpoint of the trial was a composite of two clinical measures – forced vital capacity and the 6-min walk test. Patients receiving the weekly dosing regimen of idursulfase showed a statistically significant improvement in the primary efficacy endpoint (P < 0.005) compared with placebo. Patients receiving the alternate-week dosing regimen of idursulfase also showed a statistically significant improvement (P < 0.05) compared with placebo [19]. Treatment with idursulfase was generally well tolerated. The most common adverse events observed were associated with the clinical manifestations of MPS II. Of the adverse events considered possibly related to idursulfase, infusion-related reactions were the most common and were generally mild. There were two patient deaths during the study, both of which were considered unrelated to treatment with idursulfase. IgG and IgM antibodies were observed in the idursulfase-treated patients at some point during the course of the study. When antibodies occurred, there was a transient increase in urinary GAGs, presumably due to a neutralising effect. This did not correlate with any of the clinical variables measured (Shire HGT, data on file). No IgE antibodies were observed and no patient withdrew from the trial due to an adverse event considered related to idursulfase [19]. Current treatment with idursulfase Idursulfase is given by weekly intravenous infusion over 3 h at a dose of 0.5 mg/kg diluted in an appropriate volume of saline (according to weight). Patients may receive premedication with antipyretics and/or antihistamines at the discretion of the prescribing physician. Patients who have experienced infusion-related reactions should be premedicated for subsequent infusions. Consideration should be given to the possibility of sodium overload in small infants, and, if necessary, the total volume of the infusion can be decreased to 50 ml. Although the treatment is generally safe and infusion-related reactions are no more frequent than with other protein-based therapies, anaphylactoid reactions have been reported. Early response to ERT The response to ERT appears to depend on the severity of the individual’s condition and the age at which the treatment begins. In our experience, the first sign of efficacy in most young (prepubertal) patients is usually an enhanced feeling of wellbeing and greater energy, manifesting as an increased ability to take part in normal daily activities. By 2 months, most patients have urinary GAG levels approaching the normal range and there is usually evidence of a reduction in size of the liver and spleen. After a further month of therapy there is often an improvement in soft-tissues joint contractures and the beginning of a growth spurt. By 6 months of treatment, most patients have improved the distance that they can achieve in the 6-min walk test and most show stabilisation (if not improvement) in pulmonary function tests. Most of the improvement occurs in the first 12–18 months of treatment. After this period of time, gains are very slow and the condition of most patients will generally have stabilised, although physicians still need to be vigilant and look actively for possible disease-related complications. ERT for particular patient groups Treating very young patients Experience of treating children under the age of 5 years with idursulfase is limited, as the clinical trials enrolled patients above this age so that the patients would be able to comply with and be able to cooperate with repeated pulmonary function and endurance testing. The following is therefore based on our personal clinical experience. Treating patients at a very young age leads to practical problems with the infusions. These may be resolved with indwelling vascular access devices. Outcomes are likely to be better in patients treated from a very young age, but there are currently no recommendations on how to monitor the response to treatment, especially in patients either too young to cooperate with the tests used in the clinical trials (pulmonary function tests and 6-min walk test) or in patients who are not yet showing symptoms of the disease. This situation would be improved by identifying a suitable biomarker that would reflect the disease burden and that would respond promptly to therapy. We believe that these challenges should not prevent the introduction of treatment in patients under the age of 5 years after discussion with parents. Careful clinical follow-up, however, will be necessary in these patients, accompanied by regular (annual) developmental assessments. As of October 2006, there were 12 patients below 5 years of age in HOS. Treating patients with severe CNS involvement Idursulfase is not expected to cross the blood–brain barrier and therefore would not be expected to lead to any improvement in CNS dysfunction in patients with severe MPS II. In most patients there is likely to be some initial benefit in terms of enhanced respiratory function due to airway improvement and reduction in size of the liver and spleen. In addition, joint mobility may improve as a result of ERT on peri-articular soft tissue. The burden of weekly intravenous therapy may be significant in some patients with very severe CNS disease. The authors believe that patients with severe CNS involvement may be offered the possibility of treatment for a ‘trial’ period of 12–18 months, after which time a decision should be made as to whether to continue. Decisions on whether to start and continue treatment should be made only after detailed discussion with the parents. Home therapy More than 90% of patients treated with idursulfase have had no infusion-related reactions and, in those who do, the reactions are mostly minor and respond promptly to reducing the rate of infusion and using antipyretics and antihistamines for future treatments. Under these circumstances, it would appear that idursulfase is a therapy that could be administered safely in the patient’s home after a minimum of 12 infusions in hospital. Depending on the labelling and clinical practices in particular countries, a logical approach would appear to be to consider home therapy for those patients with minimal or no obstructive airway disease (as judged by sleep study and pulmonary function tests). It should be stressed, however, that ERT during respiratory infections or other intercurrent illnesses are contraindicated. Treatment should be postponed under such circumstances, regardless of whether treatment is given in the hospital or home setting. Management of infusion reactions Infusion-related reactions appear to be of two types: those occurring during the infusion and ‘late’ reactions occurring 12 h or more after the infusion. The classic reactions occurring during the infusion, with fever, chills and urticaria, respond to temporarily stopping the infusion, administering paracetamol (acetominophen) and antihistamines, and restarting the infusion after at least 30 min but at a slower rate. Before subsequent infusions, patients should be premedicated with acetominophen and antihistamines 1 h before the infusion. If the reactions continue despite this premedication, consideration should be given to pretreatment with corticosteroids; for example, prednisolone, 1 mg/kg, 12 h and 1 h before infusion. Late reactions typically consist of a sunburn-type rash and mild wheezing. The rash can be managed as above, but the wheezing requires bronchodilator treatment and, possibly, oxygen supplementation. All patients who have significant infusion-associated reactions should have specific immune testing. Reducing the rate of infusion is mandatory in these patients. To assess the efficacy of ERT in individual patients and to ensure the appropriate management of possible infusion-related reactions, it is recommended that the treatment of patients receiving ERT is overseen by experienced physicians in centres with expertise in treating lysosomal storage diseases. Haematopoietic stem cell therapy The potentially beneficial effect of HSCT on lysosomal storage diseases is thought to be due to the replacement of deficient macrophages with marrow-derived donor macrophages (Kupffer cells – pulmonary, splenic, nodal, tonsillar and peritoneal macrophages – and microglial cells) which constitute an ongoing source of enzyme capable of gaining access to various storage sites. HSCT has been successful in modifying the course of the disease in patients with severe MPS I and MPS VI. In a single small study of HSCT in MPS II, however, the results of treatment were not positive, with nine of ten patients continuing to exhibit progressive neurodegeneration [36]. The future Whilst ERT is likely to benefit many patients with MPS II, the problems associated with severe CNS involvement are unlikely to be solved by intravenous ERT. Other methods of treatment, including alternative routes of enzyme administration, will therefore need to be studied. Direct administration of enzyme into the cerebrospinal fluid, either into the ventricle or lumbar space, has not prevented progressive neurological deterioration in another lysosomal storage disorder, type II Gaucher disease [28]. This approach has not been attempted in MPS II, and formal studies are required, perhaps with the addition of immunosuppression to prevent antibody formation. Currently, there are no suitable methods for infusion directly into the brain, and repeated intracerebral injections would not be practical. It may be that to treat CNS disease more effectively, peripheral ERT may have to be combined with other approaches, such as cell-based therapies, perhaps involving gene enhancement or replacement. Gene therapy is the logical approach for treating disorders of the CNS, and animal work on other MPS disorders is beginning to show promise [5, 30]. Choice of vector, the use of immunosuppression and the route of administration are all issues that need addressing before the first human clinical trials of gene therapy involving MPS disorders are performed. Use of nanoparticles containing engineered myoblasts expressing I2S has been reported. Scaling-up studies, however, are required to evaluate the feasibility of such devices [11]. Conclusion MPS II (Hunter syndrome) is a severe progressive multisystemic disorder that has the potential to cause disease in most body systems and is usually fatal in the second or third decade of life. Ideally, management should be centralised in major medical centres with access to all medical specialties. Management is multidisciplinary and a holistic approach to the patient is required, especially for those who have severe neurological involvement. ERT is an important new therapy that has the potential to help many patients, providing that it is started early in the course of the disease. CNS disease remains a major challenge and an innovative approach to treatment will be needed if this is to be addressed fully.

Breast_Cancer_Res_Treat-3-1-2001219

Letrozole in advanced breast cancer: the PO25 trial

Tamoxifen has been a standard first-line endocrine therapy for post-menopausal women with hormone-responsive advanced breast cancer, but more than half of patients fail to respond and time to progression is less than 12 months in responders. The third-generation aromatase inhibitors were developed to provide more effective alternatives to tamoxifen. In the Femara Study PO25, post-menopausal women with advanced breast cancer were randomized to receive letrozole 2.5 mg (n = 453) or tamoxifen 20 mg (n = 454) given orally daily until progressive disease occurred. Patients were permitted to cross over to the other treatment at progression. In the primary efficacy analysis, median time to progression (TTP) was significantly longer with letrozole than with tamoxifen (9.4 months vs. 6.0 months, respectively; P < 0.0001). The objective response rate (ORR) was significantly higher for letrozole than for tamoxifen (32% vs. 21%; P = 0.0002). Prospectively planned analyses of the intent-to-treat population showed that letrozole significantly improved overall survival (OS) compared with tamoxifen over the first 24 months of the trial. An exploratory analysis of patients, who did not cross over, indicated a median OS benefit of 14 months for letrozole compared with tamoxifen. Letrozole is the only third-generation aromatase inhibitor that has demonstrated significant improvements in ORR, TTP, and early OS. Introduction and rationale The treatment goals for advanced or metastatic breast cancer (MBC) are to delay disease progression and to prolong survival [1, 2] and to optimize patient care in terms of ameliorating symptoms, thereby improving or maintaining quality of life [3–5]. Although treatment may include surgery and radiation therapy for the treatment of locally advanced tumors or isolated metastases, systemic therapies (endocrine, cytotoxic, biologic, and palliative) are the foundation of disease management [6, 7]. Systemic therapy for patients with advanced breast cancer should be tailored according to specific tumor biology, particularly with respect to hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status, the growth rate of disease, presence of visceral metastases, history of prior therapy and response, susceptibility to treatment-related toxicity, and individual patient preference [7–14]. Systemic therapy can prolong survival and enhance patient quality of life but is not curative [1]. Consequently, minimally toxic endocrine therapies are generally preferred to cytotoxic therapy as initial therapy for patients with hormone-responsive tumors [6, 15]. Since the 1980s, endocrine therapy with tamoxifen was well established as a standard first-line treatment for post-menopausal women with advanced breast cancer, even though estrogen receptor (ER) expression was not always used routinely to select patients for endocrine therapy [16–18]. The first-generation aromatase inhibitor aminoglutethimide or a progestin such as megestrol acetate has provided a reasonable second-line alternative [19–22]. The objective response rate (ORR) to tamoxifen was shown to be in the range of 25%–45% [16, 17, 19, 21, 23–32], indicating that more than half of the patients with advanced breast cancer are intrinsically resistant to tamoxifen. Furthermore, the short median time to treatment failure (TTF), in the range 6–8 months, demonstrates a relatively rapid emergence of resistance in patients initially sensitive to tamoxifen [19, 27]. Loss of ER expression appears to be the dominant mechanism of de novo resistance, and most ER/progesterone receptor negative (PgR−) tumors do not respond to tamoxifen [18, 33–36]. However, the majority of patients who develop acquired tamoxifen resistance still express ER at the time of progression [37, 38] and may respond to alternative endocrine therapies [39]. The third-generation aromatase inhibitors letrozole, anastrozole, and exemestane were developed in the search for more effective therapeutic alternatives to tamoxifen. Aromatase inhibitors prevent estrogen synthesis by potently inhibiting the aromatase enzyme, which converts androgens to estrogen [40]. Unlike tamoxifen, the aromatase inhibitors do not have any partial estrogen-agonist activity [41] and are less susceptible to the emergence of resistance associated with long-term estrogen deprivation [42]. The development and mechanism of action of aromatase inhibitors is described in detail in the article by Dr. Bhatnagar in this supplement. Studies of aromatase inhibitors in the second-line setting The initial randomized controlled trials of third-generation aromatase inhibitors were conducted in patients with advanced breast cancer in whom tamoxifen had failed (i.e., second-line setting). Letrozole, anastrozole, and exemestane all demonstrated evidence of clinical superiority to megestrol acetate in the second-line setting [43–47]. Thus, the individual trials demonstrate a trend or even a significant difference in favor of the third-generation aromatase inhibitors in one or more efficacy end points; in addition, the aromatase inhibitors were shown to be associated with improved tolerability versus comparator endocrine therapy in these randomized trials. One trial demonstrated a significantly higher ORR for letrozole (2.5 mg dose) compared with megestrol acetate (24% vs. 16%, respectively; P = 0.04) and a trend toward longer time to progression (5.6 vs. 5.1 months, P = 0.07) [45]. In this trial, low-dose letrozole (0.5 mg) was associated with similar efficacy outcomes compared with megestrol acetate. However, in another similarly designed trial with letrozole versus megestrol acetate, overall response rates with the two doses of letrozole (0.5 and 2.5 mg) and with the comparator were similar (21%, 16%, and 15%, respectively). In this trial, low-dose letrozole was superior to megestrol acetate in terms of time to progression (TTP) (P = 0.044) and survival (P = 0.053). Differences in the distribution of baseline variables may explain the different outcomes in the two trials in terms of the superiority of letrozole over megestrol acetate according to dose [48]. Letrozole was significantly better tolerated than megestrol acetate, specifically in terms of serious adverse experiences, discontinuation due to poor tolerability, cardiovascular side effects, and weight gain [45]. Third-generation aromatase inhibitors have demonstrated greater potency and selectivity than the first-generation compound aminoglutethimide [49]. Two doses of the most potent aromatase inhibitor letrozole (2.5 mg and 0.5 mg) [49] were compared with aminoglutethimide in a randomized controlled trial in the second-line setting and demonstrated superior efficacy and improved safety [50]. The higher dose of letrozole showed a trend (P = 0.06) toward superior ORR (19.5%) compared with aminoglutethimide (12.4%). Letrozole 2.5 mg was also significantly superior in TTP, TTF, and overall survival (OS). Fewer patients taking letrozole experienced adverse events than those taking aminoglutethimide (33% vs. 46%) [50]. Letrozole has also been compared with anastrozole in a randomized, unblinded trial in the second-line setting in patients with MBC. The trial showed that letrozole was associated with a statistically higher ORR than anastrozole (19.1% vs. 12.3%, respectively; P = 0.013), whereas TTP (the major end point), TTF, and clinical benefit and duration of response were similar between the two agents [51]. Both letrozole and anastrozole were well tolerated, and a similar incidence of adverse events was observed in the two groups. These studies generated the hypothesis that letrozole might have superior efficacy to tamoxifen as first-line therapy for advanced breast cancer. A large clinical trial (Femara Study PO25) was therefore conducted to compare the efficacy and tolerability of letrozole with those of tamoxifen as first-line therapy in post-menopausal women with advanced breast cancer [52]. This review will describe the results of the PO25 trial, highlighting the evidence for the superiority of letrozole over tamoxifen as first-line endocrine therapy in this setting. Trial design and patients The Femara Study PO25 was the largest phase 3 trial conducted in the advanced breast cancer setting [52, 53]. This randomized, double-blind, double-dummy trial was powered for superiority and needed to enroll approximately 900 patients to demonstrate a 20% reduction in the risk of progression with the more effective treatment. To achieve the recruitment target, the trial was conducted in 201 centers in 29 countries. Local ethics review boards approved the protocol, and all patients gave written informed consent before study enrollment. Randomized trial design Patients were randomized to receive letrozole 2.5 mg or tamoxifen 20 mg given orally daily until progressive disease occurred. Patients were permitted to cross over from 1 treatment arm to the other in a double-blind fashion if their first-line treatment was discontinued because of progressive disease or for any other reason (Fig. 1). Patients in whom endocrine therapy was discontinued were subsequently treated as clinically indicated, using chemotherapy, trastuzumab, and bisphosphonates. The crossover design was an integral part of the study, and it probably affected the assessment of OS. Fig. 1Study design Patient population Post-menopausal women with advanced breast cancer, defined as stage IIIB locally advanced disease, locoregionally recurrent disease that was not amenable to surgery or radiotherapy, or metastatic disease, were eligible for inclusion in the trial. All patients presented with measurable or assessable tumors and were candidates for endocrine therapy. Patients had estrogen receptor-positive (ER+) and/or progesterone receptor-positive (PgR+) tumors or unknown HR status. One prior chemotherapy regimen for the treatment of metastatic disease was permitted, but recurrence during or within 12 months of adjuvant antiestrogen therapy and any prior endocrine therapy for advanced breast cancer precluded enrollment. End points The primary end point was TTP, defined as the interval between date of randomization and the earliest date of disease progression. Disease progression was determined on the basis of tumor progression (an increase of 25% or more in measurable lesions, an estimated increase of the same magnitude of nonmeasurable lesions, or the appearance of new lesions), treatment discontinuation with evidence of clinical deterioration due to breast cancer, death due to breast cancer, or death of unknown cause (with documented evidence of clinical deterioration due to breast cancer) while receiving treatment or within 6 weeks of discontinuation of treatment. The secondary end points were ORR, duration of overall response, rate and duration of clinical benefit, TTF, time to response (TTR), time to chemotherapy (TTC), safety, and OS. ORR was defined as the proportion of patients who achieved a complete response (CR) or a partial response (PR), confirmed by a second evaluation 1–3 months later. The duration of overall response was defined for patients with CR or PR, as the interval between date of randomization and the earliest date of disease progression. The rate of clinical benefit was defined as proportion of patients who achieved CR or PR or who stabilized (NC) for at least 24 weeks; the duration of clinical benefit was defined for patients who achieved CR or PR or NC as the interval between date of randomization and the earliest date of disease progression. TTF was defined as the interval between date of randomization and the earliest date of disease progression, withdrawal, lost to follow-up, or death. TTR was defined for CR or PR patients as the interval between randomization and the earliest documentation of response, and TTC was defined as the total duration of endocrine therapy. The duration of OS was defined as the interval between randomization and death for any reason. Exploratory analyses of OS were performed. The first analysis included all patients with censoring at crossover, whereas the second included only patients with no crossover. The latter group predominantly comprised of patients with “nonresponsive” disease (patients who responded to first-line therapy are more likely to be crossed over later at progression), whereas the former included “nonresponsive” as well as “responsive” patients. Efficacy The characteristics of the 907 patients included in the intent-to-treat (ITT) population were well balanced between the letrozole and tamoxifen arms. The median age of the patients was 65 years (range 31–96 years) in the letrozole arm and 64 years (range 31–93 years) in the tamoxifen arm. Patients were predominantly white (86%), and 92% had Karnofsky performance status (KPS) scores of 80–100. The majority (93%) of the study population had metastatic disease. Soft tissue lesions were the dominant metastatic site in one quarter of patients and were present in 63% and 61% of patients in the letrozole and tamoxifen arms, respectively. Bone metastases were the dominant metastatic site in approximately 30% of patients and were present in 54% and 50%, respectively. Visceral metastases were the dominant site in 43% of patients in the letrozole arm and 46% of the patients in the tamoxifen arm. Most patients (71% in the letrozole arm and 66% in the tamoxifen arm) had not received any prior chemotherapy, and few had received chemotherapy for advanced disease (9% and 11%, respectively). The majority of patients (109 of 167) treated with adjuvant tamoxifen received at least 2 years of therapy, and the treatment-free interval between stopping adjuvant therapy and entering the study was more than 2 years in 126 of 167 patients. Of the 907 patients included in the ITT efficacy population, 467 crossed over to the other treatment arm, 75 continued on first-line therapy without progression, and the remainder terminated first-line treatment without crossover (Fig. 2). Fig. 2Patient disposition Letrozole was superior to tamoxifen for all primary and secondary efficacy end points, including a prospectively planned survival analysis at 1- and 2-year follow-up [53]. Time to progression In the primary efficacy analysis, the median TTP was significantly longer with letrozole than with tamoxifen (9.4 months vs. 6.0 months, respectively; P < 0.0001) (Fig. 3) [53]. Of patients in the letrozole arm, 359 (79%) progressed, compared with 387 (85%) in the tamoxifen arm. The hazard ratio of 0.72 represents a 28% reduction in the risk of disease progression with letrozole (P = 0.0001). Fig. 3Time to progression at median follow-up of 32 months for patients on first-line letrozole versus tamoxifen. Reprinted from ref. [53] with permission from the American Society of Clinical Oncology The significant improvement in TTP with letrozole was confirmed in supportive multivariate analysis of prospectively defined baseline covariates, including receptor status, prior adjuvant tamoxifen therapy, and dominant site of metastatic disease [52, 54]. The analysis showed that the risk of progression was increased by the presence of either visceral or bone metastases as the dominant site of metastatic disease compared with soft tissue as dominant site. In the multivariate analysis, the significant improvement in TTP with letrozole over tamoxifen (hazard ratio 0.70; 95% confidence intervals [CI] 0.60, 0.81; P = 0.0001) was similar to the benefit observed in unadjusted analysis and was significant for each individual covariate (P = 0.0001) [53]. Median TTP values for letrozole and tamoxifen in the different subgroups are shown in Table 1. In patients with nonvisceral metastases, the risk for progression was 25% lower with letrozole than with tamoxifen, whereas in patients with visceral metastases, excluding the liver, the risk for progression was 34% lower and the median TTP was almost twice as long with letrozole than with tamoxifen [54]. Although TTP was shortest for patients with liver lesions, the risk for progression was still 36% lower with letrozole than with tamoxifen in this subgroup [54]. Table 1Time to progression in different patient subgroups [57]SubgroupLetrozoleTamoxifenDominant disease site: soft tissuen113115Median TTP12.1 months6.4 monthsDominant disease site: bonen145131Median TTP9.5 months6.3 monthsDominant disease site: visceran195208Median TTP8.3 months4.6 monthsPatients who had n9483    received priorMedian TTP8.9 months5.9 months    antiestrogenHazard ratio (95% CI)a0.60 (0.43, 0.84)HR-positiven294305Median TTP9.4 months6.0 monthsHazard ratio (95% CI)a0.69 (0.58, 0.83)HR-unknownn159149Median TTP9.2 months6.0 monthsHazard ratio (95% CI)a0.77 (0.60, 0.99)TTP, time to progression; CI, confidence interval; HR, hormone receptoraHazard ratios < 1.0 indicate superiority for letrozole relative to tamoxifen Patients with prior adjuvant antiestrogen therapy benefited from letrozole in line with the total group, as did patients irrespective of positive or unknown receptor status of the primary tumor. A prospectively planned analysis by patient age (<70 years and ≥70 years) also demonstrated that median TTP was significantly longer for letrozole than for tamoxifen in both age groups (8.8 months vs. 6.0 months, respectively, in the younger group and 12.2 months vs. 5.8 months in the older group) [55]. Response to therapy Letrozole was associated with a significantly better response to therapy compared with tamoxifen [52, 53]. ORR was significantly higher for letrozole than for tamoxifen (32% vs. 21%; P = 0.0002), and the corresponding rate of CRs was also significantly higher for letrozole (9% vs. 3%; P = 0.0004). The rate of treatment failure was lower with letrozole (75%) than with tamoxifen (85%), and median TTF was significantly prolonged (9.0 months vs. 5.7 months, respectively; P < 0.0001). A supportive multivariate analysis of ORR, adjusted for the same covariates as used for the TTP analysis, showed that prior adjuvant tamoxifen, as well as visceral or bone metastases as the dominant site of metastases, significantly decreased the probability of achieving a response. The analysis also confirmed that letrozole significantly increased the probability of achieving a CR or PR compared with tamoxifen (odds ratio 1.80, 95% CI 1.32–2.47; P = 0.0002) and that the superiority of letrozole remained statistically significant for each of covariates (P = 0.001) [52]. ORRs achieved with letrozole and tamoxifen in the different subgroups are shown in Table 2. Table 2Objective response rate in different patient subgroups [57]SubgroupLetrozoleTamoxifenDominant disease site: soft tissuen113115ORR50%34%Dominant disease site: bonen145131ORR23%15%Dominant disease site: visceran195208ORR28%17%Patients who hadn8483    received priorORR26%8%    antiestrogenOdds ratio (95% CI)a3.85 (1.50, 9.60)HR-positiven294305ORR33%22%Odds ratio (95% CI)a1.78 (1.20, 2.60)HR-unknownn159149ORR30%20%Odds ratio (95% CI)a1.79 (1.10, 3.00)ORR, objective response rate; CI, confidence interval; HR, hormone receptoraOdds ratios >1.0 indicate superiority for letrozole relative to tamoxifen Overall survival The median OS was 34 months for the letrozole group and 30 months for the tamoxifen group (P = 0.53). Prospectively planned analyses of the ITT population showed that letrozole significantly improved OS compared with tamoxifen over the first 24 months of the trial [53]. A Kolmogorov-Smirnov analysis to compare the survival distributions in the 2 arms [56] demonstrated a significant difference in favor of letrozole between 6 and 20 months (P = 0.003) and showed that the maximum difference in survival occurred at 14 months; at this time point, there were 85 deaths (19%) in the letrozole arm compared with 132 deaths (29%) in the tamoxifen arm. In addition, repeated log-rank tests performed at 6-month intervals indicated that survival was significantly greater with letrozole between 6 and 24 months (6 months: P = 0.0167; 12 months: P = 0.0038; 18 months: P = 0.0010; 24 months: P = 0.0246) (Fig. 4). The OS curves for the letrozole and tamoxifen groups crossed at around 36 months, at which time point most patients had either crossed over to the other study drug or had switched to different second-line treatments [53]. Fig. 4Letrozole versus tamoxifen: patients alive at 6-month intervals. Reprinted from ref. [48] with permission from Elsevier Additional exploratory analyses were therefore performed to determine the influence of crossover on OS. The crossover design was an integral part of the trial and, as with all crossover designs, had a confounding influence on the assessment of OS. Second-line endocrine therapy is generally less effective than first-line treatment in patients responsive to first-line therapy [16]; therefore, evaluation of OS may be impaired if the second-line treatment is actually more effective than the original first-line treatment. Furthermore, patients who are responsive to first-line therapy are more likely to cross over than are patients with nonresponsive disease who do not obtain benefit from first-line therapy. Approximately 50% of patients crossed over to the other treatment arm (Fig. 2), and almost all of the crossovers had occurred by 36 months. The median time to crossover was longer for patients initially randomized to the letrozole arm (17 months for letrozole to tamoxifen vs. 13 months for tamoxifen to letrozole). The median OS from initial randomization, censoring time to death at crossover, was 42 months (95% CI 36 months to not estimable) for letrozole and 30 months (95% CI 27 to <36 months) for tamoxifen [53]. The superior efficacy of letrozole compared with tamoxifen was also indicated by an analysis of mortality rates and OS following crossover to the alternate treatment. The analysis showed that the mortality rate was substantially reduced (47% vs. 63%, respectively), and OS improved in patients who crossed over to second-line letrozole compared with those who crossed over to second-line tamoxifen (31 months; 95% CI 22–40 months vs. 19 months; 95% CI 17–24 months, respectively) [53]. This OS analysis included all patients censored at the time of crossover (i.e., both “nonresponsive” and “responsive” patients). A second exploratory efficacy analysis of OS included only patients who did not cross over to the other arm and thus predominantly comprised patients with nonresponsive disease. This second analysis, limited to the patients who did not cross over to the alternate drug at progression, indicated a median OS benefit of 14 months for letrozole (35 months; 95% CI 29–43 months) compared with tamoxifen (20 months; 95% CI 16–26 months) [57]. Time to chemotherapy Hormone therapy is the preferred treatment strategy for patients with hormone-responsive advanced breast cancer, except for those individuals with rapidly progressive disease for whom initial chemotherapy is indicated [15]. Extending the TTC is thus an important goal with hormone therapy and can maintain quality of life without having a detrimental effect on outcome. In the PO25 trial, TTC was significantly longer for patients whose initial treatment was letrozole compared with those initially randomized to receive tamoxifen (16.3 vs. 9.3 months; P = 0.005). Safety Both letrozole and tamoxifen were well tolerated [52, 53]. The incidence of adverse effects related to study drug during first-line treatment was similar for letrozole (38%) and tamoxifen (37%). Hot flushes (16% and 13%, respectively), nausea (6% and 6%, respectively), and hair thinning (5% and 3%, respectively) were the most common treatment-related adverse events reported. Bone fractures of any etiology occurred in 5.3% of patients in the letrozole group, compared with 4.2% in the tamoxifen arm, resulting in fracture rates per patient-year of treatment of 0.0427 and 0.0451, respectively [52]. A quality-adjusted time without symptoms or toxicity (Q-TWiST) follow-up study assessed the trade-offs between progression-free survival and toxicity in the ITT population from the PO25 trial [58]. The Q-TWiST approach quantitatively adjusts periods in which treatment toxicities or symptoms of disease progression are present to reflect the potentially reduced value for the patient; this methodology divides the survival time of the patient into various health states, assigns utility states to each, and compares treatments based on OS experience [59]. The Q-TWiST analysis of the clinical trial data from the PO25 trial showed that the longer TTP with letrozole compared with tamoxifen is achieved without increased time with adverse events (2.2 vs. 2 months, respectively), resulting in a significantly greater quality-adjusted survival for patients on letrozole (2.5-month advantage; P < 0.0001) [58]. Time to worsening of KPS (decrease of ≥20 points) was significantly delayed for first-line letrozole compared with first-line tamoxifen (hazard ratio 0.62; P = 0.001) [54]. A subset analysis according to sites of metastases demonstrated that in patients with visceral metastases without liver involvement (mostly lung metastases), significantly fewer letrozole patients (14%) than tamoxifen patients (30%) experienced deteriorations in their KPS scores by ≥20 points [54]. However, KPS was relatively insensitive to change in these first-line patients. Cost-effectiveness In addition to its clinical superiority over tamoxifen, economic analyses have also shown that letrozole is highly cost-effective as first-line endocrine therapy in post-menopausal women with advanced breast cancer [60–62]. A follow-up analysis of patient data from the PO25 trial calculated the cost-effectiveness of first-line letrozole and tamoxifen by determining the ratio: difference in costs of breast cancer care to the difference in life years (LYs) between the two treatments [60]. The mean costs of care were $7323 and $5468 for letrozole and tamoxifen, respectively, representing $1855 in incremental costs with first-line letrozole. Mean LYs to death or to the end of first- or second-line hormonal therapy were 1.54 and 1.29 for patients randomized to first-line letrozole or tamoxifen, respectively. Thus, the incremental cost per LY saved with first-line letrozole vs. tamoxifen was $7420 (1855/0.25 = 7420) (2.5–97.5 percentiles $6470–$14,865). In another economic analysis conducted in the United Kingdom, data from the PO25 trial were used to estimate the effectiveness of treatment [61]. The analysis showed that the mean cost of providing first- and second-line hormonal therapy was GBP4765 for first-line letrozole and GBP3418 for first-line tamoxifen (a difference of GBP1347). Since patients receiving first-line letrozole gain an additional 0.228 LYs, or 0.158 quality-adjusted life years (QALYs), the cost-effectiveness analysis showed that first-line hormonal therapy with letrozole gains additional LYs at a cost of GBP5917, whereas the cost per additional QALY gained is GBP8514, which is well within the accepted cost range. The PO25 trial data were also used in a Canadian analysis that compared the cost-effectiveness of letrozole, anastrozole, and tamoxifen [62]. The analysis showed an incremental cost per quality-adjusted progression-free year of CAN$12,500 and CAN$19,600 for letrozole and anastrozole, respectively, relative to tamoxifen. The authors concluded that both letrozole and anastrozole are economically acceptable alternatives to tamoxifen. Conclusions The Femara Study PO25 has provided evidence from a well-powered, randomized, controlled trial to show that letrozole provides a significant advantage in OS compared with tamoxifen as first-line treatment of patients with advanced breast cancer [53]. Letrozole is the only aromatase inhibitor to demonstrate consistent superiority over tamoxifen in this setting [53, 54]. Randomized first-line therapy trials of anastrozole, as part of the TARGET study [63–66], and exemestane in the EORTC study [67, 68] have provided evidence of clinical equivalence or superiority to tamoxifen in post-menopausal women with advanced breast cancer. However, none of these trials demonstrated statistically significant improvements in all three end points (ORR, TTP, and OS) for the aromatase inhibitor compared with tamoxifen. The PO25 study was the largest of these randomized trials in the first-line setting and demonstrated extremely strong clinical benefits, evidenced by significant superiority in TTP and ORR, with letrozole compared with tamoxifen as first-line hormone therapy. The benefits of letrozole were observed in all patient subgroups, defined by prior antiestrogen therapy, dominant site of metastatic disease, HR status (positive or unknown), and age [52–55]. Furthermore, letrozole is the only aromatase inhibitor associated with an OS advantage for the first-line setting indication at 1-year and 2-year follow-up [53]. As demonstrated in the exploratory analysis of patients who did not cross over to the alternative treatment arm, letrozole prolonged OS by 14 months compared with tamoxifen. Thus, for every 100 patients treated with hormone therapy, eight more will be alive at 1 year if they receive letrozole instead of tamoxifen. In conclusion, third-generation aromatase inhibitors are effective and well tolerated. Letrozole should be considered as the first-line endocrine treatment in post-menopausal women with hormone-sensitive advanced or MBC. Of the available agents, only letrozole has demonstrated significant improvements in ORR, TTP, and early OS.

Matern_Child_Health_J-2-2-1592157

Prevalence of Risk Factors for Adverse Pregnancy Outcomes During Pregnancy and the Preconception Period—United States, 2002–2004

Objectives: To assess the prevalence of risk factors for adverse pregnancy outcome during the preconception stage and during pregnancy, and to assess differences between women in preconception and pregnancy. Methods: Data from the 2002 and 2004 Behavioral Risk Factor Surveillance System, United States, were used to estimate the prevalence of selected risk factors among women 18–44 in the preconception period (women who wanted a baby in the next 12 months, and were not using contraception, not sterile and not already pregnant) with women who reported that they were pregnant at the time of interview. Results: Major health risks were reported by substantial proportions of women in the preconceptional period and were also reported by many pregnant women, although pregnant women tended to report lower levels of risk than preconception women. For example, 54.5% of preconception women reported one or more of 3 risk factors (frequent drinking, current smoking, and absence of an HIV test), compared with 32.0% of pregnant women (p < .05). The difference in the prevalence of these three risk factors between preconception and pregnancy was significant for women with health insurance (52.5% in preconception vs. 29.4% in pregnancy, p < .05), but not for women without insurance (63.4% vs. 52.7%, p > .05). Conclusions: Women appear to be responding to messages regarding behaviors that directly affect pregnancy such as smoking, alcohol consumption and taking folic acid, but many remain unaware of the benefits of available interventions to prevent HIV transmission and birth defects. Although it appears that some women reduce their risk for adverse pregnancy outcomes after learning of their pregnancy, the data suggest that a substantial proportion of women do not. Furthermore, if such change occurs it is often too late to affect outcomes, such as birth defects resulting from alcohol consumption during the periconception period. Preconception interventions are recommended to achieve a more significant reduction in risk and further improvement in perinatal outcomes. Introduction Although the US has achieved substantial gains in improving the health of women and children, and infant mortality has reached record low levels, the US presently ranks 27th among established market economies in infant mortality [1]. Four recurring causes account for more than half of all infant deaths: birth defects, disorders related to short gestation and preterm birth, maternal complications of pregnancy (including complications of the placenta, cord and membranes), and sudden infant death syndrome [2]. In recent years some causes of infant mortality have increased, particularly in the percentage of births that were preterm and of low birth weight [3]. In 2002, congenital anomalies, low birth weight, preterm delivery, and maternal complications of pregnancy accounted for 14,263 (50.9%) of the 28,034 infant deaths [2]. Relatively little is known about the risk factors underlying the continued increase in these adverse outcomes. Adequate prenatal care has long been considered as an opportunity to reduce such risks. However, despite increases in access to and utilization of early prenatal care, interventions and efforts directed at addressing such risk factors fall short of their goal. Indeed, the effects of such efforts may have reached their peak, and new approaches may be necessary. Reviews on selected risk factors indicate that a large proportion of women enter pregnancy with pre-existing risks for adverse pregnancy outcomes. Although some women tend to take action to reduce their risk as soon as they learn that they are pregnant, the extent of pregnancy related change in risk factors varies considerably and often does not occur early in pregnancy when teratogenic effects are more pronounced. Moreover, post-pregnancy relapse is high [4]. For example in the period 1996–1998 the reported reduction in the use of alcohol, tobacco and illicit drugs in the first trimester was 46, 28, and 28% respectively [4]. Such information is important to the emerging emphasis on preconception care as a complementary approach to reduce risks to pregnancy. This paper provides nationally-representative estimates on risks during the preconception period and describes the apparent reductions in risk achieved during pregnancy for all known risk factors for which data are available. Data The estimates presented herein are based on data from the 2004 Behavioral Risk Factor Surveillance System (BRFSS) [5]. However, because not all risk indicators that were considered were included in the 2004 BRFSS, 2002 data were used for a small number of risk indicators for which the 2002 BRFSS provided the most recently available data. The BRFSS is an ongoing annual telephone survey of the non-institutionalized adult civilian population aged 18 years and older conducted in each state. The survey obtains information on a wide range of modifiable risk behaviors. In 2004 the median response rate for the BRFSS state surveys was 52.7 percent. This rate represents an estimate of the percentage of eligible respondents that completed telephone interviews, and is computed based on procedures recommended by the Council of American Survey Research Organizations (http://www.casro.org/resprates.cfm.) In comparison with other national surveys, BRFSS data appear to be of good quality [5]. Additional technical information is available online at http://www.cdc.gov/brfss/. The survey includes core modules asked in all states, rotating modules asked every second year in all states, and optional modules that are included only in some states. Being a general survey, the BRFSS lacks information on some factors of interest to the present topic, for example month of pregnancy for pregnant women, or information on prenatal care. Further, the BRFSS is not designed specifically to study all known risk behaviors at preconception or pregnancy. In addition, as an interview survey, the survey relies on self-reported data which contain an unknown level of reporting error. Identification of women in the preconception group in the BRFSS is made possible by questions introduced in the “family planning section” of the questionnaire. The availability of these questions makes it possible to compare risk behaviors among women about to become pregnant with those who are already pregnant on a large and nationally-representative sample of women. Women in the preconception period were identified as those who reported that they wanted a baby in the next 12 months, were not using contraception, were not sterile and were not already pregnant. Women who were pregnant were identified based on the response to the question “To your knowledge are you now pregnant?” Age of gestation was not assessed in this survey. Data were aggregated across states to make national estimates. For 2004 this resulted in a total national U.S. sample of 70,917 women aged 18 to 44 years, of whom 2308 (3.4%) were classified into the preconception period, and 2998 (4.7%) reported that they were pregnant at the time of their interview. The corresponding number of women in the 2002 are 61,284 women 18–44, with 2204 (3.7%) in the preconception period, and 2556 (4.6%) pregnant women. The analysis on folic acid and vitamins are based on surveys in 12 states in 2004. The results for these items are generalizable to the populations of these 12 states (shown in Table 2) but not to the entire United States population. Table 1Comparison of age and race/ethnicity, 2004 Women in preconception and pregnant (BRFSS) and Births (Vital Statistics data)BRFSSPreconception womenPregnant womenVital statisticsPercentCIPercentCIbirths PercentAge group 18–192.00.97.52.27.1 20–2416.33.326.53.026.1 25–2923.43.126.52.727.9 30–3428.43.126.22.824.4 35–3918.52.510.21.512.0 40–4411.42.23.10.92.6100.0100.0Race/ethnicity NH White64.93.960.13.456.8 NH Black11.92.511.51.913.6 Hispanic16.03.523.03.422.5 Others7.22.15.51.67.2100.0100.0100.0Note. For BRFSS estimates, 95% confidence interval = Percent +/− CI. Vital statistics data: Hamilton BE, Martin JA, Ventura SJ, Sutton PD, Menacker F. Births: Preliminary data for 2004. National vital statistics reports; vol 54 no 8. Hyattsville, Maryland: National Center for Health Statistics. 2005.Table 2Health risk indicators by pregnancy status Women 18–44, 2004 and 2002 BRFSSPreconception womenPregnant womenPercentCIPercentCI2004 data–nationwidea General Health  Poor/fair general health status8.32.16.41.7  14+ days in past month mental health not good*12.82.49.61.9  No health plan*18.83.511.92.3  No dental visit past year28.33.230.23.1  Told had diabetes*2.00.90.70.4 HIV  Don’t know about prevention of MTC HIV transmission38.23.534.13.0  Never tested for HIV*34.93.424.43.0  1 or more HIV risk category*4.31.46.91.8 Alcohol/smoking  Any alcohol in past month*53.93.710.71.8  Average 1 or more drink per day, past month*6.02.00.80.5  Binging: Any occasions of 5+ drinks in past month*10.72.11.90.9  Frequent drinking: binging or 1 or more drinks per day*12.92.52.20.9  Current smoker*19.42.78.41.5 Obesity  Overweight, body mass index (BMI) >2546.03.6NA  Obese, BMI >3022.43.1NA2004 data, 12 states onlyb,c Folic acid  Don’t know about folic acid for birth defects prevention46.16.938.45.6  Don’t take vitamins of any kind*36.97.110.23.6  Don’t take folic acid or multivitamin*38.47.017.54.5  Don’t take folic acid or multivitamin daily*44.86.919.94.7Nutrition (2002 BRFSS)d Fewer than 5 servings/fruit and vegetables*74.93.164.73.5 Fewer than 1 servings/fruit and vegetables3.31.12.91.3Note. 95% confidence interval = Percent +/− CI.aUnweighted number of observations: Preconception women (2308), Pregnant Women (2998).bUnweighted number of observations: Preconception women (607), Pregnant Women (756).cStates: AZ, CO, FL, KY, MN, MT, NC, ND, NE, TX, VA, WI.dUnweighted number of observations: Preconception women (2204), Pregnant Women (2556).*p < .05, preconception vs. pregnant women.**HIV risk: Any statement is true: in past year illegal drug injection, treated for STD, exchange for money/drugs, uprotected anal intercourse. Statistical methods The percentage of women exposed to 21 risk indicators was identified for women in 2 groups: women in the preconception period, and pregnant women (Table 1). The categories of risk indicators for which data are available include perceived general and mental health, lack of medical insurance, knowledge of HIV prevention and practices, alcohol and tobacco use, obesity, nutrition including folic acid uptake for the prevention of birth defects, and presence of chronic medical conditions. Because these are not longitudinal data it was not possible to observe behavior change in individual women. Rather the prevalence of risk factors was compared between preconception and pregnant women as a proxy measure of pregnancy-related risk reduction [6], although is is possible that other factors could influence the differences between preconception and pregnant women. The statistical significance between preconception and pregnant women of the percentage reporting each risk factor, was assessed using a t-test. All statistical analysis was based on weighting factors designed to produce unbiased estimates, and statistical tests were adjusted for complex sample design using the software for survey data analysis (SUDAAN, Research Triangle Institute, Research Triangle Park, NC). Results Comparisons with vital statistics data on births in 2004 indicate that the 2004 BRFSS respondents in the preconception stage and pregnant when interviewed were distributed very similarly by age and race-ethnicity to women giving birth in 2004 (Table 1). Overall, for many risk factors, pregnant women reported lower prevalence of risk than preconception women (Table 2). Percentages reporting alcohol consumption and smoking were much lower for pregnant women than preconception women. For example, 53.9% of preconception women reported any use of alcohol in the past month, compared with 10.7% of pregnant women, 19.4% of preconception women reported current smoking in contrast to 8.4% of pregnant women, and, in the 12 states that included the question, 44.8% of preconception women reported not taking vitamins with folic acid versus 19.9% of pregnant women. For some risk indicators, no statistically significant difference was observed between women in the preconception period and pregnant women. The lack of awareness about methods to prevent mother-to-child HIV transmission (PMTCT) was not significantly different between preconception women and pregnant women (38.2% vs. 34.1%). However, a small but significantly higher percentage of pregnant women reported HIV risk (6.9%) compared to preconception women (4.3%), which is perhaps related to sexual risk behaviors being causally linked to both HIV and to unplanned pregnancies. Of concern, is that although a lower percentage of pregnant women compared with preconception women had never been tested for HIV, about one-fourth of pregnant women remained untested, despite national recommendations that call for all pregnant women to be tested for HIV [7]. In the case of chronic diseases such as diabetes, the lower level of disease among pregnant women may indicate self-selection of diabetic women to avoid pregnancy. Of note, nearly one fifth of the women in preconception period were obese (defined as having a body mass index or BMI of greater than 30) and twice that number were overweight (BMI of greater than 25) (Table 2). Most preconception and pregnant women did not report consuming the recommended 5 servings of fruits and vegetables per day [8], although the percentage was lower for pregnant women (64.7%) compared with women in the preconception period (74.9%). Indicators of general well being and recent dental care were not different between women in preconception period and pregnancy. A composite measure of 3 basic pregnancy risks was formed, frequent alcohol consumption, current smoking and lack of an HIV test, in order to use the BRFSS data to illustrate the size of the target populations for preconception care. These are risk factors for which we have data that are generalizable to the US population, and for which interventions or information are widely available or promoted. The results indicate that 54.5% of preconception women reported one or more of these 3 risk factors, which represents approximately 1 million preconception women (Table 3); 32.0 percent of pregnant women had one or more of these risks, which is equivalent to 835,000 pregnant women. Table 3 also shows this composite risk factor by 2 measures of access to health care, whether the woman had a health plan or insurance and whether she had a personal physician. Pregnant women had a lower percent at risk in all categories except for those without health insurance. Among women without insurance, the percentage reporting risk was not significantly lower for pregnant women, compared with preconception women. This suggests that women with poorer access to health care, as indicated by no health insurance, are not as likely as others to adopt healthier behaviors when they become pregnant. Table 3Percentage and estimated number reporting any of 3 risks for pregnancy outcomea for preconception and pregnant women aged 18–44 Women 18–44, 2004 BRFSSPreconception women (Estimated number)Currently pregnant women (Estimated number)PercentCI1000sCINPercentCI1000sCINTotal54.5*3.71,02494228632.03.18351012949No health plan/insurance63.411.72225232852.710.315650303Others52.5*3.780279195729.43.2679882646No personal doctor64.1*9.02785944846.39.223473467Others51.6*3.974573183728.62.9601712482Note. 95% confidence interval = Percent +/− CI and Estimated number +/− CI. Estimated number is the estimated number of women in the category with one of the risk factors.aAny of 3 risk factors: frequent drinking, current smoker, no HIV test.*difference between preconception and pregnant women significant, p < .05. Discussion The 2004 BRFSS provides an opportunity to measure the extent of risk behaviors on a relatively large sample of women who are planning to become pregnant. These nationally-representative survey data indicate that major health risks were reported by substantial proportions of US women in the preconception period. The data suggest that among those at risk during preconception, the majority continued the risk into pregnancy. Women appear to respond to messages regarding behaviors that can improve pregnancy outcomes such as consuming of folic acid, and the reduction in smoking, alcohol consumption, but many remain unaware of the benefits of available interventions to prevent HIV transmission and birth defects. The fact that more than one-third of preconception and pregnant women were not aware of methods to prevent mother-to-child HIV transmission and did not know about the benefits of taking folic acid during pregnancy suggests that education and public information programs could have some effect in reducing risk behaviors. Risk reduction appears to be lower among women who have no health insurance. Other data are not available for comparison of the overall burden of risk for adverse pregnancy outcomes among women in the preconception period or pregnancy. However, data on selected indicators presented here for pregnant women are comparable to the reported range from other studies [4, 6, 8–11]. The finding that the percentage who report some risk behaviors is significantly lower among pregnant women compared with women in the preconception period may indicate the desire of many pregnant women to adopt healthier behaviors to achieve the best possible outcome for their pregnancy. Examination of three well known risk factors for which interventions are available (frequent alcohol consumption, current smoking, or never having been tested for HIV) illustrates that risks for adverse pregnancy outcomes exist among more than half of women in the preconception period and among one-third of pregnant women. Nationwide, this represents approximately one million women in the preconception period and 835,000 pregnant women. The data from the BRFSS are subject to potential errors associated with survey research such as exclusion from the sampling frame, non-response and reporting errors. BRFSS is a telephone-based survey and might not be representative of the small percentage of the population without telephones. The BRFSS methodology contains steps to ensure accurate estimation, including the use of weighting factors to compensate for non-response rates. In addition, estimates from BRFSS data have been found to be consistent with data from other surveys [5]. The preconception and pregnant women identified were distributed very similarly to women giving birth in 2004 by age group and race/ethnicity, providing support for the representativeness of the data. In this report, the reported risk behavior of women in the preconception stage and pregnant women have been compared as a proxy for the type of behavior change occurring between preconception and pregnancy, but other factors could affect the actual change in behavior among individual women. For known risk factors, progress in efforts to reduce such risks among pregnant women has been slow. For example, as indicated in data collected on earlier rounds of the BRFSS, alcohol consumption among pregnant women has remained relatively steady over the past decade [10]. Even among those who reduce consumption of alcohol, tobacco, or drugs, post pregnancy relapse of such behaviors have been found to be very high [4]. Of the 17 maternal and infant health objectives included in the Healthy people 2010 objectives, progress has been made toward the target in 8 objectives [12]. Little positive progress has occurred in the areas of maternal death, fetal alcohol syndrome, and low birth weight. To close the gap in reduction of poor maternal and child health outcomes, current maternal and child health initiatives should be complemented with alternate approaches, including more emphasis on preconception health promotion strategies among women of childbearing age. Recent experience in the prevention of alcohol-exposed pregnancy and prevention of neural tube defects through folic acid supplementation has shown that such efforts are useful. In 2003, the Project CHOICES Research Group reported the findings of a feasibility study designed to provide prevention counseling to women at high risk for an alcohol–exposed pregnancy [13]. Brief interventions were found to be effective in reducing hazardous alcohol use in adults in a number of well-controlled studies. Interventions generally consist of advice, feedback, goal setting, and follow-up for further assistance and support [14]. The use of a counseling style referred to as “motivational interviewing” has also proven to be effective in reducing problem drinking [15]. Results of the initial follow-up assessment, suggest that this approach can be effective among women of childbearing age [13], and a larger efficacy trial is now underway. To summarize, our results were consistent with a high prevalence of risk factors in the preconception period, and a high degree continuation of risk into pregnancy. This underscores the need for new approaches to complement current prevention efforts. Given that women of childbearing age in the United States have on an average 6.4 visits to physicians each year [16], it may be feasible to inform them and engage them for interventions. Such contacts may provide an opportunity to assess and advise on many elements of health mentioned here. Our finding of high risk levels among women who had no insurance or no personal doctor strongly suggests that ensuring access to such services will remain the key determinant of maximum uptake of known intervention services. Medicaid and other funding programs can play an important role to address the access gap—whether during, before, or after pregnancy. Attempts to improve the health of would-be mothers not only improve pregnancy and infant health, but also help reduce the long-term impact of many risk factors on the women themselves.

Dev_Genes_Evol-4-1-2265772

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis. Introduction Some types of annelids exhibit remarkable regenerative abilities that are comparable to planarians. However, the regeneration mechanisms are thought to be quite different between planarians and annelids. Whereas planarians regenerate via totipotent stem cells (neoblasts) that are widely distributed throughout their bodies (Reddien and Alvarado 2004), annelid regeneration is thought to occur mainly through the dedifferentiation and subsequent redifferentiation of cells without any contribution from totipotent stem cells (Thouveny and Tassava 1998). In this respect, annelids regenerate in a manner similar to that of amphibians (Carlson 1998) but on a much larger scale. The elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates. In our recent study, we have proposed the oligochaete annelid Enchytraeus japonensis as a new model system for regeneration studies (Myohara et al. 1999). E. japonensis has striking features that are highly advantageous in this regard: (1) It reproduces asexually by dividing its body into several fragments, which then regenerate a complete individual within 4–5 days, (2) artificially amputated fragments can also regenerate new individuals in the same manner, and (3) both asexual and sexual reproduction can be artificially induced in the laboratory (Myohara et al. 1999). As an initial strategy toward the elucidation of the molecular mechanisms underlying annelid regeneration, we previously performed complementary deoxyribonucleic acid (cDNA) subtraction cloning using regenerating fragments of E. japonensis and intact animals (Myohara et al. 2006). Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones we isolated contained genes that were upregulated during regeneration. These clones hybridized with probes derived from regenerating fragments but not with probes from intact worms. Of these clones, 279 were then sequenced and found to contain 165 different sequences (79 known and 86 unknown). Among these 279 regeneration-upregulated clones, as many as 13 (4.7%) were found to encode glutamine synthetase, a ubiquitous and key enzyme in nitrogen metabolism (Kumada et al. 1993). Because of its abundance in our subtracted cDNA library, we cloned the full-length sequence of E. japonensis glutamine synthetase (EjGS). By reverse transcriptase–polymerase chain reaction (RT-PCR), we subsequently revealed that the expression levels of the glutamine synthetase gene (gs) were consistently high during the regenerative process, with a gradual decline seen only in the very late stages, and our preliminary analysis by whole mount in situ hybridization further revealed strong gs expression in the blastemal tissues of the regenerating fragments (Myohara et al. 2006). As these previous results suggested an important role for gs in annelid regeneration, in the present study, we have examined the detailed expression pattern of gs in regenerating fragments and intact worms and considered the possible roles of this enzyme in the biology of E. japonensis. This is the first detailed report of the sequence and expression of gs in annelids. Materials and methods Animals E. japonensis worms were reared at 24°C in 1.1% (w/v) plain agar medium in disposable Petri dishes and were fed with rolled oats as previously described (Myohara 2004). Under these conditions, the worms grow continuously to about 10 mm long, consisting of 50–60 segments, and reproduces asexually by fragmentation every 2 weeks. To induce sexual reproduction, starved worms were cultured in wet leaf mold and refed with rolled oats as previously described (Myohara 2004). Molecular analysis The full-length cDNA of EjGS (accession number AB109095) has been isolated in our previous study (Myohara et al. 2006). Multiple alignments of the entire amino acid sequences of the glutamine synthetase proteins from representative species, which were obtained through GenBank/EMBL/DDBJ Data Bank and chosen to cover a wide range of phylogenic groups, were performed using ClustalW software (Thompson et al 1994). To avoid errors resulting from mixing of possible pseudogenes, only data originating from full-length messenger ribonucleic acid (mRNA) sequences were used. In situ hybridization Both sense and antisense digoxigenin (DIG)-labeled RNA probes were synthesized using the DIG RNA labeling kit (SP6/T7, Roche) as previously described (Myohara et al. 2006). Whole-mount in situ hybridizations were also performed as previously described (Myohara et al. 2006) with some modifications to enhance the signals. Briefly, hybridization was carried out overnight with 1 μg/ml of gs DIG-labeled RNA probe at 55°C. An anti-DIG alkaline phosphatase (AP) conjugate antibody (Roche) was preabsorbed by incubation for 2.5 h with fixed and rehydrated worms and used at a dilution of 1:2,000. In situ hybridization of paraffin sections (6 μm) was carried out according to Hoshino et al. (1999) and Abe et al. (2003) with a few modifications. Briefly, sections were hybridized with DIG-labeled probes at a concentration of 100 ng/ml in probe diluent (Genostaff, Tokyo, Japan) at 60°C for 16 h. Washes were then carried out using 5× Hybri-wash (Genostaff), and an anti-DIG AP conjugate antibody (Roche Applied Science, Tokyo, Japan) was then applied at a 1:1,000 dilution in Tris-buffered saline with 0.1% Tween 20 for 2 h. After an overnight coloring reaction with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Roche), the sections were counterstained with a Kernechtrot stain solution (Muto Pure Chemicals, Tokyo, Japan), dehydrated, and mounted in Malinol (Muto Pure Chemicals). Results and discussion Comparisons between E. japonensis glutamine synthetase and other known glutamine synthetases A full-length 2,717-bp cDNA encoding gs was previously isolated from regenerating E. japonensis in our laboratory (Myohara et al. 2006). The sequence of this gene was found to contain a putative open reading frame of 360 amino acids with a calculated molecular mass of 40,509 Da. Because this was the first report in annelids of a full-length sequence for gs, we performed a database search in the present study to determine whether similarities existed with the gs genes from other organisms. BLASTP analysis revealed that the deduced glutamine synthetase protein in E. japonensis (EjGS) was highly similar to known class II glutamine synthetases along its entire amino acid sequence (Fig. 1), which is well conserved across species (Kumada et al. 1993). Within the EjGS sequence, we identified a beta-grasp (catalytic) and a noncatalytic domain, five conserved regions that are common to both prokaryotes and eukaryotes, ligands for Mn2+ ions, and an adenosine triphosphate-binding site (Pesole et al. 1991). An amino acid alignment between EjGS and the gs proteins from five invertebrates, sea anemone (Cnidaria), C. elegans (Nematoda), Pacific oyster (Mollusca), fruit fly (Arthropoda), sea urchin (Echinodermata), and four vertebrates (zebrafish, clawed frog, chicken, and mouse) showed that the overall amino acid identities between EjGS and its counterparts in various other animal species were within a range of 61–71% (Fig. 1). Fig. 1Amino acid alignment of E. japonensis glutamine synthetase (EjGS) with representative GSs from various other species. The species and protein ID of the GSs listed here are as follows: Enchytraeus japonensis (BAE93509.1), sea anemone Aiptasia pallida (AAR36878.1), nematode Caenorhabditis elegans (NP_001041010.1), Pacific oyster Crassostea gigas (CAD90162.1), fruit fly Drosophila melanogaster (AAZ41780.1), sea urchin Paracentrotus lividus (AAC41562.1), zebrafish Danio rerio (AAH66735.1), clawed frog Xenopus laevis (AAH46681.1), chicken Gallus gallus (AAA48783.1), and mouse Mus musculus (AAH15086.1). Amino acid residues are indicated in blue when greater than or equal to 70% of these residues are identical in the ten representative species. The beta grasp domain, catalytic domain, and the regions that are conserved in prokaryotes and eukaryotes are underlined by broken single lines, broken double lines, and thick single lines, respectively. Ligands for Mn2+ ions are indicated by arrowheads. The ATP-binding sites are boxed. The protein length and overall amino acid identities with EjGS are indicated at the end of each sequence Expression of the gs gene in the blastema of regenerating E. japonensis By whole-mount in situ hybridization analysis, we detected strong gs expression in the blastemal region in regenerating fragments of E. japonensis (Fig. 2a). This expression was detectable from the early stages of the regenerative process in the cell layer covering the wound (Fig. 2b,k) and was found to persist in the epidermal cells during the formation and elongation of the blastema (Fig. 2c,d). There was also a notable concentration of this transcript in the region corresponding to the prostomium anlagen in the anterior blastema (Fig. 2e) and in the growth zone in the posterior blastema (Fig. 2l). In situ hybridization analyses with paraffin-embedded sections additionally revealed that the gs expression pattern was mainly restricted to the epidermal cells in the early blastema (Fig. 2h) but did occur also in the inside tissues of the elongated blastema, where it was detected in the presumptive regions of the brain, ventral nerve cord, and buccal cavity (stomodeum), in addition to the prostomium and epidermal cells (Fig. 2i). Fig. 2Glutamine synthetase mRNA expression during regeneration in E. japonensis. Transcripts were detected using whole-mount in situ hybridization (a–g and k–o) or in situ hybridization analysis of paraffin-embedded sections (h–j) using an antisense gs riboprobe, with the exception of the lower specimen shown in a. The anterior is to the left, and the ventral side is down in each image. Arrowheads indicate the amputated sites. a Whole-body images of regenerating fragments at about 1 day after amputation with the anterior blastema and an intact tail. The lower specimen was hybridized with a sense probe. b–f Lateral views of anterior blastemas at about 1 (b), 1.5 (c), 2 (d), 2.5 (e), and 4 days (f) after amputation. g Lateral view of an intact head. h–i Sagittal sections of anterior blastemas at about 1.5 (h) and 2 days (i) after amputation. j Sagittal section of an intact head. k–m Lateral views of posterior blastemas at about 0.5 (k), 1 (l), and 1.5 days (m) after amputation. n–o Ventral views of intact tails in an actively growing (n) and a full-grown individual (o). ba Brain anlage, br brain, bc buccal cavity, ch chloragocyte, ea esophageal appendage, ep epidermal cell, es esophagus, gl gut lumen, gz growth zone, m mouth, pc pharyngeal cavity, pp pharyngeal pad, pr prostomium, py pygidium, st stomodeum, va ventral nerve cord anlage, vn ventral nerve cord. Scale bars, 50 μm In the later stages of E. japonensis regeneration, by which time segmentation and organogenesis have almost completed, we observed that the gs expression levels in the blastemal regions had become reduced (Fig. 2f,m). In the fully formed intact head, however, the expression of this gene was detectable in the prostomium, brain, anterior end of the ventral nerve cord, epithelial cells lining the buccal and pharyngeal cavities, pharyngeal pad, and esophageal appendages (Fig. 2g,j; for E. japonensis morphology, see Schmelz et al. 2000). In intact tails, gs expression was also evident in the growth zone in actively growing worms (Fig. 2n) but was absent in full-grown individuals in which the growth zone had become indistinguishable (Fig. 2o). These observations are in agreement with the RT-PCR data included in our previous report, which showed that the expression levels of gs were maintained at high levels during the early stages of regeneration, decreased temporarily in the later stages of this process, and were upregulated again in the fully formed intact worms (Myohara et al. 2006). Expression of the gs gene in nonblastemal regions In accordance with the abovementioned RT-PCR results, we found by in situ hybridization analysis in the present study that gs expression was not restricted to the blastemal regions (Figs. 2a and 3a) but that less intense but clear expression could be observed also in the nephridia (Fig. 3a,b), chloragocytes (Figs. 2a, 3a–c), gut epithelium (Fig. 3c), epidermis of the nonblastemal regions of regenerating fragments (Fig. 3a), and also in intact worms (Fig. 3d,e). Nephridia are the excretory organs of oligochaete annelids and the chloragocytes (chloragogen cells) are specialized peritoneal cells of the intestine that participate in the metabolism, storage, and excretion of glycogen and lipids, thus resembling the vertebrate liver in terms of function (Jamieson 1981; Edwards and Bohlen 1996). The gs expression levels in these tissues were observed to be variable among the specimens examined and also among regions within the same specimen (Figs. 2a and 3c). We were unable to find any correlation between this pattern and a specific gut region or specific condition of our specimens, although an uneven distribution of glutamine synthetase has been reported previously in the mammalian and fish digestive tracts (James et al. 1998; Mommsen et al. 2003). Intense gs expression was also found in the spermatids in the seminal vesicles and in oocytes in the ovisacs in sexually mature worms (Fig. 3e). Fig. 3Glutamine synthetase mRNA expression in nonblastemal tissues. Transcripts were detected by in situ hybridization on paraffin-embedded sections (a–c and e) or by whole-mount in situ hybridization (d) using an antisense gs riboprobe, except for the right-hand specimen in a. The anterior is to the left, and the ventral side is down in each image. a Sagittal sections of regenerating fragments at about 0.5 days after amputation. The right-hand specimen was hybridized with a sense probe. Arrowheads indicate the amputated sites. b–c Sagittal sections of intact worms. d Whole-mount in situ hybridization analysis of an intact worm showing gs expression in the epidermal cells. e Sagittal section of a sexually mature worm. ch Chloragocyte, ep epidermal cell, ge gut epithelium, gl gut lumen, ne nephridium, oo oocyte, sc spermatocyte, st spermatid, sp sperm. Scale bars, 50 μm General comparisons between the distribution of glutamine synthetase in different organisms Glutamine synthetase is found in a wide range of organisms, including microorganisms, plants, and animals and is known to be expressed in a tissue-specific and developmentally controlled manner. In murine organs, glutamine synthetase activity is highest in the liver, followed in descending order by the epididymis, testes, uterine tube, stomach, kidney, brain, and adipose tissues (van Straaten et al. 2006). High glutamine synthetase expression in the brain, kidney, and liver has also been reported in fish (Murray et al. 2003). In Xenopus, gs transcripts have been detected in the ovary, kidney, eye, muscle, and embryo but not in the brain, heart, liver, stomach, or testis (Hatada et al. 1995). In the case of invertebrates, little information is currently available regarding the expression profile of this enzyme and is restricted to arthropods; gs expression has been reported in mosquito midgut epithelium, ovary, and fat body (Smartt et al. 2001) and in crustacean neural tissues (Linser et al. 1997; Allodi et al. 2006; Sullivan et al. 2007). Our present study, therefore, provides novel information regarding the expression of this enzyme in nonarthropod invertebrates, and our results are in general agreement with those of previous reports; that is, in full-grown E. japonensis, gs is expressed in (1) chloragogen tissues, which correspond to the vertebrate liver in terms of functionality (Jamieson 1981), (2) nephridia, which are the annelid counterparts of the kidney and uterine tube in vertebrates (Jamieson 1981), (3) the brain and ventral nerve cord, (4) the prostomium where large numbers of sensory cells including photoreceptor cells are present (Jamieson 1981; Edwards and Bohlen 1996), (5) the epithelium of the digestive tract, and (6) spermatids and oocytes. Possible functions of glutamine synthetase in E. japonensis Regeneration The possible involvement of glutamine synthetase in the regeneration of damaged tissue has been reported in only a few cases. These include the observed increase in gs expression after peripheral motor nerve injury in mouse (Toki et al. 1998) and also in Xenopus tail regeneration in which gs expression was found to be upregulated and could be detected in the terminal vesicle of the regenerating spiral cord (Tazaki et al. 2005). Our present results, together with our previously reported findings, strongly suggest the involvement of glutamine synthetase also in annelid regeneration. In the previous study, we have shown that glutamine synthetase is one of the most abundantly expressed upregulated genes during regeneration in E. japonensis (Myohara et al. 2006), and in the present study, our detailed expression analysis by in situ hybridization has revealed strong gs expression in the blastemal regions of regenerating E. japonensis. It has been reported previously in Xenopus that embryos abundantly express glutamine synthetase in the developing nervous system during the gastrula and neurula stages (Hatada et al. 1995). In accordance with this finding, we observed in the present analyses that gs is expressed in the presumptive regions of the brain and ventral nerve cord anlagen in the blastema during E. japonensis regeneration. This further suggests the involvement of glutamine synthetase in annelid neural regeneration. Nerve function Glutamine synthetase has been shown to play an important role in glutamate transmitter degradation in the nervous system (Derouiche and Frotscher 1991) and is used as a specific glial cell marker in the neural retina (Vardimon et al. 1993). In intact E. japonensis, the expression of this enzyme was evident in the brain and ventral nerve cord, suggesting that it has a nervous system function. Its expression in the prostomium may also be attributable to the distribution of numerous sensory cells, probably including photoreceptor cells, in this region (Jamieson 1981; Edwards and Bohlen 1996). Cell proliferation Glutamine synthetase is the only enzyme that can synthesize glutamine, which provides nitrogen for the synthesis of amino acids, purines, pyrimidines, and other compounds required in many critical cellular events including proliferation (Meister 1980). In the rat intestinal epithelium, high concentrations of glutamine synthetase protein and mRNA have been found in the highly proliferative crypt region (Roig et al. 1995). Hence, the high gs expression levels that we observed in blastemal cells and in the growth zone in actively growing worms could be explained, at least in part, by a high demand for glutamine during the intense cell proliferation that would be ongoing in these areas. Detoxification Glutamine synthetase is also important for the detoxification of excess glutamate and ammonia by converting them to glutamine, which is nontoxic and can be stored in tissues or circulate throughout the body without causing any harm (Meister 1980). A comparison between the cellular concentrations of glutamine synthetase in various murine organs in a previous report has also revealed that the highest concentrations of this enzyme positively correlate with the cells that have detoxifying functions (van Straaten et al. 2006). The gs expression that we observed in the nephridia and chloragocytes in E. japonensis herein could therefore be ascribed to the involvement of these tissues in excretion of nitrogenous waste in oligochaete annelids (Jamieson 1981). Macromolecule synthesis Glutamine is known to be required for the synthesis of certain macromolecules that are present in mucus, such as the hexosamines that lubricate and protect the gut mucus (Weiss et al. 1999). The mucus secreted by epidermal cells covering the earthworm body is also essential for locomotion, gas exchange, and protection in these animals (Jamieson 1981), and hexosamines have been found to be a component of their cuticles (Muir and Lee 1970). We speculate therefore that the gs transcripts in the E. japonensis epidermal cells have a role in mucus and/or cuticle synthesis in intact and regenerating individuals. In addition, the gs expression found in epithelial cells of the buccal and pharyngeal cavities may be attributed to cuticle synthesis in these tissues (Jamieson 1981). Gametogenesis Glutamine synthetase expression in the ovary has been reported in Xenopus (Hatada et al. 1995) and in the yellow fever mosquito (Smartt et al. 2001). In oligochaete annelids, the testes and ovaries are small organs located in the ventral part of the genital segments (Edwards and Bohlen 1996). The spermatogonia that are formed in the annelid testes pass into the seminal vesicles, which contain male cells at all stages of development. Oogonia in annelids are formed in the ovaries and divide to form oocytes, which are shed from the ovaries into the ovisacs. In E. japonensis, gs transcripts were detectable in spermatids in the seminal vesicles and in oocytes in the ovisacs but not in the testes or ovaries, suggesting that glutamine synthetase may have a role in the later stage of gametogenesis. In summary, in the present study, we have characterized an annelid gs gene and shown that it is expressed in a tissue-specific manner in regenerating and intact E. japonensis. To further elucidate the actual functions of this gene, additional studies including RNAi gene knockdown experiments will be needed in the future. Ongoing investigations in our laboratory of other regeneration-associated upregulated genes, several of which are novel (Myohara et al. 2006), should also shed important new light on the network of biological pathways involved in the regenerative processes in E. japonensis.

Clin_Rheumatol-4-1-2367388

Antioxidant intervention in rheumatoid arthritis: results of an open pilot study

There is evidence that reactive oxygen species play a causal role in auto-immune diseases, such as rheumatoid arthritis (RA). Despite the supporting evidence for a beneficial effect of antioxidants on clinical characteristics of RA, the right balance for optimal effectiveness of antioxidants is largely unknown. To determine the potential beneficial effects of an antioxidant intervention on clinical parameters for RA, an open pilot study was designed. Eight non-smoking female patients with rheumatoid factor + RA and a Disease Activity Score (DAS 28) higher than 2.5 were enrolled in the study. Patients had to be receiving stable non-steroidal anti-inflammatory drug treatment and/or ‘second line’ medication for at least 3 months. The pilot group consumed 20 g of antioxidant-enriched spread daily during a period of 10 weeks. The intervention was stopped after 10 weeks and was followed by a ‘wash-out’ period of 4 weeks. At t = 0, t = 10 weeks and t = 14 weeks, patients’ condition was assessed by means of DAS. In addition, standard laboratory analyses were performed, and blood-samples for antioxidants were taken. The antioxidant-enriched spread was well tolerated. All laboratory measures of inflammatory activity and oxidative modification were generally unchanged. However, the number of swollen and painful joints were significantly decreased and general health significantly increased, as reflected by a significantly improved (1.6) DAS at t = 10 weeks. The antioxidant effect was considered beneficial as, compared to the scores at t = 0, the DAS significantly reduced at t = 10 weeks. Increase of the DAS (0.7) after the “wash-out period” at t = 14 confirmed a causal relation between changes in clinical condition and antioxidants. This open pilot study aimed to assess the clinical relevance of an antioxidant intervention as a first step in assessing potential beneficial effects of antioxidants on rheumatoid arthritis. These conclusions need to be validated in a larger controlled study population. Introduction In spite of the great advances that have been made in the development of new drugs for the treatment of patients with rheumatoid arthritis (RA), many patients are interested in alternative treatments like dietary therapy. Although the aetiology of rheumatoid arthritis is still unknown, the inflammation resulting from the immunological reaction is quite well described. It is known that neutrophil granulocytes, macrophages and lymphocytes are activated, and that reactive oxygen and nitrogen species (RS) are produced [1, 2]. These RS can react with lipid, protein and nucleic acids and are thought to be of importance for the aetiology and chronicity of the inflammatory rheumatic diseases [3, 4]. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. There is some evidence for a positive effect of antioxidants on clinical symptoms of RA [5, 6]. However, this evidence is weak, and information about the most effective antioxidants, antioxidant doses or combinations is lacking The intervention studies that have been conducted have tested vitamin E exclusively [6, 7]. Against this background, an open (uncontrolled) pilot study was designed to assess if effects of antioxidants on the clinical parameters of patients with RA could be confirmed and if these effects would associate with changes in a selected number of molecular markers related to human antioxidant defence system status. Further, a mix of antioxidants was to be tested rather than a single high-dosed compound. Materials and methods The open study was approved by the ethical committee. Eight patients with rheumatoid factor + RA, as defined by the ARA revised criteria were enrolled in the study. To avoid influences of gender, only female patients participated. Inflammatory disease activity was defined as a Disease Activity Score (DAS) higher than 2.5. Inclusion criteria included non-smoking and no serious co-morbidity. Patients had to be receiving stable non-steroidal anti-inflammatory drug (NSAID) treatment and/or ‘second line’ medication for at least 3 months. Those taking antioxidant supplements were excluded from the study. During a period of 10 weeks, patients consumed 20 g of antioxidant-enriched margarine daily. The spread contained a mix of a-tocopherol (400 mg), lycopene (10 mg), palm oil carotenoids (5 mg; mainly α-carotene) and lutein (10 mg). Further, patients received vitamin C (200 mg daily) as a supplement. The intervention of 10 weeks was followed by a ‘wash-out’ period of 4 weeks. At t = 0, t = 5 weeks, t = 10 weeks and t = 14 weeks, patients’ condition was assessed by means of morning stiffness, visual analogue scale pain, general health score and DAS 28-score (including the following parameters: pain, swelling of joints, erythrocyte sedimentation rate (ESR), general feeling of well-being). At t = 0, t = 10 weeks and at t = 14 weeks, blood was collected, and standard clinical laboratory analyses (ESR, C-reactive protein, hemoglobin, leucocytes, thrombocytes, serum values of creatinine and transaminases) were performed. In addition, blood antioxidant status and markers of antioxidant capacity [ferric reducing ability of plasma (FRAP)] and oxidative stress (8-epi F2-isoprostane) levels were determined. Determination of vitamin A, not present in the spread, was used as a control. Serum was collected immediately after blood sampling and stored in liquid nitrogen. The potential antioxidant effect was defined as beneficial if the DAS score would significantly improve >1.2 at t = 10 weeks as compared to the scores at t = 0. At t = 14 weeks, a ‘wash-out” effect was assessed using the same parameters. Return of the clinical condition of the pilot group to that at t = 0 may indicate a causal relation between changes in clinical condition and antioxidant intake, although a placebo-controlled intervention would be better required for this assessment. Statistical analyses were done using a paired t test. Results The antioxidant-enriched spread was well tolerated. None of the patients withdrew from the study because of adverse reactions. The number of swollen and painful joints were significantly decreased, and general health significantly increased at t = 10 weeks. This was shown by a significantly decreased DAS (−1.6) at 10 weeks. After the “wash-out period”, the DAS score was increased again by 0.6 (Figs. 1 and 2). Fig. 1Clinical condition by Disease Activity ScoreFig. 2Mean Disease Activity Score The standard laboratory measures of inflammatory activity were relatively unchanged. Levels of plasma vitamin E, lutein, lycopene and alpha-carotene were significantly increased at 10 weeks. No significant changes were seen in vitamin A and vitamin C levels (Fig. 3). Fig. 3Plasma antioxidant status in eight patients Markers of antioxidant capacity and oxidative stress displayed trends for differences, but none of these reached statistical significance (Fig. 4). Fig. 4Markers of antioxidant capacity and oxidative stress Discussion This pilot study was conducted to obtain some first indications for potential beneficial effects of an antioxidant intervention on clinically relevant parameters for rheumatoid arthritis. In our present study, a significant reduction in Disease Activity Score (DAS) was observed following antioxidant intervention of 10 weeks. This was accompanied by significant increases in blood levels of antioxidants administered. The reduction of DAS is remarkable as all patients had an active (high initial mean DAS of 5.8) longstanding disease and had tried all kinds of disease-modifying anti-rheumatic drugs, including combination therapy. The unchanged level of vitamin A, not present in the spread, reinforces the significance of the increased levels of the blood antioxidant status. As NSAIDs can influence the absorption of vitamin C, this could explain the non-significant changes of the serum concentration of this antioxidant. A limited number of clinical intervention studies is available which support the therapeutic or prophylactic activity of antioxidants in the pathogenesis of RA. These studies were all conducted with an oral intake of 1,200 mg/day of vitamin E. Edmonds et al. [6] reported that the antioxidant a-tocopherol significantly reduced pain parameters in a placebo-controlled double-blind trial following a 3-week supplementation period. Results from a randomised double-blind parallel group comparison study with a-tocopherol and diclophenac showed that the clinical parameters assessed, e.g. morning stiffness, Ritchie joint index, grip strength and pain, were significantly reduced by vitamin E, with similar effectivity and less side effects as compared to regular drug therapy by diclofenac [7]. Results from other intervention studies have generally been in line with these observations [8, 9]. In our study, not only the effect of vitamin E on the inflammatory response and clinical symptoms was evaluated but also the interactions with bioavailable natural antioxidants such as carotenoids, vitamin C and others. In view of the limited number of controlled studies, the supporting evidence for beneficial effects of antioxidants on clinical characteristics of rheumatoid arthritis may be considered limited but promising. Another point is that the potential mechanism of action of antioxidants in rheumatoid arthritis needs further attention. The aspect of damage by RS and loss of critical functions can be analysed by use of ‘molecular markers’. These markers may include antioxidant status, products which arise as a consequence of oxidative damage to lipid, protein and DNA and tissue damage (as assessed by lactic dehydrogenase release) or cytokine levels (cq anti-TNFα). Although markers are very informative from a mechanistic point of view, a drawback is that none of them has been validated against clinical conditions of patients with RA. In this study, a trend for reduced levels of measures of antioxidant capacity and of ‘footprints’ of oxidative stress was observed, e.g. statistically non-significant increase in FRAP and reduction in F2-isoprostane levels. Furthermore, these markers showed a trend for correlation with clinical conditions. The data indicate that statistically significant effects may be observed when the number of patients enrolled is increased. This open pilot study therefore should be viewed as a first step in assessing potential beneficial effects of the antioxidants tested on rheumatoid arthritis. The data indicate there may be potential in using mixes of antioxidants in the treatment of RA alongside drug treatment so that drug dosage may be reduced while hopefully retaining efficacy. Further, multi-drug/antioxidant therapy could be used to reduce drug dose and thus side effects of treatment. In conclusion, our present pilot study indicated that intervention with antioxidant-enriched margarine in RA results in consistent and significant relief of clinical symptoms. Furthermore, increases in blood antioxidant status and indications for effects on oxidative stress markers were observed. These data are promising and indicate need for a double-blind, placebo-controlled randomised human trial to establish effect and demonstrate causality.

Mcgill_J_Med-11-1-2322926

Anterior cruciate ligament reconstruction: a look at prosthetics - past, present and possible future

Biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus/gracilis tendons has become the most popular procedure in surgical treatment of a ruptured ACL. This article provides a review of the history of the use of prosthetics with respect to ACL reconstruction grafts including Carbon Fibre, Gore-Tex and Dacron prosthetics as well as the Leeds-Keio Artificial Ligament and the Kennedy Ligament Augmentation Device (LAD). Emphasis is placed on the Ligament Advanced Reinforcement System (LARS) as preliminary investigations of its use have been encouraging. Significant progress has been made recently with respect to the understanding of ACL anatomy, composition, biomechanics, and healing processes, leading to innovative techniques using approaches based in tissue engineering principles and computer – assisted surgery. While research into improved ACL treatment options continues, the synthesis of recent advancements provides a new optimism towards the regeneration of an ACL mirroring its original stability, function, and longevity. INTRODUCTION The anterior cruciate ligament (ACL) is the most frequently injured ligament in the knee and consequently, the majority of research into knee ligament injuries has been directed towards the ACL. While the collateral knee ligaments exhibit strong healing potential and generally respond well to conservative treatment, the anterior cruciate ligament has a poor intrinsic healing ability due to the fact that it is enveloped by synovial fluid and lacks significant vascularization (1). Surgical reconstruction is therefore the most frequent mode of treatment pursued when the ACL is torn. The patients who experience ACL injuries are significantly younger and more active than those who experience many other orthopaedic injuries. The need for reconstruction options that exhibit longevity in the face of great stresses is therefore imperative (2). Historically, options for surgical treatment have included primary repair with or without synthetic augmentation and reconstruction using either biological tissue grafts or prosthetic ligaments. Primary repairs with or without augmentation have tended to fail at restoring stability to the knee and are not a common treatment option today (3). Likewise, prosthetic replacements have traditionally tended to be inadequate due to post-surgical complications arising from wear and degeneration. Hence, biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus/gracilis tendons has become the most popular procedure in surgical treatment of an ACL rupture. However, the frequency of significant anterior knee pain post-surgically and high occurrence of flexion contracture and crepitation in tendon autografts have kept research interests focused on the further development of prosthetic ligament implants. A significant number of early research endeavours into prosthetic ACL replacements failed due to a poor understanding of the biomechanical and physiological properties of the ACL. Tremendous gains in understanding have been made in these areas, with significant progress being made in understanding the inter-dependence between ACL anatomy, tissue composition, biomechanics, and the healing processes. Unfortunately, to date, no prosthesis has proven itself as a viable alternative to the patellar or hamstring tendon autografts, currently used in over 90% of ACL reconstructions. This article reviews some of the major historical milestones in ACL reconstruction technology and looks forward to the continuing evolution of this technology. The literature used for this review was obtained using the PubMED database with keywords “anterior cruciate ligament” searched in tandem separately with “prosthetics” and “tissue engineering.” It should be noted that many of the studies cited in this review obtained funding from orthopaedic and biomaterial companies that sponsored the implant. SURGICAL REPAIR: ADVANTAGES AND DISADVANTAGES OF BIOLOGICAL TISSUE GRAFTS Bone-patellar tendon-bone (BPTB) and semitendinosus/gracilis tendon autografts are currently the most common grafts used in ACL reconstruction. While the BPTB autografts were the former “gold standard”, recent studies have identified the quadruple semitendinosus graft as a comparable option for ACL reconstruction (4). Advances in hamstring graft fixation have led to similar strengths of fixation between BPTB and semitendinosus/gracilis grafts (5). Both techniques now offer a high degree of strength and stiffness in the reconstructed ligament. Additionally, use of the patient’s own tissue eliminates the risk of rejection and good to excellent results are reported in 85–90% of patients receiving either of these grafts for ACL reconstructions. Nonetheless, with patellar tendon autografts, many patients experience impaired function and significant morbidity at the donor site including secondary anterior knee pain, patellar tendonitis, infrapatellar contracture, and patellar fracture. Likewise, hamstring weakness and saphenous nerve injury can be seen secondary to hamstring harvest in semitendinosus/gracilis autograft ACL reconstruction (6). To avoid complications resulting from donor site morbidity in autograft reconstructions, allograft reconstruction has also been examined. While eliminating the donor site morbidity, the use of allografts is not currently considered advantageous due to a limited donor tissue supply, delayed biological incorporation, risks of disease transmission and tissue rejection. In an attempt to overcome these concerns, research into the use of synthetic prosthetic ligaments began over 30 years ago and continues today in hopes of eliminating donor site morbidity and reducing the risk of disease transmission and supply shortages. One of the main obstacles in finding an adequate prosthetic replacement for the ACL is the longevity of the graft, with autogenous tissue consistently proving to be a more durable and long-lasting replacement than the many biomaterials that have been applied to ACL replacement (7). PROSTHETIC BEGINNINGS AND PAST USE The first attempts at a synthetic ACL reconstruction were conducted by Alwyn-Smith in 1918 using silk sutures; they however failed within 3 months (8). Reconstruction with a prosthetic ligament made of polytetrafluoroethylene (PTFE) with embedded carbon or aluminum oxide fibers (Proplast) was first reported in 1973 (9). Results with this system yielded an average time to breakage of just over 1 year. A report by James et al. suggests that only 52% of reconstructions utilizing this graft yielded satisfactory results (10). Satisfactory results include all grafts that patients were content with and had not ruptured at time of lastest follow-up. Current classification systems for synthetic replacements of the ACL include grafts, ligament augmentation devices, or total prosthetics. Grafts (polyethylene, PTFE), typically fixed at both ends, were the initial focus of synthetic ACL replacement and were meant to provide stability to the ACL-deficient-knee until secondary reconstruction procedures gained popularity (11). Similarly, ligament augmentation devices (polypropylene, polyester) were intended to provide immediate protection for autogenous tissue grafts until revascularization was complete and the ingrown tissue was capable of withstanding local tensile and compressive forces. Unfortunately, these devices may in fact accomplish the opposite of their intended purpose by stress-shielding autogenous tissue, preventing it from developing adequate mechanical strength. Fixed at both ends like a graft, the total prosthesis was intended as a permanent ACL replacement without any soft-tissue ingrowth that would allow the patient to begin aggressive rehabilitation immediately as no tissue maturation or revascularization was required. While the implantation of a full-strength ACL replacement generally led to excellent short-term results, long-term efficacy results were poor due to wear and ensuing rupture of the prosthesis (11). Combining the mechanical focus provided by these prior prosthetic approaches with tissue engineering principles has led to the development of a scaffold design intended to serve as a foundation for soft tissue in-growth. Scaffolds may be permanent or may be 31 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics intended to gradually degenerate as the host tissue replaces them. Problems associated with the biological incorporation of scaffolds include variability of tissue in-growth, immature degeneration of the implant and insufficient maturation of the host tissue resulting in an inability of the scaffold to withstand inherent mechanical stresses placed on the ACL. Current directions in this area involve the use of tissue engineering focusing on developing a mechanically and biologically functional matrix for the scaffold and the use of in vitro mechanical signals to guide new tissue development (11). Table 1 summarizes the advantages of the various prosthetic ACL graft options that have been attempted and are discussed below. CARBON FIBRE PROSTHETICS Some of the earliest attempts to discover an appropriate alternative to the biological graft resulted in a variety of carbon fibre prostheses that varied greatly with respect to material and implantation technique. In 1977, Jenkins et al. were the first to use a flexible carbon fibre implant, and suggested that the carbon fibre induced the formation of tendon in animals and humans (12). However, subsequent studies reported the generation and migration of carbon wear particles into the joint space and regional lymph nodes following implantation of the prosthesis (12). To combat this problem, attempts were made to coat the carbon fibre with collagen and absorbable polymers. Of particular interest was a carbon fibre implant proposed by Alexander et al. coated with a co-polymer of polylactic acid (PLA) and polycapralactone (13). The carbon filaments were thought to act as a scaffold for tissue ingrowth by evenly distributing and reducing the interfacial stress between the implant and soft tissue attachment, while the PLA/polycapralactone would protect the fibre during implantation. Over time, the PLA was meant to resorb and the carbon fibres degrade as new tissue developed, thereby encouraging normal tissue regeneration without permanently replacing it (13). A 24-month study involving 82 patients was conducted by Weiss et al. to further examine the PLA coated prosthesis using a variety of subjective and objective measures (pain, stability, function, and isokinetic strength testing), which revealed significant improvements over the duration of the study (14). Arthroscopic evaluation demonstrated collagenous tissue ingrowth, confirmed by histological studies, to be composed of Type I and III collagen in similar proportions to that found in normal healing ligamentous tissue (14). A similar design was used in the Surgicraft ABC prosthetic ACL (Surgicraft Ltd., Redditch UK) composed of carbon and polyester fibres oriented in a partial braid by a zig-zag pattern. However, a study by O’Brien et al. with an average follow-up of 34 months showed only 11 of 31 knees (41%) had good results defined as a Lysholm score greater than 76. The Lysholm knee score is an outcome measure that assesses knee function on a 100 point scale. The authors also noted unacceptable stretching and complete rupture as major complications and concluded that the implant is unsuitable for clinical use (15). GORE-TEX PERMANENT PROSTHESIS The Gore-Tex ligament prosthesis is composed of a single long fiber of expanded polytetrafluoroethylene (PTFE) arranged into loops. Extensive mechanical testing has shown that the resulting ultimate tensile strength is about 3 times that of the human ACL and the results from cyclical creep tests and bending fatigue testing seem to identify Gore-Tex as the strongest synthetic ACL replacement in terms of pure material stability (16). Bolton and Bruchman reported that 129 out of 130 patients receiving a high strength PTFE ligament showed improved knee stability at 15 months or less (16). Glousman et al. reported an initial improvement upon physical examination and subjective scores. However, at mean follow-up of 18 months, they reported a progressive loosening of the prosthesis (17). Similarly, Woods et al. presented 2- and 3-year follow-up of Gore-Tex ACL reconstructions and showed a similar pattern of early improvement post-operatively, but deterioration over time. They reported an overall failure rate of 33% at 3-year follow-up (18). Indelicato et al.’s follow-up of Gore-Tex implant ACL reconstructions showed a 90% success rate at 2 years versus only a 76% success rate at 3 years or more (19). Despite similar reports describing complications with the Gore-Tex ACL prosthesis, subjective results in several studies remain acceptable at 60–80 % (17, 19). The Gore-Tex ACL prosthesis is currently FDA approved for use in patients who have had a failed autogenous intra-articular graft procedure. DACRON With its success as a vascular surgery implant (20), various forms of Dacron grafts have been developed as a scaffold for ACL replacements. The implant is a composite of four tightly woven polyester strips wrapped in a sheath of loosely woven velour, designed to minimize abrasion of the graft and act as a scaffold for fibrous tissue ingrowth. A report by Lukianov et al. reviewed the short-term follow-up (mean 28 months) of 41 patients who underwent ACL reconstruction with the Stryker Dacron ligament prosthesis (3). Seventy-five percent of the patients were found to have a negative Lachman, anterior drawer, and pivot shift at their most recent follow-up. However, Richmond et al. reported failure rates of 37.1% in a study of Dacron reconstruction with mean long-term follow-up of 50 months (21). Likewise, Barrett et al. reported higher failure rates of 47.5% after a four-year follow-up period (22). A clinical study by Lopez-Vazquez et al. examining ACL reconstruction with a Dacron prosthesis showed a similar deterioration of results after the first post-operative year (23). With the initial short-term strength shown by these grafts offset by their poor long-term stability, Dacron grafts should not be considered a viable alternative for ACL reconstruction. LEEDS-KEIO ARTIFICIAL LIGAMENT With the desire to design a graft that combined the properties of a permanent prosthesis and a tissue-promoting scaffold, Fujikawa and Seedhom developed the Leeds-Keio artificial ligament: a polyester mesh-like structure anchored to the femur and tibia with bone plugs (24). This mesh was intended as a scaffold for soft tissue ingrowth through the intra-articular and extra-articular sections of the ligament, eventually uniting the bone plugs. The implant was considered sufficiently flexible to be effective with a maximal tensile strength of approximately 2100 Newtons (N), which significantly exceeds that of the average young adults’ natural ACL (about 1730 N) (24). Initial descriptions from the inventors also described minimal articular wear with the ligament (24). The inventors of this graft have reported successful clinical results with arthroscopic observations documenting neoligamentous tissue within the implanted Dacron scaffold. Other investigators, however, have reported the ingrowth of non-aligned fibrous tissue (i.e. non-neoligamentous tissue) within the device after implantation and suggested that the Leeds-Keio ligament did not serve as a true scaffolding graft, but instead behaved as a permanent load-bearing prosthesis, subject to long-term failure in the joint (25). McLoughlin and Smith presented a 3.8 year follow-up study of 25 patients implanted with the Leeds-Keio ligament for chronic ACL instability. They reported a low complication rate and considerable success in the elimination of instability after finding good post-operative results with the anterior drawer test using an arthrometer at 90° of flexion (26). Nevertheless, ensuing long-term follow-up studies showed a deterioration of results after the first post-operative year and a large number of long-term graft ruptures despite excellent early results in stability testing and on the Lysholm scale (27). These findings were similar to earlier results reported by Schindhelm, who found that good early results in a sheep model were not maintained (28). Due to the number of long-term graft ruptures and the lack of long-term stability provided, the Leeds-Keio ligament is no longer suitable for reconstruction of the human ACL (27, 28). KENNEDY LIGAMENT AUGMENTATION DEVICE (LAD) Kennedy et al. introduced the concept of the Ligament Augmentation Device (LAD) in 1980 (29). The graft, composed of a band-like braid of polypropylene, was originally developed to reinforce the area of pre-patellar tissue considered to be a weak area of autogenous patellar tendon grafts. Use of this prosthesis employed the MacIntosh/Marshall transfer of a portion of the rectus femoris tendon, pre-patellar tissue, and central third of the patellar tendon in an over-the-top fashion (30). Originally, the graft was developed to protect the autogenous tissue graft from excessive stresses during the initial remodelling phase (characterized by degeneration and revascularization), allowing for earlier resumption of pre-operative activity levels (29). Research has shown that the percentage of load accepted by the LAD varies according to the type of graft employed and method of reconstruction. Comparisons of the patellar tendon and semitendinosus/gracilis LAD composite grafts revealed that the LAD will accept approximately 28% and 45% of the applied load, respectively (31). To prevent excessive stress shielding that would otherwise prevent the autogenous tissue from developing a normal functional tensile strength, the LAD was attached to the bone at only one end. Despite a promising beginning, the suture interface between the LAD and the graft was identified as the weak link of the composite. Whether collagen fibres become truly incorporated into the LAD remains a controversial issue. Most histological evidence has been derived from animal studies and thus may not be fully indicative of results in humans. Nevertheless, this evidence seems to point to adequate longitudinal collagenization of the graft with inconclusive results on whether or not collagen ingrowth within the LAD has occurred (32). Furthermore, as an intra-articular foreign body, the LAD has been reported to induce an inflammatory response characterized by foreign body giant cells and macrophages in the surrounding tissue. A review by Kumar et al. reported that the majority of complications seen following use of the LAD were characterized as effusions and reactive synovitis, likely a result of LAD-induced inflammatory response (23). The decline in use of the aforementioned MacIntosh/Marshall transfer, combined with the weak graft-prosthetic suture interface and propensity of the LAD to cause high rates of post-operative synovitis have resulted in a lack of widespread use of the device. THE FUTURE: LIGAMENT ADVANCED REINFORCEMENT SYSTEM (LARS) ARTIFICIAL LIGAMENT The Ligament Advanced Reinforcement System (LARS) (Arc-sur-Tille, France) artificial ligament consists of fibres made of polyethylene terephthalate (PET). An intra-osseous segment is composed of longitudinal fibres bound together by a transverse knitted structure while an intra-articular segment is composed of parallel longitudinal fibres twisted at 90°. The main innovation of this artificial ligament lies in its ability to mimic the natural ligamentous structure and reduce shearing forces by orientating the free fibers of the intra-articular portion of the graft clockwise or counter-clockwise for use in right and left knees, respectively. Furthermore, the PET fibres of the intra-articular segment are designed to encourage tissue ingrowth due to the porosity of the material, allowing ingrowth from the surrounding osseous tunnels. Ideally, such tissue ingrowth between the ligament fibres would contribute to the viscoelasticity of the graft and protect against friction at the opening of the bony canal and between the fibres themselves (1). A study by Lavoie et al. examined ACL reconstruction with the use of the LARS artificial ligament. Thirty-eight of forty-seven patients suffered from chronic ruptures of the ACL, while nine others presented with acute or subacute ruptures at a mean follow-up of 21.9 months. Six patients had previously had an unsuccessful ACL reconstruction. The Knee Osteoarthritis Outcome Score (KOOS) was administered to assess patients’ opinions regarding their knee, while a modified International Knee Documentation Committee (IKDC) scoring system was used to examine knee stability. The IKDC form initially consisted of seven knee-related parameters that were each rated as either normal, nearly normal, abnormal, and severely abnormal. The worst score amongst the seven categories determined the final score. The form was later modified in 2001 to include subjective factors such as symptoms, sports activities, and ability to function. The Tegner activity scale, a subjective ten point activity scale, was obtained to assess patient activity levels. A Telos radiographic stress system was used to examine anterior knee displacement, which involves obtaining radiographs with the knee in ninety degrees of flexion and a posterior force applied to the knee. When results are compared to the normal knee, this test helps in the assessment of post-operative laxity in the reconstructed knee which may indicate that the graft is failing. None of the patients presented symptoms of synovitis, but longer follow-up time is required to properly draw conclusions with respect to patient outcome. Although Tegner scores improved significantly following surgery, no patients returned to pre-injury activity levels. Data obtained by the KOOS demonstrated patient satisfaction ranged from 73.5 to 93.0 %. In comparison to the uninjured knee, post-operative Telos stress radiography and the Lachman test indicated an average posterior-anterior displacement of 7.3 mm for the involved knee (28). A more complete examination of the LARS artificial ligament was provided in a subsequent study by the same authors. Nau et al. conducted a two-year follow-up randomized controlled trial that compared the BPTB autograft with the LARS artificial ligament method of ACL reconstruction in 53 patients with chronic instability (1). Like the preceding study, assessments included clinical examination, anterior laxity testing, as well as the KOOS, IKDC, and Tegner scores. In particular, IKDC evaluation revealed little significant difference between the two methods. Follow-up values for instrumented laxity testing were greater in the LARS group. While similar overall results were obtained for both groups, these results may suggest that a full return to activity may be hastened by using the LARS artificial ligament rather than the conventional BPTB technique (1). Another recent study by Talbot et al. examined the use of the LARS artificial ligament for ligament reconstruction in knee dislocations (2). Twenty patients were included with a mean follow-up of 27.4 months. Each patient was evaluated using the Lysholm score, underwent clinical examination to identify ligamentous laxity and range of motion, and completed the ACL quality of life (ACL-QoL) questionnaire. The mean Lysholm score was 71.7, which is lower than scores reported by several other studies (range of 74.7 to 91.3) investigating the outcomes of knee dislocations treated with surgery as reported by Fanelli (35). Following the same trend, Telos radiometry revealed a mean residual laxity of 5 mm in patients post-surgery, which is also greater than these previously reported results. The average range of motion post-surgery was 118° with a mean fixed flexion contracture of 2° (2). Although preliminary investigations into the use of the LARS artificial ligament have been encouraging, concerns regarding the risk of rupture remain and must be addressed through long-term follow-up studies. TISSUE ENGINEERING ADVANCES Permanent synthetic prostheses are capable of duplicating the mechanical and structural properties of the ACL. However, they generally tend to lose strength with time. Tissue-based or tissue-aided implants offer the additional possibility of the restoration of normal joint kinematics while the mechanical behaviour of these implants is expected to improve over time as tissues are remodelled within the knee (36). An ideal ACL scaffold must meet the immediate functional mechanical demands within the reconstructed knee, however, they must also degrade at a rate similar to that of tissue ingrowth. Accordingly, the ACL scaffold should lose its mechanical integrity while allowing the remodelled tissues to gain strength and accept an increasing amount of the mechanical demands placed on the ACL. Current research into this novel tissue-engineering approach has focused on seeding either collagen-based scaffolds or synthetic biodegradable polymers with a variety of different cell types. In hopes of stimulating early healing, reducing biomaterial-related inflammatory response and improving neoligament formation, several researchers have sought to adopt a cell sheeting technique to improve the performance of the synthetic ACL scaffold. Several groups have conducted experiments on this tissue-engineered approach to ACL ligament reconstruction using both fibroblast-seeded synthetic scaffolds and collagen-based prosthetics (37, 38). Bellincampi et al. measured the ingrowth characteristics of rabbit fibroblasts on skin and ACL scaffolds (39). Subsequent in vivo studies suggested that fibroblast-seeded collagen scaffolds were viable after re-implantation into the donor rabbit. The major limitations of these approaches are the allogenicity of the collagen scaffolds, often leading to further complications. Collagen-based constructs also suffer from batch-to-batch variability, making consistent reproduction of these prostheses difficult. Collagen does not offer the same flexibility for modification that is reported with the technology of biodegradable polymers (6). As an alternative to the scaffolds made of non-degradable polymers, investigators have begun to examine biodegradable materials that would provide immediate stabilization to the repaired ligament but would also act as a scaffold for the ingrowth and/or replacement by host cells. Cao et al. described the generation of neo-tendons in a nude mice model by implanting polyglycolic acid (PGA) scaffolds seeded with bovine tendon fibroblasts in the subcutaneous space of athymic mice (40). Using a similar system, Koski et al. reported the formation of ligament-like structures when fibroblasts isolated from bovine cruciate ligaments were seeded onto PGA scaffolds and implanted subcutaneously in nude mice (6). In both studies, the tissue developed histological characteristics similar to normal tendon and ligament over time. Ouyang et al. reported that bone marrow stromal cells (bMSCs) seeded onto poly-lactic and -glycolic acid (PLGA) scaffolds grew as a multi-layer of cells intertwined in a collagen matrix synthesized by the cells 35 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics themselves. They also noted that the cell sheet formed faster than scaffolds seeded with terminally differentiated cells, such as fibroblasts and smooth muscle cells (41). They reported that degradation of the scaffolds occurred over time, as indicated by a decrease in failure load. Altman et al. seeded 6-cord silk scaffolds with human bone marrow stromal cells and cultured them for 14–21 days. Their results similarly suggested a slow degradation of the scaffolds. However, fatigue analysis and subsequent regression analysis revealed an expected matrix lifetime equivalent to 1 year in vivo. While these reports are an estimate, the authors suggest that this is a marked improvement compared to similar studies using collagen scaffolds (36). The future of tissue engineering may also require a significant contribution from cell-specific growth factors influencing the maturation and homeostasis of the healing response of ligament tissue. Studies have suggested that individual growth factors may have an important effect on cell division and enhance ligament healing, while others have pointed to the synergistic effect of a combination of growth factors as important to cell outgrowth in ACL explants (42, 43). In a study observing the effects of several growth factors on the cell migration, proliferation and collagen production in human ACL cells, Murray et al. suggested that specific dose-response relationships may exist for the optimal activity of each growth factor (44). These authors observed that the addition of transforming growth factor (TGF)β-1 led to an increased cell population, as well as increased collagen and smooth muscle actin production in human ACL cells cultured on top of a collagen-glycosaminoglycan scaffold. Despite the complex nature of the inter-dependent factors at play during the recovery and rehabilitative period following an ACL reconstruction, much of the literature today suggests that tissue engineering techniques will lead to a new generation of ACL replacements, capable of regenerating a mechanically robust and natural ACL. COMPUTER-ASSISTED ACL RECONSTRUCTION In an attempt to improve the accuracy and reproducibility of ACL graft placement, several groups have turned to computer-assisted surgery in an attempt to reduce the incidence of graft failure (45, 46). These systems are capable of modeling the placement and predicting the impingement of an ACL graft based on intra-operative anatomical landmarks and signals received at an opto-electric camera. These studies reported that experience level did not affect the placement of the tunnels. Computer-assisted ACL replacement reduces variance in tunnel placement and allows residents and less experienced surgeons to limit complications and control tunnel positioning (46). Based on the success of navigation systems for total knee and hip replacements, the use of computer-assisted ACL reconstruction may lead to similarly dramatic improvements in technical and functional outcomes. CONCLUSION Satisfactory prosthetic replacement of the ACL has been a focus of orthopaedic research endeavours for the past thirty years. Desires to provide immediate mobility and strength to the ACL-deficient knee while avoiding the donor site morbidity caused by the commonly used autograft surgical techniques continually drive new research initiatives. Most of the grafts that have been developed to date have failed due to unsatisfactory long-term physiologic and functional performance. Most permanent ACL prostheses are prone to creep, fatigue, and mechanical failure within several years after implantation (40). Tissue ingrowth scaffolds and ligament augmentation devices require further refinement to provide effective mechanical support while avoiding stress-shielding of the host tissue. In view of these factors, prosthetics are not widely used today in ACL reconstruction, and autogenous tissue grafts remain the gold standard used by the majority of surgeons. Perhaps development of resorbable, tissue-inducing and cell-seeded biomaterials will improve the long-term biomechanical performance of the reconstructed anterior cruciate ligament. Advances in tissue engineering combined with developments in molecular biology and gene therapy may couple with the rapid gains in computer-assisted surgery to provide improved options for the ACL-deficient knee, with a greater potential to restore its pre-injury state.

Eur_J_Appl_Physiol-3-1-2039775

Distribution of motor unit potential velocities in short static and prolonged dynamic contractions at low forces: use of the within-subject’s skewness and standard deviation variables

Behaviour of motor unit potential (MUP) velocities in relation to (low) force and duration was investigated in biceps brachii muscle using a surface electrode array. Short static tests of 3.8 s (41 subjects) and prolonged dynamic tests (prolonged tests) of 4 min (30 subjects) were performed as position tasks, applying forces up to 20% of maximal voluntary contraction (MVC). Four variables, derived from the inter-peak latency technique, were used to describe changes in the surface electromyography signal: the mean muscle fibre conduction velocity (CV), the proportion between slow and fast MUPs expressed as the within-subject skewness of MUP velocities, the within-subject standard deviation of MUP velocities [SD-peak velocity (PV)], and the amount of MUPs per second (peak frequency = PF). In short static tests and the initial phase of prolonged tests, larger forces induced an increase of the CV and PF, accompanied with the shift of MUP velocities towards higher values, whereas the SD-PV did not change. During the first 1.5–2 min of the prolonged lower force levels tests (unloaded, and loaded 5 and 10% MVC) the CV and SD-PV slightly decreased and the MUP velocities shifted towards lower values; then the three variables stabilized. The PF values did not change in these tests. However, during the prolonged higher force (20% MVC) test, the CV decreased and MUP velocities shifted towards lower values without stabilization, while the SD-PV broadened and the PF decreased progressively. It is argued that these combined results reflect changes in both neural regulatory strategies and muscle membrane state. Introduction Diverse laboratory conditions have been used in surface electromyography (sEMG) studies in order to gain insights into the neural regulatory strategies and muscle membrane alterations. The influence of force load on sEMG can be investigated by using force tasks or position tasks. The majority of studies have been performed as force tasks, which means that the subject controls the effort by maintaining a target force while the limb position is fixed. During the position tasks, in contrast, an inertial load is applied while the subject controls a target limb position. Both force and position tasks can be performed in static or dynamic conditions. Examples of the static force tasks are the well-known isometric experiments, with higher and lower force levels. In the recent past, force tasks in dynamic conditions have been performed sporadically, such as the cycling experiments by Pozzo et al. (2004) and Farina et al. (2004). Since Hunter et al. (2002) found that, with the same load torque, position tasks resulted in a shorter endurance time than force tasks, suggesting different regulatory mechanism for the both type of tasks, the position task studies gained field. Most position task experiments have been performed in static conditions, evaluating the underlying physiological phenomena during force versus position tasks (Hunter et al. 2002, 2003; Hunter and Enoka 2003; Rudroff et al. 2005, 2007). Potvin (1997) has described the changes in the sEMG during position tasks in dynamic conditions. Previous findings suggest that, as compared with force tasks, position tasks induce greater synaptic input into the motor neurons (Mottram et al. 2005a) and greater adaptation in the motor unit discharge (MacGillis et al. 2003). Changes in muscle activity during (static and dynamic) position tasks have been assessed using two of the three traditional sEMG parameters, the power spectrum and the global sEMG amplitude. However, the third parameter, the mean muscle fibre conduction velocity (CV), has been lacking. Spectral estimates are generally accepted in fatigue experiments as equivalents of CV because they highly correlate with the CV’s changes (Bigland-Ritchie 1981; Eberstein and Beatie 1985; Arendt-Nielsen and Mills 1985). But this correlation holds true only for the constant forces and isometric conditions; thus, a replacement of CV by power spectrum assessments does not always seem feasible (Farina et al. 2002; Broman et al. 1985). The favour of CV above power spectrum is, furthermore, that it renders direct and absolute values of conduction velocity, and is less sensitive to the anatomical local relationships, such as depth of the motor unit (MU) in relation to the muscle and skin surface (Farina et al. 2002). Yet the limitation of these global CV measurements remains the lack of sensitivity to the changes at the level of an individual motor unit potential/ motor unit. To accommodate with these limitations of CV, researchers have recently been trying to disentangle the propagation velocities of individual motor unit potentials (MUPs) from sEMG. One of the methods is the inter-peak latency method (IPL) proposed by Lange et al. (2002). The principle comprises calculating conduction velocities of the MUPs from the latencies between paired MUPs of two differential sEMG signals obtained parallel to the muscle fibres, and the distance between the recording electrodes. The negative peaks of two paired MUPs are then the elements determining the interpeak latency. As motor unit propagation velocity reflects the intrinsic physiological properties of a MU, such as fast-twitch or slow-twitch type (Buchthal et al. 1973; Andreassen and Arendt-Nielsen 1987), the IPL method renders many diverse MUP velocities. Lange at al. (2002) proposed using the standard deviation of MUP velocities as an additional measure that offers information about muscle fibre properties. Changes in these velocities during prolonged effort may indicate, for example, slowing/fatigue of the activated motor units and/or appearance of fast/newly recruited motor units. Such shifts in the activated MUs’ populations were shown by Houtman et al. (2003) by eliciting the MUP velocities with the IPL method and presenting their distribution in histograms. The IPL method has not been applied much. It yields insights into the diversity of MUP velocities and thereby the underlying changes in the MU activity. The method is simple and does not require expensive apparatus or software. When compared with techniques that assess the propagation patterns of MUPs by multi-channel/spatial resolution sEMG (Masuda and Sadoyama 1986; Rau et al. 1997), the IPL method is unable to distinguish and follow individual MUPs belonging to the specific MUs. In the present study, the sEMG signal was described with four parameters derived from the IPL method: (1) the mean muscle CV which was the average of the obtained MUP velocities; the two statistical distribution variables, which were: (2) the within-subject MUP velocities’ skewness [Sk-peak velocity (PV)] and (3) the within-subject MUP velocities’ standard deviation (SD-PV), and (4) the peak frequency (PF), a variable expressing the amount of MUP activity (number of peaks = MUPs) per second. The aim of the present study was to investigate with the four parameters the changes in MUPs’ velocities of the biceps brachii muscle (BB) during prolonged dynamic position tasks, in dependence of (low) force and duration. It is chosen for the dynamic position tasks as study design because their physiology promised the finding of a large variety of MUP velocities (great amount of activity due to the position tasks character, and diversity because of the recruitment/derecruitment changes within the dynamic cycle). Whole cycles of movement with their concentric and eccentric phases were analyzed together in order to evaluate a total of the sEMG activity with its evolution over time. To highlight the initial changes with the effect of force on it, the changes during the first 14.4 s of the dynamic tasks were evaluated separately. Additively, short static position tasks were performed in order to show the (early) changes on force, without any influence of movements on the signal. Methods Subjects The study involved short static and prolonged dynamic experiments. Forty-one healthy and physically active males (24.8 ± 6.7 years, from 17 to 48) (mean ± SD) volunteered for the first experiment and 30 randomly chosen subjects from that group (25.4 ± 7.6 years, from 18 to 42) participated in both experiments. Exclusion criteria were drug abuse and the practice of bodybuilding. Three from a total of 44 subjects were excluded because of the impossibility to obtain a required correlation coefficient between the sEMG signals used to estimate the parameters’ values. The experimental protocol was conducted according to the Helsinki Declaration and approved by the local ethics committee. All participants gave written informed consent. Experimental set up Maximal voluntary contraction (MVC) of elbow flexors was measured at least 5 days before the experiment with a hand-held dynamometer (Lameris Instruments, Utrecht, The Netherlands). During the MVC measurements subjects were sitting upright. The shoulder was slightly abducted and flexed at 45°, the elbow was firmly sustained and flexed at 90°, and the forearm was supinated. The dynamometer was applied to the wrist by the break method (van der Ploeg and Oosterhuis 1991). The peak hold was switched off and the force was kept for at least 3 s. The mean of three maximal values was taken as a MVC. The MVC was assessed with elbow at 90°, although the tests were performed at the elbow angle of 135° (Philippou et al. 2004). During the experiment subjects were sitting in a chair. The upper arm was slightly abducted and comfortably supported at 45° of the shoulder flexion, the forearm was free. When the elbow was stretched, the line of the upper arm-forearm was at 45° in relation to horizontal. When the elbow was flexed to the angle of 135°, the forearm was horizontal. The forearm was supinated during static and dynamic tests. Static tests Subjects were asked to hold the forearm horizontally (elbow angle was then 135°). A visual bar helped to maintain the correct (horizontal) position of the forearm. In the loaded tests a sack filled with lead and sand was placed in the palm. Three levels of force were applied in blocks that were 3 min apart: unloaded, loaded 10 and 20% MVC. A block consisted of three tests (three repetitions at the same level of force); every test lasted for 3.8 s and was within a block separated 30 s from one another. Dynamic tests All participants of the dynamic tests underwent previously static tests, separated by 5 min. Subjects were asked to swing the forearm from the stretched (elbow angle 180°) to horizontal position (elbow angle 135°), thus moving over an angle of 45°. They did it within a rate of 40 beats per minute (one up-and-down movement in one beat), given by a metronome sound. The visual bar indicated the horizontal position to which the lower arm returned after being stretched. Four force levels were applied: unloaded, and loaded 5, 10 and 20% MVC. The tests lasted for 4 min and were separated by 5 min. EMG recording Measurements were performed on the short head of the BB of a dominant arm. A surface electrode array consisted of three golden-coated electrodes (Harwin, P25-3526), diameter 1.5 mm, insulated in synthetic material plate, with a 10 mm distance between the electrodes (Sadoyama et al. 1985). The skin was cleaned with 95% ethanol. The electrode array was placed parallel to the muscle fibres (Sollie et al. 1985). The proximal electrode was positioned exactly on the distal one-quarter point of the upper arm, measured between the coracoid and the elbow crease. This place of the electrode was about halfway between the endplate zone and the tendon, securing a sufficient distance from the endplate (Sadoyama et al. 1985; Masuda and Sadoyama 1987). Bipolar derivation was made from the proximal to distal direction. The optimal electrode position was controlled by both the observation of the signal on the monitor and the estimation of a correlation coefficient (CC) of the two sEMG signals, which was accepted at r > 0.7 for unloaded, r > 0.85 for 5% MVC and r > 0.9 for higher loaded tests. (During the static and dynamic tests, the maximal CC for unloaded arm was usually lower than that for loaded arm, which was consistent with Hogrel et al. 1998, who have estimated for an unloaded arm r > 0.7). The ground electrode was placed on the lateral upper arm, slightly proximal from the derivation electrode. The temperature sensor was medial on the upper arm. Two obtained signals were differentially amplified (gain 2,000–10,000×) and band pass filtered at 2–250 Hz by EMG apparatus (Viking IV, US). Data processing The signals were simultaneously A/D converted (sampling 10 kHz, 12 bits acquisition). Data were stored on a personal computer. The signal was analyzed with LabVIEW 6.1 that also facilitated a partial on-line analysis. The peak selection and the correlation coefficient assessments for both static and dynamic tests were performed on 0.2 s epochs. In the static tests, measurements were taken every second during 0.8 s (comprising 4 epochs of 0.2 s). A static test was of 3.8 s duration and was repeated three times for each force level. The statistical analyses were performed on the data of these three repeated tests taken together. In the dynamic tests, the data were assembled every 30 s during 14.4 s (comprising 72 epochs of 0.2 s). The test duration was 4 min. Peak selection The basic principle was that of Lange et al. (2002). The software was custom designed and written in LabView. The orientation of the signals is up-negative. 1st step: finding zero line. 2nd step: finding the peak-to-peak amplitude of the largest MUP in an epoch of 0.2 s. 3d step: finding a peak-decline structure. The peak-decline is defined as a structure with a decline of ≥20% compared with the largest MUP amplitude of a 0.2 epoch, over ≤4 ms (≤40 sample points). 3d step: finding a peak. A peak is the highest (most negative) point previous to the decline, and must be ≥10 μV (the threshold of the noise level). 4th step: finding a pair peak. A pair peak is a peak in the second signal with the properties as previous, found in a time window between 1.49 and 4 ms after the peak of the first signal. This window is chosen assuming the physiological CV values of 2.5–6.67 m/s. 5th step: excluding double peak. If a peak from the first signal matches two different peaks from the second signal, then the first peak from the second signal is true. If during the fatiguing tests the CV severely diminishes, then the low limit of velocity is put at 1.3 m/s, making a window of 1.49–7.68 ms. As an objective pragmatic criterion to this change a lowering of the peak frequency with ≤30% was assumed, as compared with the first value of the 20% MVC test. The parameters The following calculations were performed: (1) basic calculation of PV, following the IPL method; (2) mean CV, expressed as an average value of the PVs; (3) within-subject skewness of the peak velocities (Sk-PV), expressed as a skewness of a PVs’ population of a subject; (4) within-subject standard deviation of the peak velocities (SD-PV), expressed as a standard deviation of a PVs’ population of a subject; and (5) peak frequency (PF), expressed as a number of peaks per second. Statistics One-way analysis of variance (ANOVA) with repeated measures on force was used to compare the dependent variables for static tests (three levels) and for the initial values of the dynamic tests (four levels). A two-way ANOVA with repeated measures on force ( four levels) and time (nine levels) was used to compare variables during the dynamic tests. In the case of interactions between force and time, to describe changes over time, when appropriate, four separated ANOVAs were performed with smaller time windows of 0–60, 60–120, 120–180 and 180–240 s. To be assured that the dependent variables met parametric assumptions, plots of residues were produced with SPSS program, model control as suggested by Kutner et al. (2005, p. 1157). No relevant deviations of model were detected. Pearson correlation coefficients were calculated to evaluate associations between variables. A level of P < 0.05 was used to identify statistical significance. Results Subjects’ physical characteristics are presented in the Table 1. The force of the biceps correlated positively with the upper arm circumference (r = 0.484, P < 0.01). No association was found between either force or upper arm circumference and the sEMG variables. The average skin temperature increased during the dynamic tests by 1.65°C (P < 0.001); it did not change during the static tests. Table 1Characteristics of 41 participants to the static tests; 30 of them participated in the dynamic tests as wellNMinimumMaximumMeanSDAge (years)41164824.76.7Height (cm)41166.0197.0183.28.0Weight (kg)4160.095.074.39.2Force (N) right38148.5346.5246.141.9Force (N) left (for left-handed)3262.3267.3265.12.5Skin thickness (mm)411.44.22.4.7Circumference upper arm (cm)4124.031.027.52.0Length of biceps (cm)a4130.5039.5035.42.0Length lower arm/radius (cm)b3324.030.026.41.6Length lower arm/palm (cm)c3330.036.033.11.6Initial skin temp. (˚C)4130.833.832.3.8Temp. upper arm before dyn. tests (˚C)3030.834.232.3.9Temp. upper arm after dyn. tests (˚C)3032.235.733.91.1Room temp. (˚C)4120.024.022.31.0a From the coracoid to the elbow creaseb From the lateral epicondyle to the wrist creasec From the lateral epicondyle to the middle of the palm Static tests Mean muscle fibre conduction velocity (CV) Histograms in Fig. 1 show an example of a PVs’ population in one subject during the static tests at three levels of force: unloaded, 10 and 20% MVC. Figure 2a presents the averages of the CVs over 41 subjects, calculated from the subject’s PVs. The CV of the unloaded test was the lowest (3.92 ± SD 0.27 m s−1) and it increased with augmenting force levels (effect of force P < 0.001). A positive correlation existed between the CVs of unloaded test with 10% MVC, and 10% with 20% MVC (all r > 0.505, P < 0.05). Fig. 1Distribution of peak velocities of one subject during short static tests at three force levels: unloaded, and loaded 10 and 20% MVC. Note the shift of the velocities as a whole to the higher values with increasing level of forceFig. 2Behaviour of peak velocities (PVs) as effect of force, expressed with four parameters. a Mean conduction velocity (CV); b skewness of within-subject PVs; c SD of within-subject PVs (SD); and d number of peaks per second (peak frequency = PF). Averages and standard errors are given, obtained from 41 subjects in short static tests at three levels of force: unloaded, and loaded 10 and 20% of maximal voluntary contraction. With increasing forces, the CV and amount of activity (PF) increases, accompanied with augmenting proportion of fast peaks (the skewness value diminishes). However, the spread of peak velocities within an individual (SD) does not change Skewness of peak velocities (Sk-PV) Figure 2b presents the averages of the within-subject PVs’ skewnesses over 41 subjects (see also the histograms of PVs in Fig. 1). In all the tests a moderate positive skewness was found, which indicates a relative excess of lower velocities at the distribution scale. With increasing force the Sk-PV significantly diminished, indicating that the proportion of higher velocities increased (effect of force P < 0.05). The PVs’ population as a whole shifted towards higher values with increasing forces too, which can be seen on the histograms in Fig. 1. Standard deviation of peak velocities (SD-PV) The averages of the within-subject’s standard deviations of PVs are shown in Fig. 2c. The SD-PV did not change when the force levels increased (effect of force n.s.). Peak frequency (PF) Figure 2d shows the averages of PF in the static tests. The PF increased with increasing forces (effect of force P < 0.001). Dynamic tests Muscle fibre conduction velocity (CV) Histograms in Fig. 3 show the PVs estimated from one subject during dynamic tests at four levels of force: unloaded, loaded with 5, 10 and 20 MVC. Figure 4a shows the averages of CV calculated from the subject’s PVs over 30 subjects. As in the static tests, the initial CV of the dynamic tests increased with level of force (effect of force P < 0.001). Further, a positive correlation was found between the CV of the static tests and the initial CV of the respective dynamic tests (all r > 0.409, P < 0.05). The lowest initial CV was that of the unloaded test with about 4 (3.6–4.5) m s−1 and the highest was at 20% MVC with approximately 4.6 (4.0–5.15) m s−1, increasing from the unloaded to 20% MVC test with 14 ± 9.6%. During all the dynamic tests the CV significantly declined (effects of time for the unloaded test P < 0.02; for other tests P < 0.001), whereby the decline was steeper with larger forces (interaction between force and time, P < 0.001). At the three lowest force levels (unloaded, and loaded 5 and 10% MVC), the CV had two phases: a decline phase lasting for about 120–150 s and a steady phase continuing to the end of a test. The steady state values approached the level of the unloaded test. During the 20% MVC test, however, the CV continued to decline, without a stable phase. Twelve of 30 subjects (40%) reported fatigue during the 20% MVC test and terminated the task prematurely between 90 and 210 s. The CV decreased for the fatigued subjects from 4.6 ± 0.3 (4.2–5.0) to 3.6 ± 0.3 (3.0–4.1) m s−1 and for the continuing subjects from 4.5 ± 0.3 (4.0–5.15) m s−1 to 3.8 ± 0.4 (3.0–4.7) m s−1. Neither the absolute initial CV nor the end CV differed significantly between the groups (P = 0.497 and P = 0.124, respectively). However, the relative decline of the CV tended to be larger for the fatigued subjects compared with the continuing subjects, for fatigued being about −20 (−7 to −35)% and for continuing −15 (+7 to −28)%; P = 0.074. Fig. 3Changes in the distribution of peak velocities (PVs) over time for different levels of force. The PVs are obtained from one subject (the same as in Fig. 1 for static tests) during prolonged dynamic contractions at four levels of force: unloaded, and loaded 5, 10 and 20% of maximal voluntary contraction (MVC). Every histogram represents a number of PVs within a period of 14.4 s. Initially (at time zero), a global shift of peak velocities is visible towards higher values when forces augment. During the unloaded, and loaded 5 and 10% MVC tests, slower peaks are moderately increasing and faster peaks are diminishing. During the 20% MVC test, the peak velocities shift considerably as a whole towards lower regions, and the amount of peaks visibly diminishesFig. 4Effects of force and time on the behaviour of peak velocities (PVs), expressed with four parameters. a Mean conduction velocity (CV); b skewness of within-subject PVs; c SD of within-subject PVs (SD); and d number of peaks per second (peak frequency = PF). Averages and standard errors are given, obtained from 30 subjects during prolonged dynamic tests at four levels of load: unloaded, and loaded 5, 10 and 20% of maximal voluntary contraction (MVC). Note the difference in the decline pattern of the CV and the PF: the mean muscle conduction velocity declines over time for all levels of force, while the amount of activity remains stable for the three lower force level tests (unloaded, and loaded 5 and 10% MVC). In the higher force level test (20% MVC), the CV starts declining immediately, while the PF declines first gradually and later on steeply. Note the stable SD values from about 90 s for the three lower force tests, while the SD of the higher force test (20% MVC) clearly increases Skewness of peak velocities (Sk-PV) Histograms in Fig. 3 show the distribution of PVs of one subject and Fig. 4b presents the averages of the within-subject’s skewnesses of the PVs over 30 subjects. In the initial phase, consistent with the static tests, the PVs were most positively skewed (=skewed in favour of lower velocities) in the unloaded test, and the skewness diminished with increasing forces (effect of force P < 0.001). That means that lower PVs dominated in the unloaded test and the proportion of higher PVs increased when forces augmented. Thus, in the 20% MVC test, the initial PVs approached a normal distribution. In addition, with increasing forces the PVs as a whole group seem to shift towards higher values, as can be seen in the histograms Fig. 3a–d at time zero. During the tests, the skewness increased again, except for the unloaded test, indicating growing proportion of lower PVs over time and decreasing amount of higher PVs (histograms Fig. 3a–c). The larger the forces the steeper increase of skewness (for all tests together: effect of time P < 0.001; interaction between force and time P = 0.005; effect of time in unloaded test P = n.s.; for the tests 5, 10 and 20% MVC interaction between force and time P < 0.001). During the last 2 min of the 5 and 10% MVC tests the Sk-PV stabilized. In the 20% MVC test, however, the positivity still tended to increase up to the end of the test (over the time windows 120–180 and 180–240 s: effect of time for the 5, 10 and 20% MVC tests, n.s.; interaction between force and time, P = 0.106; for the 5 and 10% MVC tests the effect of time, n.s.; for 20% MVC test over 120–180 s n.s., over 180–240 s, P = 0.052). At the end of the 20% MVC test, the whole population of PVs appeared to shift towards the lower values too, as can be seen at the last two histograms in the Fig. 3d. Taken together, in the initial phase of activity, the proportion of fast peaks increased with increasing force. In the prolonged tests loaded up to 10% MVC, the proportion of fast peaks declined again over the first 2 min and then stabilized at about the level of the unloaded test. During the 20% MVC test, however, the proportion of fast peaks still tended to decline up to the end of the test, accompanied with a growing amount of slow peaks. Standard deviation of peak velocities (SD-PV) The averages of SD-PVs of 30 subjects are presented in Fig. 4c. The initial SD-PV was for all force levels similar (P = 0.65), which resembled the static tests. The values in the dynamic tests were significantly higher compared with those of respective static tests (paired sample t test for the unloaded, 10 and 20% MVC tests, respectively P = 0.014; P = 0.027 and P = 0.001). During the tests the SD-PV changed significantly with time, depending on the force level (for all tests effect of time P = 0.011, interaction between force and time P < 0.001). The course of the SD-PV had two phases which were different for the three lower force levels (unloaded, 5 and 10% MVC), compared with 20% MVC. In the three lower force levels, the SD-PV first declined over about 90 s and then stabilized (interaction between force and time over 0–240 s P = n.s.; effect of time over the time windows 0–60 s P < 0.001, 60–120 s P = 0.075; 120–180 and 180–240 s for both P > 0.343). However, during the 20% MVC test, the decline, which lasted for approximately 60 s, was followed by an extreme increase (effect of time over 0–240 s P < 0.001; effect of time over 0–60 s P = 0.019; over 60–120 s, which was in opposite direction, P = 0.019, and 120–180 and 180–240 s P < 0.05). This pattern of results can also be seen in the histograms Fig. 3d. Peak frequency (PF) Figure 4d shows averages of PF over 30 subjects. The initial PF rose with increasing force levels (effect of force P < 0.001). Then, during the tests at three lowest force levels (unloaded, 5 and 10% MVC) the PF remained stable. But during the 20% MVC test the PF significantly diminished, at the beginning gradually and from about 120 s steeply (interaction between force and time for all the four tests, P < 0.001; for the three lowest force levels, P = 0.234; effect of time for the three lowest force levels P = 0.541; effect of time for 20% MVC P < 0.001). There was much variability among subjects in the size of decline in 20% MVC test. For those who were able to complete the test, the PF continued to decline up to the end, with exception of one subject in whom the PF increased instead. At 240th s the PF of the continuing subjects was reduced by −35 (−78 to +3)%. Discussion Changes in the distribution of MUP velocities as an effect of (low) force and duration were described with four parameters: (1) the global parameter of mean CV, (2) the within subject skewness of a population of MUP velocities; (3) the within-subject standard deviation of MUP velocities and (4) the amount of MUP activity, expressed as MUP frequency. First we will comment on the four parameters. Next, using these parameters, we will discuss the main findings. The four parameters The CV parameter renders a mean value of the motor unit potentials’ propagation velocities. The CV will increase or decrease, depending on the type of the activated (fast-twitch and slow-twitch) motor units. It will also change with the alterations in muscle membrane potential, which influences the depolarisation/repolarisation processes. For example, it decreases in muscular fatigue (Stalberg 1966; Milner-Brown and Miller 1986), and increases with a smaller interstimulus interval, such as that due to the rising rate coding (Gydikov and Christova 1984; Radicheva et al. 1986; Nishizono et al. 1989). Because of the lack of studies, it is not possible to compare the CV values with those of any other position tasks experiments. However, the estimates are consistent with those of the studies using static and dynamic force tasks, especially with those of Lange et al. (2002), obtained with the inter-peak latency method. Skewness is used as an sEMG parameter in the present study for the first time. This statistical measure of deviation from a normal distribution, in this case expresses the proportion between slower and faster MUPs within an individual. It will increase with the growing proportion of slow/tonic/fatigue resistant MUs and will decrease with the augmenting proportion of fast/phasic/fatigable MUs. All the estimates were moderately positively skewed, which indicates a relative excess of lower MUP velocities. The within-subject standard deviation of MUP velocities, a variable introduced by Lange et al. (2002) shows the spread of MUP velocities. For a fresh and healthy muscle, it will render information about diversity of the participating MUs. In a fatigued muscle, when membrane propagation is slowing, the SD-PV will broaden as a result of the temporal dispersion of velocities. Further, the SD-PV can be expected to narrow when the same velocities repeat, such as in a higher discharge rate of a certain group of MUs. In the brief static position tasks, the standard deviations were larger than those previously estimated by Lange and colleagues (2002) in the staticforce tasks, with respectively 0.55–0.62 and 0.3–0.52 m s−1. This difference can be due to the different type of tasks, as data are available suggesting that different excitatory/inhibitory inputs to the motor neurons play a part in the position tasks and the force tasks (Rudroff et al. 2005). In the present study, the SD-PVs of the dynamic tests were larger than those of the static tests. This difference can have different explanations. With every contraction of a dynamic cycle, the muscle fibres’ diameter increases, leading to higher fibre propagation velocities (in a part of a cycle) (Arendt-Nielsen et al.1992). This problem was partially restrained by using a small movement angle of 45°. However, the most important role in the increase of SD-PVs’ during dynamic contractions might be played by the cyclic changes in the motor units’ discharge characteristics. Several studies deliver the supporting data. For example, during dynamic contractions the amount of activity differs between the concentric and eccentric phases, suggesting different regulatory strategies for the two phases (Potvin 1997). Previous studies have also shown that the rate of MU discharge is related to the movement’s velocity, and the (angle) velocity alters depending on the elbow angle (Gillis 1972; Milner-Brown et al. 1973a; Potvin 1997). Thus, the discharge rates will alter through a cycle. In addition, eccentric movements are shown to further the activation of high-threshold (fast propagating) motor units (Komi and Tesch 1979; Nardone et al. 1989). Peak frequency (MUP frequency) expresses the amount of MUPs in a time. To our knowledge, it is used as sEMG parameter in the present study for the first time. The PF is comparable with the zero crossing parameter (Lynn 1979; Masuda et al. 1982; Hagg 1981). It is argued that the diminishing zero crossings’ number during prolonged exercises indicates decrease in MUs’ activity, as a sign of fatigue (Inbar et al 1986; Hagg and Suurküla 1991). Lange et al. (2002) mentioned a number of MUPs obtained during 1.5 s measurements in static force tasks, which renders the frequency of about 4–5 MUPs/s for 10% MVC test, and about 8 MUPs/s for 20% MVC test. These values are much lower than ours with 37, 47 and 50 MUPs/s (for respectively unloaded, loaded 10 and 20% MVC tests) in static position tasks. The findings are consistent with the interpretation that during position tasks more motor units are being recruited compared with force tasks, and the discharge rate of MUs is higher (Mottram et al. 2005a). The initial changes on increasing force levels (in the static and dynamic tests) The effects of force on the behaviour of MUPs in the short static tests and the initial phase of the prolonged dynamic tests were similar. With increasing forces the CV grew higher and MUP frequency increased. In the population of MUP velocities, not only the proportion of fast MUPs increased (see the skewness in Fig. 2b), but also the velocities as a whole shifted towards higher values (histograms in Figs. 1, 3a–d at time zero). Despite of the changes in the skewness, the standard deviation of MUP velocities remained unaltered. The increases of the CV with increasing force are in accordance with the previous findings inforce tasks (Naeije and Zorn 1983; Broman et al. 1985; Sadoyama and Masuda 1987; Zwarts and Arendt-Nielsen 1988; Lange et al. 2002). It is generally accepted that these increases are caused by activating high threshold/fast/phasic motor units when demands of force are augmented (Henneman et al. 1965; Milner-Brown et al. 1973b; Gantchev et al. 1992; Gazzoni et al. 2001). This explanation was supported by the increasing proportion of fast MUPs found in the present study. However, the global shift of MUP velocities towards higher values may be induced by either replacing slow MUs by fast ones, or by increasing the propagation velocity of the muscle membrane due to the rising rate coding (Radicheva et al. 1986; Nishizono et al. 1989; Van der Hoeven and Lange 1994). Little is known about changes in the within-subject standard deviation of MUP velocities. Only Lange and colleagues (2002) mentioned (inforce tasks), contrary to our results, increases between 10 and 50% MVC (and no increases between 50 and 100%). The experiments of Lange et al. and the present short static experiments were both isometric, and the duration of the contraction did not differ much (our 3.8 vs. their 1.5 s). In fact, the two studies only differed in the type of task (position tasks applied by us vs. force tasks by Lange et al.). This task difference may play a part in the discrepancy of the standard deviation, as the excitatory and inhibitory inputs for the two tasks are supposed to be different (Rudroff et al. 2005). Thus, for the two tasks different types of motor units (with different velocities) may be activated. In short, increases of mean CV with increasing forces in the initial phase of muscle activity may be a result of both recruitment of fast/phasic motor units and faster membrane propagation. Changes in the prolonged dynamic tests Tests loaded below 20% MVC The main feature of the prolonged tests loaded 5 and 10% MVC were changes in the CV, skewness and standard deviation over the first 90–120 s, followed by stabilizing. Thus, the CV first declined (with steeper decline for higher forces) and then stabilized at approximately the level of the unloaded test (Fig. 4a). The MUP velocities, which at the beginning of tests were shifted towards the higher values with increasing forces, re-shifted over the tests back to the lower values (skewness variable in Fig. 4b and histograms in Fig. 3a–c). Subsequently, the MUP velocities stabilized nearly at the level of the unloaded test too. The standard deviation narrowed first and later on stabilized at a new level (Fig. 4c). The MUP frequency held steady over these tests at the primary level determined by the used force (Fig. 4d). We suggest that this pattern of results may reflect an emerging equilibrium between phasic and tonic MU activity. No comparison is possible between the parameters used here and those in any other prolonged position tasks study. The decline of CV during contractions at low forces was in contradiction with the studies in static force tasks, which reported increases during sustained isometric contractions at forces of 10–25% MVC (Zwarts and Arendt-Nielsen 1988; Arendt-Nielsen et al. 1989; Krogh-Lund and Jorgensen 1991; Krogh-Lund and Jorgensen 1992; Krogh-Lund 1993). This discrepancy may be caused by either/or both the different contraction types (static vs. dynamic) or different tasks (force vs. position tasks). Increases of the CV in prolonged isometric contractions at low force levels are supposed to be due to the recruitment of fast (anaerobic) MUs in response to the hindered blood flow (Crenshaw et al. 1997; Zwarts et al 1987). In the dynamic conditions, the blood supply is assumed to be undisturbed, so the aerobic MUs can be activated. The decline of the CV followed by stability, along with the changes in the skewness, suggest that, within the cyclically fluctuating activity, the global amount of initially recruited fast/fatigable/anaerobic MUs may successively diminish and the proportion of slow/fatigue resistant/aerobic MUs may augment. The maintaining activity of slow MUs is in accordance with the hypothesis that tonic/fatigue resistant (aerobic) MUs remain active through the whole muscular action (Grimby and Hannerz 1968; Hagg and Suurkula 1991). On the other hand, the position character of the present tasks may have contributed to the discrepancy between the decline of CV in the present experiments and increases in previous studies, as firstly, the discharge characteristics of the same motor unit differ between the force and position tasks, and secondly, motor units show greater discharge adaptation during the position tasks (Mottram et al. 2005a, b; MacGilles et al. 2003). The evolution of the standard deviation parameter, with its narrowing followed by stabilizing, fits in with the idea that the rate coding may temporarily increase and consecutively adapt, resulting in a new balance. The MUP frequency variable, expressing the amount of MU activity produced as a result of recruitment and rate coding did not change during these tests. This suggests that all the changes in recruitment and rate coding do not, in principle, affect the total amount of MU activity. All subjects were able to complete the tests, and the sEMG parameters became stable in the course of time as well. Thus, one can assume that the three tests at lowest force levels were non-fatiguing. Taken together, the results of these apparently non-fatiguing dynamic position tasks suggest that following the initially increased activation of fast MUs, the proportion shifts after about 2 min in favour of slower MUs. The amount of activity seems to remain stable throughout the duration of the tests. The test at 20% MVC The changes encountered during the prolonged test at 20% MVC differed clearly from those at lower forces (Fig. 4). During the 20% MVC test, the CV dropped below the level of the unloaded test. At the same time the proportion of low MUP velocities increased (skewness increased), and finally the velocities’ population made a global move from higher towards lower values, while their standard deviation broadened clearly. The MUP frequency, in contrast with that of non-fatiguing tests, progressively diminished (histogram in Figs. 3d, 4d). The CV’s decreases were in accordance with those shown by Farina et al. (2004) during fatiguing dynamic force tasks of vastus medialis muscle. The behaviour of the skewness and standard deviation suggests that a large majority of the MUPs became extremely slow at the end. This general slowness best matches the slowing of the muscle membrane propagation, which is generally accepted as sign of muscular fatigue (Milner-Brown et al. 1986; Miller et al. 1987). The diminishing MUP frequency can be due to the synchronisation of discharges (Datta and Stephens 1990; Semler and Nordstorm 1999) and diminishing motor unit activity (Hagg 1981; Hagg and Suurkulla 1991). The non-linear decline pattern of MUP frequency, first slow and later on steep, suggests successively exhausting available MUs’ reserves, resulting in lower firing frequencies and failing recruitment. Accordingly, many subjects reported fatigue and stopped exercising. In short, during these apparently fatiguing dynamic position tasks, a global slowing of MUP velocities appears, suggesting a fatigued muscle membrane. The amount of MU activity seems to diminish progressively and finally the recruitment stops. Some aspects of the method must be explained. The load was applied to the palm, whereas the MVC was assessed from the wrist. By applying load to the palm, we intended to mimic natural circumstances, such as holding something in the hand. Assessment of the MVC from the palm was not feasible, however, due to the relative weakness of the wrist’s flexors compared with the elbow flexors, which influenced the estimates. A reasonable alternative was to measure the MCV from the wrist. Distances measured over the forearm and the palm (Table 1) enabled calculation of the real exerted load torque, which was about 20–25% larger than the used one. The MVC was assessed at the elbow angle of 90° despite performing the tests at the angle of 135°. The reason was that, when applying the dynamometer at the wrist with high forces, the elbow angle being at 135°, subjects tended to overstretch the wrist and experienced pain. This was not the case at the 90° angle. The MVC values at 135 and 90° were similar, which was consistent with the findings of Philippou et al. (2004), so it was chosen for assessments at 90°. In conclusion, we present a set of parameters derived from the interpeak latency method, which yields information about changes in MUP velocities’ distribution and amount of MUP activity. Skewness, standard deviation and peak frequency parameters appear to corroborate the results of a global muscle conduction velocity. Together they could contribute to quantifying the dynamics of motor unit activity and membrane fatigue. The interconnected results may be useful in ergonomics (for assessment of fatigue) and in sports (for eliciting specific capabilities, such as explosive or endurance capabilities).

J_Abnorm_Child_Psychol-3-1-1915644

Multiple Determinants of Externalizing Behavior in 5-Year-Olds: A Longitudinal Model

In a community sample of 116 children, assessments of parent-child interaction, parent-child attachment, and various parental, child, and contextual characteristics at 15 and 28 months and at age 5 were used to predict externalizing behavior at age 5, as rated by parents and teachers. Hierarchical multiple regression analysis and path analysis yielded a significant longitudinal model for the prediction of age 5 externalizing behavior, with independent contributions from the following predictors: child sex, partner support reported by the caregiver, disorganized infant-parent attachment at 15 months, child anger proneness at 28 months, and one of the two parent-child interaction factors observed at 28 months, namely negative parent-child interactions. The other, i.e., a lack of effective guidance, predicted externalizing problems only in highly anger-prone children. Furthermore, mediated pathways of influence were found for the parent-child interaction at 15 months (via disorganized attachment) and parental ego-resiliency (via negative parent-child interaction at 28 months).   Externalizing behavior problems – including aggressive, destructive, and delinquent behavior – represent the most common type of mental health problems in children. Longitudinal studies have shown that the stability of externalizing problems is relatively high from preschool age into adolescence (Moffit, Caspi, Dickson, Silva, & Stanton, 1996; Smith, Calkins, Keane, Anastopoulos, & Shelton, 2004). Moreover, early externalizing problems are predictive of other forms of psychopathology and often interfere with the child’s personal, social and academic development (Campbell, 1995, 2002; Moffitt, 1993). Although developmental psychopathologists now generally assume such problems to have their roots in children’s early developmental histories, early identification of children at serious risk of developing such pathology is hampered by a lack of insight into the determinants leading to later maladaptive outcomes (Tremblay, 2006). Although various theoretical models of the development of externalizing problems in the very first years of life (Campbell, Shaw, Gilliom, 2000; Moffitt, 1993, Greenberg, 1999; Sameroff & Chandler, 1975) differ in the emphasis they place on the role of certain etiological factors, they agree in the assumption that multiple factors from various domains additively and interactively contribute to the emergence and maintenance of externalizing problems. Taken together, four domains of factors have been proposed to contribute to their development: (a) parent-child interaction and parent-child attachment, (b) child characteristics (e.g., temperament and cognitive abilities), (c) parental characteristics (e.g., personality), and (d) contextual characteristics (e.g., socioeconomic status, partner support, and stressful life events). To date, very few studies – all focusing on high-risk samples – have longitudinally examined the joint contribution of the various key factors to the onset of externalizing problems in the very first years of life (Aguilar, Sroufe, Egeland, & Carlson, 2000; Erickson, Sroufe, & Egeland, 1985; Lyons-Ruth, Alpern, & Repacholi, 1993; Shaw, Owens, Vondra, Keenan, & Winslow, 1996). Ours is the first study to assess predictors hypothesized to be the most important from all of the aforementioned domains both in infancy (i.e., at age 15 months) and in toddlerhood (i.e., at age 28 months) to predict the emergence of later externalizing behavior problems (i.e., at age 5 years) in a non high-risk community sample. All of the abovementioned earlier studies of the multiple determinants of externalizing behavior used samples characterized by one or more risk factors. The sample studied by Shaw et al. (1996) included only low-SES families. Aguilar et al. (2000) and Erickson et al. (1985) studied low-SES samples as well, but these were also characterized by high degrees of life stress and a large proportion of single-parent families. And the low-SES sample studied by Lyons-Ruth et al. (1993) was characterized by such additional risk factors as a relatively high incidence of a history of maternal psychiatric hospitalization, single parenthood, and child maltreatment. It remains to be seen whether similar results will also be found in studies with community samples. That is mainly because earlier research has shown that the predictive power of risk factors may increase in the presence of other risk factors (Farrington, 1995). Consequently, it is possible that factors that predict externalizing problems in samples with one or more risk factors fail to do so in samples with lower levels of risk, such as the present sample. In light of this, studying the early predictors of externalizing problems in a community sample constitutes an important extension of the existing research evidence collected in high-risk samples. The criteria on which we based our selection of the various predictors and our hypotheses regarding their joint contribution to the development of externalizing problems – led by various interrelated theoretical models – are described per domain in the following paragraphs. Parent-child interaction and parent-child attachment From a transactional perspective (Sameroff & Chandler, 1975), externalizing problems are assumed to emerge and to stabilize or change in children’s continuous interactions with their immediate environment, particularly in the interactions with their primary caregivers. Repeated observations of parental and child behaviors during parent-child interactions are thus indispensable to gain more insight into the early development of externalizing behavior problems. Multiple dimensions of parental behavior in parent-child interaction have been found to be associated with behavior problems in children at various ages. A lack of positive parenting behaviors, such as the expression of sensitivity, warmth, involvement, acceptance, and positive guidance, was reported to be related to externalizing problems in preschoolers (Brophy & Dunn, 2002; Côté, Vaillancourt, LeBlanc, Nagin, & Tremblay, 2006; Gardner, 1987; Pettit, Bates, & Dodge, 1997). In addition, high levels of negative parental control such as harsh discipline, intrusiveness, negativity, and hostility also proved associated with externalizing problems (Belsky, Woodworth, & Crnic, 1996; Rubin, Burgess, Dwyer, & Hastings, 2003; Campbell, Pierce, Moore, & Marakovitz, 1996). Based on the above evidence, we expected both a lack of positive parenting behaviors and negative parent-child interactions to predict externalizing behavior problems at age 5. Another frequently mentioned risk factor and closely related to the quality of parent-child interaction, is the quality of attachment between infant and caregiver. By the end of the infant’s first year, four main patterns of infant-parent attachment – one secure and three insecure – can be distinguished that have been found to reflect the history of the caregiver’s responses to the child’s attachment behaviors (Ainsworth, Blehar, Waters, & Wall, 1978; Main & Solomon, 1986, 1990). Ainsworth and colleagues (1978) originally proposed three “organized” patterns of attachment. Presumably as a result of a history of caregiver sensitive responsiveness to their signals and needs, securely attached (B) infants use the caregiver as a secure base from which to explore the environment. Avoidant (A) infants are characterized by a tendency to minimize their attachment behaviors while under stress in the presence of the caregiver, probably resulting from earlier experiences with a caregiver who tended to reject or ignore the child’s expression of attachment behaviors. Infants with a resistant attachment (C), featuring both clinging and angry behaviors towards the caregiver when under stress, are presumed to maximize the expression of attachment behavior as an adaptation to their caregiver’s inconsistent responsiveness. Main and Solomon (1990) later added a fourth disorganized (D) pattern typified by seemingly contradictory attachment behaviors that is thought to reflect the breakdown or absence of a strategy for the infant to use the caregiver as a secure base in times of stress (Main & Solomon, 1990). Disorganized attachment is thought to ensue from extremely unpredictable or frightening behavior on the part of a maltreating and/or traumatized parent (cf. Lyons-Ruth & Jacobvitz, 1999). With regard to the predictive value of attachment security versus insecurity for the later occurrence of externalizing problems, the empirical evidence is rather inconsistent. Although attachment insecurity was found to be associated with externalizing problems in high-risk samples as well as in community samples in studies that did not include the disorganized attachment classification (Erickson et al., 1985; Fagot & Kavanagh, 1990; Munson, McMahon, & Spieker, 2001; Shaw et al., 1996), other studies did not report such a relationship (Bates, Maslin, & Frankel, 1985; Goldberg, Corter, Lojkasek & Minde, 1990). However, in contrast to the other insecure attachment categories, disorganized attachment has consistently been found to be related to the later development of externalizing problems (for reviews, see Lyons-Ruth and Jacobvitz, 1999, and Van IJzendoorn, Schuengel, and Bakermans-Kranenburg, 1999; also see Lyons-Ruth et al., 1993; Vondra, Shaw, Swearingen, Cohen, & Owens, 2001). In view of the above, we expected infant attachment insecurity and particularly attachment disorganization to be related to elevated levels of externalizing behavior at age 5. Considering that attachment patterns have been found to reflect the history of parent-child interactions, we expected the association between the early parent-child interaction and later externalizing problems to be at least partially mediated by the quality of the infant-parent attachment. Child characteristics Several child characteristics can prompt the development of externalizing behavior, both directly and indirectly by affecting the quality of parent-child interactions. A considerable body of research has shown associations between externalizing problems and child temperamental characteristics with a crucial role for negative emotionality denoting negative mood, irritability, and high-intensity reactions like anger (for a review, see Sanson, Hemphill, & Smart, 2004). In contrast, temperamental inhibition and fear/shyness may act as a protective factor as these characteristics have been demonstrated to be negatively associated with later externalizing problems (Lacourse, Nagin, Vitaro, Côté, Arsenault, & Tremblay, 2006; Sanson, Oberklaid, Prior, Amos, & Smart, 1996; Schwartz, Snidman, & Kagan, 1996). Nevertheless, the relationship between child temperament and externalizing behavior is not always clear or direct. Etiological models of externalizing behavior increasingly propose that children with temperamental difficulties are more vulnerable to negative rearing influences than children without such difficulties (Belsky, 1997; Moffitt, 1993). Several recent studies have substantiated this latter assumption (Bates, Pettit, Dodge, & Ridge, 1998; Belsky, Hsieh, & Crnic, 1998; Hemphill & Sanson, 2001; Lacourse et al., 2006; Leve, Kim, & Pears, 2005). In line with these findings, we anticipated high levels of child negativity and low levels of fearfulness in infancy and toddlerhood, particularly in interaction with parenting, to predict the development of externalizing problems. Another contributory child characteristic is low cognitive or linguistic ability (Burt, Hay, Pawlby, Harold, & Sharp, 2004; Dekker, Koot, Van der Ende, & Verhulst, 2002; Moffitt, 1993). Therefore, we hypothesized that child cognitive ability at 15 months would be negatively related to the occurrence of externalizing problems at age 5 years. Boys and girls have been found equally likely to exhibit externalizing behavior problems up until the age of about four years. By school entry, however, boys tend to exhibit up to 10 times the rate of externalizing problems for girls (Côté, et al, 2006; Mesman, Bongers, & Koot, 2001; Rubin et al., 2003). Accordingly, at age 5 we expected the boys in our sample to score higher on externalizing behavior than the girls. Parental characteristics Little research has focused on parental personality as a potential predictor of externalizing problems in children. The available studies only focused on aspects of parental psychopathology like antisocial personality (Shaw, Vondra, Hommerding, Keenan, & Dunn, 1994) or maternal depression (Cummings & Davies, 1994; Shaw, Gilliom, Ingoldsby, & Nagin, 2003). Less attention has been paid to the effects of more or less adaptive parental characteristics, which is why we included parental ego-resiliency as a potential predictor of externalizing problems. Block and Block (1980) defined ego-resiliency as a “resourceful adaptation to changing circumstances,” “flexible invocation of the available repertoire of problem-solving strategies,” and “the ability to maintain integrated performance while under stress” (p. 48). As such, ego-resilient individuals should be particularly well-equipped to cope with the often stressful task of parenting. And – as outlined before – higher quality parenting is hypothesized to predict lower levels of externalizing behavior in children. In the 15-month assessment (Van Bakel & Riksen-Walraven, 2002a), parental ego-resiliency was found to be related to the infants’ socioemotional development, partially via the quality of the parent-infant interaction. Based on the above and in line with the 15-month findings, we expected higher levels of parental ego-resiliency to predict lower levels of externalizing behavior in the children at age 5, and we expected this relation to be mediated by lower quality parent-child interactions. Contextual characteristics Besides parental and child attributes, specific characteristics of the social and economic context in which the parent-child interaction is embedded may also contribute to the development of externalizing problems (Belsky, 1984; Greenberg, Speltz, & DeKlyen, 1993). We included three characteristics of the child-rearing context that earlier studies have shown to be associated with externalizing problems: (1) high incidence of stressful life events (Aguilar et al., 2000), (2) a lack of partner support (Jouriles, Murphy, Farris, Smith, Richters, & Waters, 1991; Shaw et al., 1996), and (3) low socio-economic status (SES; Côté, et al, 2006; Shaw et al., 1996). It needs to be stressed, though, that in previous research the effects of sociodemographic factors like SES proved modest compared to the effects of parental or child risk factors (for a review, see Loeber and Dishion, 1983; also see Shaw et al., 1996). Nevertheless, based on earlier findings, we expected stressful life events, partner support, and SES to contribute to the development of externalizing behavior problems. In sum, the aim of the present study was to predict the occurrence of externalizing behavior problems in a sample of 5-year-old children on the basis of various parental, child, dyadic, and contextual factors assessed at 15 and 28 months and at age 5. We expected the following specific factors to independently or interactively contribute to the development of externalizing problems: (a) a low quality of parent-child interaction at 15 and 28 months; (b) disorganized parent-infant attachment; (c) child temperament (i.e., high anger proneness and low fearfulness) at both 15 and 28 months and particularly in interaction with low quality parenting; (d) low levels of child cognitive ability at 15 months; (e) low parental ego-resiliency; (f) high incidence of stressful life events between 15 months and 5 years; (g) lack of partner support; and (h) low SES. Furthermore, the contribution of parental ego-resiliency to child externalizing behavior was expected to be mediated by the quality of parent-child interaction, and the contribution of the early parent-child interaction by the quality of infant-parent attachment. Method Participants The original 15-month sample (M = 15.1; SD = 0.25) consisted of 129 healthy children (67 boys, 62 girls) and their primary caregivers. The children were recruited on the basis of the records from health-care centers in the Dutch city of Nijmegen. During nine consecutive months, all families (n = 639) with a 15-month-old baby from various socioeconomic backgrounds were sent a recruitment letter with a brief description of the research procedures and an invitation to participate in this study that aimed to “gain more insight into children’s development in the first years of life”. If they met the two specified eligibility criteria (i.e., sufficient fluency in Dutch and child without serious health problems) and were interested in participation, they were requested to return a response card. Of the 174 families who responded, 129 parent-child dyads (the maximum attainable given the time and resources available for the project) were randomly selected for the study. Of the infants, 73 were first-borns and 56 had one or more older siblings. The sample included 123 two-parent families and six single-parent families. In three families, the father was the primary caregiver and in the remainder of the families this was the mother. The percentages of single parents and fathers acting as primary caregivers are representative of families in the Netherlands with children in this age group. The primary caregivers were between the ages of 22 and 47 years at the time of recruitment. The level of education for the primary caregivers was indicated on a seven-point scale from low (elementary school) to high (college degree or more) with a mean of 4.95 (SD = 1.77); the category equivalent of 5 is high school degree. Of the 129 15-month-olds, 114 children (61 boys, 53 girls) participated in the second wave of measurements at age 28 months (M = 28.3; SD = 0.30) and 116 (62 boys, 54 girls) in the third assessment at age 5 years (M = 63.6 months; SD = 1.16). At this third assessment, there were 107 two-parent families, nine single-parent families, and again three families with the father as the primary caregiver.1 To determine whether there was selective drop-out or not, the 116 families participating in the 5-year assessment were compared to the 13 families that did not take part. Independent t-tests for all the major 15-month study variables revealed a significant difference for parental ego-resiliency only, t(126) = 2.08, p < .05, with a tendency for primary caregivers with lower levels of ego-resiliency to drop out. Procedure The 15-month data were available from an earlier study focusing on the determinants of parenting and infant development (Van Bakel & Riksen-Walraven, 2002a). The 15-month assessment involved data collection during a single home visit and a single visit of the primary caregiver and infant to the research center. During the home visit, the primary caregiver completed a Q-sort and a set of questionnaires assessing child temperament, parental ego-resiliency, partner support, and additional background information. Next, the parent-child interaction was videotaped during the performance of four instructional tasks lasting three to four minutes each. At the subsequent assessment at the research center, the child’s cognitive ability was assessed and the quality of the infant-parent attachment with an abbreviated version of the Strange Situation (Ainsworth et al., 1978). For a more detailed description of the 15-month data collection, see Van Bakel & Riksen-Walraven (2002a, 2002b) The 28-month assessment was conducted during a single home visit. The primary caregiver was interviewed about stressful life events since the first assessment and asked to complete a questionnaire to assess child temperament. The parent-child interaction was videotaped during four instructional tasks comparable to those performed at 15 months. At the age-5 assessment, the primary caregiver was interviewed at home about stressful life events since the previous assessment and asked to complete a questionnaire to assess child behavior problems. The child’s teacher completed a questionnaire to assess any behavior problems and asked to return this by mail, which was done by all but one teacher. Instruments and measures Quality of parent-child interaction (at 15 and 28 months) The videotaped parent-child interaction episodes were rated using five 7-point scales (Erickson et al., 1985) to assess the quality of parental interactive behavior: (1) supportive presence or the provision of emotional support; (2) respect for the child’s autonomy or nonintrusiveness; (3) effective structure and limit setting; (4) quality of instructions; and (5) hostility. Subsequently, child interactive behavior was rated on four 7-point scales (Erickson, et al., 1985): (I) negativity or anger, dislike, and hostility towards the parent; (II) avoidance of interaction with the parent; (III) compliance to suggestions and directions given by the parent; and (IV) positive affect expressed towards the parent. Each interaction episode at 15 months was rated independently by two trained observers and each interaction episode at 28 months by four independent observers. Based on 25 cases (19%) for the 15-month assessment and 20 cases (18%) for the 28-month assessment, the interrater reliabilities expressed as adjusted kappas were all above 0.83. Evidence for the validity of the scales has been provided in various studies (see Van Bakel & Riksen-Walraven, 2000a, for a review). To obtain robust dyadic measures for parent-child interaction, the ratings of parental and child interactive behavior were factor analyzed together, for the 15- and 28-month assessment separately, using varimax rotation. Scree plots of eigenvalues indicated the emergence of two clear and comparable factors at both ages. The two factors explained 68% of the variance in parent-child interaction at 15 months, and 71% of the variance at 28 months. The first factor, labeled effective guidance, was marked by high loadings on the following parent-child interaction variables (factor loadings for 15 and 28 months within parenthesis): effective structure and limit setting (.89; .95), high quality of instructions (.62; .76), and supportive presence (.79; .48) on the part of the parents; and compliance (.53; .84) and low avoidance (−.52; −.65) on the part of the child. The second factor, labeled negative interaction, was characterized by high loadings on the following variables (factor loadings for 15 and 28 months within parenthesis): parental hostility (.74; .85); low respect (−.63; −.78); child negativity (.85; .67); and an increasing lack of positive affect in both partners as evident from low parental supportive presence (−.48; −.77) and low child positive affect (−.35; −.54). Quality of parent-child attachment (at 15 months) An abbreviated version of the Strange Situation Procedure (Ainsworth et al., 1978), i.e., including one as opposed to two separation-reunion situations, was used to assess the quality of infant-parent attachment. A similarly abbreviated version of the Strange Situation has been found to be valid for the assessment of attachment quality both within normal (Lewis, Feiring, McGuffog, & Jaskir, 1984; Waters, Wippman, & Sroufe, 1979) and within clinical samples (Willemsen-Swinkels, Bakermans-Kranenburg, Buitelaar, Van IJzendoorn, & Van Engeland, 2000). Two trained coders (the second author and D.C van den Boom from the University of Amsterdam) rated the videotaped separation-reunion episodes and classified the infants as Secure (B), Avoidant (A), Resistant (C), or Disorganized/ disoriented (D) consistent with the directions provided by Ainsworth et al. (1978) and by Main & Solomon (1990). Intercoder reliability on 20 cases (16%) was adequate, with 95% agreement on the main classifications. Child temperament (at 15 and 28 months) Child temperament was evaluated using the Toddler Behavior Assessment Questionnaire (TBAQ; Goldsmith, 1994) The TBAQ comprises 111 items, organized in five 7-point scales. For the present study, two scales were used that have earlier been found associated with externalizing problems, i.e., Anger proneness (28 items, Cronbach’s alpha .89 and .88 for 15 and 28 months, respectively) and Social fear (19 items, Cronbach’s alpha .77 and .84 for 15 and 28 months, respectively). Cognitive ability (at 15 months) A Dutch version of the Bayley (1969) Mental Scale of Infant Development (Van der Meulen & Smrkovsky, 1983) was used to assess the child’s level of cognitive functioning at 15 months, expressed in the standardized Mental Developmental Index (MDI, M = 100; SD = 15). Parental ego-resiliency (at 15 months) Primary caregivers rated their own ego-resiliency using a Dutch translation of the California Adult Q-set (CAQ; Block, 1961, 1978) The CAQ consists of 100 descriptive statements that sample a broad domain of personal and interpersonal characteristics and functioning. The primary caregivers were asked to sort each statement into one of nine possible categories ranging from “least characteristic” to “most characteristic” in terms of salience for themselves. An ego-resiliency score was then attained by correlating the Q-sort description for each parent with the criterion profile provided by experts for a prototypically ego-resilient person (see Block, 1991). The ego-resiliency scores could range from +1.00 (very ego-resilient) to –1.00 (very ego-brittle). Stressful life events (at 28 months and 5 years) A semi-structured interview was used to obtain information regarding stressful life events occurring between the three assessments. The questions/items were derived from Saranson, Johnson, and Siegel’s (1978) Life Experiences Survey and Coddington’s (1972) Life Events Scale for Children. Both measures have sound psychometric properties and have been widely used in international research (Abela, 2001; Johnston, 1996). Stressful events that were likely to have a negative impact on the child’s development were selected for inclusion (e.g., loss of a loved one, serious physical or mental illness on the part of a parent or the child, divorce). All items required a ‘yes’ or ‘no’ response. The total number of stressful life events between 15 and 28 months and 28 months and age 5 were taken together to constitute the stressful life event scores between 15 months and age 5. Partner support (at 15 months) A subscale of a Dutch questionnaire to assess family problems (VGP; Koot, 1997) was used to assess the support the primary caregiver received from the partner. The subscale comprises five statements gauging partner support during child rearing, such as “My partner supports me in my role as a parent” and “My partner and I agree about child rearing.” Cronbach’s alpha was 0.82. Single parents were also asked to complete the questionnaire when they were still in contact with the other parent or were living with a new partner. Otherwise, they were assigned a minimum score. Evidence supporting the validity of the subscale has been reported in various studies (Van Bakel & Riksen-Walraven, 2002a; Van Zeijl et al., 2006). Socioeconomic status (SES at 15 months) SES scores were assigned on the basis of the level of education (along a 7-point scale) and level of occupation (along a 6-point scale) for both parents. SES scores based on education and occupation of both parents have frequently been used in other studies (e.g., Shaw et al., 1996). The levels of education and occupation for the two parents were first standardized and then summed to derive a single SES score. For single parents, the level of education and occupation for the primary caregiver were summed to compute the SES score (cf. Shaw et al., 1996). Externalizing problems as rated by parents and teachers (5 years) To assess externalizing problems at age 5, the parents completed the Dutch version of the Child Behavior Checklist for ages 4–18 (CBCL/4–18; Achenbach, 1991a; Verhulst, Van der Ende, & Koot, 1996). The Aggressive behavior subscale (20 items) and the Delinquent behavior subscale (13 items) from the CBCL/4–18 were summed (combined α = .86) to determine the externalizing score for the child at 5 years. Teachers completed the teacher version of the CBCL (TRF, Achenbach 1991b); the externalizing score was based on the scores for the subscales Aggressive behavior (25 items) and Delinquent behavior (9 items) which were summed (combined α = .94) to derive an externalizing score for each child. Results Results are presented in two sections. In the first section we report the results of preliminary analyses, i.e., the distributions of the various predictor and outcome variables as well as their intercorrelations, and the parental and teacher ratings of the children’s externalizing behaviors are compared and related to each other. The second section reports the main results of the study, namely the prediction of externalizing behavior at age 5 from the various predictors at earlier ages. First, hierarchical regression analysis is conducted to estimate the unique and interactive contributions of the multiple predictors to the variance in Externalizing scores at age 5. Next, the hypothesized mediated pathways are tested. Finally, path analysis using AMOS 5.0 (Arbuckle, 2003) is applied to test the complete longitudinal model comprising all the direct, moderated, and mediated pathways of influence between the predictors at the various ages and age 5 externalizing behavior. Preliminary analyses Table 1 presents the means and standard deviations for the study variables as well as their intercorrelations. The distribution of children across attachment categories (64% B, 14% A, 8% C, and 14% D) did not differ from the distribution reported by Van IJzendoorn et al. (1999) for “normal”, i.e., middle-class, non-clinical groups in North America. The scores for child cognitive ability were normally distributed, with a mean score close to the population mean of M = 100. The distribution of the scores for ego-resiliency was in line with the findings of earlier studies and the distribution of scores for partner support was mildly to moderately skewed to the left but showed sufficient variation. Table 1 also shows significant stability for the two parent-child interaction measures as well as for child social fear and anger proneness from 15 to 28 months. For the predictor variables, no differences were found between boys and girls. Table 1Intercorrelations, means and standard deviations for the study variables1234567891011121314151617181915-month predictors1. Effective guidance–2. Negative interaction.00–3. B vs. non-B attachmenta.38**-.46**–4. A vs. non-A attachmenta−.27**.22*−.55**–5. C vs. non-C attachmenta−.05.11−.40**−.12–6. D vs. non-D attachmenta−.22*.33**−.53**−.16−.12–7. Child social fear.42**-.03.19*−.16−.06−.05–8. Child anger proneness.04.02.02.06−.00−.09.35**–9. Child cognitive ability.31**−.09.09−.07.05−.08.18.12–10. Child sex.10−.01.10.03−.15−.05.08.07.15–11. Parental ego-resiliency.23*−.07.17.07−.28**−.09−.00−.07.19*−.05–12. Partner support.16−.03.16.02−.21*−.07−.06−.01.12.05.17–13. SES.05−.17−.02.02.10−.07.11−.02.14−.02.17−.07–28-month predictors14. Effective guidance.32**−.06.21*−.23*.15−.18.25*.10.41**.04.09−.07.19–15. Negative interaction−.09.63**−.34**.11−.05.41**−.11−.02−.07−.08−.28**−.10−.29**.00–16. Child social fear.21*.14−.04−.15.10.13.47**.16.03.15−.15−.17.05.17.02–17. Child anger proneness−.01−.05−.02.15.01−.13.23*.56**.09.07−.02−.15.08−.05−.15.14–5-year predictor18. Life events 15 mo-5 yr.02.19*−.06−.15.10.16−.04.12−.12−.03−.25*−.35**−.10.10.19.16.22*–5-year outcome19. Externalizing behavior−.22*.31**−.33**−.02.05.45**−.18−.02−.09−.25**−.25**−.27**−.18−.17.51**−.11.11.23*–M0.00b0.00b0.640.140.080.143.683.38103.900.470.448.320.00c0.00b0.00b3.383.611.480.00cSD1.001.000.480.350.270.340.850.7216.930.500.192.052.811.001.000.800.721.311.73Note. N = 105–111.aAttachment was dummy coded as B (=1) vs. ACD (=0); A (=1) vs. BCD (=0); C (=1) vs. ABD (=0); and D (=1) vs. ABC (=0).bThe parent-child interaction variables are factor scores with a mean score of 0.00 and a standard deviation of 1.00.cSES and Externalizing behavior are composed variables which were first standardized and then summed.*p < .05. **p < .01Table 2Hierarchical regression results for predicting age 5 externalizing scores (Total Model)Hierarchical regression resultsBlockBSE BβΔR2(%)1D versus non-D attachmenta1.880.42.36**22.2Negative interaction 28 months0.650.15.35**10.7Sex (male)b0.610.26.18*4.3Anger proneness 28 months0.470.18.20**4.4cPartner support−0.140.06−.17*2.62Interaction termsEffective guidance × Anger proneness 28 months−0.330.14−.19**3.3R2 final model =47.6%F(6,94) final model =14.21**aD vs. non-D coded as D = 1 and non-D = 0.bSex was coded as 0 (girls) or 1 (boys).cAlthough anger proneness was not significantly correlated with externalizing behavior, it had a significant Beta weight. This effect seems due to a suppressor variable. As recommended by Tabachnick & Fidell (1989), predictors were systematically deleted from the regression equation to identify which variable is the suppressor, indicating that D versus non-D attachment acted as the suppressor variable.*p < .05. **p < .01. The mean score on the Externalizing scale was 9.97 (SD = 6.35; N = 111) for the CBCL/4–18 with 22% (n = 24) of the children scoring in the clinical range (T-score of ≥63; Achenbach, 1991a). For the TRF, the mean score was 7.50 (SD = 9.10; N = 111) with 16% (n = 18) of the children scoring in the clinical range (T ≥ 63; Achenbach, 1991b). Both the CBCL and TRF Externalizing scores were significantly higher than those reported for 5-year-olds in the Dutch norm population for the CBCL/4–18 (M = 8.23, SD = 6.37; Verhulst et al., 1996) and the TRF (M = 4.99, SD = 6.58; Verhulst, Van der Ende, & Koot, 1997), t (185) = 2.26, p < .05 for the CBCL/4–18, and t (185) = 2.51, p < .05 for the TRF. A paired-samples t-test showed parents to report significantly more externalizing behaviors than school teachers, t (110) = 3.24, p < .01. The correlation between the CBCL and TRF scores was .51, p < .001. To obtain a robust measure of externalizing behavior, a composite Total Externalizing score was computed by first standardizing and then summing the children’s Externalizing scores on the CBCL and TRF. For sex differences on this measure, a t-test revealed a significantly higher score for boys on Total Externalizing (Mboys = 0.41, SD = 1.93; Mgirls = −0.44, SD = 1.35, t (109) = 2.70, p < .01). Predicting age 5 externalizing scores Hierarchical regression analysis To examine the unique and interactive contributions of the predictors to the variance in Externalizing scores at age 5, hierarchical multiple regression analysis was conducted with Total Externalizing as the dependent variable and the predictors at the different ages as the independent variables. The predictors were entered in the first block. In the second block, the interaction terms hypothesized in the introduction to predict Externalizing scores were entered, i.e., child temperament (2 measures) × parent-child interaction (2 measures) at both 15 and 28 months, and child cognitive development × parent-child interaction (2 measures) at 15 months. The interaction terms were computed according to the procedure recommended by Aiken and West (1991). To limit the number of interaction terms in the regression analysis, only those interaction terms that significantly predicted externalizing behavior at age 5 when examined in isolation were included in the regression analysis (see also Leve et al., 2005), i.e., Effective guidance × Anger proneness at 28 months and Negative interaction × Anger proneness at 28 months. Table 2 presents the results for the final model. The overall model proved significant and explained 47.6% of the variance in the Total Externalizing scores. In the first block, D versus non-D attachment at 15 months explained a significant 22% of the variance in the Total Externalizing scores at age 5. Negative interactions at 28 months explained an additional 11% of the variance, child sex accounted for another 4%, just like anger proneness at 28 months, and partner support at 15 months contributed another 3% to the regression equation. In the second block, Effective guidance × Anger proneness at 28 months explained another 3% of the variance in Total Externalizing scores at age 5. Fig. 1Child externalizing behavior scores in relation to effective guidance for children high and low on anger-proneness To examine the nature of the interaction effect of effective guidance and anger proneness at 28 months on externalizing behavior at age 5, the relationship between effective guidance and externalizing behavior was determined for children with high (1 SD above the mean) and low (1 SD below the mean) levels of anger proneness, following the procedures of Aiken and West (1991). The regression lines for high and low anger prone children are plotted in Fig. 1. The simple slope of effective guidance was significant at high levels of anger proneness (B = −.55, t(107) = −2.56, p < .05), but not at low levels of anger proneness (B = .19, t(107) = 1.01, ns.). Thus, a lack of effective guidance was associated with more externalizing behavior for highly anger prone children but not for low anger prone children. Mediational analyses Next, the hypothesized mediated pathways between the predictors and age 5 externalizing behavior were tested using regression analysis, according to the procedure outlined by Baron and Kenny (1986). According to this procedure, three conditions must hold in order to establish mediation: (a) the predictor is significantly associated with the outcome, (b) the predictor is significantly associated with the mediator, and (c) the mediator is significantly associated with the outcome. If these conditions hold in the predicted direction, mediation is proven when the effect of the predictor on the outcome is shown to decrease when the mediator is also entered as a predictor in the regression equation. First, we tested whether D versus non-D attachment mediated the effect of the parent-child interaction (two measures) at 15 months on age 5 externalizing behavior. For both hypothesized pathways, the three abovementioned conditions for establishing mediation were met (see Table 1 for correlations). Furthermore, the effect of effective guidance at 15 months on externalizing behavior (β = −.22, p < .05) dropped to a nonsignificant level (β = −.13, ns) when D versus non-D attachment was controlled for (Sobel test: z = −2.11, p < .05), showing that D versus non-D attachment completely mediated the effect of effective guidance at 15 months on externalizing behavior. The effect of negative interactions at 15 months on externalizing behavior (β = −.31, p < .001) decreased but remained significant when D versus non-D attachment was controlled for (β = −.18, p < .05); Sobel’s test showed the decrease to be significant (z = 2.77, p < .01). These findings indicate that negative interactions at 15 months are associated with age 5 externalizing behavior both directly and via D versus non-D attachment. Next, we tested whether the effect of parental ego-resiliency on child externalizing behavior was mediated by the quality of the parent-child interaction (two measures) at 15 and 28 months. As evident from Table 1, the three conditions for establishing mediation only held for one of the four possible pathways, namely for the pathway mediated by negative interactions at 28 months. For this pathway, multiple regression showed the effect of parental ego-resiliency on externalizing behavior (β = −.25, p < .01) to decrease (Sobel test: z = −2.63, p < .01) to a nonsignificant level (β = −.12, ns) when negative interactions at 28 months was controlled for. These results indicate that the effect of parental ego-resiliency on children’s externalizing behavior was completely mediated by negative parent-child interactions at 28 months.Fig. 2Final path model summarizing the pattern of direct, moderated, and mediated relations between predictors at 15 and 28 months and age 5 externalizing behavior. Values given are standardized path coefficients. *p < .05. **p < .01 Path analysis Finally, path analysis using AMOS 5.0 (Arbuckle, 2003) was applied to test the complete longitudinal model for predicting age 5 externalizing behavior including: (1) all the direct and moderated relations between the predictors and age 5 externalizing behavior found in the regression analysis; (2) the three significant mediated pathways resulting from the mediational analyses; and (3) the longitudinal relations between the 15- and 28-month assessments of the two parent-child interaction factors (i.e., effective guidance and negative parent-child interactions). A good model fit is indicated by a nonsignificant χ2 statistic, a Bentler comparative fit index (CFI) of .95 or above, and root mean square error of approximation (RMSEA) of .05 or below. The analysis of the initial model yielded a significant fit (χ2 = 21.86, df = 23, p = .53, CFI = 1.00, RMSEA = .00), indicating that the model fits the data well.2 The final model, including the standardized path coefficients, is depicted in Fig. 2. Discussion The present study is one of the first attempts, and the first with a community sample, to include the theoretically most important parental, child, dyadic, and contextual characteristics, longitudinally assessed in both infancy and toddlerhood, in the prediction of externalizing behavior problems at age 5. Data were gathered using multiple sources (parents and teachers) and multiple methods, including questionnaires, standardized tests and extensive observations of the parent-child interaction. Predictors from all four domains were found to be associated with children’s externalizing behavior at age 5, which is in accordance with the results of earlier studies using high-risk samples (Aguilar et al., 2000; Erickson et al., 1985; Lyons-Ruth et al., 1993; Shaw et al., 1996). The longitudinal model that emerged from our analyses provides more insight into the pattern of direct, moderated and mediated influences that work together to shape the development of externalizing behavior problems across the first five years of life. As emphasized in the introduction, the present study was conducted on a non high-risk sample, in contrast to the above prior longitudinal studies that were all conducted on high-risk samples. After recruitment, when the children were 15 months of age, the sample appeared to be representative of the Dutch population of families with young children and therefore was not considered as a high-risk sample (see Van Bakel & Riksen-Walraven, 2002a). At the 5-year assessment, the selective drop-out of families with relatively low ego-resilient parents seemed to make the risk status of the remaining sample even lower. In this light, our finding that the children had higher CBCL and TRF externalizing scores than the relevant normative Dutch samples is puzzling. It may be that the level of externalizing behavior problems has increased over the last few years; unfortunately, no recent normative data are available. An alternative explanation may be that in middle-class communities, families with concerns about their children’s development are more likely to volunteer for research studies. Because relevant information is not available for the present sample, this remains an issue for further study. The only hypothesized predictors that were not associated with later externalizing behavior were SES and child temperament and cognitive ability at 15 months. The lack of association between SES and externalizing behavior is in line with earlier studies that also reported parental and child characteristics to be more consistently associated with externalizing behavior than contextual characteristics (Shaw et al., 1996). Restriction of range in SES scores in our sample is not a likely explanation for the null findings given that the sample seems to be fairly representative of the Dutch population of families with children in the same age range, as indicated above. Yet, it may be that the variation of SES in the Netherlands is less than in other countries, such as the USA. Further research is recommended to examine how well the model that our analyses generated applies in community samples from other countries or for ethnically diverse samples of parents and children. The present nonsignificant relations between infant temperament and cognitive ability on the one hand and externalizing behavior on the other are in line with the results of two other recent longitudinal studies, both using high-risk samples (Aguilar et al., 2000; Brennan, Hall, Bor, Majman, & Williams, 2003), that also failed to find an association between early temperamental and neuropsychological risks on the one hand and persistent aggressive behavior on the other. Given that – as argued in the introduction – the predictive power of risk factors may be higher in high-risk samples than in lower-risk samples, it is not surprising that variables that failed to predict externalizing behavior in high-risk samples also failed to predict such problems in a lower-risk sample like ours. With regard to the predictive power of risk factors in relation to sample risk status, it should be noticed that comparing the predictive power of risk factors in studies differing in sample risk status is not the most elegant way to address this question. A more direct way to test whether risk factors interact with sample risk status is by testing for interactive effects in a sample with a greater range of risk (see, for example, Lacourse et al., 2006). Although temperament assessed in infancy did not interact with the parent-child interaction in the prediction of externalizing behavior, temperament assessed in toddlerhood did. More specifically, a parent-child interaction characterized by a lack of effective guidance in toddlerhood predicted externalizing behavior only in highly anger-prone toddlers but not in less anger-prone toddlers. This finding seems to support Belsky’s (1997) differential susceptibility hypothesis that not all children are similarly affected by the same rearing experience. It should be kept in mind, however, that our effective guidance factor not only includes parental behavior, but child behavior as well. With the same reserve, our findings appear to also be in line with the results of studies that showed child negative emotionality to interact with parenting in predicting externalizing problems (Belsky et al., 1998; Leve et al., 2005). Rather than an association with negative parent-child interactions and hence in contrast to earlier reports, our findings showed temperament to interact with a different dimension of the parent-child interaction, namely a lack of effective guidance. This is not to say that negative parent-child interactions as such carried no weight in the prediction of externalizing problems in the present study. Quite the contrary, the factor proved to be one of the most powerful predictors of externalizing behavior problems in the present study and this was true for all the children in our sample and not just for a subsample of temperamentally difficult children. Our results on the interaction between temperament and parenting in the development of externalizing problems extend those of earlier studies by showing that a specific temperament by parenting interaction may work particularly in a specific developmental phase. That the interaction between anger proneness and effective guidance proved to predict externalizing problems particularly for toddlers makes sense when interpreted from a developmental psychopathology perspective: establishing autonomy and learning to comply with social rules and expectations are major developmental tasks for toddlers that challenge the parents’ ability to provide effective guidance by imposing structure and setting limits while at the same time remaining emotionally supportive of the child. As a result of the child’s striving for autonomy, the second year is marked by parent-toddler conflicts. Toddlers that are prone to show angry behaviors in such discord situations are particularly in need of effective guidance in order to prevent their lack of emotional and behavioral self-control to further escalate and evolve into externalizing behavior problems. Our finding that D attachment was among the strongest predictors of externalizing behavior at age 5 supports the results of previous research (Lyons-Ruth et al., 1993; Munson et al., 2001; Shaw et al., 1996; Vondra et al., 2001). Disorganized attachment has been found to reflect a history of disturbed parent-child interaction on the one hand and to predict later externalizing problems on the other (cf. Lyons-Ruth & Jacobvitz, 1999). The results of our path analysis have shed more light on the nature of the contributions of the parent-child interaction and parent-child attachment to the development of externalizing behavior problems. First of all, our path analysis showed D attachment to mediate the relations between the two aspects of parent-child interaction (i.e., effective guidance and negative interaction) at 15 months and externalizing behavior at age 5. In addition, the results showed negative parent-child interactions at 28 months to contribute significantly to later externalizing problems beyond D attachment at 15 months. These findings are in line with earlier findings by Erickson et al. (1985) and support Greenberg’s (1999) model that considers parent-child interaction and parent-child attachment as two different domains that contribute independently to a child’s development but that also reciprocally influence one another. The results of this study may have clinical and policy implications. Many studies have shown that children born in disadvantaged environments are at risk for developing externalizing problems later in life. The present study showed that, also for children living in presumably lower-risk families, certain characteristics in infancy and toddlerhood indicate an increased risk of externalizing problems at preschool age. It should be kept in mind, however, that our sample may have been more at risk than originally thought, because a relatively large proportion of children turned out to have externalizing behavior scores above the subclinical cutoff. An important practical implication of our findings is that one of the most powerful predictors identified in the present study, i.e., negative parent-child interactions, is relatively easy to observe already in infancy and proves to be highly stable, which makes it an interesting candidate for inclusion in early community mental health screening procedures. And the specific interaction of the other parent-child interaction factor (i.e., effective guidance) with temperamental anger proneness in toddlerhood in predicting later externalizing problems also suggests that paying more attention to early parent-child interactions may be useful in early detection of children at risk and in preventive intervention programs for parents in community mental health care. But more research is needed, of course, to further explore these possible applications. It is important to also point out limitations of the present study. First, although it is a strong point that data were collected using multiple methods and multiple sources, parental report was the sole source of information regarding four different predictors of externalizing problems, namely infant temperament, parental ego-resiliency, partner support, and stressful life events. Thus, response bias may have partially accounted for the results. What speaks against this, however, is that the four predictors were mostly uncorrelated with each other (see Table 1). Only stressful life events was significantly correlated with two other predictors (i.e., ego-resiliency and partner support), which may explain why negative life events did not independently contribute to the explanation of later externalizing problems beyond the other two predictors. Another limitation is that the present study focused on the role of children’s experiences with their primary caregivers in the development of externalizing problems, which leaves questions regarding the possible effects of the children’s experiences with other caregivers and with their peers, for example in child-care centers (cf. Gevers Deynoot-Schaub & Riksen-Walraven, 2006). A third limitation of the study is that we did not have additional information about earlier externalizing behavior and about characteristics of the children and families prior to our first 15-month assessment at our disposal. Future research that includes observations earlier in infancy may further improve our understanding of the mechanisms involved in the early development of externalizing problems. A fourth limitation lies in the relatively small sample size. The sample was small by epidemiological standards, particularly with regard to evaluating mediator and moderator effects. And as a fifth and final limitation of the present study it should be mentioned that we based our assessments of the quality of infant-parent attachment on an abbreviated version of the Strange Situation that includes one as opposed to two separations from the parent. Despite the evidence supporting the validity of the abbreviated procedure (see Method section), it is possible that we have underestimated the number of D children given that in the regular Strange Situation Procedure children may be classified as disorganized based on their reunion behavior after the second separation. Anyhow, the present study shows that D attachment, even when assessed with the abbreviated Strange Situation, was a powerful predictor of later externalizing behavior problems. In sum, the longitudinal model our analyses generated provides more insight into the complex interplay among parental, child, dyadic and contextual characteristics that together shape the development of externalizing behavior from age 15 months onwards. Future research should reveal whether the predictive power of the model can be improved, for instance by including earlier measurements and by taking into account the children’s early experiences with the other parent and with their caregivers and peers in child-care facilities.

Eur_Radiol-4-1-2270369

Prevalence of anatomical variants and coronary anomalies in 543 consecutive patients studied with 64-slice CT coronary angiography

The aim of our study was to assess the prevalence of variants and anomalies of the coronary artery tree in patients who underwent 64-slice computed tomography coronary angiography (CT-CA) for suspected or known coronary artery disease. A total of 543 patients (389 male, mean age 60.5 ± 10.9) were reviewed for coronary artery variants and anomalies including post-processing tools. The majority of segments were identified according to the American Heart Association scheme. The coronary dominance pattern results were: right, 86.6%; left, 9.2%; balanced, 4.2%. The left main coronary artery had a mean length of 112 ± 55 mm. The intermediate branch was present in the 21.9%. A variable number of diagonals (one, 25%; two, 49.7%; more than two, 24%; none, 1.3%) and marginals (one, 35.2%; two, 46.2%; more than two, 18%; none, 0.6%) was visualized. Furthermore, CT-CA may visualize smaller branches such as the conus branch artery (98%), the sinus node artery (91.6%), and the septal branches (93%). Single or associated coronary anomalies occurred in 18.4% of the patients, with the following distribution: 43 anomalies of origin and course, 68 intrinsic anomalies (59 myocardial bridging, nine aneurisms), three fistulas. In conclusion, 64-slice CT-CA provides optimal visualization of the variable and complex anatomy of coronary arteries because of the improved isotropic spatial resolution and flexible post-processing tool. Introduction Since the beginning of the 1990s, a variety of non-invasive techniques have been introduced in coronary artery imaging in an attempt to replace invasive conventional coronary angiography (CCA). These techniques have shown promising results, although they were considered inadequate for large-scale clinical implementation. Furthermore, advanced modalities such as magnetic resonance (MR) and electron-beam computed tomography (EBCT) are still not widely available on the territory [1–6]. The introduction of multislice computed tomography coronary angiography (MSCT-CA) allowed the detection of significant coronary artery stenosis. Improved performance of 64-slice CT equipment, characterized by isotropic spatial resolution and faster temporal resolution, provided a valid alternative to CCA in selected patient populations [7–11]. MSCT-CA is currently considered the ideal tool to three-dimensionally visualize the complex and tortuous anatomy of coronary arteries [12, 13]. Previous studies with four- and 16-slice CT-CA demonstrated that anomalous coronary arteries may be defined [14–16]. However, to the best of our knowledge, 64-slice studies with large patient series have not yet been published. In the present study, a large patient population who underwent 64-slice CT-CA was reviewed to assess the prevalence of coronary artery variants and anomalies. Materials and methods Population A total of 543 consecutive patients (389 male, 154 female, mean age 60.5 ± 10.9, range 21–87 years), who underwent 64-slice CT-CA from 27/07/2004 to 28/02/2006 in our department, were reviewed for coronary artery variants and anomalies. The majority of patients (n = 476) were scheduled for CCA because of suspected or known coronary artery disease (CAD). The other 67 patients were addressed to 64-slice MSCT-CA after CCA to determine the three-dimensional image of a suspected origin or course anomaly. The indication for MSCT-CA were: atypical angina (n = 111), typical angina with inconclusive stress test (n = 162), presence of risk factors and high risk of major coronary events (n = 46), proximal stent patency follow-up (n = 112) and by-pass patency follow-up (n = 45). The Institutional Review Board approved the study protocol. CT scan and reconstruction parameters All examinations were performed with a 64-slice CT scanner (Sensation 64, Siemens, Forcheim, Germany) with the following parameters: slices/collimation 32/0.6 mm, rotation time 330 ms, effective temporal resolution (with 180° algorithm) 165 ms, 120 kv, 900 mAs, table feed/s 11.63 mm, effective slice thickness 0.6 mm, reconstruction increment 0.3 mm, field of view (FOV) 140–180 mm, isotropic voxel resolution of 0.4 × 0.4 × 0.4 mm. Patients with heart rate >70 bpm received 100 mg of metoprolol per os 1 h prior the examination. A bolus of 100 ml of high iodinated contrast material (400 mg/ml iomeprol, Iomeron 400, Bracco, Milan, Italy) was injected into an antecubital vein of the right arm with a flow rate of 5 ml/s, followed by a 40-ml saline chaser. A bolus-tracking technique was used for the synchronization between arterial passage of contrast material and MSCT-CA. Data were reconstructed by retrospective gating in end-diastolic phase (from -300 to -450 ms before the peak of the subsequent R wave) or end-sistolic phase to better image the right coronary artery (RCA). Image and data analysis All CT examinations were reviewed by three radiologists (L.L.G., R.M. and F.A.) with a level 3 expertise in cardiac CT [17], who loaded the datasets off-line into a dedicated workstation (Leonardo, Siemens, Germany). A total number of 559 examinations were performed and only 13 scans were considered not assessable by the readers in consensus due to poor image quality (severe breathing, triggering or motion artefacts). All data were analysed with post-processing tools such as multiplanar reconstructions (MPR), curved MPR (cMPR), maximum intensity projections (MIP), and volume rendering (VR) to three-dimensionally image the complex anatomy of the coronary artery tree. Disagreement was solved by a consensus reading. Segments were classified according to the American Heart Association (AHA) scheme. Variants considered were: the coronary dominance (right, left, balanced), the variable origin of the conus branch and sinus node artery, the left main (LM) length, the presence of the intermediate branch, the number of diagonal and marginal branches. Anomalies of origin and course, intrinsic coronary anomalies (myocardial bridging, aneurisms >1.5 mm) and termination anomalies (fistulas) were checked. Prevalence data of single coronary artery variants and anomalies were collected. Results Our cohort results were heterogeneous because of the multi-ethnic Dutch population (immigrants 11%, Table 1). Table 1Our population resulted heterogeneous because of the multiethnic Dutch populationEthnic group% (n)The Netherlands88.95 (483)Middle East Asia3.31 (18)South-East Asia2.94 (16)East Europe1.84 (10)South Europe1.10 (6)South America0.92 (5)Africa0.92 (5) Most of coronary segments were identified, although with a variable rate due to different diameters (Table 2). The anatomical variants of the coronary artery tree are extremely frequent (Table 3). According to the literature, the dominance was right in 86.6% (n = 470), left in 9.2% (n = 50), balanced in 4.2% (n = 23) [18]. The LM trunk (segment 5) presented a variable length (mean 112 ± 55 mm, range 17–601 mm, median 106 mm): <1 cm (n = 226, 41.6%), 1–2 cm (n = 257, 47.3%), and >2 cm (n = 38, 7%) (Fig. 1). The LM trunk was absent in 22 cases (4.1%) due to split origin of the left coronary artery (LCA) (n = 18, 3.3%) or other origin anomalies (n = 4, 0.7%). A variable number of diagonal branches was observed: one diagonal branch in 136 cases (25%), two diagonal branches in 270 cases (49.7%), and more than two in 130 cases (24%) (Fig. 2). No diagonal branches were visualized in just seven cases (1.3%). Marginal branches of the left circumflex (LCX) artery were observed in the 99.4% (n = 540): one in 191 cases (35.2%), two in 251 cases (46.2%), and more than two in 98 cases (18%) (Fig. 2). Table 2Segments visualized according to the American Heart Association classificationSegments% (n)199.8 (542)299.3 (539)397.8 (531)492.4 (502)595.9 (521)6100 (543)7100 (543)897.8 (531)998.7 (536)1073.7 (400)11100 (543)1299.4 (540)1397.2 (528)1464.3 (349)1572.4 (393)16a21.9 (119)aSegment 16 refers to the intermediate branchTable 3Prevalence of coronary artery variants (RCA right coronary artery, LAD left anterior descending artery, LCX left circumflex, LM left main, ND not detected)Variants Patients % (n)Conus branchFrom proximal RCA64.1 (348)From ostial RCA22.3 (121)From aorta11.6 (63)ND2 (11)Sinus node arteryFrom RCA65.4 (355)From LCX16.6 (90)From RCA and LCX9.2 (50)From LCX and pulmonary artery0.2 (1)From aorta0.2 (1)ND8.4 (46)LM length<1 cm41.6 (226)1–2 cm47.3 (257)>2 cm7 (38)Intermediate branch21.9 (119)Diagonal branches from LADND1.3 (7)125 (136)249.7 (270)>224 (130)Septal branches from LAD93 (505)Marginal branches from LCXND0.6 (3)135.2 (191)246.2 (251)>218 (98)Fig. 1a LM length. b The separate origin of the LAD and LCX might cause technical difficulties during coronary angioplasty due to poor visualization. c–e The LM may present variable lengthFig. 2Variable number and course of diagonal (a–c) and marginal branches (d–f).The intermediate branch courses along the anterior wall of the left ventricle with a variable pattern (g–i) When the intermediate branch supplies the vascularization of the antero-lateral wall of the left ventricle, a decreased number of diagonal branches (segments 9 and 10) was observed: one, 38.6%; two, 43.7%; more than two, 14.3%; none, 3.4% (Fig. 2). Furthermore, CT-CA may visualize smaller branches, such as the conus branch artery (532/543, 98%), the sinus node artery (497/543, 91.6%), and the septal branches (505/543, 93%). The conus branch artery may arise from the right coronary artery (RCA) (64.1%), in proximity with the RCA ostium (22.3%) or from the aorta (11.6%). The sinus node artery may originate from the RCA (355/543, 65.4%), from LCX (90/543, 16.6%), from RCA and LCX (50/543, 9.2%), from LCX and pulmonary artery (0.2%), or from aorta (0.2%) (Fig. 3). Fig. 3The variable origin of the conus branch artery (arrow): from RCA (a), in proximity with the ostium (b), and from aorta (c). The variable origin of the sinus node artery (arrowhead): from RCA (d), from LCX (e), or both pathways may be present (f) Coronary anomalies were observed in the 18.4% of our population (n = 100). Patients presented single or associated coronary anomalies (Table 4). Table 4Prevalence of coronary artery anomalies (LM left main artery, PDA posterior descending artery)Coronary anomaliesPatients % (n)Myocardial bridging10.9 (59)Absent LM3.3 (18)Rotation of the aortic root with normal coronary origin from the sinuses of Valsalva2.6 (14)Coronary aneurysms1.6 (9)Anomalies of origin and course1.5 (8)Fistulas0.5 (3)Early take-off of PDA0.5 (3) Single or multiple myocardial bridging was visualized in 59 patients (10.9%) (Fig. 4). Forty-three anomalies of origin and course were found with the following distribution: absence of left main artery occurred in 18 patients (3.3%), origin anomalies of the RCA and the LCA caused by rotation of the aortic root between 45° and 90° (with normal coronary origin from the sinuses of Valsalva) in 14 patients (2.6%), anomalies of origin and course (with anomalous origin from the sinuses of Valsalva) in eight patients (1.5%), early take-off of the posterior descending artery in three patients (0.5%). The anomalies of origin and course encountered were: three retroaortic LCX (two arising from RCA, one from the right sinus of Valsalva), two inter-arterial LCAs from RCA, two inter-arterial RCAs from the left sinus of Valsalva, one septal RCA from left sinus of Valsalva (Fig. 5). Fig. 4Examples of myocardial bridging (arrowhead). Myocardial bridging of mid-LAD displayed by MPR (a) and VR (b) images. Another case of myocardial bridging depicted by conventional angiogram in systole (c), not visualized in diastolic image (d), and clearly displayed by VR image (e)Fig. 5Anomalies of origin and course (arrowhead). VR (a) and MIP (b) images of a LCA arising from the RCA with a septal course as confirmed by conventional angiogram (c). VR (d) and MIP (e) images of a RCA arising from the left sinus of Valsalva with an interarterial course, and corresponding conventional angiogram (f). VR (g) and cMPR (h) images of a stented retroaortic LCX arising from the right sinus of Valsalva and corresponding conventional angiogram (i) Coronary aneurisms (>1.5 mm, if compared with the normal vessel diameter) were identified in nine patients (1.6%) (Fig. 6). Coronary fistulas were observed in three patients (0.5%) (Fig. 7). Fig. 6Examples of coronary aneurisms. Aneurisms of LCX and LAD displayed by VR (a) and MIP (b) images, and corresponding conventional angiogram (c). Aneurisms of the RCA depicted by VR image (d) and vessel tree isolation (e), confirmed by the conventional angiogram (f)Fig. 7Abnormal termination of coronary arteries. Fistula between the RCA and the coronary sinus depicted by VR (a), coronary tree isolation (b), and MPR images (c). Fistula between the LAD and the right ventricle displayed by VR (d) and coronary tree isolation images (e), and corresponding conventional angiogram (f) Discussion The wrong interpretation of a coronary variant or anomaly might cause technical difficulties during interventional procedures or lead to clinical misdiagnosis or major complications might occur during graft surgery. The need for an accurate anatomical evaluation of the coronary artery tree is relevant during angioplasty, due to revascularization purposes [19]. Coronary anomalies are often asymptomatic and may be accidentally discovered. Given the increase of interventional procedures, the detection of coronary anomalies is becoming of major clinical importance [20]. The coronary anomalies cannot be considered just rare aspects because they may often lead to relevant clinical consequences [21]. In an attempt to clarify the variability of the coronary artery tree, Angelini et al. [18] proposed these definitions: normal, any morphological feature observed in >1% of an unselected population; normal variant, an alternative, relatively unusual, morphological feature seen in >1% of the population; and anomaly, a morphological feature (number of ostia, proximal course, termination) rarely encountered (<1%) in the general population. However, the incidence of coronary anomalies is relevant not only for conceptual and educational purposes but, more importantly, for public health issues, given that 5.6% of the total American population could have some kind of coronary anomaly [18]. Moreover, the 19% of sudden deaths in young athletes are related to these anomalies [22]. That is a reason why the diagnosis of coronary anomalies should be a healthcare priority. To date, despite some limitations, CCA has been the “gold standard” for the diagnosis of coronary anomalies. Selective catheterization and subsequent interpretation of vessel anatomy may be difficult in CCA because the operator is not aware of an atypical location of the vessel orifice. Therefore, the diagnosis of a coronary anomaly is often established on the impossibility of finding the coronary arteries in their normal anatomical position. Finally, interpretation of the courses of anomalous coronary arteries may be erroneous because CCA is two-dimensional and cannot provide enough information about the complex three-dimensional vessel anatomy [18, 23, 24]. The study with the largest number of cases, performed in North America at the Cleveland Clinic on 126,595 patients who underwent coronary angiography, reported a 1.3% incidence [25]. In recent years, other techniques in cardiologic diagnostic imaging have been developed, such as trans-thoracic echocardiography (TTE), trans-oesophageal echocardiography (TEE), magnetic resonance angiography (MRA), EBCT and MSCT [1–4]. TTE, which is used mainly in paediatric radiology, does not always provide reliable diagnostic results. When performed on adult patients, it proves difficult to obtain diagnostic images owing to the interposition of the bones of the ribcage (ribs and sternum), pulmonary parenchyma and subcutaneous adipose tissue [5, 26]. Data reported in the literature suggest that TEE is more sensitive than TTE in identifying coronary anomalies and assessing their course, although it remains an invasive technique (i.e. insertion of a probe down the oesophagus and a varying degree of sedation according to patient tolerance required to perform the examination) characterised by a significant level of operator dependence and therefore impossible to perform as a screening test [6]. In addition, both echocardiography techniques are able to assess only the proximal tract of the coronary arteries, and therefore, their diagnostic capabilities are limited to only a part of coronary arteries [6]. MRA is a highly promising technique since no ionising radiation is used. In the study of the origin of the coronary arteries, MRA can provide more complete information than CCA, particularly in patients with other concomitant congenital cardiac anomalies [1, 27]. The main limitation of MRA is incomplete visualisation of the coronary vessels, particularly their distal tracts. This limits the diagnostic capabilities for the assessment of fistulas, the origin of coronary arteries other than the aortic sinuses (i.e. from the pulmonary artery) and collateral vessels. However, MRA provides optimal functional assessment of complex congenital heart disease including anomalies of great vessels, and cardiac chambers and valves (the procedures may include evaluation of ventricular mass and volumes, quantification of valvular disease, and contrast enhancement) [12]. Ropers et al. [2] firstly studied the ability of contrast-enhanced EBCT to identify anomalous coronary arteries and their course with good accuracy. Recent advances in MSCT-CA equipments have continuously improved the quality of non-invasive coronary artery imaging. Various studies have demonstrated a high accuracy of coronary angiography with MSCT-CA for the diagnosis of CAD. In particular, the high negative predictive value of 64-slice CT allows to reliably exclude significant coronary artery stenoses [7–11]. Given the high sensitivity and negative predictive value of the technique, MSCT could represent a non-invasive alternative to CCA in patients prior to cardiac valve surgery. By selecting only those patients with coronary significant lesions to undergo CCA, MSCT-CA could avoid cardiac catheterization in a large number of patients without CAD [28]. The latest advance of CT technology is represented by the dual-source CT coronary angiography (DSCT-CA), with improved temporal resolution of 83 ms. Scheffel et al. [29] firstly demonstrated that DSCT-CA provides high diagnostic accuracy for assessment of CAD in a high pre-test probability population with extensive coronary calcifications and without heart rate control. The advantages of MSCT lie primarily in its high level of diagnostic and anatomical accuracy. The technique offers excellent spatial resolution with the possibility of performing a flexible post-processing (i.e. MPR, MIP, and VR). Previous studies with four- and 16-slice CT-CA confirmed that the complex and tortuous coronary anatomy can be readily visualized and anomalous coronary arteries may be defined [14–16]. To the best of our knowledge, that is the first 64-slice CT-CA with large patient population, which evaluates the prevalence of coronary arteries variants and anomalies. The AHA classification is the currently used scheme to identify the coronary artery segments and refers to 16 segments, in the attempt to standardize the remarks [30]. Another classification was reported by the BARI group, with emphasis to the coronary dominance pattern [31–33]. According to literature data, the dominance was right in 86.6% (n = 470), left in 9.2% (n = 50) and balanced in 4.2% (n = 23) [18]. The majority of coronary segments was identified according to the American Heart Association scheme. The intermediate branch was present in 119 patients (21.9%), slightly less than reported in the literature [34]. However, the need for reporting the intermediate branch is stressed by the correlation between its presence and the decreased number of diagonal branches observed. Furthermore, the septal branches of the left anterior descending artery (LAD) were detected in the 93% (505/543). The opportunity of reporting septal branches of LAD must be taken into account because of the hemodinamic relevance of these vessels. The prevalence of coronary anomalies was estimated at the 18% (100/543) in our population. This prevalence rate is higher than reported in the literature. However, our department is a major centre for cardiovascular pathologies and many patients were enrolled in non-invasive cardiovascular research projects. Myocardial bridging occurred in the 10.9%, a prevalence more comparable with autopsy rates, than with angiographic series [35]. The split of LCA also had a prevalence higher than reported in the literature [18]. Eighty-five patients with not significant CAD presented 14 coronary anomalies (16.5%). Four hundred and fifty-eight patients with significant CAD (with >50% stenosis, during stent or by-pass follow-up) presented 86 coronary anomalies (18.8%). Therefore, the prevalence was similar in the two groups. However, a bias is given by the fact that 67 patients were addressed to 64-slice CT-CA after CCA to determine the three-dimensional imaging of a suspected anomaly. After excluding these 67 patients, the prevalence of coronary anomalies was higher in the CAD patients (11.4% vs 4.2%). The prevalence of myocardial bridging resulted higher in the CAD group (7.4% vs 2.8%). There are several limitations in our study. The first one is inherent to the heterogeneous population consisting of various ethnic groups (immigrants 11%) with a substantial age range (21–87 years). The age may affect the development of the collateral vascularization and influence the percentage of segments visualized. A second limitation is related to the high prevalence of origin anomalies, due to the fact that 67 patients were addressed to 64-slice CT-CA after CCA to determine the three-dimensional imaging of a suspected anomaly. As opposed to MRA, which also permits the non-invasive evaluation of coronary anomalies (proximal tracts), MSCT-CA requires radiation and a contrast agent. However, the high spatial and temporal resolution make it reasonable to use MSCT-CA as one of the first-choice imaging modalities in the work-up of known and suspected coronary anomalies [12, 13]. The high radiation exposure should be a matter of concern and debate in young patients. In these patients, the first-choice imaging modality could be MRA. In the case of suspected complex congenital heart disease (including anomalies of great vessels, and cardiac chambers and valves), the use of MRA would be highly advisable, since the optimal evaluation of ventricular and valvular function is provided [12, 13]. Compared with 16-slice CT-CA, 64-slice CT-CA provides improved temporal resolution and isotropic spatial resolution which allow optimal three-dimensional visualization of the variable and complex anatomy of coronary arteries. Sixty-four-slice CT-CA may non-invasively define normal anatomical variants from potentially dangerous anomalies and support the clinical management of referring cardiologists and cardiac surgeons.

Pediatr_Nephrol-4-1-2335291

Treatment of the neurogenic bladder in spina bifida

Renal damage and renal failure are among the most severe complications of spina bifida. Over the past decades, a comprehensive treatment strategy has been applied that results in minimal renal scaring. In addition, the majority of patients can be dry for urine by the time they go to primary school. To obtain such results, it is mandatory to treat detrusor overactivity from birth onward, as upper urinary tract changes predominantly start in the first months of life. This means that new patients with spina bifida should be treated from birth by clean intermittent catheterization and pharmacological suppression of detrusor overactivity. Urinary tract infections, when present, need aggressive treatment, and in many patients, permanent prophylaxis is indicated. Later in life, therapy can be tailored to urodynamic findings. Children with paralyzed pelvic floor and hence urinary incontinence are routinely offered surgery around the age of 5 years to become dry. Rectus abdominis sling suspension of the bladder neck is the first-choice procedure, with good to excellent results in both male and female patients. In children with detrusor hyperactivity, detrusorectomy can be performed as an alternative for ileocystoplasty provided there is adequate bladder capacity. Wheelchair-bound patients can manage their bladder more easily with a continent catheterizable stoma on top of the bladder. This stoma provides them extra privacy and diminishes parental burden. Bowel management is done by retrograde or antegrade enema therapy. Concerning sexuality, special attention is needed to address expectations of adolescent patients. Sensibility of the glans penis can be restored by surgery in the majority of patients. Introduction The incidence of spina bifida worldwide still ranges from 0.3–4.5 per 1,000 births. Renal scarring and renal failure have been important issues in spina bifida literature over the years, with reported death due to renal failure up to 20% in the first year of life. The incidence of renal damage is nearly 100% in patients with an overactive pelvic floor (detrusor/sphincter dyssynergia; DSD) when not adequately treated. It is important to realize that renal damage starts early in life, within the first 6 months. In contrast to a few decades ago, we can now assure parents of children with a neurogenic bladder that renal function can be preserved provided they comply with the treatment regime [1–14]. To prevent renal damage, urological and nephrological treatment of a neurogenic bladder should start immediately after birth. Modern treatment provides the opportunity to convert a high-pressure bladder with functional urethral obstruction based on neuropathic detrusor/sphincter dyssynergia into a low-pressure reservoir that is safe for the upper urinary tracts [15, 16]. The objectives in the urological management of patients with spina bifida are (1) preservation of renal function; (2) quality of life, preferably with urinary dryness by school age; and (3) independence at an older age with respect to bladder and bowel management. Finally, sexuality in the spina bifida patients is an underestimated problem. Initial approach for the management of spina bifida To preserve renal function, low bladder pressure must be maintained from birth. The status of pelvic floor activity must be assessed shortly after birth to ascertain whether a child is at risk for high detrusor pressures. At first presentation after birth, inspection of the anal sphincter, closed or open, gives an impression of the status of the pelvic floor: overactive or paralyzed. Approximately 50% of children with spina bifida aperta and 25% of children with occult spinal dysraphism have a detrusor/sphincter dyssynergia that carries a serious risk of early upper urinary tract damage by high bladder pressures and urinary tract infections (UTIs). It is important to realize that after closure of the back, pelvic floor behavior can change from paralyzed to overactive in the first 2–3 months of life. That is a reason to delay the first urodynamic study (UDS) until 2 months after birth. Clean intermittent catheterization In principle, all newborn patients are put on clean intermittent catheterization (CIC), oxybutynin, and chemoprophylaxis (trimethoprim 2 mg/kg once daily) immediately after closure of the back. By doing so, safe pressures in the lower urinary tract can be obtained in most patients. In most patients, bladder volume and compliance remain satisfactory for years. In patients with DSD, this approach has also reduced the need for bowel augmentation of the bladder from 90% to less than 5%. Of course, it is still a matter of discussion whether it is necessary to perform CIC from birth onward in all patients. In patients with proven pelvic floor paralysis, it is also possible to wait until after surgery to make the patient continent. However, it can be a tedious affair to teach CIC to an 8-year-old. Parents of these children are advised to catheterize at least twice daily to maintain the habit of CIC. CIC was introduced for neurogenic bladders in 1972 [17–20]. Parents and other caregivers carry out CIC in the first 8–9 years of life: if the children show sufficient dexterity, they take up this task themselves after this age. After birth, CIC is carried out with self-lubricating 8-F catheters. The size of the catheter depends on the patient’s age; the aim is always to use the largest possible catheter to obtain optimal bladder emptying. Antimuscarinic therapy Oxybutynin is best started together with CIC immediately after closure of the back. Other antimuscarinic agents have not yet been registered for pediatric use. This subject is more elaborately described in the section “Overactive detrusor”. Infection prophylaxis In principle, all patients are put on low-dose chemoprophylaxis, mostly trimethoprim 2 mg/kg per day. In case of breakthrough infections, nitrofurantoin or ciprofloxacin can be used. Symptomatic infections are treated intravenously with amoxicillin/clavulanic acid and gentamicin until the result of bacterial culture is known. Many centres stop prophylaxis after the age of 1 year, and approximately 50% of patients seem to do well without prophylaxis. Scientific proof is lacking on this subject. To obtain such proof, we are doing a multicenter randomized study with half of the patients on prophylaxis and half without. Specific needs Overactive pelvic floor From birth, patients with an overactive pelvic floor generally become dry with the help of oxybutynin and CIC. Every year, UDS are carried out to check bladder activity, capacity, and compliance. Patients are given ultrasound scans for upper-tract dilatation and renal growth. A wait-and-see policy is adopted when bladder capacity is normal for age and end-filling detrusor pressures are 30 cms H2O or less. When bladder compliance is insufficient, with end-filling pressures of more than 30 cms, an autoaugmentation or detrusorectomy can reduce the pressure. Augmentation by ileocystoplasty or colocystoplasty is inevitable in patients with serious overactivity in combination with poor bladder compliance and low capacity. Both operations can be performed at any age and can be combined with other procedures to obtain better urinary continence to facilitate CIC and/or to reduce UTIs (reflux) [21]. Paralyzed pelvic floor Patients with a paralyzed pelvic floor are incontinent for urine. Their upper urinary tracts are safe as long as the paralytic pelvic floor is left untreated. They need bladder-neck surgery to become dry. If bladder capacity and compliance are insufficient, surgery can be combined with autoaugmentation of the bladder (detrusorectomy) or (rarely) with clam ileocystoplasty or colocystoplasty. Overactive detrusor muscle If UDS reveals detrusor overactivity, patients are treated with antimuscarinics to increase bladder capacity, even if detrusor pressures are safe. Life-long suppression of detrusor overactivity is required in patients with an overactive neuropathic bladder. In some patients, overactivity can be treated surgically by detrusorectomy or ileocystoplasty. In spina bifida, rhizotomies (interruption of spinal roots) are not yet being used routinely to cure overactivity, but they seem to be a promising alternative. Ideally, we would like to start a protocol with neonatal rhizotomies during the first back closure, but so far, practical and ethical considerations have prevented us from doing this. Antimuscarinic therapy is the gold standard for pharmaceutical therapy of neuropathic detrusor overactivity. Oxybutynin has proven to be inexpensive and effective and can be taken orally, intravesically, and transdermally. Several new antimuscarinic agents have been introduced, which may prove valuable in the future. Repeated injection therapy of the bladder with 300 U of botulinum toxin can be an alternative to antimuscarinic therapy. This therapy effectively suppresses detrusor contractions for 6–9 months. Injections need to be repeated at a 6- to 9-month interval. Adverse effects, even after repeated injection therapy, have not been reported. The long-term effects have yet to be established [22, 23]. Surgical procedures Timing of surgery There is no age-related contraindication for any operation. Thus, indication for surgery is made in mutual agreement with the child’s parents or with the patients themselves after the age of 11 years. The combination of high bladder pressures and vesicoureteral reflux can sometimes force intervention as early as the first few months of life. We combined antireflux surgery, bladder autoaugmentation, and transvaginal sling suspension in a 3-month-old girl with febrile breakthrough infections, with good clinical and urodynamic result after follow-up for more than 10 years. Initially, reserves existed on doing sling suspensions before puberty, especially in boys, out of fear of introducing obstruction during puberty based on prostate growth. Over the years, it has been proven that puberty can safely be passed after sling suspension of the bladder neck, both in girls and boys. Parental burden can be an indication for performing a catheterizable stoma. If a child weighs 20 kg, five daily transfers for CIC can be too much for a parent with lower back pain. Finally, the patient’s privacy can be an important argumentation for construction of a stoma for CIC. Parents are increasingly reluctant for any caregiver to help their child perform CIC transurethrally, as this involves exposure of the genitalia to strangers several times a day. Surgery for incontinence Patients with a paralytic pelvic floor need bladder-neck surgery to achieve continence. There are many surgical options. Our standard approach is an abdominoperineal puboprostatic sling procedure in boys and a transvaginal sling procedure in girls. If there is some persistent leaking after a sling procedure, this is cured by injecting a bulking agent in the bladder neck. Optimal results are obtained if the injection needle is passed into the bladder neck by means of a suprapubic puncture with transurethral endoscopic visual control. So far, we have mainly used silicon grains in povidone (Macroplastique®) as a bulking agent. The polymer Deflux® offers a good alternative because it is easier to inject. However, in our experience, Deflux does not work if CIC needs to be carried out through the same channel. A recent survey, yet unpublished, of the results of 76 sling suspensions revealed an 80% success rate. Sling suspension in boys There is a great variety in the reported results regarding sling suspension of the bladder neck in boys with neuropathic sphincter incompetence, especially when compared with girls. The plane between the bladder neck/prostate and the rectum must be developed to allow the sling to be wrapped around the bladder neck. There are three possible techniques, each with their specific advantages and disadvantages. First, from the abdominal wound, the pelvic diaphragm can be opened left and right so that a pathway can be bluntly dissected for the sling around the bladder neck. A disadvantage of this technique is the risk of passing into the prostatic urethra with the right angle that is used to feel the way around during surgery. Experienced urologic surgeons do not take this argument seriously, but in practice, most surgeons perform this operation with the relatively low frequency of only a few cases a year. The second possible technique is the abdominoperineal approach to develop the plane between the bladder neck and the urethra. The plane between the rectum and the bladder neck is exposed through a perineal incision. The most important advantages of this procedure are the short time, approximately 20 min, needed to develop the plane around the bladder neck and the fact that the bladder does not need to be touched at all [24]. A third method to find the path around the bladder neck has been proposed by Lottmann et al. The complete bladder is dissected from the peritoneum and the rectum until only the urethra and both ureters with the vascular pedicles are left. The sling can easily be passed around the bladder neck, with direct visual control and exactly at the level of the bladder neck [25]. It is difficult to determine which of these three approaches is the best. All three have enthusiastic followers. At our department, we have had two urethral defects treated by two experienced surgeons in a small series of the abdominal approach going directly around the bladder neck. We have had one urethral defect leading to bladder-neck closure in 76 patients operated on abdominoperineally. Our experience with the Lottmann approach is limited to three successful cases and, because of operation time and for fear of limited bladder vascularization, we abandoned this approach. Another important subject is the amount of tension that needs to be put on the sling. In the past, we tried to regulate tension by measuring urethral and leak-point pressures during surgery, but there was no significant positive outcome. Nowadays, we determine sling tension by measuring the ability to pass a relatively large Foley catheter past the sling; for example, a 12-F catheter in a 7-year-old boy. In some patients with anal atresia after prior rectal pull-through surgery, it may be necessary to adopt the abdominal approach to pass around the bladder neck. Abdominoperineal operations in 14 patients, published in 1999, led to continence in all 14 patients; one patient required a subsequent injection of a bulking agent to help achieve continence [24]. It is important to note that in this series, the erectile function of the penis proved to be preserved after sling suspension. False routes are a risk after sling suspension. Out of approximately 50 male patients, we had to construct a catheterizable stoma in five: four as a result of a false route, and one because of a huge congenital prostatic cyst. Sling suspension in girls In general, sling suspension of the bladder neck in girls leads to good results concerning urinary continence. There are also several ways to find the way around the bladder neck in girls. The standard well-known technique is to identify the level of the bladder neck by feeling the balloon of a transurethral Foley catheter, and, subsequently, developing the plane between the bladder neck and the anterior vaginal wall from above. This procedure can be very difficult in girls with severe spine deformities. There is always a certain risk of entering the urethra or the bladder neck when passing around. This is also the case in patients with a normal anatomy. To avoid this risk, in postpuberty female patients, many surgeons opt for incising the vaginal wall at the level of the bladder neck and bringing the sling around under direct visual control. We chose to put the sling through the vagina, first only in prepubertal girls and later in all patients. This method has two major advantages: there is no risk of bladder-neck lesions and only a small risk of sling erosion [26]. In spina bifida patients, the risk of sling erosion is higher than in patients with stress incontinence, because the sling must be tightened more strongly in patients with neuropathic sphincter incompetence. Alternatives There are many surgical alternatives for reaching urinary continence. Kropp and Pippi Sale developed operations to create a flap valve from the bladder neck. Young/Deese/Leadbetter, Tanagho, and Mitchell procedures lengthen the urethra into the bladder. Several authors prefer American Medical Systems (AMS) artificial sphincters to help reach continence. The majority of patients still need CIC after AMS sphincter prosthesis [27]. We found that Burch-type colposuspension was insufficient for spina bifida girls to reach urinary continence. This is related to the huge intra-abdominal pressure that can result from transfers from the wheelchair. Several groups do not opt for urinary continence at an early age and try to maintain safe leak-point pressures by regular dilatation of the female urethra, sometimes also of the male urethra, after making this possible with a perineal urethral stoma. In some patients, they construct a cutaneous vesicostomy for the first few years and perform an ileocystoplasty when the stoma is closed. Alternatives in dealing with a neurogenic bladder When cerebral function is bad and the patient has no dexterity, diapers can also be chosen. For this choice, a low-pressure bladder is necessary. Good results are obtained by cutaneous vesicostomy. In girls, low pressures can be achieved by making a vesicovaginal fistula by endoscopic cutting into the vagina between the ureteric orifices. Temporary low pressures can be achieved by overdistension of the female urethra. In boys, endoscopic external sphincterotomy in the 12 o’clock position produces low outlet resistance for 2–3 years and needs to be repeated when bladder pressures rise. Catheterizable stomas The need for transfers from a wheelchair to do CIC is the most important indication to make a catheterizable stoma. Sometimes in boys, the impossibility of carrying out CIC transurethrally after false routes results in the need for a stoma. Parents increasingly indicate their child’s privacy as a reason for asking for a catheterizable stoma. In patients with a large bladder capacity for their age, a continent stoma can be constructed from a bladder tube. Patients with a normal bladder capacity can be treated by cutaneous appendicovesicostomy or by an ileal tube (Monti procedure) [28]. The direction of peristalsis from the appendix (Fig. 1) needs to go in the direction of the bladder to avoid mucus spotting in the clothes. When ileal or colonic bladder augmentation is performed at the same time, the continent stoma can be constructed from the side or middle part of the gut used for the augmentation. In some patients, a ureter can be used as a catheterizable stoma. The intravesical tunnel of the tube in the bladder should be at least 2 cm long, as there is a risk that the tunnel will end up being too short, especially in case of an extravesical implantation of an appendix. Fig. 1The appendix can be used to make a stoma to the bladder for intermittent catheterization In the literature, after construction of a catheterizable stoma, complications are described in as many as 50% of patients. These complications are mostly temporary. Stomal stenosis, mostly at skin level, is a frequently occurring temporary complication that can be avoided with a silicon ace stopper left behind in the skin portion of the stoma for 6 months between catheterizations. Stomal leakage of urine can be a frustrating complication. Endoscopic treatment with bulking agents can be tried, but lengthening of the intravesical tunnel or reimplantation of the stoma will often be needed. Leakage of the stoma occurs more often in midline stomas (umbilicus) than in stomas placed on the right side of the lower abdominal wall. The reason is probably that the lateral stoma passes through the rectus muscle and is thus occluded during increases in abdominal pressure due to rectus contraction. In midline stomas we now also pass the stoma through the median margin of one rectus muscle. Stoma stenosis at the level of entrance into the bladder can sometimes be treated endoscopically with success. In those patients, formal reimplantation of the stoma into the bladder will frequently be needed as well. Autoaugmentation or detrusorectomy of the bladder In general, not too much extra capacity should be expected, but high-end filling pressures based on low bladder compliance can be reduced to safe values by detrusorectomy. In selected patients that undergo surgery to become dry, we also do an autoaugmentation (Fig. 2) to try and get patients off antimuscarinic therapy, with success in approximately 50% of cases. An important factor for success is identification, opening, and marking the adventitial layers of the bladder separately before the detrusorectomy starts, and the meticulous closure of this layer at the end of the procedure. This means that before the start of the detrusorectomy, the detrusor has been freed of all adventitial layers. If a mucosal leak occurs during the operation, this is closed with Tissuecoll®. To prevent shrinkage and scarring, cycling of the bladder has to start immediately after detrusorectomy. This can be done by maintaining a 20-cm H2O pressure on day 1, 30 cm on day 2, followed by clamping the catheter on day 3 for 2 h, and finally by opening the catheter every 3 h to empty the bladder on following days. In this way, optimal expansion of the detrusorectomized bladder can be obtained [21]. Several authors have reported success by expanding the bladder over a balloon for several days while ensuring drainage of urine by ureteral catheters. We have no experience with this procedure but assume it could work well if there is leakage. Others have successfully covered the bare part of the mucosa with a demucolized patch of sigmoid colon. Covering with peritoneum or omentum has been reported to fail. Fig. 2The bladder can be augmented by removing the detrusor muscle. A sling is used to improve bladder outlet resistance Clam cystoplasty Clam cystoplasty (Fig. 3) is indicated when the bladder capacity is too small to reach acceptable numbers of catheterization per day and/or end-filling pressures are too high to be safe for the upper tracts. Clam cystoplasty is performed with either ileum or colon. In general, 25 cm of ileum are used, opened antimesenterically, and constructed into a U-shaped cap. The last 20 cm of the ileum are not used, to prevent malabsorption of vitamins. If there is a short ileal mesentery, the sigmoid colon can easily be used. It is important to avoid the formation of an hourglass bladder by opening the clam anteriorly up to the bladder neck and posteriorly up to the trigone. In children, we prefer to bring the augmented bladder extraperitoneally by closing the peritoneum around the vascular pedicle. This extraperitonealization of the bladder may initially make it more difficult to reach sufficient bladder capacity, but it seems to reduce the risk of spontaneous perforation of the bladder, which is a prominent risk in children because of their tendency toward bad CIC compliance during puberty. During a period of several years, we had two spontaneously leaking ileal bladders out of six that had not been placed extraperitoneally, and no spontaneously leaking/rupture in more than 40 other patients with extraperitoneal ileal bladder. Fig. 3The bladder can be augmented by using a bowel segment Bowel management and surgery For the first 2 years of life, a wait-and-see policy is adopted in bowel management, often supported by chronic use of laxatives. At the age of 3 years, bowel management can be performed successfully in the vast majority of patients with retrograde colonic enemas. The rectum is filled with 20 ml/kg tap water through a plastic cone that passes approximately 2 cm into the anus. In the rare case where patients have trouble emptying their rectums with retrograde enemas, we offer an antegrade colonic enema stoma [29, 30]. A patient’s choice can be an indication for an antegrade colonic enema stoma. As a separate procedure, the simplest way to carry out an antegrade colonic enema stoma is to use laparoscopy to bring the tip of the appendix to the abdominal skin (Fig. 4). Alternatives are an open procedure with open abdomen for other surgery or, in the absence of an appendix, to create a transverse tube from the colon (left or right) and a submucosal tunnel to prevent leakage. Many pediatric surgeons and pediatric gastroenterologists have had good experiences with several types of buttons that are put into the colon. Fig. 4The appendix can be used to make an antegrade colonic enema stoma If these procedures are carried out, treatment of spina bifida patients can be very successful. In our experience between 1988 and 2001, the incidence of renal scarring in 144 patients was 2.1%, and urinary dryness in patients who opted to be dry was nearly 80% [14]. Surgery to improve sexuality in male patients Sexuality can be frustrating for male spina bifida patients, because in most cases, they can have intercourse but penis sensibility is absent. To overcome this, in patients with a lesion under the level of L1, we have connected the ilioinguinal nerve with the dorsal nerve of the penis, resulting in glans sensibility and erogenous feeling in most patients. Patients report a positive effect on sexuality. Our experience with the first three patients has been published, and by now, of 15 cases operated, 12 report normal sensibility of the glans penis [31]. Conclusion Kidney-function preservation and early urinary dryness are important factors for optimal quality of life for spina bifida patients. Another important factor is patient independence regarding his or her bladder and bowel management. For optimal treatment of this difficult group of patients, a multidisciplinary team is needed consisting of members from all the necessary medical specialties, including rehabilitation specialists, physical therapists, specialized nurses, and social workers. Questions (Answers appear following reference list) Concerning renal scarring and renal failure in spina bifida literature: Reported death rate due to renal failure is up to 20% in the first year of lifeReported renal failure rate is up to 20% in the first year of lifeReported death rate due to renal failure is up to 20% in pubertyReported renal failure rate is up to 20% in pubertyConcerning clean intermittent catheterization (CIC): CIC is best started immediately after closure of the spinal defectCIC is best started immediately after the age of 8–9 yearsCIC is best started not earlier than after bladder neck surgeryCIC is best started not earlier than after several urinary infections and reported renal scarringInfection prophylaxis in spina bifida patients: All patients need low-dose chemoprophylaxisFifteen percent of all patients need low-dose chemoprophylaxisScientific proof of the need for infection prophylaxis is still lackingNo patients need low-dose chemoprophylaxisIn case of hyperactive detrusor muscle in a patient with spina bifida: Central side-effects occur less frequently if antimuscarinic therapy is administered orallyAntimuscarinic therapy is the gold standard for pharmaceutical therapyRepeated injection therapy of the bladder with only 3 U of botulinum toxin can be an alternative to antimuscarinic therapyAntimuscarinic therapy has positive effects on perspiration and body-temperature regulationIn modern management of spina bifida, endpoint targets are: Kidney-function preservationDrynessFecal continenceAll the above Electronic supplementary material Below is the link to the electronic supplementary material. ESM 1 (MPG 72.8 MB)

J_Med_Internet_Res-1-suppl1-1761819

SYD1/444: Assessing the Readership of the UK National Database of Telemedicine

Introduction The National Database of Telemedicine (NDTM) is a Web site containing information resources on UK telemedicine projects and related work. Its purpose is to provide a source of information to anyone researching the field or proposing a trial or a larger scale implementation of Telemedicine. The NDTM Web site was launched on 27th October 1998 and publicised extensively. Before and after its launch, the Web site has been kept up to date with information about new projects and modifications to the details kept about existing ones. It is clearly important for such a resource to keep track of who is reading it. We may need to tailor information to a particular audience, or bring the Web site to the attention of people who may not yet have found it. This paper describes a mechanism for tracking usage of the Web site that provides more information than conventional approaches.

Intensive_Care_Med-3-1-2040486

The first demonstration of lactic acid in human blood in shock by Johann Joseph Scherer (1814–1869) in January 1843

Lactic acid was first found and described in sour milk by Karl Wilhelm Scheele (1742–1786) in 1780. The German physician–chemist Johann Joseph Scherer (1841–1869) demonstrated the occurrence of lactic acid in human blood under pathological conditions in 1843 and 1851. In this article we honour the forgotten observations by Scherer and describe the influence of Scherer's finding on further research on lactic acid at the end of the 19th century. We conclude that Scherer's 1843 case reports should be cited as the first description of lactic acid in human blood after death and also as the first demonstration of lactic acid as a pathological finding in septic and haemorrhagic shock. Carl Folwarczny was, in 1858, the first to demonstrate lactic acid in blood in a living patient. Introduction Lactic acid was first found and described in sour milk by the Swedish chemist Karl Wilhelm Scheele (1742–1786) in 1780 [1]. The Swedish chemist Jöns Jakob Berzelius (1779–1848) found lactic acid in fluid extracted from meat in 1808 [2, 3], and the German chemist Justus von Liebig (1803–1873), who established the world's first school of chemistry at Giessen, proved that lactic acid was always present in muscular tissue of dead organisms [4]. In 1859, Emil Heinrich du Bois-Reymond (1818–1896) published several articles on the influence of lactic acid on muscle contraction [5–9]. Araki and Zillessen found that if they interrupted oxygen supply to muscles in mammals and birds, lactic acid was formed and increased [10–14]. This was the first demonstration of the relationship between tissue hypoxia and the formation of lactate. The occurrence of increased lactic acid in blood (hyperlactataemia) nowadays reflects severe illness, in which the increased blood lactate levels may result from both anaerobic and aerobic production or from a decreased clearance. It was the German physician–chemist Johann Joseph Scherer who first demonstrated the occurrence of lactic acid in human blood under pathological conditions after death in 1843 and 1851 [15, 17], and Carl Folwarczny in 1858 who first demonstrated lactic acid in blood of a living patient. In this article we wish to honour Scherer's forgotten observations and describe the influence of his finding on further research on lactic acid at the end of the 19th century. Biography of Johann Joseph Scherer Born on 18 March 1814 in Aschaffenburg, Germany, Scherer studied medicine, chemistry, geology and mineralogy at the university of Würzburg between 1833 and 1836. He obtained his PhD in medicine and surgery in 1838 with a thesis entitled “Versuche über die Wirkung einiger Gifte auf verscheidene Thierclassen” (Experiments on the action of some poisons on several classes of animals). He practised medicine in Wipfeld, but inspired by the chemist Ernst von Bibra (1806–1878) he completed his studies in chemistry at the University of Munich between 1838–1840 [18]. In 1840 he was employed at the laboratory of Justus Liebig at Giessen, and became professor at the medical faculty in 1842, professor of organic chemistry in 1847, and later professor of general, anorganic and pharmaceutical chemistry. His work especially concerned quantitative research on blood and urine in pathological conditions. In 1843 he published his book ‘Chemische und Mikroskopische Untersuchungen zur Pathologie angestellt an den Kliniken des Julius-Hospitales zu Würzburg’ (Chemical and microscopic investigations of pathology carried out at the Julius Clinic at Würzburg) [15] (Fig. 1), in which he described 72 case reports, giving details on clinical course, diagnosis, and results obtained during autopsy and analysis of body fluids. Scherer died on 17 February 1869 [18]. Fig. 1Title page of Scherer's 1843 book The 1843 cases In one chapter in his 1843 book entitled 'Untersuchungen von krankhaften Stoffen bei der im Winter 1842–1843 in Würzburg und der Umgegend herrschenden Puerperal-Fieber-Epidemie' (Investigations of pathological substances obtained during the epidemic of puerperal fever which occurred in the winter of 1842–1843 in and around Würzburg) Scherer described the cases of seven young women who all died peripartum. One of the women, the 23-year-old primipara Eva Rumpel, gave birth to a healthy child on 9 January 1843. The same night she developed a painfully swollen abdomen and became ill, feverish, and sweaty, with rapid pulse and severe thirst. The initiated treatment was bloodletting and clystering. The next evening she deteriorated, became delirious, with anxious breathing, a tense abdomen, cold extremities and rapid pulse, finally losing consciousness. Again, bloodletting followed. At 4:30 a.m., 36 h after the onset of the first symptoms, she died. During autopsy, severe purulent endometritis, vaginal pus, pulmonary oedema, and shock liver and shock spleen were found. The blood that was obtained directly from the heart was chemically analysed, in which lactic acid was found. Most likely this unfortunate woman had died from a fulminant septic shock caused by group A haemolytic streptococci (Streptococcus pyogenes). Scherer diagnosed this case as perimetritis with secondary peritonitis. Another patient, the 28-year-old, 7 months pregnant (second pregnancy) Margaretha Glück, was, after being icteric, nauseous, vomiting and complaining about epigastric pain for 8 days, admitted to the lying-in birth clinic on 6 February 1843. Four days later she was transferred to the hospital with severe nosebleeds and generalised exanthema or purpura. In the evening she suffered from severe gastric bleeding and epistaxis, showing rapid pulse, cold extremities and dizziness. The next morning, she was transferred back to the birth clinic, where she gave birth to a premature child (30 weeks) and suffered from a severe post-partum fluxus. She was again transferred to the hospital with the following symptoms: cold clammy skin, tachycardia, severe lochia and persistent exanthema or purpura, but without signs of an acute abdomen. During the night of February 11, she became aphasic and restless, followed by chills and profound sweating. On the morning of February 13, she further deteriorated and bilirubinuria was detected. The next day she was comatose, finally developed rattling breathing and convulsions. Death occurred during the following night. Autopsy revealed a small intracerebral haematoma, normal lungs without pulmonary oedema, ascites and an anaemic, foul smelling uterus filled with purulent and decayed tissue and pus. Blood was also obtained directly from the heart during autopsy and lactic acid was found. In this case we could think of a haemorrhagic shock and cerebral haemorrhage due to clotting disorders possibly resulting from either acute fatty liver of pregnancy/HELLP syndrome, idiopathic thrombocytopenic purpura, thrombotic microangiopathy (TTP/HUS) or DIC. The case was most likely complicated by a sepsis (endometritis). Scherer himself diagnosed this case as septic endometritis. In the conclusions of his 1843 book, Scherer attached high importance to the fact that he found lactic acid in cases of puerperal fever, which he had not found before in healthy persons. He held the opinion that lactic acid was formed in blood during bodily deterioration in severe diseases like puerperal fever. Lactic acid was thus described for the first time in human blood and was demonstrated for the first time as a symptom of septic and haemorrhagic shock. In the same period a junior obstetrician in Vienna, Ignaz Philipp Semmelweis (1818–1865), discovered in 1847 that physicians carried infectious particles on their hands from the mortuary to the obstetrical clinic, causing puerperal fever and puerperal sepsis, and he introduced a successful method for its prevention. Louis Pasteur (1822–1894) found in 1879 that infection with streptococci was the most important cause of puerperal fever [16]. The 1851 article Scherer worked closely with the famous pathologist Rudolf Virchow (1821–1902) on several projects (Fig. 2). In 1851 Virchow performed an autopsy on a patient who had died from leukaemia and offered Scherer blood from this patient for analysis. The results of this analysis were published the same year in the ‘Verhandlungen der Physikalisch-Medicinischen Gesellschaft in Würzburg’  [17]. Virchow and Scherer had previously studied the spleens of patients who died from leukaemia, and were curious if they could find the same results in the blood. Scherer reached the conclusion that: the blood of this patient contains: “Ameisensäure, Essigsäure und Milchsäure, die gleichfalls von mir schon früher als in der Milzflüssigkeit vorkommend bezeichnet wurden” (Formic acid, acetic acid, and lactic acid, as also found by me previously in fluids from the spleen). Fig. 2Johann Joseph Scherer (left) and Rudolf Virchow (right) in 1849 Further research Scherer's observations inspired others to conduct further research, primarily in patients with leukaemia [19–22], but also in patients with other conditions and diseases and in animal experiments with dogs and rabbits [23]. While Scherer found lactic acid in blood obtained after death during autopsy, Mosler and Körner [19] mention an observation made by Carl Folwarczny, published in the Allgemeinen Wiener Medicinischen Zeitung in 1858, where blood was withdrawn from a leukaemia patient during life, analysed according to Scherer's method, and found positive for lactic acid. In addition, Carl Folwarczny described in 1863 in his 'Handbuch der Physiologischen Chemie' [24] that lactic acid can be found in the blood of patients with leukaemia, septicaemia (pyaemia) and in conditions leading to septicaemia like puerperal fever, the latter probably after Scherer's observations. In an extensive article, the Berliner physician Georg Salomon [25], who had serious doubts that the occurrence of lactic acid in blood was mostly related to leukaemia, proved in 1878 that lactic acid was also present in the blood of patients who were suffering and died from other diseases. He studied blood obtained during autopsy from cadavers, but also blood from patients obtained by bloodletting or cupping, and in some cases he compared the blood before and after death. He was able to demonstrate lactic acid in the blood of patients suffering from leukaemia, (pernicious) anaemia, congestive heart failure, chronic obstructive pulmonary disease, pleuritis, pericarditis, pneumonia and several solid malignant tumours. Gaglio [26] is often erroneously mentioned as the first author to find lactic acid in blood [27–29]. He was able to demonstrate lactic acid in fresh arterial blood withdrawn from dogs and rabbits after bloodletting. Berlinerblau [30] confirmed these observations in mammalian and venous human blood. Both Gaglio and Berlinerblau, however, neglected previous research, as indignantly described by Salomon in 1888 [“Ich erlaube mir, den Inhalt meiner Arbeiten, die von Gaglio nur ganz flüchtig, von Berlinerblau gar nicht berührt sind, in Kürze zu reproduciren” (I take the liberty of summarizing the contents of my work, which was mentioned only briefly by Gaglio and not at all by Berlinerblau)] [31]. The Japanese chemist Trasaburo Araki showed that the amount of lactic acid in exhausted muscle results from muscle activation [11]. Irisawa [32], inspired by the results obtained by Salomon and Gaglio, obtained fresh blood of 11 dying patients with serious conditions. In six cases he found hyperlactataemia, in four cases normal values. He speculated on the aetiology of hyperlactataemia, the most plausible cause being the severe hypoxia during the dying process. In an experiment in which he made a dog anaemic for several days, he found a rise in lactic acid levels during the time leading up to death. In Cambridge (UK), Walter Morley Fletcher (1873–1933) and Frederick Gowland Hopkins (1861–1947) worked together on the metabolic changes occurring in muscular contractions and rigor mortis under anaerobic conditions, and found that lactate was the product of carbohydrate metabolism [33]. Their classic 1907 paper demonstrated rigorously that muscle contraction is accompanied by the anaerobic formation of lactic acid, which is removed aerobically, at a rate depending on the level of exposure to oxygen [34]. Poul Astrup and John Severingshaus mentioned Scherer's 1851 article as first demonstration of lactic acid in blood, but overlooked the 1843 cases and Folwarczny's work [35]. In conclusion, Scherer's 1843 case reports [15] should be cited as the first description of lactic acid in human blood and also as the first demonstration of lactic acid as a pathological finding in septic and haemorrhagic shock. Folwarczny, in 1858, was the first to demonstrate lactic acid in blood in a living patient.

Anal_Bioanal_Chem-4-1-2287205

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence. Introduction In recent years, the presence of endocrine-disrupting compounds (EDCs) in the environment has become a major issue of concern in view of their effects on both human health and ecosystem integrity. The classes of substances that possess estrogenic activities include diverse manmade and natural chemicals (e.g., pesticides, plasticizers, polychlorinated biphenyls, phytoestrogens, natural hormones) and pharmaceuticals (e.g., ethinylestradiol, tamoxifen) [1, 2]. EDCs can interfere with the endocrine system by binding to the estrogen receptors α (ERα) or β (ERβ) and mimicking the action of the naturally produced hormone estrogen or blocking the receptor for this hormone in target cells, thereby preventing its action [3]. Therefore, it is important to screen compounds and compound mixtures for their ability to bind to one of these receptors. Alternatively, the task of screening for compounds containing estrogenic and antiestrogenic properties is also very important from medical and pharmaceutical points of view [4]. Several biodetection methodologies have meanwhile been developed to determine the bioaffinities of ligands towards ERα. These assays include relatively cumbersome radioligand binding assays [5], fluorescence enhancement based assays [6] and fluorescence polarization (FP) based binding assays [7]. A major drawback of these assays is that, in the presence of cross-reactive compounds, the response provided by the bioassay reflects the sum of the concentrations of all individual compounds and their affinities for the ERα. Moreover, matrix components, which are sometimes present in far higher concentrations than the analyte(s), interfere with the binding assay signals, thereby leading to false-positive or false-negative results [8]. Therefore, none of these methods can be applied for the identification of individual ERα binding ligands in mixtures of compounds, as is the case with natural compound extracts, biological samples containing different EDCs, and drug metabolite mixtures. However, the introduction of the on-line high-resolution screening (HRS) technology enabled the screening of individual compounds in mixtures by coupling a separation technology, usually gradient HPLC, to post-column biochemical detection assays on-line [9–14]. Oosterkamp et al. [10] developed a HRS system to screen mixtures for compounds with an affinity for ERα. A similar setup has been used to screen natural plant product extracts for ERα and ERβ binding activity [15] and to identify and screen cytochrome P450-derived metabolites of tamoxifen with ERα affinity [2, 16]. The reported HRS ERα bioaffinity assays are based on the increase of the fluorescent signal of the tracer compound coumestrol upon binding to the ERα ligand binding domain (LBD). When the tracer is displaced by a compound with ERα affinity that elutes from the HPLC column, it is seen as a negative peak in the baseline of the ERα bioassay. Coumestrol, the fluorescent probe, has short excitation (340 nm) and emission (410 nm) wavelengths when bound to the ERα LBD [6]. Since there are many potentially interfering fluorescent compounds that can be excited at 300–350 nm and emit at 360–430 nm in biomatrices, coumestrol is not appropriate for the precise and selective detection of EDCs in biosamples containing phytoestrogens such as flavonoids, coumestans or lignans. One solution to this problem is to use another tracer compound, thus making it possible to monitor the bioassay signal at higher excitation and emission wavelengths. However, no tracer compounds are currently available which also display the necessary fluorescence enhancement upon binding to the ERα, thus making it impossible to use them in a HRS ERα setup. Another option is fluorescence polarization (FP) detection. FP is based upon the principle that interaction of a receptor with a fluorophore-containing ligand increases the effective size of the fluorophore through the formation of a receptor ligand complex. The increase in effective size reduces the rate of fluorophore rotation and increases the polarization over that of the unbound fluorophore [7]. FP instruments measure the rate of fluorophore rotation in solution rather than fluorescence intensity [17]. The polarization value is independent of the fluorophore concentration and intensity and has the advantage that it is minimally affected by autofluorescence, solution opacity or color, and is more tolerant to fluorescence quenching and light scattering [18]. The objective of the present study was to develop and evaluate the use of FP detection in combination with the on-line HRS technology to provide information about the ERα affinities of individual compounds in complex mixtures. The assay is based on competition for the ERα LBD between eluting compounds and a fluorescein-labeled estradiol derivative (E2-F), which was synthesized based on tracer structures described by Parker et al. [7] and Ohno et al. [19] for off-line FP-based ER assays. After synthesis of the tracer E2-F, the ERα binding assay was first optimized in FP platereader format and subsequently transferred to flow-injection analysis (FIA) mode with FP detection by using an on-line FP detector cell, which was recently constructed by Kool et al. [20] to measure GPCR-mediated modulation of cAMP production. Finally, after optimization of the assay in FIA mode, the on-line FP detection system was coupled to a gradient HPLC system. In this so-called HRS setup we separated mixtures of known estrogenic compounds—e.g., 17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone—and screened the individual compounds for ERα affinities. We also showed that this HRS ERα-FP system was suitable for screening off-line-generated cytochrome P450 (Cyt P450) metabolites of zearalenone for ERα affinity. We conclude that we were able to incorporate an on-line FP detector cell into a HRS system to screen for the ERα affinities of individual compounds in complex mixtures without incurring interference from the autofluorescence of the compounds. Materials and methods Materials Tween 20, 17β-estradiol (E2), estrone (E1), 17α-ethinyl estradiol (EE2), diethylstilbestrol (DES), estriol, zearalenone, fluorescein-5-isothiocyanate (5-FITC) and coumestrol were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). ELISA blocking reagent was purchased from Roche (Mannheim, Germany). Dry THF was freshly distilled from LiAlH4. All other chemicals were of analytical grade and obtained from standard suppliers. Synthesis Fluorescein-labeled estradiol derivative (E2-F) The fluorescein-labeled estradiol derivative (E2-F) was synthesized in analogy to methods described previously [7, 19] from estrone as the starting material (Fig. 1). To a solution of estrone (1, 700 mg, 2.59 mmol) in dry DMF (3 mL), imidazole (250 mg) was added. Subsequently, tert-butyl dimethylsilyl chloride (tBDSC) dissolved in dry THF (2 mL) was slowly added and the reaction mixture was stirred at room temperature for 3 h. The resulting cloudy white mixture was extracted with ethyl acetate (EtOAc). The organic extract was washed with water once, with brine twice and then with water once again. The organic extract was dried over MgSO4, filtered, and concentrated by rotary evaporation at 45 °C. Recrystallization was performed using a mixture of 15 mL MeOH and 5 mL DCM to afford 2 (667.2 mg, 67%). Under an atmosphere of dry nitrogen, 3-butyn-1-ol (1.35 g) was dissolved in dry THF (27 mL) and cooled to −78 °C. n-Butyllithium (BuLi) in hexane (16.9 mL, 0.027 mol) was added dropwise at −78 °C. Compound 2 (3.60 g, 9.36 mmol) dissolved in dry THF (25 mL) was carefully added dropwise at −78 °C and the solution was allowed to warm to room temperature and was thereafter stirred at 40 °C for an additional 60 min. The remaining BuLi was neutralized by carefully adding 5 mL EtOAc/MeOH (1:1). Extraction was performed with EtOAc, as described above. The crude product was purified by column chromatography on silica gel (10% EtOAc in hexane) to afford 3 (1.62 g, 38%). To a solution of 3 (1.6 g, 3.52 mmol) dissolved in dry THF (25 mL) was added triethylamine (TEA, 718 μL) followed by MeSO2Cl (395 μL, 5.1 mmol) while stirring at 0 °C. Afterwards the solution was allowed to warm to room temperature. After 10 h the reaction mixture was extracted with DCM. The organic extract was treated as described above and concentrated by rotary evaporation to afford 4 (1.80 g, 96%). Under an atmosphere of dry nitrogen, NaN3 (8.0 g) was added to a solution of 4 (1.80 g, 3.38 mmol) dissolved in dry DMPU (25 mL) while stirring rapidly. After stirring the solution for three days, the resulting reaction mixture was extracted with EtOAc as described above to afford 5 (1.36 g, 84%). To a solution of 5 (1.36 g, 2.83 mmol) in MeOH (70 mL) and TEA (16 mL), 1,3-propane dithiol (12 mL) was added. The solution was stirred o/n at room temperature and was concentrated by rotary evaporation. The concentrated solution dissolved in 100 mL hexane was purified by column chromatography on silica gel. Elution of the purified product 6 (1.08 g, 84%) was performed using EtOAc/MeOH (1:1). To remove the protective tert-butyl dimethylsilyl group, TBAF.H2O (300 mg) was added to a solution of 6 (258 mg, 0.57 mmol) dissolved in dry THF (4 mL) at 0 °C. After stirring for 1 h and allowing the solution to warm to room temperature, the crude product was extracted with DCM. The product was purified with Flash Master chromatography with an EtOAc/MeOH gradient to afford 7 (176 mg, 91%). Fluorescein-5-isothiocyanate (5-FITC, 7 mg, 17.8 μmol) was added to a solution of 7 (5 mg, 14.7 μmol) dissolved in dry DMF (500 μL) and dry pyridine (25 μL). The solution was stirred o/n in the dark at room temperature. The crude product was extracted with DCM and purified first with Flash Master chromatography (silicagel-based; EtOAC/MeOH gradient) and then with preparative C18 HPLC to afford 8 (E2-F, 8.02 mg, 75%). 1H NMR (400.13 MHz, CD3OD): δ 8.04 (1 H, d, J = 1.9 Hz), 7.70 (1 H, dd, J = 8.2, 1.9 Hz), 7.10 (1 H, d, J = 8.2 Hz), 7.01 (1 H, d, J = 8.5 Hz), 6.89 (1 H, d, J = 8.9 Hz), 6.88 (1 H, d, J = 8.9 Hz), 6.66 (2 H, d, J = 2.3 Hz), 6.58 (2 H, dd, J = 8.9, 1.9 Hz), 6.47 (1 H, dd, J = 8.5, 2.6 Hz), 6.42 (1 H, d, J = 2.6 Hz), 3.80 (2 H, t, J = 6.6 Hz), 2.70 (1 H, d, J = 1.9 Hz), 2.65 (2 H, t, J = 6.6 Hz), 2.1–2.3 (3 H, m), 1.9–2.0 (2 H, m), 1.65–1.85 (4 H, m), 1.25–1.50 (5 H, m), 0.85 (3 H, s). ESI-MS: m/z 729 (M + H)+. Fig. 1Synthesis of a fluorescein-labeled estradiol (E2-F) derivative (for abbreviations, see “Experimental” section) 7-Hydroxy-3-p-hydroxyphenylcoumarine (coumarol) The synthesis of coumarol was analogous to the synthesis of benzene-hydroxylated-3-aryl-coumarins described by Buu-Hoi et al. [21]. Briefly, 2,4-dimethoxybenzaldehyde was reacted with p-methoxyphenyl-acetonitrile to form α-p-methoxyphenyl-β-(2,4-dimethoxyphenyl)-acrylonitrile. The formed product formed was demethylated using pyridine hydrochloride to form the final product coumarol. 1H NMR (400.13 MHz, DMSO-d6): δ 10.57 (1 H, s), 9.68 (1 H, s), 8.00 (1 H, s), 7.56 (1 H, d, J = 8.6 Hz), 7.53 (2 H, d, J = 8.7 Hz), 6.81 (2 H, d, J = 8.7 Hz), 6.80 (1 H, dd, J = 10.8, 2.7 Hz), 6.73 (1 H, d, J = 2.2 Hz). ESI-MS: m/z 255 (M + H)+. Biomaterials Microsomes Rat liver microsomes were prepared from phenobarbital (PB)-induced rat liver, as described previously by Roosenboom et al. [22]. Estrogen receptor α The ERα ligand binding domain (LBD) was expressed in Escherichia coli according to the method of Eiler et al. [23], but without estradiol in the medium. The concentration of ERα LBD (250 nM) was measured by determining the Bmax value (by titration with radiolabeled estradiol) [24]. The Bmax value was measured as the maximum amount of ligand binding extrapolated to a very high concentration of ligand. Microsomal incubations Microsomal PB-induced rat liver incubations were carried out in a total volume of 500 μL. The reaction mixture consisted of 2.5 mM MgCl2 in 100 mM potassium phosphate buffer (pH 7.4), 100 μM zearalenone and 2.6 mg/mL rat liver microsomes. The reactions were initiated by the addition of a NADPH regenerating system, resulting in final concentrations of 0.1 mM NADPH, 0.3 mM glucose-6-phosphate, and 0.4 U/mL glucose-6-phosphate dehydrogenase. Incubation was carried out at 37 °C for 120 min and the reaction was terminated by the addition of 50 μL 10% HClO4. Following centrifugation at 14,000 rpm for 10 min, 450 μL of supernatant was applied to a strata_X 33 μm Polymeric Sorbent 200 mg / 3 mL (Phenomenex, Amstelveen, The Netherlands) column for SPE sample preparation. Before sample application the column was conditioned with 2 mL MeOH and subsequently equilibrated with 2 mL H2O. The sample was washed with 2 mL 5% (v/v) of aqueous methanol and elution was performed using 2 mL 50% (v/v) of MeOH in ACN. After evaporation, the residues were dissolved in 1 mL 40% (v/v) of aqueous methanol. Instrumentation A NOVOstar+ microplate fluorometer from BMG LABTECH GmbH (Offenburg, Germany) was used for the FP-based microplate reader assay. The excitation wavelength was set at 485 nm, the emission wavelength at 520 nm. Black-bottomed PP-96-well microtiter plates from Greiner Bio-one (Alphen a/d Rijn, The Netherlands) were used for all microplate reader experiments. The setup we used was adapted from the homogeneous coumestrol-based ERα affinity detection system described by Kool et al. [2]. For the on-line FP assay, two Knauer K-500 HPLC pumps (Berlin, Germany) were used to control the 120-mL superloops, made in-house, containing receptor (ERα) and tracer ligand (E2-F), respectively, and one Knauer K-500 HPLC pump was used to deliver the injected samples. The ERα and E2-F solutions were prepared in sodium phosphate buffer (10 mM; pH 7.4) containing 150 mM NaCl and 0.4 mg/mL ELISA blocking reagent. Both superloops were kept on ice. Flow restrictors were inserted between the pumps and the superloops to ensure proper operation of the pumps at low flow rates. The flow restrictors consisted of natural PEEK tubing and resulted in back pressures of approximately 50 bar. The pressure limits of the pumps were set 20 bar higher than the working pressure, and VICI Jour backpressure regulators (Schenkon, Switzerland) were inserted after the superloops to prevent damage from possible clogging of the system. A Gilson 234 autoinjector (Villiers-le-Bel, France) equipped with a Rheodyne (Bensheim, Germany) six-port injection valve (different injection loops) was used to inject samples. To maintain the reaction coils at a constant temperature, a Shimadzu CTO-10AC column oven (Duisburg, Germany) was integrated into the system. An Agilent 1100 (Waldbronn, Germany) series fluorescence detector (λex 498 nm; λem 522 nm) was used to monitor fluorescence polarization, as described by Kool et al. [20]. Briefly, the standard 8 μL FLD cell was modified with parallel-placed (both mounted in the detector cell) fluorescence polarization filters from Melles Griot (Didam, The Netherlands). One filter (12.5 mm visible pol. 350–650 nm 03FPG019) was placed in the pathway of the excitation beam and one (Mount. Sheet Pol. 20.D- 16.9CA 03FPG001) in the pathway of the emission beam. All hardware was integrated into one HRS system by Kiadis B.V. (Groningen, the Netherlands), and was controlled by software developed by Kiadis B.V. Coumestrol-based microplate reader assay of ERα affinity The microplate reader assay for determining ERα binding based on the competition between fluorescent coumestrol and estrogenic compounds previously described by Gurer-Orhan et al. [6] was used to plot a competitive binding curve for E2-F. FP-based microplate reader assay of ERα affinity The microplate reader assay was used to optimize the FP ERα receptor affinity detection (RAD) system in FIA and HPLC modes. This assay is based on the competition between estrogenic compounds and the newly synthesized fluorescein-labeled estradiol derivative (E2-F) for binding to the ERα LBD. Displacement of E2-F was measured as the decrease in fluorescence polarization intensity. Different concentrations of E2-F tracer were tested first. Since Tween 20, a detergent also used by Kool et al. in their coumestrol-based HRS ERα assay [2], can improve the resolution of the assay in HRS mode, and since organic modifiers are necessary for the optimal performance of the gradient HPLC system, the next step in the optimization process was to vary these parameters. The experiments were carried out at 24 °C with well volumes of 250 μL binding buffer [potassium phosphate buffer (10 mM; pH 7.4) containing 0.4 mg/ml ELISA blocking reagent]. The ERα LBD was introduced into the binding assay buffer at a final concentration of 1.64 nM. The E2-F stock solution was prepared in DMSO and dilutions were made in binding buffer. Concentrations ranging from 0.42 nM to 3.31 μM were investigated for E2-F optimization. For the experiments to investigate the detergent and organic modifier effects, the final concentration of E2-F in the assay was 83.3 nM. Aliquots of 25 μL of organic modifier or detergent at various concentrations were pipetted into a 96-well plate that already contained either 75 μL binding buffer (equal to 0% displacement) or 75 μL 33.3 μM E2 in binding buffer (equal to 100% displacement). The 96-well plate was gently but thoroughly mixed after pipetting 75 μL E2-F into each well. Finally, 75 μL of the ERα LBD was pipetted into the wells. After an incubation time of 60 min at room temperature, the fluorescence polarization (FP) was recorded at 485 nm excitation and 520 nm emission. All experiments were performed in quadruplicate. The difference in FP (ΔFP) was calculated by subtracting the FP value found at 100% displacement from the FP value found when no E2 was present under different conditions. FP-based ERα receptor affinity detection (RAD) in flow-injection analysis (FIA) mode A schematic overview of the RAD system in FIA mode is depicted in Fig. 2. In this system the flow from a superloop containing ERα LBD is continuously mixed with flow from a superloop containing E2-F. Compound samples (10 μL) are introduced into the system by flow injection into a carrier solution (100 μL/min). During optimization, the carrier solution was water. Injected compounds and ERα, added to the carrier stream via an inverted Y-type mixing union at a flow rate of 100 μL/min, were allowed to bind in a Tefzel reaction coil (25 μL). This mixture was combined with the flow of the E2-F solution of 100 μL/min. The final equilibrium between ERα, compound, and E2-F was established in a knitted 0.5 mm i.d. PTFE reaction coil (250 μL). The temperatures of both reaction coils were maintained constant at 37 °C. Fluorescence polarization (FP) detection took place directly after the second reaction coil. When the binding of E2-F is decreased due to competition from an eluting ERα ligand, depolarization occurs. This decrease in fluorescence polarization is a measure of the affinity of the ligand towards ERα. Fig. 2Schematic overview of the ERα fluorescence polarization receptor affinity detection (RAD) system in high-resolution screening (HRS) mode. Samples are injected with an autoinjector (A.I.) and are separated using a gradient reversed-phase high-performance liquid chromatography (HPLC) system controlled by P1 and P2, HPLC gradient pumps. After the column the flow is split by a flow spitter, S (split ratio 1:9). HPLC elution is monitored by UV or fluorescence detection. A makeup gradient, controlled by makeup gradient pumps P3 and P4, is introduced and eluting compounds are mixed in the first reaction coil with ERα delivered by superloop-1 (SL-1). In the second reaction coil, the probe ligand E2-F is added to the mix by superloop-2 (SL-2). ERα affinity detection is performed using a fluorescence detector (FLD). In FIA mode the samples are delivered directly by a carrier solution into the bioassay FP-based ERα receptor affinity detection coupled on-line to gradient HPLC Coupling of the homogeneous ERα FP RAD assay to HPLC was performed in a similar way to that described recently by Kool et al. [14] for a Cyt P450 affinity assay. When operating the ERα FP RAD system in HPLC mode (Fig. 2), two pumps were used to control the HPLC gradient and, directly after the HPLC column, the HPLC eluent was split into a 1:9 ratio where nine-tenths of the flow was directed to either an UV (Agilent 1100 series) or fluorescence detector (Agilent 1100 series). The choice of detector depended on the compounds to be analyzed. To compensate for increasing the organic modifier concentrations before the delivery of the HPLC eluent to the ERα FP RAD system, two pumps were used to supply a makeup flow consisting of an opposite H2O/MeOH gradient compared with the HPLC gradient in order to keep the MeOH concentration in the assay constant at 10%. One-tenth of the total makeup flow was combined with one-tenth of the HPLC eluent and this flow was directed to the ERα FP RAD at 160 μl/min. The remaining part of the flow in the ERα FP RAD was the same as described above, with the exception that the HPLC flow combined with the makeup flow replaced the carrier solution flow. For HPLC analysis, test compounds were dissolved in 50% MeOH in water. For coumestrol, a linear gradient from 75% MeOH to 99% MeOH in 7.5 min, constant for 47.5 min, and back to 75% MeOH in 1 min was applied. The total flow of the HPLC pumps was 162 μL/min and detection took place by measuring UV at 254 nm. For the mixture containing estriol, coumestrol, coumarol, E2 and zearalenone, constant elution at 55% MeOH for 40 min, followed by a linear gradient from 55% MeOH to 70% MeOH in 50 min, constant for 50 min, and back to 55% MeOH in 1 min was applied. The total flow rate of the HPLC pumps was 229 μL/min and detection took place using UV at 254 nm. For zearalenone and its cytochrome P450-generated metabolites, a linear gradient from 40% MeOH to 99% MeOH in 40 min, constant for 30 min, and back to 40% MeOH in 1 min was applied. The total flow rate of the HPLC pumps was 162 μL/min and detection took place using a fluorescence detector (λex 279 nm; λem 440 nm). All HPLC separations were carried out on a 150 × 4.6 mm i.d. Luna C18(2) column protected with a 2.0 × 5.0 mm i.d. C18 guard column (Phenomenex, Amstelveen, The Netherlands). The HPLC column was thermostated at 25 °C. Results and discussion The aim of this research was to combine a recently developed on-line FP detector cell (Kool et al. [20]) with high-resolution screening (HRS) technology to develop a sensitive screening platform for measuring the ERα affinities of individual components in mixtures of compounds without incurring interference from autofluorescence at wavelengths below 450 nm. We first synthesized a suitable tracer compound and used this tracer to set up a FP-based ERα binding assay in FP microplate reader format. The optimized assay conditions for the FP microplate reader format were transferred to an on-line bioassay format in FIA mode. In this mode we subsequently optimized the on-line assay conditions and validated them by measuring IC50 values of known estrogenic compounds in both microplate reader format and in FIA mode. We used the optimized conditions in FIA mode to couple the ERα bioassay on-line to a gradient HPLC system. In HPLC mode we measured the ERα bioaffinities of different estrogenic compounds and screened mixtures with five known estrogenic compounds. Finally, we measured the individual ERα bioaffinities of zearalenone and its metabolites generated off-line by Cyt P450 in HPLC mode. Synthesis of E2-F Synthesis of the fluorescein-labeled derivative E2-F (Fig. 1) was relatively straightforward. The synthetic route described in this paper makes use of readily available chemicals. The yields of intermediate products are relatively high except for the product of the second reaction step, in which compound 2 is converted into 3. However, the required reactants for this step are not expensive, which makes it possible to perform this step on a large scale. The last and most expensive step involving the coupling of 5-FITC to 7 still gives a yield of 75% after extraction and purification. Overall, we conclude that this synthetic protocol offers a good alternative to the synthetic scheme described in the literature [7, 19] that is used to produce a fluorescein-labeled E2 derivative. We describe a seven-step synthesis with an overall yield of 12% compared with an eight-step synthesis in the literature with a total yield of 7% [19]. To test whether the newly synthesized E2-F had affinity for the ERα, a competitive binding curve for E2-F and ERα LBD was constructed (data not shown) using the coumestrol-based microplate reader assay described by Gurer-Orhan et al. [6]. E2-F was able to displace coumestrol from the ERα binding site, resulting in an IC50 value of 28.2 nM. This value is consistent with KD values found for similar tracers, which were also in the nanomolar range [7, 19]. Therefore, we concluded that the synthesized E2-F had affinity for the ERα and could be used to further develop a FP-based microplate reader assay. FP-based microplate reader assay of ERα affinity The first step in the optimization of the FP-based microplate reader ERα affinity assay was the investigation of the influence of E2-F tracer concentration upon the FP-based microplate reader assay response of ERα-ligands. To achieve this we compared the FP signal obtained with 0% ligand displacement (no E2 present in the incubation) with the FP signal with 100% ligand displacement (33.3 μM E2 present in the incubation). We also incubated different concentrations of E2-F tracer with a fixed concentration of ERα. The difference in fluorescence polarization [ΔFP in millipolarization (mP)] was plotted against decreasing E2-F concentrations (Fig. 3). From this figure it can be seen that ΔFP increases with decreasing tracer E2-F concentrations until it reaches a maximum at 166 nM. However, it should also be noted that when the E2-F tracer concentration decreases, the error bars increase, which means that the S/N ratios become worse. For the ERα-binding assay, we preferred to use a tracer concentration which is not too high, because high tracer concentrations make it necessary to use high concentrations of test compounds as well in order to displace the tracer from the receptor, which results in a decreased assay sensitivity. However, if we use a tracer concentration which is too low, this will also result in unfavorable S/N ratios. Based upon our results and the KD values of 10.4 and 3 nM found by Ohno et al. [19] and Parket et al. [7] respectively for structurally similar fluorescein-labeled E2 derivatives, we decided to use an E2-F tracer concentration of 83.3 nM for further optimization experiments. At this concentration the ERα response was high, which makes it easier to detect the tracer–receptor complex in an on-line assay, with a low S/N ratio, and with the concentration in the same range as the KD value, which makes it possible to determine IC50 values more accurately. Fig. 3FP-based microplate reader tracer concentration optimization. Incubations in the presence of ERα ligand E2 (33.3 μM; equal to 100% tracer displacement) were compared with incubations without E2 (equal to 0% tracer displacement) at decreasing E2-F tracer concentrations The second step in the optimization process was the investigation of the influence of the detergent Tween 20 and the solvents methanol (MeOH), acetonitrile (ACN) and isopropanol (iPrOH) on assay performance using the conditions as optimized in the previous step. Experiments revealed that Tween 20 was not compatible with the ERα FP assay (data not shown). At very low concentrations (0.04%), the presence of Tween 20 in the assay caused a large decrease in ΔFP between E2-F bound to ERα (0% displacement; absence of E2) and E2-F displaced by 33.3 μM E2 (100% displacement). Although the addition of Tween 20 can give improved resolution in terms of peak width [25], we decided not to use it due to the large decrease in the difference in polarization. In Fig. 4, the effect of increasing concentrations of MeOH, ACN and iPrOH on ΔFP is shown. It can be seen from this figure that ΔFP decreases with increasing organic modifier concentrations. ΔFP decreased to almost zero for ACN at the highest concentration. For MeOH and iPrOH, however, a signal difference between the E2-F bound to ERα and displaced by E2 could still be observed at the highest modifier concentration. Oosterkamp et al. [10] have stated that denaturation of the estrogen receptor occurs upon prolonged contact with organic solvents such as MeOH and ACN. It is therefore reasonable to assume that when the organic modifier is present at higher concentrations, the denaturation process for ERα LBD increases, which results in smaller fluorescence polarization signal differences. Fig. 4FP-based microplate reader solvent optimization. Incubations in the presence of ERα ligand E2 (33.3 μM; equal to 100 % tracer displacement) were compared with incubations without E2 (equal to 0% displacement) at increasing MeOH, ACN and iPrOH concentrations at an E2-F concentration of 83.3 nM FP-based ERα receptor affinity detection (RAD) in flow-injection analysis (FIA) mode The optimized microplate reader assay conditions were transferred to the on-line ERα RAD system in FIA mode (Fig. 2). In this system, compounds introduced by a carrier solution are allowed to bind to the ERα LBD in a first reaction coil, and the E2-F tracer is introduced after this coil. The tracer compound can interact with the unbound receptor in a second reaction coil and detection takes place after the second reaction coil using FP detection. In a FIA setup, carrier solution replaces the gradient HPLC system, thereby allowing the direct injection of compounds and the optimization of on-line assay conditions before final on-line coupling to HPLC. The influence of the ERα LBD and E2-F tracer concentrations and the corresponding excitation and emission wavelengths were investigated in FIA mode in order to optimize the performance of the FP ERα RAD system. The influences of ACN and MeOH, later used in HPLC mode too, on the FP ERα RAD system performance were also tested in the FIA mode. All measurements were performed in duplicate. ERα LBD and E2-F tracer optimization Different receptor concentrations (ERα LBD) were tested in combination with different tracer concentrations (E2-F) by varying the flows of SL-1 (containing 10 nM ERα in binding buffer) and SL-2 (containing 1 μM E2-F in binding buffer). The total flow rate was kept constant by introducing a compensating superloop containing only binding buffer. Water was used as carrier solution in order to inject 10 μL 100 μM E2 in 50% MeOH samples (100% ligand displacement) in duplicate followed by 10 μL injections of 50% MeOH (0% displacement) at a flow rate of 250 μL/min. The results obtained with these experiments are depicted in Fig. 5. This figure shows that as the E2-F tracer concentration increases the FP signal increases too, until it reaches a maximum. Using the present FIA FP ERα RAD setup, we were able to determine the KD and maximum response (Rmax) values for the E2-F tracer compound (Table 1) at different ERα concentrations. The KD values found were of the same order as observed by Ohno et al. [19] and Parker et al. [7] for an analogous FP tracer compound. It can be seen that Rmax increases linearly with higher ERα concentrations, as was expected. Based upon these results, we conclude that the FP ERα RAD in FIA mode worked well, and we continued by investigating the influence of the organic modifier upon the assay performance. Fig. 5Determination of KD and maximum response (Rmax) for E2-F in FIA mode. Different concentrations of E2-F were incubated with three different ERα concentrationsTable 1KD and maximum response (Rmax) determination for E2-F in FIA mode[ERα] (nM)KD (nM)Rmax (AUC)a0.743.6 ± 12.42.17 × 1061.526.4 ± 5.04.04 × 1063.034.5 ± 5.39.38 × 106aRmax was determined by integrating the decrease in FP signal, and arbitrary units were chosen to display this decrease Organic modifier concentration Although we also investigated the influence of iPrOH upon the ERα bioaffinity assay performance in the FP-based microplate reader format, we decided to evaluate only MeOH and ACN in FIA mode based upon the fact that we planned to use only these two solvents in HPLC mode. Different concentrations of MeOH and ACN were used as the carrier solution in order to inject 10 μL 100 μM E2 ligand in 50% MeOH samples (100% ligand displacement) in duplicate followed by 10 μL injections of 50% MeOH (0 μM E2 ligand; 0% displacement) into the system. The ERα LBD concentration in the second reaction coil was 2.7 nM, the E2-F tracer concentration 110 nM and the total flow rate 300 μL/min. Increasing the concentration of MeOH decreased the S/N ratio in FIA mode (Fig. 6). A similar effect was observed with ACN. A possible explanation for this decreasing effect is the partial denaturation of the estrogen receptor due to the presence of high concentrations of organic modifier. Oosterkamp et al. [10] have described this phenomenon during the optimization of the ERα coumestrol-based RAD system when a decrease in response was observed upon the contact of the ERα with organic solvents such as MeOH and ACN. One advantage of a higher organic modifier concentration is that higher flow rates can be used in HPLC gradients, which may result in shorter analysis times and higher resolutions. Another advantage is that high concentrations of organic modifier prevent lipohilic compounds from adsorbing to the walls of the reaction coils and other tubing, which may increase resolution. Based upon our results during FP FIA setup, we concluded that both MeOH and ACN might be used in the on-line gradient HPLC system. Fig. 6Effect of MeOH and ACN reaction coil concentrations upon the assay performance in FIA mode. The S/N ratios of the E2 injections equalling 100% tracer displacement are displayed Excitation and emission wavelengths When the excitation and emission wavelengths were varied, the optimal FP signal responses in FIA mode were obtained at an excitation wavelength of 498 nm and an emission wavelength of 522 nm (data not shown). These wavelengths were therefore used to further optimize the on-line FP system in FIA mode. Competitive receptor binding curves To validate the ERα FP RAD system, competitive binding curves for five known estrogenic test compounds—DES, E2, EE2, tamoxifen and coumestrol—were constructed using the FP-based microplate reader assay, and these values were compared with the IC50 values obtained using the ERα FP setup in FIA mode. The ERα LBD concentration was 1.6 nM and the E2-F tracer concentration was 80 nM for the microplate reader assay. In FIA mode, the ERα LBD concentration in the second reaction coil was 2.7 nM and the E2-F concentration was 110 nM. All five test compounds displayed sigmoidal concentration–response curves during both the microplate reader setup and in FIA mode, and from these curves we were able to determine the IC50 values (Table 2). The values found in the two different setups were similar for each individual compound and in agreement with values obtained from the literature [7, 26]. We thus demonstrated that the FP-based microplate reader assay and the ERα FP setup in FIA mode could be employed to determine the IC50 values of a wide range of typical ERα-binding ligands accurately. The next step in the process of developing the HRS platform was to couple the FIA ERα FP system to a gradient HPLC system on-line. Table 2IC50 values determined in FIA mode and in microplate reader assay formatCompoundFIA mode IC50 (M ± SEM; n = 3)Microplate setup IC50 (M ± SEM; n = 3)Literature data IC50 (M)DES6.9 ± 0.3 × 10−96.1 ± 0.6 × 10−93.5 × 10−9 aTamoxifen1.2 ± 0.1 × 10−71.3 ± 0.4 × 10−71.9 × 10−7 aCoumestrol1.8 ± 0.2 × 10−72.5 ± 0.3 × 10−73.6 × 10−7 bE26.7 ± 0.8 × 10−93.3 ± 0.3 × 10−95.6 × 10−9 aEE21.6 ± 0.2 × 10−93.5 ± 0.8 × 10−92.3 × 10−9 bIC50 values of five estrogenic compounds towards the ERα, as measured with the RAD system in FIA mode and with the microplate reader assay format. Data from literature a[7], b[26] are also shown. DES, diethylstilbestrol; E2, 17β-estradiol; EE2, 17α-ethinyl estradiol FP-based ERα receptor affinity detection coupled on-line to HPLC Following assay optimization in the microplate reader and FIA modes, the ERα FP RAD assay was coupled on-line to gradient HPLC. In this HRS setup the flow eluting from an HPLC column was coupled directly to the FP RAD system, which enables compound separation prior to the screening of individually eluting compounds for their ERα affinities. This allowed us to screen for compounds with ERα affinities in mixtures. To test the performance of the on-line HRS FP ERα system, different concentrations of the phytoestrogen coumestrol were injected first. A negative peak in the ERα bioaffinity trace was observed at a retention time of 40 min, which is the exact time when coumestrol eluted from the HPLC column (data not shown). As expected, the negative bioaffinity peaks became larger upon injections of higher concentrations of coumestrol. The highest negative peaks were observed when all of the E2-F was displaced by coumestrol. Higher concentrations of coumestrol only caused more tailing of the bioaffinity peaks. These measurements illustrate that the ERα bioaffinity of the phytoestrogen coumestrol could be measured sensitively and efficiently with the new E2-F based HRS ERα FP system, which is not possible due to the occurrence of autofluorescence using the coumestrol-based ERα HRS system developed by Oosterkamp et al. [10]. Representative superimposed chromatograms of different concentrations of a mixture of five known estrogenic compounds—coumestrol, coumarol, estriol, 17β-estradiol and zearalenone—are depicted in Fig. 7. Although some baseline drift in time is observed over two hours, it is quite possible to detect the ERα bioaffinities of all five individual peaks in the mixture. The explanation for the baseline drift is most likely that the post-column makeup HPLC gradient does not totally compensate for the effect of variations in the organic modifier concentration. The FP ERα RAD system is sensitive to changes in the organic modifier concentration, as shown during FIA optimization, and this can lead to baseline drifts. Fig. 7ERα FP RAD trace of a mixture of five estrogenic compounds injected into the ERα FP RAD HPLC system (injected compounds are as follows: estriol (1) [3.75 nmol; 49.5 min], coumestrol (2) [3.75 nmol; 53.7 min], coumarol (3) [37.5 nmol; 80.4 min], 17β-estradiol (4) [3.75 nmol; 102.8 min] and zearalenone (5) [12.5 nmol; 112.5 min] To illustrate another application of the on-line HRS ERα FP system, metabolites of the phytoestrogen zearalenone, which were anticipated to show affinity for the ERα, were analyzed in the present FP-based HRS ERα system. For this purpose, zearalenone was metabolized by phenobarbital (PB)-induced rat liver microsomes in an off-line fashion, followed by analysis with the FP-based HRS system. The results are depicted in Fig. 8. The UV trace of the t = 0 min incubation shows that zearalenone (Z) elutes at 51.4 min. The corresponding bioaffinity trace shows that zearalenone has affinity for ERα. The FLD trace of the t = 120 min incubation shows that zearalenone is metabolized by rat liver microsomes into at least three major metabolites (M1: 33.5, M2: 39.5 and M3: 43.2 min). The corresponding bioaffinity trace demonstrates that all three major metabolites (M1–3) have affinity for ERα. LC/MS measurements were performed to identify the three major metabolites. M1 had a molecular ion at an m/z value of 333, which likely corresponds to a monohydroxylated zearalenone metabolite. M2 had a molecular ion at an m/z value of 317, which is the exact same m/z value as that of zearalenone itself. This can be explained by the relocation of the double carbon–carbon bond next to the aromatic ring. However, more research must be performed to confirm this. M3 had a molecular ion at an m/z value of 319, which corresponds well to the m/z value of the zearalenone metabolite zearalenol described in the literature [27]. The metabolism of zearalenone in vitro by rat liver microsomes has been described very recently by Pfeiffer et al. [28], and they confirm the findings of M1 and M3. The present Cyt P450 metabolism experiment illustrates that it is possible to screen metabolic mixtures of phytoestrogenic compounds for ERα affinity using the present FP-based HRS ERα setup. However, no further structural elucidation of the zearalenone metabolites has been performed, because the goal of the present experiment was only to show that the on-line FP HRS ERα assay can be used to screen for parent compounds and metabolites of phytoestrogens with ERα affinity. Fig. 8a–ba HPLC FLD chromatograms of zearalenone (Z) incubated for 0 and 120 min with PB-induced rat liver microsomes. M1, M2 and M3 are metabolites formed during the 120-min incubation period. b The corresponding ERα FP affinity traces. The structure of zearalenone is depicted in the figure We conclude that we succeeded in developing, for the first time, an on-line FP-based HRS platform to screen for ERα affinity using the novel on-line FP detector cell recently described in a FIA mode by Kool et al. [20]. The sensitivity of the FP-based HRS platform is comparable to that of the coumestrol-based HRS platform, and no interference from fluorescence was observed at wavelengths below 450 nm. The novel on-line FP detector cell can in principle be applied to other HPLC-based bioaffinity assays which require FP detection. The present HRS ERα FP system offers a novel technology for investigating the metabolic profiles of drugs and other compounds such as phytoestrogens which cannot be measured by the coumestrol-based HRS system due to the occurrence of autofluorescence from the test compounds.

Obes_Surg-4-1-2226018

Psychosocial Functioning, Personality, and Body Image Following Vertical Banded Gastroplasty

Background In addition to increased risks of morbidity and mortality, extreme obesity is substantially associated with psychosocial problems. Therefore, the ultimate goal of bariatric surgery should not only be reducing weight and counteracting comorbid conditions but also improving psychosocial functioning. In addition to being an important goal of bariatric surgery, enhanced psychosocial functioning may motivate patients to adhere to adequate health behavior to maintain the surgically established weight loss. Introduction Obesity is a major public health concern. Its prevalence is increasing globally [1]. In addition to an increased risk of morbidity and mortality caused by medical conditions [2], extreme obesity is associated with debilitating psychological and social consequences especially in patients seeking surgery [3–6]. Psychological consequences of extreme obesity are, among others, anxiety, depression, low self-esteem, and negative body image [5–10]. Socially, the extremely obese have to deal with prejudice, discrimination, and social isolation [11]. Because only surgical treatment results in long-term weight reduction and the improvement or cure of comorbidities, including reduction of psychopathology [3–6], the number of procedures performed has more than doubled within a few years [12]. However, studies show great variation in outcome. In contrast to research showing large and long-lasting improvements, other studies suggest that postoperative improvements lag behind functioning of normative groups or show a decline over the years [4, 5]. Enhanced psychosocial functioning is an important goal of bariatric surgery. It may motivate patients to adhere to adequate health behavior to maintain the surgically established weight loss [13]. In addition to medical–somatic and surgical–technical factors, success of bariatric surgery is especially influenced by the extent of successful adjustment of eating patterns, which, for a substantial part, can be attributed to psychological factors. Therefore, success after bariatric surgery should not only reflect weight loss and improvement or cure of comorbid conditions but also improvements in eating behavior, psychosocial variables, and quality of life [14]. However, in contrast to physical changes, psychosocial changes are not studied as systematically or diligently yet [15]. To facilitate the identification of the psychological variables that are related to success and the development of treatment interventions to enhance adjustment and success, a better understanding of patients’ psychosocial functioning after bariatric surgery is needed. Materials and Methods The present study was designed to increase our understanding of patients’ changes in psychosocial functioning, personality, and body image during the first 2 years after vertical banded gastroplasty (VBG). The main research questions were: what specific improvements can be achieved, is postoperative functioning noticeable different from functioning in relevant normative groups, and do all patients profit? These questions were examined based on data collection preoperatively and 6, 12, and 24 months after VBG. Procedures The restrictive operation technique used in our hospital is hand-assisted laparoscopic vertical banded gastroplasty, with a complete division between the vertical staple lines. VBG is a relative simple, safe, and cost-effective intervention with fewer side effects than other procedures [16]. Not withstanding this, mixed long-term results, such as weight regain and dietary problems, have been reported [17]. Contrary to the situation in the USA, in The Netherlands, the numbers of VBGs that have been performed in the last 5 years is rather stable, despite the fact that the number of adjustable gastric banding procedures is showing an enormous increase. Materials Earlier studies of psychosocial factors in the surgical treatment of extreme obesity have not provided consistent findings, which may, in part, be caused by the employment of a wide variety of non-standardized assessment procedures of questionable reliability and validity [11]. In the present study, we assessed preoperative as well as postoperative psychosocial functioning, personality, and body image using a semi-structured interview and three standardized questionnaires. As interview-based psychological studies may be questionable, we will report here only on the questionnaire data. Preoperatively as well as on three follow-up assessment points, patients filled in the Symptom Checklist-90 (SCL-90) [18], the Dutch Personality Questionnaire (NVP) [19], and the Body Attitude Test (BAT) [20]. The SCL-90 is a multidimensional psychopathology indicator with eight subscales and one summarized score (psychoneuroticism); its psychometric qualities are satisfactory to good [21]. In addition to neuroticism, the NPV measures six personality characteristics; its psychometric qualities are also satisfactory to good [22]. The BAT measures subjective body experience and the attitude towards one’s own body; it has three subscales and one summarized total score, is commonly used in Europe, and is a reliable and valid questionnaire [20]. Patients In the context of standard preoperative evaluation, 131 patients who underwent VBG in our hospital between January 2001 and October 2004 were psychologically assessed. In addition to the inclusion criteria determined by IFSO [23], we required 10% preoperative weight loss with a minimum of 12 kg. From these 131 patients, 104 (79.4%) could be included in our study; others did not respond to our repeated requests for follow-up or sending back questionnaires. The studied patient group consisted of 91 (87.5%) female and 13 (12.5%) male patients. Preoperatively, they had a mean age of 38.4 ± 8.3 years, an average body mass index (BMI) of 45.4 ± 5.1, and a mean percentage excess weight of 104.5 ± 22.1%. Statistical Analyses Statistical analyses were performed using the SPSS 14.0-packet (SPSS for Windows, Rel. 14.0. 2005, SPSS Inc., Chicago). To study changes over time in weight, psychosocial functioning, personality, and body image, mixed between-within subjects analyses of variance for repeated measures were performed (ANOVA; time as within-subjects factor, gender as between-subjects factor). To identify waning of changes, Bonferroni post hoc equations were used. To compare patients’ questionnaire scores to normative data, we used one-sample t tests. To identify heterogeneity in postoperative psychosocial functioning, descriptive statistics were used using summarized scales whenever possible for reasons of clarity. In reporting excess weight (EW) and excess weight loss (EWL), we followed the guidelines of Deitel and Greenstein [24]. Results Weight Loss Over time, using ANOVA, substantial and significant changes in mean BMI, EW, and EWL were found. From preoperative status to 2 years after surgery, BMI and EW significantly (P ≤ 0.001) decreased 13.5 points and 58.6%, respectively, which represent a large size (partial η2 = 0.83) [25]. After 2 years, using the criterion that an operation can be conceived of as successful when EWL ≥50% [26], 61% of the patients had a successful operation. Preoperatively, 91.3% of the patients were extremely obese; in contrast, 2 years after surgery, only 12% was extremely obese. Psychosocial Functioning Our earlier literature review suggests that psychopathology is related to extreme obesity, manifesting itself mostly as depression or anxiety [11]. In assessing psychosocial functioning, we used the SCL-90 to assess short-term psychosocial functioning [18] and the NPV neuroticism scale to measure long-term neuroticism [19]. With respect to all aspects, except for hostility, significant changes over time were found (for most of the comparisons, P ≤ 0.001; for neuroticism, P ≤ 0.01; for sleeping problems, P ≤ 0.05). For the majority of alterations, large effect sizes were found [25]. Post hoc analyses showed that compared to the preoperative status, for most aspects of psychosocial functioning, scores were significantly lower on all follow-up assessment points, reflecting better psychosocial functioning. However, depressive symptoms and neuroticism were only significantly lower 6 months and 1 year after surgery; sleeping problems were lower only 6 months after surgery. For most aspects of psychosocial functioning, there was only a significant decrease in scores from preoperative status to 6 months postoperatively, however, not between 6 months and 1 year or between 1 and 2 years postoperatively (Table 1). Table 1Postoperative changes in psychosocial functioning (mean ± SD)SCL-90Preoperative6 months1 year2 yearsPartial η2Anxiety14.3 ± 5.011.7 ± 2.4***12.1 ± 3.912.6 ± 5.6.23Phobic complaints9.3 ± 3.67.4 ± 1.1***7.5 ± 1.67.9 ± 3.1.20Depression24.8 ± 8.620.8 ± 6.3***20.9 ± 7.123.1 ± 11.3.18Somatic complaints21.2 ± 7.916.2 ± 3.5***17.0 ± 5.217.9 ± 6.7.29Obsessive15.0 ± 5.011.6 ± 2.7***12.0 ± 3.112.7 ± 5.1.28Sensitivity28.7 ± 10.423.0 ± 6.4***22.8 ± 6.124.2 ± 10.8.25Hostility7.3 ± 1.77.2 ± 2.07.1 ± 1.57.3 ± 2.4.06Sleeping problems5.7 ± 2.94.6 ± 2.3*4.9 ± 2.55.5 ± 3.1.09Psychoneuroticism139.5 ± 41.0113.0 ± 22.7***114.8 ± 26.4122.8 ± 45.2.30N93939393Neuroticism (NPV)11.98 ± 8.98.5 ± 7.4**8.0 ± 6.69.3 ± 8.8.15N94949494Because of listwise exclusion, n = 93 (SCL-90) or 94 (NPV) on all moments; Obsessive: obsessive compulsive behavior; Sensitivity: mistrust and interpersonal sensitivity.***P < 0.001**P < 0.01*P < 0.05 Furthermore, we compared patients’ mean scores on psychosocial functioning to corresponding scores obtained in relevant normative groups, reflecting the general population [18, 19]. Compared to those normative groups, mean preoperative scores for psychosocial functioning were significantly higher, except for hostility. However, with respect to preoperative neuroticism, no significant difference was found. Postoperatively, there was a mixed picture. In spite of these inconsistencies, the overall picture revealed that on most subscales and most follow-up assessment points, patients’ mean scores were not significantly different from the corresponding scores obtained in the normative groups. Two years after surgery, patients’ mean scores on measures of sleeping problems and somatic complaints were again significantly higher than the normative group’s scores. As to neuroticism, patients’ postoperative mean scores were significantly lower than the normative group’s scores on all follow-up assessment points. Despite these differences, all postoperative patients’ scores fell in the below mean, mean, or above mean categories of the normative groups’ scores. Personality Because personality substantially influences health behavior, personality characteristics may be relevant for adjustment to surgery [27]. According to earlier literature reviews, extremely obese individuals can be described as, among others, immature persons having a poor impulse control and a low self-esteem [28]. With respect to postoperative personality change, mixed results have been reported. For instance, some studies suggest that patients still have more pathology than comparison groups [13], whereas other research suggests that personality remains largely unchanged [3]. In assessing personality with the NPV [19], over time, we only found, with a large effect size [25], a significant (P ≤ 0.01) change in self-esteem. Post hoc analyses revealed that compared to the preoperative status, self-esteem was significantly higher 1 year postoperatively. Six months after surgery, there was a tendency to significance (P = 0.06). Two years postoperatively, however, no significant difference was found. Also, no significant differences were found between follow-up assessment points (Table 2). Table 2Postoperative changes in personality characteristics (mean ± SD)NPV subscalesPreoperative6 months1 year2 yearsPartial η2Social anxiety8.9 ± 7.96.8 ± 6.57.0 ± 6.57.5 ± 7.1.0.07Rigidity24.4 ± 7.324.0 ± 7.924.2 ± 7.423.7 ± 7.80.03Hostility14.0 ± 8.514.1 ± 8.614.0 ± 8.915.3 ± 9.30.05.Egoism9.7 ± 4.710.6 ± 5.19.9 ± 4.810.4 ± 5.00.04Dominance13.9 ± 6.415.3 ± 7.515.3 ± 7.215.0 ± 7.30.06Self-esteem27.9 ± 6.029.7 ± 5.329.9 ± 5.5*28.6 ± 6.70.14N94949494Because of listwise exclusion, n = 94 on all moments.*P < 0.01 We did not find many significant personality changes, but compared to normative data [19], we did find some significant differences. Compared to a relevant normative group, reflecting the general population, patients’ mean preoperative scores on social anxiety, dominance, and self-esteem were not significantly different. However, they were after surgery, albeit not on all follow-up assessment points and not always for both sexes. Postoperatively, patients’ scores on social anxiety were lower, whereas their scores on dominance and self-esteem were higher than the corresponding scores obtained in the normative group. On scales of Hostility and Egoism, preoperatively, patients had significant lower mean scores; postoperatively, this was only the case for some small subgroups. On the Rigidity measure, patients had significantly lower mean scores on all measurement moments preoperatively as well as postoperatively. All patients’ mean scores fell in the below mean, mean, or above mean categories of the normative group’s scores. Body Image Some studies suggest that especially for female patients, the desire to improve their physical appearance and avoidance of embarrassment are the most common motivators for bariatric surgery [29]. Also, improved body image would be one of the main reasons for post-surgical psychological improvement, better social integration, and enhanced quality of life [30]. Weight loss after bariatric surgery leads to marked improvements in body image and attractiveness and to less shame. However, with time, some patients still feel overweight, are discontent with the increasing skin folds [13], or still may have more concern with their body shape and size than controls [31]. In our earlier study, we found that preoperatively, patients reported significantly higher body dissatisfaction and negative body image than general population samples [28]. In the present study, using the BAT [20], over time, we found significant (P ≤ 0.001) changes in negative appreciation of body size, lack of familiarity with one’s own body, general body dissatisfaction, and the summarized BAT score, reflecting improved body image and large effect sizes [25]. Post hoc analyses showed that compared to the preoperative status, scores were significantly (P ≤ 0.001) lower on all three follow-up assessment points. We found significant decreases between preoperative status and 6 months postoperatively, however, not between 6 months and 1 year or between 1 and 2 years after surgery (Table 3). Also, in contrast to the preoperative situation, on all three follow-up assessment points, body image was significantly correlated to BMI, reflecting a more positive body image when BMI was lower (r varied from 0.37, 6 months postoperatively, to 0.29, 1 year postoperatively, and 0.31, 2 years after surgery). Table 3Postoperative changes in body image (mean ± SD) Preoperative6 months1 year2 yearsPartial η2Negative appreciation of body size26.2 ± 5.814.9 ± 7.1*14.4 ± 8.214.2 ± 8.20.59Lack of familiarity with one’s own body16.5 ± 7.27.5 ± 4.7*7.1 ± 4.87.5 ± 6.10.52General body dissatisfaction15.2 ± 3.68.1 ± 4.6*8.0 ± 4.68.3 ± 5.00.61Total BAT score64.0 ± 15.436.9 ± 15.6*35.8 ± 16.235.9 ± 17.40.63N97979797Because of listwise exclusion, n = 97 on all moments.*P < 0.001 Although we found significant improvements in body image over time, on all moments, preoperatively as well as postoperatively, and on all scales, patients’ mean scores were significantly (P ≤ 0.001 or P ≤ 0.01) higher than those of (female students) normative groups [20, 32]. Heterogeneity in Postoperative Psychosocial Functioning Most studies report broad psychosocial improvements after bariatric surgery. However, a significant minority of extremely obese patients do not benefit from surgery [4]. Some patients may even have difficulties in adapting psychosocially to the various consequences of bariatric surgery [13]. In the present study, in addition to a large variation in EWL, we found substantial variance with respect to changes in psychosocial functioning. Especially with respect to psychoneuroticism and neuroticism, most patients reported postoperative improvements; however, this was not the case for all patients. For instance, in the case of psychoneuroticism, 77 out of 99 patients (77.8%) reported improvement after 6 months; in contrast, 21 patients (21.2%) had higher scores, and one patient did not change. After 24 months, approximately two thirds of the patient group (64.7%) still had better scores on psychoneuroticism, whereas one third (31.4%) showed signs of an increase in psychoneuroticism. In the case of personality characteristics, a mixed picture was found; however, in general, there were only small differences between patients reporting positive or negative changes. Regarding body image, almost all patients reported improvements, and these were quite stable over time. Discussion After vertical banded gastroplasty, in addition to substantial and significant weight loss, patients also changed for the better in body image and most domains of psychosocial functioning. With respect to personality, only a significant improvement in self-esteem was found. Unfortunately, there was substantial variance in improvements, and some improvements waned over time. Most of our findings are in line with other studies, with respect to weight loss [33], as well as regarding improvements in psychosocial functioning and body image [7, 8]. The fact that we could not demonstrate many significant changes in patients’ personality is in contrast to the results of some studies [34], but is consistent with the suggestion that there is no obese personality [35]; furthermore, because of the stability of personality, these findings might be expected. The impact of bariatric surgery seems to be more in the areas of symptoms of psychopathology. Just like others, we found a decline of some psychosocial improvements over the years [13]. Various explanations have been suggested for waning psychosocial improvements, for example, stabilization or rising of patients’ weight, disappointment that life did not improve dramatically after substantial loss of weight, or difficulties in adapting psychologically to the consequences of bariatric surgery [13]. Also, our results concerning patients’ postoperative heterogeneity are in accordance with earlier findings [36]. These outcomes underscore the suggestion that vertical banded gastroplasty, just like other methods of bariatric surgery, is not equally effective for each patient. Although our study has some assets, such as the employment of standardized, reliable questionnaires and the focus on psychosocial functioning in a wide variety of domains of life, some weaker points could also be indicated. For instance, we were not able to retain all patients of our preoperative sample. This problem is also identified by others who suggest that patients’ participation in psychological follow-up is much lower than in follow-up visits for other specialists of the bariatric team [37]. Although we found mixed results, the overall psychosocial improvements provide additional justification for vertical banded gastroplasty as a surgical treatment option for extreme obesity. However, as some improvements wane over time and not all patients profit in the same way, surgery alone may not be sufficient to sustain success. Consequently, additional behavioral treatment may be necessary.

Osteoporos_Int-3-1-1820757

Links between cardiovascular disease and osteoporosis in postmenopausal women: serum lipids or atherosclerosis per se?

Introduction and hypothesis Epidemiological observations suggest links between osteoporosis and risk of acute cardiovascular events and vice versa. Whether the two clinical conditions are linked by common pathogenic factors or atherosclerosis per se remains incompletely understood. We investigated whether serum lipids and polymorphism in the ApoE gene modifying serum lipids could be a biological linkage. Introduction Osteoporotic fractures and acute cardiovascular events remain the predominant contributors to morbidity and mortality among the elderly. Postmenopausal women with osteoporosis are at increased risk for acute cardiovascular events independent of their age and cardiovascular risk profile, and the increase in risk is proportional to the severity of osteoporosis at the time of the diagnosis [1]. Bone mineral density (BMD) at the hip is inversely correlated with the severity of aorta calcification (AC) and hence low hip BMD can be a surrogate marker of the atherosclerotic burden in elderly women [2]. Large cohort studies demonstrate that the rate of bone loss in metacarpals, proximal femur, and calcaneus is proportional to the progression of AC [3–5]. Finally, advanced AC is associated with increased risk of osteoporotic fractures [5, 6]. Collectively, numerous epidemiological observations document an overlap between the pathogenesis of the two diseases [7, 8]. An attractive concept is that cardiovascular disease and osteoporosis are linked by a common denominator acting in parallel on both vascular and bone cells [9, 10]. Plausible candidates of such linking factors are serum lipids, which in addition to their established role in atherogenesis, were also shown to act on osteoblasts [11–13] and osteoclasts [14] under experimental conditions. Although some smaller cross-sectional studies found associations between lipid profile and BMD in humans [15–17], neither large population-based studies (e.g., NHANES III) nor prospective studies support these initial observations [18, 19]. A possible explanation the discrepancies could be that the true driver of the association is obstructive vascular disease, meaning that the association of lipids to bone is not detectable before the severity of intraluminal lesions reaches a critical grade when blood supply to the anatomical region is considerably hampered. Until now there have been no epidemiological studies that investigated associations of lipids and lipoprotein components with bone independent of a direct surrogate of the atherosclerotic burden. The aim of the present study was to test this hypothesis and investigate associations of a wide array of serum lipids and lipoproteins as well as of a genetic determinant of lipid profile with different surrogate measures of osteoporosis (BMD, fractures) and cardiovascular disease (AC, acute events) in 1176 Danish postmenopausal women providing full-set of data for such analyses. Subjects and methods Study population The study population consisted of 1176 generally healthy postmenopausal women participating in the PERF study. Details on recruiting have been described elsewhere [20]. This subpopulation was selected based on those whose BMD and body composition were measured by the same DXA scanner. The population characteristics were not statistically significant from the total PERF population indicating that the results reported herein are generally applicable to elderly Caucasian women 60–85 years old. None of the women received any approved antiresorptive treatment or any other drugs with known effect on bone mass for more than 1 year. Forty-two patients reporting ongoing treatment with lipid-lowering drugs was excluded when analyzing associations of allelic variation in the ApoE gene with lipid profile. All women gave written their informed consent to participation and the study was carried out in compliance with the Helsinki Declaration II and the European Standards for Good Clinical Practice. The study protocol was approved by the local ethical committee. Demographic characteristics, risk factors, and clinical events Body weight and height were measured to the closest 0.1 kg and 0.1 cm, respectively, in women wearing light indoor clothing and no shoes. Body mass index (BMI) was calculated as body weight (in kg) divided by height squared (in m). Information about age, years since menopause, smoking habits, regular alcohol and coffee consumption, weekly exercise, prevalent diabetes and cardiovascular diseases (stroke, coronary events, intermittent claudication, unstable angina) were collected during personal interview using a questionnaire [20]. Bone mineral density and fat mass measurement Bone mineral density (BMD) of the lumbar spine L1-4 and total hip was measured by dual energy X-ray absorptiometry (DEXA) using a QDR-2000 scanner (Hologic Inc., Waltham, MA, USA). Daily phantom scans were performed each morning for proper quality control. Body composition also was measured by the same whole-body scanner. The two components measured were total body fat mass (TFM) in kilograms and total lean mass in kilograms [21]. Total body fat mass was divided into two depots: central fat mass (CFM, subcutaneous and visceral fat mass of the trunk) and peripheral fat mass (PFM, subcutaneous fat mass of the legs and arms). Body fat distribution was expressed by the CFM/PFM ratio, analogous to the well-known waist-to-hip ratio [22]. Fracture diagnosis Lateral X-rays of the thoracic and lumbar spine were performed using standard X-ray equipment. Vertebral deformities from T4 to L4 were assessed by digital measurements of vertebral deformations using the Image Pro Image Analyzer software (version 4.5 for windows, Media Cybernetics Inc., Silver Spring, MD, USA). The ratio of the anterior and posterior heights of each vertebral body was determined and a difference between the anterior and posterior edges exceeding 20% was considered as a radiographic vertebral fracture. Information on prevalent non-vertebral fractures (wrist, hip, humeral fracture, rib, ankle, and foot) was collected during personal interview and later verified by X-rays or hospital discharge summaries. Grading of aorta calcification AC was assessed on lateral radiographs. Briefly, calcified deposits in the lumbar aorta adjacent to each lumbar vertebra (L1–L4) were assessed separately for the anterior and posterior wall of the aorta using the midpoint of the inter-vertebral space as the boundaries. Each wall of each segment was graded for the presence of calcified deposits with a score from 0 to 3 (0: no deposits, 1: less than one-third of the aortic wall, 2: one-third to two-thirds of the aortic wall. 3: more than two-thirds of the aortic wall covered with calcified deposits). The sum of the scores of individual aortic segments both for the anterior and posterior walls, termed as anterior-posterior severity score and was used to describe the overall severity of AC in the lumbar aorta. Maximum score possibly given was 4×2×3=24. The same investigator, who was blinded for all other results of the individual participants, carried out the evaluations. Intra-rater correlations between repeated measurements were in the range of r=0.92–98 (n=50). Laboratory parameters Blood samples were collected in the morning after fasting overnight (>12 h). Serum total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined by enzymatic assay using a Vitros-250 (Johnson & Johnson, Taastrup, Denmark). LDL cholesterol was calculated by Friedewald formula (LDL−C = TC−HDL−C−0.45 × TG). Lipoprotein(a), ApoA1, and ApoB were measured by the Cobas Mira (Hoffman-La Roche, Mannheim, Germany) automatic blood analyzer. Genotyping DNA was isolated from peripheral blood samples. ApoE epsilon 2, epsilon 3 and epsilon 4 alleles were determined by enzymatic amplification of genomic DNA followed by restriction enzyme treatment of the amplified products (Ossendorf and Prellwitz, Qiangen News 2000, issue1, 11–13). Briefly, amplified products were treated with AflII and HaeII, respectively, and subjected to electrophoretic separation in agarose gels. The sequences of the amplification primers were 5’-ACTGACCCCGGTGGCGGAGGAGACGCGTGC-3’ and 5’-TGTTCCACCAGGGGCCCCAGGCGCTCGCGG-3’. One of these primers was deliberately designed with a mismatch (underlined) to create a non-polymorphic AflIII site permitting assessment of the efficiency of the digestion process. Statistical analysis Results are presented as mean ± SD, unless otherwise indicated. Confounders of hip BMD and AC were identified by univariate correlation analysis. We classified ApoE genotypes into two categories marked by absence or presence of the ɛ4 allele. General linear models (GLM) tested the differences in selected dependent variables after adjustment for possible confounders. Similar approach was used to identify factors showing differences between women with or without fractures. Logistic regression models were established to assess the age-dependent and age-independent relative risk of wrist, hip, or vertebral fractures in women with advanced aorta calcification or cardiovascular disease. Statistical analyses were carried out using the SPSS data analysis software (version 12, SPSS Inc., Chicago, IL). All statistical tests were two-tailed and differences were considered significant if the p-value was less than 0.05. Results Demographic characteristics Demographic characteristics of the total population are shown in Table 1. Less than one fifth of the population with an average age of 69.3 years had one or more manifestations of osteoporosis in form of a low BMD T-score equal or below −2.5 and/or a fracture. In terms of cardiovascular status, the mean severity score of AC was 3.0 ± 3.8 and the number of women with history of an acute cardiovascular event was 50 (4.3%). Table 1Demographic characteristics in the study populationCharacteristics and equivalent valuesNo. of women1176Age (years)69.3 ± 6.4Age at menopause (years)48.7 ± 5.0BMI (kg/m2)26.4 ± 4.4Current smoking (%)24.6%Regular intake of alcohol (%)55.2%Serum total cholesterol (mmol/l)6.3 ± 1.0Serum triglyciders (mmol/l)1.4 ± 0.7Serum HDL (mmol/l)1.7 ± 0.4Serum LDL (mmol/l)4.0 ± 0.9Serum ApoA1 (mmol/l)203.9 ± 37.1Serum ApoB (mmol/l)120.3 ± 24.5Serum Lp(a) (mmol/l)31.9 ± 34.6ApoE ɛ4 allele carrier367, 31.2%Total hip BMD (g/cm2)0.80 ± 0.1Lumbar spine BMD (g/cm2)0.92 ± 0.2Hip BMD T-score ≤−2.5 (no. of women,%)147, 12.5%Spine BMD T-score ≤−2.5 (no. of women,%)179, 15.2%Vertebral fracture (no. of women, %)204, 17.6%Hip fracture (no. of women,%)16, 1.4%Aorta calcification3.0 ± 3.8Cardiovascular disease (no. of women,%)50, 4.3%Data shown are mean±SD. The ApoE genotype frequencies were 0.9% (n = 11) for ApoE ɛ2-2, 12.7% (n = 149) for ApoE ɛ2-3, 2.5% (n = 29) for ApoE ɛ2–4, 55.2% (n = 649) for ApoE ɛ3–3, 26.6% (n = 313) for ApoE ɛ3–4, and 2.1% (n = 25) for ApoE ɛ4–4, respectively. When classifying ApoE genotypes into absence of ApoE ɛ4 allele and presence of ApoE ɛ4 allele, 31.2% (n = 367) of women were ApoE ɛ4 allele carriers. These genotype proportions did not deviate significantly with those expected under conditions of Hardy-Weinberg equilibrium (X2 = 4.55; df = 3; p = 0.21), and they were similar to those previously reported for Danish populations [22, 23]. Correlates of hip and spine BMD and AC In univariate analyses, summarized in Table 2, AC showed direct correlation with age, CFM/PFM ratio, smoking, treated hypertension, and inverse association with physical exercise, regular alcohol consumption, BMI, and TFM%. On the other hand, BMI, TFM% and CFM/PFM ratio were significant direct, whereas age, years since menopause, and smoking were inverse correlates of both spine and hip BMD. Treated hypertension was significantly associated with spine BMD, whereas the severity score of AC was a significant inverse correlate of hip BMD (p < 0.05). Table 2Independent correlates of aorta calcification, hip BMD, and spine BMD (multiple regression models) Aorta CalcificationHip BMDSpine BMDAge0.35 (p < 0.001)−0.23 (p < 0.001)−0.03 (p = 0.45)YSM−0.06 (p = 0.16)−0.06 (p = 0.10)−0.08 (p = 0.04)BMI−0.09 (p = 0.07)0.48 (p < 0.001)0.45 (p < 0.001)TFM%−0.04 (p = 0.36)−0.10 (p = 0.04)−0.21 (p < 0.001)CFM/PFM ratio0.08 (p = 0.02)−0.004 (p = 0.85)0.05 (p = 0.21)Smoking0.19 (p < 0.001)−0.05 (p = 0.10)−0.01 (p = 0.66)Exercise−0.03 (p = 0.25)0.04 (p = 0.17)0.04 (p = 0.21)Treated hypertension0.08 (p = 0.006)0.006 (p = 0.81)0.05 (p = 0.09)Alcohol consumption−0.08 (p = 0.007)0.06 (p = 0.04)0.07 (p = 0.02)Apo e4−0.009 (p = 0.77)0.01 (p = 0.76)−0.03 (p = 0.22)Triglyceride0.02 (p = 0.63)0.03 (p = 0.43)0.04 (p = 0.32)HDL-C0.05 (p = 0.48)−0.06 (p = 0.29)0.02 (p = 0.74)LDL-CExcl.0.02 (p = 0.73)−0.10 (p = 0.10)Total cholesterol0.05 (p = 0.43)Excl.Excl.ApoA1−0.10 (p = 0.04)0.05 (p = 0.36)0.05 (p = 0.39)ApoB0.10 (p = 0.14)−0.03 (p = 0.60)0.10 (p = 0.85)Aorta Calcification−0.09 (p = 0.002)0.04 (p = 0.20)R = 0.44, SEE = 3.32 P < 0.001R = 0.55, SEE = 0.09 P < 0.001R = 0.34, SEE = 0.13 P < 0.001Excl.: excluded from the model due to strong co-linearity. The independent role of the ApoE ɛ 4 allele Results of comparing lipids and lipoproteins between women with or without the ApoE ɛ4 allele are summarized in Table 3. After adjustment for confounders, women with an ApoE ɛ4 allele had significantly higher serum total cholesterol, triglycerides, LDL-C, and ApoB levels than women without this allele. Furthermore, presence of the ApoE ɛ4 allele was associated with significantly lower HDL-C. In contrast, the differences in serum ApoA1 and Lp(a) did not reach statistical significance (Table 3). Finally, there were no significant differences in adjusted spine or hip BMD or the severity of AC (p > 0.05). Table 3Lipid and bone profile in women with or without an ApoE epsilon 4 allele Absence of ApoE epsilon 4 allele (n = 809)Presence of ApoE epsilon 4 allele (n = 367)P valueSerum total cholersterol* (mmol/l)6.24 ± 0.036.51 ± 0.05<0.001Serum triglycides* (mmol/l)1.36 ± 0.021.48 ± 0.030.005Serum HDL* (mmol/l)1.72 ± 0.011.65 ± 0.020.006Serum LDL* (mmol/l)3.91 ± 0.034.19 ± 0.05<0.001Serum ApoA1* (mmol/l)205.8 ± 1.3201.3 ± 2.00.06Serum ApoB* (mmol/l)117.3 ± 0.8127.2 ± 1.3<0.001Serum Lp(a)* (mmol/l)31.9 ± 1.329.2 ± 2.00.24Total hip BMD (g/cm2)*0.803 ± 0.0040.804 ± 0.0050.89Spine BMD (g/cm2)*0.924 ± 0.0050.915 ± 0.0080.32Hip fracture1.7%0.5%0.17Vertebral fractures16.4%19.3%0.24Severity score of AC**2.81 ± 0.142.97 ± 0.210.13Cardiovascular disease4.0%4.9%0.44Values are mean±SEM.*Adjustment for age, years since menopause, BMI, TFM%, CFM/PFM ratio, current smoking, physical exercise, and regular alcohol consumption.**Adjustment for the aforementioned confounders + treated hypertension. Associations of lipids with BMD and AC In univariate analysis (Table 2), total cholesterol and triglycerides were common correlates of spine BMD, hip BMD, and AC. HDL-C and ApoA1 were common correlates of AC and hip BMD (p < 0.05), whereas LDL-C, but not ApoB, was a common correlate of AC (p < 0.05). To obtain insights into the direct contribution of lipids to the variation of hip and spine BMD and the severity of AC, we established multiple regression models. The independent contributors to the variation in these surrogate markers are indicated in Table 2. Lipids were not independent contributors to the variation in BMD when assessed in the presence of the severity of AC and other confounders. However, ApoA1 levels did seem to contribute to the variation in AC. Table 4Characteristics of women with or without at least one vertebral fracture Without vertebral fractureWith vertebral fracturep-valueAge68.9 ± 6.471.3 ± 6.4<0.001YSM12.7 ± 8.415.5 ± 8.6<0.001BMI26.4 ± 4.426.3 ± 4.4NSTFM%41.3 ± 7.640.7 ± 7.8NSCFM/PFM ratio0.88 ± 0.230.90 ± 0.24NSRegular exercise73.8%67.6%0.08ApoE ɛ4 allele +30.5%34.8%NSTotal cholesterol6.32 ± 1.006.31 ± 1.04NSTriglycerides1.42 ± 0.691.31 ± 0.540.02HDL-C1.68 ± 0.431.71 ± 0.39NSLDL-C3.98 ± 0.934.01 ± 0.96NSApoA1203.8 ± 37.8204.6 ± 33.4NSApoB120.6 ± 24.5119.1 ± 24.5NSLp(a)31.7 ± 34.932.5 ± 33.5NSSpine BMD0.93 ± 0.150.87 ± 0.14<0.001Hip BMD0.82 ± 0.110.75 ± 0.11<0.001Hip fracture1.0%2.9%0.04Wrist fracture11.4%14.6%0.04AC score2.8 ± 3.73.5 ± 3.40.03CVD3.6%7.4%0.02There were no significant differences in the frequency of smoking, regular alcohol, coffee, milk or seasonal vitamin D consumption, type 2 diabetes, treated hyperlipidemia or treated hypertension Comparison of women with or without fractures There were altogether 293 subjects who had an osteoporotic fracture after the menopause (i.e., prevalent hip, vertebral, or wrist fracture). Of the 204 women with at least one vertebral fracture, 33 also had a history of wrist fracture and 16 reported a history of hip fracture. Prevalence of non-vertebral fractures was significantly higher in women with compared with those without vertebral fracture(s). Characteristics of women with or without vertebral fractures are summarized in Table 4. Women with fractures were on average older and had a tendency for being physically less active. However, no significant differences in BMI, body adiposity, or body fat distribution were apparent. In addition, no significant differences in a wide array of lifestyle factors were apparent. When comparing the lipid profile of women with or without osteoporotic fractures after adjustment for potential confounders (i.e., age, years since menopause, BMI, TFM%, CFM/PFM ratio, smoking, ApoE ɛ4 allele, physical exercise, alcohol consumption, and severity score of AC), only triglycerides showed significant differences between the two groups (p < 0.05). Thus, triglycerides in women with and without fractures were 1.31 ± 0.04 mmol/l and 1.42 ± 0.03 mmol/l, respectively. When analyzing fracture types separately, the difference between women with or without hip or wrist fractures were not statistically significant. The relative risk of hip or vertebral fracture in women with severe AC or CVD To assess the implications of atherosclerotic vascular disease for osteoporotic fracture risk, we calculated the odds ratio of the different types of fractures in women with severe AC (≥6) and/or manifest CVD. As indicated in Table 5, after adjustment for age, the risk of hip fractures but not wrist or vertebral fractures was increased in subjects with advanced vascular disease. Table 5Relative risk of different types of osteoporotic fractures in women with severe aorta calcification (score ≥ 6) and/or manifest cardiovascular disease (n = 282)  Odds ratio (95% CI)p-valueHip fracture N = 16Crude4.4 (1.6–12.9)0.001Age-adjusted3.0 (1.1–8.8)0.04Vertebral fracture N = 204Crude1.2 (0.9–1.7)0.27Age-adjusted1.0 (0.7–1.4)0.93Wrist fracture N = 145Crude1.4 (1.0–2.1)0.06Age-adjusted1.2 (0.8–1.7)0.46 Discussion In the present study, we investigated whether serum lipid and lipoproteins represent a biological linkage between osteoporosis and cardiovascular disease. The main findings were as follows: 1) neither allelic polymorphism in the ApoE gene nor serum lipids per se were independent correlates of BMD at any skeletal site, 2) ApoA1 levels were independent correlates of AC, which seem to play a role in the determination of hip BMD, 3) decreased triglycerides were independently associated with vertebral fractures, but not with non-vertebral fractures (hip and wrist), and 4) advanced vascular disease was a significant contributor to risk of hip fractures, but not to vertebral or wrist fractures. These observations suggest that the role of lipids as linking factors between cardiovascular disease and osteoporosis is indirect via promotion of atherogenesis, which in turn can contribute to bone loss, especially at skeletal sites with end-arterial blood supply. Antiatherogenic measures can also be expected to contribute to decreasing the burden of hip fractures among the elderly. Univariate analysis pointed out several common correlates of BMD and atherosclerosis. Of these factors, aging, the duration of the menopause, BMI, and TFM% seemed to have reciprocal impact on BMD and AC. In multiple regression models, whereas BMI continued to correlate directly, TFM% became an inverse correlate of BMD. Accordingly, while weight-bearing may have a favourable impact on bone metabolism, adipose tissue accumulation exert an adverse influence. These findings corroborate the recent findings of Hsu YH et al. [24]. Although traditional views considered obesity a protective factor due to endogenous estrogen production, these more recent findings emphasize that other factors deriving from adipose tissue, e.g., adipokines, inflammatory cytokines, and perhaps even lipids/lipoprotein metabolism are also to be taken into account. In addition, these findings also draw attention to the need of proper adjustment for overall adiposity and body fat distribution when addressing the direct implication of selected potential mediators. To address whether serum lipids represent a biological linkage between osteoporosis and vascular disease, one approach is to investigate whether polymorphism in a gene with known modulator effects on serum lipid profile is associated with variation in the severity of AC and BMD. Similar to previous observations [25, 26], we found that presence of the ɛ4 variant allele in the ApoE gene was independently associated with higher levels of total cholesterol, LDL-C and ApoB. In addition, we also observed subtle, yet statistically significant differences in serum triglycerides and HDL-C levels. Nevertheless, we were not able to document significant associations of the ɛ4 allele with either BMD or AC score. In male or mixed populations, the ɛ4 allele was reported as a risk factor for atherosclerosis [27, 28], suggesting that the association might be gender-specific for males. Total cholesterol and LDL-C are important predictors of cardiovascular risk in men, but less so in postmenopausal women in whom triglycerides and HDL-C carry greater importance [29, 30]. Another explanation to the apparent discrepancies could be geographic variation in the relative importance of allelic variation in the ApoE gene. In support, a study in Finnish men reported higher importance of the ɛ3 allele for atherogenesis compared with the ɛ4 allele [27]. Similar discrepancies exist concerning the relative importance of this gene polymorphism for the determination of bone mass. Although there are reports suggesting that presence of the ɛ4 allele is a contributor to accelerated bone loss and/or increased risk of osteoporotic fractures [31, 32], not all studies confirm this finding, particularly those undertaken in women [33–35]. Collectively, ApoE polymorphism with primary influence on total cholesterol, LDL-C, and apoB levels does not seem to provide a major biological linkage between the pathogenesis of osteoporosis and CVD. Experimental observations proposed LDL-C and HDL-C as common modulators of bone cells and vascular smooth muscle cells [11–14]. On the clinical side, smaller cross-sectional studies found associations between lipid profile and BMD [15–17]. However, none of these studies adjusted systematically for a direct DEXA measure of body fat distribution and/or smoking habits, which are important confounders of lipid profile. Our multiple regression analyses could not reveal significant independent associations of any of the considered lipid and lipoprotein components with hip or spine BMD. These findings are in line with the largest population-based observational study, the NHAEMS III survey including more than 13,000 subjects [19]. In further support, a recent longitudinal analysis of results from the Framingham study cohort reported insignificant direct influence of serum lipids for changes of BMD during a 25-year observational period [18]. Collectively, these observations do not seem to nurture the concept that serum lipids and lipoproteins exert clinically detectable direct effects on bone mineralization in elderly women. To further assess the potential implications of serum lipids and lipoproteins for osteoporosis, we also compared the profile of postmenopausal women with or without osteoporotic fractures. Interestingly, serum triglycerides were significantly lower in women with at least one vertebral fracture compared with those with no fractures, while no differences were seen between those with or without an osteoporotic non-vertebral fracture (i.e., hip or wrist fracture). This finding is similar to that reported by Yamaguchi et al. [15]. Since triglycerides were not associated with spine BMD, their direct contribution to fracture risk, if any, is via modulation of bone quality or bone strength. Given the cross-sectional design of this study, we cannot exclude the possibility that a confounder or confounders of triglyceride levels, which was not considered herein is the actual modulator of bone fragility and hence fracture risk. Important finding of the multiple regression analysis was that the severity of AC was an independent correlate of hip BMD with no simultaneous independent contribution of lipids. This observation provides evidence that obstructive vascular disease rather than lipids/lipoproteins per se that drive bone loss from the hip. In a previous study, we showed that women with intermittent claudication have not only severe AC, but also very low BMD [2]. The direct contribution of atherosclerosis is also supported by the observation showing that asymmetric vascular disease is accompanied by asymmetric demineralization of this skeletal site [36]. Moreover, studies show that the rate of demineralization at the hip is significantly associated with the rate of atherogenesis [5] and even future risk of cardiovascular events [37]. On the other hand, we recently demonstrated in a large population-based cohort of postmenopausal women that accelerated atherogenesis is an independent predictor of hip fracture risk. Histological investigations of explants from elderly patients with femoral neck fractures indicate that both the larger (a. iliaca ext and a. femoris profunda) and the small vessels in the ligamentum teres supplying this skeletal site are frequently obstructed by atheromatous lesions [38, 39]. These observations provide strong support for the important role of atherosclerosis for the pathogenesis of osteoporosis in the proximal femur. In summary, the results of the present observational study provide further evidence for the independent association of peripheral vascular disease with osteoporosis in the proximal femur. Since the association of lipids and lipoproteins to BMD and non-vertebral fractures is not independent of the severity of AC, it seems unlikely that these metabolites exert direct and clinically significant effects on bone turnover in postmenopausal women. Their contribution is via promotion of atherogenesis, in which regard ApoA1 levels seem to take a leading role. The remaining issue to be clarified is which genetic or environmental factors underlie the association of low triglycerides levels to vertebral fractures.

J_Neurol-3-1-1915648

Does modafinil enhance activity of patients with myotonic dystrophy?

We performed a double-blind placebo-controlled crossover study in 13 patients with myotonic dystrophy to address the question whether modafinil, known to improve hypersomnolence in myotonic dystrophy, may improve levels of activity as well. We used the Epworth Sleepiness Scale as a measure of hypersomnolence and a structured interview of the patient and the partner or housemate as a measure of activity. We additionally used a restricted form of the RAND-36 to relate a possible improvement of activity to perceived general health. We confirmed earlier positive findings of modafinil regarding reduced somnolence (p = 0.015), but no significant effects were seen regarding activity levels (p = 0.2 for patients’ self-reports and 0.5 for partners’ reports). Introduction Myotonic dystrophy (MD) is a multi-system disorder, the most well known symptoms being muscle weakness and myotonia. Distressing daytime sleepiness and diminished spontaneous activity, often referred to as inertia, reduced initiative, inactivity or apathy, are frequently reported. These latter symptoms often seem to cause more hindrance in daily life, both to patients and their spouses, than muscle weakness itself. Hypersomnia can even be present when there is virtually no weakness [1]. Recent reports [2, 3, 4] have demonstrated that modafinil has a beneficial effect on daytime sleepiness in MD. Although the relationship between excessive sleepiness and the lack of spontaneous activity is not clear, it seemed reasonable to suppose that increasing vigilance might result in an increase in spontaneous activity. The present study intended to answer the following questions: firstly, do patients with MD undertake more activities when using modafinil; secondly, does any such improvement relate to a change in somnolence or to another disease characteristic? Patients and methods Thirteen outpatients (5 males) participated in a randomized double-blind crossover placebo controlled study. Their mean age was 43.5 years (SD: 13.9 years). Age of onset of symptoms was before 12 years of age in 3 patients; all three lived independently at the time of study, although they needed professional social support with respect to their household or daily activities. Three patients were employed in highly responsible jobs. The remaining patients had been considered unfit for normal paid employment for reasons related to their disorder. They were mainly involved in housekeeping. With the exception of two elderly men with considerable weakness of the legs, weakness had little impact on activities of daily living (ADL) functions. Even these two men were ambulant, although they used a wheelchair regularly. Twelve patients had a partner or housemate. Medication was given during two periods of 14 days, separated by a one-week washout period. The study was preceded by a two-week period free of all drugs with an exception being made for contraception. Patients were randomized for either placebo first or modafinil first. The modafinil dose was 200 mg per day for the first week. The patients were instructed to double the dose during the second week of each period if they perceived an insufficient effect. The main outcome measure was an increase in spontaneous activity, assessed using a novel structured interview of both the patient and the partner or housemate, if present. These interviews took place at home after the first period, and by telephone after the second one. After the first medication period the patient and the partner/housemate were asked to compare the level of activity with that of the preceding two (-baseline-) weeks. After the second medication period they were asked to compare that period with the baseline weeks. The following issues were addressed, and scores attributed: have you, during the past two weeks, been more active than during the baseline period?no (0 points)to some degree (1 point)definitely (2 points)can you give one (1 point), two (2 points) or three (3 points) substantial and observable examples of activities/specific actions you undertook that you would otherwise not have done? The range of the score was therefore zero to five points. The partner was asked the same questions with respect to the patient. During the interview patients and partners were asked whether they had guessed which medication, modafinil or placebo, the patient had used in the past period. If so, they were asked what made them believe so. The answers were not used in the assessment of medication effects, but served to estimate possible unblinding. The RAND-36 questionnaire was used to measure possible changes in subjective general health [5]. This was filled in before the start and after the end of each medication period. The questionnaire was restricted to issues considered relevant to the study: General Health, Role Limitations by emotional and physical problem, Social Functioning, Vitality, and Mental Health. The Epworth Sleepiness Scale (ESS, [6] was used to assess possible changes in hypersomnolence. After completion of the trial the remaining capsules in the medication boxes were counted to assess compliance. The institutional Committee of Medical Ethics had approved the study. Patients gave written informed consent after study information was provided orally and in writing at the patients’ home. Results All patients completed the trial. Medication compliance was good: only three patients had omitted one dose each. The only reported side effect was slight headache in one patient using modafinil. Ten patients doubled the dose of both modafinil and placebo after the first week, meaning that results largely concern a daily dose of 400 mg modafinil. More often than not both patients (67%) and their partners/housemates (77%) correctly guessed when they had been taking either modafinil or Placebo, usually on the basis of a ‘decreased sleepiness’ and/or ‘increased activity’. The structured interview regarding activity and actions did not show significant differences between modafinil and placebo (p = 0.2 for patients and p = 0.5 for partners/housemates). The RAND-36 questionnaires revealed a poor perception of general health for the whole group with a mean value of 29 points out of 100 (range 0–50) on the General Health rating. The ratings were virtually identical for each patient over the four assessments (p = 1,Wilcoxon test). The perception of Role Limitations varied widely: mean 66 out of 100 (range 0–100). A medication related change was not observed (p = 0.7, Wilcoxon test). This also held for the perception of Social Functioning (p = 0.6), Vitality (p = 0.2) and Mental Health (p = 0.5). The ESS revealed a significant improvement with modafinil, in that the mean score decreased from 10.5 (range: 3–18) to 6.8 points (range: 1–15); for placebo the corresponding values were 10.5 (range: 3–18) and 10.7 (range: 2–17) points (p = 0.015, Wilcoxon test). There was no suggestion of a difference in outcome between patients with high and those with low scores. There was no significant relationship between the increase in activity/actions as perceived by the patient/ partner and indicated by the structured interview, and changes in perceived hypersomnolence as measured by the ESS (p = 0.38, Spearman’s test). Discussion The present study confirmed the beneficial effect of modafinil on excessive sleepiness in MD, but did not detect a concomitant effect on spontaneous activity as measured by a structured interview of the patients and their partners. This interview, not formally validated, was designed to reflect a clinically relevant and observable increase in daily activity by asking for specific actions. Examples might be that patients went to the theatre after a busy day, when they would otherwise have postponed such a visit, or cleaning up the shed. By asking for specific actions we hoped to distinguish actions from the mere feeling of being active or the intention to become so. The study was small, leaving open the possibility that minor changes have been missed. The study was also focused on short-term effects and thus it is not able to detect changes of behaviour that take more time to become manifest, but we believe that a fortnight is long enough to detect relevant improvements in activity as defined above. A further consideration is the unblinding we have observed, which was most probably due to a correct perception of an effect on somnolence. This might have confounding effects on the interpretation of intended double-blind studies of modafinil on symptoms other than hypersomnolence. In the present study this does not seem to have happened, as the effect on hypersomnolence was neither related to perceived improved activity, nor to perceived aspects of general health. That many patients and partners reported more activity in addition to less sleepiness when asked why they thought that modafinil or placebo had been used, might be the result of the expectations implied in the aim of the study as discussed with the participants. The structured interview did not detect this increased activity, which we feel speaks in favour of its validity. In a previous study of 11 patients modafinil improved excessive daytime sleepiness in MD, measured with the Multiple Sleep Latency Test and the Epworth Sleepiness Scale [2]. Possible effects on spontaneous activity were not considered. MacDonald et al. [3], in a double-blind cross-over placebo-controlled study of 40 patients with a time-scale identical to ours confirmed the reduction of somnolence as measured by Epworth and Stanford Sleepiness Scales. They also found modafinil-induced decreased fatigue-inertia, and increased vigor-activity, as measured by the Profile of Mood States. The latter findings might predict improved observed activity, but the study did not include this issue and our study did not demonstrate such effect. Using the RAND-36 they also observed enhanced measures of energy and perception of health, but no changes in the other items of the test; the former effects were not confirmed in our study. Talbot et al. [4] performed a similar double-blind cross-over study of 19 patients selected for hypersomnolence (ESS 10 or more points), using the ESS, a Modified Maintenance of Wakefulness test (MWT), a steering simulator, the Short Form 36 and an “activity diary” as measures. They found a reduction of sleepiness, especially in the MWT, less convincingly in the ESS. The other tests did not show significant changes. Data from the activity diaries are not given. It is apparent that the symptoms referred to as inertia, reduced initiative, inactivity or apathy are hard to define and even harder to measure. Recently, van der Werf et al. inferred that the lack of correlation between fatigue scores and sleepiness in MD suggests that different pathophysiological mechanisms underlie these clinical manifestations [7]). We believe that our findings point in the same direction.

Antonie_Van_Leeuwenhoek-3-1-2140093

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP. Introduction Mycoplasma mycoides subsp. mycoides small colony type (SC) is an extra-cellular pathogen living in close association with host cells. It causes contagious bovine pleuropneumonia (CBPP), an acute, subacute or chronic infection of the respiratory system in cattle with a mortality rate of up to 30%, causing severe losses in livestock production, in particular on the African continent (Provost et al. 1987; Food and Agriculture Organization of the United Nations 2003). Lipoproteins are usually strongly antigenic membrane proteins known to play a central role in interactions between bacteria and eukaryotic cells, particularly with respect to adhesion, and to stimulate the release of pro-inflammatory cytokines (Mühlradt and Frisch 1994; Herbelin et al. 1994; Brenner et al. 1997; Marie et al. 1999; Calcutt et al. 1999; Belloy et al. 2003). Lipoproteins have been put forward as possible virulence factors of pathogenic mycoplasmas (Dyson and Smith 1997; Vilei et al. 2000; Pilo et al. 2003). Membrane lipoprotein LppQ is the predominant antigen of M. mycoides subsp. mycoides SC and shows the strongest signal on immunoblots containing total antigen from this pathogen reacted with serum from cattle that have suffered from CBPP. It induces a specific, early and persistent immune response in infected animals (Abdo et al. 2000). LppQ is encoded as a precursor (of 445 amino acids) with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site (Figure 1). The leader sequence of LppQ shows a typical transmembrane structure with a significant helix formation capacity (Abdo et al. 2000). LppQ was shown to be a membrane protein by Triton X-114 phase partitioning and lipidation was demonstrated by palmitic acid radiolabelling (Abdo et al. 2000). The C-terminal part of LppQ was found to possess repeated integral membrane structures rich in hydrophobic and aromatic amino acids, which have pore formation potential, and immunoblot analysis showed that the C-terminal domain possesses no particular immunogenicity, as serum derived from cattle naturally infected with M. mycoides subsp. mycoides SC did not react against it (Abdo et al. 2000). In contrast, the N-terminal domain of LppQ has three strongly hydrophilic domains and was shown to be the origin of the strong antigenic response against LppQ in naturally infected cattle. From these data, Abdo and collaborators deduced that the N-terminal part of LppQ is exposed at the outer surface of M. mycoides subsp. mycoides SC (Abdo et al. 2000). Fig. 1Structure of lipoprotein LppQ. (Panel A) LppQ protein sequence. Repeats are indicated by dashed arrows. The symbol # corresponds to the signal peptidase II cleavage site, which allows for the obtainment of mature LppQ protein. (Panel B) The 10 C-terminal repeats rich in hydrophobic and aromatic residues, as identified with the MEME software, which build the transmembrane region of LppQ. Black background indicates conserved residues. The consensus sequence is shown. (Panel C) Model for protein topology of LppQ in the membrane lipid-bilayer of M. mycoides subsp. mycoides SC. Repeat numbers and amino acid positions for the beginning of mature LppQ (28), the beginning of C-terminal portion (193) and the protein end (445) are indicated. The N-terminal part is supposed to be anchored in the lipid-bilayer by the lipid anchor of the Cys28 residue. The three hydrophilic domains of the N-terminal part, which were found with significant scores for coiled-coil tertiary structure (Abdo et al. 2000), are depicted as globular items. The C-terminal part consists of a transmembrane region built up of the 10 repeated integral membrane structures, whose consensus sequence is shown at the bottom Due to the strong antigenicity of the extracellular N-terminal part of LppQ and its specificity for M. mycoides subsp. mycoides SC, a recombinant peptide comprising amino acids 22-218 of the LppQ precursor protein was used to develop a specific and sensitive ELISA assay for the serological detection of CBPP in cattle (Bruderer et al. 2002). Furthermore, LppQ seems to be involved in the inflammatory processes of M. mycoides subsp. mycoides SC, as cattle immunized with the recombinant peptide showed an increased susceptibility to infection with M. mycoides subsp. mycoides SC and exhibited more severe symptoms of CBPP than unvaccinated animals (Nicholas et al. 2004). As LppQ is apparently involved in the molecular mechanisms of pathogenicity of M. mycoides subsp. mycoides SC (Nicholas et al. 2004; Pilo et al. 2006), we have investigated the topology of LppQ in M. mycoides subsp. mycoides SC by means of high resolution field emission scanning electron microscopy (SEM), with particular respect to the predicted localization of LppQ on the mycoplasmal cell surface. This technique allows proteins to be localized on mycoplasmas unequivocally and with high accuracy and resolution (Pilo et al. 2005). Here, we provide evidence that the N-terminal part of LppQ is located on the surface of the mycoplasmal cell membrane and is accessible from the extracellular side while the C-terminal part of LppQ is not exposed. Materials and methods Bioinformatic analysis Prediction of membrane topology was carried out by using the softwares TMpred (Hofmann and Stoffel 1993; http://www.ch.embnet.org/software/TMPRED_form.html), TopPred (von Heijne 1992; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) and Phobius (Käll et al. 2004; http://phobius.cgb.ki.se/). Protein secondary structure was predicted with the MINNOU software available on the web page http://minnou.cchmc.org/ (Cao et al. 2006). Determination of the C-terminal repeats of lipoprotein LppQ was assessed by the MEME software version 3.5.4 at http://meme.sdsc.edu/meme/meme.html (Bailey and Elkan 1994). M. mycoides subsp. mycoides SC strain and sample preparation M. mycoides subsp. mycoides SC strain Afadé was used in the present study. This strain was isolated in 1968 at Farcha Laboratory, N’Djamena, Chad, from a cow from Afadé, Northern Cameroon, which suffered from acute CBPP. The strain Afadé is known to be highly virulent (Vilei and Frey 2001) and was used previously for experimental infection studies (Abdo et al. 1998). It was grown in standard mycoplasma medium (Axcell Biotechnologies, St. Genis l’Argentière, France) by inoculation of 20 μl of a frozen stock culture into 3 ml of medium and incubation for 3 days at 37°C. Growth and handling of live M. mycoides subsp. mycoides SC were performed in a biological safety laboratory fulfilling the BSL-3 containment safety standards. Crude lysates were prepared by resuspending harvested mycoplasmas in TES buffer (10 mM Tris–HCl, 1 mM EDTA, 0.8% NaCl, pH 8.0) at a concentration of approximately 109 cells/ml (Abdo et al. 2000). Production of antibodies against recombinant LppQ peptides The polyhistidine-tagged recombinant peptides LppQ-N′ (amino acids 22 to 218) and LppQ-C′ (amino acids 214 to 422) from the N- and C-terminal portions of LppQ, respectively, were produced in Escherichia coli strain BL21(DE3) harbouring the respective plasmids as reported previously (Abdo et al. 2000; Bruderer et al. 2002). Recombinant peptides were polyhistidine-tagged at both their N- and C-termini. Purification of the peptides was performed by Ni2+ chelation chromatography and the purity of the peptides was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal monospecific sera directed against LppQ-N′ and LppQ-C′ were obtained by subcutaneous immunization of rabbits with 160–200 μg of purified recombinant polyhistidine-tagged peptides in 500 μl of 50 mM PBS buffer (50 mM Na2HPO4/NaH2PO4, 140 mM NaCl, pH 8.0) mixed with 500 μl of Adjuvant 10 (Gerbu Biotechnik GmbH, Gaiberg, Germany). Booster immunizations with 40 and 20 μg of protein were performed 2- and 4-weeks later, respectively. The rabbits were bled 10 days after the last booster immunization and antisera were stored at −20°C. Immunoglobulin G (IgG) fractions were purified from the two rabbit antisera with the HiTrap Protein G kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. Immunoglobulins from pre-immune sera were also purified. The purity of all IgGs was analyzed by SDS-PAGE. Protein concentrations were determined by using the method of Bradford (1976). Immunoblot analysis Whole cell antigens of strain Afadé were separated by 5–15% gradient SDS-PAGE (Ausubel et al. 1999) and blotted onto a nitrocellulose membrane with a pore size of 0.2 μm (Bio-Rad Laboratories AG, Reinach, Switzerland). The membrane was blocked with 1% skim milk powder in TBS buffer (100 mM Tris–HCl, 150 mM NaCl, pH 7.5) for 60 min at room temperature. Strips were incubated with corresponding IgGs at a concentration of 1–2 μg/ml (diluted 1:2000) overnight at 4°C and washed three times with TBS buffer for 5 min. Serum obtained from a cow that was infected experimentally with M. mycoides subsp. mycoides SC strain Afadé (Abdo et al. 1998; diluted 1:100) or previously prepared (Abdo et al. 2000) rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ (diluted 1:1000) were used as controls. The strips were incubated for 90 min at room temperature with alkaline phosphatase-labelled goat anti-rabbit IgG (H + L) (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) diluted 1:2000 or monoclonal antibody anti-bovine IgG (Sigma, Buchs, Switzerland) diluted 1:5000 in skim milk buffer. The strips were washed three times with TBS buffer. The colour reaction was initiated with 0.3 mg/ml nitroblue tetrazolium (NBT) (Roche Diagnostics, Rotkreuz, Switzerland) and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics) in alkaline buffer (7 mM Na2CO3, 3 mM NaHCO3, 1 mM MgCl2, pH 9.6) and stopped with distilled water. Preparation of M. mycoides subsp. mycoides SC for immunogold labelling Coverslips that had been coated with gold or platinum and treated with poly-l-lysine (Sigma) to promote cell adhesion were immersed into 3 ml of fresh standard medium in multiwell plates. Thereafter, 100 μl of mycoplasma culture were added and the cultures were incubated for 5 to 6 days at 37°C until stationary phase. Coverslips with adhering mycoplasmas were washed three times with PBS buffer (2.9 mM NaH2PO4, 7.1 mM Na2HPO4, 137 mM NaCl, pH 7.4) at 37°C and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After fixation, samples were washed with PBS buffer and blocked with 1% BSA and 0.2 M glycine in PBS for 15 min at room temperature. Thereafter, mycoplasma cells were incubated with anti-LppQ-N’ or anti-LppQ-C’ IgGs diluted 1:100 (30 μg/ml) in PBS containing 1% BSA overnight at 4°C. After another washing step with PBS buffer for 5–10 min, mycoplasmas were labelled with 15 nm colloidal gold-conjugated goat anti-rabbit antibody (British Biocell International, Cardiff, UK) diluted 1:50 in PBS for 90 min at room temperature. Coverslips were then washed with 0.1 M cacodylate buffer (pH 7.4) and processed for SEM. Scanning electron microscopy Coverslips were incubated in 1.33% osmium tetraoxide with 0.11% ruthenium red in 0.13 M cacodylate buffer (pH 7.4) for 15 min, washed with 0.1 M cacodylate buffer, dehydrated through an ascending ethanol series and dried by evaporation of hexamethyldisilazane (Sigma) as described previously (Stoffel et al. 1993; Stoffel et al. 2002; Stoffel and Friess 2002). Finally, they were mounted on metal stubs with a conductive adhesive (Provac AG, Balzers, Liechtenstein). Samples were examined without further metal coating. Secondary electron micrographs and corresponding backscattered images were obtained with a fully digital field emission scanning electron microscope DSM 982 Gemini (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV, a working distance of 6–8 mm and a magnification from 50,000 to 100,000×. Control experiments included omission of primary antibody as well as the use of a rabbit anti-calcitonin antibody (Anawa, Zurich, Switzerland) and of IgGs from rabbit pre-immune sera as irrelevant substitutes for the anti-LppQ primary antibodies. Results Structure of the C-terminal domain of lipoprotein LppQ Conventional predictors of membrane topology available online did not envisage the C-terminal domain of LppQ to be membrane-associated. However, determination of repeats in the LppQ protein sequence revealed that its C-terminal domain, represented by the last 250 amino acids, consists of a region built up of 10 repeated units rich in hydrophobic and aromatic residues (Figure 1). These repeat units of 25 amino acids were all found to adopt a particular secondary structure consisting of helices of approximately 10 amino acids flanked upstream and downstream by amino acids forming random coils (not shown). Monospecificity of IgG preparations On immunoblots containing total antigen of M. mycoides subsp. mycoides SC strain Afadé, purified IgG preparations directed against the N-terminal and against the C-terminal halves of LppQ each identified a single protein band with an apparent molecular mass of 48 kDa, which corresponds to the calculated size of 49.1 kDa of LppQ (Figure 2). Reference rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ collected almost a decade ago gave identical results. Control experiments with IgGs purified from pre-immune sera did not detect any corresponding antigen (Figure 2). Fig. 2Immunoblot analysis of total antigen of M. mycoides subsp. mycoides SC with anti-LppQ-N′ and anti-LppQ-C′. Approximately 10 μg of total antigen of strain Afadé per lane was separated by 5–15% gradient SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the serum from a cow infected experimentally with strain Afadé (Abdo et al. 1998), or with IgGs from rabbit pre-immune sera or from antisera against LppQ-N′ and LppQ-C′, respectively. Reference rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ obtained almost a decade ago (Abdo et al. 2000) were also used. The arrowhead indicates the position of LppQ on the immunoblots Morphology of the mycoplasmas Electron microscopy showed cultivated M. mycoides subsp. mycoides SC to be basically spherical in shape. They usually occurred as single cells but pairs or small groups of up to five organisms were also noted. Cell diameter varied between 200 and 500 nm and the cell surface was slightly grainy and irregular (Fig. 3A, C, E). Between fully grown microorganisms, small granular debris were observed. Fig. 3Scanning electron micrographs of M. mycoides subsp. mycoides SC with anti-LppQ-N′ and anti-LppQ-C′ antibodies. Secondary electron microscope images (A, C, E) reveal the spherical shape and rough cell surface of the mycoplasmas. Corresponding backscattered electron micrographs (B, D, F) show indirect immunolabelling with 15 nm colloidal gold antibodies directed against the N-terminal extracellular domain of LppQ (B). In contrast, LppQ is not detected with anti-LppQ-C′ antibody (D). Labelling is abolished when an anti-calcitonin antibody is used as an irrelevant primary antibody (F) Immunogold labelling Labelling of mycoplasmas on coverslips with anti-LppQ-N’ antibodies yielded a SEM signal of moderate to high density at the cell surface (Fig. 3B). Specific labelling of granular debris was also present. In contrast, labelling with anti-LppQ-C′ antibodies was absent in most of the cases (Fig. 3D) and appeared to be insignificant and coincidental in few cases (Table 1), as also observed in the three control experiments whereby labelling was investigated in the absence of primary antibodies and when pre-immune serum or anti-calcitonin antibodies were used (Fig. 3F). Overall, there were significant differences between the number of particles labelled per cell by using anti-LppQ-N’ antibodies and those labelled in the other four experiments (P < 0.0001; Table 1) Table 1Statistical analysisStatistical dataTreatmentAnti-LppQ-N’Anti-LppQ-C’Anti-calcitoninPre-immune serumNo primary antibodySingle cells investigated2748192023Labelled particlesTotal33317675Min20000Max314332Average per cella12.33 ± 7.580.35 ± 0.840.32 ± 0.820.35 ± 0.810.22 ± 0.52P-valueb<0.0001<0.0001<0.0001<0.0001a± Standard deviationbCalculated by the analysis of variance (anova) method: single factor. Differences between the number of particles labelled per cell by using anti-LppQ-N’ antibodies and those labelled in the other four experiments were analysed Discussion The goal of the present study was to discriminate the distribution of extracellular vs. integral membrane domains of lipoprotein LppQ in M. mycoides subsp. mycoides SC at the ultrastructural level. Amino acid sequence analysis for hydrophobicity by the method of Hopp and Woods (1981) revealed the N-terminal portion of LppQ to be particularly hydrophilic (Abdo et al. 2000). Thus, the lipidated N-terminal domain was predicted to be exposed at the extracellular surface of the plasma membrane (Abdo et al. 2000). On the other hand, the C-terminal portion consists of a region built up of 10 hydrophobic repeated units, and not nine as reported previously (Abdo et al. 2000). The repeat units were found to consist each of approximately 10 amino acids forming helices delimited on both sides with random coils, i.e. conformations without a regular secondary structure, and were not predicted to be membrane-associated by bioinformatic analysis. Since transmembrane helices are mostly composed of hydrophobic amino acid and the degree of hydrophobicity of the C-terminal domain of LppQ is clearly significant, the C-terminal portion was expected to be an integral membrane domain (Fig. 1). It has to be noted at this point that lipoproteins generally do not possess additional membrane-spanning domains (MSD) beyond their N-terminal lipid anchors. However, there are relevant examples from Gram-positive bacteria, which contradict this general statement about lipoproteins and substantiate instead the occurrence of such additional MSDs. These examples include lipoproteins CtaC and QoxA of Bacillus subtilis (Bengtsson et al. 1999; Antelmann et al. 2001), as well as the proven lipoprotein of Mycoplasma pneumoniae F0F1 ATPase (Pyrowolakis et al. 1998). High resolution scanning electron microscopy in combination with immunogold labelling revealed extracellular epitopes with antibodies against LppQ-N′ but no signal above the background of the three negative controls was detected with the antibody against LppQ-C′. These results substantiate the predicted localization of the two domains, as they unambiguously corroborate the accessibility of the N-terminal domain at the extracellular side of the plasma membrane. The failure to immunolabel the C-terminal epitopes is an indication that they may be imbedded in the membrane. However, one cannot rule out the possibility that such epitopes (i) are buried in the centre of a globular protein, (ii) are masked by other surface components or (iii) are not recognized in their native forms by rabbit antibodies produced by inoculation of the recombinant peptide. Beyond providing ultrastructural evidence for the location of the hydrophilic and hydrophobic domains of LppQ, these results also demonstrate that immunogold labelling in scanning electron microscopy allows for subtle topographical discrimination and that selective visualization of peptides being exposed at the outer surface of microorganisms can be achieved with high accuracy and reliability. The domain analysis of LppQ, which demonstrates the outstanding surface localization of the strongly hydrophilic N-terminal part of the lipoprotein, may explain the strong humoural response elicited by this lipoprotein during infection of cattle by M. mycoides subsp. mycoides SC (Abdo et al. 2000). It has to be demonstrated yet whether LppQ is also able to induce any cell-mediated immune response, as it is more often the case for cytoplasmic proteins. Absence of a cell-mediated immune response would explain the lack of protection in animals immunized with purified recombinant LppQ. In this respect, it should be noted that immunization of cattle with the purified recombinant N-terminal domain of LppQ elicited inflammatory processes of M. mycoides subsp. mycoides SC and exacerbated the clinical signs of CBPP after experimental infection compared to non-immunized animals (Nicholas et al. 2004). Conclusions Direct visualization of the N-terminal part of LppQ at the outer surface of M. mycoides subsp. mycoides SC, as demonstrated herein, provides evidence for the contention that LppQ is located to the extracellular side of the plasma membrane of mycoplasmas and that it is easily accessible by the humoural immune system of infected animals upon contact of host cells with M. mycoides subsp. mycoides SC.

Doc_Ophthalmol-3-1-1896293

ISCEV standard for clinical pattern electroretinography—2007 update

The pattern electroretinogram (PERG) is a retinal response evoked by viewing a temporally alternating pattern, usually a black and white checkerboard or grating. The PERG is important in clinical and research applications because it provides information both about retinal ganglion cell function and, because the stimulus is customarily viewed with central fixation, the function of the macula. The PERG can therefore facilitate interpretation of an abnormal pattern VEP by revealing the retinal responses to a similar stimulus to that used for the VEP. However, practitioners may have difficulty choosing between the different techniques for recording the PERG that have been described in the literature. The International Society for Clinical Electrophysiology of Vision published a standard for clinical PERG recording in 2000 to assist practitioners in obtaining good quality reliable responses and to facilitate inter-laboratory communication and comparison. This document is the scheduled revision of that standard. Introduction The pattern electroretinogram (PERG) is a retinal biopotential evoked when a temporally modulated patterned stimulus (checkerboard or grating) of constant mean luminance is viewed. The PERG is most often evoked by alternating contrast-reversal of a black and white square checkerboard pattern with central fixation. It may be altered in dysfunction confined to the macula or to the retinal ganglion cells, which do not significantly affect the a- and b-waves of the conventional full-field ERG, and thus the PERG receives clinical and research attention in both neurological and ophthalmological practice. Clinically, the PERG can be used in a patient with an abnormal visual evoked potential to establish whether a retinal (macular) disorder is present, and thus differentiate between macular and optic nerve dysfunction as a cause for the VEP abnormality. It can also directly demonstrate retinal ganglion cell dysfunction. However, the PERG is a very small signal, typically in the region of 2–8 μV across a normal population, and PERG recording is technically more demanding than standard flash ERGs. The amplitude values recorded will depend upon stimulus characteristics and the electrodes being used. The recordings published in the literature vary considerably in technique and technical quality; this document is intended to guide new or existing users to the most appropriate techniques for recording a standard clinical PERG. The International Society for Clinical Electrophysiology of Vision (ISCEV) initially published “PERG Guidelines” [1], intended as a guide to practice and to assist in interpretation of PERGs. Those guidelines were subsequently revised to a PERG standard [2] and the present document is the scheduled revision of that standard. The standard PERG represents a minimum protocol for recording a PERG with straightforward technical procedures that should allow reproducible responses to be recorded under defined conditions. As a minimum, it is intended that the standard method and responses be widely used, but in addition to rather than to the exclusion of other paradigms. Common additional PERG techniques are also briefly described in this document, and individual laboratories are encouraged to use additional stimuli and protocols tailored to their own requirements. The standard is based upon equipment and analytic capabilities currently available in most neurophysiological or ophthalmological electrodiagnostic clinics. This document addresses recording conditions and technology specific to the PERG, and presumes that the reader already has basic understanding and skills in clinical electrophysiology. Although much of the document will apply equally to adults and children, the standard is not necessarily appropriate to paediatric applications. The standard will be reviewed by ISCEV in a further four years. Waveform nomenclature and measurement The waveform of the PERG evoked by pattern-reversal stimuli depends on the temporal frequency of the stimulus. By convention, positivity is displayed upward. Transient PERG The ISCEV standard PERG is a transient response i.e. a response that is effectively complete before the next contrast reversal. Transient recording allows separation of the PERG components. At low temporal frequencies (<6 reversals per second (rev/s); equivalent to <3 Hz) transient PERGs are obtained (Fig. 1). The PERG waveform in normal subjects usually consists of a small initial negative component with a peak time of approximately 35 ms, N35, which is followed at 45–60 ms by a much larger positive component (P50). This positive component is followed by a larger negative component at 90–100 ms (N95). Fig. 1A normal PERG. The amplitude of P50 in a normal subject is usually between 2.0 and 4.0 μV For the transient PERG, amplitude measurements are made between peaks and troughs: the P50 amplitude is measured from the trough of N35 to the peak of P50. In some patients the N35 is poorly defined; in these cases N35 is replaced by the average baseline between time zero and the onset of P50. The N95 amplitude is measured from the peak of P50 to the trough of N95. It should be recognised that measured in this way, N95 amplitude includes the P50 amplitude and P50 that of N35. It can be argued that baseline to peak measurements may be more meaningful. However, the P50 and N95 components may not be independent. At the time of writing there are no peer-reviewed data attempting to resolve these issues, and given the wealth of data already published using peak-to-peak measures, they remain the standard measures for PERG amplitudes. The times to peaks in the waveform (implicit time) should be measured from the onset of the contrast-reversal to the peak of the component concerned; it should be noted that the highest absolute amplitude point on a waveform will not always be appropriate for the definition of the peak if there is contamination from muscle activity or other artifacts. The peak should be designated where it would appear on a smoothed or idealised waveform (see Fig. 1). The peak times or implicit times are often (erroneously) referred to as latencies. Correctly, latency refers to the delay prior to the onset of a response, not to the maximum activity of a peak. Basic technology Standard equipment for visual stimulus generation, amplification of physiological signals, and the recording and storing of electrophysiological data, is required for PERG testing. Information about the calibration of equipment and measurement of the parameters specified in this standard appears in the ISCEV Calibration Guidelines [3]. Electrodes Recording electrodes Clinical ERG electrodes that contact the cornea or nearby bulbar conjunctiva should be used as the active electrodes to record standard PERGs. Electrodes that degrade image quality on the retina (this includes all contact lens electrodes) must not be used. Thin conductive fibres and foils can usually be positioned without topical anaesthesia. Electrode integrity should be checked prior to insertion, to meet guidelines for each electrode type. It is recommended not to measure impedance in situ unless explicitly specified by the particular equipment manufacturer. Electrodes should be carefully positioned to minimise instability (a major source of artifact or interference). Those who perform the test should be aware of possible causes of artifact.Fibre electrodes are best positioned in relation to the upper margin of the lower eyelid. Some place the electrode in the lower conjunctival fornix (under the lower eyelid); such a position may reduce trial-to-trial variability but will also result in a lower amplitude PERG. Optimum stability is achieved by tethering the electrode at the nasal canthus.Foil electrodes should be positioned directly under the centre of the pupil so that there is minimal or no movement of the electrode when the patient blinks. This is best achieved by having the foil curve over the lower eyelashes without contacting them, and then tethering the lead to the cheek. The junction of the electrode and lead should form as straight a line as possible and the junction should not touch the skin.Loop electrodes should be hooked into the lower fornix. Loops should be folded so that the contact windows on otherwise insulated wire are positioned on the bulbar conjunctiva, about 5 mm under the limbus. Loop electrodes should not touch the cornea. To achieve this, the limbs of the loop should diverge widely (15–20 mm) before entering the fornix. The lead is then taped to the cheek. The appropriate techniques for individual electrode types are very important to achieve stable and reproducible PERG recordings. Additional sources should be consulted in relation to the specific electrode used. Surface (skin) active electrodes should not routinely be used for recording the standard PERG; a surface electrode positioned on the lower eyelid will record PERGs of lower amplitude than those recorded from an electrode in contact with the eye. Surface electrode recordings may however be useful when a corneal electrode is contraindicated or in paediatric practice. The use of a surface electrode to record the PERG is a variation from the standard and should be noted in the report. Reference electrodes Separate surface reference electrodes should be placed on the skin near the ipsilateral outer canthus of each eye. Mastoid, earlobe or forehead locations may result in contamination of the PERG from cortical potentials or the fellow eye. If monocular PERG recording is performed, the electrode in the occluded eye may be used as a reference. Ground electrodes A separate surface electrode should be attached and connected to the amplifier “ground input”; the forehead would be a typical location, but other locations are acceptable as the location of the ground electrode should not affect the standard PERG. Surface electrodes The skin should be prepared with a suitable cleaning agent, and a suitable conductive paste used to ensure good electrical connection. The impedance between the skin electrodes used for reference and ground, measured on the subject, should be less than 5 kΩ. Since the electrode in the eye will have very low impedance, a low impedance of the reference electrode is also important to obtain recordings as free as possible from mains (line frequency) interference. Electrode cleaning and sterilisation Electrodes should be cleaned and sterilised according to local health and safety regulations. The ISCEV Standard for full-field flash ERGs describes the appropriate care of ERG electrodes [4]. Stimulus parameters This standard specifies the protocol for basic clinical PERG recording. Laboratories may choose to test more conditions or parameters than are described herein. Field and check size A black and white reversing checkerboard should be used for the standard PERG. It is not necessary to use a square stimulus field, but the aspect ratio between the width and the height of the stimulus field should not exceed 4:3. The mean of the width and the height of the stimulus field should be 15° (±3°) with a check size of 0.8° (±0.16°). Luminance PERGs are difficult to record with low stimulus luminance, and a photopic luminance level for the white areas of greater than 80 cd · m−2 is required. The mean luminance of the stimulus screen must be constant during checkerboard reversals (i.e. no transient luminance change). Contrast The contrast between black and white squares should be maximal (close to 100%) for the standard PERG and not less than 80%. The contrast and luminance used should be reported. Frame rate Raster-based CRTs are typically used to present the pattern stimuli. The frame rate of the CRT is a significant stimulus parameter for PERGs, and a frequency of 75 Hz or greater should be used. Background illumination The luminance of the background beyond the checkerboard field is not critical when using the standard PERG technique providing dim or ordinary room lighting is used; ambient lighting should be the same for all recordings. Care should be taken to keep bright lights out of the subjects’ direct view. Pupil diameter should be recorded. Reversal rate The standard transient PERG should be obtained using a reversal rate of 4 rev/s ± 0.8 rev/s (i.e. 2 Hz ± 0.4 Hz). Calibration All stimulus parameters including luminance and contrast should be calibrated either locally or by the manufacturer and regular recalibration is advised [3]. Recording equipment Amplification systems AC-coupled amplifiers with a minimum input impedance of 10 MΩ should be used. Amplification systems must be electrically isolated from the patient according to the current safety standards for medical recording systems. The frequency response of bandpass amplifiers should include the range from 1–100 Hz; analogue notch filters (that suppress signals at the alternating current line frequency) should not be used. Some users may encounter severe electromagnetic interference from the stimulus display that makes it difficult to obtain satisfactory recordings with these filter settings. Ideally, such interference should be eliminated by shielding or modifying the equipment; rearranging the electrode leads may be of benefit. Averaging and signal analysis Signal averaging is necessary because of the small amplitude of the PERG. The analysis period (sweep time) for the standard PERGs should be 150 ms or greater. Some laboratories assess baseline stability by displaying two successive responses on the same trace. Artefact rejection Computerised artifact rejection is essential. The limits for rejection should be set at no higher than ± 50 μV, and preferably lower. The amplifiers must return to baseline rapidly following artifactual signals to avoid inadvertent storage of non-physiological data. Sampling rate A minimum sampling rate of 1,000 Hz (1 ms per point) is recommended. See the calibration standard (3) for further information. Data display systems Display systems must have adequate resolution to represent accurately the characteristics of this small amplitude signal. Ideally the recording system provides simultaneous display of the input signal and the accumulating average. In the absence of a simultaneous display, the system should allow a rapid alternation between displaying the input signal and displaying the current average so that the quality of the input signal can be adequately monitored. Even with a computerised artifact rejection system, it is important that the input signal be continuously monitored for baseline stability and the absence of amplifier blocking. Clinical protocol Preparation of the patient Positioning The patient should be as comfortable as possible with their head in a stable position against a head-rest. A chin rest is inappropriate and should not be used. Pupils The PERG should be recorded without dilatation of the pupils, to preserve accommodation and thus retinal image quality. Fixation A fixation mark in the centre of the screen at a node of the checkerboard is essential. If there are any doubts about the quality of fixation in an individual patient, an effective method is to give the patient a (laser) pointer and have them point at the middle of the screen throughout. Excessive blinking during recording should be discouraged, pauses may be advantageous. Refraction Because of the nature of the stimulus, PERG examination should be performed with optimal visual acuity at the testing distance. Patients should wear the appropriate optical correction for the test distance. Monocular and binocular recording Proper positioning of recording and reference electrodes will permit either monocular or binocular recording of the PERG. Binocular recording is recommended for the standard PERG because it is generally more stable, it reduces examination time and it allows fixation by the better eye in cases of asymmetric visual loss. Monocular stimulation is required to record the PERG and the VEP simultaneously. Recording A minimum of 100 artefact-free sweeps should be collected and averaged for a standard PERG. More sweeps, perhaps as many as 300, will be needed when the PERG is small or undetectable or in a “noisy” subject. At least two trials for each stimulus condition should be obtained to confirm reproducibility (i.e. at least one replication). It may be beneficial to superimpose repeated PERG recordings to evaluate quality and reproducibility. PERG reporting Reporting It is recommended that all reports contain measurements of P50 and N95 amplitude (see above), and P50 peak time (the peak of N95 is often rather broad precluding accurate peak time measurement of this component). All reports should also contain the stimulus parameters (luminance, contrast, and field size), and normal ranges for the laboratory concerned. Pupil size should be noted. Whenever practical, reporting of PERG results should include representative waveforms with appropriate amplitude and time calibrations, marks for the N35, P50 and N95 components, and should show replications. Normative data/reference ranges At present there are no standard international reference ranges for PERG measurements. Each laboratory should establish normal values for its own equipment and patient population. It should be noted that there are PERG changes with age. Additional tests Large field PERGs For some applications, such as glaucoma assessment, a larger field, such as 30°, may be more appropriate. Further, some practitioners find the improved signal-to-noise ratio obtained with a larger field may have advantages; users may wish to consider recording a large field PERG in addition to the standard response. Steady-state PERG At higher temporal frequencies, i.e. above 10 rev/s (5 Hz), the successive waveforms overlap and a “steady-state” PERG is evoked. There are situations in which the steady-state PERG is useful and some laboratories favour it for glaucoma studies. Since little extra time is required, laboratories may wish to consider recording it in addition to the transient response. For steady-state PERG, a reversal rate of approximately 16 rev/s (8 Hz) is recommended. The steady-state PERG waveform is roughly sinusoidal, and interpretation of steady-state PERGs requires measurement of amplitude and phase shift (relative to the stimulus) of the second harmonic by Fourier analysis. The presence of a significant first harmonic indicates technical problems. For correct interpretation the analysis period must be an integer number of stimulus cycles, preferably greater than 6. Steady-state PERG recording without the capability for such analysis is not recommended. Reporting of steady-state PERGs should include the amplitude and phase shift of the PERG at the reversal rate (i.e. at the second harmonic of the stimulus rate in Hz).

Anal_Bioanal_Chem-4-1-2045123

A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action. Introduction There is concern that chemicals in our food, water, and environment affect human health by disrupting normal endocrine function, possibly leading to reproductive failure in humans and tumors in sensitive tissues [1, 2]. This relates to chemicals with previously unknown hormonal properties, like certain pesticides and plasticizers, but also to compounds used in pharmaceutical preparations, eg., oral contraceptives and tablets for hormone-replacement therapy, the endogenous steroids excreted in urine of man and domestic animals and potentially also compounds used for their growth-promoting properties in animals. Of all endocrine disruptors, environmental estrogens are the most studied [3]. However, recent studies show a crucial involvement of the androgen receptor in abnormal sex development. The presence of pollutants with adverse effects on human androgen receptor (hAR) has been reported from paper-mill effluents and as a result of intensive farming [4, 5]. Xenoandrogenic exposure-related disorders include testicular cancer, hypospadias, cryptorchidism, and poor sperm and very recently prepubertal gynecomastia was linked to both estrogenic and antiandrogenic effects of lavender and tea tree oil [6]. Chemical and immunological methods are commonly used to detect steroid hormones in food, clinical practice, environmental samples, or doping control. Owing to the great variety of chemicals with hormonelike activity, these methods have the drawback that they only quantify the compound of interest and are not able to determine biological activity of unknown compounds and their metabolites, this in contrast to biological assays. Receptor-based transcription activation assays can be used to detect all compounds having affinity for a given receptor [7, 8]. In contrast to receptor binding assays, receptor gene bioassays also include the transactivation step and can distinguish between receptor agonists and receptor antagonists [9]. This feature is very helpful in detecting both known and unknown compounds. Several assays have been developed for this purpose, using both mammalian and yeast cells. In general, transcription activation assays based on mammalian, or more particular human, cell lines have been shown to be more sensitive than yeast-based assays, and may be able to identify compounds that require human metabolism for activation into their active state. Metabolic conversion can either activate or inactivate some compounds [10], whereas the relatively low metabolic capacity of yeast ensures that the test reflects the activity of the original compound. In addition, yeast-based assays have several other advantages. These include low costs, easy handling, lack of known endogenous receptors that may compete with the receptor activity under investigation (no crosstalk), and the use of media that are devoid of steroids [11–13]. Furthermore, yeast cell assays are extremely robust and survive extracts from dirty sample matrices such as sediments, urine, and feed [14–16]. Especially in the case of androgens, the lack of known endogenous receptors in yeast is a great advantage compared with mammalian cell lines, as androgen responsive elements (AREs) can also be activated by the progesterone receptor and the glucocorticoid receptor. To avoid potential crosstalk in mammalian cell lines, a lot of efforts was expended to construct an ARE that is specific and no longer inducible by the progesterone and glucocorticoid receptor [17–19]. However, up till now such an ARE does not exist and it is doubtful whether it will be found, as the consensus progesterone responsive element/glucocorticoid responsive element is equal to the consensus ARE. Moreover, the glucocorticoid receptor is normally expressed in all mammalian cell types. So far this has resulted in cell lines that are not specific for androgens and that also respond to gestagens or glucocorticoids [20–22]. This paper reports the development of a new yeast androgen bioassay by creating a stably transfected yeast strain that expresses yeast enhanced green fluorescent protein (yEGFP) as a measurable reporter protein in response to androgens. The lack of known endogenous receptors in yeast enabled us to use the strong nonspecific consensus ARE sequence, which is actually a common hormone responsive element that is recognized by the androgen, progesterone and glucocorticoid receptors and can therefore not be used in mammalian cell lines expressing more than one of these receptors. Exposures to 17β-testosterone, 17β-estradiol, progesterone, dexamethasone, and other compounds were performed in 96-well plates in order to demonstrate the suitability and specificity of this new yeast androgen bioassay. Additionally, flutamide and several brominated flame retardants were tested for their antagonistic mode of action and the results were compared with a yeast androgen bioassay expressing β-galactosidase as a reporter protein. Materials and methods Chemicals Chemicals and methods to prepare the growth media, to perform PCR, to isolate DNA, and to transform bacteria and yeast were as described earlier [23]. Corticosterone, dexamethasone, 17α-estradiol, 17β-estradiol, estrone, flutamide, 4-hydroxytamoxifen, medroxyprogesterone 17-acetate, and progesterone were obtained from Sigma (St. Louis, MO, USA). The following compounds were obtained from Steraloids (Newport, RI, USA): 17β-boldenone, diethylstilbestrol, 5α-dihydrotestosterone, 17α-ethynylestradiol, 17β-testosterone and 17β-trenbolone. Tetrahydrogestrinone (THG) was a gift from M. Thevis (DSHS, Cologne, Germany). Copper sulfate and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany) and methyltrienolone was obtained from PerkinElmer (USA). All restriction endonucleases and corresponding buffers were obtained from New England Biolabs (Hitchin, UK) and the yeast β-galactosidase assay kit was from Pierce Biotechnology (Rockford, IL, USA). 2,4,6-Tribromophenol (TBP), BDE-39, and the hydroxyl derivative 4-OH-BDE-17 were synthesized at the Wallenberg Laboratory (Stockholm University, Sweden). Yeast strains The yeast Saccharomyces cerevisiae (CEN.PK 102-5B, K20, URA3−, HIS3−, LEU−) host strain was a gift from H. Silljé (University of Utrecht, The Netherlands). The yeast androgen bioassay with β-galactosidase as a marker was kindly provided by D.P. McDonnell (Duke University, USA). Plasmids The p403-GPD and p406-CYC1 yeast expression vectors were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The pyEGFP3 plasmid was a gift from A.J. Brown (Stanford University, USA). Construction of the p403-GPD-hAR expression vector The yeast cells provided by McDonnell were grown overnight and chromosomal DNA was isolated. This DNA was used to serve as a template for the PCR to obtain the complementary DNA (cDNA) of hAR. Full-length hAR cDNA was obtained using the Expand High Fidelity PCR system (Boehringer Mannheim) and an Eppendorf Mastercycler gradient. The sequence of the 5′-primer was 5′-GCTCTAGAATGGAAGTGCAGTTAGGGCTGGG-3′, containing a restriction site for XbaI just before the ATG start codon. The sequence of the 3′-primer was 5′-GCGGATCCTCACTGGGTGTGGAAATAGATGGG-3′, containing a restriction site for BamHI just after the TGA stop codon. This PCR generated a full-length double-stranded (ds) cDNA of 2,763 bp of the hAR gene with a 5′-XbaI and a 3′-BamHI restriction site. The 2,763-bp full-length hAR PCR product was isolated from a 1% low-melt agarose gel, cleaved with XbaI and BamHI and ligated into the corresponding site of the p403-GPD yeast vector. Plasmid digestion and PCR controls revealed several good clones. Construction of the p406-ARE2-CYC1-yEGFP reporter vector A set of complementary oligonucleotides (a and b), each with two consensus ARE sequences (in bold), were synthesized. A solution with both cDNA oligonucleotides, 2.5 μM of each, was heated at 95 °C and cooled down to room temperature in 2 h. This set gave a ds DNA with a 5′-SacI sticky end and a 3′-MscI blunt end.Sa: 5′-AAAGTCAGAACAGCATGTTCTGATCAAATCTAGAAGATCCAAAGTCAGAACAGCATGTTCTGATCAAACTCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGG-3′Sb: 5′-CCATCCACGCTATATATACACGCCTGGCGGATCTGCTCGAGTTTGATCAGAACATGCTGTTCTGACTTTGGATCTTCTAGATTTGATCAGAACATGCTGTTCTGACTTTAGCT-3′. This ds DNA was cloned into the corresponding site of the p406-CYC1 vector. Subsequently, yEGFP [24] obtained from a HindIII/SalI double digestion of pyEGFP was cloned in the corresponding HindIII/SalI sites of the p406-ARE2-CYC1 reporter construct. Plasmid digestion and PCR controls revealed several good clones. Transformation of yeast cells Transformation of yeast K20 host strain (Ura−, His−, and Leu−) was performed by the lithium acetate protocol as described earlier [23]. First, the yeast was transformed with the p406-ARE2-CYC1-yEGFP reporter vector, integrated at the chromosomal location of the uracil gene via homologous recombination. Therefore, prior to transformation, the reporter vector was linearized by cutting with StuI, which has a unique restriction site in the URA3 marker gene. Transformants were grown on minimal medium plates containing l-leucine and l-histidine (MM/L plates). This yeast reporter strain was then transformed with the p403-GPD-hAR expression vector, which was linearized by cleavage with NsiI, which has a unique restriction site in the HIS3 marker gene (histidine). Transformants were grown on MM/L plates and PCR controls were used to select clones that contain the p406-ARE2-CYC1-yEGFP reporter and the p403-GPD-hAR expression construct. PCR controls PCR controls were performed on the reporter-receptor transformants. Yeast chromosomal DNA of transformants was isolated and PCR controls were performed. PCR I was performed with a 5′-primer on the backbone of the reporter plasmid and a 3′-primer on the ARE2 sequence. The sequence of the 5′-primer was 5′-AGCGAGTCAGTGAGCGAGGAAG-3′ and the sequence of the 3′-primer was 5′-TGCTGTTCTGACTTTGGATC-3′. PCR II was performed with a 5′-primer on the CYC1 (cytochrome c oxidase) promoter of the reporter plasmid and a 3′-primer on the CYC1 terminator. The sequence of the 5′-primer was 5′-TCTATAGACACACAAACACAA-3′ and the sequence of the 3′-primer was 5′-GGGAGGGCGTGAATGTAAG-3′. PCR III was performed with the primers that were also used to obtain the full length cDNA of the hAR (see “Construction of the p403-GPD-hAR expression vector”). Streamlined yEGFP assay with the yeast androgen bioassay The day before running the assay, a single colony from a MM/L agar plate was used to inoculate 10 mL of the selective MM/L medium. This culture was grown overnight at 30 °C with vigorous orbital shaking. At the late log phase, the yeast androgen receptor biosensor was diluted in the selective MM/L medium to an optical density (OD) at 604 nm between 0.08 and 0.12. For exposure, aliquots of 200 μL of this diluted yeast culture were pipetted into each well of a 96-well plate and 2 μL of a 17β-testosterone or other stock solution in DMSO was added. DMSO-only controls were included in each experiment and each sample concentration was assayed in triplicate. Exposure was performed for 24 h at 30 °C and orbital shaking at 125 rpm. Fluorescence was measured at 0 and 24 h directly in a CytoFluor multiwell plate reader (Series 4000, PerSeptive Biosystems) using excitation at 485 nm and measuring emission at 530 nm. The fluorescence signal was corrected with the signals obtained with MM/L containing DMSO solvent only. Densities of the yeast culture were determined by measuring the OD at 630 nm, but this was only done to check whether a sample was toxic for the yeast cells. For the calculation of the relative androgenic potency (RAP) of the compounds in the yeast androgen bioassay, the data of a complete dose–response curve were fitted using the equation (Slide write Plus, version 6.00). This is equivalent to , where response is the measured fluorescence signal, [agonist] is the concentration of the test compound, and EC50 is the concentration of the test compound giving half-maximum response. lacZ-based yeast androgen bioassay: β-galactosidase assay An agar plate containing the selective growth medium, consisting of a yeast nitrogen base with dextrose (2%), lysine (36 mg/L), tryptophan (48 mg/L), uracil (24 mg/L), and adenine (41 mg/L), was inoculated with the yeast androgen receptor cytosensor from a frozen -80 °C stock (20% glycerol v/v). The plate was incubated at 30 °C for 24–48 h and then stored at 4 °C. The day before running the assay, a single colony of the yeast was used to inoculate 10 mL of the selective growth medium. This culture was grown overnight at 30 °C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast androgen receptor cytosensor was diluted in growth medium to an OD of 0.06 at 604 nm, and CuSO4 (0.05 mM) was added to induce the expression of the hAR. For exposure in 96-well plates, aliquots of 200 μL of this diluted yeast culture were pipetted into each well and 2 μL of stock solutions in DMSO was added. Exposure was performed for 24 h at 30 °C and 125 rpm, and the β-galactosidase activity was measured with a commercial yeast β-galactosidase assay kit from Pierce (Rockford, IL, USA). This kit uses o-nitrophenyl β-d-galactopyranoside as a substrate, and the solution turns yellow upon hydrolysis of β-d-galactopyranoside to o-nitrophenol and galactose. The yellow o-nitrophenol is measured in a Biotek (Winooski, VT, USA) model ELx 808 series ultra microplate reader at 405 nm. Densities of the yeast culture were determined by measuring the OD at 630 nm. The measured response at 405 nm was corrected for the OD at 630 nm. Results and discussion A recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Both the receptor construct as well as the reporter construct were stably integrated into the yeast genome by the use of yeast-integrating plasmids. For the construction of the reporter vector the p406-CYC1 plasmid, containing the URA3 marker gene, was used. Two consensus AREs were placed in the SacI/MscI site of the truncated CYC1 promoter in a way that the −254 to −147 XhoI-SphI part of the CYC1 promoter was restored [23]. High expression levels of the androgen receptor were obtained by placing the cDNA of the hAR gene behind the strong constitutive yeast glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter in the p403-GPD plasmid. This plasmid contains the HIS3 marker gene. Transfected strains were checked with PCR. The correct and specific functioning of the yeast androgen bioassay was studied by exposures to 17β-testosterone and other compounds and the results were compared with results obtained with the lacZ-based yeast androgen bioassay provided by McDonnell. In addition, the antiandrogenic properties of several brominated flame retardants were investigated. PCR controls A number of different PCR controls were carried out to check the integration of the vectors into the yeast genome. Figure 1 shows the gel electrophoresis results of these PCR controls. PCR I (Fig. 1a) was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. As expected, it gave the specific 360-bp band with the p406-ARE2-CYC1-yEGFP reporter vector and the DNA that was isolated from the yeast androgen biosensor. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host (the nontransfected yeast cell), showed no PCR bands. PCR II (Fig. 1a) was performed with primers on the CYC1 promoter and the CYC1 terminator. As expected, it gave the specific 873-bp band with the reporter vector and the DNA that was isolated from the biosensor, because both contain the reporter construct with the yEGFP that was ligated between the CYC1 promoter and CYC1 terminator. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host, did not show the reporter-specific 873-bp band. However, this PCR generated a 435-bp band with the DNA of the empty yeast host and the biosensor. This 435-bp band corresponds to the CYC gene of the yeast host itself and is therefore also a specific band. PCR III (Fig. 1b) was performed with the primers on the hAR gene. As expected, it gave the specific 2,763-bp band with the p403-GPD-hAR expression vector and the DNA that was isolated from the biosensor. In the negative control, performed with the DNA that was isolated from the yeast host, the receptor-specific 2,763-bp band was not present. These PCR controls demonstrate that all specific PCR bands can be seen, thus demonstrating that our yeast androgen bioassay contains the p403-GPD-hAR expression vector and the p406-ARE2-CYC1-yEGFP reporter vector, both stably integrated in the yeast genome. Fig. 1PCR controls. The PCR controls were performed as described in “PCR controls.” a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. Lanes 2–5 are PCR I on the p406-ARE2-CYC1-yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7–11 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2–4 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein Dose–response curves obtained with the new yeast androgen bioassay The dose–response curves for several natural and synthetic androgens are shown in Fig. 2. 5α-Dihydrotestosterone, 17β-testosterone and 17β-boldenone caused a dose-related increase in the production of yEGFP, demonstrating that these compounds are potent androgens. The bioassay showed a limit of detection of 3 nM for 5α-dihydrotestosterone with a dynamic range from 3 to 500 nM and very low standard deviations (les than 3%). The figure also shows that 17β-estradiol and progesterone give a response. The female hormone 17β-estradiol gives a full dose–response curve, but the maximum of the curve is reached at a 500 times higher concentration than that of 17β-testosterone and is less steep. Progesterone gives a response, but the maximum response is only about 35% of that of 17β-testosterone and is reached at a 25 times higher concentration. Both 17β-estradiol and progesterone are known to possess androgenic properties. Progesterone displays low binding to the androgen receptor [25] and shows androgenic effects in vivo [26]. According to [27], 17β-estradiol and progesterone showed androgenic activity in ten out of 11 and seven out of nine mammalian cell reporter gene (MCRG) systems, respectively. The corticosteroids corticosterone and dexamethasone showed no response in our assay. Fig. 2Response of the yeast androgen biosensor to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in dimethyl sulfoxide (DMSO). Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the standard deviation (SD). 17β-T 17β-testosterone, DHT 5α-dihydrotestosterone, Prog progesterone, Dex dexamethasone, 17β-E2 17β-estradiol, Bold 17β-boldenone Table 1 shows the calculated EC50, ie., the concentration giving a half-maximum response, and the RAP, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, for several compounds. The yeast androgen bioassay showed good sensitivity towards all androgens tested, with the following range of potencies: 5α-dihydrotestosterone > 17β-trenbolone > methyltrienolone > tetrahydrogestrinone > 17β-testosterone > 17β-boldenone > medroxyprogesterone acetate > 17β-estradiol > progesterone. Steroids representative for other hormone receptors, like estrone, 17α-estradiol, 17α-ethynylestradiol, and diethylstilbestrol for the estrogen receptor and corticosterone and dexamethasone for the glucocorticoid receptor, showed no agonistic response. Only 17β-estradiol, progesterone and medroxyprogesterone acetate gave a clear agonistic response. However, these compounds are known to exert androgenic effects. Table 1EC50 concentrations and relative androgenic potencies (RAP) of compounds in the yeast androgen biosensor expressing yeast enhanced green fluorescent protein in response to androgensCompoundQualitative response for AR agonismaCommentsbEC50 (nM) in the yeast androgen bioassaycRAPd17β-Testosterone Positive (11/11)Strong AR agonist761.05α-DihydrotestosteronePositive (21/21)Strong AR agonist, weak ER agonist332.317β-Boldenone5100.1517β-TrenbolonePositiveBinds strongly to AR521.5MethyltrienolonePositive (8/8)AR agonist541.4TetrahydrogestrinoneAR agonist651.217β-EstradiolPositive (10/11)AR agonist and antagonist, strong ER agonist9,0000.0084EstronePositive (2/2)AR agonist, strong ER agonistNRNR17α-EstradiolNegative (1/1)ER agonistNRNR17α-EthynylestradiolNegative (1/1)Strong ER agonistNRNRDiethylstilbestrolNegative (2/2)Strong ER agonistNRNR4-HydroxytamoxifeneNegative (1/1)ER antagonistNRNRProgesteronefPositive (7/9)1,7000.045Medroxyprogesterone acetate Positive (4/4)Weak AR agonist1,5000.051CorticosteroneNegative (1/1)Binds weakly to ARNRNRDexamethasonePositive (3/4)AR agonistNRNRFlutamideNegative (5/5)AR antagonistNRNR2,4,6-Tribromophenol NRNRBDE-19NRNR4-OH-BDE-17NRNRAR androgen receptor, ER estrogen receptor, NR no responseaQualitative response for AR agonism across all mammalian cell reporter gene studies (data obtained from [27])bComments obtained from [27]cThe EC50 is the concentration giving half-maximum response.dThe RAP is defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound.eThis compound was toxic to yeast above 30 μM.fThese compounds reach a maximum response that is lower than 70% of the maximum response obtained with 17β-testosterone. The maxima obtained with 4-androstenedione and progesterone are about 40 and 35%, respectively. Compared with the NIH publication [27], there are a few discrepancies. According to [27], estrone is an androgen receptor agonist that showed androgenic activity in two out of two MCRG systems. However, some mammalian cells are able to convert estrone into 17β-estradiol and vice versa. This conversion is ascribed to 17β-hydroxysteroid dehydrogenase 3 and this enzyme is responsible for the high relative estrogenic potency (REP) of estrone in the estrogen bioassay with the T47-D breast cancer cells (ER-CALUX test). In that test, the estrogenic potency of estrone was equal to that of 17β-estradiol and a REP of 1.0 was reported for estrone [10]. This probably explains why estrone gave a positive result for androgenic activity in two MCRG systems, but gave a negative result in our yeast androgen bioassay. Yeast is obviously not able to convert estrone into 17β-estradiol. The reported REP of 0.2 in our yeast estrogen biosensor corresponds nicely with the in vivo potency of this compound [28]. Dexamethasone was also described as an androgen receptor agonist in the NIH publication, showing androgenic activity in three out of four mammalian assays, but gave a negative result in our yeast androgen bioassay. However, these MCRG systems use an MMTV-Luc reporter construct or an ARE-Luc reporter construct. Both the MMTV and the ARE sequence are recognized by the glucocorticoid receptor and this means that the response found in these MCRG systems is probably due to crosstalk, as the glucocorticoid receptor is normally expressed in all cell types. Dose–response curves obtained with the lacZ-based yeast androgen bioassay Figure 3 shows the dose–response curves for several natural and synthetic androgens obtained with the lacZ-based yeast androgen bioassay. The assay was simplified in our laboratory by scaling it down to a 96-well format and the use of a β-galactosidase assay kit. With increasing β-galactosidase activity, the density of the yeast culture, measured at 630 nm, dropped to about 50% (data not shown). Therefore, the measured â-galactosidase activity was corrected for the OD of the yeast culture. Table 2 shows the calculated EC50 and the corresponding RAP values. The data demonstrate that there is a good correlation between the EC50 values for androgens determined in our laboratory and those reported by Gaido et al. [29] in 1997. It seems that the lacZ-based yeast androgen bioassay, in terms of EC50 values, is 5–10 times more sensitive than our new bioassay. However, there was almost no difference in the limit of detection. Although the curves in the β-galactosidase assay go up at around 0.3 nM and in the yEGFP assay at around 1 nM, the limit of detection is about 3 nM for 5α-dihydrotestosterone in both assay types. This is mainly because the standard deviations are much higher in the β-galactosidase assay. However, the curves in the β-galactosidase assay are much steeper and are thus responsible for the lower EC50 values. The steeper curves are probably due to using an enzyme as a marker, β-galactosidase compared with a yEGFP marker protein, and the expression of the RSP5 cofactor that enhances transcription activation in the lacZ-based yeast androgen bioassay. The dynamic range for 5α-dihydrotestosterone in the McDonnell assay was from 3 to 100 nM and is slightly smaller than the range of the new bioassay (3–500 nM). There were also no great differences in the RAP values determined, although methyltrienolone was more potent in the lacZ-based bioassay. However, the new yEGFP bioassay is less sensitive for 17β-estradiol and progesterone: RAPs of 0.008 and 0.045, respectively, and for the latter no full dose–response curve, compared with RAPs of 0.1 and 0.068 and full dose–response curves in the lacZ-based bioassay. This means that our new bioassay is more specific for detecting compounds with a pure androgenic mode of action. The main difference between the lacZ-based and our yEGFP biosassay is that protein RSP5, which is a counterpart of the mammalian RPF1, is overexpressed in the lacZ-based bioassay in order to enhance transcriptional efficacy. However, this cannot explain the observed difference with methyltrienolone , as according to Imhof and McDonnell [30], this did not alter the potency or specificity of the assay. In addition and in contrast to the lacZ-based yeast androgen bioassay there were no differences in the density of the yeast culture measured at 630 nm upon exposure to different compounds that induced yEGFP expression, which indicates a decreased growth of yeast cells exposed to androgens in the lacZ-based yeast androgen bioassay. The only other known yeast androgen bioassay, one that uses luciferase as a reporter protein, displays similar characteristics in terms of specificity and was 5–10 times more sensitive in terms of EC50 values [15]. However, this assay needs the correction of the same yeast strain that only and stably expressed luciferase as an external control to correct for and normalize the aspecific responses caused by variation in cell vitality due to matrix and analyte toxicity. Unorrected dose–response curves for 17β-testosterone displayed more than 10 times higher EC50 values. An earlier assay described by Lee et al. [31] in 2003 uses β-galactosidase, but only expresses the hinge-ligand binding domain of the androgen receptor. Fig. 3Response of the McDonnell yeast androgen bioassay to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in DMSO. The β-galactosidase activity was determined after 24 h and corrected for the optical density at 630 nm as described in “lacZ-based yeast androgen bioassay: β-galactosidase assay.” Signals are the mean of a triplicate with the SDTable 2EC50 concentrations and RAPs of compounds in the yeast androgen bioassay expressing β-galactosidase in response to androgensCompoundEC50 (nM)a by Gaido et al. [29]EC50 (nM)b in our laboratoryRAP17β-Testosterone4.711.51.05α-Dihydrotestosterone3.54.92.317β-BoldenoneND700.217β-TrenboloneND130.9MethyltrienoloneND3.73.1TetrahydrogestrinoneND11.51.017β-Estradiol 86.1950.117α-EstradiolNDNRNRProgesterone89.31700.068CorticosteroneNDNRNRDexamethasoneNDNRNRND not determinedaValues obtained by Gaido et al. [29], using the McDonnell yeast androgen bioassaybValues determined in our laboratory, using the McDonnell yeast androgen bioassay (see “Dose–response curves obtained with the lacZ-based yeast androgen bioassay”) Antiandrogenic activity The specificity of the new yeast androgen bioassay was further demonstrated by the ability of antiandrogens to suppress the induction of yEGFP. Figure 4 shows the antiandrogenic activity of the known antagonist flutamide and three brominated flame retardants, BDE-39, TBP, and 4-OH-BDE-17. The antagonistic properties were examined by coexposure with a concentration of 5α-dihydrotestosterone that induced a submaximal response (50 nM). None of these four compounds were able to show an agonistic response (Table 1), but Fig. 4 clearly shows that all three were able to inhibit the response induced by 5α-dihydrotestosterone . The IC50 value was about 1 μM for flutamide, and TBP and 4-OH-BDE-17 were about as potent, while BDE-39 was clearly less antiandrogenic. Similar results were obtained with a human cell line. Only TBP was less potent in that test, but cytotoxicity of TBP could not be excluded [32]. Fig. 4Inhibition of a submaximal response obtained by 5α-dihydrotestosterone with flutamide and three brominated flame retardants (BFRs). Coexposure to a concentration of 5α-dihydrotestosterone that induced a submaximal response was started by adding to 200 μL of a yeast culture, 1 μL of a 5α-dihydrotestosterone and 1 μL of the BFR stock solution in DMSO. Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the SD. TBP 2,4,6-tribromophenol Conclusions A recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Compared with other yeast androgen bioassays, this new biosassay showed a similar limit of detection and dynamic range. However, the measurement of the fluorescence (yEGFP) can be followed as a function of incubation time and is easier, quicker, and cheaper than the measurement of the β-galactosidase or the luciferase activity, which needs cell wall disruption and/or the addition of expensive substrates. Owing to the ease of the yEGFP measurement, standard deviations are generally less than 3%. Moreover, the assay seems to be more robust and more specific for detecting compounds with a pure androgenic mode of action. Brominated flame retardants with suspected antiandrogenic properties were able to inhibit the response obtained with 5α-dihydrotestosterone, the most potent endogenous androgen, demonstrating that this yeast androgen bioassay is suited to detect compounds with both agonistic and antagonistic characteristics. As all compounds tested were able to show either their agonistic or their antagonistic properties, neither the cell wall nor the cell membrane seemed to be an obstacle. As for the yeast estrogen bioassay, we validated this new yeast androgen bioassay according to international criteria, eg., the determination of the decision limit (CCα) and the detection capability (CCβ). The decision limit is then used to distinguish negative and suspect samples [16] (unpublished results). The assay was proven to be useful to detect the new designer steroid tetrahydrogestrinone in human urine [33] while prohormones with an androgenic mode of action, e.g., dehydroepiandrosterone, are not active in yeast-based bioassays and need metabolic activation before they can be detected [34]. Future work will include the validation of the assay for urine and feed and the screening of prohormones with and without metabolic activation. To mimic the in vivo metabolic activation, liver slices, liver cell lines, liver S9 enzymes, and pure enzymes will be used.

Anal_Bioanal_Chem-2-2-1764597

Advances in analytical techniques for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like PCBs

Analytical techniques for the determination of polychorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and dioxin-like PCBs (DLPCB) are reviewed. The focus of the review is on recent advances in methodology and analytical procedures. The paper also reviews toxicology, the development of toxic equivalent factors (TEF) and the determination of toxic equivalent quantity (TEQ) values. Sources, occurrence and temporal trends of PCDD/PCDF are summarized to provide examples of levels and concentration ranges for the methods and techniques reviewed. Introduction In this review, we describe the current state-of-the-art for the determination of the chlorinated dibenzo-p-dioxins (dioxins), chlorinated dibenzofurans (furans), and dioxin-like polychlorinated biphenyls (DLPCBs, or coplanar PCBs). Only a brief summary of the historical development of methods for these compounds is given here; all of the most important earlier work has been summarized in previous reviews [1–4]. The characteristics of modern methods for dioxin/furan/DLPCB determination are derived from several factors related to their chemical, physical and toxicological properties. The exceptionally high toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) meant from the outset that very low detection limits (DLs) were required; consideration of their hydrophobic nature and concern over chronic rather than acute toxicity effects meant that bioaccumulation was of increased concern, which pushed the need for even lower DLs. An understanding of the environmental levels, transport and fate of these compounds is important if we wish to understand the significance of these compounds as well as the low detection limits and broad range of detection required; therefore, a section on levels, transport and fate of these compounds is included in this review. A variety of different analytical methods and techniques have been developed to increase sensitivity and selectivity and to reduce analysis times. To fully understand the analytical methods developed and their application, the references cited in Table 5 should be examined. Table 5 also illustrates the wide range of sample types and concentration ranges of dioxins and DLPCBs detected in the global environment, which explains why so many variations on the basic analytical methodology exist. Another factor in method development and application was the large amount of litigation that occurred because of the detection of these compounds in humans as well as the environment. Methods of analysis were required to generate data that could withstand scrutiny in a court of law, which led to the necessity of using methods based on isotope dilution–HRMS techniques. In addition, the rapid growth in high-quality standards and reference materials contributed to substantial improvements in the accuracy and precision of modern methods. The development of analytical methods was also influenced by the need for regulators to be able to evaluate the combined toxicity of a number of compounds that have similar toxic properties to 2,3,7,8-TCDD, but widely varying potencies. This development is so important to the development of analytical methods that we have included a section below on toxicology and TEFs. The bottom line for dioxin methods was that definitive separation of the 2,3,7,8-substituted dioxins/furans and DLPCBs from a large number of other congeners with almost identical physical and chemical properties, as well as numerous other potentially interfering compounds, was essential. Brief background on dioxin determination Chlorinated dibenzo-p-dioxins (dioxins) have been of concern for decades because of their toxic properties, as described below. A structurally similar series of compounds, the chlorinated dibenzofurans (furans), have similar chemical properties and toxic effects, and are generally determined as a group with the dioxins. In recent years there has been a growing trend to include a specific subgroup of the polychlorinated biphenyls (PCBs): the so-called dioxin-like PCBs (DLPCBs). This has been added to methods along with the dioxins and furans. Figure 1 shows the structures of 2,3,7,8-TCDD, 2,3,7,8-TCDF and PCB-126. The carbon numbering system is marked next to each carbon to indicate substitution positions. Dioxin and furan congeners substituted in the 2,3,7 or 8 position are toxic, while PCBs substituted in the 3,3′,4,4′,5 or 5′ position and no or only one 2- or 2′-substitution are considered to be dioxin-like and to exhibit dioxin toxicity (see Table 1). Fig. 1Basic structures of the chlorinated dibenzo-p-dioxins (dioxins), chlorinated dibenzofurans (furans) and polychlorinated biphenyls (PCBs). For the dioxins and furans, congeners with chlorine substitution at the 2,3,7 and 8 positions are considered toxic. Of the 75 possible dioxin and 135 possible furan structures, only 17 have 2,3,7,8-substitutionTable 1World Health Organization toxic equivalent factors (TEFs) for humans/mammals, fish and birds [37]CongenerHumans/MammalsFishBirds2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)1111,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDD)1111,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.50.051,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.10.011,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDD)0.10.10.11,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD)0.010.001<0.0011,2,3,4,5,6,7,8-Octachlorodibenzo-p-dioxin (OCDD)0.0001<0.00010.0012,3,7,8-Tetrachlorodibenzofuran (TCDF)0.10.0511,2,3,7,8-Pentachlorodibenzofuran (PeCDF)0.050.050.12,3,4,7,8-Pentachlorodibenzofuran (PeCDF)0.50.511,2,3,4,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF)0.10.10.12,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF)0.10.10.11,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF)0.010.010.011,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF)0.010.010.011,2,3,4,5,6,7,8-Octachlorodibenzofuran (OCDF)0.0001<0.00010.0013,4,4′,5-Tetrachlorobiphenyl (PCB 81)0.00010.00050.13,3′,4′,4′-Tetrachlorobiphenyl (PCB 77)0.00010.00010.052′,3,4,4′,5-Pentachlorobiphenyl (PCB 123)0.00010.0000050.000012,3′,4,4′,5-Pentachlorobiphenyl (PCB 118)0.0001<0.0000050.000012,3,4,4′,5-Pentachlorobiphenyl (PCB 114)0.0005<0.0000050.00012,3,3′,4,4′-Pentachlorobiphenyl (PCB 105)0.0001<0.0000050.00013,3′,4,4′,5-Pentachlorobiphenyl (PCB 126)0.10.0050.12,3′,4,4′,5,5′-Hexachlorobiphenyl (PCB 167)0.00001<0.0000050.000012,3,3′,4,4′,5-Hexachlorobiphenyl (PCB 156)0.0005<0.0000050.00012,3,3′,4,4′,5′-Hexachlorobiphenyl (PCB 157)0.0005<0.0000050.00013,3′,4,4′,5,5′-Hexachlorobiphenyl (PCB 169)0.010.000050.0012,3,3′,4,4′,5,5′-Heptachlorobiphenyl (PCB 189)0.0001<0.0000050.00001 Originally, attention was focused on the most toxic member of these substances, 2,3,7,8-TCDD. Gas chromatographic columns were soon developed that could separate 2,3,7,8-TCDD from the other 21 TCDD isomers in sample extracts [5], and other columns that could fortuitously separate the groups of congeners from each other by degree of chlorination (i.e., all tetrachlorinated dioxins/furans were eluted, followed by all of the pentachlorinated dioxins/furans, followed by the hexachlorinated, then the heptachlorinated, then the octachlorinated [6]. Many early publications reported a so-called congener group determination, where the total concentrations of all of the isomers from each group containing the same number of substituent chlorines were quantified and summed. Under this approach, 11 concentrations would be reported: total tetrachlorinated dioxins (T4CDDs), total pentachlorinated dioxins (P5CDDs), total hexachlorinated dioxins (H6CDDs), total heptachlorinated dioxins (H7CDDs), the octachlorinated dioxin (OCDD), the same dibenzofuran groups (T4CDFs, P5CDFs, H6CDFs, H7CDFs, OCDF), and 2,3,7,8-TCDD. This type of determination is often incorrectly termed a total homolog determination. Although “homolog” is still used today to refer to dioxins or furans that have different numbers of chlorine atom substituents, the chlorinated dioxin or furan series are not homologous, because the different members of the series are not formed by adding the same structural unit (Cl atom), but by substituting Cl for H in higher members of these series. The various chlorinated dioxins are correctly termed “congeners” (members of a like series), as are the chlorinated dibenzofurans. It was clear over 30 years ago that gas chromatography–mass spectrometry (GC–MS) was the instrumental method of choice for dioxin and furan determinations. The great tunable selectivity of mass spectrometry achieved by the ability to monitor specific characteristic ions in the mass spectrum of a compound combined with the retention time-matching ability of a gas chromatogram made GC–MS far superior to other detection systems. Even with the use of GC–MS, the exceptionally great toxicity of 2,3,7,8-TCDD meant that equally exceptionally low detection limits were required for this compound in real environmental samples. At such DLs as parts-per-trillion (ppt:10−12 g 2,3,7,8-TCDD per g of sample) or parts-per-quadrillion (ppq: 10−15 g 2,3,7,8-TCDD per g of sample), the principal barrier to successful analysis of samples was the separation of TCDD from the bulk sample matrix and from all other organic chemicals in the matrix. The keys to success in this endeavor were the number of separation steps in the entire analytical scheme, and their effectiveness. These separation steps included the following: Representative sampling (not included in this review);Separation of TCDD from the sample matrix (extraction); transfer of TCDD to an appropriate organic solvent;Separation of TCDD from all other organic coextractives (clean-up; can involve several distinct steps);Separation of TCDD from other relatively nontoxic tetrachlorinated dioxin isomers (gas chromatography);Separation and recording of characteristic TCDD molecular fragments using a mass spectrometer. Although an impressive degree of optimization of each of these steps has occurred over the past decade, this basic approach was formulated in the mid-1970s. A paper published in 1973 showed how high-resolution mass spectrometry (HRMS) was used to determine 2,3,7,8-TCDD in various sample types at the 1.0 ppt DL [7]. Modern dioxin/furan determination In recent years, methods have built on the early developments referred to above, and have achieved a high degree of sophistication and greatly improved accuracy and precision. The various stages of development of dioxin/furan determination can be generically described as follows: 1970s to 1980s The principal concern was to ensure correct identification of TCDD; the majority of analyses reported total concentrations of dioxin and furan (tetrachlorinated to octachlorinated) congener groups, and/or 2,3,7,8-TCDD; very few reliable analytical standards or certified reference materials were available; few laboratories worldwide could perform this work; 1980s to 1990s Fused silica open tubular GC columns predominantly used for this application; quality control for GC–MS methods enhanced greatly by the use of the isotope-dilution technique; GC-HRMS was accepted as “gold standard” method; introduction of “toxic equivalent factors (TEFs)” made it imperative to improve speciation of all dioxins/furans with chlorine substitution at the 2,3,7 and 8 ring positions; 10–20 or so laboratories worldwide were proficient in this work; 1990s to 2000s 13C-isotope labeled standards of all 2,3,7,8-substituted dioxins/furans available, as well as wide array of certified or consensus real-matrix reference materials; reliable methods available for precise, accurate isomer-specific determination of all 2,3,7,8-substituted dioxins/furans at ppt to ppq concentrations in virtually any sample matrix; most results for regulatory work reported as “toxic equivalent quantity (TEQ)” concentrations; coplanar PCBs began to be included in TEQ calculations; >100 laboratories worldwide are proficient in this work; Future focus Detection limits are low enough for most regulatory purposes, but may need to be pushed lower for bioaccumulation studies and determination of subtle effects from these compounds like changes in sex ratios; future improvements will be difficult due to ubiquitous background levels of these compounds and potentially interfering compounds at such low DLs; biggest challenges could be to lower the high cost and to improve the speed of analysis; bioanalytical methods may help, but are still of limited success after over 20 years of development; multidimensional orthogonal methods, such as two-dimensional chromatography (GC×GC, LC×GC ) or tandem mass spectrometry (MS/MS), may increase selectivity and also reduce detection limits, allowing for an increased number of analytes in a single analysis. Toxicology and TEFs Dioxin-like halogenated aromatic hydrocarbons (HAHs) provoke an extraordinarily broad spectrum of toxic effects in vertebrate laboratory animals. Exposure to a few micrograms per kg body weight of the most potent congener, 2,3,7,8-TCDD, leads to (among numerous effects) loss of body weight, liver necrosis, immune impairment, reproductive toxicity, teratogenesis and cancer in many laboratory species [8–11]. A single TCDD dose of 1 μg/kg is lethal to guinea pigs. In human populations exposed to HAHs by occupation or by industrial accident, the reported adverse health effects include reproductive disorders [12], developmental toxicity [13, 14], insulin insensitivity [15] and cancer [16, 17]. However, there is considerable controversy and uncertainty about the degree of risk to human health from dioxin-like compounds, particularly regarding cancer risk [18, 19]. There is no question, however, that TCDD can cause the severe skin disorder known as chloracne in some highly-exposed individuals [20]; chloracne is considered to be the “hallmark” of human dioxin toxicity. If we understand the mechanism by which a chemical produces its toxic effects, we are in a better position to assess the risk that the chemical poses to humans and to other species. Extensive research over the past quarter-century has demonstrated that all major toxic effects of dioxin-like chemicals are mediated by their binding to a soluble intracellular protein, the aryl hydrocarbon receptor (AHR) [21–23]. The AHR’s normal function is to regulate transcription of multiple genes that are important in development, physiologic function and adaptive responses to xenobiotic chemicals [24, 25]. Dioxin-like chemicals appear to exert their toxicity by dysregulating expression of key genes that are under control of the AH receptor [23]. Throughout most of its history, the science of toxicology has needed to cope with the effects of only one chemical at a time. HAHs present an unusual challenge to the toxicologist because “real-world” HAH exposure is not to single compounds; rather, HAH exposure involves complex mixtures of dozens to hundreds of congeners from the dioxins (75 possible), furans (135 possible) and PCBs (209 possible). The fact that dioxin-like compounds work through a common mechanism, the AHR [10, 23], fostered development of the “toxic equivalent factors” (TEF) approach to assessing the toxicological potency of HAH mixtures [26]. The TEF approach is based on a proposal first made by the Ontario Ministry of the Environment in 1984 [27]. In this approach, 2,3,7,8-TCDD, the most potent congener, is assigned a TEF of 1.0, and all other congeners are assigned TEFs that reflect their toxic potency relative to that of TCDD. For example, 2,3,7,8-tetrachlorodibenzofuran and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) each are assigned a TEF of 0.1, whereas octachlorodioxin has a TEF of 0.0001, reflecting its very low toxicity compared with that of 2,3,7,8-TCDD. The toxicity of a mixture is stated as TEQ (TCDD equivalents) and is assumed to be equal to the sum of the concentration of individual congeners multiplied by their potencies (TEFi) [28]: Note that the TEF approach applies only to agents that are dioxin-like in that their toxic effects are mediated by the AHR. Noncoplanar PCBs have different mechanisms of action and are not accommodated in the TEF approach. For simplicity it is assumed that all dioxin-like congeners produce toxic responses that are qualitatively the same and that the congeners differ only in their potency to produce these adverse effects, but this simplifying assumption should not be viewed as being true in a literal sense. It is true, however, that the toxic potencies of congeners within the dioxin-like category vary over several orders of magnitude. By definition, in order to calculate the total TEQ of a mixture we need to know the concentration of each dioxin-like component in the mixture. Hence the need for sensitive and reliable congener-specific chemical analyses. Regulatory decisions and risk management need to proceed even in the absence of perfect information. The TEQ approach is a useful simplifying strategy to allow risk assessment to move forward for exposures that involve complex mixtures, but it includes several assumptions [28–30]. There are two key issues: What is the proper TEF for each congener?What is the predominant mode of interaction between congeners when present in a mixture? The first question—what is the proper TEF for a particular congener?—has not been as straightforward as was initially envisioned. The main difficulty is that the estimated relative potency (REP) of congeners differs depending on what endpoint is measured, in what tissue and in what animal species. The relative potency estimates that underlie TEFs are derived from highly heterogeneous data sets [27] that require judgment and interpretation rather than providing numbers that can simply be plugged into an equation. The TEFs currently used in risk assessment are not based on a single biological endpoint. Rather, current TEFs were assigned in 1997 by an expert panel of the World Health Organization (WHO) who took into account multiple biochemical and toxicologic endpoints. The highest weight was given to studies done in mammalian systems in vivo (rather than in vitro), to chronic studies (rather than acute), and to studies that measured a toxic outcome (rather than a biochemical response). The relative potency estimates and resulting TEFs derived by the WHO panel are now undergoing refinement [27]. The second question—how do components in a complex HAH mixture interact?—is also not easily answered. The central premise in the equation used to calculate TEQ is that the toxic effects of components in the mixture are additive; i.e., that the overall toxicity of the mixture is the sum of the concentration of each congener multiplied by its TEF. There is experimental evidence to support the assumption of additivity [31, 32]. However, principles of receptor pharmacology predict that compounds which compete for the same receptor site may antagonize each other so that the overall response is less than additive. From the viewpoint of risk, of course it would be desirable for the toxicity of mixtures to be less than the sum of toxicities of individual components. Several studies have found antagonism between chemicals that act at the AH receptor site [29, 33, 34]—in other words, the standard TEQ approach in these circumstances overestimates the toxic potency of the mixture. It also is possible that components in a mixture might synergize to produce a response that is greater than that predicted by the additive model. Fortunately there is limited evidence of such an unwanted scenario for the toxicity of dioxin-like chemicals [35]. It is important to recognize that TEF values for some congeners can differ by tenfold or more among different animal species. For example, the TEF for 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin is ten-fold higher in fish than in birds (Table 1) [37]. When assessing risks to wildlife from exposure to HAH mixtures, we need to be aware that TEF values for most HAH congeners have been rigorously determined within only a very few nonhuman, nonrodent species. Risk estimates may be erroneous or misleading if TEF values from one animal species are assumed to be the same for other species. Congener-specific chemical assays for dioxin-like compounds have dramatically improved in sensitivity and reliability over the past two decades. Nonetheless, such assays remain expensive. Therefore, economical alternatives have been sought which would permit estimates of the potential toxicity of mixtures to be made. Bioassays such as the “chemically activated luciferase gene expression” assay (CALUX) assess the overall biological activity of a mixture and do not require knowledge of the identity or concentration of individual components of that mixture. The CALUX cell bioassay is predicated on the AHR being the mediator of toxicity. It tests sample extracts for their ability to increase expression of a luciferase reporter gene that is under the control of the AH receptor and thereby provides an index to the total equivalents of dioxin-like chemicals (CALUX–TEQ). As with chemical assays, sample extraction and sample clean-up are critical stages in bioassays such as CALUX. For some purposes, bioassays such as CALUX can provide a useful complement or alternative to congener-specific chemical assays. See Windal et al. [36] for an excellent review of the properties and applications of CALUX. Modern methods of analysis The “gold standard” for the analysis of dioxins and furans uses classical extraction techniques such as Soxhlet [38], liquid/liquid extraction [38], solid-phase extraction (SPE) [40, 41], or the more recent pressurized fluid extraction [42–44]. Once the extract has been transferred to a suitable solvent, a three-stage (silica, alumina and carbon) open-column clean-up is followed by GC–HRMS (employing isotope dilution) as the analytical finish. Minor variations on this method have been used since the late 1970s and it continues to be the method of choice for regulatory and legal purposes. A number of these methods are listed in Table 2. Table 2Regulatory methods for the analysis of dioxins, furans and DLPCBsMethodAnalytes/CommentsReferenceUSEPA 1613Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in water and wastewater. Uses isotope dilution–GC–HRMS[38]USEPA 1668a209 PCB congeners. 12 WHO dioxin-like PCBs by GC–HRMS, the remaining 197 by GC–MS[39]USEPA 23Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in incinerator stack gasses. Uses isotope dilution–GC–HRMS[45]USEPA 8290 (SW-846)Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in materials and waste. Uses isotope dilution–GC–HRMS[46]ISO 18073Equivalent to USEPA 1613, also allows GC–MS as an alternate detection method[47]ISO 17858Twelve WHO dioxin-like PCBs in environmental matrices by GC–HRMS [48]EN 1948Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in stationary sources by isotope dilution–GC–HRMS[49]MOE 3418Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals and 12 WHO dioxin-like PCBs by GC–HRMS. Uses isotope dilution–GC–HRMS[50]ENVCAN 1/RM/19Seventeen 2,3,7,8-substituted dioxins and furans and congener group totals in pulp and paper effluents by isotope dilution–GC–HRMS[51]JIS K0312Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals in wastewater by isotope dilution–GC–HRMS[52]JIS K0311Seventeen 2,3,7,8-substituted dioxins and furans including congener group totals in incinerator stack gasses by isotope dilution–GC–HRMS[53] Overview of analytical process The generic steps of the analytical process were listed above in the “Introduction”, except for the objectives, and data evaluation. These are mentioned here because of their great importance, but will not be discussed in detail. It is important to be clear about the objectives of dioxin/furan/DLPCBs determinations, because there are several choices in methodology—associated with substantially different costs—that generate data of differing precision and accuracy [54]. For example, for rapid on-site analysis for the purpose of directing a site remediation project, a substantially different methodology can be used compared to that required for legal action. Data evaluation will also not be discussed, but the authors would simply like to make the point that in spite of significant advances in software and data systems, the low-level determination of dioxins, furans and DLPCBs still requires detailed manual examination by experienced analysts of the raw data generated by GC–HRMS systems. A discussion of sampling is beyond the intended scope of this review, but it may often be the case that the imprecision of sampling is similar to or significantly greater than the combined imprecision of the remaining steps of the analytical process. Sample extraction methods See [55]. Classical extraction techniques like liquid/liquid extraction and Soxhlet extraction are described in detail in the methods listed in Table 2 and will not be addressed in this review. A number of alternative extraction techniques are summarized in Table 3. Supercritical fluid extraction (SFE) uses a gas above the critical point (the combination of temperature and pressure where the gas has liquid-like properties enabling enhanced extraction capability) to extract analytes from the matrix. The main advantage with SFE is that the extracting gas can be evaporated and so solvent disposal is not required. SFE behaves significantly differently to classical solvent extraction and recoveries can be reduced or enhanced. Pressurized liquid extraction (PLE), also known as accelerated solvent extraction (ASE) evolved from SFE. Solvents were added to SFE extractions as modifiers in order to mimic classical liquid extractions. SFE without extraction gas and only solvent modifier best matched classical extraction recoveries. This led to the development of PLE. Microwave-assisted extraction (MAE or MASE) uses closed vessels to increase pressure and extraction efficiency. Polar solvents like acetone or water are required to supply heat for extraction. Sun et al. [56] reported the analysis of wet samples by MAE, indicating that significant time was saved by the reduced sample drying requirements. Table 3Alternative extraction techniquesMatrixTechnique ReferenceSoil/sediment/solidPLE[4244]SFE[58]MASE[59]AqueousSPMD, passive sampling[60, 61]SPE[40, 41]AirSwipe/biofilms[62]Passive sampling[63, 64]Solid-phase extraction can significantly reduce analysis times and solvent usage for the extraction of water and waste water samples, even with significant particulate loadings [40]. Particles collected on C18 extraction disks can be extracted quantitatively without Soxhlet or PLE extraction. Water samples have also been analyzed using semi-permeable membrane devices (SPMD). SPMD sampling is a form of passive sampling that can be used for water, air or sediments. Namiesnik [57] has recently reviewed passive sampling techniques in environmental samples. Passive sampling is based on the free flow of analytes from the sampling medium to the collecting medium. This technique eliminates the requirement for power, and is a composite sampling procedure that can reduce analysis cost because of reduced sampling events and analyte loss during shipping and storage as the analyte is on a trapping medium. Swipe tests of biofilms on surfaces can also be used to monitor ambient conditions, especially after fires or from fugitive emissions. Sample extract clean-up methods Sample preparation of dioxin extracts is one of the most challenging in analytical chemistry. Many coextractable and potentially interfering compounds present in the raw extract must be removed in order provide maximum concentration factors (106 to 108) to meet DLs. A number of classical adsorbents can be used, including silica, alumina, Florisil and carbon. Most current procedures are based on the “Dow” [65] and “Smith-Stallings” [66] procedures. These procedures are described in detail in the references given in Table 2 above. Disposable C18/silica SPE cartridges [67] have been used for rapid clean-up of biological materials.The analysis of dioxins is very costly and time-consuming. Over the past 20 years, a number of modifications have been made to automate sample extraction and sample extract preparation to reduce analysis times and to attempt to reduce costs. Focant [42] has recently reviewed the automated sample preparation of biological samples. Multicolumn automated systems like the FMS (fluid management system) automated Power Prep system were developed to prepare human serum extracts, and have subsequently been used in a number of applications, including biota, water and food. Interchangeable cartridges (silica, alumina, carbon) can be used in different configurations [68, 69]. The addition of a PLE interface preceding the Power Prep enables the sample to be extracted and cleaned in the same automated run. Food samples (three) have been extracted, cleaned and analyzed in ten hours using this system [68]. An alternate method reported by Thal [70] used an automated gel permeation chromatographic (GPC)/carbon system. The GPC removes coextractables like lipids from the extract, which is then processed on activated carbon to separate the planar from the nonplanar compounds. This is based on a system reported by Norstrom et al. [71]. Van Beuzekom used an automated microwave solvent extraction system (MASE) coupled to a liquid chromatograph for the analysis of fine particles from fire emissions [59]. Nording [72] was able to modify a PLE extraction cell to insert the sample matrix above AX21 carbon packing in order to perform extraction and clean-up in the cell as a screening method. Results compared well with GC/HRMS confirmatory analysis.Sample preparation schemes are developed to remove most interferences from the sample extract. Unfortunately, because many compounds have similar physical and chemical properties, a considerable number of interferences often still remain in the cleaned sample extracts. Some examples of interferences on selected DLPCBs using a 5% phenyl column are listed below [50]. PCB congenerPotential interferencePCB 81PCB 87PCB 77PCB 110PCB 123PCB 149PCB 126PCBs 178 and 129PCB 156PCB 171PCB 157PCB 201 Gas chromatography columns and methods PCB interferences can be eliminated or removed physically as in MOE Method 3418, or by analyzing extracts on multiple columns (US EPA Method 1668). A number of analyte-specific columns have been developed to reduce both dioxin and PCB interferences and reduce the need for multicolumn analysis. The standard 5% phenyl columns exhibit multiple coelutions for PCBs (see above) and dioxins. The Rtx-Dioxin2 [73, 74] column has very few interfering compounds. BPX-DXN exhibits very low bleed and enhanced separation over 5% phenyl columns [75]. Liquid crystal columns [76, 77] provide outstanding separation for 2,3,7,8-substituted dioxins and furans, but suffer from high bleed and low column temperature maximum limits.Fast GC is accomplished by reducing inner column diameters and phase thickness. The number of theoretical plates per meter is increased significantly. If the phase ratio (inner column diameters/phase thickness) remains constant, the chromatography does not change and separations in micobore columns can be achieved much faster because of the shorter column lengths [78–80]. MacPherson [81] reported the use of simultaneous dual parallel column analysis of dioxins/furans/coplanar PCBs (Fraction A) on a 40 m DB-5 column and ortho-substituted PCBs (Fraction B) on a 20 m DB-5 column in under 45 min. Extracts were separated using activated carbon/silica. This combination can meet GC–HRMS QC specifications (e.g., US EPA 1613).Two-dimensional chromatography [82–84] has been investigated by a number of researchers in order to solve coelution issues from single-column analysis. A number of column combinations have been used to solve coelutions for dioxins and PCBs [85, 86]. GC×GC produces very narrow peaks that can provide significantly enhanced sensitivity, increased column capacity and reduced analysis times. It requires fast detection which pushes the limits of HRMS capability. A number of applications have been reported using TOFMS [85, 86], ECD [87, 88] ITMS [89] LRMS [90] and HRMS [178] as detectors for GC×GC. The potential for enhanced chromatographic selectivity and reduced analysis times has created a lot of interest in this area. Mass spectrometry detection methods See [91, 92]. HRMS has been the definitive detection method since the beginning of dioxin analysis (in 1973) [7]. Many detection techniques have been investigated [93], but none can match the selectivity and sensitivity of HRMS [94–97]. Tandem mass spectrometry as hybrid/MS [98], triple quadrupole MS/MS [99, 100] or ion trap MS/MS [85, 101] have been used to analyze dioxins and furans. MS/MS is more selective than HRMS for dioxins in most cases, because the parent molecule loses COCl, weighing 63 amu. No other halogenated organic has been shown to fragment in this way. The sensitivities of MS/MS instruments are typically less than HRMS, but with slight adjustments to sample size and final extract volume, GC–HRMS QC specifications can typically be achieved. Eppe [102] achieved 5:1 signal to noise with the injection of 200 fg of 2,3,7,8-TCDD. Unfortunately, the enhanced selectivity observed with MS/MS analysis of dioxins is not experienced with PCBs. The loss of Cl2 from the parent molecule is not unique to polychlorinated compounds and typically interfering peaks can be detected in the MS/MS chromatograms of PCBs.Electron ionization (EI) with reduced electron energy (∼35 eV) is the typical method of ionization used in dioxin/DLPCB analysis. The reduced electron energy focuses ion current towards the molecular ion, reduces the number of fragment ions, and subsequently enhances sensitivity. Negative ion chemical ionization (NICI or NCI) is also a low-energy ionization technique [103], which produces mass spectra with little fragmentation, resulting in enhanced parent ion signals. NCI analysis of dioxins, furans and PCBs has recently been investigated by Chernestsova using a variety of reagent gases [104]. Detection limits for PCBs are equivalent to or lower than for EI with a trend of increasing signal strength with increasing degree of chlorination. Similar results were observed for dioxins, except for 2,3,7,8-TCDD which is up to two orders of magnitude less sensitive in NCI than EI [105]. The molecular anion of 2,3,7,8-TCDD readily fragments to the chloride anion. Unfortunately, many other coextractables also fragment to the chloride anion, significantly reducing selectivity, and as a result NCI has not been used routinely for the analysis of dioxins and furans. Alternate methods of analysis Due to the challenging sample preparation, selectivity and ultra-low detection requirements, the cost of dioxin analysis is much higher than any other analytical method. Researchers have been searching for alternate low-cost methods for years. Immunoassay [106–110] and bioassay [111–114] methods can reduce costs by 50% or more and have been used to analyze food samples and samples collected from hazardous sites where very fast analysis is needed. The relative potencies for bioassays and cross-reactivities for immunoassay methods roughly mimic toxic equivalent factors used in mass spectrometric methods, enabling results to be obtained in TEQ directly. The main disadvantages of these types of methods are the inability to use labeled internal standards to correct for recovery and the determination of congener profiles for source apportionment. Spiked samples, similar to standard addition quantitative analysis, permit recoveries to be estimated, overcoming this problem. Recent publications have shown that bio/immuno methods compare well with standard GC/HRMS methods in terms of sensitivity, selectivity and accuracy [115–118].Most analytical laboratories are now being challenged to analyze for multiple analyte groups, including polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes, as well as dioxins, furans and DLPCBs. Multicomponent methods have been developed for a variety of matrices [119–122] in order to reduce costs accrued by multiple separate extractions, clean-up and instrumental analyses. Analysts must exercise caution when adding additional analyte groups to existing methods. Modifying methods to increase the number of analytes can result in compromised (less than ideal) conditions for a number of analytes, giving rise to potential interferences and/or reduced recoveries for these analytes. Quality control and data reporting Dioxin methods inherently include a number of quality control standards and samples. Isotope dilution (ID) methods are typically used with 13C-labeled internal standards. ID standards are added prior to extraction, allowing correction for recovery over the multistage clean-up procedures. The isotopically labeled standards can also act as markers for the identification of native analytes in samples. As the ID standards are often added at concentrations 10–100 times those expected for the analytes, the standards can act as “carriers” to improve recoveries of analytes at ultra-trace concentrations and can offset losses due to adsorption. The isotopically enriched standards aid in method development/validation, as they can be added at various stages of the analytical process to track down problems associated with analyte recovery or method bias.Until recently very few reference materials were available for method validation and performance evaluation. CRMs for sediments [123] and biota [124, 125] are now available.Uncertainty in analytical data is an important method attribute. The calculation of uncertainty is carried out using data from a number of sources, including instrument precision, linearity, fortified matrices, MDLs, spiked samples and interlaboratory data [126]. Uncertainties for dioxins, furans and dioxin-like PCBs are listed in Table 4 in percent (%) [127]. Uncertainties for dioxins and furans range between 15 and 20% for most analytes. The two most significant errors in the analysis are the error in the standards (±10%) quoted by the manufacturer and the error of calibration (typically ±20%) that is accepted by the analyst. Other errors, such as mass and volumetric determinations, should be less than 5%. Uncertainties are elevated for water samples over soil and biota due to the greater amount of labware that contacts the sample and the influence of particles on the homogeneity of the sample. Similar levels of uncertainty are observed for dioxin-like PCBs, except for PCB 105 and 118. These congeners are typically present at background levels of 20 and 50 pg, respectively, in the sample. Analyte losses from the extraction and sample preparation steps are corrected by the isotope dilution analytical technique and should not be significantly greater than the error in the addition of the surrogate internal standard. Table 4Uncertainty of dioxin, furan and DLPCB measurements in selected matrices, reported in percent (%) [127]ParameterCAS No.Soil N=59Biota N=34Water N=35Veg N=122,3,7,8-TCDD1746-01-6202619211,2,3,7,8-PeCDD40321-76-4251714211,2,3,4,7,8-HxCDD39227-28-6151318161,2,3,6,7,8-HxCDD57653-85-7171830291,2,3,7,8,9-HxCDD19408-74-3192131241,2,3,4,6,7,8-HpCDD35822-46-9122238261,2,3,4,5,6,7,8-OCDD3268-87-9151831202,3,7,8-TCDF51207-31-9152337231,2,3,7,8-PeCDF57117-41-6152032232,3,4,7,8-PeCDF57117-31-4171631271,2,3,4,7,8-HxCDF70648-26-9131529171,2,3,6,7,8-HxCDF57117-44-9141334211,2,3,7,8,9-HxCDF72918-21-9161335222,3,4,6,7,8-HxCDF60851-34-5211631241,2,3,4,6,7,8-HpCDF67562-39-4161332191,2,3,4,7,8,9-HpCDF55672-89-2111537161,2,3,4,5,6,7,8-OCDF39001-02-021271533N=56N=26N=10PCB 77 3,3’4,4’-TCB32598-13-3241414PCB 81 3,4,4’,5-TCB70362-50-423169PCB 105 2,3,3,’4,4’-PeCB32598-14-4287127PCB 114 2,3’,4,4’,5-PeCB74472-37-0191717PCB 118 2,3’,4,4’,5-PeCB31508-00-6259049PCB 123 2’,3,4,4’,5-PeCB65510-44-3232214PCB 126 3,3’,4,4’,5-PeCB57465-28-8281516PCB 156 2,3,3’,4,4’,5-HxCB38380-08-419239PCB 157 2,3,3’,4,4’,5’-HxCB69782-90-7322210PCB 167 2,3’,4,4’,5,5’-HxCB52663-72-6211213PCB 169 3,3’,4,4’,5,5’-HxCB32774-16-6251511PCB 189 2,3,3’,4,4’,5,5’-HpCB39635-31-9141712When assessing the overall error and/or uncertainty for determinations of dioxins and DLPCBs, two important factors are often overlooked. First, the sampling error is not investigated in many studies. This is very difficult to assess properly because it requires many more analyses to be performed, and the cost of this work is often prohibitive. Second, a growing trend is to report results as total TEQs, and sometimes the concentration data for individual congeners—used to perform the TEQ calculation—may not be reported at all. Also, more than one set of toxic factors exist, and some papers do not report which set of factors were used or the format (e.g., ND=0, ND=1/2) in the TEQ calculation. Baccarelli [128] has recently reviewed the reporting of TEQ values where non-detect values are used in the TEQ calculation. It should be remembered that the toxic factors used are themselves approximations, and may contribute significantly to the degree of uncertainty in the interpretation of results. Another unfortunate trend is that reconstructed chromatograms and/or congener patterns from analyses are being reported less and less. These patterns can be useful when assessing possible sources of the dioxins/DLPCBs detected. Sources, occurrence and temporal trends in PCDD/Fs Alcock and Jones [129] have previously reviewed the occurrence and temporal trends in PCDD/Fs in industrialized countries including Germany, the United States, Sweden, the Netherlands and the United Kingdom. This review covered trends in sediments, air, vegetation, soils, sewage sludge, livestock tissue and milk, wildlife and human tissues. The authors reported a general global temporal trend in PCDD/F inputs to the environment; PCDD/F contamination increased in the 1930s/1940s, reaching a maximum in the 1960s/1970s, and subsequently declined into the 1990s as a result of actions to reduce emissions. There is general agreement in the temporal trend data between European and North American studies; however, some European studies have not determined the post-1970 declines in PCDD/F contamination evident in North America. The US EPA [130] estimated a 75% decrease in PCDD/F emissions over the period 1987–1995. Päpke [131] reported declines ranging from 50–70% for PCDD/F levels in human adipose tissue, blood and breast milk in Germany over the period 1980–1996. There is also now a considerable body of literature reporting the presence of preindustrial baseline trace levels of PCDD/F contamination in soils and sediments prior to the turn of the nineteenth century [132–138]. Natural sources of PCDDs/Fs can include forest fires, volcanic activity, and other natural combustion processes [139, 140]. Although natural formation of PCDD/Fs had been postulated much earlier, some previous studies may have been compromised as a result of extraneous contamination of field samples or in the laboratory [133, 135, 136]. These measurements are significant in determining the relevance of natural sources vs. anthropogenic sources in order to assess requirements for further reductions in global PCDD/F emissions. However, it is also evident that any PCDD/F loadings from natural sources are dwarfed by contemporary emissions from anthropogenic sources. PCDD/F contamination in the North American Great Lakes has been recently reviewed by Norstrom [141], including occurrence, geographical distribution and temporal trends in air, water, sediments, fish, seabirds, snapping turtles and humans. Patterns and concentrations of PCDD/Fs in sediment indicate that atmospheric inputs dominate in Lake Superior, lower Lake Michigan and Lake Erie. Inputs from the Saginaw River to Lake Huron, and from the Fox River to upper Lake Michigan, are responsible for additional PCDD/PCDF loading to these areas beyond atmospheric deposition. Lake Ontario continues to be heavily impacted by input of PCDD/Fs, particularly 2,3,7,8-TeCDD, from the Niagara River [142]. According to Alcock and Jones [129], studies of sediment cores from the Great Lakes represent the bulk of sediment temporal trend data for PCDD/Fs (e.g., [143]); sediment cores are particularly useful when studying the chronology of PCDD/F contamination (e.g., [144–147]). North American Great Lakes sediment core and biomonitoring data generally show that PCDD/F contamination peaked in most lakes in the late 1960s to early 1970s, followed by rapid order of magnitude declines in the mid to late 1970s [144, 146–148]. The downward trend leveled off in some lakes in the 1980s, but appears to have continued after the late 1990s, presumably in response to remedial actions and reductions in PCDD/F emissions to the atmosphere [141]. Pearson et al. [148] reported that PCDD/F accumulation rates in the early 1990s ranged from <0.03 ng cm−2 y−1 for Lake Superior to 0.39 ng cm−2 y−1 for Lake Ontario; these rates represent 30–70% of maximum accumulation in the 1960s to 1970s. In his review, Norstrom noted the lack of comprehensive congener-specific PCDD/F data for Great Lakes sediment cores [141]. Cohen et al. [149] modeled atmospheric transport and deposition of PCDD/Fs to the Great Lakes using a United States and Canadian air emissions inventory based on data from 5,700 point sources and 42,600 area sources (Fig. 2). They identified municipal waste incineration, iron sintering, medical waste incineration and burning of hazardous waste in cement kilns as the most significant sources based on 1996 emissions data. Source areas up to 2,000 km removed from the Great Lakes were significant contributors. Combustion sources continue to be the predominant contributor to global ambient air levels of PCDD/Fs [150]. Subsequent long-range transport of combustion emissions are thought to be primarily responsible for the global ubiquity of PCDD/Fs [144, 151]. Fiedler recently reviewed global inventories of PCDD/Fs using national air inventories from 23 countries to formulate a global air flux estimated range of 7,500–13,000 g TEQs per annum [152]. Brzuzy and Hites estimated total global deposition from the atmosphere to land at 12,500±1,300 kg y−1 [151]. A study published in 2000 by Wagrowski and Hites using tree bark and soil samples estimated global deposition from the atmosphere to land at 2–15 t y−1 [153]. Fig. 2Geographical distribution of United States and Canadian dioxin emissions for 1996. Reproduced with permission from Cohen et al. [149] The recent report by Fiedler also showed sources of PCDDs/Fs in developed countries to be different from developing countries [152]. In developed countries, industrial activities are the primary source category, while open-burning activities are the dominant source category in developing countries. However, burn barrels and other household garbage burning methods are estimated to emerge as the greatest source of PCDD/F emissions in North America subsequent to implementation of new industrial air emission standards [154]. Open-burning processes include forest fires, pre- and post-harvest burning in agriculture, burning in landfills, and backyard burning. High releases were also estimated from incineration of medical waste. Emissions from municipal solid waste incineration (MSWI) were estimated to account for 70% of the PCDD/F burden from industrial sources in the United Kingdom in 1995 [129]; other significant sources included emissions from metal industries and coal combustion. Industrial emissions account for roughly 90% of the total inventory in the United Kingdom [155]. Other combustion sources contributing to PCDD/F emissions in industrialized countries include metal reclamation, wood burning, chemical fires, automobile exhaust, cement kilns and cigarette smoke. Non-combustion sources include chemical production (pesticides, perchloroethylene, pentachlorophenol), metal production, and pulp and paper mills. Recent data on PCDD/F emissions for individual industrialized countries has been reported for the Netherlands [156], the United Kingdom [157], the United States [130] and Canada [158]. A global PCDD/F emission inventory was reported by the United Nations Environmental Program [159]. The multimedia occurrence of PCDD/Fs globally in industrialized countries is fairly well documented; however, data are generally far less common than for other chemicals such as PCBs and organochlorine pesticides. UNEP [160] has produced a report on global trends in persistent toxics, including PCDD/Fs. Table 5 shows selected global data for levels of PCDD/Fs in a variety of matrices, based on information cited in the UNEP report. Table 5Selected global environmental concentrations of PCDD/FsMatrixRegionConcentration RangeReferenceRemarksAirEurope<1 fg TEQ/m3Typical ruralAirEurope15,000 fg TEQ/m3Contaminated areaAirAsia7–1,486 fg TEQ/m3AirSouth America3–394 fg TEQ/m3FreshwaterKorea0.001–1.061 pg TEQ/LFreshwaterJapan0.012–48 pg TEQ/L2,116 sitesSeawaterJapan0.005–3.9 pg TEQ/m312 sitesSeawaterJapanND–0.4 pg TEQ/m321 sitesSoilSeveso, Italy0.91–16 pg TEQ/gSoilGermany1–5 pg TEQ/g[143]RuralSoilGermany30,000 pg TEQ/g[143]ContaminatedSoilGermany10–30 pg TEQ/g[143]UrbanSoilThe Netherlands2.2–16 pg TEQ/g[143]RuralSoilThe Netherlands98,000 pg TEQ/g[143]ContaminatedSoilUnited Kingdom<1–20 pg TEQ/g[143]RuralSoilUnited Kingdom1,585 pg TEQ/g[143]ContaminatedSoilNew Zealand0.17–1.99 pg TEQ/g[143]RuralSoilNew Zealand260–6,670 pg TEQ/g[143]UrbanSedimentsLake Ontario91 pg TEQ/g[165]Lakewide averageSedimentsPo River1–11 pg TEQ/g[166]SedimentsLake Baikal0.03 pg TEQ/gSedimentsJapan16.1–50.7 pg TEQ/gSedimentsNew Zealand0.081–2.71 pg TEQ/gMarine sedimentsNorth Sea5.5–17.2 pg TEQ/gMarine sedimentsGulf of Finland101,000 pg TEQ/gHighly contaminatedMarine sedimentsCatalan coast0.4–8 pg TEQ/g[167]Marine sedimentsJapan0.012–49.3 pg TEQ/gVegetationGermany0.53–1.64 pg TEQ/gPine needlesVegetationAustria0.3–1.9 pg TEQ/gSpruce needlesBirds and animalsIndia19–24 pg/g fat[168]EagleBirds and animalsIndia150–200 pg/g fat[168]OspreyBirds and animalsIndia9.2–270 pg/g fat[168]Spotted OwletBirds and animalsIndia1,300–2,700 pg dioxins/g fat[168]Spotted Owlet liverBirds and animalsIndia620-1,000 pg furans/g fat[168]Spotted Owlet liverFishGreat Lakes<1–63 pg TEQ/g[169]FishGreat Lakes<1–59 pg TEQ/g[170]Dioxin TEQFishGreat Lakes<1–182 pg TEQ/g[170]PCB TEQFishFinnish coast165–329 pg TEQ/g lipid[171]Herring muscleFishAdriatic Sea0.23–329 pg TEQ/g lipid[171]Mackerel>Red mullet>AnchovyFishJapan Sea0.10–0.95 pg TEQ/gCodMarine mammalsBird Island2 pg TEQ/g blubber[172]Fur sealsMarine mammalsCanadian Arctic2–23 pg TEQ/g ww[173]Polar bearsMarine mammalsJapan Sea0.71–13 pg TEQ/g wwWhaleMarine mammalsJapan Sea17–360 pg TEQ/g wwWhale fatMarine mammalsAustralia0.1–2.6 ng/g wwBottlenose dolphinFoodEurope0.3–2.5 pg TEQ/g fat[174]Milk and dairy productsFoodNorth America0.3–0.9 pg TEQ/g fat[174]Milk and dairy productsFoodSouth America0.01–2.8 pg TEQ/g fat[174]Milk and dairy productsFoodAsia0.3–1.8 pg TEQ/g fat[174]Milk and dairy productsHuman milkThe Netherlands17.09–21.29 pg TEQ/g fatHuman milkUkraine8.38–10.16 pg TEQ/g fatHuman milkNorway7.16–7.43 pg TEQ/g fatHuman milkCzech Republic7.44–10.73 pg TEQ/g fatHuman milkBulgaria5.08–7.11 pg TEQ/g fatBlood/serum/plasmaGreat Lakes27.5 ng TEQ/L[175]Recreational fishersBlood/serum/plasmaIsrael26.6–32 ng TEQ/g fat[176]Blood/serum/plasmaA So, Vietnam16.6–45.9 pg TEQ/g lipid[177]Blood/serum/plasmaNew Zealand12.8 pg TEQ/g serum fat[143]1,834 samplesAll citations are UNEP (2003) [160] or individual UNEP Regional Assessments unless otherwise noted. ND denotes “not detected”; ww denotes “wet weight” Profiles of PCDD/Fs in abiotic matrices including air, soils, and sediments in some areas can reflect the source emission profile. However, a confounding factor in attributing PCDD/F contamination to specific sources can be a contrast in profiles between source samples and corresponding profiles in environmental sinks, e.g., soils and sediments, due to degradation, weathering, or transformation processes [161]. Eitzer and Hites observed a “consistent and systematic change” from a combustion source profile to a sediment profile as a result of long-range transport and depositional processes [162]. Tysklind et al. attributed differences in PCDD/F source profiles and air profiles to transformation processes including photolytic degradation or hydroxyl radical reactions [163]. Koester and Hites reported that wet and dry deposition are important mechanisms for scrubbing PCDD/Fs from the atmosphere, and that these mechanisms contribute to the predominance of OCDD in sediments [164]. Ultimately, the PCDD/F profile in environmental samples can be influenced by both source type and environmental processes. Studies of congener and homolog profiles for source apportionment have been carried out in abiotic matrices; biota have a tendency to accumulate primarily the 2,3,7,8-substituted compounds due to non-2,3,7,8-substituted congeners being metabolized [129, 161]. There are numerous examples of studies of the variation of PCDD/F congener and homolog profiles with source (e.g., [161, 144–147]); these studies have also illustrated how different sources of PCDD/Fs have influenced temporal trends, primarily according to production, use and disposal of chlorinated organics. Principal component analysis (PCA) of profiles of source and sink samples have also proven to be a valuable tool for source apportionment [146, 147, 161, 163].

Clin_Auton_Res-3-1-1797061

Diversity of sympathetic vasoconstrictor pathways and their plasticity after spinal cord injury

Sympathetic vasoconstrictor pathways pass through paravertebral ganglia carrying ongoing and reflex activity arising within the central nervous system to their vascular targets. The pattern of reflex activity is selective for particular vascular beds and appropriate for the physiological outcome (vasoconstriction or vasodilation). The preganglionic signals are distributed to most postganglionic neurones in ganglia via synapses that are always suprathreshold for action potential initiation (like skeletal neuromuscular junctions). Introduction The sympathetic nervous system supplies blood vessels throughout the body with vasoconstrictor axons that control local blood flow and contribute to total peripheral resistance. Signals arising in the central nervous system send specific controls to different tissues according to the functional response for each organ by means of reflexes and patterned regulation from higher centres [14]. The sympathetic outflow arises in the thoracolumbar cord, where it is particularly vulnerable to spinal cord injuries, being damaged directly if the lesion involves regions between T1 and L3 or the ventral roots at these levels, and being disconnected from the higher control centres by injuries to descending pathways in the cervical cord. The degree of disordered visceral and vascular function that ensues depends on the level and the severity of trauma in each individual. The great majority of all sympathetic preganglionic neurones are vasoconstrictor in function and make synapses in ganglia of the paravertebral chain from which they project through grey rami and spinal nerves to the vasculature of the head, neck, trunk and limbs. Because the sympathetic outflow is restricted to thoracolumbar segments, preganglionic neurones in the upper thoracic segments control vessels in the head and neck and those in the lowest thoracic and upper lumbar segments control vessels in the pelvic organs and hindlimbs. Thus, for example, postganglionic neurones in the superior cervical ganglia receive inputs from T1-4 segments [25] whereas inputs to neurones in the lower lumbar paravertebral chain projecting to the hindlimbs mostly arise in the last two segments of the thoracolumbar sympathetic outflow [1]. Many visceral vasoconstrictor neurones also lie in the paravertebral chain and send their axons to the viscera via the various splanchnic nerves. The properties of these pathways supplying the vasculature and their modification after spinal cord injury (SCI) are the main topics of this review. Patterns of natural activity in sympathetic vasoconstrictor pathways Impulses travelling in sympathetic vasoconstrictor pathways arise in preganglionic neurones by integration of excitatory and inhibitory signals that arise supraspinally, intersegmentally, and segmentally. In vasoconstrictor pathways, the patterns of ongoing or tonic firing of preganglionic neurones originate in the rostral ventrolateral medulla [3]. However many descending excitatory glutamatergic axons and inhibitory axons containing gamma-amino-butyric acid and/or glycine that arise both locally and supraspinally impinge on preganglionic neurones [17]. These inputs may be modulated by amines and peptides co-released from these pathways [26]. The preganglionic neurones integrate reflex signals from e.g. arterial baroreceptor and chemoreceptors via the brainstem, cutaneous and visceral nociceptors at spinal levels, signals arising from higher levels of the CNS, etc. Strong reciprocal links between vasomotor and respiratory centres in the brainstem, together with blood pressure swings related to breathing, mean that vasoconstrictor activity tends to be linked to the respiratory cycle as well as the cardiac cycle [8, 10, 19, 32]. The final output frequency and pattern of discharge in individual sympathetic axons is modulated by the membrane properties of the preganglionic neurones. Action potentials in these neurones are triggered by the summed depolarization produced by many small excitatory inputs [5, 12, 29] combined with inhibitory synaptic potentials arising from both local spinal interneurones and direct descending inhibitory pathways [6, 7]. The frequency of preganglionic discharge is limited to short bursts at <10 Hz by a large Ca2+-activated K+ conductance (and ensuing afterhyperpolarization) that follows each action potential, together with a prolonged transient (A-type) K+ current [4, 29]. The average ongoing preganglionic discharge in vasoconstrictor pathways in anaesthetized animals is 0.5–1 Hz [14]. The natural activity of individual postganglionic neurones projecting to different target tissues has been described by recording extracellularly spontaneous and evoked signals from sympathetic axons in peripheral nerves projecting to a known target tissue [14, 35]. Ongoing activity of individual units is recognized by the spike configuration and the unit functionally characterized by its responses to natural stimuli. Physiologically relevant responses are recorded from e.g. presumed sudomotor axons in cutaneous nerves immediately preceding secretion from sweat glands. Perhaps surprisingly, when simultaneous recordings are made from skin and muscle vasoconstrictor axons, the responses are generally opposite. For example, baroreflex inhibition is powerful in muscle vasoconstrictor (MVC) axons but weak or absent in cutaneous vasoconstrictor (CVC) axons. Chemoreceptor activation stimulates MVCs but inhibits CVCs, nociceptor stimuli excite CVCs but inhibit MVCs [13, 14]. These differences reflect what is known about blood flow through skin and muscle, with the former involved primarily in temperature regulation and the latter in regulation of peripheral resistance at rest. Similar differences in skin and muscle vasoconstrictor axons are observed in microneurographic recordings from conscious humans in which the excitatory effects of emotion on CVCs and mental stress on MVCs are revealed [33, 34]. The importance of these observations is that they make the concept of a generalized sympathetic vasoconstrictor “tone” inappropriate. When this could be studied, the patterns and frequencies of preganglionic discharge have convincingly parallelled those of the postganglionic projections in the same pathway. Thus the average postganglionic discharge frequency in vasoconstrictor pathways is 0.5–1 Hz [13, 14]. Transmission of vasoconstrictor signals through sympathetic ganglia The similarity between pre- and postganglionic discharge raises questions about the purpose of the synapses in sympathetic ganglia. Each preganglionic vasoconstrictor neurone branches to form synapses with multiple postganglionic neurones in several adjacent ganglia in the paravertebral chain. However only a subset of inputs (generally those that arise in one spinal segment) transmits the centrally derived command signal to the postganglionic neurones. This results because, in individual postganglionic neurones, transmission occurs in an all-or-none manner from a single input, as at the skeletal neuromuscular junction [21]. By recording intracellularly, and recruiting preganglionic inputs using graded stimuli, each postganglionic neurone can be shown to receive one “strong” excitatory input (rarely 2 or 3) that is always suprathreshold for action potential initiation, as well as several subthreshold excitatory inputs. (There are no inhibitory synapses in sympathetic ganglia.) As the safety factor for transmission at most ganglionic synapses is very high, high concentrations of nicotinic antagonists are necessary to block ganglionic transmission. The important role of the strong input has been confirmed in intracellular recordings in vivo [22, 30], which show that subthreshold inputs rarely summate, due to both the low preganglionic firing frequency and the large afterhyperpolarization after the action potential (again due to a Ca2+-activated K+ conductance) triggered by the strong input. In practice, subthreshold inputs appear largely redundant. The total number of preganglionic inputs parallels the number of dendrites of the postganglionic neurone and these increase with animal size [27]. It is anticipated that each human paravertebral neurone receives at least 20 synaptic inputs, but that, in most cases, only one of these is functionally effective. The connectivity described above means that ganglionic synapses in vasoconstrictor pathways simply relay the centrally-derived signals to the periphery without alteration. Thus the ganglia act primarily as distributors of the central pattern of impulses, each preganglionic axon activating many postganglionic neurones. Teleologically, this can be seen to reduce the need to provide large numbers of preganglionic neurones in the spinal cord to control the extensive vasculature throughout the body. However, although the preganglionic axons diverge to contact many ganglion cells (∼150 in the rat, [28], the redundancy of many of these contacts means that the functional autonomic equivalent to the motor unit is much smaller (only ∼15 in rat, perhaps >100 in humans). Changes in vasoconstrictor outflow after spinal cord injury In microneurographic recordings from skin and muscle nerves in conscious relaxed intact humans (Fig. 1A), recordings from sympathetic axons in muscle nerves show bursts of activity in rhythm with the arterial pulse wave that wax and wane with respiration [31], reflecting the cyclic inhibition of MVCs by baroreceptor input. Such bursts are rarely recorded in CVCs where ongoing activity is largely unsynchronized. During simultaneous recordings from skin and muscle fascicles, brief electrical stimulation of skin leads to a burst of activity in the CVCs but inhibition of ongoing bursts in MVCs. This reflects the reflex activation of CVCs and inhibition of MVCs by nociceptor stimuli. Fig. 1Extracellular recording of multiunit sympathetic activity in skin and muscle nerves of conscious humans. (A) from intact individual; (B) from a subject with complete spinal cord transection at C6. At arrows, the skin over the forearm was stimulated electrically. Note the absence of ongoing activity in both nerves and the synchronous activation by the stimulus after spinal cord injury. Modified from Stjernberg et al. (1986) Such paired recordings from leg nerves of people with thoracic spinal cord injury, showed completely different responses (Fig. 1B). The cardiac related bursts were absent and the signals from both nerves showed little background activity. Electrical stimuli to the skin produced identical brief bursts of activity in both skin and muscle nerves. These observations reflect the removal of both descending sympathetic drive with periodic baroreceptor inhibition and the conversion of the nociceptor-evoked reflex in MVCs from inhibitory to excitatory. This reversal of the spinal nociceptor reflex has also been demonstrated in chronic spinal cats [15]. It should be noted that sympathoexcitation following nociceptor stimulation was transient without evidence of enhancement or prolongation of the reflex sympathetic discharge although the vasoconstriction was prolonged. Changes in connectivity in paravertebral ganglia after destruction of preganglionic neurones The consequences for the remaining sympathetic pathways of damage to preganglionic neurones or their axons by a spinal injury can be studied in experimental animals by partially denervating the paravertebral ganglia and observing the types of connection made by the remaining terminals. Extensive collateral sprouting of the remaining preganglionic axons occurs [16, 23]. Transection of the descending lumbar paravertebral chain just above the last segmental preganglionic outflow left postganglionic neurones in the next more caudal lumbar ganglion with <1 input, the great majority being subthreshold (Ireland 1999); the residual preganglionic input in the last sympathetic segment formed strong inputs on only 10% of the neurones. After 4–5 weeks, however, 70% of neurones in the same ganglion had a large strong input as well as ∼2 subthreshold inputs. Thus. the remaining preganglionic neurones had sprouted and preferentially formed strong inputs, even on neurones that were completely denervated by the lesion. New strong inputs must have formed on some neurones with a peripheral function that differed from that of the original preganglionic input [16]. The formation of such aberrant strong inputs after SCI might explain more generalized vasoconstriction, or the activation of sudomotor neurones by reflexes normally directed at peripheral vessels. Autonomic dysreflexia While modified vascoconstrictor reflexes in spinally-injured people can be mimicked in experimental animals, they do not predict the dramatic and life-threatening rises in arterial blood pressure that are known as autonomic dysreflexia. These uncontrolled hypertensive episodes are usually triggered by overdistension of bladder or bowel but many also arise from nociceptor activation from a pressure sore or other unheeded injury below the spinal cord injury [20, 36]. Such episodes are usually relieved by removal of the stimulus but, if the trigger is unknown, treatment with vasodilators requires hospitalisation which is inconvenient and expensive. Other symptoms of dysreflexia are excessive sweating and muscle spasms. Dysreflexia is typically seen only if the lesion is at or above T5-6. It is generally believed that the unrestrained hypertension results from the absence of descending inhibitory control by baroreceptors and clearly the loss of this reflex control is highly relevant. The reason that T5 is critical as the cut-off for dysreflexic episodes is presumably because baroreflex vagal slowing of heart rate and inhibition of intact sympathetic pathways only to the head, neck and arm vasculature are not sufficient to buffer increases in vascular resistance below the injury. The simultaneous activation of both cutaneous and muscle vascular beds below the injury would contribute a large increase in peripheral resistance, and the effect would be more dramatic if the visceral vasculature is also excited reflexly. There is a prevailing view that the splanchnic circulation must be involved as it is controlled largely from below T5 [36]. Animal experiments have suggested that primary afferent nociceptor inputs (containing calcitonin gene-related peptide but not Substance P) sprout within the cord around the lesion [36], probably due to the release of neurotrophins by inflammatory cells in the segments around the lesion site. However, as mentioned above, sympathetic discharge following brief stimuli in spinally-injured people is not particularly large or prolonged, suggesting that the expansion of afferent connections is not responsible for much enhancement of discharge. Thus the mechanisms underlying the pronounced vasoconstriction during AD have been unclear. Changes in vascular reactivity after spinal cord injury If the sympathetic discharge after SCI is very much reduced and the reflex discharges provoked by e.g. bladder stimulation are not particularly marked, why does dysreflexia develop? The prolonged vasoconstriction might be explained if neurally evoked vasoconstriction was in some way potentiated. This might result from the potentiated release of transmitter per impulse, as has been shown at ganglionic [9] and central [24] synapses when ongoing activity is blocked. To test this idea, isolated vessel segments were mounted in a myograph and contractile responses evoked by short trains of supramaximal transmural electrical stimuli at 0.1–5 Hz, frequencies within the physiological range. Vessels were isolated from rats with spinal transection at T4 or T8, leaving all parts of the sympathetic pathways intact, and compared with those from age-matched and sham-operated controls. After 2 or 8 weeks, the responses of cutaneous and splanchnic arteries to stimulation of the perivascular nerves were greatly enhanced (Fig. 2). Fig. 2Contractile responses of segments of (A) cutaneous (tail) artery and (B) splanchnic (mesenteric) artery isolated from (above) control rats and (below) rats 7–8 weeks after spinal transection at (A) T8 and (B) T4. Trains of the same number of transmural electrical stimuli at different frequencies (indicated by bars at bottom) were presented to each vessel to activate the sympathetic postganglionic terminals. Responses in vessels from spinalized rats were greatly enhanced compared with controls. Redrawn from (A) Yeoh et al. (2004a) and (B) Brock et al. (2006) Transmission in small diameter blood vessels is known to occur primarily at neurovascular junctions on the outer layers of smooth muscle cells [11, 18]. Even when the relation between the postganglionic varicosities and the effector tissue is not direct, it has been shown in all preparations so far investigated that responses to nerve-evoked and to exogenous transmitters do not involve the same receptors or postreceptor mechanisms [11]. The enhanced effect of endogenous neurotransmitters on blood vessels after spinal transection might involve one or more of a number of mechanisms (increased norepinephrine (NE) release, decreased NE clearance, increased junctional receptor sensitivity, increased postreceptor responsiveness), only some of which can be tested directly. Rather unexpectedly, the cellular mechanisms underlying the enhanced responsiveness to NE released from postganglionic nerve terminals were not the same for each vessel. In the rat tail artery, in which contractile responses to nerve stimulation were enhanced more than 10-fold, most of the changes in neurovascular transmission were postsynaptic, involving an increased reactivity of the smooth muscle [38]. There was some maintained increase in sensitivity of α2- but not α1-adrenoceptors to exogenous NE. In contrast, in the mesenteric artery, where transmission was increased 5-fold, most of the changes were presynaptic and involved a decrease in the activity of the NE transporter that takes the released NE back up into the postganglionic terminals [2]. Differences between the vessels in the ability of α-adrenoceptor and purinoceptor antagonists to inhibit nerve-evoked responses imply that the mechanisms of neurovascular transmission are selective for different vascular beds. To test whether the enhanced responsiveness resulted from the removal of ongoing activity in the postganglionic axons, activity to the tail artery was prevented in normal animals by cutting the lumbar paravertebral chain to disconnect the preganglionic input to the postganglionic neurones (decentralization, Fig. 3(2)). Neurovascular transmission in the tail artery was enhanced after decentralization in the same way as after spinal transection [37]. Notably, sensitivity of the vascular muscle to α1-adrenoceptors was not modified. Even after denervation of the tail artery (by cutting the postganglionic nerves, Fig. 3(3)) to remove the perivascular terminals (and thus the NE transporter), the responses to phenylephrine were much more enhanced relative to control than those to another α1-agonist, methoxamine, that is not a substrate for the NE transporter. These observations emphasize the significant role of the postganglionic nerve terminals in reducing the effective concentration of catecholamines applied to the outside of vascular preparations. Fig. 3Diagram showing sites of lesions used to study long term effects on neurovascular transmission in arterial vessels of rats. (1) Transection of thoracic spinal cord without damage to preganglionic neurones. (2) Transection of paravertebral chain to remove preganglionic inputs (decentralization). (3) Transection of postganglionic nerves to denervate artery. Segments of artery were removed from the animals after 2–8 weeks and contractile responses to stimulation of perivascular sympathetic nerves were recorded during exposure to adrenoceptor antagonists and other drugs. that selectively interfere with neurovascular mechanisms Overall, the data so far suggest that blood vessels in spinalized animals would constrict more powerfully to even short bursts of sympathetic activity evoked reflexly below the lesion. As well as being consistent with the complaints of many spinally-injured people about cold feet and legs, and the common difficulties in healing pressure sores, this modified vascular reactivity could contribute strongly to the development of autonomic dysreflexia in humans. Conclusions Despite many years of investigation, we still know remarkably little about the way in which neurally-released transmitters, including NE, lead to constriction of vascular smooth muscle. It is now clear that, as for many autonomically-innervated tissues, the mechanisms are not the same as those activated by exogenous transmitters. The work discussed here emphasizes the diversity of mechanisms that underlie the neural control of arterial vessels in different vascular beds and the plasticity of both innervation and effector tissues after lesions to the nervous system. Marked changes can occur even when both pre- and postganglionic components of the sympathetic innervation are undamaged. While the findings in experimental animals need to be confirmed in spinally injured people, they may help us to devise better ways to improve their rehabilitation and long-term maintenance. In addition, we may need to revise our thinking about the role of sympathetic activity in the regulation of the cellular processes involved in neurovascular transmission, even in intact individuals.

Soc_Psychiatry_Psychiatr_Epidemiol-3-1-2039804

Prevalence and predictors of health service use among Iraqi asylum seekers in the Netherlands

Background A long asylum procedure is associated with higher prevalence rates of psychiatric disorders, lower quality of life, higher disability and more physical health problems. Additional knowledge about health seeking behavior is necessary to guide governments and health professionals in their policies. Introduction Mental health service use differs among populations and geographical areas [3, 15, 25]. Refugees and asylum seekers have high prevalence rates of psychiatric disorders [6, 12] and adequate use of health services is important. Literature on this issue is growing, but still limited [8, 9, 16]. Health policies, including accessibility of service for asylum seekers differ between European Union countries [19]. In the Netherlands refugees have direct access to the general practitioner, while asylum seekers can only enter the curative health care system after a screening by a nurse or doctor in the asylum seeker center. This system has been criticized [18], but others claim that these professionals, involved in triage after receiving training in cultural competence, may prevent inadequate referrals. Health service use, according to the Anderson behavioral model [1, 2], is a function of three sets of variables: predisposition, enablement and need. Predisposition includes demographic factors such as age, gender, religion, cultural factors, social network, and support. Enabling factors facilitate service use and include individual social functioning, availability of services, and costs. The third set, i.e. the need variables, consists of health related factors. Both objective (type and severity of a health problem), and subjective health indicators (disability and perceived health) belong to the need variables. McCracken et al. [15] used the above model in a community survey on depression in five European countries, and found that severity of depression, lower perceived health status, social dysfunctioning, and low level of social support were significant predictors of use. Kamperman et al. [11] used the model in a study among migrants in Amsterdam and found that there were migrant-specific mechanisms in health care consumption. Higher levels of acculturation and lower level of cultural traditionalism increased the use of mental health care facilities. In these health service use studies, a variety of potential predictors, such as psychopathology, physical diseases, physical, and mental well-being, have been included. But disability and quality of life were not included. Nor is there a study that compared groups from the same country of origin, that differ in length of stay in a host country. In a national community study among Iraqi asylum seekers (n = 294) in the Netherlands [12–14] we measured prevalence rates of psychiatric disorders, quality of life, disability, and physical health problems in relationship with pre- and post-migrations stressors. The study focuses on the risks of a long asylum procedure and showed that asylum seekers who stayed more than 2 years in the Netherlands had significantly higher overall prevalence rates of psychiatric disorders (66.2%), than those who arrived recently (42.0%). A long asylum procedure was an important independent risk factor for a psychiatric disorder with an odds ratio of 2.16 (CI 1.15–4.08). This ratio was higher than the odds ratio for premigration stressors, such as exposure to human rights violations. In addition the ‘long stay’ group had significantly lower quality of life, higher disability, and higher levels of physical health complaints. In the Anderson model these health indicators can be considered as need factors. The aim of this paper is to examine patterns and predictors of health service use and their relationship with length of stay. The research questions are: (a) what is the prevalence of service use of Iraqi asylum seekers in the Netherlands, (b) is a long asylum procedure associated with a higher prevalence of service use, (c) what is the relationship between psychopathology and service use, and (d) which predisposing and other need variables predict service use. We hypothesize that a long asylum procedure is associated with a higher prevalence of service use and that higher levels of psychopathology are related to higher levels of service use. Methods A comprehensive description of used methods is provided in a previous article [12]. A summary is given below. From the entire population of adult Iraqi asylum seekers, two groups were selected based on their length of stay in the Netherlands. Personal data on these groups was obtained from the Agency for the Reception of Asylum Seekers (COA). Group 1 was selected on the criterion that persons had been living in the Netherlands for less than 6 months (between September 2000 and November 2001). From the randomly selected 362 respondents, data could be used from 143 interviews. Group 2 was selected on the criterion that they were living in the Netherlands for at least 2 years. On the chosen date of May 31st, 1999, the COA found that 2,352 Iraqi asylum seekers fulfilled this criterion. From the randomly selected 474 respondents, data could be used from 151 interviews. The questionnaire about service use—in the 2 months prior to the interview—included regular services as well as alternative services. The studied regular services are: out-patient services: preventive healthcare (nurse/doctor in center), primary healthcare (general practitioner), generic healthcare (medical specialist, non-psychiatric), social care (social worker), psychiatric services (mental health professional); in-patient services: hospital admission physical health, hospital admission mental health; use of drugs (any drugs, hypnotics, anxiolytics and analgetics). Examples of drug names were given to be sure the drugs were put in the right category. As mentioned above, in the Netherlands medical staff is present in all asylum seekers centers. They perform health assessments of recently arrived asylum seekers, facilitate entry into primary health care, and refer to the general or mental health services. Their services can be classified as gateway services [7]. The studied alternative services are: use of religious helpers or rituals, and herb-doctors or herbs. Respondents’ predisposition to service use was measured by age, gender, religion, ethnicity, and length of stay (membership Group 1 or Group 2). Enabling factors were not measured: regular health services for asylum seekers are available and accessible in the Netherlands without financial obstacles. Need factors include: psychiatric disorders, physical health, quality of life, and disability, while post-migration living problems (PMLP) were added as a special set of need variables. Psychiatric disorders were measured with the WHO-CIDI, version 2.1 [28] and cluster diagnoses were used in the analyses. Physical health was assessed with 22 items, dealing with: perceived physical health, physical handicaps, chronic physical diseases (e.g. lung disease, epilepsy, diabetes), and chronic physical complaints (e.g. stomach problems, joint problems, headache more than 3 months). Quality of life (Qol) was assessed with the WHOQOL-BREF [22]. The first two single questions i.e.: ‘How would you rate your quality of life?’ (‘overall Qol’) and ‘How satisfied are you with your health?’ (‘Qol perceived general health’) were used in the current study. Disability (Brief Disability Questionnaire, VonKorff [26] was measured in two dimensions: total disability (total of score of 11 items on physical and social role impairments), and the total number of days with serious impairment in the last month (BDQ-days). Post-Migration Living Problems (PMLP) were assessed with a checklist, adapted from Silove et al. [21]. The 24 items were clustered, based on a factor analysis [13] as family issues, discrimination, asylum procedure, socioeconomic living conditions, and religious aspects. The items ‘lack of work’ and ‘work below level’ loaded on different factors and were analyzed as separate items. The used Iraqi-Arabic composite questionnaire is based on a Palestinian-Arabic version [4, 5] and was culturally validated and translated with the help of a focus group. Oral interviews were taken by trained Iraqis. Statistics Differences between the two groups with respect to socio-demographics, psychiatric disorders, service use, and drug consumption were calculated with the χ2-test. Univariate relationships between predisposing and need variables were assessed with a correlation matrix. Ethnicity, religion, and marital status did not show a significant (P < 0.05) correlation with any of the health services. All other variables were entered into multivariate logistic regression analyses as: predisposing variables (study group, sex and age), need variables (one or more psychiatric disorders, overall quality of life, perceived quality of general health, disability (2 items), physical health (2 items), and a special set of need variables: the PMLP (7 variables). The same independent variables were used in each analysis, in line with the Anderson model. The dependent/outcome variables were: use of preventive service (nurse/doctor in the center), general practitioner, medical specialist (non-psychiatrist), social worker, mental health professional, and use of any drugs. We used a three-step procedure: in the first step each set of variables (predisposing factors, need factors and PMLP) was analyzed separately; in the second step all variables entered one analysis, that way the risk of one variable was corrected for the risks of all other variables. In step one and two, the entire dataset (n = 294) was used. In the third step the analysis was done for Group 1 and Group 2 separately in order to assess the differences of predictors between asylum seekers that had recently arrived (Group 1) and those who had stayed for more than 2 years in the asylum procedure (Group 2). Adjusted Odds Ratios (ORs), 95% confidence intervals (CIs), and P values were calculated (only the ratios with confidence intervals higher or lower than 1 are shown in the tables). Differences were considered significant at P < 0.05. All analyses were performed with SPSS version 10 [20]. Results Socio-demographics, health and health related variables The two study groups differed on several socio-demographic characteristics (Table 1). Group 1 contained more subjects younger than 24 years of age, and more females. The average time of stay in the Netherlands of Group 2 was more than 3 years. On the characteristics literacy, social status in Iraq, and psychiatric problems in the family, the two groups did not differ (not shown in the table, c.f. [12]. Group 2 had higher scores on prevalence of psychiatric disorders, disability and physical complaints, and lower quality of life score (Table 1). Except social-religious items, all clustered and non-clustered post-migration living problems were significantly higher in Group 2. Table 1Socio-demographic, health characteristics and post-migration living problems in at random samples of Iraqi asylum seekers arrived <6 months (Group 1) versus >2 years (Group 2) in the Netherlands, 2000–2001VariablesGroup 1 (n = 143)Group 2 (n = 151)Total (n = 294)P-valueStay in months (mean, SD)2.51 (1.16)36.77 (6.30)20.12 (17.76)P < 0.0005, t(292) = 63.66Sex (%)P < 0.0005, χ2(1) = 27.31    Male49.778.864.6    Female50.321.235.4Age (%)P = 0.003, χ2(4) = 16.35    18–24 years21.79.315.3    25–34 years42.049.045.6    35–44 years14.725.820.4    >45 years21.715.818.7One or more psychiatric disorder (%)42.066.254.4P < 0.0005, χ2(1) = 17.44Overall quality of life (mean, SD)a2.88 (0.99)2.23 (1.14)2.55 (1.11)P < 0.0005, Z(294) = −5.29Perceived Qol general health (mean, SD)b3.06 (1.15)2.74 (1.27)2.89 (1.22)P = 0.017, Z(294) = −2.39Physical and role disability (mean, SD)c17.31 (7.43)19.25 (6.77)18.30 (7.15)P = 0.020, t(292) = −2.34Days of disability (mean, SD)d5.37 (8.24)7.68 (9.17)6.56 (8.80)P = 0.024, t(292) = −2.27Physical diseases (mean, SD)e0.85 (1.18)0.84 (0.98)0.85 (1.08)n.s.Physical complaints (mean, SD)e0.83 (1.38)1.62 (1.58)1.23 (1.54)P = 0.0005, t(292) = 4.52Post-migration living problems    Family related issues (mean, SD)f36.54 (26.55)52.81 (24.24)44.90 (26.62)P < 0.0005, t(292) = −5.49    Discrimination (mean, SD)f2.05 (7.92)11.17 (21.01)6.74 (16.65)P < 0.0005, t(292) = −4.87    Asylum proc. related issues (mean, SD)f48.58 (25.35)60.13 (23.23)54.51 (24.97)P < 0.0005, t(292) = −4.07    Socioeconomic living cond. (mean, SD)f22.35 (19.16)32.48 (21.08)27.55 (20.76)P < 0.0005, t(292) = −4.30    Socio-religious aspects (mean, SD)f12.96 (17.46)14.48 (17.19)13.74 (17.31)n.s.n.s.: not significant P ≥ 0.05aScale of 1 (very bad)–5 (very good)bScale of 1 (very bad)–100 (very good)cRange: 11 (no impairment at all)–33 (serious impairment)dRange: 0–31 (number of days of serious impairment in last months)eRange: diseases: 0–12; complaints: 0–6fRange: 0 (not worried)–100 (extremely worried about the issues) Health service use Table 2 shows the prevalence of service use in the 2 months prior to the interview. Overall, the most frequently used service was the preventive healthcare service, followed by the general practitioner. Sixteen (5.4%) respondents visited a mental health professional. Group 1 visited the preventive healthcare services in the center more often than Group 2 (72.0% vs. 39.7%) and Group 2 visited the mental health services more often than Group 1 (9.3% vs. 1.4%). More than 39% of the respondents used drugs, 31.0% used analgetics. Overall drug consumption was higher in Group 2 (45.7% vs. 32.2%). Use of alternative services and treatments was very low, and there was no difference between the two groups: contact religious helper (2.7%), use of religious rituals or treatment (5.1%), contact with herbal doctor (0.7%), use of herbal treatment (0.3%). Table 2Health service use and other help seeking behavior in at random samples of Iraqi asylum seekers arrived <6 months (Group 1) and >2 years (Group 2) in the Netherlands, 2000–2001Use of services last 2 monthsGroup 1 n = 143 (%)Group 2 n = 151 (%)Total n = 294 (%)P valueUse of any health servicea76.966.271.4P = 0.042, χ2(1) = 4.119bUse of any out-patient (o-p) service74.152.362.9P < 0.005, χ2(1) = 14.992bUse of any o-p curative service38.536.437.4n.s.Use of preventive o-p service    Nurse/doctor in center72.039.755.4P < 0005, χ2(1) = 31.004bUse of o-p curative service    General practitioner32.925.829.3n.s.    Medical specialist in hospital12.617.915.3n.s.    Social worker5.66.66.1n.s.    Mental health worker1.49.35.4P = 0.003, χ2(1) = 8.846Use of in-patient service    Hospital admission physical health1.44.02.7n.s    Hospital admission mental health0.00.70.3n.sUse of any drugs32.245.739.1P = 0.018, χ2(1) = 5.643    Use of anxiolytics10.522.516.7P = 0.006, χ2(1) = 7.649    Use of hypnotics11.921.216.7P = 0.032, χ2(1) = 4.578    Use of analgetics23.837.731.0P = 0.010, χ2(1) = 6.707n.s.: P > 0.05aIncludes all regular and alternative services, including drugs, religious rituals/treatment and herbsbGroup 1 more than group 2, see text Relationship between psychopathology and service use Table 3 shows the univariate relationships between having ‘one or more psychiatric disorder’ and service use. Overall, respondents with psychopathology used significantly more services (70.0% vs. 54.5%), both curative and preventive ones. There was no significant difference in use of the services of the general practitioner, this was the case in both groups. Respondents with psychopathology visited a medical specialist (non-psychiatrist) much more often in Group 1, but not in Group 2 (P > 0.05). Table 3Use of services in respondents with and without one or more psychiatric disorder in Iraqi asylum seekers arrived <6 months (Group 1), and >2 years (Group 2) in the Netherlands, 2000–2001One or more psychiatric disorderGroup 1 n = 143Group 2 n = 151Total n = 294Yes n = 60, %No n = 83, %Yes n = 100, %No n = 51, %Yes n = 160, %No n = 143, %Use of services last 2 months    Use of any health servicea88.368.7*76.047.1*80.660.4*    Use of any out patient (o-p) service86.765.1*60.037.3*70.054.5*    Use of any o-p curative service53.327.7*43.023.5*46.926.1*Use of preventive o-p service    Nurse/doctor in center83.363.9*47.025.5*60.649.3Use of o-p curative service    General practitioner41.726.529.019.633.823.9    Medical specialist (non-psychiatrist)26.72.4*21.011.823.16.0*    Social worker8.33.610.00.0*9.42.2*    Mental health worker1.71.213.02.0*8.81.5*Use of any drugs45.022.9*58.021.6*53.122.4*    Anxiolytics18.34.8*31.05.9*26.35.2*    Hypnotics18.37.2*29.05.9*25.06.7*    Analgetics31.718.148.017.6*41.917.9**P < 0.05aIncludes all regular and alternative services, including drugs, religious rituals/treatment and herbs The use of drugs in respondents with psychopathology was higher in both groups, compared to those without psychopathology. Almost 60% of the respondents in Group 2 with psychopathology used drugs, versus 21.6% of those without psychopathology. Also the use of analgetics was higher, especially in Group 2 (48% vs. 17.6%). Predictors of service use In the ‘step 2’ analyses (Table 4), with all the independent variables in one analysis, the use of preventive healthcare services was predicted by Group 1 membership, low perceived quality of general health, and physical diseases. Visits to a general practitioner were predicted by age, low perceived quality of general health, and days of disability in the last month. Use of the services of a medical specialist (non-psychiatrist) was predicted by having one or more psychiatric disorders, high overall quality of life, low perceived quality of general health and physical diseases. Use of a social worker was predicted by low total disability and days of disability in the last month. Two variables predicted service use of a mental health worker: Group 2 membership, and low perceived quality of general health. Use of any drugs was predicted by age, and low perceived quality of general health. Use of analgetics (not in table) was predicted by age, lower perceived quality of general health, and physical diseases. The variables sex, physical complaints, and post-migration living problems (PMLP) did not predict any type of service use in the step 2 analyses (Group 1 and 2 together). Table 4Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker in the Netherlands (N = 294), 2000–2001ServiceNurse/Doctor centerGeneral practitionerMedical spec. (non-psychiatry)Social workerMental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors    Group 2 membershipb0.180.08–0.375.561.08–28.69    Age (older age)1.031.00–1.051.051.02–1.08Need factors    One or more psychiatric disorder1.341.18–2.76    Overall Quality of life1.801.18–2.76    Perceived quality of general health0.440.30–0.640.510.36–0.730.540.35–0.840.350.17–0.770.490.33–0.71    Disabilityc0.890.80–0.99    Disability daysd1.041.001–1.081.071.01–1.15    Physical diseases1.481.01–2.161.061.02–1.112.071.41–3.05PMLPaOR: Odds ratios are adjustedbGroup 2 membership >2 year in asylum procedurecDisability: total score of Brief Disability QuestionnairedDisability days in last month Tables 5 and 6 show the ‘step 3’ analyses for Group 1 (<6 months) and Group 2 (>2 years in the Netherlands) separately. In both groups a lower score on perceived quality of general health predicted almost all types of health services as well as drug use. In Group 1, but not in Group 2, use of the services of a medical specialist (non-psychiatrist) was also predicted by physical diseases, and by having a psychiatric disorder (Table 5). In Group 2, but not in Group 1, two PMLP (asylum procedure and socio-economic living conditions) predicted visits to the nurse/doctor in the center, the general practitioner, and/or a medical specialist (Table 6). In Group 2, but not in Group 1, number of days of disability in the last month and physical complaints predicted the use of analgetics (not in table). Table 5Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker, <6 months in the Netherlands (Group 1: N = 143), 2000–2001ServiceNurse/doctor centerGeneral practitionerMedical spec. (non-psychiatry)Mental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors    Age (older age)1.061.01–1.10Need factors    One or more psychiatric disorder1.931.18–3.16    Overall quality of life2.491.05–3.163.161.29–7.75    Perceived quality of general health0.250.11–0.560.550.31–0.970.410.18–0.960.290.12–0.74    Disability daysb1.081.01–1.15    Physical diseases2.651.33–5.282.711.33–5.54PLMPaOR: Odds ratios are adjustedbDisability days in last monthTable 6Multivariate logistic regression of predisposing and need (incl. PMLP: post-migration living problems) variables related to service use of Iraqi asylum seeker, >2 years in the Netherlands (Group 2: N = 151), 2000–2001ServiceNurse/doctor centerGeneral practitionerMedical spec. (non-psychiatry)Mental health workerUse of any drugsORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)ORaCI (95%)Predisposing factors    Age (older age)1.041.00–1.09Need factors    Perceived quality of general health0.54034–0.870.430.25–0.720.540.31–0.960.410.18–0.880.580.37–0.93    Disabilityb0.920.86–0.99    Disability daysc1.071.01–1.13    Physical diseases0.560.32–0.992.161.26–3.79PMLP    Asylum procedure1.381.04–1.830.680.51–0.92    Socio-economic l.c.1.191.02–1.381.231.00–1.50aOR: Odds ratio’s are adjustedbDisability: total score of Brief Disability QuestionnairecDisability days in last month Discussion The main findings of this study are: There is a high overall service use among Iraqi asylum seekers in the Netherlands. The hypothesis that a long asylum procedure is associated with higher levels of service use, is not confirmed by the results, except for mental health service use and drug use. Psychopathology is related to a higher level of service use (second hypothesis), but when corrected for the influence of other predisposing and need factors, other factors, such as: high role and functional disability, and low perceived quality of general health, are more important predictors. Moreover having one or more psychiatric disorder(s) predicts the use of a medical specialist (non-psychiatrist), but does not predict mental health service use. The overall use of mental health service use is very low compared to the high prevalence of psychiatric disorder: over 80% of the asylum seekers with a psychiatric disorder used any health service, but only 8.8% visited a mental health service. Next paragraphs will discuss the four research questions. Prevalence of service use, relationship with length of stay The preventive healthcare services are the most frequently visited services in both groups. As explained earlier (see “Methods”), in the Netherlands medical staff is present in all asylum seeker centers. Shortly after arrival, all asylum seekers are supposed to get a preventive medical screening by the doctor in the center doing triage for e.g. tuberculosis and AIDS. This probably explains the higher use of this service by Group 1. Gerritsen et al. [9] found that 63.7% of the asylum seekers from Iran, Afghanistan and Somalia reported a visit to the preventive healthcare services in the center. Their average length of stay was 3.4 years, comparable with Group 2. The rate of the Iraqi group in this study is much lower: 39.7%, suggesting a difference in use of this service between asylum seekers with different origins. In contradiction to the hypothesis, the use of a general practitioner in Group 2 was not higher compared to Group 1. The use (30%) is even lower compared to the general Dutch population, which is 42%, and even more so compared to immigrants: 51% [27]. Also Van Oort et al. [24] found that asylum seekers visit the general practitioner less often than the general Dutch population: number of contacts per year 3.5 vs. 4.5. So, despite the higher prevalence of health problems, asylum seekers make less use of the general practitioner. Furthermore, use of a medical specialist is not higher in Group 2. This is explained by the fact that in the Dutch system a patient can only visit a medical specialist after referral by a general practitioner. Drug consumption is significantly higher in Group 2 (45.7%), compared to Group 1 (32.2%), confirming the hypothesis. Gerritsen et al. [9] found an even higher rate (57.8%). The high use of analgetics is striking (see later). Use of alternative services is very low. Maybe Iraqi asylum seekers are not interested in these services, but even in case they would be, these services are probably not easily available and accessible. We found no other studies on this issue among Iraqi refugees/asylum seekers. Relationship psychopathology and service use The findings indicate a huge unmet need for mental health care. About 30% of the asylum seekers with a psychiatric disorder did not visit any service, and more than 90% of the asylum seekers with a psychiatric disorder did not visit a mental health service (Table 3). However 60.6% visited a nurse/doctor in the center and 33.8% the general practitioner. Both services are important in the pathway to mental health care (see later). We hypothesized that higher levels of psychopathology would be related with higher service use. In the univariate analyses this hypothesis stands, except for use of a general practitioner. Also, there are differences between the groups. The hypothesis is strongly confirmed when we consider drugs use. The high use of analgetics in Group 2 might be explained by the high levels of pain disorders (11.3%) and physical health complaints (66.2%) in this group. However other explanations are possible. Van Dijk et al. [23] did a qualitative study among 22 asylum seekers and concluded that it seems that “the prescription of paracetamol has become a symbol for the lack of interest of and the rejection by the health care system”. Their study reports dissatisfaction with the services of the nurse/doctor in the center, as well as with the services of the general practitioner. Predictors of service use After correcting the risks for all other included risk factors in multivariate analyses, low perceived quality of general health was the only significant predictor for mental health service use. Psychopathology, disability, nor physical complaints were significant predictors, while these factors were found to be important predictors in other studies (e.g. [8, 17]). Psychopathology, however, was a predictor for higher use of a medical specialist (non-psychiatrist). Our findings lead to the following hypothesis: (1) asylum seekers present themselves with physical rather than with mental problems (we know [14] that the level of physical complaints is high), (2) the staff in the center and the general practitioner do not recognize the mental health problems, and if they do (3) only a few patient are referred for adequate mental health care. This hypothesis is supported by the findings in the study of Van Oort et al. [24]. They found that in only 6% of the cases ‘mental health problems’ was recorded as reason of visiting the medical staff in the center, and only 2% of the referrals to the general practitioners were because of mental health reasons. The general practitioner diagnosed a mental health problem in 10% of the cases, and of those only 21.4% were referred to a mental health service (while 33.3% was referred to a medical specialist). The mismatch between type of health problem and type of health service use seems to be less pronounced in Group 2: in the analyses per group (Tables 5, 6) psychopathology did not predict the use of a medical specialist in this group. A curious finding is that overall quality of life has a positive relationship with the use of medical specialist. There might be a parallel with the phenomena that low social support is a risk factor for psychopathology [10], but high social support predicts health service use in some studies [15]. Several post-migration living problems (PMLP) predict health service use in Group 2. Especially worries about socio-economic living conditions increase the need for healthcare. The findings do not support the idea that asylum seekers look for recognition of their health problems in order to get a resident permit: there is no relationship between PMLP and mental health service use and there is a negative relationship with use of a medical specialist. Conclusion This study has shown that Iraqi asylum seekers have a low level of mental health service use, despite the high levels of psychiatric disorders and other health indicators, especially within the group that stayed in the asylum procedure for over 2 years. Moreover, there is a mismatch between the type of health problem and the type of health service use: asylum seekers with a psychiatric disorder make more use of non-mental health service. There is room for improvement of the ‘gate way’ preventive services in detection and referrals of patients with mental health problems. The study results suggest that this service is a barrier rather than a facilitator in the pathway to mental health care. The general practitioner should be more involved and consulted. Mental health institutions are recommended to start and/or improve consultation and (assertive) care programs.

Ann_Hematol-4-1-2324130

Immunoglobulin and free light chain abnormalities in Gaucher disease type I: data from an adult cohort of 63 patients and review of the literature

Gaucher disease type I, the most common lysosomal storage disorder, is associated with immunoglobulin abnormalities. We studied the prevalence, risk factors, pathogenesis, and effect of enzyme relation therapy (ERT) on gammopathies in an adult Gaucher disease type I cohort (N = 63) and related the results to a review of the currently available literature. Polyclonal gammopathies and monoclonal gammopathy of undetermined significance (MGUS) in our adult GD I cohort were found in 41% and 19% of patients. These results are similar to the data from the literature and correspond to the increased risk of multiple myeloma (MM) that has been described. The prevalence of MGUS in our cohort increased with age but was not associated with disease severity or exposure time. The serum levels of free light chains of immunoglobulins were measured and were not found predictive for the development of MGUS or MM. Levels of pro- as well as anti-inflammatory cytokines, growth factors, and chemokines, especially those involved in inflammation and B-cell function, are disturbed in GD I, with the most impressive and consisting elevations for interleukin-10 and pulmonary and activation-regulated chemokine. A beneficial effect of ERT on the occurrence and progression of gammopathies was suggested from longitudinal data. Introduction Gaucher disease type I (GD I, OMIM #230800) is the most common lysosomal storage disorder. The disease is characterized by a deficiency of the lysosomal enzyme glucocerebrosidase (glucosylceramidase), which leads to the accumulation of glucocerebroside in macrophages. The lipid laden macrophages are called Gaucher cells and are mainly found in liver, spleen, and bone marrow, resulting in the most important clinical features hepatosplenomegaly, skeletal disease, and cytopenia [1, 2]. Since 1991, GD I can be effectively treated with recombinant glucocerebrosidase (enzyme replacement therapy, ERT, imiglucerase; Genzyme, Cambridge, MA, USA). More recently, substrate reduction therapy (miglustat, Actelion Pharmaceuticals, Basel, Switzerland) has been registered for more attenuated disease. An associated feature of GD I is the high frequency of polyclonal and monoclonal gammopathies [3–22]. In the general population, polyclonal hypergammaglobulinemia is a common clinical finding, especially in the setting of chronic inflammation [23], resulting from an overproduction of immunoglobulins by plasma cells or B-lymphocytes. An abnormal increase of monoclonal immunoglobulins or fragments thereof (M protein) can be found in pre-malignant conditions such as MGUS (monoclonal gammopathy of undetermined significance) and malignancies of the B-cell lineage such as multiple myeloma (MM), primary amyloidosis, Waldenström macroglobulinemia, chronic lymphocytic leukemia, and B-cell lymphoma. MGUS is found in approximately 3.2% of adults over the age of 50, increasing to 7.5% at the age of 85 [24]. Of these individuals, 1% per year shows progression to MM or a related malignancy. Accordingly, in GD I the risk of MM is strongly increased, as has been reported in recent studies [25–29]. The risk increases with advancing age [19, 26], but whether disease severity is a risk factor is currently unknown. The mechanism explaining the relationship between GD and immunoglobulin abnormalities is far from understood. Studies have mainly focused on cytokines as the mediators between Gaucher cells, surrounding macrophages and B cells. Plasma levels of several pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6, as well as the anti-inflammatory IL-10, were found to be increased in GD to variable extents [4, 30–33]. IL-6 and IL-10, as well as hepatocyte growth factor (HGF), are also involved in the pathogenesis of MM [34]. Other interesting factors, known to be highly elevated in GD patients, are pulmonary and activation-regulated chemokine (PARC) [35], which is involved in initiating the adaptive immune response. Two studies have reported on the effect of ERT, with some decrease in polyclonal gammopathies [6] and a decline in monoclonal M-protein level in a single case [36]. A relatively new method to identify and monitor monoclonal gammopathies is by quantifying immunoglobulin free light chains (FLC), circulating κ and λ chains that are not bound to the immunoglobulin heavy chains, using a highly sensitive automated immunoassay [37–39]. An abnormal FLC ratio is an important independent risk factor for progression of MGUS to MM or a related disorder [40, 41]. In addition, measuring FLC is a sensitive additional tool to identify monoclonal gammopathies [42]. In GD I, in which the incidence of MGUS and MM is highly increased [26], the FLC assay may provide a useful diagnostic tool for early detection of MGUS and MM. In this study, we present new data on the prevalence of polyclonal and monoclonal gammopathies from a large adult GD I cohort and review the currently available literature. Second, we studied whether age, disease severity, and exposure time are risk factors for the development of monoclonal gammopathies, and if this risk is subsequently decreased by the administration of ERT. In addition, for the first time, we investigated the role of FLC as a predictor of the development of monoclonal gammopathies in GD I. Finally, the pathogenesis of immunoglobulin abnormalities in GD patients was studied by means of pro- and anti-inflammatory cytokine, chemokine, and growth factor levels and is discussed in relation to previous literature. Materials and methods Patients and study design The files were reviewed and archived serum samples were collected of all adult GD type I patients that were known from the outpatient clinic for inherited metabolic disorders at the Academic Medical Centre (AMC), Amsterdam, The Netherlands (N = 63). The diagnosis GD was based on measurement of deficient glucocerebrosidase activity in leukocytes and genotyping [43]. Of the 63 patients, 50 received ERT according to an individualized dosing protocol [44] and two patients received substrate reduction therapy. Data on age, sex, splenectomy, genotype, severity score index, (SSI [45]), creatinine, urea, chitotriosidase, and the presence and type of immunoglobulin class of monoclonal proteins were collected. In patients with a monoclonal gammopathy, exposure time was defined as the number of years between the diagnosis GD I and the diagnosis monoclonal gammopathy or start of ERT, whichever came first. In patients without a monoclonal gammopathy, exposure time was calculated as the number of years between the diagnosis GD I and start of therapy, or, in patients not receiving therapy, the first visit to our clinic. MGUS was defined as the presence of a monoclonal immunoglobulin component in the serum (as determined by protein electrophoresis in agar/agarose gels and immunofixation) at a concentration of less than 30 g/l (IgG, IgA, or IgM) and normal serum calcium and creatinine [46]. Although histology is required to distinguish osteolytic lesions caused by MM from those caused by GD, we decided to classify long-existing lesions as being GD related. Bone marrow examinations were only performed when a previously unknown M protein was discovered or in case of a sharp rise in the levels of immunoglobulins or FLC. In case of MM, stage was determined according to the Durie–Salmon [47] and International Staging System (ISS) criteria [48]. We investigated whether levels of FLC could be predictive for the development of MGUS and whether these levels were related to disease severity. Therefore, we measured FLC levels in serum before start of enzyme replacement therapy in ten patients with mild GD (SSI ≤ 8) and ten patients with severe GD (SSI ≥ 9), all without a monoclonal gammopathy. In addition, we studied whether changes in cytokine levels and in FLC and immunoglobulins at baseline and during ERT were related to the presence or absence of a monoclonal gammopathy. Therefore, the group of patients with a monoclonal gammopathy was matched for age, sex, spleen status, SSI, and the use of ERT to a control group of GD patients without a monoclonal gammopathy. Serial measurements of levels of IL-6, IL-10, and PARC at baseline and after 12, 24, and a median of 114 months of ERT (range 40–143, whatever was the last determination) were performed. These were related to changes in immunoglobulins and free light chains. Hepatocyte growth factor (HGF) was measured only at baseline. One patient did not receive therapy; the first hospital visit was used as T0. Assays The standard enzyme activity assay for chitotriosidase with 4-MU-chitotriose (Sigma, St. Louis, MO, USA) as a substrate was performed at pH 5.2 [49]. Serum κ and λ FLC were measured using FREELITE reagents (The Binding Site Ltd, Birmingham, England) on a BNII nephelometer (Dade-Behring, Deerfield, IL, USA) [37–39]. Monoclonal FLC were identified as values for κ or λ that exceeded the reference ranges (κ 6.2–30.2 mg/l, λ 9.1–40 mg/l) and produced an abnormal κ/λ ratio (<0.3 or >1.57). Elevated concentrations of FLC without an abnormal κ/λ ratio indicate a polyclonal or oligoclonal increase of FLC. Levels of IgG, IgA, and IgM were measured by immunoturbidimetric assay (Roche Tina-quant). IL-6, IL-10, HGF, and PARC were measured by specific enzyme-linked immunosorbent assays (ELISAs), according to the instructions of the manufacturer (IL-6 and IL-10: Sanquin, Amsterdam, The Netherlands; HGF: R&D Systems, Minneapolis, MN, USA; PARC: Biosource International, Camarillo, CA, USA). Statistics Values in patients are given as medians and ranges. Differences between patient groups were analyzed by the Mann–Whitney U test or by χ-square test. Linear mixed models were used to assess longitudinal changes. Correlations were tested by the rank-correlation test (Spearman coefficient, ρ). P < 0.05 was considered to represent a statistical difference. Search strategy To compare the prevalence of poly- and monoclonal gammopathies in our cohort with earlier case series and to study the relative risk of MM, we performed a MEDLINE search combining the MESH terms Gaucher disease with either hypergammaglobulinemia, paraproteinemias, and/or multiple myeloma. Literature on cytokines, chemokines, and growth factors in relation to the development of immunoglobulin abnormalities in GD were searched combining the MESH terms Gaucher disease with cytokines and/or chemokines. Case reports were excluded and only studies that included at least ten patients were selected. Results Patients The 63 patients had a median age of 53 years (range 25–83). Thirty-two of patients (51%) were men, and 23 (37%) were splenectomized. At baseline, median SSI was 8 (range 3–19). The majority of patients had a genotype containing N370S (95%), with N370S/L444P being the most common combination (40%). All patients had creatinine and urea values within the normal range. At the first assessment, 24 (38%) patients had normal immunoglobulin levels, 26 (41%) patients had a polyclonal gammopathy, 12 (19%) patients had MGUS, and one patient had MM (Table 1, #2). During follow-up (range 5–16 years), none of the patients with normal immunoglobulin levels or a polyclonal gammopathy developed a monoclonal gammopathy. Table 1Characteristics of Gaucher disease type I patients with a monoclonal gammopathyPatient no.SexSxAge in 2007 or at deathTherapyMG typeIg type1MN55ERTMGUS, progression to amyloidosis and MMFree κ2MY71ERTMMIgGκ3FN46ERTMGUS, progression to amyloidosisIgGλ4MY60ERTMGUSIgGκ5MY67ERTMGUSIgGλ6MY59ERTMGUSIgAλ, IgGκ7FN67ERTMGUSIgAκ8MN56ERTMGUSIgGλ9MY65ERTMGUSIgGκ, IgGλ, IgAκ10MN56ERTMGUSIgMκ, IgMλ11FN63NoMGUSIgGλ, IgMκ12FY75ERTMGUSIgGκ13MN51ERTMGUSIgGλM Male, F female, Sx splenectomy, ERT enzyme replacement therapy, MG monoclonal gammopathy, MM multiple myeloma, MGUS monoclonal gammopathy of undetermined significance, Ig immunoglobulin Of the 12 patients with MGUS, two developed MM and/or amyloidosis after approximately 2 years of ERT (#1 and 3). Patient #1 was diagnosed with MM and amyloidosis 25 months after start of ERT. Pulse therapy with dexamethasone was started. Three months after the diagnosis the patient died from cardiac failure. At 24 months after start of ERT, patient #3 was diagnosed with amyloidosis. She received melphalan and prednisone, but died 12 months later from cardiac failure. Patient #2 was diagnosed with MM stage IA before start of ERT. During follow-up, IgG levels gradually increased. After 7 years of ERT, treatment with reduced dose melphalan and prednisone was started, which resulted in unacceptable cytopenia. Subsequently, thalidomide, 100 mg daily, was started with beneficial effect and stable disease parameters for the last 3 years. Of the ten remaining patients with non-progressive MGUS, six had a monoclonal, three had a biclonal, and one a triclonal gammopathy. The most common immunoglobulin type was IgGκ (Table 1; results previously presented in part in [26]). Patients with a monoclonal gammopathy were significantly older than patients without a monoclonal gammopathy (Table 2). Exposure time to GD, defined as time from diagnosis until first assessment at our clinic, and severity of disease measures, assessed by SSI and chitotriosidase at baseline, were comparable. Table 2Baseline characteristics of Gaucher type I patients with a monoclonal gammopathy vs patients without a monoclonal gammopathy Without MGWith MGPNo. of patients5013 Age in 2007 or at death51 (25–83)60 (46–75)0.003No. of male patients23 (46%)9 (69%)NSYears of exposure11 (0–37)18 (1–44)NSNo. of splenectomies17 (34%)6 (46%)NSSSI at baseline7 (3–19)12 (4–16)NSChitotriosidase (nmol/ml h) at baseline16,703 (5,409–132,199)22,534 (6,417–62,122)NSData reflect absolute numbers (and percentage) or median (and range).MG Monoclonal gammopathy, NS not significant, SSI severity score index A search for studies on monoclonal and polyclonal gammopathies in GD I resulted in five series [4, 6, 16, 19, 21]. Polyclonal gammopathies were reported in 14–64% and monoclonal gammopathies in 1–35% of GD I patients (Table 3). Table 3Studies on the prevalence of monoclonal and polyclonal gammopathies in type I Gaucher diseaseReferenceNo. of Gaucher patientsAgePolyclonal gammopathiesMonoclonal gammopathiesde Fost et al. [26]6325–8326 (41%)12 (19%)Pratt et al. [19]169–706 (38%)4 (25%)Shoenfeld et al. [21]2524–7815 (60%)2 (8%)Marti et al. [16]2341.8 ± 1810 (43%)8 (35%)Allen et al. [4]2223–6514 (64%)3 (14%)Brautbar et al. [6]50716–8114–25%5 (1%)Data reflect absolute numbers (and percentage), or, in the study of Marti et al., mean ± standard deviation. Five studies have described the prevalence and/or relative risk of MM in GD patients [25–29]. Table 4 summarizes the data from these studies. The frequency of MM in these cohorts was 0.4–4.0%, with significantly elevated relative risks of 5.9–51.1. Table 4Studies on the prevalence and relative risk of multiple myeloma in type I Gaucher diseaseReferenceNo. of patientsEthnic backgroundAgeControl groupNo. of patients with MMRR of MMde Fost et al. [26]131Mixed50 ± 14Dutch Cancer Registry2 (1.5%)51.1 (95% CI: 6.2–184)Lee [25]239MixedNot givenNone5 (2%)NDShiran et al. [28]48Jewish54 ± 20511 individuals from the same region2 (4%)NDZimran et al. [29]505Jewish38 ± 21Israeli Cancer Registry2 (0.4%)NDRosenbloom et al. [27]2,510Mixed33US Cancer Registry10 (0.4%)5.9 (95% CI: 2.8–10.8)Data on age reflect mean ± standard deviation; data on number of patients with multiple myeloma reflect absolute numbers (and percentage).MM Multiple myeloma, RR relative risk, 95% CI 95% confidence interval, ND not done Immunoglobulin and FLC levels In 20 GD patients from the Dutch cohort without a monoclonal gammopathy (ten with mild disease, SSI ≤ 8, and ten with severe disease, SSI ≥ 9), FLC were measured before start of therapy (Fig. 1a). One patient was found to have a slightly abnormal ratio, and six patients had an increase in one or both FLC, with a normal ratio. There were no significant differences in κ or λ FLC-levels between patients with severe- and patients with mild GD I. During follow-up (range 10–16 years), none of the patients developed a monoclonal gammopathy. Fig. 1FLC levels in Gaucher disease type I patients without a monoclonal gammopathy with mild disease (SSI ≤ 8) and severe disease (SSI ≥ 9) (a). FLC levels in Gaucher disease patients with a monoclonal gammopathy and matched Gaucher disease controls (b). MG Monoclonal gammopathy. The normal range for κ was 6.2–30.2 mg/l and for λ was 9.1–40 mg/l. The normal ratio for κ/λ was 0.3–1.57 FLC levels were measured in all GD I patients with a monoclonal gammopathy (Table 1, #1–13) and matched GD I controls (#co1–co13). No serum was available of patient #12, resulting in two groups of 12 patients. Baseline immunoglobulin levels were not available in one patient with a monoclonal gammopathy (#11) and five patients from the control group (#co4, #co5, #co7, #co11, and #co13). At baseline, the patients who already had or would develop MM and/or amyloidosis (#1–3) had strongly abnormal FLC κ/λ ratios (Fig. 1b). Of the nine patients with MGUS, one (#7) had an abnormal FLC κ/λ ratio and four showed elevated levels of FLC κ or λ, but with a normal FLC ratio. The remaining four patients with MGUS had both FLC levels as well as a FLC κ/λ ratio within the normal range. Six of the patients from the control group had FLC levels and a FLC κ/λ ratio within the normal range, and six had elevated levels of one or both chains of whom only one (#co6) patient showed an abnormal FLC κ/λ ratio. During follow-up (range 6–15 years), none of the patients from the control group developed a monoclonal gammopathy. Cytokines, chemokines, and growth factors At baseline, IL-6 levels were within the normal range in all but four patients (Fig. 2). There was no significant difference in IL-6 levels between patients with or without a monoclonal gammopathy (median (range) 5.9 pg/ml (1.2–118.4) and 2.2 pg/ml (1.0–57.2), respectively). The majority of patients (17/24) showed elevated IL-10 levels, especially in the group of patients with a monoclonal gammopathy, although not significantly different from the patients without a monoclonal gammopathy (17.1 pg/ml (3.9–419.8) and 6.9 pg/ml (1.4–299.3), respectively, P = 0.2). PARC levels were elevated in all GD patients, without a difference between patients with or without a monoclonal gammopathy (1,400.0 pg/ml (348.9–2817.3) and 1,078 pg/ml (411.4–3,552.0), respectively). HGF was within the normal range in most (16/22) patients, without a difference between patients with or without a monoclonal gammopathy (1,751 pg/ml (926–4,791) and 936 pg/ml (424–9,382), respectively). Fig. 2Baseline levels of IL-6, IL-10, PARC, and HGF in Gaucher disease patients with a monoclonal gammopathy and matched Gaucher disease controls. Patients with multiple myeloma and/or amyloidosis are marked by an arrow. Dotted lines reflect the upper limit of the normal range. IL Interleukin, HGF hepatocyte growth factor, PARC pulmonary and activation-regulated chemokine, MG monoclonal gammopathy A literature search resulted in seven studies on cytokines, chemokines, and growth factors in GD I patients (Table 5). In one of these studies, mRNA levels were measured [32], while in the remaining studies, plasma levels were studied. If possible, the number of patients with cytokine levels exceeding the reference range was determined. In studies in which normal ranges were not provided [4, 32], we defined the normal range as mean ± 2SD based on the values of the control group. This was not possible in the study of Barak et al. [30], in which no detailed data were provided. Table 5Studies on plasma levels of cytokines, chemokines, and growth factors in Gaucher type I patientsReferenceNo. of GD I patientsAgeCytokinesResults, no. of patients (%) with elevated levelsP value for difference with mean values of the control groupde Fost et al. [26]2225–83IL-64/24 (17%)NDIL-1017/24 (71%)PARC24/24 (100%)HGF6/22 (27%)Michelakakis [33]25NGTNF-α16/25 (64%)NDAllen et al. [4]2223–65IL-1βNot detectableNSTNF-α4/11 (36%)NSIL-619/22 (86%)P = 0.0001IL-1013/13 (100%)P < 0.0001Lichtenstein et al. [32]18NGIL-1β mRNA5/19 (26%)P = 0.0337TNF-α mRNA2/19 (11%)NSIL-6 mRNA3/19 (16%)NSIL-8 mRNA3/19 (16%)NSHollak et al. [31]2916–66M-CSF24/28 (86%)P < 0.001sCD1425/27 (93%)P < 0.0005IL-825/27 (93%)P < 0.0005IL-60/27 (0%)NSTNF-α0/27 (0%)NSBarak et al. [30]215–39IL-1βMean levels elevatedP = 0.01IL-1RAMean levels elevatedP = 0.01sIL-2RMean levels elevatedP < 0.001IL-6Mean levels elevatedP < 0.01IL-8Not elevatedNSTNF-α3/21 (14%)NSBoot et al. [35]5512–67PARC55/55 (100%)P < 0.0001Altarescu et al. [56]1211–67TNF-α1/12 (8%)NSNG Not given, ND not done, NS not significant Longitudinal changes In general, at no time point was there a statistically significant difference in plasma levels of any of the factors (i.e., IgG, IgA, IgM, FLC κ and λ, IL-6, IL-10, and PARC) between patients with and patients without a monoclonal gammopathy (Fig. 3). In addition, using mixed model analysis, in neither of the two patient groups, a significant decrease or increase could be established in plasma levels of any of the measured factors. The only exception was PARC, which decreased in all patients, except for one patient who did not use therapy (#11). Changes in PARC levels could not be related to changes in levels of immunoglobulins or free light chains. There were no strong correlations (i.e., rho > 0.6) between any of the factors. Fig. 3Longitudinal changes in plasma levels of immunoglobulin heavy and free light chains, IL-6, IL-10, and PARC. Dotted lines reflect the normal range. Numbers reflect Gaucher disease patients with a monoclonal gammopathy as described in Table 1 (#1–13) and matched Gaucher disease controls (#co1–co13) without a monoclonal gammopathy. IL Interleukin, PARC pulmonary and activation-regulated chemokine More specifically, immunoglobulin levels remained stable or decreased in all patients, except for patient #11, who showed an increase in IgM. This patient was different from other MGUS patients because she did not receive therapy. Free light chain levels were more variable, but clearly FLC levels of patients who were to develop or already suffered from MM and/or amyloidosis remained high during ERT (#1–3). Although in most patients no trends could be distinguished, a consistent decrease in free light chains and immunogobulins was seen for example in patient 7, who had an IgAκ M protein. This patient responded very well to ERT, but other patients with excellent clinical responses did not always show a similar decrease in M-protein levels. No relationship was found between clinical response and decrease in either levels of immunoglobulins or FLCs. The course of cytokines did not show a relationship with changes in either FLC or immunoglobulin levels, although in the untreated patient with increasing levels of IgM (#11), also an increase in IL-6 and PARC was noted. On the contrary, a spontaneous decrease in IL-6 was found in patient #2, without therapy for his MM. In patient #1, IL-10 showed a clear decrease after start of ERT but before the diagnosis amyloidosis and MM was made. Discussion In addition to the classical symptoms of GD I, such as hepatosplenomegaly, skeletal disease, and cytopenia, there is increasing evidence for existence of co-morbidities, including increased risk of cancer and abnormal immunoglobulin profiles. In fact, polyclonal gammopathies and MGUS occurred in 41% and 19%, respectively, of our adult Gaucher cohort, which is in line with findings from the literature (14–64% and 1–35% of patients [4, 6, 16, 19, 21]). In addition, we established that the relative number of biclonal (3/10) and triclonal (1/10) gammopathies within the MGUS patients was especially high considering that in general, biclonal gammopathies occur in 3% of subjects with MGUS [24], while triclonal gammopathies occur only very sporadically [50]. The types of immunoglobulin heavy and light chain immunoglobulins were comparable to the findings for MGUS in a non-GD population, with a predominance of IgGκ [24]. As in the general population, the prevalence of MGUS in our cohort increased with age, but there was no relationship with disease severity measures, as assessed by exposure time, baseline SSI, and chitotriosidase levels. A relationship with age was first suggested by Pratt et al. [19], who investigated 16 patients aged 9 to 70 years and established an MGUS in four patients, all being over 50 years of age. Indirectly, he also showed an association with severity of disease, since all patients with splenomegaly had either a monoclonal or polyclonal gammopathy. Considering the relatively high risk of monoclonal gammopathies in GD, the question was raised whether sensitive measuring of FLC would be useful for the early detection of monoclonal gammopathies in GD I patients. In a non-GD population, MGUS patients showed an abnormal FLC ratio in 44% of cases, which was an important risk factor for progression to MM or a related disorder [40, 41]. We found that the three GD patients who had or would soon develop a B-cell malignancy could easily be identified by their strongly abnormal FLC κ/λ ratios. It was however, not possible to identify GD patients with MGUS on the basis of abnormal FLCs since 44% of GD patients with MGUS had both FLC levels as well as a FLC κ/λ ratio within the normal range. A slightly abnormal ratio was not predictive for progression to MM or MGUS in the single patient without a monoclonal gammopathy, even after 15 years of follow-up. None of the patients had a decrease in renal function, implicating that any FLC increase was indeed due to increased production by plasma cells. MGUS patients are at risk for developing MM at a rate of 1% of patients per year. Accordingly, the prevalence of MM in GD I cohorts of 0.4–1.5% clearly exceeds the general prevalence of 0.02% (deduced from The Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute (NCI)). The assumed diversity in relative risk of developing MM (5.9 and 51.1) can simply be explained by age differences in the cohorts: the highest number of MM patients was clearly established in the highest age groups. In a recent consensus report on hematological manifestations of Gaucher disease, the higher incidence of multiple myeloma in Gaucher disease is acknowledged, and it is recommended that Gaucher patients should have their immunoglobulin profile determined at diagnosis and monitored every 2 years (patients <50 years) or every year (patients >50 years) [51]. Hypotheses on the pathogenesis of immunoglobulin disorders in GD have mainly focused on the accumulated glucocerebroside as the causative agent for chronic stimulation of macrophages surrounding Gaucher cells. Cytokines of macrophagic origin could in turn stimulate B cells, leading to a polyclonal gammopathy which may eventually transform, directly or via MGUS, into MM. Interestingly, plasma cells can be found in close contact with Gaucher cells, suggestive of local interaction (Fig. 4). Fig. 4Bone marrow aspirate showing plasma cells surrounding a Gaucher cell in a patient with Gaucher disease type I and multiple myeloma In our study, we measured IL-6, IL-10, and HGF, three factors important for differentiation and/or proliferation of plasma cells. In addition, IL-6 and IL-10 are also involved in inflammation and in abnormal bone remodeling in MM, aspects that are also present in GD. We found elevated IL-10 levels in most patients, especially those with a monoclonal gammopathy, and predominantly normal levels of IL-6 and HGF. Previous studies have also described increased IL-10 levels [4]. However, findings on IL-6 in GD I have been variable [4, 30–32]. Inconsistent results have also been reported for the pro-inflammatory cytokines TNF-α and IL-1β and the chemokine IL-8 [4, 30–33, 52]. Although not included in this study, increases have been described for the growth factor M-CSF (monocyte/macrophage-colony stimulating factor), the monocyte/macrophage activation marker sCD14 (soluble CD14) [31], sIL-2R (soluble IL-2 receptor), and IL-1RA (IL-1 receptor antagonist) [30]. The most impressive increase, both in our study as well in as in the study by Boot et al. [35], has been found for PARC, a chemokine that is assumed to be involved in B-cell differentiation by recruiting T cells and CD-38 negative mantle zone B lymphocytes to antigen-presenting cells. In addition to the in vivo studies, it has been described that murine macrophages that were stimulated in vitro by glucocerebroside release the pro-inflammatory lymphocyte-activating factor (LAF = IL-1) in a dose-responsive manner. This effect was not seen on incubation with galactocerebroside, sphingomyelin, and ceramidetrihexoside [53]. Possibly, only minor glycolipid accumulation is sufficient for the development of B-cell derangement. In fact, a glucocerebrosidase deficient mouse (L444P homozygote) showed evidence of B-cell proliferation, as well as elevated serum IgG levels, even though storage cells were not found [54]. The heterogeneity in plasma cytokine levels that have been found could be explained by differences in patient populations and cytokine assays. In addition, it is not known whether plasma levels adequately reflect tissue levels. Nevertheless, clearly both pro- as well as anti-inflammatory cytokine levels are disturbed in GD I. It is unlikely that Gaucher cells directly induce inflammation, since Gaucher cells have a phenotype resembling anti-inflammatory alternatively activated macrophages. More likely, pro-inflammatory cytokines are produced by surrounding macrophages, with Gaucher cells compensating for the inflammatory compounds [55]. Now that it is generally accepted that ERT effectively reduces hepatosplenomegaly, cytopenia and skeletal disease, the focus should be shifted to new challenges, including the prevention and treatment of Gaucher associated co-morbidities. Novel data from our study suggest a beneficial effect of ERT on the occurrence and severity of gammopathies in GD patients. First, none of the patients developed MGUS during ERT. We did see progression of MGUS to amyloidosis and MM in two patients. However, since both patients developed these diseases relatively shortly after start of ERT, one could envision that irreversible changes leading to malignant transformation had already taken place, and could not be corrected by ERT. Second, we found that immunoglobulin levels either decreased or remained stable in both patients with and without a monoclonal gammopathy. The only patient with a strong increase in immunoglobulins (IgM) was a patient not using ERT. A clear influence of ERT on levels of FLC, IL-6, and IL-10 was not found in our study. A previous report also found a decrease in immunoglobulin levels during ERT, although only in patients with a polyclonal gammopathy, and not in patients with a monoclonal gammopathy [6]. Decreases in the levels of IL-10 [4], M-CSF, sCD1431, and PARC [35] have been described. Nevertheless, to draw firm conclusions on a possible positive effect of ERT on gammopathies in GD I, our presumptions should be confirmed in larger studies. This would require an international multicenter effort that should aim to establish whether indeed these long-term complications can be prevented by ERT and whether this would justify the early start of treatment. In summary, there is a high prevalence in GD I of both polyclonal as well as monoclonal gammopathies, including MM. The risk of these diseases increases with age. Mechanisms causing gammopathies remain to be elucidated, but include the disturbance of cytokine levels involved in inflammation and B-cell function. ERT is likely to have a beneficial effect in preventing the occurrence and the progression of gammopathies.

Cancer_Causes_Control-2-2-1705538

Breast cancer risk factors in relation to breast density (United States)

Objectives Evaluate known breast cancer risk factors in relation to breast density. Introduction Numerous studies have shown that breast density, as assessed through mammography, is an important breast cancer risk factor [1–5]. Relative to the lowest classification of breast density (fatty tissue), women with the highest classification (extreme density) may have a 2- to 6-fold increased risk of breast cancer [5–8]. In addition to its role in breast cancer risk, breast density reduces mammographic accuracy [9–12], potentially increasing the risk of a later stage breast cancer diagnosis. Previous studies indicate that established breast cancer risk factors, including family history of breast cancer, age at first birth, parity, and postmenopausal hormone use, have similar associations with breast density. In contrast, the influence of age at menarche, which in most studies is inversely related to breast cancer risk [e.g., 13–15], remains uncertain. Some studies have found positive associations [6, 16, 17], at least one suggests an inverse association [18], and others found no relation between age at menarche on breast density [19, 20]. In an effort to clarify inconsistent results from previous studies, we evaluated established breast cancer risk factors in relation to breast density in a large population of women enrolled in a statewide mammography registry. Our intention was to determine whether characteristics associated with breast cancer risk were also related to breast density, a finding that would be consistent with the notion that density mediates breast cancer risk. We were particularly interested in assessing the influence of menarcheal age. Methods The New Hampshire Mammography Network (NHMN) registers all consenting women who undergo mammography at participating mammographic facilities in our state. Details of the registry have been described previously [21, 22]. For the present study, potentially eligible women were NH residents of ages 30–89, who had at least one mammogram registered in the NHMN from 1 May 1996 to 20 June 2002. The epidemiological data used in this analysis arose from three sources: a self-administered questionnaire completed by the patient, a patient intake form administered face-to-face by the radiologic technologist, and a standardized clinical assessment form completed by the radiologist. The questionnaire collected height, weight, place of birth, ethnicity, marital status, education, insurance coverage, reason for the current visit, past history of clinical breast examinations and mammography, age at menarche, parity, and age at first birth. The questionnaire also queried women regarding the date of their last menstrual period and history of gynecological surgery. This information was used to classify women as premenopausal (still having periods naturally) or postmenopausal (periods had stopped permanently) either naturally, because of chemotherapy/radiation, or surgery). The patient intake form obtained date of birth, family history of breast cancer (in the subject’s mother, sister, daughter, or other relative), personal history of breast cancer, history of breast procedures, type of exam conducted at current visit, examination outcomes, recommendation for further work-up or follow-up, and current use of postmenopausal hormone therapy (HT). The clinical assessment form obtained the type of exam conducted at current visit, breast density, examination outcomes and recommendation for further work-up or follow-up. All three forms are completed during the woman’s first NHMN mammography visit. Patient intake and clinical assessment forms are also completed at subsequent mammography visits, and the questionnaire is updated as possible. Breast density, the outcome variable, estimates the proportion of fibroglandular tissue in the breast, relative to fat. Breast density was recorded on the standardized clinical assessment form by interpreting radiologists using the American College of Radiology Breast Imaging Reporting and Data System® (BI-RADS®) classification (1 = fatty, 2 = scattered density, 3 = heterogeneously dense, and 4 = extremely dense) [23]. In the event of discordance in the density of the right and left breast, the woman was classified according to the higher density classification. Breast density readings were available for 162,933 (95.4%) of the 170,815 women who had at least one mammogram recorded in the registry. To optimize temporal correspondence between the women’s characteristics and the classification of breast density, the statistical analyses were, when possible, based on the woman’s breast density on the date of the first recorded mammogram. When data for variables (other than HT use) were unavailable for the date of the mammogram, we searched forward in the NHMN records to retrieve replacement information corresponding to a subsequent mammography visit. Informative forward searches retrieved information from subsequent mammographic encounters occurring, on average, within 24 months of the index mammogram, and reduced missing values by 3–9%. Current body mass index (BMI; kg/m2) was missing for 18,195 women, and the analyses were confined to 144,018 women for whom this measure was available. Included in the analytic sample were 131,480 (91%) women with a screening mammogram, 10,885 (8%) with a diagnostic mammogram, and 1,653 (1%) for whom the reason for the mammogram was not recorded. The majority of women, 136,283 (95%) had no personal history of breast cancer, 6,033 (4%) had a prior history of breast cancer based on NHMN records or the patient intake form, and 1,702 (1%) had unknown breast cancer status. We used unconditional logistic regression analyses to generate odds ratios (OR) and 95% confidence intervals (CI) [24] for the association between factors and breast density, dichotomized as heterogeneously/extremely dense (dense) or fatty/scattered density (not dense). Statistical significance required a probability value of <0.05 (two-sided test). OR were computed using the cutpoints shown in the tables. Tests of trend and the corresponding OR were based on the categorical (age at menarche) or the continuous form of the variable (age, BMI, age at first birth, parity), in accordance with the method of data collection. Because breast density was inversely associated with age (p for trend <0.0001) and current BMI (p for trend <0.0001), terms for these variables, using the continuous form, were included in all models. We found no evidence of confounding by the other variables shown in Table 1 (fully adjusted OR were within 10% of those adjusted for age and BMI). Model building began with terms representing the main effects, and included interaction terms involving age, BMI, and menopausal status as suggested by visual inspection of the stratified analyses. The presence of statistical interactions was formally tested using likelihood ratio tests. The interaction term representing BMI was defined as BMI ≥30 (high BMI), versus <30 (low BMI). The final multivariable model, based on all women, contained terms for age and BMI in their continuous form, BMI (high, low), family history of breast cancer, age at menarche, age at first birth, parity, menopausal status, current use of HT, and terms for the interactions involving BMI (high, low) and age at menarche, age at first birth, and parity, a term for the interaction involving age and current HT use, and a term for the interaction between age at first birth and menopausal status. We repeated the analyses in parous women, in women with a screening mammogram, and in women who did not have a personal history of breast cancer. Table 1Distribution of women’s characteristics by breast density, dichotomized as dense versus not denseCharacteristics of study sample (n = 144,018)Not denseDensen%n%Age (years)    30–396,728810,01116    40–4924,9703129,95448    50–5921,5372713,59722    60–6915,160195,4339    70–7910,349133,0395    80–892,47637741    All women81,2205662,79844Education    <High school6,88782,7034    High school graduate27,4853417,29128    College graduate35,2704331,11350    Post graduate9,9711210,68117    Missing1,60721,0102Marital status    Not married25,2353116,90527    Married54,1816744,59871    Missing1,80421,2952Current BMI    <202,64036,47910    20–22.499,8481216,57026    22.5–24.9915,4801916,06026    25–27.4914,266189,81216    27.5–29.9912,689166,35310    30–34.9915,197195,3268    35+11,100142,1984Family history breast cancer    No55,5916841,23466    Yes25,6293221,56434Age at menarche    <116,13083,0945    1114,028179,32815    1220,9282615,83325    1322,0242718,28129    149,430128,53414    15+7,41396,83011    Missing1,26728981Age at first birtha    <2015,537218,55415    20–2430,5744118,67434    25–2916,6942214,27626    30–346,03886,70512    35+1,97232,4784    Missing4,52665,0269Parity    05,87987,08511    17,02397,66512    219,7912418,26229    317,6522213,03221    411,723146,90511    5+13,795175,8969    Missing5,35773,9536Menopausal status    Premenopausal29,1323637,55460    Postmenopausal46,8745821,89535    Missing5,21463,3495HT useb    No32,8296312,86751    Yes16,3383110,72242    Missing2,92161,6557aAge at first birth in parous womenbA small proportion of women (5.5%) who reported whether they used HT did not give their menopausal status Results In all women, scattered density (45%) was most frequently recorded, followed by heterogeneous density (34%), fatty breasts (12%), and extremely dense breasts (10%). The distribution of factors by breast density, classified as not dense versus dense, is shown in Table 1. In general, based on the cutpoints shown, there was a tendency for younger women, those with higher education, and married women to have denser breasts. Women with lower BMI also had greater breast density, consistent with our use of BI-RADS categories, which assess the proportion of fibroglandular tissue (versus fat) in the breast. Women with a family history of breast cancer, later menarcheal age, later age at first birth, and low parity had a tendency toward higher breast density. High breast density was more common in premenopausal than in postmenopausal women, and in women currently using HT compared to nonusers. Age-stratified analyses, adjusted for age and BMI, showed a weak, positive influence of family history of breast cancer across all age groups assessed (Table 2). A small positive association between age at menarche and breast density appeared to vary by age, with weaker effects in the older and youngest age groups. There were no clear age-related patterns for either the positive effect of age at first birth or the inverse effect of parity or menopausal status. Current HT use, compared to nonuse, was inversely associated with breast density in the younger age groups, and positively associated in women of age 50 or more. In women of age 70 or more, those using HT, compared to nonusers, had twice the odds of having dense breasts. Table 2Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to age groupCharacteristicaAge group<4040–4950–5960–6970+n = 16,739n = 54,924n = 35,134n = 20,593n = 16,628OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)Family history breast cancer    NoReferenceReferenceReferenceReferenceReference    Yes1.06 (0.99, 1.13)1.10 (1.06, 1.14) 1.07 (1.02, 1.13) 1.10 (1.03, 1.18) 1.11 (1.02, 1.20) Age at menarche    Overall OR (95% CI)b1.04 (1.01, 1.06)1.06 (1.05, 1.08)1.03 (1.02, 1.05)1.01 (0.99, 1.03)1.01 (0.98, 1.03)    p for trendb0.002<0.00010.00010.310.70    <11ReferenceReferenceReferenceReferenceReference    110.99 (0.85, 1.18)1.14 (1.04, 1.24)1.28 (1.15, 1.42)1.06 (0.90, 1.24)1.11 (0.89, 1.38)    121.04 (0.89, 1.21)1.15 (1.06, 1.25)1.26 (1.15, 1.39)1.12 (0.96, 1.30)1.18 (0.96, 1.45)    131.15 (0.98, 1.35)1.25 (1.16, 1.36)1.27 (1.15, 1.40)1.20 (1.03, 1.39)1.00 (0.82, 1.23)    141.21 (1.02, 1.44)1.34 (1.22, 1.46)1.37 (1.23, 1.53)1.23 (1.05, 1.46)1.22 (0.99, 1.51)    15+1.29 (1.08, 1.55)1.45 (1.32, 1.60)1.36 (1.21, 1.52)1.05 (0.89, 1.25)1.15 (0.92, 1.43)Age at first birthc    Overall OR (95% CI)d1.08 (1.06, 1.10)1.05 (1.04, 1.06)1.07 (1.06, 1.08)1.07 (1.05, 1.09)1.06 (1.05, 1.08)    p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001    <20ReferenceReferenceReferenceReferenceReference    20–241.06 (0.95, 1.19)1.14 (1.08, 1.21)1.21 (1.14, 1.29)1.23 (1.12, 1.34)1.07 (0.93, 1.24)    25–291.09 (0.98, 1.22)1.19 (1.12, 1.26)1.40 (1.30, 1.51)1.51 (1.36, 1.68)1.28 (1.11, 1.49)    30–341.35 (1.18, 1.53)1.23 (1.15, 1.36)1.58 (1.42, 1.76)1.64 (1.39, 1.95)1.42 (1.18, 1.70)    35+1.67 (1.30, 2.16)1.48 (1.35, 1.63)1.85 (1.58, 2.17)1.90 (1.44, 2.49)1.47 (1.14, 1.91)Parity    Overall OR (95% CI)d0.86 (0.84, 0.88)0.91 (0.90, 0.93)0.90 (0.86, 0.91)0.89 (0.87, 0.90)0.87 (0.85, 0.89)    p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001    0ReferenceReferenceReferenceReferenceReference    10.74 (0.65, 0.86)0.90 (0.83, 0.87)0.87 (0.79, 0.97)0.87 (0.73, 1.05)0.88 (0.73, 1.05)    20.58 (0.52, 0.66)0.73 (0.69, 0.78)0.77 (0.71, 0.85)0.77 (0.67, 0.89)0.66 (0.57, 0.78)    30.48 (0.42, 0.55)0.69 (0.65, 0.74)0.67 (0.61, 0.73)0.65 (0.57, 0.76)0.59 (0.51, 0.69)    40.49 (0.42, 0.57)0.61 (0.57, 0.66)0.61 (0.55, 0.67)0.55 (0.48, 0.64)0.49 (0.42, 0.58)    5+0.38 (0.33, 0.45)0.60 (0.55, 0.66)0.52 (0.47, 0.58)0.49 (0.42, 0.56)0.39 (0.33, 0.46)Menopausal status    PremenopausalReferenceReferenceReference    Postmenopausal0.69 (0.60 , 0.79 )0.71 ( 0.68, 0.75)0.71 (0.67, 0.75)N/AN/AHT use    NoReferenceReferenceReferenceReferenceReference    Yes0.69 (0.57, 0.84)0.80 (0.75, 0.86)1.39 (1.32, 1.46)1.82 (1.70, 1.95)2.02 (1.84, 2.22)aAdjusted for age and current BMI as continuous variablesbBased on the cutpoints showncAmong parous womendBased on the continuous form of the variable Analyses stratified on BMI suggested that the influence of some factors was less evident in women of BMI ≥30 (high BMI) than in those of BMI <30 (low BMI) (Table 3). In particular, the positive influence of age at menarche and age at first birth, and the inverse influence of parity were least apparent in the highest BMI group. The data suggested an inverse effect of being postmenopausal, relative to premenopausal, which decreased consistently across the BMI groupings, but the change in OR from the lowest to the highest BMI group was slight. Although the influence of current HT use fluctuated somewhat over BMI groups, there were no obvious patterns. Table 3Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to BMI groupCharacteristicaCurrent BMI <22.522.5–25.4925.5–27.4927.5–29.99≥30n = 35,547n = 37,153n = 18,744n = 18,775n = 33,799OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)OR (95% CI)Family history of breast cancer    NoReferenceReferenceReferenceReferenceReference    Yes1.09 (1.03, 1.14)1.03 (0.99, 1.08) 1.08 (1.01, 1.15) 1.15 (1.08, 1.23) 1.09 (1.03, 1.15) Age at menarche    Overall OR (95% CI)b1.04 (1.03, 1.06)1.05 (1.03, 1.06)1.04 (1.02, 1.06)1.03 (1.01, 1.05)1.01 (0.99, 1.03)    p for trendb<0.0010.0010.0010.0040.25    <11ReferenceReferenceReferenceReferenceReference    111.25 (1.09, 1.43)1.17 (1.05, 1.31)1.13 (0.98, 1.31)1.20 (1.05, 1.38)1.10 (0.99, 1.22)    121.36 (1.20, 1.55)1.26 (1.13, 1.40)1.10 (0.96, 1.27)1.16 (1.01, 1.32)1.09 (0.98, 1.21)    131.43 (1.26, 1.63)1.33 (1.20, 1.48)1.16 (1.01, 1.33)1.21 (1.06, 1.38)1.06 (0.94, 1.16)    141.54 (1.34, 1.75)1.44 (1.28, 1.61)1.25 (1.08, 1.45)1.19 (1.02, 1.38)1.16 (1.02, 1.31)    15+1.52 (1.32, 1.75)1.43 (1.27, 1.61)1.26 (1.07, 1.48)1.33 (1.13, 1.56)1.06 (0.93, 1.21)Age at first birthc    Overall OR (95% CI)d1.08 (1.07, 1.09)1.07 (1.06, 1.08)1.07 (1.06, 1.09)1.06 (1.04, 1.07)1.03 (1.02, 1.04)    p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001    <20ReferenceReferenceReferenceReferenceReference    20–241.21 (1.13, 1.31)1.20 (1.13, 1.28)1.15 (1.05, 1.26)1.13 (1.03, 1.23)1.04 (0.96, 1.11)    25–291.39 (1.29, 1.51)1.34 (1.25, 1.44)1.21 (1.10, 1.34)1.31 (1.19, 1.45)1.10 (1.01, 1.19)    30–341.45 (1.32, 1.60)1.55 (1.42, 1.70)1.40 (1.23, 1.59)1.42 (1.25, 1.63)1.11 (0.99, 1.24)    35+1.79 (1.56, 2.06)1.84 (1.61, 2.09)1.88 (1.56, 2.28)1.62 (1.33, 1.97)1.29 (1.08, 1.53)Parity    Overall OR (95% CI)d0.86 (0.85, 0.87)0.88 (0.87, 0.89)0.90 (0.88, 0.92)0.91 (0.89, 0.93)0.93 (0.92, 0.95)    p for trendd<0.0001<0.0001<0.0001<0.0001<0.0001    0ReferenceReferenceReferenceReferenceReference    10.75 (0.67, 0.84)0.80 (0.73, 0.89)0.93 (0.81, 1.08)1.13 (0.98, 1.30)0.95 (0.85, 1.07)    20.61 (0.55, 0.66)0.67 (0.62, 0.73)0.74 (0.66, 0.84)0.90 (0.79, 1.02)0.85 (0.78, 0.94)    30.52 (0.48, 0.58)0.58 (0.53, 0.63)0.68 (0.60, 0.77)0.80 (0.71, 0.91)0.81 (0.73, 0.89)    40.44 (0.39, 0.48)0.52 (0.47, 0.57)0.60 (0.52, 0.69)0.71 (0.62, 0.81)0.75 (0.68, 0.84)    5+0.37 (0.34, 0.41)0.44 (0.40, 0.48)0.52 (0.45, 0.60)0.64 (0.56, 0.74)0.66 (0.59, 0.74)Menopausal status    PremenopausalReferenceReferenceReferenceReferenceReference    Postmenopausal0.67 (0.63, 0.72)0.68 (0.64, 0.72)0.71 (0.65, 0.77)0.73 (0.67, 0.79)0.75 (0.70, 0.80)HT use    NoReferenceReferenceReferenceReferenceReference    Yes1.23 (1.15, 1.31)1.33 (1.26, 1.42)1.34 (1.23, 1.46)1.41 (1.30, 1.55)1.34 (1.25, 1.45)aAdjusted for age and current BMI as continuous variablesbBased on the cutpoints showncAmong parous womendBased on the continuous form of the variable Analyses stratified by menopausal status revealed largely similar results for most variables, but the relationship between age at first birth and breast density was stronger in the postmenopausal women (Table 4). Table 4Odds ratios (OR) and 95% confidence intervals (CI) for the association with breast density according to menopausal statusCharacteristicaMenopausal status PremenopausalPostmenopausaln = 66,686n = 68,769OR (95% CI)OR (95% CI)Family history of breast cancer    NoReferenceReference    Yes1.09 (1.05, 1.13)1.08 (1.04, 1.12) Age at first birth    Overall OR (95% CI) 1.05 (1.04, 1.06)1.03 (1.02, 1.04)    p for trend<0.0001<0.0001    <11ReferenceReference    111.12 (1.03, 1.21)1.15 (1.06, 1.25)    121.12 (1.04, 1.22)1.20 (1.12, 1.30)    131.22 (1.13, 1.32)1.21 (1.12, 1.31)    141.31 (1.21, 1.43)1.29 (1.18, 1.40)    ≥151.40 (1.28, 1.53)1.25 (1.15, 1.36)Age at first birthb    Overall OR (95% CI)c1.05 (1.05, 1.06)1.07 (1.06, 1.08)    p for trendc<0.0001<0.0001    <20ReferenceReference    20–241.07 (1.01, 1.13)1.21 (1.15, 1.27)    25–291.11 (1.05, 1.17)1.41 (1.33, 1.49)    30–341.21 (1.13, 1.29)1.57 (1.45, 1.71)    ≥351.48 (1.35, 1.62)1.77 (1.57, 2.00)Parity    Overall OR (95% CI)c0.90 (0.87, 0.91)0.88 (0.88, 0.89)    p for trendc<0.0001<0.0001    0ReferenceReference    10.87 (0.81, 0.93)0.86 (0.79, 0.93)    20.70 (0.66, 0.74)0.72 (0.68, 0.77)    30.63 (0.60, 0.68)0.62 (0.58, 0.67)    40.57 (0.53, 0.62)0.55 (0.51, 0.59)    ≥50.52 (0.49, 0.57)0.46 (0.43, 0.50)HT use    NoNAReference    YesNA1.47 (1.41, 1.52)aAdjusted for age and current BMI as continuous variablesbAmong parous womencBased on the continuous form of the variable We assessed risk factors and potential interactions in a multivariable model (Table 5). Only one factor, family history of breast cancer, which showed a weak but significant positive effect (OR 1.09; 95% CI 1.05–1.14) on breast density, was not involved in an interaction. The possible interaction involving age and age at menarche, suggested by the age-stratified analyses, was not statistically significant (p = 0.10). A statistically significant interaction was found between current HT use and age (p < 0.0001). Interactions were also present between BMI and age at menarche (p = 0.04), age at first birth (p < 0.0001), and parity (p = 0.01). We also noted a significant interaction between menopausal status and age at first birth (p = 0.004), but the coefficients were inconsistent across categories of age at first birth. When the interaction between menopausal status and age at first birth was omitted from the multivariable model, results for the remaining terms were essentially unchanged. A possible interaction involving age, menopausal status, and HT was not significant (p = 0.10). Table 5Betas (β) and standard errors (SE) for the association between factors and breast density in all women and in subgroupsaCharacteristicAll women n = 144,018Women without breast cancer n = 136,283β (SE)β (SE)Intercept4.82 (0.17)4.88 (0.17)Age−0.04 (0.001)−0.04 (0.001)Current BMI−0.12 (0.003)−0.12 (0.003)Family history of breast cancer0.09 (0.02)0.09 (0.02)BMI <30−0.53 (0.11)−0.53 (0.11)Postmenopausal−0.43 (0.09)−0.43 (0.09)HT use−1.22 (0.11)−1.25 (0.11)Age at menarche    15+−0.01 (0.10)−0.03 (0.11)    140.04 (0.10)0.01 (0.10)    13−0.01 (0.08)−0.03 (0.08)    120.08 (0.08)0.06 (0.08)    110.04 (0.08)0.02 (0.08)BMI <30 * Age at menarche    15+0.30 (0.12)0.32 (0.12)    140.24 (0.11)0.28 (0.11)    130.22 (0.09)0.24 (0.10)    12 0.12 (0.09)0.15 (0.10)    110.11 (0.10)0.14 (0.10)Age at first birth    Nulliparous0.38 (0.16)0.13 (0.17)    35+0.19 (0.22)0.25 (0.23)    30–34−0.19 (0.15)−0.18 (0.15)    25–29−0.11 (0.12)−0.12 (0.12)    20–240.14 (0.11)0.13 (0.11)BMI <30 * Age at first birth    Nulliparous0.57 (0.10)0.59 (0.11)    35+0.41 (0.18)0.40 (0.18)    30–340.31 (0.11)0.32 (0.12)    25–290.12 (0.08)0.15 (0.08)    20–240.06 (0.06)0.06 (0.06)Postmenopausal * Age at first birth    Nulliparous0.09 (0.15)0.10 (0.15)    35+−0.08 (0.19)−0.14 (0.19)    30–340.27 (0.13)0.26 (0.13)    25–290.25 (0.11)0.23 (0.11)    20–24−0.05 (0.10)−0.04 (0.11)Paritya    10.29 (0.09)0.28 (0.09)    20.22 (0.07)0.23 (0.07)    30.14 (0.07)0.17 (0.07)    40.09 (0.07)0.11 (0.08)BMI <30 * Parityb    10.30 (0.10)0.30 (0.10)    20.22 (0.07)0.20 (0.08)    30.18 (0.08)0.15 (0.08)    40.09 (0.08)0.07 (0.09)Age * HT use0.03 (0.002)0.03 (0.002)aBased on a multivariable model containing all terms shown in the tablebThe nulliparous parameter and its interaction with BMI were set to 0, since these variables were linear combinations of other variables We repeated the analyses in the subgroup of women who did not have a personal history of breast cancer, and found similar results (Table 5). The findings were also comparable when the analyses were confined to parous women, or to those with a screening mammogram (data not shown). Discussion Evidence accumulating for nearly 30 years supports the association between breast density and breast cancer [1–5]. Although the notion remains controversial, breast density may be a biomarker of risk [25]. In addition to its influence on breast cancer risk, breast density reduces the accuracy of screening mammography [11, 12, 26, 27], particularly in younger women [9] who tend to have denser breasts [28]. Perhaps as a direct consequence of reduced screening accuracy, breast density is associated with increased risk of interval breast cancers [11], with an adverse impact on breast cancer prognosis. Most studies of breast cancer risk have shown an inverse effect of age at menarche [13–15, 29], but previous studies of the relationship between age at menarche and breast density have produced inconsistent results. A positive association was seen in two studies [16, 18], including a large HMO population of nearly 30,000 women in Seattle [16]. Studies of breast cancer family members [17], Singaporean women [20], and Hispanic women [19] found no association between menarcheal age and breast density. Findings from the HMO-based study suggested the positive effect of age at menarche was stronger in the youngest and oldest age groups [16], whereas a study of nearly 5,000 women in Guernsey found significant positive effects only in postmenopausal women [6]. In contrast, our age-stratified analyses suggested weaker effects in the oldest age groups, although the interaction involving age and age at menarche was not statistically significant. Also in this study, analyses stratified on BMI suggested that age at menarche was positively associated with breast density in most BMI groups, but the association was tenuous in women with high BMI, and the interaction involving age at menarche and high BMI was statistically significant. A positive association between age at menarche and breast density, even if confined to women with lower BMI, seems paradoxical, given the usual inverse association between age at menarche and breast cancer and the strong positive association between breast density and breast cancer risk. Consistent with some [16–18, 20, 30, 31], but not all [6, 19] previous efforts, our findings show that age at first birth and parity are generally associated with breast density in a pattern resembling known associations with breast cancer risk. However, our stratified analyses indicate that the influence of the reproductive variables is less pronounced in women with high BMI, and our modeling results confirmed interactions involving BMI and both variables. These findings, along with our findings for age at menarche, are consistent with the possibility that hormonal or reproductive events are less influential in heavier women, whose circulating hormone levels may be influenced by conversion in peripheral adipose tissue. We also noted a stronger positive influence of age at first birth in postmenopausal women, although a previous study of Native American women found stronger effects in premenopausal women [30]. Our sample was large, and the multivariable results were inconsistent across categories of age at first birth; thus, it is possible the interaction between menopausal status and age at first birth was due to statistical artifact rather than true effect modification. Only one variable, family history of breast cancer, was not involved in interactions with age, BMI, or menopausal status. Although previous studies have not shown an effect of family history on breast density [3, 6, 19], this is likely due to limited power to detect a weak association. The modest inverse effect of menopausal status has been noted previously [19, 20, 30]. Most previous reports, although not all [20], found a positive association between use of HT and breast density [16, 17, 19, 30, 32, 33], resembling the well-known association between use of these hormones and breast cancer risk. The large size of our study allowed an assessment of HT use in young postmenopausal women, and these analyses showed a modest but significant inverse effect in postmenopausal women less than 50 years of age, a phenomenon that has no clear explanation. In women of age 50 or more, the positive effect of HT on density increased with age, perhaps reflecting a corresponding decrease in density in untreated women in the same age group. Consistent with our findings, at least two previous studies of breast density showed an increasing effect of HT use when examined over increasing age groups [16, 32]. A prospective study of breast cancer risk also noted stronger HT effects in older women [37], although this is not always seen [36]. The age-related increase observed in our study could potentially reflect a longer duration of HT use, but at least two studies have shown that most of the increase in breast density occurs soon after HT initiation [33, 34], and duration of use was not associated with increased breast density in the HMO study [16]. In contrast, the positive influence of HT on breast cancer risk is usually observed for current/recent and long-term use [35–40]. While speculative, it is possible that sustained breast density associated with long term HT use mediates the relationship between HT and breast cancer risk. Finally, our data did not indicate a stronger effect of HT on breast density in leaner woman, but a few studies [38–40], including a collaborative analysis of 51 studies [38], suggested a stronger association between HT and breast cancer risk in leaner women. Although the type of HT used (estrogen alone or estrogen combined with progesterone) was not assessed in our study, a possible role of progesterone is suggested by reports that breast density is greater during the luteal phase of the menstrual cycle [41–43]. In addition, at least two studies have found substantially greater changes in parenchymal patterns in women initiating use of a combined estrogen plus progesterone hormone regimen, as opposed to single agent estrogen [33, 34]. Results from the Women’s Health Initiative randomized clinical trials of postmenopausal hormones also indicate that the increased risk of breast cancer is due to the combined regimen [44] rather than single agent estrogen [45]. Strengths of our study include the large size of our sample, allowing analyses stratified by relatively refined age and BMI groups, which has not been possible in most previous studies, and good representation of the underlying population. Epidemiologic data were obtained on the time of the mammographic visit, ensuring updated information, and importantly, a high level of correspondence between use of hormone replacement therapy and the assessment of breast density. Limitations of our study include 11% of women for whom BMI is missing, reliance of self-report for BMI, and a lack of information regarding the type and duration of HT use. Our definition of family history of breast cancer included second degree relatives, which may have attenuated the effect of this variable. Also, the BI-RADS scores are qualitative, as opposed to digital quantification of density, and were applied by community radiologists, who despite being trained to use this system, may apply it differently. Nevertheless, our general findings in terms of the direction of effects for reproductive factors and HT use were compatible with those of most previous studies. In conclusion, our results, based on the largest study to date, confirm earlier findings that most established breast cancer risk factors behave similarly in relation to breast density, consistent with the notion that breast density mediates breast cancer risk. However, our data indicated an inverse effect of HT in younger women, and a positive influence in older women, which has not been reported previously. We also noted effect modification by BMI, in which the positive effects of age at menarche and age at first birth, and the inverse effects of parity were less apparent in heavier women. Further investigation, including biological studies, may elucidate the complex interrelationships of hormones, BMI, breast density, and breast cancer and potentially offer opportunities for breast cancer prevention.

Graefes_Arch_Clin_Exp_Ophthalmol-4-1-2367393

Posterior capsulorhexis combined with optic buttonholing: an alternative to standard in-the-bag implantation of sharp-edged intraocular lenses? A critical analysis of 1000 consecutive cases

Background Current after-cataract prevention relies on optimizing the natural barrier effect of the optic rim against lens epithelial cell (LEC) migration. However, deficiencies in circumferential capsular bag closure caused by the intraocular lens (IOL) haptic or delayed secondary re-division of the fused capsules by Soemmering´s ring formation lead to primary or secondary barrier failure. Consequently, surprisingly high posterior laser capsulotomy rates have been reported long-term, even with optimal capsular surgery and the most widespread hydrophobic acrylic IOLs, considered to be the most advanced. Intraoperative removal of the central posterior capsule has been shown to be effective in further reducing LEC immigration. However, efficacy has turned out to be limited because of the propensity of LECs to use the posterior optic surface as an alternative scaffold. Background The current concept of after-cataract prevention (“posterior capsule opacification”, or “PCO”) is based on fully exploiting the natural barrier effect of the optic rim against lens epithelial cell (LEC) migration. This barrier effect is inherent to any optic rim, even when rounded. However, it can be optimized by the following measures: (1) by providing a circumferential overlap of the optic by the anterior capsular leaf, (2) by sharpening the posterior optic edge, and (3) by enhancing the collagenous sealing of both capsular leaves along the optic rim [10]. The latter is achieved by using fibrosis-inducing optic materials, and by preserving the integrity of the LEC layer as the substrate of fibrosis. The importance of fibrotic sealing is mirrored by two facts: (1) intraocular lenses (IOLs) made of materials with a lower fibrogenetic potential, such as acrylics, undergo a sharp increase in after-cataract score after 3–5 years [27], while with those made from materials with a higher fibrogenetic potential, such as silicones, it remains fairly constant, and (2) LEC abrasion by anterior capsule polishing significantly increases regeneratory after-cataract formation and the need for Nd: YAG laser capsulotomy (YAG-LCT) [11]. Implementation of all three above-mentioned parameters into modern cataract surgery has led to a drastical reduction of after-cataract and YAG-LCT rates. However, utilization of the edge barrier effect has two major shortcomings: (1) overlap of the optic by the anterior capsule leaf induces fibrotic whitening and shrinkage of the latter, thereby reducing the size of the free optic zone (this is especially true if the rhexis opening is small or decentered, be it because of inadequate dimensioning or due to subsequent contraction), and (2) the barrier function can be lacking from the very beginning, or wear off over the years, even when all the three above-mentioned requirements are fully met. The barrier cannot build up when fusion of both capsular leaves and consecutive bending of the posterior capsule at the posterior optic edge is not initiated. Such “primary barrier failure” can be limited to sectors or comprise the whole circumference of the optic. By nature, capsular fusion is impeded at the haptic-optic junction. While with looped haptics the barrier at the junction may remain fully preserved, broad-based haptics, including those used with the currently popular one-piece IOLs, inevitably disrupt the barrier at the optic insertion [24]. As a result, a gateway is opened up for LECs migrating from the germinative equatorial zone inwards. Deficiencies of capsular bag fusion may also arise independent of the haptic junction. When the reason is not evident, these have been termed “idiopathic primary barrier failure”. Most often, however, it is caused by ovalization of the capsular bag by overly long and rigid haptics, which impedes capsular fusion along the longitudinal IOL axis. Even when primary capsular fusion has been complete, the barrier may eventually fail. As Soemmering´s ring formation sets in, the two capsular leaves are increasingly redivided from the capsular equator inwards. If collagenous sealing of the capsule leaves along the optic rim is insufficient, these are increasingly forced apart, and the bend at the posterior optic edge is finally annihilated. Dormant LECs may thus become reactivated [15] and access the retrolental space. This phenomenon has been termed “secondary barrier failure”. These barrier failures are the reason why in the long run YAG-LCT is still required in a considerable proportion of cases, even with optimized IOL designs and appropriate surgery. For the Acrysof 3-piece IOL, the reported YAG-LCT rate at 5 years ranges between 16% [2] and 27% [own data in publication]. After 10 years, a cumulative YAG-LCT rate of 42% was found (L. Vock, R. Menapace R. Long-term YAG laser capsulotomy and after-cataract rates with the sharp edge Acrysof and round edge PhacoFlex intraocular lenses: 10 year results. Abstract XXVth Congress of the ESCRS, 8.−12.9.2007, Stockholm). Thereby it must be kept in mind that the YAG-LCT rate only reflects those cases where central pearl formation is significant enough to interfere with vision. When also considering those eyes where LEC invasion remains limited to the periphery of the retrooptical capsule, the rate of barrier failure is significantly higher. Therefore, the potential for avoiding after-cataract formation by optimizing the edge barrier effect is limited, even when all relevant parameters are fully exploited. In addition, even in those cases where the sharp edge forms a permanent circumferential barrier, visibility of the peripheral retina may be significantly reduced by pearl formation in the intercapsular space outside of the optic rim. The sharp edge as such also has the severe disadvantage of immanently causing dysphotopsia. Negative dysphotopsia is especially annoying for the patient. In the consultation section of the April 2005 issue of the Journal of Cataract and Refractive Surgery, dysphotopsia has been classified the “problem number one of modern cataract surgery” [16]. Interestingly, this problem is not limited to acrylic IOLs with double square-edged (truncated) optics, but is also experienced with frosted edges, and even with rounded anterior edges. Also, it has been found with both acrylics and silicones [3]. Rationale The still significant percentage of after-cataract formation requiring YAG-LCT in the longer run, and the problem of dysphotopsia, have fostered the search for better alternatives. Thereby, a new approach was required, since the concept of optimizing the migrational barrier effect of the optic edge is largely exploited. As one option, intraoperative removal of the central posterior capsule by performing a primary posterior continuous curvilinear capsulorhexis (PPCCC) seemed promising, since the posterior capsule serves as a scaffold for centrally migrating LECs. As such, it is not an alternative, but much more supplements the current sharp edge concept by providing a “second barrier of defence” when the optic edge barrier is overcome by migrating LECs. However, even though it has been shown to be safe [26, 4], the efficacy of adding a PPCCC is also limited. Firstly, the residual peripheral capsule can still be invaded and opacified by LECs which reduces the free optic zone. Secondly, similar to the anterior LEC ongrowth inside the ACCC rim often observed with acrylics [1], equatorial LECs can also grow over the PPCCC rim unto the free posterior optic surface, as they may utilize the latter as an alternative scaffold. Incidence and severity vary depending upon surface tension, wetability, and contact angle of the optic material, as well as the individual biological responsiveness, and is more pronounced in eyes with a defective blood–aqueous barrier [25]. We performed a randomized prospective intraindividual comparison study including 29 patients with bilateral PPCCC, whereby one eye received a silicone, the other a hydrophilic acrylic IOL. Since round-edged optic IOLs were used at that time, this made the full impact of a PPCCC more obvious. After 2 years, partial reclosure of the PPCCC opening due to peripheral LEC ongrowth was found in 55% of the acrylic, and in 28% of the silicone IOL eyes. Total reclosure of the PCCC was observed in two eyes of the acrylic and in one eye of the silicone IOL group [5] (Fig. 1). When evaluated with an objective grading system, after-cataract formation within the PPCCC opening exceeded 2 and 3 out of 10 possible grades for the silicone and acrylic IOL respectively. Fig. 1Primary posterior capsulorhexis decreases YAG laser capsulotomy rate by removing scaffold for LEC migration; however, residual capsule may still opacify, and LECs may alternatively grow upon optic surface Thus, a PPCCC constitutes a useful additive surgical measure, but does not fully prevent after-cataract formation. One promising alternative concept is combining a PPCCC with posterior buttoning-in of the optic (“posterior optic buttonholing”, or POBH), a technique which has been reserved for pediatric cataract surgery [6]. By doing so, the posterior capsule ends up lying on top of the optic. Instead of hindering the LECs from migrating, these are diverted towards the anterior optic surface, thus bypassing the optic edge. By doing so, migrating LECs, by principle, can no longer access the retrolental space. In order to elucidate the suitability of the posterior optic buttonholing (POBH) technique as a routine procedure for senile cataracts, an extensive prospective study comprising a large number of eyes was initiated. In several substudies, a prospective randomized bilateral design was used to elucidate the risk profile compared to the standard in-the-bag implantation of IOLs in detail. For this purpose, the surgical technique of POBH was revised and optimized to be then systematically investigated. From September 2004 to June 2007, over 1000 eyes underwent this POBH procedure. Patients were randomly taken from the waiting list. All prospective comparison studies were approved by the Medical University of Vienna Ethics Committee. Technique The surgical technique comprises an additional step of sophisticated, but perfectly controlled capsular and viscosurgery. After performing a standard 5–6 mm anterior continuous curvilinear capsulorhexis (ACCC), and after removing the lens contents with phacoemulsification and meticulously cleaning the capsular bag, the posterior capsule is flattened and the capsular fornix collapsed by pushing the iris and anterior capsule backwards with a low-viscosity cohesive ocular viscoelastic device (OVD, preferably Healon®). Thereby, the pupil is additionally widened. Due to this pupil-dilating effect, all eyes with a pupil size larger than 4 mm under full pharmacological dilatation were amenable to the procedure. After incising the center of the posterior capsule with a 30-gauge hypodermic needle (Sterican® manufactured by B. Braun, Melsungen, or Fine-Ject® by Henke-Sass-Wolf, Tuttlingen, Germany), a small amount of OVD is injected through the capsular opening. Then, the edge of the incised capsule is taken up by Utrata forceps and the incision extended peripherally to create a well-centered 4–5 mm PPCCC opening. When after the first pass one quadrant of the PPCCC has been completed, OVD is again injected in order to separate the underlying anterior hyaloid surface from the posterior capsule. After completion of the PPCCC and removal of the flap, OVD is again injected, this time in order to ensure full circumferential separation of the anterior hyaloid membrane from the residual posterior capsule up to the very periphery. Adequate viscoseparation of capsule and hyaloid is considered crucial to allow safe buttoning-in of the optic, and will be addressed in more detail in the Discussion Section below. Then, an open-loop IOL is implanted through an injector and fixated in the capsular bag fornix. When both loops are placed in the bag, the optic is buttoned-in into the PPCCC opening by gently pressing it down. As the elastic posterior capsule thereby wraps around the optic periphery in between the haptic junctions, the distended capsule and the optic form a mechanically stable and watertight diaphragm (Figs. 2 and 3). Finally, the OVD is aspirated from the anterior chamber, the globe is tonisized, and the paracenteses are hydrated. The cataract incision is left unsutured. The surgery is performed under topical plus optional intracameral anesthesia (Lidocaine 4% plus Lidocaine unpreserved 1%) to ensure bright and stable retroillumination during the PPCCC manoeuvre. The HOYA AF-1 with a 6-mm optic is preferably used due to its particular haptic design (continuous optic-haptic transition: Fig. 4; “hopper loop”). Should the PPCCC result to be larger than the optic, as may occur in eyes with lax zonules or a large capsular bag, a 6.5-mm optic IOL should be resorted to, in order to ensure sealing of the diaphragm by sufficient circumferential capsular overlap. Additional implantation of a capsular tension ring facilitates estimation and performance of an appropriately sized and centered PPCCC in these eyes [9]. Fig. 2Schematic of interrelationship between capsule leaves and IOL. Loops residing in capsular fornix, optic buttoned-in in PPCCC. Posterior capsule lying on top of optic (crescent-shaped blue areas); this precludes optic contact and thus fibrosis of the anterior capsule (red lines: rim of ACCC). If the ACCC is smaller than the optic (bold line), anterior capsule fibrosis limited to area adjacent to the haptic junction (red area); if larger than the optic (hatched line), no fibrosis also in this areaFig. 3Retroillumination photography of buttoned-in IOLs (HOYA AF-1): note perfect autocentration of optic and run of posterior capsule rimFig. 4HOYA AF-1-type IOL preferred because of continuous haptic–optic junction and “hopper loop” haptic. POBH causes optic to position (by approximately 1 mm) more posteriorly (compared to standard in-the-bag placement), thereby stabilizing the vitreous body Approximately 150 eyes underwent additional anterior capsule polishing with a special LEC aspiration curette [13] (Fig. 5), the fibrosis-preventing efficacy of which had been substantiated in an earlier clinical study [19]. In this subgroup of eyes, the impact and potential advantages of this additional treatment was evaluated. Polishing was performed after completion of cortex aspiration and meticulous peeling of any residual lens fibers adhering to the peripheral posterior capsule. Fig. 5“Aspiration curette” for effective and safe debridement of anterior LEC layer additionally reduces capsular fibrosis Results Surgical complications In a preliminary evaluation of the first 376 consecutive cases performed during the first year (Menapace R. Routine posterior optic buttonholing for after-cataract prevention: The better alternative to the sharp edge optic? Breaking News Session, XXIIIth Congress of the ESCRS, 10.−14.9.2005, Lisbon) no case of cystoid macular edema (CME), and only one case of retinal detachment (RD) were encountered. Since the latter occurred only 4 months after surgery in a highly myopic eye with an intact anterior hyaloid surface, the causative role of POBH is questionable. The RD emerged from a group of small round holes in the superotemporal quadrant, and was repaired by cryopexy and gas injection. In only three cases could buttoning-in not be performed as planned because of a too small or decentered PPCCC. However, even with a suboptimally centered PPCCC the optic always perfectly centered permanently. During surgery, there was no case of vitreous prolapse into the anterior chamber, and therefore no need for an anterior vitrectomy. Postoperatively, four eyes presented with a minor, and two eyes with a major vitreous entrapment without apparent vitreous traction. These cases happened at a time when the surgical procedure was still being elaborated, and the importance of performing a complete circumferential viscodissection of anterior hyaloid and posterior capsule prior to the optic buttoning-in manoeuvre to preclude vitreous incarceration was not yet fully recognized. One of the two patients with major entrapment underwent bimanual anterior vitrectomy in topical anesthesia through the pre-existing paracentesis openings while the optic was temporarily desenclavated. The other patient declined the proposed reintervention. No CME or RD ensued in any of these eyes in the 3-year follow-up period. The technique, results, and complications of the first 500 consecutive cases have been extensively published in a peer-reviewed journal [12]. In a later sub-series which investigated the effect of creating an ACCC opening larger than the optic diameter to further reduce anterior capsule fibrosis especially at the haptic–optic junction, two other cases of delayed vitreous herniation into the anterior chamber occurred when a relatively large but still overlapping PPCCC was secondarily distended by posterior capsule contraction due to fibrosis emerging from the edge of the ACCC rim outside the optic, thus exposing the optic edge and creating a gap in the capsular diaphragm along the optic rim (Fig. 6). Consequently, due to the saccadeous eye movements, mobile vitreous incrementally crept into the anterior chamber. Using bimanual vitrectomy, with the infusion inserted through one of the pre-existing paracentesis openings and the vitrectome through a pars plana sclerotomy, the floppy vitreous strand was retracted into the vitreous cavity and excised. Except for these few cases, no further cases of vitreous entrapment or herniation were encountered in the second 500 consecutive cases. In two other cases of the above-mentioned sub-series, spontaneous desenclavation of the optic without vitreous presentation was noted the day after surgery due to a too largely dimensioned PPCCC. There was no case of endophthalmitis in the 1000 cases performed up to June 2007. The results of the first 150 cases—which may represent the learning curve—and the subsequent 850 cases are summarized in Table 1. Fig. 6Retraction of scarcely overlapping PPCCC rim from optic rim due to fibrosis emerging from ACCC larger than optic allows for delayed vitreous herniationTable 1ComplicationsResultsFirst 150 cases: • vitreous presenting in AC during surgery: none • vitreous entrapment detected postoperatively  • minor: four eyes  • major: two eyes (→ translimbal AVE in one eye) • CME / RD: noneCases # 151 to >1000: • oversized PPCCC with incomplete diaphragm: four eyes  • secondary desenclavation : two cases  • delayed vitreous herniation: two cases (→ single-port ppVE) • retinal detachement: one eye  • highly myopic 58 yr-old male, AL>26.5 mm, 4 months postop: Cryo+gas • CME: none • endophthalmitis: none After-cataract preventive effect After a maximum follow-up exceeding 3 years, there was no case of retrolental after-cataract formation. The optic inside the capsular edges remained completely clear in all cases (Figs. 7right and 8). When the anterior capsule was left unpolished, the posterior capsule overlying the optic periphery was often covered with a thin layer of translucent regeneratory LECs that formed into delicate pearls. These, however, did not relevantly compromise visualization of the peripheral retina. In some cases, localized Soemmering´s ring formation was observed (Fig. 8left). Additional anterior capsule polishing significantly reduced regeneratory LEC proliferation on the residual posterior capsule, thereby optimizing visibility of the peripheral retina (Fig. 8right). Fibrotic after-cataract formation depended upon whether anterior polishing had been performed or not. If so, no fibrosis was observed at all. If not, fibrosis of the anterior capsule was essentially restricted to the area adjacent to the haptic junction, where the undercrossing posterior capsule allowed an anterior capsule smaller than the optic diameter to establish direct contact with the optic (Fig. 9 and 10). From there, limited encroachment of the fibrosis unto the neighboring capsular areas was often noted. Typically, some amount of fibrosis was observed to have spread out along both the anterior and posterior capsulorhexis edges. In the areas between the haptic junctions where the posterior capsule overlapped the optic and had been sandwiched between the latter and the anterior capsule, however, both capsules remained essentially clear and transparent. If the anterior rhexis was larger than the optic, no fibrosis formed, also adjacent to the haptic junctions, even without capsular polishing due to the lack of contact to the optic. However, fibrosis extending from the ACCC edge unto the posterior capsule retracted the latter from the optic edge in two cases with scarce optic overlap, resulting in gaping of the capsular diaphragm and delayed vitreous herniation as reported above. Fig. 7Right. Two years after POBH: optic within capsule edges completely clear, posterior capsule overlying optic shows thin layer of regeneratory LEC proliferation with scarce small pearls. Left. Contralateral eye with in-the-bag placed IOL: regeneratory LEC proliferation on posterior capsule behind optic with abundant large pearlsFig. 8Right. Two years after POBH with polishing: significantly lower amount of regeneratory LEC proliferation—compared to contralateral eye (left) having undergone POBH without polishing Fig. 9Posterior capsule sandwiched between anterior capsule and optic precludes direct contact which catalyses anterior LEC transdifferentiation leading to fibrosis. Residual fibrosis essentially confined to haptic–optic junctionFig. 10Schematic close-up view highlighting capsule–IOL interplay at haptic junction. Undercrossing of PPCCC edge allows for localized anterior capsule contact with optic, resulting in fibrosis adjacent to haptic junction which may spread out along rhexis rim Detailed risk profile Several substudies were initiated to exactly determine potential risks compared to the standard in-the-bag implantation technique. All were prospective randomized intraindividual comparison studies. There was no statistically significant difference in the early postoperative pressure course, either with or without the use of pressure-lowering drops [21]. This was also true for the inflammatory response as measured with a laser-flare-cell-meter, even after inadvertent puncturing of the anterior hyaloid surface [20]. Regarding the postoperative axial shift of the IOL optic and its final position, there was a significant difference. While during the first 4 weeks the bag-fixated optics generally moved forward to finally settle more anteriorly, the buttoned-in optics immediately stabilized and did not move thereafter. This resulted in a difference of mean anterior chamber depth of approximately 1 mm. Accordingly, there was a significant mean myopic shift after bag-fixation, while refraction remained unchanged after buttoning-in of the optic [22]. Though no clinically apparent CME was reported, subclinical postoperative changes had to be ruled out. This was done by investigating the thickness and morphology of the central retina using a last-generation optical coherence tomography (OCT) device. In a series of 50 bilateral cases, no difference could be found in any of these parameters (Menapace R, Stifter E. Macular integrity after cataract surgery with combined primary posterior capsulorhexis and posterior optic buttonholing as evaluated by optical coherence tomography. Abstract XXVth Congress of the ESCRS, 8.−12.9.2007, Stockholm), [23]. Another ongoing study is proving the clinically obvious lack of optic decentration and tilt even within a suboptimally centered PPCCC. Discussion The advantages, possible downsides, and specific features of the technique are addressed in the following: The advantages of PPCCC fixation of the optic as compared to the standard in-the-bag fixation were found to be: Eradication of after-cataract formation behind the optic by diverting migrating LECs to bypass the optic rim towards the anterior optic surface. When the optic is buttoned-in, the posterior capsule leaf is relocated to lie on top of the anterior optic surface (Figs. 2, 3 and 8). By principle, migrating LECs can no longer access the retrolental space. At the optic junctions where the posterior capsule leaf undercrosses the haptic base, the high pressure exerted by the distended posterior capsule ring blocks LEC migration beneath the junctions.Significant reduction of capsular fibrosis without polishing. Myofibroblastic transdifferentiation is the substrate of capsular whitening and shrinkage. It mainly affects the anterior capsule, and is the cause for numerous complications including rhexis contraction (“rhexis phimosis”) and rhexis retraction with secondary extrusion of the optic out of the bag (“anterior optic buttonholing”), with possible decentration and tilt [10]. Direct contact between the anterior capsule and the optic is the trigger mechanism. The optic material then catalyses the myofibroblastic transdifferentiation of the anterior LECs on the back side of the anterior capsule, with consecutive capsule contraction and collagen deposition [8]. Buttoning-in causes the posterior capsule to be sandwiched between the anterior capsule and the optic, avoiding direct contact between the two. Consequently, LEC transdifferentiation of the anterior LEC layer is not initiated (“no contact - no fibrosis”). Only along the capsulorhexis edges some residual fibrosis may be seen. The usually seen extensive fibrosis of the whole anterior LEC layer remains restricted to the area of direct optic contact inside the optic junction.Reduced anterior optic ongrowth without polishing. After standard in-the-bag fixation, ongrowth of LECs unto the free optic zone is regularly observed with acrylates to a variable extent [1]. Usually transient, it may also be permanent. Though mostly limited to the periphery, it may in cases cover the whole free optic zone. With POBH this complication is abolished, since the crescent-shaped projection of posterior capsule forms a buffer zone between the anterior rhexis edge and the free optic. As with fibrosis, the optic ongrowth is restricted to the areas of direct anterior capsule contact to optic inside the haptic-optic junction.Eradication of residual fibrosis and ongrowth with additional polishing. Additional polishing of the anterior capsule specifically avoids fibrosis or ongrowth inside the haptic-optic junction, and fully excludes residual fibrosis in the areas where both capsules overlap.Capsular polishing without negative impact of retrolental regenerate formation. With bag-fixated IOLs, the strength and permanence of the barrier effect at the optic edge is largely dependent upon fibrosis [10]. Polishing, though effectively reducing fibrosis, therefore results in a significant increase of the YAG-LCT rate due to weakening collagenous edge sealing [11]. Since with POBH the migrating LECs are redirected to the anterior optic surface instead of being halted at the optic rim, the after-cataract preventive effect is completely independent of fibrosis. Polishing, therefore, does not promote retrolental LEC ingrowth when combined with POBH.Autocentration of optic. Even with a suboptimally centered PPCCC, the optic will automatically center within the buttonhole and align with the anatomical axis of the eye (Fig. 11), since the centering force is exerted by the loops which reside in the capsular bag fornix. As opposed to standard capsular bag fixation, centration of the optic is also independent of the size and centration of the ACCC.Forgivingness of undersized PPCCC. Due to the extreme stretchability and elasticity of the posterior capsule, POBH will still be feasible with a PPCCC opening smaller than 4 mm in diameter (Fig. 12). If the PPCC opening should happen to be too small to allow for buttonholing, the PPCCC can be either secondarily enlarged (similar to an ACCC), or the optic is simply placed in the bag without being buttoned in. In fact, this only happened in the early series in cases with a poorly dilating pupil.Immediate stability of capsular diaphragm and implant. Due to the lack of forward movement of the optic as seen after in-the bag fixation due to the loss of haptic memory and the distension of the capsule diaphragm as the capsules seal and fibrose, no myopic refractive shift occurs. While delayed by up to 4 weeks after bag-fixation depending upon the haptic design and material, final refraction is thus instantly attained, allowing prescription of final glasses 1 week after surgery, when the patient is seen by the referring ophthalmologist.Posterior positioning of the IOL optic by about 1 mm compared to bag-fixation significantly increases the interspace between the iris and optic. This avoids chronic iris chafing when a sulcus-fixated add-on IOL is secondarily implanted in order to correct for an erroneous IOL power selection, or to add astigmatic correction or multifocality.Fig. 11Bag-fixated haptics compensate for decentration of PPCCC openingFig. 12Exquisite stetchability and elasticity of posterior capsule compensates for undersizing of PPCCC opening Two advantages of POBH deserve special mentioning: 1. After-cataract prevention is entirely independent of the optic edge design. Thus, the design of the optic rim can be optimized in order to minimize reflectivity and thus dysphotopsia [16] without compromising the after-cataract performance of the IOL.2. After-cataract prevention is entirely independent of the optic material. Thus, the choice of optic material can be solely driven by achieving optimum biocompatibility, leading to minimum cell and bacteria adhesion. This may bring about a renaissance of the round-edged optics. Also, efforts in improving IOL materials may solely concentrate on optical performance, biostability and biocompatibility irrespective of their LEC inhibiting properties. As opposed to PPCCC with bag-fixation of the optic, POBH creates a stable and watertight diaphragm. Vitreous prolapse or entanglement will not occur should the anterior chamber flatten after retracting the aspiration–infusion tip following removal of residual OVD from the anterior chamber. The POBH technique as such is safe and highly effective with regard to after-cataract prevention. It is not only feasible, but highly recommended to perform it under topical anesthesia in order to guarantee bright and stable retroillumination when performing the PPCCC. No special or extra instruments or implants are required. Interestingly, no case of endophthalmitis occurred in the whole series, despite the fact that no intracameral or postoperative antibiotic prophylaxis had been performed. Though this series is not large enough to draw a meaningful conclusion, it may be attributed to the fact that buttonholing avoids seclusion of bacteria within the retrolental space. Other than with bag-fixation, the optic and capsule surface are fully exposed to the aqueous humor circulation from both sides. In case of an endophthalmitis, the vitreous cavity may be easily accessed for translimbal tapping and bimanual vitrectomy after temporary desenclavation of the IOL optic. Four crucial surgical steps deserve in-depth discussion: Sizing of the ACCC. Apart from capsular polishing, formation of residual fibrosis, in particular adjacent to the haptic-optic junction, can also be avoided by making the ACCC larger than the optic diameter (Fig. 13right). However, combining a standard 5 mm ACCC with anterior capsule polishing is definitely recommended over creating an overly large ACCC,because of the following downsides of the latter. Appropriate dimensioning of the PPCCC is rendered more difficult since the ACCC can no longer be utilized as a ruler, making unintentional under- or oversizing more likely. Should a PPCCC result to be much larger than the optic, alternative buttoning-in of the IOL optic through the ACCC opening can then no longer be resorted to. Also, a posterior capsule that only slightly overlaps the optic may be pulled off its edge as fibrosis of the anterior capsule sets in. Delayed gaping of the capsule-IOL diaphragm along the optic rim, with possible consecutive vitreous herniation, may result, as has happened in two cases. Therefore, a 5-mm ACCC combined with anterior capsule polishing is definitely preferable over creating an oversized ACCC.Viscodissection of the central hyaloid-capsular interspace (Berger´s space and Wieger´s ligament).In the young eye, the anterior hyaloid surface is separated from the posterior capsule by an interspace with two concentric compartments: Berger´s space in the center, and Girard´s space in the periphery. These two compartments are separated by a concentric circular ring along which both membranes are attached to each other, the Wieger´s ligament, which has a diameter of 8–9 mm and a width of 1–2 mm (Fig. 14). In practice, a great anatomical variability is experienced while performing a PPCCC in adult or senile eyes. In some eyes, attachment along Wieger´s ligament is no longer present, with the two united compartments thus forming one commun patent interspace. In others, there is hardly any interspace present, and viscoseparation of the strongly adhering membranes may be difficult to perform. This may be very difficult or not at all anticipated using the slitlamp with a cataractous lens. Therefore, utmost care must be used when centrally incising the posterior capsule, and inadvertent puncturing of the anterior hyaloid membrane still cannot be fully excluded. If in such a case larger quantities of OVD were injected through the capsular opening immediately after puncturing, the OVD might find its way through the punctured hyaloid surface and spread behind it within the vitreous body. This is excluded by performing part of the PPCCC beforehand. Only then, OVD is injected into Berger´s space to ensure that separation of the anterior hyaloid surface from the capsule has been attained. Viscodissection is then continued until Wieger´s ligament, if still adherent to the capsule, is severed and Girard´s space peripheral to it is accessed.Sizing and centration of the PPCCC.The posterior capsule being extremely stretchable and elastic, buttoning-in will also be feasible when a PPCCC turns out to be smaller than 4 mm. Also, the forces of the haptics will center the optic unto the anatomical axis within a suboptimally centered PPCCC. Therefore, though a 5 mm diameter and perfect centration should always be aimed at, deviation from this target will be tolerated. However, with a too large PPCCC that evades the optic rim, even when only along part of optic circumference, the IOL must be buttonholed through the ACCC with the loops either placed in the sulcus or capsular bag equator in order to avoid delayed vitreous prolapse. Therefore, the ACCC should be well-centered and no larger than 5–6 mm in diameter, to preserve the option of anterior rhexis fixation of the optic. Apart from providing an alternative for safe IOL fixation, an appropriately dimensioned ACCC also serves as a ruler for adequate sizing of the PPCCC.Sizing of the PPCCC may be difficult in eyes with a large capsular bag or lax zonules. This is heralded by wrinkles in the posterior capsule when flattened by OVD, and by traction folds when trying to create a central puncture or when pulling on the capsule edge when starting out with the PPCC. In such a case, implantation of a capsular tension ring remedies the problem as it puts the posterior capsule on radial stretch, creating uniform vector forces along the whole circumference. Such a ring can still be safely implanted when the PPCCC has already been started. However, bimanual tangential insertion should be preferred over injector implantation, and a smaller ring with a low resilience should be chosen. This minimizes capsular stress and distortion of the anterior segment [9], which could potentially lead to extension of the PPCCC or protrusion of the vitreous surface.Viscodissection of the peripheral hyaloid-capsular interspace (Girard´s space).Before implanting the IOL, the peripheral interspace between posterior capsule and anterior hyaloid must be circumferentially dissected with OVD in preparation of optic buttonholing. If not appropriately performed, the hyaloid surface may be shoveled up and damaged by the rim of the optic.Selection of IOL design. Though 1-piece IOLs with broad loops have been successfully used for the procedure [12], 3-piece IOLs with slim loops are definitely preferable. Of these, the HOYA AF-1 is most appropriate, since its continuous transition between haptic and optic allows the rim of the PPCCC to smoothly slide along it while the IOL is buttoned-in and centers itself within the PPCCC opening (Fig. 15)Fig. 13Schematic detailing capsular interplay with ACCC smaller (left) and larger than optic (right). Creating an ACCC larger than the optic precludes contact and thus fibrosis at the haptic–optic junction (green circle), but may retract a scarcely overlapping PCCC and thus expose optic rimFig. 14Schematic depicting interrelationship between posterior capsule and anterior hyaloid (Courtesy of Dr. T. Miyoshi). In practice, Wieger´s ligament may be completely detached; conversely, hyaloid may be closely adhering to capsule over extended areasFig. 15Continuous haptic-optic transitions allow PPCCC rim to smoothly slide along IOL circumference as optic is buttoned-in and centered within PPCCC by loops residing in capsular bag fornix Are there any drawbacks of the PCCC plus POBH concept long-term with regard to delayed after-cataract formation or retinal complications? Delayed after-cataract formation. In principle, equatorial LECs might access the retrolental space beneath the haptic junction where the PCCC rim under-crosses the haptic base. This has been observed in some cases, but was always minimal and limited to the very periphery. The explanation may be that the stretched PPCCC rim firmly adheres to the haptic base, and LEC migration is very much limited, due to the narrow door for potential invasion and the lack of capsular as a scaffold for migration should the barrier fail. Moreover, because of the lack of collagenous sealing between the capsular leaves LECs might theoretically invade the exposed posterior capsule and form pearls that accumulate at the anterior capsular rim or protrude into the anterior chamber, as is seen along the edges of YAG-LCT openings. This, however, has also not been observed to a clinically significant extent. LEC ongrowth unto the exposed posterior capsule, if at all present, was always flat with no or only hinted pearl formation not exceeding the PPCCC rim. Both observations have been recently confirmed by a pertinent publication on the eyes of children who had undergone a similar procedure 5 to 12 years earlier [7], which may be explained by the proliferation-inhibiting effect of the aqueous. Considering the fact that children’s eyes exhibit a much higher proliferative potential, long-term results in adults may be even better.Retinal complications. An increased incidence of CME and RD rate has been a concern. An earlier report did not find an increased incidence with PPCCC alone [26]. In our series of over 1000 POBH cases performed so far, not a single case of clinically significant CME was reported, and our OCT study revealed no difference in macular thickness or morphology. CME is provoked by the dissipation of cytokines released in the anterior segment, and by vitreous traction exerted by the anterior displacement of the vitreous body following exchange of the natural lens volume by a thin artificial lens. In these regards, POBH has certain advantages over standard in-the-bag IOL implantation. Firstly, as opposed to a sole PPCCC, optic buttoning-in creates a watertight diaphragm blocking the posterior dissipation of cytokines. In addition, viscodissection of the posterior capsule and anterior hyaloid creates a contiguous cushion of OVD behind the capsule–IOL diaphragm including the zonular region which precludes access of cytokines to the posterior segment until it is eventually resorbed. The more posterior positioning of the buttonholed optic together with the OVD cushion behind it, and the lack of subsequent anterior movement of the optic, prevent both immediate and delayed anterior displacement of the vitreous body. Severing the attachments between the posterior capsule and the hyaloid, namely Wieger´s ligament, may help to avoid traction on the vitreous when the chamber happens to flatten during OVD aspiration, or thereafter. Regarding RD, one case was encountered 4 months after surgery in a young male with high axial myopia. For standard cataract and IOL surgery, a recent study reported a 5-year RD incidence of 0.7% and 21%, depending on the absence or presence of lattice degenerations [18]. In the 5 years following standard cataract surgery, such posterior vitreous detachment occurs in about four out of five eyes with the vitreous still attached. This is triggered by the anterior positioning of a bag-placed optic. Though our follow-up with POBH surgery is shorter than 5 years, the excessively low RD rate of only 0.1% overall seems to indicate a significantly lower risk. This may explained as follows: as an IOL optic buttoned-in into a PCCC is positioned 1 mm more posteriorly compared to bag-fixation [22] (Fig. 4bottom), it inherently stabilizes the vitreous, and thus may avoid or delay posterior vitreous detachment. Severing of all adhesions between the posterior capsule and the anterior hyaloid and creating a cushion of OVD behind the capsule-IOL diaphragm, which is only slowly resorbed, may serve as an additional protective factor. A longterm study, including the incidence of vitreous and retinal detachment following POBH, is required to establish possible advantages of this technique over standard in-the-bag implantation of IOLs. If all the precautions detailed above are carefully met, PPCCC with POBH is a very controlled and safe procedure which carries the potential of becoming a routine procedure. In any case, apart from pediatric cases, it may be considered in following clinical situations: pseudoexfoliation syndrome because of the pronounced tendency of capsular shrinkage; high myopia because of the significantly greater inclination to develop after-cataract; and peripheral retinal pathologies requiring controls and treatment (myopia, diabetes) because of the unimpeded insight for diagnosis and therapy. Two potential future applications may be mentioned separately: Toric IOLs. Overly long plate and z-shaped open-loop haptics have been used to provide for rotational stability. However, these cannot completely avoid rotation and may interfere with capsular bag closure and thus after-cataract performance. Posterior rhexis fixation makes postoperative optic rotation impossible without the need for special haptics (Fig. 16).Accommodative shift IOLs. Currently marketed passive-shift IOLs have been designed to move anteriorly within the capsular bag upon accommodative effort [14]. The fact is that any bag-fixated IOL is immobilized due to fibrotic shrinkage. Instead of moving the optic, movement of the whole IOL-capsule complex is therefore more promising. However, fibrotic distension of the zonules would again obviate axial movement. POBH drastically reduces capsular fibrosis, as the sandwiched posterior capsule precludes the contact between the anterior capsule and the optic as the prerequisite for LEC transdifferentiation. Additional polishing of the anterior capsule, with special regard to the areas adjacent to the haptic–optic junctions, has been shown to keep the capsule fully transparent and possibly elastic enough to allow axial movement of the optic under ciliary muscle contraction. This may fulfil the conditions of sufficient passive axial mobility, as postulated by Preussner, to allow for clinically relevant shift-induced accommodation when embedded in his magnet-driven concept [17] (Figure 17). Instead of implementing the magnets into a special CTR as suggested by Preussner, these could be directly mounted to the optic periphery. A study investigating passive mobility under pharmacological stimulation with cycloplegics and pilocarpine is ongoing. Fig. 16Rhexis-fixation precludes postoperative IOL rotation in an oversized bag and delayed optic rotation by haptic compression arising from capsular bag shrinkageFig. 17Repelling magnets located in the optic periphery (open square at 3/9 o´clock) and under the superior/inferior rectus muscle insertions. Upon ciliary muscle contraction/zonular relaxation, the optic is pushed forward, increasing the power of the IOL optic within the eye Table 2 summarizes current and potential future indications. Table 2IndicationsSummary of indications[Pediatric cataracts]PEX-SyndromaHigh myopia (reduced barrier effect)Peripheral retinal disease: axial myopia, diabetes (improved insight for diagnosis & therapy)Multifocal IOLs (patients more susceptible to capsular opacification)→ToricIOLs→Accommodativelens system In conclusion, primary posterior capsulorhexis combined with posterior optic buttonholing is a well-controlled, safe, and highly effective procedure with a steep learning curve, thus carrying the potential of becoming a routine alternative to standard in-the-bag implantation of intraocular lenses. As opposed to standard in-the-bag placement, its effectiveness is completely independent of optic material and optic edge design. This technique is meant for the skilled and dedicated surgeon, and must be carefully approached. It should be reserved to large pupil eyes. Should the pupil come down during the surgery, one may switch to standard in-the-bag surgery, optionally combined with a small PPCCC. Thorough viscodissection of anterior hyaloid and posterior capsule is crucial. Additional anterior capsule polishing is highly recommended, as it abolishes any residual fibrosis without any risks or downsides. Though long-term results in children's eyes support sustained efficacy and lack of complications, 5-year results should be awaited until the technique may be considered or recommended as a routine alternative to standard in-the-bag fixation of the IOL. For the time being, it is recommended with pseudoexfoliation syndrome, high axial myopia, peripheral retinal disease, multifocal IOLs, and toric IOLs when made available.

Psychopharmacologia-4-1-2244695

Selective PDE inhibitors rolipram and sildenafil improve object retrieval performance in adult cynomolgus macaques

Rationale Selective phosphodiesterase (PDE) inhibitors improve the formation of hippocampus-dependent memories in several rodent models of cognition. However, studies evaluating the effects of PDE inhibition on prefrontal cortex-dependent cognition and in monkeys are rare. Introduction Second messenger cyclic nucleotides, i.e., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), have been strongly implicated in processes of synaptic long-term potentiation (LTP; Frey et al. 1993; Zhuo et al. 1994) and have cognition-enhancing properties (Bernabeu et al. 1996; Blokland et al. 2006; Prickaerts et al. 2002a; Prickaerts et al. 2005). cAMP and cGMP are hydrolyzed by phosphodiesterase (PDE) enzymes, and inhibitors of PDEs (PDE-Is) produce cognition-enhancing effects in animal models of cognition. In this field, research has focused on PDE4 and PDE5 inhibitors (Blokland et al. 2006; Rose et al. 2005; Rutten et al. 2006) and more recently PDE2 (Boess et al. 2004; Rutten et al. 2007b) and PDE10 (Rodefer et al. 2005) inhibitors. For example, inhibition of PDE2, PDE4, and PDE5 can improve memory in the object recognition task in rodents (Boess et al. 2004; Prickaerts et al. 2004; Rutten et al. 2007b). Moreover, the cAMP-selective PDE4-I rolipram is an effective performance enhancer in the passive avoidance task (Egawa et al. 1997; Imanishi et al. 1997), fear-conditioning test (Barad et al. 1998), and radial arm maze (Zhang and O'Donnell 2000). The cGMP-selective PDE5-I sildenafil has been shown to enhance performance in the object recognition task, in the T-maze, and spatial plus maze (Devan et al. 2006; Devan et al. 2004; Patil et al. 2006; Prickaerts et al. 2004). In addition, sildenafil improved object recognition memory in mice (Rutten et al. 2005), and another PDE5-I, zaprinast, improved performance in a passive avoidance task in neonatal chicks (Campbell and Edwards 2006). Taken together, these results confirm the beneficial effects of PDE inhibition on cognition involving the hippocampus. The present study assesses the effects of PDE inhibition on object retrieval (OR) performance, a task of prefrontal cognition, in monkeys. OR (also known as the detour reaching task) involves attention, response inhibition, and planning (i.e., executive function; Diamond et al. 1989). Because the prefrontal cortex is not well represented in rodents (especially the dorsolateral prefrontal cortex) and considerable debate exists on whether the rodent prefrontal cortex subserves the same behavioral functions as the primate/human prefrontal cortex (Brown and Bowman 2002; Uylings et al. 2003), tests of prefrontal functioning (i.e., executive function) are preferentially carried out in monkeys. Successful completion of the OR task requires various hypothetical steps. An animal must appreciate the contradiction between visual and tactile input, ignore the incorrect input, remember the previous incorrect approach, and formulate a novel approach to obtain the reward, which acts against the innate tendency of perseveration toward sustained sensory stimulation (Lipina and Colombo 2001). Direct support for the involvement of the frontal cortex in OR comes from studies in which lesions of the frontal cortex impaired, while lesions of the hippocampus did not impair, performance in the task (Diamond et al. 1989; Wilkinson et al. 1997). Furthermore, deficits in OR tasks have been documented in monkeys after treatment with methylphenyl tetrahydropyridine (Schneider and Roeltgen 1993; Taylor et al. 1990a; Taylor et al. 1990b), phencyclidine (Jentsch et al. 2000; Jentsch et al. 1999a; Jentsch et al. 1999b), and after excitotoxic lesioning with quinolinic acid of the striatum (Roitberg et al. 2002). The effect of PDE-Is in the OR task have, to our knowledge, never been evaluated before in unimpaired monkeys. As mentioned above, improvement in hippocampus-dependent memory tasks in rodents is well established through PDE inhibition. However, no evidence exists on the effects of rolipram or sildenafil on prefrontal cortex-dependent tests of executive function. As the PDE4 enzyme is omnipresent throughout the brain and PDE5 is expressed in the hippocampus, cortex, and cerebellum (van Staveren et al. 2004), we hypothesized that OR performance would be improved by the PDE4 and PDE5 inhibitors rolipram and sildenafil, respectively. Materials and methods Subjects Fourteen adult (age 5–12 years; weights 6–9 kg) male cynomolgus macaques (Macaca fascicularis) were housed in same-sex pairs in a colony room maintained at 21 ± 2°C, 40 ± 10% humidity, and on a normal 12-h light/dark cycle (lights on at 7:00 a.m.). Food (Purina High Protein no. 5045) and water were available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Roche Palo Alto and were in accordance with National Institutes of Health guidelines. Object retrieval task The OR task has been previously described by Diamond et al. (1989). Briefly, this task requires a monkey to reach into a clear acrylic box (dimensions = 5 × 5 × 5 cm) with one open side, to retrieve a food treat (cubes of apple or pear, 1–2 cm2). The box was positioned in front of the monkey and outside of the home cage, with the open side facing left, right, or toward the monkey. Food treats were placed on the outer edge, inner edge, or deep within the box. A test session consisted of 17 trials with nine “easy” food retrievals (i.e., placement of the food reward on the inner or outer edges of the box or when the opening is toward the monkey) and eight “difficult” food retrievals (i.e., placement of the food reward deep within the box and the open side facing left or right, see Fig. 1). The order of presentation never varied (Table 1), there were no contingencies for incorrect reaches (i.e., monkeys typically acquired the treat after the incorrect reach) or dropped treats, and trials were terminated if there were no reaches within 3 min. The box was cleaned diligently between trials to minimize cues that could influence the task and subsequent task performance. After an initial period (1 week) to acclimatize the monkeys to the apparatus and procedure, tests were conducted twice a week. Fig. 1A schematic overview of possible trials in the object retrieval (detour-reaching) task. The transparent box containing a food reward is depicted form the monkey’s point of view. The top five orientations depict easy trials, and the bottom two orientations depict difficult trialsTable 1Primate object retrieval: trial orderTrial numberDescriptionLevel1LOS—line of sightEasy2LOS—line of sightEasy3RO—right outsideEasy4RD—right deepDifficult5RO—right outsideEasy6RI—right insideEasy7RD—right deepDifficult8LO—left outsideEasy9LD—left deepDifficult10LO—left outsideEasy11li—left insideEasy12ld—left deepDifficult13ld—left deepDifficult14rd—right deepDifficult15ld—left deepDifficult16rd—right deepDifficult17los—line of sightEasy Drugs Rolipram (0.003, 0.01, 0.03, and 0.1 mg/kg; Sigma Chemicals, St Louis, MO) and sildenafil (0.3, 1, and 3 mg/kg; Sequoia Research Products Limited, Pangbourne, UK) were prepared fresh daily in a suspension of 10% cremaphore/90% saline and administered 30 or 60 min before testing, respectively. Drugs were administered via intramuscular injection (i.m.) in a volume of 0.1 ml/kg. Compounds were typically administered on Tuesday and Friday each week. Weekly test sessions were comprised of a vehicle session and a drug test session. Drug administration and behavioral measurement were completed blind, and each drug–dose combination was tested once. Drug doses were administered in a pseudorandom order. Data analysis The mean percent correct first reaches for easy and difficult food retrievals were analyzed with a one-factor (Dose) repeated-measures analysis of variance. When significant effects were demonstrated, a post-hoc Dunnett analysis comparing dose conditions to vehicle performance was performed. For all tests, the significance level was 0.05 (two-tailed). Results During vehicle sessions, monkeys readily reached for the food treats, successfully acquiring the treat on the first reach 100 ± 0% during easy trials and 52 ± 3% during difficult trials. Over the course of the experiment (2 months), there were no changes in vehicle performance. The PDE4 inhibitor rolipram (0.003–0.1 mg/kg, i.m.) dose-dependently increased correct first reaches during difficult trials (F[3, 55] = 13.5, p < 0.05), reaching significance at 0.01 mg/kg (Fig. 2a). At the highest dose tested without side effects (0.03 mg/kg), the percentage correct first reaches were increased approximately 20 to 71 ± 3%. Note that at 0.1 mg/kg (data not shown), emesis was observed in all but two monkeys; these results were therefore excluded from analysis. Fig. 2The effects of PDE-I on object retrieval (OR) performance (mean values and SEM) a The effects of the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil (b) on the percentage correct first reaches on difficult trials in the OR task. Asterisks indicate significant differences from baseline (P < 0.05) The PDE5 inhibitor sildenafil (0.3–3 mg/kg, i.m.) dose-dependently increased correct first reaches during difficult trials (F[3, 50] = 8.6, p < 0.05), reaching significance at 1 mg/kg (Fig. 2b). At the highest dose tested (3 mg/kg), correct reaches were increased approximately 20 to 73 ± 3%. No side effects were observed. Neither rolipram nor sildenafil altered performance during the easy trials, with the exception of the high dose of rolipram (0.1 mg/kg), in which monkeys failed to perform the task (data not shown) because of emetic side effects. Discussion The present study demonstrates performance-enhancing effects of two selective PDE-Is on OR performance in monkeys. OR is subserved by the prefrontal cortex and/or fronto-striatal pathways, within which modulation of dopamine and acetylcholine transmission are involved in attention, response inhibition (i.e., executive function), and working memory in rodents and primates (Jentsch et al. 2000; Lipina and Colombo 2001; Palfi et al. 1996; Ramos et al. 2003; Wilkinson et al. 1997). To our knowledge, this is the first study to investigate the effects of PDE5 inhibition on executive function in monkeys. Of note, two previous studies have investigated the effects of sildenafil on cognition-related variables in humans. In these studies, sildenafil was shown to enhance simple reaction times and to some extent enhancement of focused attention (Grass et al. 2001; Schultheiss et al. 2001). However, these findings have not been further described in the literature. The effects of the PDE4 inhibitor rolipram on executive function have yet to be assessed in humans. Thus, the current results complement and extend to the cognition-enhancing effects of PDE inhibition. Previous studies have repeatedly shown that PDE-Is can have cognition-enhancing effects, mainly in hippocampus-dependent memory tasks, in rodents. For example, the PDE4 inhibitor rolipram improved long-term memory in the object recognition task, in passive avoidance learning, and fear conditioning (Barad et al. 1998; Rutten et al. 2006; Zhang et al. 2005). In addition, rolipram had performance-enhancing effects in a prefrontal cortex-dependent working memory task, i.e., delayed alternation, in young rats and young monkeys (Ramos et al. 2003). Results from the present study corroborate the cognition-enhancing effects of low-dose (0.01 mg/kg) rolipram treatment in a prefrontal cortex-dependent task, although the present OR task does not involve working memory but requires attention and response inhibition (i.e., executive function; Diamond et al. 1989). Furthermore, in aged mice, rolipram ameliorated the age-related deficits in the passive avoidance task, a test of hippocampus-dependent memory. In contrast, rolipram impaired prefrontal cortex-dependent working memory performance in aged rodents and aged monkeys. With advancing age, opposite profiles between the function of protein kinase A (PKA) in the hippocampus and prefrontal cortex were suggested to explain these results; that is, the prefrontal cortex showed indices of increased PKA activity, while the hippocampus exhibited evidence of decreased PKA activity (Ramos et al. 2003). Although in the present study, cognition-enhancing effects on executive function were observed after rolipram treatment in young monkeys, the possible cognition-impairing effects of rolipram on prefrontal cortex-dependent tests in aged monkeys should be further investigated. Compared to PDE4 inhibition, the cognition-enhancing effects of PDE5 inhibition have not been studied as extensively. However, a growing number of studies have shown cognition-enhancing effects of PDE5-Is in multiple tests and in multiple species. PDE5-Is improved cognitive performance in object recognition and inhibitory and passive avoidance tasks (Baratti and Boccia 1999; Prickaerts et al. 2005; Prickaerts et al. 2004; Prickaerts et al. 2002b; Rutten et al. 2005; Shafiei et al. 2006; Singh and Parle 2003). In addition, the inhibition of PDE5 reversed memory deficits induced by scopolamine, diabetic neuropathy, or nitric oxide (NO) synthase inhibitors in rats (Devan et al. 2006; Devan et al. 2004; Patil et al. 2004; Prickaerts et al. 1997). To our knowledge, no literature exists on the possible cognition-enhancing effects of PDE5 inhibitors in nonhuman primates. Furthermore, the effects of PDE5 inhibition on prefrontal cortex-depending cognition, i.e., working memory and executive function, are unknown. The underlying mechanisms of PDE-Is and cognition enhancement are still elusive, but several possible pathways have been described. Possible mechanisms of action for rolipram and sildenafil are based on the proposed underlying signaling pathways of LTP. Both cAMP and cGMP have been strongly implicated in hippocampal LTP (Frey et al. 1993). Activation of LTP-related signaling pathways of cAMP/PKA/cAMP response element-binding protein (CREB) and cGMP/protein kinase G (PKG)/CREB have been implicated as the underlying mechanisms for the cognition-enhancing effects of PDE4 and PDE5 inhibitors (Bernabeu et al. 1996; Blokland et al. 2006; Lu and Hawkins 2002; Prickaerts et al. 2002a; Rutten et al. 2007b). A very recent study suggested another pathway through which cGMP can influence cognitive processes. This study showed that the hippocampal NO/cGMP pathway directly stimulates the postsynaptic cAMP/PKA/CREB pathway (Matsumoto et al. 2006). Alternatively, cGMP has also been found to maintain LTP via a presynaptic cGMP/PKG pathway (Zhuo et al. 1994). However, the present study involves prefrontal cortex-dependent behavior, and it remains to be proven that the above mentioned hippocampal LTP mechanisms of synaptical strengthening are also applicable in the prefrontal cortex. Of note, the effects of rolipram on prefrontal cortex performance were explained via activation of the postsynaptic PKA pathway (Ramos et al. 2003). Thus, both cGMP and cAMP can activate cellular cascades resulting in CREB phosphorylation, which could induce long-lasting changes in neuronal signaling and may thereby explain the performance enhancement observed in the present study. Apart from LTP-related mechanisms of action, the effects of rolipram and sildenafil on prefrontal functioning in the OR may be explained by their stimulatory effect on neurotransmission. Because rolipram is independent of a specific receptor system, it is suggested that rolipram may ameliorate physiological abnormalities that occur secondary to alterations in dopaminergic, glutamatergic, serotonergic, and/or adrenergic transmission (Maxwell et al. 2004; Rutten et al. 2007a). Elevated cAMP levels are thought to excite noradrenergic and dopaminergic neurotransmitter systems (Schoffelmeer et al. 1985), thus enhancing their availability, hence also enhancing synaptic transmission. Similar to rolipram, sildenafil is a drug that is not linked to specific neurotransmitter systems. Modulatory activity of sildenafil was suggested on central dopaminergic pathways (Ferrari et al. 2002). Dopaminergic dysfunction in prefrontal cortex may subserve at least a component of the impaired OR/detour performance in deficit models (Jentsch et al. 1999a). Moreover, serotonin depletions of the prefrontal cortex in the common marmoset result in deficits in the acquisition of the OR task (Walker et al. 2006). Hence, activation of cGMP or cAMP may improve dopaminergic turnover and prefrontal functioning in the OR task. Thus far, it remains unclear which of these abovementioned mechanisms are involved in the prefrontal cortex and how they explain cognition enhancement by inhibition of PDEs in the OR task. To summarize, previous research demonstrated the cognition- and largely memory-enhancing effects of PDE4 and PDE5 inhibition in rodents. The results from the current study extend these cognition-enhancing effects of PDE-Is in rodents to nonhuman primates. More specifically, rolipram and sildenafil improve OR performance, thus demonstrating that PDE4 and PDE5 inhibition enhance performance in a cognitive domain involving executive function (and attention) mediated by the prefrontal cortex.

Behav_Genet-4-1-2257997

Speed, Variability, and Timing of Motor Output in ADHD: Which Measures are Useful for Endophenotypic Research?

Attention-Deficit/Hyperactivity Disorder (ADHD) shares a genetic basis with motor coordination problems and probably motor timing problems. In line with this, comparable problems in motor timing should be observed in first degree relatives and might, therefore, form a suitable endophenotypic candidate. This hypothesis was investigated in 238 ADHD-families (545 children) and 147 control-families (271 children). A motor timing task was administered, in which children had to produce a 1,000 ms interval. In addition to this task, two basic motor tasks were administered to examine speed and variability of motor output, when no timing component was required. Results indicated that variability in motor timing is a useful endophenotypic candidate: It was clearly associated with ADHD, it was also present in non-affected siblings, and it correlated within families. Accuracy (under- versus over-production) in motor timing appeared less useful: Even though accuracy was associated with ADHD (probands and affected siblings had a tendency to under-produce the 1,000 ms interval compared to controls), non-affected siblings did not differ from controls and sibling correlations were only marginally significant. Slow and variable motor output without timing component also appears present in ADHD, but not in non-affected siblings, suggesting these deficits not to be related to a familial vulnerability for ADHD. Deficits in motor timing could not be explained by deficits already present in basic motor output without a timing component. This suggests abnormalities in motor timing were predominantly related to deficient motor timing processes and not to general deficient motor functioning. The finding that deficits in motor timing run in ADHD-families suggests this to be a fruitful domain for further exploration in relation to the genetic underpinnings of ADHD. Introduction It has become apparent from twin- and adoption studies that the development of the Attention-Deficit/Hyperactivity Disorder (ADHD) (American Psychiatric Association 1994) is strongly genetically based (Faraone et al. 2005; Willcutt, in press). Overall heritability estimates exceed 0.70 (Faraone and Doyle 2000; Smalley 1997) and are fairly constant across studies conducted worldwide (Faraone et al. 2005). Research aimed at the molecular genetic basis of the disorder (genotype) has had success in identifying some susceptibility genes by using information from behaviorally observable symptoms (phenotype) (Faraone et al. 2005). However, the current knowledge about the genetic basis of the disorder is still limited and the causal pathway(s) leading from genotype to phenotype have yet to be revealed. Both issues have been the aims of investigation in endophenotypic research. Endophenotypes are defined as heritable, vulnerability traits that mark a risk for the development of the disorder (Almasy and Blangero 2001; Castellanos and Tannock 2002; Doyle et al. 2005; Gottesman and Gould 2003; Skuse 2001; Waldman 2005; Zobel and Maier 2004). They are conceptualized as forming an intermediate link between the genotype and phenotype and are presumably genetically less complex compared to phenotypic symptoms (Gottesman and Gould 2003; Waldman 2005). Because of these characteristics, it is thought that, compared to phenotypic symptoms, endophenotypes are more suitable for detecting disease genes and for unraveling the modes of actions of these disease genes. Several criteria have been proposed to discriminate an endophenotype from other biological markers that are not causally involved in the disorder but are merely associated with the disorder (Durston et al. 2004; Gottesman and Gould 2003). Although these criteria do not appear to be universally agreed upon, several key criteria have emerged from the literature (Almasy and Blangero 2001; Castellanos and Tannock 2002; Doyle et al. 2005; Gottesman and Gould 2003; Skuse 2001; Waldman 2005; Zobel and Maier 2004). First, an endophenotype should co-occur with the disorder, although given the heterogeneity of ADHD, it is unlikely that a single endophenotype will occur in all patients with ADHD (Doyle et al. 2005). Second, non-affected relatives should also exhibit the endophenotype to some extent, indicating that the endophenotype contributes to a familial susceptibility for the disorder. Because non-affected relatives share, on average, 50% of their genes with the affected family member, it is theorized that they also carry some of the susceptibility genes of ADHD which translate into subtle abnormalities in the endophenotype (Gottesman and Gould 2003; Waldman 2005). Third, the endophenotype should show familial resemblance, reflected by significant sibling correlations for the endophenotypic measure. In addition to these criteria, several other important characteristics of an endophenotype have been put forward, such as reliability of measurement, stability over time (i.e. expressed regardless whether or not the disorder is currently manifested), and acting as a mediator and/or moderator between genes and disorder (Doyle et al. 2005; Waldman 2005). The focus of our study lies on the first three key criteria of an endophenotype. Possible endophenotypic candidates might be found in the deficits in motor output associated with ADHD. Fine motor coordination problems as well as gross motor coordination problems are frequently observed in co-occurrence with ADHD (Carte et al. 1996; Korkman and Pesonen 1994; Marcotte and Stern 1997; Piek et al. 1999; Pitcher et al. 2003; Whitmont and Clark 1996). Because of this highly frequent co-occurrence, it is feasible that motor problems might be genetically related to the risk for developing ADHD (Gillberg 2003), which has indeed been reported recently (Martin et al. 2006). This might make motor measures useful endophenotypic candidates, which was underlined by the findings of motor control difficulties in non-affected siblings of children with ADHD (Rommelse et al. 2007a; Slaats-Willemse et al. 2005). A specific aspect of motor output that might serve as a candidate endophenotype is temporal organization (Castellanos and Tannock 2002; Waldman et al. 2006). Temporal organization of motor output refers to the timing of movements (i.e. motor timing) and seems to be predominantly mediated by the cerebellum and basal ganglia and their reciprocal connections with the cerebral cortex (Handy et al. 2003; Harrington et al. 1998; Ivry 1996; Nenadic et al. 2003). The timing of motor output is hypothesized to consist of two components: a clock component, which reflects central time keeping operations, and a motor delay component, which reflects random variability due to response implementation processes (Harrington et al. 1998; Keele et al. 1985; Wing and Kristofferson 1973). Children with ADHD seem to be predominantly impaired in the clock component, as evidenced by paradigms specifically assessing timing operations independent of motor operations, such as duration discrimination tasks (Keele et al. 1985; see for review Toplak et al. 2006). The motor component is possibly best assessed using simple reaction time tasks and free Tapping tasks in which timing is minimized. Some have found no impairments in children with ADHD (Kalff et al. 2003; Seidman et al. 1997, 2000), others have (Kalff et al. 2005). By combining both components, one can assess timing that is predominantly related to motor output, which reflects the interplay between timing and motor skills. Studies using these motor timing tasks have, however, not revealed consistent results of impairments in children with ADHD, possibly due to inconsistencies in methodological approaches across studies (Toplak et al. 2006). However, greater variability in motor output when a timing component is required is a frequently reported finding in children with ADHD (Leth-Steensen et al. 2000; Pitcher et al. 2002; Rubia et al. 1999, 2003; Toplak et al. 2006; Van Meel et al. 2005). None of these studies, however, used a measure of basic motor speed/variability (without requirements regarding timing/rhythm) in addition to the motor timing measure. Therefore, it remains unclear whether the variability in motor timing is primarily due to variability of motor processes, or due to variability of timing processes or both. Here, we administered two simple motor tasks in addition to the motor timing task, in order to investigate whether deficits were specifically related to the timing of motor output or whether deficits were also observed in tasks that do not require timing of motor output. The current study is, to our knowledge, the first to examine motor timing abilities in not only children with ADHD, but also in their siblings, in order to investigate whether motor timing measures might be suitable endophenotypic candidates. Previous studies on related topics have shown that non-affected siblings of children with ADHD have comparable problems in time reproduction skills as their affected siblings (Rommelse et al. 2007b) and that variability in reaction time shows familial overlap with ADHD (Andreou et al. 2007), giving support to the hypothesis that motor timing measures may be suitable endophenotypic candidates. It was expected that (1) children with ADHD would be impaired on motor timing measures and not (or to a lesser extent) on motor measures without a timing component, indicating an association between ADHD and motor timing deficits. Furthermore, we expected (2) to find similar findings in the non-affected siblings of the children with ADHD, suggesting motor timing deficits are not merely associated with the disorder. Last, we expected (3) to find correlations between siblings indicating familial resemblance on motor timing measures. Method Participants Families with at least one child with the combined subtype of ADHD (proband) and at least one additional sibling (regardless of possible ADHD-status) were recruited in order to participate in the Dutch part of the International Multicenter ADHD Genes study (IMAGE). The IMAGE project is an international collaborative study that aims to identify genes that increase the risk for ADHD using QTL linkage and association strategies (Brookes et al. 2006). Additional control families were recruited from primary and high schools from the same geographical regions as the participating ADHD-families. Controls and their first degree relatives had no formal or suspected ADHD diagnosis. A total of 238 ADHD-families and 147 control-families fulfilled inclusion and exclusion criteria. Within the ADHD-families, 238 probands (all with combined subtype of ADHD), 112 affected siblings (64 with combined subtype, 28 with inattentive subtype and 20 with hyperactive-impulsive subtype of ADHD) and 195 non-affected siblings participated. Control-families consisted of 271 children. For 51 control children, no additional control sibling could be recruited for the study (see for an overview Tables 1, 2). Table 1Distribution of family sizesNumber of siblings within a familyADHDControlFamilies (n)Individuals (n)Families (n)Individuals (n)100515121773547214435315920604832416Total238545147271Note: ADHD = Attention-Deficit/Hyperactivity DisorderTable 2Distribution of affected and non-affected siblings within ADHD-familiesTotal number of children within a familyDiagnostic statusADHD-families (n)Proband (n)Affected siblings (n)Non-affected siblings (n)211–621–1115312–7111241–222413–2121111241–31Total238Note: ADHD = Attention-Deficit/Hyperactivity Disorder All children were between the ages of 5 and 19 years and were of European Caucasian descent. Participants were excluded, if they had an IQ < 70, a diagnosis of autism, epilepsy, brain disorders or known genetic disorders, such as Down syndrome or Fragile-X-syndrome. Within an ADHD-family, both proband and siblings were similarly screened using the standard procedures of the IMAGE project described by Brookes et al. (2006). Briefly, screening questionnaires (parent and teacher Conners’ long version rating scales [Conners 1996] and parent and teacher Strengths and Difficulties Questionnaires [SDQ, Goodman 1997]), were used to identify children with ADHD symptoms. T-scores ≥ 63 on the Conners’-N-scale (DSM-IV total symptom score) and scores > 90th percentile on the SDQ-hyperactivity scale were considered as clinical. For all children within a family scoring clinically on any of the questionnaires, a semi-structured, standardized, investigator-based interview was administered separately for each child: the Parental Account of Children’s Symptoms (PACS; Taylor 1986). The PACS covers DSM-IV symptoms of ADHD, conduct disorder, oppositional defiant disorder, anxiety, mood, and other internalizing disorders. The section on autistic behaviour traits was administered, if a clinical score (raw score ≥ 15) was obtained on the Social Communication Questionnaire (SCQ; Berument et al. 1999). A standardised algorithm was applied to the PACS and parent rated Conners’ to derive each of the 18 DSM-IV ADHD items, providing operational definitions for each behavioural symptom. These were combined with items that were scored 2 (‘pretty much true’) or 3 (‘very much true’) in the teacher rated Conners’ ADHD subscale to generate the total number of hyperactive-impulsive and inattentive symptoms of the DSM-IV. Situational pervasiveness was defined as at least one symptom occurring within two or more different situations as indicated by the parents in the PACS interview as well as the presence of at least one symptom scoring 2 or 3 on the ADHD subscale as indicated by teachers on the Conners’. Siblings were regarded as non-affected, if they obtained scores in the non-clinical range on both the parent and teacher questionnaires (Conners’-N-scale: T-score ≤ 62, SDQ < 90th percentile). No PACS interview was administered concerning non-affected siblings. The Conners’ long version for both parents and teachers was completed for control children. Control children had to obtain non-clinical scores on both the parent and teacher version (Conners’-N-scale: T-score ≤ 62). Table 3 provides the characteristics of the four groups. Table 3Sample characteristicsProbandsAffected siblingsNon-affected siblingsNormal controlsF3,812Contrastsn = 238n = 112n = 195n = 271MSDMSDMSDMSDAge in years12.02.512.03.411.53.611.63.2ns% Right handed91.187.589.285.5nsa% Male84.556.345.140.6113.9*a1 > 2,3,42 = 3 & 2 > 43 = 4Estimated full scale IQ97.913.0100.710.6103.810.9106.010.223.5*1 = 2 & 1 < 3 = 42 = 3 & 2 < 43 = 4Conners’ parent DSM-IV    Inattentive71.18.466.011.647.97.046.54.8585.4*1 > 2 > 3 = 4    Hyperactive-impulsive79.19.267.813.649.06.947.35.1767.3*1 > 2 > 3 = 4    Total76.98.668.311.648.26.846.54.5875.7*1 > 2 > 3 = 4Conners’ teacher DSM-IV    Inattentive66.09.161.710.248.36.046.44.6386.3*1 > 2 > 3 = 4    Hyperactive-impulsive70.210.763.513.348.36.547.25.0378.1*1 > 2 > 3 = 4    Total69.89.863.811.448.35.846.44.5485.8*1 > 2 > 3 = 4ADHD diagnosis    Inattentive–28––    Hyperactive-impulsive–20––    Combined23864––Note: 1 = Probands; 2 = Affected siblings; 3 = Non-affected siblings; 4 = Normal controlsADHD = Attention-Deficit/Hyperactivity Disorder; DSM-IV = Diagnostic and Statistical Manual for Mental Disorders (4th edition)* P < 0.001; a χ2; Contrasts based on p-values of 0.05 Measures Motor Timing Task This task was designed to measure the accuracy and variability of motor timing (Van Meel et al. 2005). Subjects were instructed to press a button with their preferred index finger when they thought a 1-second time interval had elapsed. The start of the interval was announced by a tone. After the subject’s response, visual feedback concerning the accuracy of the response was presented on the screen, indicating whether the response was correct, too short or too long. A response was regarded as correct, if it fell between the lower and upper boundary set by a dynamic tracking algorithm. Boundaries were set at 500 to 1,500 ms at the beginning of the task. If the response fell within these boundaries, the boundaries of the subsequent trial were narrowed by 100 ms. Likewise, the boundaries of the subsequent trial were widened with 100 ms, if the response on the previous trial fell outside the boundaries. The practice session consisted of 20 trials, the experimental session of 80 trials. Both sessions were preceded by presenting 10 times a cartoon figure for exactly 1 s on the screen to demonstrate the duration of 1 s (Van Meel et al. 2005). Dependent measures were accuracy (median of productions in ms, which reflects under- versus over-production) and variability (SD of productions in ms). Baseline Speed task This task was designed to measure the speed and variability of motor output in response to an external cue and comparable to a simple reaction time task (De Sonneville 1999). Subjects were required to press a key as quickly as possible, when a fixation cross in the centre of a computer screen changed into a white square. Immediately following the response, the white square changed back into the fixation cross. The time interval between a response and the emergence of the next white square varied randomly between 500 and 2,500 ms in order to prevent anticipation strategies. A practice session (10 trials) and an experimental session (32 trials) were administered for both hands separately. The task was first practised and executed with the index finger of the non-preferred hand, thereafter practised and executed with the index finger of the preferred hand. Dependent measures were the speed (mean reaction time in ms) and variability (SD of reaction times in ms) of responses. Tapping task This task measured the speed and variability of self-generated motor output (without internal or external cues) (De Sonneville 1999). Subjects were required to tap as frequently as possible within an interval of 18 s. The beginning and end of the interval were announced by a tone. During tapping, the number of taps was continuously counted and displayed on the screen. A practice session (5 s) and an experimental session (18 s) were administered for both hands separately. The task was first practised and executed with the index finger of the non-preferred hand, thereafter practised and executed with the index finger of the preferred hand. Dependent measures were speed (mean intertap interval in ms) and variability (SD of intertap intervals in ms) of motor output. Intelligence Full-scale IQ was estimated by four subtests of the WISC-III (Wechsler 2002) or WAIS-III (Wechsler 2000) (depending on the child’s age): Vocabulary, Similarities, Block Design and Picture Completion. These subtests are known to correlate between .90–.95 with the Full-scale IQ (Groth-Marnat 1997). Procedure Testing of ADHD children and their siblings took place at the VU University Amsterdam or at the Radboud University Nijmegen Medical Centre and was conducted simultaneously for children within a family. Psychostimulants were discontinued for at least 48 h before testing took place (Pelham et al. 1999). Participants that took other medication than stimulants to suppress their symptoms of ADHD were also off medication during testing. The medication of these children was gradually decreased in line with standard procedures to allow for sufficient wash-out. Children were motivated with small breaks. At the end of the session, a gift worth approximately € 4, was given. Control children were tested in a similar way in a quiet room at their school. The study had medical-ethical approval. Analyses The percentage of missing data was less than 5% for each of the dependent variables. Missing data were replaced by using the Estimation Maximization procedure (Tabachnick and Fidell 2001). None of the dependent variables was normally distributed. Therefore, variables were successfully normalized by applying a Van der Waerden transformation (Statistical Package for the Social Sciences [SPSS] version 14). The Van der Waerden transformation transforms raw scores into z-scores corresponding to the estimated cumulative proportion of the distribution corresponding to a particular rank. It is defined by the formula r/(w + 1), in which w is the sum of the case weights and r is the rank, ranging from 1 to w (Lehmann 1975). Cases are given different weights by means of simulated replication. The value of the new standardized variable equals the sum of case weights (SPSS version 14). This transformation has two important advantages: It handles the (extreme) influence outliers may have on the data, by ranking them as (very) high or low within the normal distribution, and the comparison between the variables was facilitated since the variables were all depicted on the same scale. Homogeneity of variance was tested by calculating Fmax (ratio of the largest cell variance to the smallest). Since sample sizes were relatively equal (i.e. within a ratio of 4 to 1 or less), an Fmax of 10 and lower was acceptable (Tabachnick and Fidell 2001). For all six normalized variables, the ratio was well within acceptable limits (all below 1.37). Alpha was set at .01 for all tests. Following Cohen’s guidelines (Cohen 1988), effect sizes were defined in terms of the percentage of explained variance: 1, 9 and 25% were used to define small, medium, and large effects. These figures translate into η2-values of 0.01, 0.06 and 0.14. Linear mixed models were used for the analyses. The linear mixed model expands the general linear model so that the data are permitted to exhibit correlated variability. This model allows for the investigation of group differences while correcting for the non-independency of data (i.e. more than one child participated per family, which resulted in related measurements within groups and between groups). In first instance, we tested the main effects of possible confounders (hand, gender, IQ, and age) on performance on the three different tasks. This was done within the control group to avoid dependency with the factor group. Thereafter, interactions between group and the confounders were examined to investigate whether effects of possible confounders were comparable across groups. In second instance, we investigated whether group differences existed for each task measure. Group was used as factor (four groups: proband, affected sibling, non-affected sibling, and control), age (linear and/or curvilinear) as covariate(s), and family as random effect to account for within family correlation. Pairwise comparisons were used to compare groups and it was analyzed whether a linear trend was present in polynomial group contrasts. It was expected that probands and affected siblings performed worse than controls (mainly on the motor timing measures but not or to a lesser extent on the other motor measures), indicating an association between motor timing deficits and ADHD. It was also expected that the non-affected siblings performed worse than controls and formed an intermediate group in between their affected siblings and controls, suggesting motor timing deficits were related to a familial susceptibility to the disorder. The Conners’ Total ADHD scale (averaged across parents and teachers) was used as an additional covariate in the analyses to rule out that possible deficits in the non-affected siblings group could be attributed to sub-clinical ADHD symptoms in this group. Correlations (with 95% confidence intervals) were calculated between siblings to test the familial resemblance of the motor measures (Statistical Analysis for Genetic Epidemiology [S.A.G.E] 5.3.1, 2007). Results Testing of possible confounders Main effects of hand, gender, IQ, and age We tested for the effects of hand, gender, IQ, and age within the control group to avoid dependency with the factor group. No significant effect of hand was found on the Baseline Speed task (F(1, 271.0) = 0.05, P = .83, ηp2 < .01), but there was a significant effect on the Tapping task (F(1, 271.0) = 5.99, P = .02, ηp2 = .02). Control children were faster and less variable, when performing the Tapping task with their right hand than their left hand, likely reflecting an effect of hand dominance. The Motor Timing task was only performed with the preferred hand. Gender had no effect on the Motor Timing task and Baseline Speed task (F(1, 241.5) = 0.03, P = .87, ηp2 < .01 and F(1, 229.5) = 0.36, P = .55, ηp2 < .01, respectively), but had an effect on the Tapping task (F(1, 225.1) = 14.91, P < .001, ηp2 = .05). Control boys were faster and less variable in their tapping performance than control girls. No effect of IQ was found on the Motor Timing task, Baseline Speed task, or Tapping task (F(1, 258.4) = 0.20, P = .66, ηp2 < .01, F(1, 254.8) = 0.06, P = .81, ηp2 < .01 and F(1, 259.9) = 0.95, P = .33, ηp2 < .01, respectively). In order to assess whether age could be best modeled in linear and/or curvilinear terms, we first analyzed the linear effect of age and then in a second model analyzed the quadratic effect of age, while keeping the linear term for age in the model. In this manner, the incremental contribution of the curvilinear relation with age over and above the linear relation was tested. Age (in linear terms) had a strong effect on the Motor Timing task, Baseline Speed task, and Tapping task (F(1, 252.7) = 26.65, P < .001, ηp2 = .22, F(1, 239.1) = 292.38, P < .001, ηp2 = .57, and F(1, 244.5) = 278.31, P < .001, ηp2 = .57, respectively). Older control children were more accurate, faster and less variable in their motor output. Results indicated that when both age terms were implemented in the model, no curvilinear effect of age was present on the Motor Timing task (F(1, 259.3) = 1.34, P = .25), but there was on the Baseline Speed task and Tapping task (F(1, 257.8) = 20.59, P < .001 and F(1, 258.3) = 12.47, P < .001, respectively). No significant interactions were present between gender and age, whether age was modelled in linear or curvilinear terms. Interactions between group and possible confounders Group did not interact with hand on the Baseline Speed task (F(3, 816.0) = 1.14, P = .33, ηp2 < .01), but did interact marginally significantly with hand on the Tapping task (F(3, 816.0) = 2.73, P = .04, ηp2 = .01). Group did not interact with gender on the Motor Timing, Baseline Speed, or Tapping tasks (F(3, 762.9) = 1.96, P = .12, ηp2 < .01, F(3, 743.3) = 0.52, P = .67, ηp2 < .01, and F(3, 751.8) = 0.53, P = .66, ηp2 < .01, respectively) nor with IQ (F(3, 730.1) = 2.01, P = .11, ηp2 = .01, F(3, 714.3) = 1.28, P = .28, ηp2 = .01, and F(3, 727.2) = 2.11, P = .09, ηp2 = .01, respectively), nor with linear age (F(3, 732.0) = 1.67, P = .17, ηp2 = .01, F(3, 731.1) = 0.88, P = .45, ηp2 < .01, and F(3, 755.2) = 1.36, P = .26, ηp2 = .01, respectively), nor with curvilinear age (F(3, 727.1) = 1.61, P = .19, ηp2 < .01, F(3, 733.2) = 0.51, P = .67, ηp2 < .01, and F(3, 754.4) = 1.21, P = .31, ηp2 < .01, respectively). Based on the results of these analyses, it was decided to average the measures across hands to simplify results, since no group differences were found for the percentage of right- and left-handed (Table 3) and since there was only a marginal significant interaction of small effect between group and hand for one of the tasks (Tapping). Furthermore, not included as covariates were IQ (had no effect on motor performance and did not interact with group) and gender (had only a small effect on one of the tasks and even in the opposite direction i.e. boys performing better than girls, and gender did not interact with group). Both the linear and curvilinear effects of age were included as covariates in the analyses for the Baseline Speed task and Tapping task. Only the linear effect of age was included as covariate in the analyses for the Motor Timing task. Raw means and SDs are presented in Table 4. Table 4Means and standard deviations of the motor measures in msDependent variableProbandAffected siblingNon-affected siblingControlηp2ContrastsMSDMSDMSDMSDMotor timing    Accuracy98199997961,0071141,020100.031 = 2 > 3 = 4    Variability389265375265344260295218.101 = 2 > 3 > 4Baseline Speed    Speed35575356853538135178.021 = 2 > 3 = 4    Variability13285131871238211775.031 = 2 > 3 = 4Tapping    Speed23939248482565124948.01ns    Variability4620482345204418.01nsNote: 1 = Probands; 2 = Affected Siblings; 3 = Non-Affected Siblings; 4 = Controls. ns = not significantScores were averaged across hands for the Baseline Speed and TappingOutliers (|z| > 3) were removed Endophenotypic analyses Motor Timing task A significant small effect of group was found for accuracy (F(3, 532.9) = 7.21, P < .001, ηp2 = .03). Pairwise comparisons indicated that probands and affected siblings did not differ from each other (P = .21). Both differed significantly from controls (P < .001 and P = .02, respectively): probands and (to a lesser extent) affected siblings tended to under-produce the 1,000 ms interval (M = 981 ms and M = 997 ms, respectively) compared to controls (M = 1,020 ms). A tendency to under reproduce appeared to be associated with ADHD, but was not convincingly related to a familial predisposition for the disorder, since non-affected siblings did not show this tendency: they differed significantly from probands (P < .001), marginally significantly from affected siblings (P = .02) but not from controls (P = .08) (see Fig. 1). Using the Conners’ Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P = .04). However, a polynomial group contrast indicated a linear trend to be present (Contrast Estimate [CE] = 0.29, P < .001), suggesting probands performed most abnormal, followed by affected siblings, then non-affected siblings and then controls. Siblings marginally significantly resembled each other (r = .11 confidence interval [CI = .02–.19], P = .02). Fig. 1Accuracy and variability of motor timing (adjusted for the linear effect of age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean Groups also differed with respect to the variability of motor timing (medium effect) (F(3, 524.6) = 25.12, P < .001, ηp2 = .10). Pairwise comparisons indicated that probands and affected siblings were equally variable (P = .45) and both were more variable than controls (both P < .001), suggesting ADHD and variability in motor timing were associated. Moreover, variability in motor timing appeared related to a familial predisposition for the disorder, since non-affected siblings formed an intermediate group: They significantly differed from probands, affected siblings, and controls (P < .001, P = .009 and P < .001, respectively). Using the Conners’ Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P = .001). A linear group contrast was present (CE = −0.49, P < .001). A significant familial resemblance was found for variability (r = .29 [CI = .20–.38], P < .001). These findings lend support for variability of motor timing as endophenotype, though the accuracy of motor timing appears only to be associated with ADHD and not conclusively related to a familial susceptibility for the disorder (Fig. 1). Baseline Speed task A small but significant effect of group was found for speed (F(3, 537.2) = 6.92, P < .001, ηp2 = .02). Pairwise comparisons revealed that probands and affected siblings did not differ from each other (P = .82) and both were slower than controls (P < .001 and P = .007, respectively), indicating a relationship between speed and ADHD. It appeared that speed was not related to a familial vulnerability for ADHD, since non-affected siblings differed from probands and affected siblings (P < .001 and P = .01, respectively), but not from controls (P = .84). Using the Conners’ Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P = .99). Nevertheless, polynomial group contrasts indicated a linear trend (CE = −0.14, P = .001), suggesting probands to perform most abnormal, followed by affected siblings, then non-affected siblings, and then controls. A significant correlation between siblings was found (r = .29 [CI = .20–.38], P < .001). Groups differed somewhat (small effect size) in the variability on the Baseline Speed task (F(3, 555.1) = 6.96, P < .001, ηp2 = .03). Pairwise comparisons indicated that probands and affected siblings were equally variable (P = .74) and both groups were more variable than normal controls (P < .001 and P = .003, respectively), signaling an association between ADHD and variability in self generated motor output. Again, non-affected siblings did differ from probands (P = .003) and affected siblings (P = .04), but not from controls (P = .38). Using the Conners’ Total ADHD score as covariate did not change the difference between non-affected siblings and controls (P = .41). Nevertheless, a linear group contrast was present (CE = −0.22, P < .001). Variability correlated between siblings (r = .16 [CI = .07–.25], P < .001). These findings suggest slow and variable motor output in response to an external cue is associated with ADHD, but probably not related to a familial vulnerability for ADHD, since motor output of non-affected siblings resembles that of normal controls more than that of their affected siblings (Fig. 2). Fig. 2Speed and variability of Baseline Speed (adjusted for the linear and curvilinear effects age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean. Scores were averaged across hands Tapping Task No significant effect of group was found for speed (F(3, 547.4) = 1.88, P = .13, ηp2 = .01) or variability (F(3, 553.6) = 1.95, P = .12, ηp2 = .01). No significant linear trend was present in polynomial group contrasts for speed or variability (CE = 0.10, P = .04 and CE = −0.12, P = .03, respectively). Siblings did resemble each other in the speed and variability of tapping (speed: r = .27 [CI = .18–.36], P < .001; variability: r = .18 [CI = .09–.27], P < .001). These findings indicated that speed and variability in self generated motor output were not familially associated with ADHD (Fig. 3). Fig. 3Speed and variability of tapping (adjusted for the linear and curvilinear effects of age) in probands, affected siblings, non-affected siblings and control children. Error bars represent 1 standard error from the mean. Scores were averaged across hands Since there were group differences for speed and variability of externally cued motor output (Baseline Speed task), the issue was raised whether the deficits found on the Motor Timing task were primarily related to these group differences in basic motor output. Therefore, analyses were undertaken whereby the speed on the Baseline Speed task was used as an additional covariate in the analyses on accuracy on the Motor Timing task. The variability on the Baseline Speed task was used as additional covariate for the analyses on variability on the Motor Timing task. The effect of group on accuracy on the Motor Timing task remained significant after accounting for speed on the Baseline Speed task (F(3, 532.4) = 7.51, P < .001, ηp2 = .03). The unadjusted means of the raw (unstandardized) data of the accuracy on the Motor Timing task for probands, affected siblings, non-affected siblings, and controls were: 981, 997, 1,007, and 1,020 ms, respectively. The adjusted means after covarying for speed on the Baseline Speed task were: 979, 998, 997, and 1,018 ms, respectively. The same was true for variability of motor timing: the medium effect of group remained significant after accounting for variability on the Baseline Speed task (F(3, 528.2) = 21.72, P < .001, ηp2 = .09). The unadjusted means of the raw (unstandardized) data of the variability on the Motor Timing task for probands, affected siblings, non-affected siblings, and controls were: 389, 375, 344, and 295 ms, respectively. The adjusted menas after covarying for the variability on the Baseline Speed task were: 438, 428, 424, and 316 ms, respectively. Group contrasts for accuracy and variability as reported above also remained unchanged. These findings suggest that the deficits found on the Motor Timing task can not be explained by the deficits found in basic motor output. Discussion There was investigated whether accuracy and variability of motor timing were viable endophenotypic candidates as reflected by poor performance on these measures in children with ADHD (i.e. indicating an association between the deficits and the disorder), reflected by poor performance in non-affected siblings in between their affected siblings and controls (i.e. suggesting a relation between the deficits and a familial susceptibility for the disorder), and reflected by sibling correlations (i.e. signalling familial resemblance for deficits). We administered two motor tasks in addition to a motor timing task, in order to investigate whether deficits were specifically related to the timing of motor output or whether deficits were also observed in tasks requiring motor output without any timing demands. Probands and affected siblings were dissociated from controls with respect to accuracy of motor timing. Both groups tended to under-produce the 1,000 ms compared to control children (who tended to over-produce the interval). This finding has been reported previously using exactly the same task (Van Meel et al. 2005) and is comparable to some other studies documenting on under estimation/(re)production in patients with ADHD compared to controls (see for review Toplak et al. 2006). These findings suggest a relation between under reproduction (possibly reflecting a somewhat speeded internal clock and/or impulsivity) and ADHD. The findings were less convincing with respect to non-affected siblings: despite a linear trend in group contrasts, their accuracy of motor timing was more like controls than that of affected siblings. Furthermore, correlations between siblings on accuracy were also modest suggesting familial resemblance for accuracy was present but not strongly. Therefore, accuracy of motor timing seems not to be a strong endophenotypic candidate. Variability of motor timing, however, convincingly met all characteristics of an endophenotype as investigated in our study: Probands and affected siblings were clearly more variable in their motor output than controls, non-affected siblings also differed significantly from controls and had variability scores in between their affected siblings and controls, and greater variability in motor timing was evidently familial. Greater variability in motor timing in children with ADHD compared to controls concurs with previous studies (Pitcher et al. 2002; Rubia et al. 1999, 2003; Toplak et al. 2006; Van Meel et al. 2005) and suggests variability in motor timing is characteristic of ADHD. Our study adds important knowledge to this topic, showing that non-affected siblings portray a similar type of variability in their motor timing and that siblings resemble each other in the variability of motor timing. These findings suggest the variability in motor timing is not only associated with the disorder, but is related to familial vulnerability for ADHD, which may make it a useful tool in future studies aimed at unraveling the genetic underpinnings of ADHD. All in all, variability of motor timing may form a fruitful endophenotypic candidate. However, group differences were also present on motor measures that did not require a timing component, suggesting motor deficits not to be specifically related to timing but to be more generalized. Probands and affected siblings were significantly slower and more variable than controls concerning motor output in response to an external cue (Baseline Speed). These findings of slow and variable responding are in line with a study using the same task in young children at risk for ADHD (Kalff et al. 2005) and suggests slow and variable responding are characteristic of ADHD. However, in contrast to variability of motor timing, non-affected siblings did not differ from controls with respect to speed and variability of externally cued motor output. So, despite the finding that slow and variable motor output is associated with ADHD, it does not appear to be convincingly associated with a familial vulnerability for the disorder. Given that poor motor output was observed in probands and affected siblings (Baseline Speed), it was surprising that no such abnormalities were found in self-generated motor output (Tapping). Probands and affected siblings had a normal speed and variability in self generated motor output. The discrepancy in results between both tasks may lie in the suggestion that Baseline Speed may have required some form of cognitive processing (i.e. registering a stimulus and responding to it), whereas Tapping only required executing a motor action. The normal performance of children with ADHD on the Tapping task is in line with some studies (Seidman et al. 1997, 2000), but in contrast with others (Toplak et al. 2006). This might be explained by the important difference in timing requirements necessary in the Tapping task used here and in some previous studies. Here, no timing was required to execute the Tapping task. The instruction was simply to press the button as often as possible within a certain time interval. However, in other studies the child was required to modify his/her tapping rate to be in synchrony with the stimulus and maintain the rhythm in the absence of the stimulus (Rubia et al. 1999; Toplak et al. 2006). These tasks load differently on timing processes. Our findings suggest that self-generated motor output does not form a viable area of endophenotypic research, even though speed and variability of self-generated motor output correlate within families. Since group differences were not only present on measures of motor timing, but also on measures of motor output (Baseline Speed), it was investigated whether motor timing impairments may be due to deficits in basic motor output. This appeared not to be the case. Even when speed and variability of basic motor output were used as covariates in the analyses on motor timing, group differences for motor timing remained. These findings suggest abnormalities in the accuracy and variability of motor timing are relatively independent of general deficits in basic motor output (Keele et al. 1985) and suggest abnormalities in motor timing are predominantly related to timing operations and not to motor functioning. Since motor timing appears predominantly regulated by the cerebellum, basal ganglia and their reciprocal connections with the cerebral cortex (Handy et al. 2003; Harrington et al. 1998; Ivry 1996; Nenadic et al. 2003), subcortical regions in addition to cortical regions might be important in the etiology of ADHD (Halperin and Schulz 2006). Limitations We did not administer the PACS interview for nonaffected siblings. This might have resulted in undetected ADHD cases in the nonaffected sibling group, which in turn might explain the deficits of this group. However, we do not believe this to be the case, because (1) all siblings were thoroughly screened and, if they scored clinically on any of the screening questionnaires, the PACS interview was administered, and (2) even when symptom severity was used as a covariate, group differences between nonaffected siblings and controls remained significant. Furthermore, including measures aimed at isolating aspects of timing performance, such as time estimation and reproduction paradigms, in addition to the measure of time production would have enhanced the comprehensiveness of our findings with respect to the internal clock. Some previous studies using time estimation and reproduction tasks have reported that children with ADHD performed abnormally (see for review Toplak et al. 2006). We documented previously on time reproduction deficits present in both children with ADHD as well as their non-affected siblings (Rommelse et al. 2007b), suggesting familial deficits in timing in ADHD generalize across timing paradigms and extent beyond motor timing as reported in the current study. Conclusions Variability in motor timing appears a useful endophenotypic candidate: It is clearly associated with ADHD, it is also present in non-affected siblings, and it correlates within families. Accuracy (under- versus over production) in motor timing appears less useful: even though accuracy is associated with ADHD (probands and affected siblings have a tendency to under-produce compared to controls), non-affected siblings did not exhibit this tendency and sibling correlations were only marginally significant. There were group differences in motor speed and variability (Baseline Speed task): probands and affected siblings were slower and more variable in their motor output as response to an external cue. Even though siblings resembled each other in their speed and variability, non-affected siblings performed more like controls. These findings suggest that speed and variability of externally cued motor output are associated with having ADHD, but probably not related to a familial vulnerability for the disorder. Interestingly, the speed and variability in self-generated motor output (Tapping) is normal in probands and affected siblings, making it unsuitable to unravel underlying vulnerabilities leading up to ADHD. Deficits in motor timing cannot be explained by deficits already present in basic motor output without a timing component (Baseline Speed), suggesting abnormalities in motor timing are predominantly related to deficient timing operations but not to deficient motor functioning. The finding that deficits in motor timing run in ADHD-families suggests this to be a fruitful domain for further exploration in relation to the genetic underpinnings of ADHD.

Biotechnol_Lett-3-1-2045120

Solute transport in orthorhombic lysozyme crystals: a molecular simulation study

Long-time equilibrium molecular dynamics simulations were performed to study the passage of a substrate, l-arabinose, through nanopores of orthorhombic hen egg white lysozyme crystals. Cross-linked protein crystals (CLPC), as novel biological nanoporous media, consist of an extensive regular matrix of chiral solvent-filled nanopores via which ions and solutes, e.g. sugars and amino acids, travel in and out. We studied the diffusive motion of arabinose inside protein channels. The computed diffusion coefficients within the crystal were orders of magnitudes lower relative to the diffusion coefficient of the solute in water. This study is valuable for understanding the nature of solute–protein interactions and transport phenomena in CLPCs and provides an understanding of biocatalytic and bioseparation processes using CLPC. Introduction Protein crystals are conventionally thought as important elements in structure determination of biomolecules as well as in protein purification (Johnson and Philips 1965; Blundell et al. 2003; Haring and Schnrier 1991). They contain pores that range from approx. 0.3 nm to 10 nm and occupy 25–75% of the crystal volume (Bishop and Richards 1968). Their porosity is comparable to that of inorganic porous catalysts and sorbents, such as zeolites and silica-gel (Bishop and Richards 1968; Vilenchik et al. 1998; Margolin and Navia 2001). More robust cross-linked protein crystals (CLPC) have been applied successfully as extremely stable biocatalysts (Vilenchik et al. 1998) and as selective (chiral) separation media (Margolin and Navia 2001). The complex crystal structure of the protein contains many functional active sites. Moreover, it resists environmental degradation once it is cross-linked (Vilenchik et al. 1998; Margolin and Navia 2001). Understanding the nature of transport of solutes in CLEC is relevant to many biotechnological processes (Margolin and Navia 2001; Margolin and Vilenchik 1996; Margolin et al. 2000; Vuolanto et al. 2002, 2004; Jokela et al. 2002). Properties of intracrystalline water molecules and ions, and their transport through the crystal, are essential to many of these applications. Apart from their practical use, protein crystals also provide a unique model to study transport phenomena in protein channels in cell membranes since the structure of water-filled pores in the crystals is determined to atomic resolution by X-ray diffraction (Morozova et al. 1996). There are effects of both the solute on protein and protein on the solute (Nagendra et al. 1998). The latter effect is more pronounced in protein crystals, where proteins make up the lining of small pores and water and ions affect the passage of substances through such pores (Nagendra et al. 1998; Eisenberg and Kauzmann 1969). Several studies have focused on the experimental determination of the solute and water transport in protein crystals (Bishop and Richards 1968; Morozov et al 1995; Velev et al. 2000; Cvetkovic et al. 2005a, b; O’Hara et al. 1995). Recent studies on diffusion in protein crystals still leave open questions concerning the mobility of solutes and protein–solute interactions near proteins that constitute the pore walls (Cvetkovic et al. 2005a, b). In addition to advanced experimental techniques, versatile computational tools are generally needed to correlate reactivity of the protein and transport of solute with nanoporosity of the enzyme crystals at atomistic level (Malek et al. 2004, 2005). Molecular dynamics simulations with explicit representation of molecules and ions should, in principle, provide realistic information about the diffusive motion of water, individual solute molecules and ions at atomic resolution. These simulations, however, are only practical at longer time and length scales. Due to heterogeneity of the protein surface, it is necessary to treat transport of molecules at the protein–water interface as a local property (Bon et al. 1999). There have been a number of such studies to determine how the water molecules on the protein surface are perturbed from the bulk (Bizzari and Cannistraro 1996; Bizzari et al. 1996). To our knowledge, there have been no such studies on dynamical motion of small solute molecules in a protein crystal. The mobility of small dipolar molecules, such as sugars and amino acids, in the vicinity and far from the protein surface in a confined biological channel, exhibits different dynamical and structural behavior from that in a free protein in solution (Velev et al. 2000; Cvetkovic et al. 2005a, b; O’Hara et al. 1995; Malek et al. 2004, 2005). In the crystalline form, proteins are in a highly ordered three-dimensional structure, where the protein molecules strongly bind to each other with specific intermolecular interactions. Protein crystals are particularly interesting materials for chiral separations (Margolin and Navia 2001; Margolin and Vilenchik 1996; Margolin et al. 2000; Vuolanto et al. 2002, 2004; Jokela et al. 2002). The crystal framework poses an asymmetric environment and is made of l-amino acids. Enzymes in crystals have also specific binding sites for the substrates and cofactors. In addition, protein crystals are nanoporous materials. Based on these properties, many CLPCs have recently been used in chiral separation of racemic mixtures (Vuolanto et al. 2004; Jokela et al. 2002). These novel insights are valuable for biotechnological applications to devise highly selective biocatalysts, biosensors and bioseparators. Here, we use orthorhombic lysozyme as a simple model enzyme to study diffusion of a chiral substrate through pore network of the enzyme crystal. Its well-known and extremely stable molecular structure makes it a good choice for computational studies. Moreover, lysozyme belongs to a large class of enzymes known as glycosidases. These are extremely efficient glycosidic hydrolysis catalysts. Biocatalysis inside the protein lattice has advanced features of product separation and catalyst recovery. Once the catalytic reaction occurs in lysozyme crystals, understanding the transport of products (sugars) through the lattice becomes an important issue. In short, the simple and well-known structure of lysozyme as model protein, the catalytic activity of lysozyme for hydrolyzing the glycosidic bond, the practical potential of protein crystals as biochemical porous media for chiral separation and as a model for biological channels, in general, were our first motivations for choosing a simulation model based on transport of sugar substrate into orthorhombic lysozyme lattice. Here we provide, for the first time, long-time MD simulations of diffusion of l-arabinose in orthorhombic hen egg white lysozyme crystals. Fundamental questions are; do solute molecules move by translation and/or rotation? How solute–protein interactions influence transport of a chiral solute through the protein crystal? What is the detailed nature of the diffusive motion of solute molecules in a lysozyme crystal and what are the length scales and the time scales (dynamics) of all those events. Computational methodology We use MD simulations to examine diffusion of l-arabinose (Ara) in a fully hydrated orthorhombic lysozyme crystal. Ara is an aldopentose (Fig. 1) that has been extensively used as a substrate to probe diffusion properties of protein channels (Nikaido and Rosenberg 1981). Lysozyme consists of 129 amino acids with 1001 non-hydrogen atoms. The crystal structures of lysozyme, entry 1AKI (Artymiuk et al. 1982), is taken from the Brookhaven Protein Database and used as a starting point. Hydrogen atoms attached to aliphatic carbon atoms are incorporated with the latter, but the remaining 342 hydrogen atoms are treated explicitly, leading to 1,343 (pseudo-) atoms in total. The simulations are done at pH 7. The amino acids Glu and Asp are taken to be deprotonated while Lys, Arg and His residues are protonated (Artymiuk et al. 1982; Stocker et al. 2000). This leads to +8 electron charges per protein molecule. Chloride ions are then added for electroneutrality. In an orthorhombic crystal, four protein molecules related by the crystallographic symmetry P212121 are placed in the unit cell with a = 5.9062 nm, b = 6.8451 nm, and c = 3.0517 nm. Repeating the unit cell along crystallographic axes generates the pore network (Fig. 2). Channels and cavities within the orthorhombic lysozyme lattices are determined by using a procedure explained elsewhere (Malek et al. 2004), based on HOLE and CHANNEL algorithms (Smart et al. 1996; Kisljuk et al. 1994). The radius of a pore is determined at any given distance along the pore axis by calculating the maximum size for a spherical probe to still fit in the pore without overlap with the van der Waals radii of atoms in the pore wall (Kisljuk et al. 1994). Fig. 1Ball-and-stick model of l-arabinose. CHn groups are represented by united atomsFig. 2Computer generated images of an orthorhombic lysozyme lattice, visualized along z- crystallographic axis. The solvent channels are shown in grey. A single unit cell is labelled by square and its sphere representation is shown in (b). Red lines represent water molecules. The surface representation in (a) was computed from the electron density calculations using the crystal structure data (1AKI) available in the Brookhaven protein data bank. Hydrophilic, hydrophobic and polar regions are shown in blue, red and green respectively Simulations are carried out using a fully atomistic 1 × 1 × 5 lysozyme lattice (a = 5.9062 nm, b = 6.8451 nm, and c = 15.2585 nm). The single unit cell in Fig. 2b is repeated five times along the z-axis, so that a long pore is constructed. We start from an initial configuration by random placing arabinose inside the lysozyme channel. The system contains 20 lysozyme molecules, 21 arabinoses, 6,239 crystallographic and non-crystallographic water molecules and 160 chloride ions, leading to 46,031 atoms in total. After that the system was equilibrated for 500 ps using harmonic position restraints (1000 kJ mol−1 nm−2). Production runs were performed for another 26 ns, the last 25 ns of which was used for the analysis. Simulations are performed using GROMOS96 force field (van Gunsteren et al. 1990, 1996). In our force field, interactions between atoms are divided into non-bonded interactions, between any pair of atoms that are within a given cut-off radius, and bonded interactions between atoms connected by chemical bonds. In case of the non-bonded interactions (electrostatic and van der Waals), a partial charge and parameters for repulsion and attraction are assigned to each atom. The bonded interaction consists of bond, angle and dihedral terms. Bond and angle bending are given by simple harmonic potentials. The torsional rotational potential for the dihedral angle is a periodic function with a 3-fold barrier. The partial charges and interaction parameters for all species are taken from GROMOS96 database (van Gunsteren et al. 1990). In our MD simulation, a cut-off of 1.4 nm is used for Van der Waals interactions, while a cut-off of 1 nm and PME with a grid spacing of 0.12 nm and fourth order interpolation are used for electrostatics interactions (Morozova et al. 1996). During the simulations, the potential energy and the total energy are monitored in order to check if the system is in equilibrium. MD simulations are performed in a canonical (NVT) ensemble at 300 K. The temperature (300 K) is controlled by the Berendsen weak-coupling algorithm, separately for protein, Ara and solvent plus ions with a time constant of 0.1 ps. During the simulations, polar hydrogen atoms are treated as dummy atoms with an increased mass of 4 Da. This allowed the integration time step to be 5 fs. Simulations are done with the GROMACS package (Lindahl et al. 2001; Berendsen et al. 1995; http://www.gromacs.org). Visualization is done by using the VMD v1.8.3 (Humphrey et al. 1996) commercial package. Results and discussions Figure 2a shows an instantaneous configuration of the atomic model of the orthorhombic lysozyme lattice, constructed of 3 × 3 × 3 unit cells, whereas a single unit cell is framed in Fig. 2b. In each unit cell, there is a main pore lies along the z-axis and there are no remarkable pores in the other directions (Malek et al. 2004; Geremia et al. 2005; Falkner et al. 2005). Figure 3a visualizes part of the main pores along the z-axis. The average pore radius is about 0.88 ± 0.02 nm. Figure 3b shows the pore radius as a function of the pore axis. The profiles show that there are constricted zones inside each pore. The pore radius slowly decreases from over 0.95 nm to slightly less than 0.82 nm at its narrowest point. The charged residues Lys1, Lys13, Lys33, Lys96, Lys97, Lys116, Arg14, Arg21, Arg114, Asp87, and Asp119 belonging to the lysozyme molecules lie in the pore space. The motion of these residues may affect the pore shape and pore size during the transport of Ara. Our analysis shows that the effect of the protein fluctuations on pore shape is negligible, particularly for solutes of sizes much smaller than the pore diameter, such as Ara. Fig. 3(a) Visualized pore along z-axis in a 1 × 1 × 5 lysozyme lattice. (b) Pore radius profile along the pore axis The averaged root mean square fluctuation (RMSF) of C-α of each residue, calculated from the distance fluctuation matrix of a 25 ns trajectory in crystalline orthorhombic lysozyme is shown in Fig. 4a. The root mean square fluctuations from the X-ray structure are averaged over 20 proteins. The fluctuation results (Fig. 4a) are consistent with previous works on fluctuations and correlations in lysozyme crystals (Héry et al. 1997, 1998). The largest fluctuations are for the α-helix loops. The β-strand residues show a low mobility with values on the order of 0.1 nm. β-Strands exhibit a high degree of rigidity with moderate correlations, while only parts of α-helices are rigid or correlated (Héry et al. 1998). Although the protein atoms fluctuate slightly during the simulation, these are too small to cause significant changes in pore size or in diffusivity. Figure 4b shows that the RMSF patterns are similar for all twenty lysozyme molecules. The residues Gly117, Gly71 and Asp101 show fluctuations above 0.10 nm. Among these, Gly117 displays the maximum fluctuations ranging from 0.15 nm to 0.25 nm. Most of the charged residues accessible from the pore space show fluctuations around 0.05 nm. The latter confirms that the pore shape and pore size are not affected by protein fluctuations during 25 ns MD simulation. Fig. 4(a) A typical root mean square fluctuation of the C-α atoms of each residue of one of the 20 lysozyme molecules in the simulation box with respect to their average position. (b) RMSD of each resides of all lysozyme molecules. The curves are on the same scale. The full lines are shifted by 0.2 nm in the vertical direction In order to study the motion of solutes inside orthorhombic lysozyme crystals, we take into account that proteins affect the dynamics of solute molecules. MD simulations provide information to the dynamic picture of the solute motion in the vicinity of protein molecules. The self-diffusion coefficient D, which has been widely used in both spectroscopic experiments and MD simulations, is a suitable parameter characterizing the dynamical behavior of solute in the water filled protein channels in protein crystals. The diffusion coefficient D, is related to the slope of the mean square displacement (MSD) of water molecules by the Einstein relation, which in d dimension is: where is the MSD of solute molecules during the time Δt, averaged over the ensemble of molecules in a d dimensional space, from the moment t0 that their motion is equilibrated in the pore space. Use of the Einstein equation for the determination of D requires a linear dependence of the MSD with time. For the determination of the solute diffusivity, the MSD–t linear relation is usually fulfilled on time scales longer than a few to hundred nanoseconds (Bizzari and Cannistraro 1996). In order to study diffusion of a solute (Ara) inside pore space of an orthorhombic lysozyme crystal, we have performed extensive MD simulations of Ara transport in a fully hydrated lysozyme crystal. Protein atoms are subject to move according to the equation of motions. Ara molecules are randomly incorporated inside the main pore along z-axis (Fig. 3a). The dynamical motion of solute molecules was sampled each 10 ps during 25 ns simulation. Figure 5a shows the displacement of nine Ara molecules along the z-axis as a function of time. The location of the solute molecules is sufficiently randomized during the simulation time, so that the diffusion properties do not depend on the arbitrary initial placement of the Ara molecules. The motion of Ara molecules displays many jumps, with little motion between jumps. A few Ara molecules travel within the pore space with displacement in the order of 5–10 nm, while a few others remain around the same axial (z) position in the pore region, moving rather in xy plane. Overall, solute molecules move freely, some travel all the way within the pore network, a few remain in the pore and some go deep into the pore and return after some time. At some points along the trajectory, a group of two or three Ara molecules establish hydrogen bonding and perform a collective motion as a united body. The latter is particularly observed in longer simulation times thereby bringing an artefact in to the diffusion analysis of single Ara molecules. To calculate the self-diffusion (D) of solute molecules in the pore, the Einstein relationship (Eq. 1) is used. Fig. 5(a) The displacement of nine arabinose molecules along the z-axis through the pore region of lysozyme crystal shown in Fig. 2a. Different colours represent different Ara molecules. (b) Mean square displacement (MSD) of Ara molecules versus time Figure 4b shows Mean square displacement values (MSD) values versus time for Ara molecules diffusing in the pore network of lysozyme crystal. Only trajectories of individual Ara molecules are included in calculation of MSD and therefore the collective motion of solute molecules is not included in Fig. 5b. Overall, the log-log behavior of MSD vs. time (for 5 ns < t < 20) is linear with a slope close to one. This shows that in fully hydrated pores of the lattice, the Einstein relation, Eq. 1, can describe the diffusion of Ara. The diffusion coefficient of Ara in pores along z-axis is calculated from the intercept of the log–log plot, that is (8.51 ± 0.064) × 10−13 m2 s−1. Recently, we have performed combined dynamic Monte-Carlo and Brownian dynamic (MC–BD) simulations to study electrostatic and steric confinement effects on the mobility of spherical probes in orthorhombic and tetragonal lysosyme crystals (Malek et al. 2004). The diffusion coefficient of a probe of size comparable to that of the hydrated Ara (0.86 ± 0.01 nm), calculated by MC–BD simulations is (8.85 ± 0.05) × 10−13 m2 s−1, which is in good agreement with the MD prediction. This indicates that size exclusion is the main reason for uncharged solute transport in orthorhombic lysozyme crystals. The diffusion of solutes within lysozyme crystals has also been investigated using different experimental techniques (Velev et al. 2000; Cvetkovic et al. 2005a, b; O’Hara et al. 1995). Velev et al. (2000) studied the diffusion of surfactants in lysozyme crystals using fluorescent probes by means of quantitative fluorescence microscopy. The diffusion coefficients obtained range from 2 to 30 × 10−14 m2 s−1. By using 3-D laser scanning confocal microscopy visualizations, Cvetkovic et al. (2005a, b) investigated detailed diffusion coefficients of some solutes, fluorescein and rhodamine B, into tetragonal, orthorhombic, and triclinic lysozyme crystals. The results showed that the transport of solute molecules depends on the chemical nature of solutes (e.g., hydrophobic vs. hydrophilic), charge and size of solutes and pore network within protein crystal. The diffusion of fluorescein, with an average diameter of 0.69 ± 0.02 nm, in orthorhombic lysozyme crystals was highly anisotropic and the diffusion coefficient was calculated as approx. 7.0 ± 0.5 × 10−13 m2 s−1. The diffusion coefficient of Ara calculated from our MD simulation is in reasonable agreement to these experiments. The diffusion rate is 4–5 orders of magnitude slower than that in free water (∼7.73 × 10−10 m2 s−1 at 298 K) (Nikaido and Rosenberg 1981; Mogi et al. 2007). The deviation is most likely as a result of single-point interactions between solute and side chains of the XI, which are usually characterized by hydrogen bindings and dipole-dipole interactions. Our preliminary analysis based on hydrogen bonds, number of contacts and minimum distance between Ara molecules and residues on the pore wall showed that the solute molecules spend considerable time in the vicinity of active site residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87. This indicates that the orientation and translational-rotational motion of Ara play an important role in its transport through channels in orthorhombic lysozyme crystals. These results suggest that in lysozyme lattice, and in contrast to the free lysozyme in solution, the interactions between dipolar solute and protein are mostly promoted by a large electric field on the residues far from the active site cleft (which are Asp52, Asp53, Glu35, and Asp99). Many of the latter charged amino acids that act as adsorption/desorption sites for the solute are, in fact, buried, while the residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87 are more easily accessible. Considerable experimental and computational evidence supports several aspects of the mechanism that has been proposed for the catalytic activity of the lysozyme in solution (Dao-Pin et al. 1989; Bottoni et al. 2005). In a manner similar to that proposed for free lysozyme, our simulation results can be validated based on finding the most favorable interaction of the substrate molecules in the mixture with the catalytic active centers of the enzyme molecules in lattice. Such qualitative agreement with experimental data corroborates predictive capabilities of our model. Moreover, our study highlights the importance of the protein–solute interactions on the transport in protein crystals. Despite its simplicity and obvious limitations, this computational study provides insight into the main features of solute transport in protein crystals. Our study allows relating transport properties of the nanopores in protein crystals to solvent and ion motion as well as to protein fluctuations. Although our findings are in good qualitative and quantitative agreement with existing experimental data, more experimental studies are still needed by which we can compare our data directly. These results in combination with experimental information provide vital insights for understanding biocatalytic and chiral separation processes in CLPC. Conclusion In the present study, we performed MD simulations to study the motion of an aldopentose molecule (l-arabinose) in nanopores of a fully hydrated orthorhombic lysozyme crystal. The electrostatic and steric interaction inside the crystal channel significantly influences the diffusion of solute. No clear adsorption site in the crystal was detected in the simulation, most likely due to the low solute concentration. However, our analysis show that the solute molecules spend considerable time in the vicinity of active site residues Lys96, Lys97, Lys116, Arg14, Arg21, and Asp87. According to our results, the average mobility of solute molecules in lysozyme crystal channels is reduced orders of magnitude compared to that in free water. This is why crystalline proteins are easily accessible to solute molecules that are smaller than the necks of the crystal channels. The results are of practical interest to evaluate the time necessary to impregnate the crystals with ligands, water and solutes and provide valuable insights of solute–protein interactions during solute transport in nanopores of protein crystals.

J_Mol_Med-4-1-2359832

Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target. Introduction The murine double minute (MDM) family members are key regulators of levels and activity of the oncosuppressor p53 [1]. MDM2, acting as specific E3 ubiquitin ligase and as transcriptional repressor, is the best known among them. MDM2 overexpression has been observed in many human tumors characterized by tumor protein 53 (TP53) wild-type (wt) status, supporting the model of multiple means of p53 inactivation in tumor cells [2]. In recent years, another member of the MDM family, MDM4 (also named MDMX) has come into the limelight and its function in the inactivation of p53 has been revealed by molecular and genetic approaches [1, 3]. In particular, MDM4 can negatively regulate p53-mediated cell cycle arrest, a role that distinguishes its function from that of MDM2, more related to the suppression of p53-mediated apoptotic response [1, 3–5]. However, others have reported on the antiproliferative and proapoptotic effect of MDM4 in the presence of wild-type TP53 under conditions such as stress [6, 7]. Studies aimed at characterizing the human MDM4 (also named HDMX) status in human tumors have shown amplification of its locus in the presence of wild-type TP53 in breast cancers, glioblastoma, retinoblastoma, and soft-tissue sarcomas [8–11], confirming that it may contribute to p53 inactivation during tumorigenesis. The impact of human MDM4 on tumor properties as well as its behavior during tumor progression however still poorly explored. In addition to the full-length (fl) protein, different splice variants of MDM4 have been described, two of which detected in human tumors. A short form named MDM4-S (a.k.a. HDMX-S) was identified first [12]. This variant derives from an alternative splicing that produces a truncated protein, containing essentially the p53-binding domain. It is a more potent inhibitor of p53 transcriptional activity than wt MDM4 and although present in both normal and tumor tissues, it is overexpressed specifically in tumor samples [11, 12]. Recently, our group has characterized another MDM4-splicing form, HDMX211 (hereafter named MDM4-211), in a thyroid tumor cell line and in some lung cancers [13]. This form derives from an aberrant splicing and produces a protein containing essentially the RING finger COOH-terminus, where the MDM2 binding site resides. As a consequence, this variant is able to stabilize MDM2 protein levels, thus contributing to inactivation of p53. These data, while underlying the complexity of MDM4 expression in human tumors, strongly encourage studies on the comparative analysis of this protein and its derivative forms in human cancer. Thyroid tumors represent over 90% of all endocrine cancers and are characterized by different genetic alterations, among which TP53 mutations are confined quite exclusively to the infrequent anaplastic and poorly differentiated histotype [14]. According to current models of human carcinogenesis that consider inactivation of the oncosuppressor p53, a common feature of almost all tumors, MDM2 amplification and/or overexpression have been reported in thyroid tumors too [15–17]. However, the overall frequency of MDM2 overexpression is only about 30%, suggesting the existence of other pathways of p53 inactivation. The aim of our study has been to analyze papillary thyroid carcinomas (PTC), the most frequent thyroid cancer, for the presence of MDM4 and its derivative forms to investigate alterations of these proteins in this tumor histotype and correlate them with histopathological features. In addition, we have analyzed MDM2 levels to assess the potential relationship between alterations of the two MDM family members. Our data have revealed the aberrant presence of variants MDM4-S and MDM4-211 messenger RNA (mRNA) in PTC as well as lack of correlation between MDM4 and MDM2 mRNA levels. Of note, levels of MDM4 mRNA were significantly downregulated in tumor samples in comparison to normal counterparts, and such downregulation appears significantly associated with tumor stage. Materials and methods Tissue samples and patients Fifty-seven papillary thyroid carcinomas and 57 matched normal thyroid tissue samples from the contra-lateral lobe (CTRL) of 57 patients were studied. In addition, to confirm statistical analyses, 26 papillary thyroid carcinomas and three normal thyroid tissues from independent individuals were analyzed. All specimens were obtained from patients undergoing surgery at the University of Perugia from 1997 to 2007. Before the surgical procedure, all patients signed informed consent forms for collection of fresh thyroid samples for genetic studies. All specimens were sampled from the primary tumor at the time of surgery, snap frozen, and stored at −80°C until use. Tumors containing at least 70% of tumor cells based on the hematoxylin–eosin staining were selected. All normal thyroid tissue from the contra-lateral lobe were histopathologically analyzed for the presence of tumor. Available medical records of the patients were consulted to gain information about the clinical features of the disease and, when possible, the tumor stage was defined according to the sixth edition of the “American Joint Committee on Cancer” Cancer Staging Manual (American Joint Committee on Cancer 2002) based on pathological tumor–node–metastasis parameters and distinguishing patients in two groups: (1) patient age < 45 years (stage I and II), (2) patient age ≥ 45 years (stage I, II, III, IV). The total of 83 papillary thyroid carcinomas included 55 of the classic variant, 20 of the follicular variant, five of other variants (tall cell, solid, diffuse sclerosing), and three showing the coexistence of PTC histology with areas of dedifferentiation. The mean period of follow-up was 45.8 ± 30.4 months. Genetic analysis of TP53 mutation, BRAF mutations and ret/PTC rearrangements Analysis of the TP53 gene status was performed as follows: exons 5, 6, 7, and 8 of p53 gene were sequentially amplified by polymerase chain reaction (PCR) assay with the use of AmpliTaq Gold (Applied Biosystems) and following primer sets: exon 5 sense: TTCCTCTTCCTACAGTACTC; exon 5 antisense: GCCCCAGCTGTTCAC; exon 6 sense: ACTGATTGCTCTTAG; exon 6 antisense: AGTTGCAAACCAGAC; exon 7 sense: AGTTGTGTTATCTCCTAG; exon 7 antisense: CAAGTGGCTCCTGAC; exon 8 sense: TCCTATCCTGAGTAG; exon 8 antisense: GTCCTGCTTGCTTAC. Purified PCR products were sequenced in both directions with the use of the BigDye terminator Cycle sequencing Kit (version 1.1, Applied Biosystems) and an ABI Genetic Analyzer (Model 3130, Applied Biosystems). Sequence data were analyzed by means of SeqScape software (version 2.1, Applied Biosystems) followed by manual review. Search for mutations of BRAF was conducted by single-stranded conformational polymorphism (SSCP) screening of real-time (RT)-PCR products of exons 15, followed by sequencing, as previously described [18]. Screening for ret/PTC 1 and ret/PTC 3 rearrangements was performed by RT-PCR using primers spanning the breakpoints, as previously described [18]. Quantitative real-time PCR Total RNA was extracted with Trizol™ (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer’s instructions. The expression of MDM2, MDM4, and MDM4-211 in tumors and matching normal thyroid samples was measured by quantitative real-time PCR (qRT-PCR), based on TaqMan methodology, using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) according to Giglio et al. [13]. Briefly, custom-designed TaqMan primers and probes (Applied Biosystems) specific for MDM4, MDM2, and MDM4-211 were used. Our results are expressed as relative units of target mRNA, referred to a sample called calibrator, chosen to represent 1× expression of the target gene. The calibrator used was the lowest value in the tissue collection under study. All analyzed samples express n-fold mRNA relative to the calibrator. Each sample mRNA was normalized relative to the β-actin mRNA. Predesigned TaqMan primers and probe (Applied Biosystems) for the housekeeping gene ß-actin were used. Each sample was treated with deoxyribonuclease (DNase) I amplification grade (Invitrogen) and tested before and after DNase treatment. Comparative analysis of flMDM4 and MDM4-S was driven by qRT-PCR according to Bartel et al., [11] using specific probes and SYBR Master mix (Applied Biosystems) with evaluation of dissociation curves. A MDM4-S-to-flMDM4 ratio of >1.5 was considered indicative of overexpressed MDM4-S. Western blot analysis Tumor and normal tissue pairs whose material was sufficient for western blot analysis were selected. All samples were homogenized with Tissue Lyser (Qiagen) and western blots were performed according to Giglio et al. [13]. Briefly, samples were lysed in radio-immunoprecipitation assay buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na desoxicholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid) supplemented with a cocktail of protease inhibitors (Boehringer). Whole lysates were boiled in SDS Laemnli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis on precast 10% gels (Invitrogen) and subsequently transferred to polyvinylidene fluoride membranes (Millipore). After protein transfer, membranes were blocked for 30 min with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk and incubated with specific antibodies. The following primary antibodies were used: rabbit anti-MDM4 polyclonal antibody R1 (raised against full-length MDM4 protein) and (Bethyl Laboratories) mouse anti-MDM2 monoclonal antibody 2A10 and monoclonal antibody Ab-1 (oncogene), mouse anti-α-tubulin monoclonal antibody DM-1A (Sigma), and mouse α-actin monoclonal antibody AC-40 (Sigma). MDM4 was probed with both Bethyl and R1 antibodies, giving similar results although, in comparison, the polyclonal antibody R1 was more sensitive. The homemade R1 antibody has been raised using the entire MDM4 protein. Experiments of epitope mapping by using MDM4-deletion mutants have shown that R1 does not recognize the NH2-terminus of MDM4. It is therefore unable to recognize the MDM4-S form. Statistical analysis Statistic analysis was carried out using the Analyze-it software for Microsoft Excel (Analyze-it Software, Ltd.). Spearman rank correlations and Kendall rank correlation (for measure of linear association) were used to evaluate the correlation between not-normally-distributed variables. To compare groups, we used the Wilcoxon Signed-Ranks test for related samples (57 tumors vs. matched thyroid normal tissues) and Mann–Whitney U test for independent samples (83 tumors vs. 60 normal thyroid samples). A probability of p < 0.05 was considered as statistically significant. Intraindividual variation was calculated by utilizing the T-to-N ratio that derives from the ratio of corrected tumor (mRNA or protein) levels to corrected normal tissue levels of MDM4 and MDM2. Results Analysis of MDM4 and MDM2 levels in papillary thyroid tumors A group of 57 thyroid tumors samples and 57 matched normal thyroid tissues from the CTRL were analyzed. All tumors were of papillary histotype (PTC). The mean age of the studied population was 49.4 ± 16.81 (SD); the female percentage was 72%. The histopathological features of the 57 tumors are shown in Table 1. Tumor stage was defined according to the sixth edition of the “American Joint Committee on Cancer.”.Table 1Histopathological features of the 57 Papillary Thyroid Tumors (PTC) Yes, n (%)No, n (%)ND, n (%)BRAF mutation35 (61)20 (35)2 (3)Ret/PTC rearrangement5 (9)50 (88)2 (3)TP53 mutation0 (0)57 (100)0Extra-thyroidal invasion15 (26)41 (72)1 (2)Multifocality24 (42)32 (56)1 (2)Nodal metastasis24 (42)33 (58)0Distant metastasis6 (10)35 (62)16 (28)Tumor stage of patients <45 years (n = 20)I15 (75)05 (25)II0 (0)15 (75)5 (25)Tumor stage of patients ≥45 years (n = 37)I8 (22)18 (48)11 (30)II2 (5)24 (65)11 (30)III3 (8)23 (62)11 (30)IV13 (35)13 (35)11 (30)HistologyClassic38 (67)19 (33)0Follicular13 (23)44 (77)0Other4 (7)53 (93)0Dedifferentiated2 (3)55 (97)0ND Not determined All samples were screened for the presence of TP53 mutations (limitedly to exons 5–8 where 80% of mutations occur), BRAF mutations (by SSCP followed by sequence analysis), and for ret/PTC 1 and ret/PTC 3 rearrangements (by RT-PCR; Table 2). MDM4 and flMDM2 mRNA levels were evaluated by qRT-PCR, using specific primers and probes. MDM4 primers were chosen in a region not present in the variant forms MDM4-S, HDMX-G [19] MDM4-211, XALT1, and XALT2 [20] to amplify specifically the fl product. Similarly, the chosen MDM2 primers do not amplify the MDM2-A, MDM2-B, MDM2-C, MDM2-D, and MDM2-E alternative spliced forms and most of the aberrantly spliced forms [21]. Table 2Tumor sample dataSampleM4M4 T/NMDM4-S/flM4MDM4-211MDM4-211 to flM4M2 T/NM4 to M2TP53 statusBRAF mutRet/PTC rearr.Patient statusaTumor stageMultifocalityN1M1Histologyb19.420.91.81.360.1411.3wt+−13−−−2218.961.35.21.42.5wt−−44+++4329.341.60.49.030.311.53.5wt−−11−−−2414.931.79.71.12wt−+11+−−154.390.30.90.41.1wt−−44−++3611.160.41.30.51.9wt−−44−++3712.600.80.78.940.710.73.9wt−−11++−184.440.40.82.10.7wt+−n.d.n.d.−−n.d.1915.511.40.61.41.6wt+−14++−1108.630.30.60.91.3wt−−n.d.n.d.+−n.d.1115.960.21.20.90.7wt−−24−+−1128.110.51.110.9wt+−31++−1136.250.41.310.161.80.8wt+−n.d.n.d.−+n.d.2146.130.41.50.80.9wt+−n.d.n.d.−−n.d.1158.340.31.60.61.2wt−−11−+−1163.820.21.41.230.320.80.5wt+−31−−−11718.8310.80.38.8wt+−24++−2186.321.20.71.11.2wt+−11−−−1193.610.21.82.60.1wt−−11−−−2205.120.330.90.8wt+−n.d.1+−−1218.40.54.20.61.1wt−+2n.d.+−n.d.1225.10.3n.d.*10.6wt−+13−−−1237.060.90.92.60.7wt+−n.d.n.d.−−n.d.1248.850.60.111.4wt+−11+−−1256.210.60.12.30.6wt−−n.d.n.d.+−n.d.1269.6110.11.21.1wt+−11−−−2277.110.30.60.41.6wt+−n.d.n.d.++n.d.1283.530.2n.d.0.21.4wt+−12−−−2295.760.42.20.71.2wt+−n.d.n.d.+−n.d.13010.7111.91.2wt+−11−−−13111.470.3n.d.0.80.9wt−+31++−13214.421.5n.d.2.41.4wt+−11−−−1333.920.30.20.41.7wt+−34−−+13411.960.710.52.8wt−−11−−−2353.690.11.81.170.320.40.7wt+−14++−2368.030.42.70.71.4wt+−11−−−13721.3320.40.85.6wt−−11++−4385.560.44.71.60.6wt+−34++−2391.120.11.12.40wt−−14−+−24013.981.41.43.50.9wt+−11+−−2415.840.33.610.6wt+−34−++1427.360.31.31.830.250.90.8wt+−11−−−1434.770.31.20.80.8wt−+3n.d.−+n.d.4442.450.12.20.70.4wt+−13−+−1455.120.30.70.80.7wt+−34−++1463.10.32.11.60.2wt+−11−−−14710.271.12.419.60.1wt+−11++−24812.131.12.41.12.2wt+−n.d.n.d.+−n.d.1497.940.81.60.82.2wt−−31++−15014.720.82.80.82.2wt+−34n.d.+−15115.671112.2wt+−12−−−15266.493.60.63.62.2wt+−1n.d.+−n.d.15328.941.41.71.42.2wt+−1n.d.−−n.d.1543.720.40.32.80.5wt−−1n.d.++n.d.4555.30.40.71.80.5wt+−11−−−1562.040.60.92.30.3wtn.d.n.d.1n.d.+−n.d.1576.870.7n.d.1.40.8wtn.d.n.d.1n.d.−−n.d.1n.d. Not determined, N1 nodal metastasis, M1 distant metastasisa1, recovered; 2, recurrence occurred, 3,disease persistence, 4, expiredb1, classic variant; 2, follicular variant; 3, dedifferentiated variant; 4, other MDM4 and MDM2 mRNA levels in normal tissues followed a normal distribution according to Kurtosis analysis (Fig. 1a,b). On the contrary, MDM4 and MDM2 values in tumor samples were not normally distributed (p < 0.0001; Fig. 1c,d). Comparative analysis by Wilcoxon signed-ranks test for paired samples revealed a highly significant difference in the distribution of MDM4 values between tumor and CTRL groups (Wilcoxon’s W  = 1,385, p < 0.0001; Fig. 1e). Conversely, comparison of MDM2 values did not show significant difference (p  = 0.61; Fig. 1f), in spite of the presence of four upper outliers in the tumor sample set. There was no correlation between MDM4 and MDM2 mRNA tumor levels indicating that the alterations of the two transcripts are independent from one another (data not shown). Fig. 1a–d Frequency histograms showing the distribution of the mRNA levels of the indicated genes in the indicated sample groups. Relative units of target mRNA are referred to a sample called calibrator, chosen to represent 1× expression of the target gene. Each sample mRNA was normalized relative to the β-actin mRNA. The superimposed line shows the normal distribution curve. For each distribution, the mean (M) ± standard deviation (SD) is shown. e, f Comparison of MDM4 and MDM2 levels, respectively, between control and tumor samples. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Wilcoxon signed-ranks test To confirm the decrease of MDM4 mRNA levels in PTC in comparison to CTRL, we analyzed these levels in an additional 26 independent PTC and three normal thyroid samples. Comparison of tumor and CTRL groups still resulted in a highly significant correlation (Mann–Whitney for independent samples U  = 634, p < 0.0001) and confirmed previous results of a strong downregulation of MDM4 expression in tumor samples. Indeed, study of prediction by receive operator characteristics curves (ROC curves) indicated that the use of MDM4 mRNA levels as potential diagnostic test has a highly significant ability to discriminate between normal and tumor tissues (area under the ROC 0.84, p < 0.0001 Fig. 2). Fig. 2Receive operator characteristics curves for MDM4 levels between normal and tumor samples, assessing variable MDM4 levels as diagnostic test. The plot shows sensitivity of the test against specificity. p value was 0.0001. For each decision threshold, the percentage of tumor cases correctly identified as such (true positives) against the percentage of normal cases incorrectly diagnosed as tumor (false positives) is shown To ascertain whether the decrease of MDM4 mRNA in tumor samples results in decreased protein levels, we analyzed whole-cell extracts from 20 available pairs of tumors and CTRLs (Fig. 3). In 16 out of 20 pairs, the tumor-to-CTRL ratio of densitometric values of the MDM4 protein levels correlated significantly with the observed ratio of the mRNA levels (Kendall tau = 0.7, p  = 0.0002). Interestingly, western blot confirmed the decrease of MDM4 mRNA in tumor relative to normal tissue in seven samples, reinforcing previous observations. Notably, in three samples (44, 27, and 33), we observed an increase of MDM4 proteins in tumor compared to CTRL despite equal or even lower levels of corresponding tumor mRNA relative to CTRL, indicating MDM4 protein stabilization independent of transcription. Western blot analysis revealed also the presence of an additional species of about 58 kDa in some tumor samples (Fig. 3, arrows). This band was recognized also by a mix of three monoclonal antibodies to MDM4 (6B1A, 11F4D, and 12G11G, data not shown), suggesting it may be a MDM4 variant. However, its size does not correspond to any of the MDM4 variant forms described so far. Fig. 3Western blot of WCE derived from 20 couples of tumor (T) and matching control (N). The values derive from the ratio of densitometric value of MDM4–actin, both at the protein and mRNA levels. Asterisks mark tumor samples expressing MDM4-211 mRNA; section signs mark tumor samples showing increased levels of MDM2 protein with no increased levels of the corresponding mRNA. Arrows mark a lower weight protein of about 58 kDa, recognized by different αMDM4 antibodies Further, western blot revealed frequent overexpression of the MDM2 protein in tumors that did not show overexpression of the mRNA (Fig. 3, samples marked by section signs). Analysis of intraindividual variation of MDM4 and MDM2 levels Previous data were obtained by comparing tumors versus CTRLs. We then analyzed the intraindividual variation of MDM4 and MDM2 mRNA levels for each tumor (T) and its matched CTRL (N) by calculating the relative ratio, T to N. The range of MDM4 T-to-N ratios varied from 0.1 to 3.6 (Fig. 4a and Table 2). The majority of tumor samples showed a decrease of MDM4 levels compared to the corresponding CTRLs (29 out of 57 samples having a ratio of <0.5), confirming the previously observed decrease of tumor MDM4 expression at individual levels too. In other tumor histotypes, amplification of MDM4 gene and overexpression of the mRNA have been reported. In this PTC group, MDM4 overexpression (as defined by a T-to-N ratio of ≥5) was not detectable; in only two cases, the ratio of T to N showed a value of ≥2 (2 and 3.6, respectively). On the contrary, comparison of MDM2 mRNA levels showed tumor values twofold higher than matching CTRL in 11 samples, among which one had a ratio of >19, suggesting an amplification event (Fig. 4b and Table 2). Moreover, only six samples (10%) showed a ratio of <0.5, indicating that downregulation of MDM2 levels in PTC tumor is a rare event in comparison to that of MDM4. Notably, in two of these tumors, western blot analysis did not confirm the mRNA MDM2 decrease (Fig. 3; samples 35, 27). Fig. 4a, b Frequency histograms showing the distribution of the T-to-N ratios derived from tumor–CTRL levels of the indicated genes. The superimposed line shows the normal distribution curve. For each distribution, the mean ± standard deviation is shown Correlation of MDM4 and MDM2 with tumor histopathological features We then investigated whether tumor MDM4 and MDM2 mRNA levels as well as the T-to-N ratio varied according to tumor properties by correlating them with the histopathological features of samples as reported in Table 1. First, we examined their association with BRAF mutations and ret/PTC rearrangements, two hallmarks of thyroid carcinogenesis. BRAF mutation were detected in 35 out of 57 patients, ret/PTC rearrangements in 5 out of 57 (Table 2). All BRAF mutations but one involved a heterozygous T > A transversion at nucleotide 1799, resulting in the prototypic valine-to-glutamic-acid substitution at position 600 (BRAFV600E). In one case, the in-frame insertion at position 1796 of an additional codon, coding for Valine, was detected (BRAFV599Ins). This was shown to be a “gain-of-function” mutation in all super imposable to BRAFV600E [22]. No association was found between MDM4 and MDM2 expression, BRAF mutations, and ret/PTC rearrangements suggesting that these oncogenic hits are independent events. We then analyzed the correlation between MDM4 or MDM2 expression with the other tumor histopathological features. This revealed association of MDM4 with multifocality of tumors: specifically, tumors presenting as multifocal show higher values than nonmultifocal (Spearman rank correlation rs = 0.29, p  = 0.032; Fig. 5a, Table 3). This association was not evident when we correlated multifocality with the MDM4 levels of matching normal tissues (p  = 0.23), indicating that this tumor property is not attributable to a patient predisposition given by individual MDM4 expression levels but rather it is a specific feature of tumor cells. Fig. 5a Vertical box-whisker plots showing MDM4 levels between nonmultifocal (no) and multifocal (yes) tumors. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman test. b Vertical box-whisker plots showing tumor–CTRL MDM4 mRNA levels in stage 1 compared to other stages of tumors. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman testTable 3Summary of significant correlations of MDM4 and MDM4 T–N values with histopathological features MultifocalityTumor StagePresenceAbsenceIII–IVn = 24n = 32n = 23n = 18MDM4 T–NMean ± SD0.9 ± 0.760.56 ± 0.430.81 ± 0.540.51 ± 0.43Median ± IQR0.63 ± 0.830.38 ± 0.60.65 ± 0.810.3 ± 0.58MDM4Mean ±SD12.4 ± 12.68.1 ± 6.410.28 ± 5.98.38 ± 5.9Median ± IQR8.74 ± 8.76.19 ± 5.18.85 ± 5.445.7 ± 8.0 We also correlated MDM4 T-to-N ratio with tumor features. We observed an even stronger association between this parameter and multifocality (Spearman correlation coefficient rs = 0.32, p = 0.01, Table 3), confirming previous observations. Further, we found a significant correlation of MDM4 T-to-N ratio with tumor stage (Spearman correlation coefficient, rs = 0.35 for n = 41, p = 0.02; Fig. 5b); in particular, stage I tumors showed the highest ratio, indicating downregulation of MDM4 levels in late-stage tumors in comparison to early-stage ones (Table 3). On the contrary, MDM2 levels as well as MDM2 T-to-N ratio did not show correlation with any histopathological parameter. Characterization of MDM4 variants in thyroid tumors We have analyzed the presence of the MDM4 variant forms, MDM4-S and MDM4-211, in the same sample group. So far, these are the only variant forms of MDM4 detected in human tumors. MDM4-S is a splicing variant present both in normal and tumor tissues; however, in tumor cells, MDM4-S is overexpressed and, as a consequence, the ratio MDM4-S to flMDM4 is >1. At first, we performed comparative analysis of flMDM4 and MDM4-S by qRT-PCR in CTRL samples. In agreement with the literature, we detected both transcripts in all samples with a ratio MDM4-S to fl-MDM4 ranging from 0.09 to 0.79 with only two samples showing values of 1.14 and 1.05 (data not shown). On the basis of these results, we considered overexpression as defined by a ratio of ≥1.5 The same analysis in PTC detected MDM4-S in all but five samples; the resulting MDM4-S-to-flMDM4 ratio was ≥1.5 in 20 out of 57 of tumors (35%; Table 2). The same percentage resulted from the analysis of the additional group of 26 tumors (9 out of 26), in agreement with other reports [11]. Statistical analysis did not reveal any significant association of MDM4-S-to-flMDM4 ratio with the features described in Table 1. The second variant form we investigated was MDM4-211 (HDMX211), previously isolated from a thyroid tumor cell line [13]. MDM4-211 mRNA was present in 7 out of 57 original tumor samples and in 8 out of 26 additional samples analyzed with an overall frequency of 18% (Table 2 and data not shown). Its mRNA was not detected in any of the CRTL samples, thus confirming its specific presence in tumor tissue. In agreement with our previous reports [13], western blot analysis of available tumor and CTRL samples confirmed enhanced levels of the oncoprotein MDM2 in tumors expressing MDM4-211 (Fig. 3, samples marked by *). In our tumor set, the presence of MDM4-211 did not correlate with any of the properties described in Table 1 nor with the expression of the form MDM4-S, indicating that the presence of these two variants is an independent event. Some authors have hypothesized that the presence of MDM4 variants may correlate with lower level of flMDM4 [11]. We therefore assessed the presence and expression of MDM4-S and MDM4-211 in correlation with flMDM4 and with the MDM4 T-to-N ratio. We observed no significant correlation but rather a slight association between low levels of both MDM4-S and flMDM4 (p = 0.056). Discussion In this study, we have analyzed the expression of MDM4 and MDM2 mRNAs in a group of 57 papillary thyroid tumors in comparison to matching normal tissues. We observed that MDM4 levels are significantly lower in tumor samples than in control. Specifically, tumor samples expressing levels of MDM4 lower than matching control are very frequent (51%) and this decrease in expression appears to correlate significantly with higher tumor stages (stage I in comparison to the other), indicating that it occurs with tumor progression. These data led us to hypothesize that a robust presence of MDM4 might exert an antagonistic role in PTC evolution. To date, the majority of the literature has reported amplification of MDM4 in the presence of wt TP53 in different tumor histotypes [3], partly in contrast with our data. However, a different status of MDM4 in some cancer has also been reported. In a gene profiling analysis, MDM4 downregulation was observed in prostate tumors with poor prognosis in comparison to those with good prognosis in agreement with the hypothesis that MDM4 may be detrimental to tumor progression [23]. Moreover, two studies reported low levels of MDM4 protein in chronic myeloid leukemia tumor samples and in tumor cell lines [24, 25]. It would be interesting to know whether those cell lines have features resembling those of advanced-stage tumors. Some authors have suggested that low MDM4 expression levels may be correlated to the presence of a mutated TP53 [11, 25]. However, lack of detection of any of the most frequent mutation of TP53 gene in this PTC group does not support this hypothesis, at least in this type of cancer. Similarly, in the prestate tumor study TP53 was found to be wild type by sequencing (A. Farsetti, personal communication). These data counteract the holistic view of MDM4 as a p53 inactivation tool and suggest that its function may be more complex than previously considered, at least in some tissues. Thus far, molecular studies indicating MDM4 functions different from negative regulation of 53 have provided evidence that MDM4 may display antiproliferative or proapoptotic activities, under stress conditions [6, 7]. Further, a recent work has shown for MDM4 a role in suppressing tumorigenesis through the control of bipolar mitosis [26]. Whether these mechanisms occur in PTC remain to be elucidated. In the analysis of tumor MDM4 expression levels, we found that high levels of MDM4 are associated with multifocal tumors. Different hypothesis have been postulated to explain the origin of these tumors. Certainly, multifocality is related to the high proliferative potential of thyrocites. MDM4 has been reported as inhibitor of p53-mediated growth arrest [1]; thus, it might be hypothesized that the presence of MDM4 may predispose to multifocality and tumor development, while a decrease in its expression may confer an advantage to tumor progression. Interestingly, one study has documented a statistically significant association of MDM4 amplification with low-grade astrocytomas, thus supporting the hypothesis that high levels of MDM4 may represent an early advantageous event for tumor development [27]. Further, the association between MDM4 levels and tumor multifocality is of particular clinical relevance. Indeed, the possibility of early diagnosis of tumor multifocality might allow discrimination between partial or total thyroid gland ablation. The extension of this type of analysis to a broader set of samples, including benign lesions, should help to confirm these findings and to investigate potential applications of this molecule in clinical practice. In addition, we have observed with high frequency the aberrant presence of MDM4 alternatively spliced forms, whose oncogenic activity has been assessed so far only in vitro. In our analysis, the aberrant presence of these variants did not correlate with any of the histopathological features. In a study with primary sarcomas, the MDM4-S-to-flMDM4 ratio correlated with enhanced cell proliferative potential and decreased patient survival [11]. The high incidence of patient survival in PTC (only three deaths in our group, Table 2) does not allow to confirm this association. For MDM4-211, this is the first frequency report in a large group of tumor samples and indicates that its presence is appreciable in PTC (18%). Interestingly, these MDM4 variants forms show in vitro an even stronger inhibitory activity toward p53 than the full-length protein. Their presence may concur to modify the activity of flMDM4, making more complex the assessment of its function in tumor cells. It has to be mentioned that in many PTC samples we observed the presence of a MDM4 minor form whose nature is unclear, supporting the hypothesis that MDM4 variants may concur to MDM4 oncogenic functions. Conversely, MDM2 mRNA levels were increased in majority of tumor samples. In particular, we observed with high frequency enhanced protein levels in tumor samples that do not show enhanced mRNA levels. This is in accord with previous reports and confirms that in thyroid tumors MDM2 overexpression takes place frequently by mechanisms other than enhanced transcription [28]. At least in some of these samples, the simultaneous presence of MDM4-211 variant may cause such upregulation. These data further confirm the general view of the antagonistic role of MDM2 toward the oncosuppressor p53. Finally, in this study, we did not find any correlation between MDM2 and MDM4 mRNA levels, suggesting that the alterations we observed occur independently from one another. Overall, our results reveal that MDM4 alterations, particularly the presence of its variant forms with p53-inhibitory properties, are present at high frequency in PTC confirming the general model of different ways of p53 inactivation in human tumors. However, our data represent the first statistical report of a strongly significant downregulation of flMDM4 expression in cancer and particularly in advanced-stage tumors, suggesting that this protein may play additional roles besides p53 inactivation at least in PTC. In view of the suggested strategies for abrogation of MDM4 in human tumors [1, 3], our findings counsel acquisition of more information on the molecular functions of this protein in different human tumors in order to carefully evaluate the application of such therapies.

Histochem_Cell_Biol-3-1-1779629

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing. Introduction Cartilage–bone transition by endochondral ossification is a highly complex process which determines not only longitudinal growth of long bones, ribs and vertebrae, but also plays a critical role in bone fracture callus healing, osteophyte formation and cartilage tissue engineering. Decisive steps in this process are proliferation, maturation and hypertrophy of chondrocytes in the growth plate which play a key role in mineralization, apoptosis and induction of resorption of hypertrophic cartilage by invading bone marrow. These events are regulated by synergistic action of several signaling pathways and transcriptions factors controlled by a multitude of growth factors and hormones. A major signaling system regulating chondrocyte proliferation and maturation is controlled by PTHrP, which is induced by Indian hedgehog, stimulates chondrocyte proliferation and delays maturation through the PTHrP receptor PTHR1 [for reviews see (Kronenberg et al. 1998; Vortkamp 2000)]. Also FGF factors stimulate proliferation and block maturation through FGF receptor 3, but their action in the growth plate is more complex with respect to the differential distribution of different FGFs and FGF receptors in the various zones of the growth plate (Ornitz and Marie 2002). The role of BMPs during chondrocyte maturation and hypertrophy is somewhat controversially discussed: several studies show that BMP factors such as BMP-6 and BMP-7 stimulate hypertrophic differentiation of chondrocytes and promote collagen X expression, thus preparing growth plate cartilage for replacement by endochondral bone (Enomoto-Iwamoto et al. 1998; Volk et al. 1998; Grimsrud et al. 2001). On the other hand, it was reported that BMP-2 and BMP-4 overexpression in developing chick limbs caused delayed hypertrophy of chondrocytes (Duprez et al. 1996). The fourth family regulating chondrocyte maturation are Wnt factors and the Wnt/β-catenin signaling system (Dong et al. 2005), also acting in diverse directions. For example, Wnt 7a inhibits chondrogenesis and Wnt 5a impairs chondrocyte hypertrophy and type X collagen (Col10a1) expression in vitro (Daumer et al. 2004), but activation of the canonical β-catenin pathway by Wnt 8c and Wnt 9a induces chondrocytes maturation, hypertrophy and calcification, associated with upregulation of Col10a1 expression (Enomoto-Iwamoto et al. 2002; Dong et al. 2005, 2006; Hu et al. 2005). Recently increasing evidence points to complex crosstalks between these signaling pathways. For example, there is experimental evidence for a coordination between Indian hedgehog and BMP signaling in chondrocyte proliferation (Minina et al. 2002). Targets of the signaling pathways in the growth plate are transcription factors such as Sox9 responsible for chondrogenic differentiation (Ng et al. 1997; Lefebvre and de Crombrugghe 1998) and cbfa1/runx2 inducing osteogenic differentiation (Karsenty et al. 1999; Ducy 2000) as well as chondrocyte maturation and Col10a1 expression (Kim et al. 1999; Enomoto et al. 2000; Zheng et al. 2003). There is ample evidence in the literature that factors regulating chondrocyte maturation and hypertrophy may be also directly involved in the regulation of Col10a1 gene expression, e.g. PTHrP (Iwamoto et al. 1994; Ionescu et al. 2001; Riemer et al. 2002; Gebhard et al. 2004), Runx2 (Enomoto-Iwamoto et al. 2001; Leboy et al. 2001; Zheng et al. 2003), BMP2 (Volk et al. 1998; Grimsrud et al. 2001), or c-fos (Thomas et al. 2000; Riemer et al. 2002). Thus, in order to elucidate the exact role of the various signaling systems and involved factors regulating endochondral ossification, it would be useful to generate a reporter gene mouse in which the expression of LacZ is under the control of the complete Col10a1 gene regulatory sequences. Previously we have reported on the generation of transgenic mouse lines in which LacZ expression is under the control of the 4.6 kb promoter of Col10a1 which includes a strong enhancer element (Gebhard et al. 2004) and runx2 binding sites (Zheng et al. 2003). We have shown that this promoter is sufficient to drive specific expression of LacZ reporter genes in hypertrophic chondrocytes of growth plate cartilage of long bones, ribs, vertebrae and sternal cartilage (Gebhard et al. 2004). Reporter gene expression was restricted to hypertrophic cartilage, but not all hypertrophic chondrocytes in the growth plate of 18.5-day embryos were found to be lacZ positive, and only weak lacZ expression was observed at early embryonic stages before day E17. Furthermore, not all transgenic founders containing the LacZ transgene showed lacZ activity in the growth plates, suggesting a high level of sensitivity to interfering activities of the genomic context of insertion or additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences. Here we report on the construction of a BAC reporter mouse expressing lacZ under the control of the Col10a1 gene. A LacZ-Neo cassette was inserted into the second exon of Col10a1 within the context of a 215 kb BAC using a phage-based homologous recombination system in E. coli (Yu et al. 2000; Lee et al. 2001). Transgenic mouse lines established with this modified BAC show specific LacZlacZ expression at high levels in hypertrophic zones of long bones, ribs, vertebrae, mandibles and sterna of transgenic mouse lines. No significant unspecific expression was detected in other chondrogenic or non-chondrogenic tissues except some transient, probably unspecific X-gal reaction in the prenatal epidermis and hair papillae. The robust and specific expression of Col10a1-based BAC recombineering vectors in transgenic mice opens new and unique possibilities to study the role of growth factors and transcription factors in chondrocyte hypertrophy and endochondral ossification, and to define further cis-acting regions in the Col10a1 gene. Materials and methods BAC clones Murine Col10a1 containing BAC clones were selected from the UCSC Bioinformatic databases (http://www.genome.ucsc.edu/) (Kent et al. 2002); clone RP23-192A7 contained 211 kb of genomic DNA flanking the Col10a1 gene derived from the L129 mouse strain. RP23-192A7 (cloned in the pBACe3.6 vector) was obtained from the BACPAC Resources Center at Children’s Hospital Oakland Research Institute, (CHORI). The presence of the full-length Col10a1 gene was verified by PCR amplification using both primers for detecting the enhancer at −4.6 kb (Enmcol10a1-3′: ATT CTC GAG CTT TGG GAA GCA TGG TG ; Enmcol10a1-5′: CAA GAA ATC TTT GGG AAA TGA ATG AAT G ) (Gebhard et al. 2004) , and for a region 2036 bp downstream of the poly A site of the Col10a1 gene (mcol10a1-psg-5′: TCT ACC AGG AGG CCT CTC TTC AAT GTT AC; mcol10a1-psg-3′: GGG AAT TCT TAC CTT AAA GTA GAT ACA TG (amplicon size 222 bp). Construction of targeting vector placH+Col10a1-lacZ-frt-neo-frt The 5′ recombination site was generated by PCR amplification of a 129 bp fragment mapping to the 113 bp 3′-end of intron 1 and 16 bp of the 5′-UTR encoded in exon 2 by using BAC RP23-192A7 as template (see Fig. 1). Terminal SalI and NcoI restriction sites were added by using primers Col10a1-5′SalI (5′-ACG CGT CGA CGA TAT CTC AGT CAT TTA AAA AAC CAT GA) and Col10a1-5′NcoI (5′-CAT GCC ATG GAT TTT CAG ATA GAT TCT GAA AAG CAG A. The product was digested with SalI and NcoI and cloned into the multiple cloning site 2 of plasmid placH (Zhou et al. 1995) kindly provided by Dr. V. Lefebvre, Cleveland) containing the lacZ gene and the 3′-end of the murine protamine 1 gene with an intron and poly A signal resulting in clone placH+5′COL10a1. Use of the NcoI site linked the lacZ reading frame to the start ATG of Col10a1. Fig. 1Generation of the BAC-Col10a1-lacZ-neo DNA for the transgenic expression of the lacZ reporter gene in hypertrophic cartilage. a Genomic structure of the murine Col10a1 gene, with exons (gray boxes) and introns. The sequences used for generation of the 5′ and 3′ homologous regions in exon 2 and intron 1 are shown in black. b The targeting vector placH- Col10a1-lacZ-frt-neo-frt contains Col10a1 derived 5′ and 3′ homology domains flanking a lacZ-frt-neo-frt cassette for homologous recombination in E. coli. The lacZ coding sequence was fused with the start ATG of the Col10a1 reading frame; shown is only the insert of the targeting vector, contained in the placH vector. c Partial map of the BAC-clone Col10a1-lacZ-frt-neo-frt clone after homologous recombination with the targeting vector placH-Col10a1-lacZ-frt-neo-frt The 3′ recombination site was generated by overlapping primers corresponding to a 57 bp sequence of exon 2. Additional HindIII sites were introduced at both ends as well as a XhoI site at the 3′-end. The frt-neo-frt cassette of the pICGN21 vector (Yu et al. 2000; Lee et al. 2001); kindly provided by Dr. Copeland) harboring the neomycin phosphotransferase gene under the SV40 promoter, an HSV thymidine kinase and a poly A signal was amplified by PCR using primer FRTNeo5´HindIII (5′-AGT CAA GCT TTA ACT GAT CGC GGC CAG CTT GAA GTT) (pIGCN21 sequence pos. 4664–4689, marked in bold) and the primer Col10a1-3′HindIII (5′-TAA AGC TTC TCG AGT TGT GTC TTG GGG CTA GCAAG TGG GCC CTT TAT GCC TGT GGG CGT TTG GTA CCG TTC TAT TCC AGA AGT AGT GAG GAG GCT TT) and plasmid pICGN21 as template (pICGN2 sequences pos. 6103 to 6123 marked in italics) and Col10a1 from exon 2 (82 to 138, underlined). The product was digested with HindIII and inserted into the multiple cloning site behind the mP1 gene of placH+5′Col10a1. The resulting plasmid placH+Col10a1-lacZ-frt-neo-frt was verified to be correct by restriction digestion and sequencing for correct insertion and composition. Homologous recombination and detection of BAC transgene BAC RP23-192A7 was electroporated into the E. coli strain EL250 (DH10B[λcl857(cro-bioA)<>araC-PBADflpe]) kindly provided by Dr. Neal G. Copeland, NCI, Frederick, MD, USA, containing a defective λ prophage which supplies essential functions (exo, bet, gam) to protect and enhance recombination of linear DNA (Lee et al. 2001). The 5.1 kb recombination cassette was excised from plasmid placH+COL10a1-lacZ-frt-neo-frt by XhoI; complete linearization with XhoI was important to avoid amplification of the circular targeting vector in E. coli. For homologous recombination 300 ng linear fragment was transformed by electroporation according to Dower (Dower et al. 1988) in 0.1 ml cuvettes containing 50 μl of ice-cold competent EL250 containing BAC RP23-192A7, using a Bio-Rad gene pulser set at 1,750 kV, 23 mF with a pulse controller set at 200 Ω. Electrocompetent bacteria were prepared as described in (Lee et al. 2001). A 15 min heat shock at 42°C was used for induction of the homologous recombination machinery according to (Yu et al. 2000; Lee et al. 2001). Transformed cells were incubated for 1.5 h at 32°C in 1 ml of LB and selected for chloramphenicol- and kanamycin-resistance. Resulting clones (RP23-192A7 + lacZ-frt-neo-frt) were screened for homologous recombination by PCR detecting the 5′ recombination event (amplicon 437 bp) using primers P1 (5′-TTT AGA GCA TTA TTT CAA GGC AGT TTC CA) and P2 (5′-CGG CAC CGC TTC TGG TGC CGG AAA CCA GGC), and the 3′ recombination event (product 378 bp) using primers P3 (5′-ACA GAA TAA AAC GCA CGG GTG TTG GGC GT) and P4 (5′-ATC ATT CCG CTG TAC TAG CTC AAG CCA ATC). Direct sequencing of BAC DNA was performed with primers P1 and P4. BAC DNA purification and generation of transgenic mice BAC-Col10a1 - lacZ-neo DNA from clone#11 was extracted from multiple E. coli EL250-BAC#11 minipreps by alkaline lysis (Sambrook et al. 2001), purified by potassium acetate precipitation, washed with ethanol and dissolved in Tris-EDTA (TE) buffer. For size and quality control, aliquots were subjected to pulsed field gel electrophoresis (PFGE) (Fig. 2). For purification, 50 μg BAC DNA of clone#11(BAC-Col10a1-LacZ-neo) were dissolved in TE buffer and concentrated to 500 μl by vacuum centrifugation. The DNA was linearized overnight with PISceI enzyme (NEBiolabs) cleaving the unique site in the BACe3.6 vector part. For chromatographic purification a 5 ml plastic pipette was packed with Sepharose CL-4B (Pharmacia) equilibrated in microinjection buffer (5 mM Tris/HCl, pH 7.4, 0.1 mM EDTA, 5 mM NaCl) (Zeilhofer et al. 2005), the linearized BAC#11-DNA was mixed with bromophenol blue and chromatographed. Half milliliter fractions were collected until the dye reached the column bottom, and BAC DNA concentration in each fraction was analyzed by PFGE using serial dilutions of BAC RP23-192A7 DNA as internal standard. The BAC DNA concentration of the main fraction was adjusted to 1.5 ng/μl, and NaCl concentration was increased to 100 mM to stabilize BAC DNA. Fractions were stored at 4°C until microinjection. Injection into fertilized oocytes and generation of transgenic mice was performed by standard techniques using oocytes from FVB mice and FVB/ FVBxC57BL6 F1 hybrids as described (Zeilhofer et al. 2005). Fig. 2Characterization of one of the BAC-Col10a1-LacZ-frt-neo-frt clones (clone#11). Digestion with PISceI (lane 3), NotI (lane 4), PmeI (lane 5) and NruI (lane 6) resulted in fragments of the expected sizes. Sizes are compared with 1 kb marker (lane 1), 1 kb extension (lane 2) and PFG marker (lane 7), and the lengths are indicated. For microinjection, BAC #11 DNA was linearized with PISceI, purified by gel chromatography on Sepharose CL4B equilibrated in microinjection buffer and analyzed and quantitated by pulsed field gel electrophoresis Genotyping of transgenic mouse lines Transgenic founder animals were selected by PCR of tail biopsies using primers pairs P1/P2 and P3/P4 (see above) detecting recombination events. Each sample was standardized by PCR using P1/P4 primer pair which generates a 538 bp fragment from the wt Col10a1 allele. Founders derived from injections of F1 hybrids were crossed with C57BL6, whereas founders derived from FVB oocytes were crossed with FVB mice. For genotyping of newborn mice and embryos DNA was extracted from skin or placenta, and transgene DNA was identified by PCR as above. Histological analysis and in situ hybridization Newborns mice were anesthetized under CO2, dissected and subjected to X-gal staining, followed by either clearing with KOH fixation, embedding into paraffin or by cryotome sectioning as described (Zhou et al. 1995). Embryos at 12.5 to 16.5 day were left undissected and stained with X-gal. Paraffin and frozen sections were counterstained with eosin. In situ hybridization for collagen X was performed as described previously (Aigner et al. 1992), but using a digoxigenin-labeled mouse Col10a1-specific anti-sense probe derived from the 3′-end of the Col10a1 gene instead of 32P-labeled dCTP. Non-radioactive in situ hybridization was done as described elsewhere (Schmidl et al. 2006), using alkaline phosphatase labeled anti-digoxigenin antibodies and BM purple (Roche) as color substrate according to the manufacturers protocol. Determination of transgene copy number by Southern hybridization For quantitative analysis of transgene copy number, genomic DNA was isolated from skin of newborns or from placenta of embryos and cleaved with BamHI. For standardization 5 μg wild-type DNA were mixed with 0.37 ng up to 18.38 ng RP23-192A7 + lacZ-frt-neo-frt DNA corresponding to 1 up to 50 copies of recombined BAC per lane. Samples for standardization and 5 μg of DNA from transgenic founders were cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized to with a 32P-labeled probe derived for from the pBACe3.6 vector, prepared by PCR using primers BAC11-5 (5′-TTTAAACGTGGCCAATATGGA) and BAC11-6 (5′-CGCGGATCCTCTCCCTAT). An 8 kb band was detected in transgenic samples and the intensity was measured in a FLA-3000 phosphoimager (Fujifilm) using the AIDA program (Advanced Image Data Analyzer, Raytest ). Results Construction of a placH-Col10a1-lacZ-frt-neo-frt targeting vector and homologous recombination The BAC clone RP23-192A7 selected from the CHORI BAC library RPCI23 contains the mouse Col10a1 gene (7.2 kb) with 171 kb of upstream and 33 kb of downstream genomic regions. For insertion of the lacZ reporter gene into the second exon of the Col10a1 gene by homologous recombination, a targeting vector was constructed containing the lacZ gene linked to a neomycin resistance cassette flanked by two frt sites. The targeting vector was flanked by a 129 bp 5′-terminal homology arm overlapping parts of the 3′-end of intron 1 and 14 bp of exon 2 including the start ATG for fusion with NcoI. The 3′ recombination site mapped to a 57 bp sequence in the 3′ part of exon 2 (Fig. 1b). The vector construct was verified by restriction mapping, Southern blotting and sequencing. Initial attempts using shorter homology regions resulted in mostly incorrect recombination events. For homologous recombination, the BAC clone RP23-192A7 was electroporated into the E. coli strain EL250, resulting in EL250/RP23-192A7. The Col10a1-lacZ-frt-neo-frt insert was excised from the targeting vector with XhoI (Fig. 1) and electroporated into electrocompetent EL250/RP23-192A7. Chloramphenicol- and kanamycin-resistant BAC-positive clones were selected and tested by PCR for homologous recombination by using two primer pairs P1/P2 and P3/P4 (see Fig. 1 and Materials and Methods) specific for the 5′- and 3′- recombination sites, respectively. To verify the integrity of the Col10a1 gene in its genomic context after homologous recombination, the presence of the enhancer at 4.6 kb upstream the transcription site and the 5′-end of a pseudogene downstream of the 3′ UTR sequences was verified by PCR with specific primers (see Fig. 1 and Materials and Methods) and sequencing of the BAC DNA. Furthermore, intact BAC ends were confirmed by PCR, using primers derived from the pBACe3.6 vector and the genomic insert. To minimize BAC degradation, DNA was prepared from BAC-Col10a1-LacZ-Neo clone #11 by alkaline lysis and ethanol precipitation, avoiding absorption to minicolumns and shear stress. Control of the BAC DNA by PFGE confirmed the expected size of 220 kb (Fig. 2). Generation of transgenic mice DNA from BAC-Col10a1-lacZ-neo (clone #11) was linearized with PISceI at the unique restriction site in the pBACe3.6 vector sequence, purified by molecular sieve chromatography, and injected into the pronuclei of fertilized oocytes of FVB mice and FVB/C59Bl F1 hybrids. Both strains were by far superior in litter size and successful raising offspring as compared to C57/Bl6 mice. Out of 70 newborn pups, 13 were found harboring the lacZ gene after PCR analysis of genomic DNA, using primer pairs P1/P2 and P3/P4 (see Fig. 1). Southern blot analysis of genomic DNA (Fig. 3a) as well as Real- time PCR analysis of genomic DNA using Col10a1 intron specific primers showed that the founders contained between one and seven transgene copies (Table 1) Fig. 3a Analysis of BAC transgene copy numbers in 13 transgenic founders by Southern hybridization. Genomic DNA was isolated from skin of newborns, cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized with a 586 bp 32P-labeled probe derived from the pBACe3.6 vector which recognizes an 8 kb band in transgenic samples. Band intensities were quantitated in a phosphoimager. Copy numbers of transgene BAC DNA (Table 1) were calculated on the basis of a standard curve established with serial dilutions of BAC DNA starting from 50 copies down to 1 copy of BAC RP23-.192A7, diluted in 5 µg of wt DNA. b Comparison of LacZ activity in the scapulae of three different BAC transgenic lines harboring different BAC copy numbers shows some correlation between staining intensity and BAC copy number. All specimen were from new born animals and stained under identical conditions with X-gal for 18 hTable 1BAC transgene copy number in 13 founders, determined by Southern hybridization (see Materials and methods)Founder #Strain sexβ-gal activityBAC copy no.800FVB, m+5986FVB, f+31501FVB, f+11504FVB, m+31508F1, fND21510F1, mND11515F1, fND41516F1, f+61520F1, m+71524F1, fND21527F1, mND11533FVB, mND11534FVB, m+4ND not determined Seven of the 13 independent founder lines were analyzed so far for LacZ activity by X-gal staining of newborns or embryos (all F1 generation). All offspring analyzed revealed strong LacZ activity after X-gal staining which was restricted to the hypertrophic zones in all growth plates of long bones, ribs, vertebrae, sternebrae and to hypertrophic cartilage in the skull in E14.5- and E16.5-day embryos and newborns (see Figs. 4, 5, 6). The X-gal staining pattern was identical in all seven lines investigated, while staining intensity varied in relation to the BAC transgene copy number (Fig. 3b). Under identical staining conditions, X-gal staining was strongest in founder #1520 (seven transgene copies), followed by #800 (five copies) and #986 (three copies) (Fig. 3b). Fig. 4Detection of lacZ expression in BAC-Col10a1-lacZ-Neo transgenic embryos by X-gal staining at E14.5 (a), E16.5 (b) and P1 (e) of the F1 generation of founder lines #1534 (a), #1504 (b), and #1520 (c). X-gal staining reveals strongest expression in chondrocytes in the hypertrophic zones of the scapula (s), humerus (h), femur (f), clavicula (cl) and all other long bones, furthermore in the hypertrophic zone of the ribs and in cervical vertebrae (c1, c2), (p pelvic bone). c, d In E16.5 embryos some X-gal staining was also seen in the epidermis of the tail (barrow) and in hair papillae of the forehead and snout, and at nostrils (n). This expression is restricted to some basal keratinocytes in the epidermis, here shown in a section of the snout (d). It disappears after birth and may be a result of unspecific β-gal activity. No LacZ activity was detected in non-hypertrophic chondrocytes or in non-chondrogenic tissues.Fig. 5X-gal staining of transgenic newborn mice of BAC-Col10a1-lacZ-neo (a, b founder line #800, F1; c–i founder line #1520, F1). All hypertrophic zones of the growth plates of long bones, ribs, and vertebrae exhibit strong LacZ activity. a Humerus, distal; b ulna, proximal; c forearm, d hand; e skull: C1 cervical vertebrae 1 (atlas); eb ecto-occipital bone; h hyoid bone; m mandibular condyle; MC Meckels cartilage; mh head of malleus; r head of ramus. f Thoracal vertebrae (left four) and cervical vertebrae (right three), view from ventral; g ribs; h spine, view form dorsal, i sternumFig. 6Paraffin sections of transgenic newborns (a–e) (founder line #1520, F1) and E16.5 day (g) (founder line #1534) embryos showing strong and specific LacZ activity in all hypertrophic chondrocytes, e.g. in the growth plate of the humerus (a) (distal), elbow joint of the radius (b), sternebrae (c), atlas (d), ribs (e) and spine (f). Comparison of the lacZ staining pattern in the elbow joint (h radius) with that observed after in situ hybridization with a Col10a1 probe (g) in a parallel section illustrates that LacZ is expressed in the BAC transgenic mice only in collagen X expressing hypertrophic chondrocytes Specific expression of the BAC-Col10a1-LacZ-neo transgene in hypertrophic cartilage Various stages of embryonic development were analyzed for expression of the BAC-Col10a1-lacZ-neo transgene by X-gal staining. In E14.5-day embryos, strong LacZ activity was seen in all ribs, the hypertrophic zones of scapula, humerus, radius and ulna in the forearm and femur, tibia and fibula of the hindleg, as well as in mandibles and cervical vertebrae (Fig. 4a). At day 16.5, the extent of X-gal staining expanded in the diaphyseal region of scapula, humerus and in the ribs, corresponding to the expansion of the hypertrophic zones at this embryonic stage. (Fig. 4b, e) Sectioning of X-gal-stained embryos revealed that the lacZ transgene expression in the skeleton occurred exclusively in the hypertrophic zone of growth plates and ribs (see Figs. 5, 6), correlating with collagen X expression as seen by in situ hybridization (Fig. 6g, h). At embryonic stages E12.5 to 16.5 X-gal staining also occurred in some regions of the epidermis (Fig. 4b, arrow, Fig. 4c, d), mainly in the papillae of the forehead in the snout (Fig. 4c), and in the skin of the tail, hindlegs, forelegs, and shoulder It was restricted to the basal layer of keratinocytes (Fig. 4d) and seen at E12.5 day before the first appearance of hypertrophic chondrocytes in cartilage, but was not detected after birth. It seemed unspecific as no α1(X) mRNA signals were seen in the epidermis or hair papillae at any stage of development by in situ hybridization (N. Adam and K. von der Mark, unpublished data). Transgenic newborn F1 offspring of all seven founders analyzed so far revealed an even and strong LacZ activity in all growth plates of long bones (Figs. 4e, 5a, b), ribs (Figs. 4e, 5g), vertebrae (Figs. 4e, 5f, h), and sternebrae (Figs. 4e, 5i). In the skull, LacZ activity was seen in mandibular condyles, in hyoid cartilage, heads of malleus and ramus, ectooccipital and basisphenoid bone anlagen, cervical vertebrae and other ossifying cartilage anlagen (Fig. 5e). The high intensity of X-gal staining allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage, for example the inner and outer aspects of the thoracal and cervical vertebrae (Fig. 5f, h). Analysis of the X-gal staining pattern in paraffin and frozen sections of transgenic embryos and newborn mice confirmed that the LacZ activity was restricted to hypertrophic chondrocytes (Fig. 6), shown here for the humerus (a), radius (b), sternum (c), atlas (d), and ribs (e, f). The X-gal staining pattern correlated well with the expression pattern of α1(X) collagen mRNA seen by in situ hybridization analysis of a similar section (Fig. 6g, h). Neither non-hypertrophic chondrocytes nor any non-chondrocytic cell types revealed detectable levels of LacZ-activity in newborn transgenic animals. After prolonged staining, a non-specific reaction appeared in osteoclasts of long bones in transgenic and wildtype animals owing to endogenous galactosidase activity. Regardless of the transgene copy number, the LacZ activity in the offspring of all founders was seen persistently in all hypertrophic chondrocytes of the developing skeleton. Discussion In a previous report on the generation of Col10a1-specific reporter gene mouse lines the expression of lacZ under the control of a 4.6 kb mouse Col10a1 promoter region was restricted to hypertrophic cartilage, indicating that the -4.6 kb mouse promoter including a 500 bp enhancer element located at the 5′-end of this region is sufficient for tissue specific expression (Gebhard et al. 2004). Also in another transgenic mouse expressing LacZ under a −4.0 kb proximal Col10a1 promoter lacking the enhancer, lacZ expression was restricted to hypertrophic cartilage, although mostly to the lower hypertrophic zone (Zheng et al. 2003). The Col10a1 enhancer has been originally described in the human COL10A1 gene; it was located between −1.9 and −2.4 kb by in vitro transfection studies in primary hypertrophic chondrocytes with various reporter gene constructs (Thomas et al. 1995; Beier et al. 1997; Chambers et al. 2002; Riemer et al. 2002). This enhancer is active in hypertrophic, but not in resting zone chondrocytes. Further upstream, a silencer element between −2.4 and −2.8 kb suppressed transcription in non-chondrogenic cells (Beier et al. 1997). The enhancer element is highly conserved in mammals with 78–85% sequence identity between human, bovine and murine Col10a1 genes (Gebhard et al. 2004) and contains a conserved AP-1 site which is essential for high transcription of enhancer- reporter genes in hypertrophic chondrocytes (Gebhard et al. 2004). In the murine gene, the enhancer is located between −4.1 and −4.6 kb due to insertion of two virus-related sequences in the promoter (Gebhard et al. 2004). Further cis-regulatory elements in the 4.6 kb promoter include an AP-1 site in the proximal promoter (Harada et al. 1996) and several runx2 bindings sites (OSE elements), (Zheng et al. 2003). Yet, although lacZ transgene expression in the 4.6 kb Col10a1–LacZ reporter mice was specific and restricted to hypertrophic chondrocytes, not all hypertrophic chondrocytes in the growth plates of 18.5-day embryos were found to be LacZ positive, and only weak LacZ expression was observed at early embryonic stages before day E17 (Gebhard et al. 2004). Furthermore, not all transgenic founders containing the lacZ transgene were stained with X-gal in the growth plates, suggesting positional effects of additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences. It may also indicate a high level of sensitivity to interfering activities of the genomic context of insertion. It is possible that the −4.6 kb promoter including the first intron contains essential silencing elements required for suppressing Col10a1 expression in non-hypertrophic cartilage and in non-chondrogenic tissues, but possibly lacks additional regulatory elements beyond the enhancer region. In contrast, in the Col10a1-BAC transgenic lines presented here, all hypertrophic chondrocytes showed strong and even LacZ expression in all zones of endochondral ossification in embryonic and postnatal transgenic animals. The LacZ reaction of hypertrophic cartilage became visible already after 3–4 h of X-gal staining, while under the control of the 4.6 kb Col10a1 promoter significant LacZ activity in transgenic mice did not become visible before 18 h staining time (Gebhard et al. 2004). Recently, evidence is accumulating that in many genes further tissue-specific enhancers are located in large distances from the start site of transcription. For example, regulatory sequences were identified 20–30 kb upstream of the transcription start site of the Myo D gene (Goldhamer et al. 1992) or the Ubx gene in Drosophila (Irvine et al. 1991), up to 270 kb downstream in the mouse BMP5 gene (DiLeone et al. 2000) or between 290K kb upstream and 450 kb downstream of the Sox9 gene (Bagheri-Fam et al. 2006). Thus we cannot exclude that additional tissue-specific regulatory elements may be present in the Col10a1 gene, either upstream in the region between −170 kb and the enhancer at −4.6 kb, or downstream of the 3′-end of the Col10a1 gene. The BAC transgenic reporter lines established here would facilitate the identification of such elements by generating further mutants using the homologous recombination technique (DiLeone et al. 2000). Reasons for the high reporter gene expression levels in the BAC-Col10a1-lacZ-neo reporter lines in comparison to the 4.6 kb Col10a1-lacZ mice are for one part the high copy number of BAC transgenes, and for the other part the favorable und undisturbed conditions for transgene expression in the BAC environment. The X-gal staining intensity in founder line #1520 harboring seven BAC transgene copies was clearly higher than that of founder line #986 harboring only three BAC copies. But in addition, the use of targeted BAC recombination permits the expression of genes in their genomic context, containing most relevant regulatory regions in the correct orientation and distance, in contrast to conventional transgenic mice generated by injection of short reporter genes. This strategy is less sensitive to unpredictable influences by regions surrounding the random integration site (Mortlock et al. 2003). The expression pattern of LacZ, however, was very similar in all seven BAC lines analyzed so far, apparently independent of the integration sites and the copy number of the BAC transgene. Most E14.5- to E16.5-day BAC-Col10a1-lacZ-neo transgenic embryos showed some X-gal staining in hair follicles of the forehead and snout, and of the epidermis of the tail, legs and shoulder, which disappears after birth. In the X-gal positive spots, staining was restricted to the basal layer of keratinocytes. Since these cells do not express type X collagen, the effect may be due to endogenous galactosidase activity, or a result of LacZ activation by other, Col10a1-unrelated regulatory elements located in the BAC clone. In addition to Col10a1, the BAC clone RP23-192A7 contains coding regions of three additional genes Tspyl-1, Tspyl1-4 and Nt5dc1. Whether these genes are expressed under their endogenous promoters in the BAC transgenic mice is currently investigated. If they are expressed, they could affect the phenotype of the established transgenic mouse lines, but no phenotypic alteration in skeletal elements nor in other tissues have been observed in transgenic embryos, newborn or adults as compared to wildtype littermates. The high intensity of X-gal staining under the control of the BAC-Col10a1 promoter allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage of anatomical structures not investigated so far in such detail and completeness, for example the skull or the inner and outer aspects of the thoracal and cervical vertebrae. The data presented in this study illustrate the superior potential of reporter gene expression analysis in combination with BAC recombineering technique as compared to conventional in situ hybridization analysis. The robust and specific expression of the lacZ reporter gene under the control of BAC-Col10a1 environment furthermore shows that the placH+Col10a1-lacZ-frt-neo-frt targeting vector may be an extremely versatile vector for the targeted overexpression of other genes in hypertrophic chondrocytes of growth plates with high efficiency and specificity. Thus, the Col10a1-based BAC vector may be a useful tool to analyze the role of effector genes in skeletal development, endochondral ossification, and fracture callus formation.

Eur_J_Nucl_Med_Mol_Imaging-3-1-1998881

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

Purpose Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. Introduction Acute renal failure (ARF) that results from ischemic or toxic insults to the kidney is usually referred to clinically as acute tubular necrosis (ATN), where tubular damage and altered glomerular hemodynamics may coexist or even lead to each other [1–3]. In some cases of either human ARF [4, 5] or experimental models of renal injury [6], a lack of correlation between histological evidence of injury and renal function has been found, and the relative contribution of functional versus structural changes to the evolving renal dysfunction has not been unequivocally substantiated. Since ATN is a dynamic clinical condition, rapidly evolving from the incipient to the established phase, its early identification is critical. However, the various currently available methods of clinical and laboratory assessment are insufficient to differentiate between ischemic ARF, resulting in acute tubular dysfunction secondary to cell injury, and prerenal azotemia, characterized by decreased glomerular filtration but no epithelial cell injury. The inability to distinguish between these situations leads to long disputes over the pathogenesis, diagnosis, and management of evolving renal failure. Renal biopsy provides limited data because of the small sample size and the very focal pattern of tubular injury [2, 7]. In addition and most importantly, it is impractical in the human syndrome. Functional data in early phases are also of little help, because prerenal azotemia becomes an important consideration at that time, and only the evolution of the clinical situation really defines the nature of the problem [8, 9]. The utilization of specific urinary biomarkers of tubular injury, such as KIM-1, is not yet considered valid or sufficiently sensitive to establish ATN [10, 11]. Moreover, these tests remain qualitative rather than quantitative markers of injury. Since therapeutic interventions might differ at various points in the evolution of ATN, advanced techniques are required to noninvasively follow the dynamic status of tubular injury. Furthermore, a technology that enables imaging endpoints, instead of time-consuming dissection and detection of renal parenchymal cellular damage, would be highly valuable. Multiple lines of evidence suggest that both apoptotic and necrotic cell death are important pathogenic mechanisms in acute renal failure [12, 13], where renal parenchymal cells are the primary target of a broad spectrum of inciting factors, ranging from ischemic and nephrotoxic agents to endotoxemia. Apoptotic cell death is a feature of both ischemia–reflow injury [14] and radiocontrast-induced acute renal failure [15], and is a documented event in transplant biopsy as well [16]. The role of apoptosis in sepsis syndrome has not been adequately explored, but there is rapidly developing evidence to suggest that increased apoptotic processes may play a determining role in the outcome of sepsis syndrome [17, 18]. Although apoptosis is recognized as playing a role in these conditions, there is a lack of sensitive markers to monitor programmed cell death in association with disease progression or regression. Recently, we developed a family of small molecule compounds, ApoSense, capable of discriminating between vital and apoptotic cells. ApoSense molecules are small non-peptidic fluorescent compounds that are capable of selective targeting, binding, uptake, and accumulation within apoptotic and/or necrotic cell cytoplasm, from the early stages of the death process. Our preliminary results from different animal models associated with apoptosis showed that the recognition of damaged cells by ApoSense is universal, irrespective of the cell type or the apoptotic/necrotic trigger. Indeed, examined in multiple animal models, ApoSense was proven to be a highly sensitive and specific sensor for in vivo detection of neoplastic, hepatic, myocardial, atherosclerotic, and neuronal apoptotic and necrotic cell death (in preparation). In the present study the potential of both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine (DDC), was explored as a diagnostic imaging agent for detection of renal tubular injury in three different in vivo animal models of ATN: renal warm ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature/perforation-induced sepsis. These different models illustrate the applicability of ApoSense as a diagnostic imaging agent enabling real-time accurate detection of renal parenchymal damage of various types, and evaluation of its distribution and magnitude. Materials and methods Male Sprague-Dawley rats weighing 180–250 g and 8- to 10-week-old C57Bl mice (Harlan Laboratories, Jerusalem, Israel) were used for these experiments. All experimental protocols were performed according to the Guiding Principles for Research Involving Animals, and approved by the local Animal Care Committee; the experiments described in this manuscript also comply with the current laws of Israel. ApoSense synthesis and radiolabeling The ApoSense compound used in the present study was N,N′-didansyl-L-cystine (DDC). Both its inherent fluorescent characteristics and its radiolabeled derivative (labeled with 3H radionuclide) were used for identification and quantification of apoptotic cells. For DDC synthesis, dansyl chloride (675 mg) and cystine (240 mg) were dissolved in water/acetone solution with potassium carbonate (550 mg), and after 1.5 h at room temperature, the aqueous mixture was extracted with ethyl acetate. The crude DDC was dissolved in 5% sodium carbonate, washed with ether, and reacidified to pH 3 to afford a yellow precipitate. The product was analyzed using 1H-NMR. Labeling of DDC with 3H was performed by consecutive addition of 0.16 mg cystine (0.65 μmol), 0.57 mg sodium carbonate (5.3 μmol), and 37 MBq (1 mCi) of 3H-dansyl chloride (Vitrax) at a concentration of 1.4×10−4 mmol. The product was purified using a Bond-Elut C-18 cartridge eluted with 25% acetonitrile in 0.1% TFA in water, then lyophilized and dissolved in 1 ml of NaPPi buffer (0.1 M, pH=7.4). Radiochemical purity was assessed by thin-layer chromatography. Evaluation of ApoSense binding to apoptotic cells in vitro Human adult T-cell leukemia Jurkat cells (clone E6-1) and cervical carcinoma HeLa cells (CCL-2.2), were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37°C in 5% CO2. For induction of apoptosis in the Jurkat cell sample, 1×106 cells/ml were treated with IgM anti-Fas (CD95) antibody, clone CH11 (Medical and Biological laboratories, Japan) at a concentration of 0.1 μg/ml for 120–180 min. In additional studies, before incubation with anti-Fas antibody, Jurkat cells were subjected to 50 μM of caspase inhibitor Z-VAD-FMK in 0.05% DMSO (Enzyme System Products, Dublin, CA). For apoptosis induction in HeLa cells, growing cells were treated with staurosporin (STS; 250 nM; Sigma) for 18 h. At the end of the incubation period, cells were harvested by trypsinization (Trypsin-EDTA, Bet-Haemek, Israel) and collected by centrifugation. For assessment of apoptosis, both fluorescence microscopy and flow cytometry were employed. Trypsinized HeLa cells were incubated with DDC (50 μM) and annexin V-FITC (Phosphatidyl Serine Detection Kit, IQP-116F) for 20 min and the staining pattern was evaluated under a fluorescence microscope (BX51TF; Olympus Optical.Co., Ltd., UK) equipped with relevant filters. For DDC staining, excitation at 365 and emission at 420 (band pass) nm was employed, whereas for FITC detection, excitation was at 488 nm and emission at 530 nm. Apoptotic Jurkat cells were detected by flow cytometry after double staining with annexin V-FITC and PI or with DDC and PI. Briefly, 30 μl of cell suspension (1×106 cells/ml) was diluted in 300 μl Hepes buffer (10 mM Hepes and 140 mM NaCl, pH 7.4) containing annexin V-FITC/PI or 50 μM of DDC/PI. Following 40 min of incubation at room temperature, the cells were subjected to analysis using FACS Vantage VE (BD Biosciences, San Jose, CA) and CellQuest software. A total of 10,000 events were collected for each sample. Evaluation of ApoSense uptake into apoptotic/necrotic cells in vivo The detection of renal parenchymal cell death by ApoSense was assessed in three animal models of ATN, generally classified under ischemic and septic etiologies [19–21]. Rat renal ischemia–reperfusion (I/R) model Operative procedures were performed in rats under general anesthesia induced by the combination of ketamine, 80 mg/kg, and xylazine, 10 mg/kg, administered intraperitoneally. Renal ischemia was induced by unilateral left renal artery clamping, using a small nontraumatic vascular clamp, for 45 min [12]. Reperfusion was initiated by removal of the clamp. The period of renal reperfusion was 24 h. Preliminary experiments revealed no difference between the I/R rat contralateral kidney and sham-operated normal rat kidneys with regard to cell death of renal tubular epithelium after 24 h. Thus, in the present study, the contralateral, untreated kidney from the same animal served as the sham-operated control kidney. During the course of reperfusion, animals were injected intravenously with 20 mg/kg of DDC and 4 h later both kidneys were excised, frozen in liquid nitrogen, and stored at −70°C until use. For microscopic analysis and fluorescence imaging, 5-μm-thick cryo-sections of both kidneys were evaluated either by fluorescence microscopy or were stained with hematoxylin and eosin (H&E) for light microscopy analysis of apoptotic and/or necrotic lesion distribution. Furthermore, the presence of apoptotic cell nuclei was confirmed by apoptosis detection kit staining (ApopTag Fluorescein Kit, Intergen Company, Purchase, NY) using the TUNEL (terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling) method. Additional ex vivo whole organ fluorescence imaging was carried out on freshly excised kidneys analyzed under a Leica (Leica MZ FL III, Leica, Switzerland) stereomicroscope. DDC was visualized using a 360Ex and >420Em nm (NU) filter set. Emitted fluorescence was collected on a Leica DC 300F digital camera and images were processed and analyzed by Leica QWin Lite software. For quantitative analysis of the extent of renal DDC accumulation, previously frozen kidneys were thawed, weighed, and homogenized in buffer containing 50 mM Tris-HCl and 0.005% Triton X-100, pH 7.4 (1:7 w/v), using Heidolph RZR 2020 homogenizer (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Solubilized tissue extract samples were centrifuged twice at 13,800 rpm (20 min, 4°C) and the supernatant fractions were analyzed using a microplate reader (GENious Fl Reader, TECAN, Grödig, Austria) equipped with optical filters for fluorescence measurements. All samples were pipetted in triplicate onto black Ritter flat bottom microplates and read at 360 nm Ex and 535 nm Em. For the calibration curve, a dilution series of DDC were prepared in the same Tris-Triton buffer, supplemented with 0.05% kidney extract of control vehicle-injected animals. The same control animal samples were also used for background values. In all samples, DDC concentration (μg/g tissue) and the percentage of the injected dose (% ID/g) were calculated.Additional biodistribution studies were carried out to assess the spread of radiolabeled DDC into different organs. Twenty-four hours after unilateral renal ischemia/reperfusion, rats were intravenously injected with 5–20 μg/kg of the tritiated form of DDC (3H-DDC) [111–370 kBq (3–10 μCi)/animal]. Four hours later, both kidneys, liver, gut, and blood samples were collected, weighed, and submitted to lyses by SOLVABLE reagent (GNE9100, Packard Bioscience), according to the manufacturer’s instructions, and sample radioactivity was determined using a beta counter (TRI-CARB 2100TR, liquid scintillation analyzer, Packard Bioscience). Both tissue uptake (% ID/g) and the ratios of the radioactivity of the ischemic kidney (DPM/g) to that of the contralateral kidney and the other non-target tissues were calculated. Rat model of radiocontrast-induced distal tubular necrosis (DTN) The selectivity of DDC uptake in injured renal tissues was evaluated in this rat model, characterized by selective medullary hypoxic tubular damage. Nephropathy was induced by the combined administration of indomethacin (Sigma Chemical Co.), 10 mg/kg, i.v., N-ω-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical Co.), 10 mg/kg, i.v., and the radiocontrast agent sodium iothalamate 80% (Angio-Conray, Mallinckrodt Inc), 6 ml/kg, i.a., as previously described [22]. The study was performed on six rats, with an additional two rats injected with vehicles serving as control (these two control animals studied are representative of at least 29 control rats tested in our previous experiments). Twenty-four hours after insult, animals were intravenously injected with 20 mg/kg DDC and 2 h later they were sacrificed. The left kidney was removed and snap-frozen for fluorescence analysis (and for H&E and TUNEL staining, as detailed above), whereas the right kidney, used for morphological evaluation, was in vivo fixed with glutaraldehyde perfusion through the abdominal aorta, as described elsewhere [22, 23]. Perfusion-fixed kidneys were immersed in buffered 2% OsO4, dehydrated, and embedded in an Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, containing cortex and medulla, including fornix and papillae. One-micrometer sections were stained with methylene blue and analyzed in a blinded fashion (i.e., without knowing the fluorescence results) for morphological alterations. As previously detailed [23], tubular necrosis was determined separately for S3 proximal tubules in the outer stripe and medullary rays, and for medullary thick ascending limbs (mTALs) in the outer, mid, and inner zones of the inner stripe of the outer medulla. The extent of damage was expressed as the percentage of necrotic tubules out of total tubules counted. Papillary necrosis was semiquantitatively assessed using a score of 0–3. Sepsis model in the mouse The sensitivity of cell death recognition by DDC was tested in the cecal ligature and perforation (CLP) model in mice, where rare, focal tubular cell damage is hardly detected by regular morphological evaluation [24, 25]. The cecum of six anesthetized mice was isolated, ligated distal to the ileocecal valve, and punctured twice with a 26-gauge needle. An additional four sham-treated animals were laparotomized, but the cecum was neither ligated nor punctured. Twenty-four hours later, mice were injected i.v. with DDC, and 2 h afterwards, kidneys were removed and subjected to fluorescence microscopy analysis as detailed above. Statistical analysis Data reported are mean±s.e.m. Student’s t test was used to assess the significance of differences between two groups. Significance was accepted at p<0.05 Results Binding of ApoSense to apoptotic cells in vitro Concomitant confocal imaging of HeLa cells undergoing apoptosis, double stained with DDC and annexin V, revealed that DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external apoptotic cell membrane (Fig. 1). Accumulation of ApoSense within apoptotic cells can contribute to amplification of the signal obtained and consequently to the high signal-to-noise ratio. Previous studies have shown that through an interaction with the death receptor CD95 induces apoptosis by formation of a signaling complex at the cell membrane and subsequent caspase-8 and caspase-3 activation [26]. Therefore, the specificity of DDC uptake was tested in apoptotic Jurkat cells that were co-treated with caspase inhibitor and analyzed by flow cytometry. As shown in Fig. 2, in viable, non-apoptotic cells (green line), no uptake of DDC was detected, except in a small population of spontaneously dying cells representing late events. Following 150 min of incubation with anti-Fas, most cells accumulated DDC and created a new peak of cells (red line) with a higher fluorescence intensity (the geometric mean fluorescence intensity value, GMFI, was 120.18) while the peak of late apoptotic cells remained unchanged. In the presence of the caspase inhibitor Z-VAD-FMK, cells lost the ability to bind DDC (black line), while their viability remained preserved, suggesting that accumulation of DDC compound is dependent on the apoptotic process itself. Upon addition of the caspase inhibitor, a marked shift towards a lower fluorescence intensity was noted, and the final intensity values were almost the same as the control cell values (GMFI 46.38 and 46.02, respectively), suggesting maximal inhibition. Fig. 1Concomitant confocal imaging of HeLa cell undergoing apoptosis by DDC (green fluorescence) and annexin V (red fluorescence). DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external membraneFig. 2Binding of DDC to apoptotic cells is caspase dependent. Jurkat cells were treated with CD95 in the absence (red line) or presence (black line) of the pan caspase inhibitor z-VAD-fmk for 150 min. Following incubation, cells were centrifuged and incubated in the presence of DDC for 40 min before FACS analysis. This histogram charts the number of cells counted (y-axis) and the fluorescence intensity (x-axis). The green line represents the control non-apoptotic sample ApoSense uptake into ischemia/reperfusion (I/R) injured kidneys Evaluation of DDC accumulation into injured tubular cells of an ischemic rat kidney was performed on the basis of its intrinsic fluorescent properties. Either ex vivo whole kidney fluorescent imaging or cryo-section fluorescent analysis was employed. As seen in Fig. 3, DDC accumulated in the injured (a) but not in the contralateral intact kidney (b), thereby exhibiting substantial capability for imaging apoptosis and/or necrosis in vivo. Microscopic analysis of corresponding kidney sections revealed intense cytoplasmatic staining of tubular epithelial cells (Fig. 3c,d) located at the corticomedullary junction. No uptake of DDC into viable renal parenchyma was detected. The extent of DDC accumulation in these cells was quantitatively determined in homogenized whole kidney extracts from five rats, using microplate reader fluorescence measurement. As shown in Fig. 3e, the average DDC uptake into the ischemic kidney (expressed as %ID/g) was 6.73±1.34 fold higher (p=0.0076) than that in the intact control kidney (0.41±0.11 and 0.06±0.02, respectively). H&E-stained histological sections of kidneys from I/R-treated rats revealed multiple apoptotic as well as necrotic cells situated along the tubule epithelium or in the tubule lumen (Fig. 4). A spectrum of changes characteristic of tubular cell apoptosis, including shrinkage of renal tubular cells, cytoplasmic acidophilia, chromatin dense condensation and margination, nuclear fragmentation, and pyknosis, is shown in Fig. 4a, left. In addition, as previously described [27–30], a large percentage of tubules in this region showed substantial tubular dilatation, epithelial necrosis, dissolving of tubular cells, luminal necrotic debris, and cast formation (Fig. 4b, left). The presence of apoptotic/necrotic cells was also defined by intensive green fluorescence of TUNEL-positive nuclei (Fig. 4c, left). As shown in Fig. 4a–c, right panel, the accumulation of DDC within the damaged areas in ischemic kidneys corresponds completely, even at the single cell level, to the injured areas detected by H&E and TUNEL staining. However, it seems that in some areas, accumulation of DDC in apoptotic cell cytoplasm occurred earlier than DNA destruction and thereby cells were not stained by TUNEL but merely by DDC (Fig. 4c, arrow). These results may indicate that DDC has the ability to identify early apoptotic cells and that use of TUNEL alone may not reveal the full spectrum of cell death stages. Fig. 3DDC uptake into renal damaged areas. a,b Ex vivo images of the left ischemic (a) and the right sham-operated (b) kidney derived from a single I/R rat (×7). Whole organs were viewed under a stereomicroscope using a NU filter set without light flux, 4 h after DDC administration. c,d Histological sections of kidneys (c, ×100; d, ×600) revealing intense uptake along the tubule epithelium or in the tubule lumen. e A quantitative estimate of DDC uptake into I/R ischemic and intact (sham) kidney from five rats, normalized to total injection dose for each gram of tissue. Values are means±s.e.m.Fig. 4Morphological evidence for renal tubular cell apoptosis/necrosis following ischemia–reflow. a Tubular cells (H&E staining) are seen with changes typical of apoptosis (left). In an adjacent 5-μm section (right), these cells take up DDC. ×1,000. b Necrotic tubular cells in tubular lumina (H&E staining; left) and corresponding DDC fluorescence of the same injured cells (right). ×400. c Co-localization of DCC fluorescence (right) with TUNEL-positive nuclear staining (left). Some cells are likely in the early stages of apoptosis and are TUNEL negative, but take up DDC (arrow). ×200 Biodistribution studies of 3H-DDC were performed in seven additional rats following the I/R procedure. The accumulation of radioactivity in the blood, gut, liver and kidney at 4 h after intravenous administration is shown in Fig. 5. Uptake in the ischemic kidney was significantly higher than that in the intact contralateral one(p<0.05) and also higher than liver, gut and blood uptake values (p<0.01). The average uptake values at 4 h, rendered as % ID/g, were 1.20±0.13 for the ischemic kidney, 0.58±0.12 for the intact kidney, 0.41±0.07 for the liver , 0.42±0.09 for the gut, and 0.28±0.01 for blood. The corresponding mean ischemic kidney-to-tissue ratios were 2.1±0.5, 2.9±0.5, 2.8±0.7, and 4.3±0.6, respectively. These results indicate that at 4 h following administration, DDC remained accumulated in the target organ, while being cleared from non-target ones. Fig. 5Biodistribution of radiolabeled 3H-DDC after renal I/R treatment. Uptake of 3H-DDC (% injected dose per gram corrected for background) in target and non-target tissues, determined 18–24 h after ischemia and 4 h after 3H-DDC administration, is shown. Data are presented as mean±s.e.m., n=7. Asterisks denote significantly higher accumulation of 3H-DDC in ischemic kidney compared with other organs (tissues): *p<0.05, **p<0.01 Radiocontrast-induced distal tubular necrosis Heyman et al. [23] reported previously that animals subjected to the radiocontrast model of DTN developed varying degrees of renal dysfunction and structural damage, but the presence, distribution, and extent of tubular injury on an individual basis could not be predicted from the magnitude of kidney dysfunction. In the present study, as well as in previously published studies [22, 23, 31], renal morphology analysis disclosed a wide-ranging extent of medullary (namely mTALs and S3 segments in the outer medulla and medullary rays) and papillary damage, with a poor overall correlation between structural damage and functional deterioration (data not shown). By contrast, as shown in Figs. 6 and 7, DDC distribution pattern and degree of deposition closely mirrored the morphological findings in the contralateral perfused kidney, suggesting that ApoSense uptake may reflect the level and degree of renal damage more closely than do functional tests. Results from both fluorescence and morphological analysis disclosed the characteristic striped pattern of injury (Fig. 6a,b), where tubules adjacent to the vasa recta are preserved while those distant from vascular bundles are injured (Fig. 7a). Homing of DDC was primarily restricted to injured regions within the outer medulla (Fig. 6c). No DDC uptake at a specified region in the absence of morphological damage was seen, as demonstrated in Fig. 7b, where necrosis of the papillary tip in one of the kidneys was clearly morphologically demarcated and DDC deposition was restricted to precisely the same injured area. Uptake occurred preferentially in apoptotic/necrotic lesions, as confirmed by co-localization with TUNEL staining (Fig. 6d). Thus, DDC was found to be a specific molecular marker for tubular injury in various segments of the kidney. Fig. 6Renal macroscopic and microscopic findings in rats subjected to the ARF protocol (indomethacin, L-NAME and iothalamate) and subsequently injected with DDC. a Macroscopic view of the characteristic striped pattern of severe hypoxic outer medullary damage in an in vivo fixed kidney. b The in vivo microscopic distribution of DDC in the contralateral kidney correlates closely with the distribution of the striped pattern of outer medullary necrosis in the ipsilateral one (×30). c Details (×100) of the cross-section, showing fluorescent imaging of DDC uptake in injured areas of both the inner strip and the forniceal region of the outer medulla. Examination of contiguous 5-μm sections revealed that the accumulation of DDC in damaged areas (d, upper panel) matched the infarct areas stained by TUNEL (d, lower panel) (×200). OM outer medulla,IM inner medulla, SP secondary pyramidFig. 7Characteristic morphological findings in rats subjected to the ARF protocol and injected with DDC. Cryo-sections from the left kidney were used for DDC fluorescence analysis, whereas the in vivo fixed right kidney was used for light microscopy morphological examination of methylene blue-stained slides. Analyses of both kidneys were performed in a blinded fashion. An example is shown of the striped pattern reflecting the damage gradient, where tubules adjacent to the vasa recta (VR) are preserved (red stars) while those distant from vasa recta are injured (black stars) (a, left panel, ×100). Note the excellent co-localization of morphologically damaged tubules with the DDC fluorescence (a, right panel, ×100) in the contralateral kidney. DDC fluorescence was selectively identified in all morphologically injured regions, including papillary tip structures (b: left panel ×40, right panel ×100) Cecal ligature/perforation-induced sepsis DDC was evaluated as a potential in vivo marker for renal tubular cell apoptosis in another animal model of ATN, in which sepsis-associated renal dysfunction was induced by cecal ligation and puncture. Morphological alterations of the kidney due to sepsis can hardly be identified using routine histological techniques [32, 33], and experimental data regarding the apoptotic cells of kidneys affected by sepsis are therefore almost non-existent. Indeed, in all septic mice we found only rare focal renal tubular cell injury, seen either in the outer medulla or in the cortex (Fig. 8a). As seen in Fig. 8b, focal renal tubular cell injury is apparently identified by DDC fluorescence, but is not clearly traced using H&E histopathological analysis; this demonstrates the high sensitivity of DDC in the detection of such renal damage. TUNEL staining confirmed the presumption that renal cells stained by DDC are indeed dying cells (Fig. 8c). Fig. 8Focal uptake of DDC in kidneys of six mice following sepsis caused by cecal ligature and perforation (CLP). Apoptotic cells, imaged by DDC, were shown either in the outer medulla (a, left) or in the cortex (a, right) of individual mice (×100). Most apoptotic cells appeared to be tubular epithelial cells (b, left, arrows). An evident correlation was demonstrated between DDC fluorescence (b, right and c, right) and apoptotic cells detected on H&E (b, left) or TUNEL (c, left) stained slides (×200) Discussion As a result of recent progress in medicine, biology, and physics, the field of diagnostic imaging is shifting from conventional anatomical imaging to the sphere of functional and molecular imaging, with the aim of imaging biological processes related to both health and disease. The implementation of the new ApoSense technology for clinical imaging of apoptosis would represent a major addition to this emerging field, allowing non-invasive imaging of cell death processes in vivo as a means to both the diagnosis of disease and the monitoring of treatment efficacy in a broad spectrum of disease states. In the present study, we have reported the successful application of ApoSense technology as a diagnostic imaging tool for the detection of apoptotic and/or necrotic cell death. ApoSense technology may be particularly useful in ARF, where current clinical and physiological assessment and complementary imaging tests are insufficient to discriminate between true renal parenchymal damage and prerenal azotemia (in which kidney filtration function is drastically reduced even though the internal structures of the kidney are intact). An inherent fluorescent property of one of the ApoSense family of compounds, DDC, was utilized for detection of tubular cell death in three animal models of ATN. The results obtained are in complete agreement with in vitro findings of DDC-specific binding to apoptotic cells (Figs. 1, 2), and thereby strongly suggest the ability of DDC to image renal tubular cell apoptosis in vivo. The three in vivo experimental models of ATN used in the present study complement each other regarding the applicability of ApoSense technology in the detection of ATN. The results obtained using the I/R model showed the qualitatively remarkable capability of ApoSense in targeting of injured tissues and ex vivo imaging of apoptosis (Fig. 3). Additionally, the model provides quantitative analysis of the extent of renal tubular cell damage based on the extent of DDC accumulation in these cells. It was shown that the uptake of DDC in the ischemic kidney was 6.73-fold higher than in the non-ischemic one. Moreover, the specificity of targeting of the injury by ApoSense was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected by H&E and TUNEL staining (Fig. 4). Furthermore, it was observed that some TUNEL-negative cells with a normal overall nuclear morphology were distinctively stained by ApoSense (Fig. 4c), conceivably representing cells in the initial phases of apoptosis. These results possibly indicate that ApoSense technology can provide a tool which will directly assess for early stages of programmed cell death in the ischemic kidney, before membrane vesicle formation and DNA degradation, particularly as measured by the TUNEL method [34]. The radiocontrast model of distal tubular necrosis provides a good example of specific targeting by ApoSense. Indeed, the presence of DDC was restricted to regions (medullary outer and inner stripe; papillary tip) that corresponded closely to the injured zones in the contralateral, perfusion-fixed kidney (Figs. 6, 7). Methylene blue-stained thin sections from those in vivo fixed kidneys enabled precise evaluation of the renal morphology and accurate identification of tubular cell types at various stages of evolving structural damage. The complete correlation between DDC fluorescence and histological evidence of damaged areas, mainly made up of apoptotic and necrotic cells, is indicative of the specificity of DDC uptake. Thus, while the degree of renal dysfunction served as an inaccurate indicator of tubular injury [4, 5], ApoSense uptake closely predicted the existence of ATN, with an absolute specificity for involved tubular segments. Our findings derived from the mouse sepsis model further illustrated the sensitivity of ApoSense in the detection of rare focal tubular cell injury that is not well defined by routine H&E staining (Fig. 8). It has to be reiterated that data regarding the morphology of sepsis-related ATN are nearly non-existent, mainly because such rare, focal tubular cell damage can hardly be detected by regular morphological evaluation [35, 36]. The inherent fluorescent properties of the ApoSense molecule DDC have been used in the present study to evaluate its specific binding to apoptotic cells. On the other hand, the structure of ApoSense molecules is modular, allowing for versatile attachment of various clinical imaging markers and, therefore, enabling their use as non-invasive imaging agents. In the current study, a 3H-labeled DDC molecule was used for biodistribution and quantitative uptake studies. Nevertheless, autoradiography with radiolabeled DDC was not performed in this series of experiments and this may be the main limitation of the present study. Moreover, additional studies based on preparation of ApoSense as a PET imaging agent (following its conjugation with 18F) are necessary before the introduction of the technology in the form of diagnostic imaging tests. These studies are currently in progress. Overall, the results presented here are promising and illustrate the high sensitivity and specificity of ApoSense in the assessment of tubular cell damage in multiple experimental templates that may have clinically relevant applications. For instance, ApoSense technology may be implemented for the in vivo assessment of the degree to which renal parenchymal cell apoptosis is contributing to renal failure in sepsis and for the differentiation of evolving ATN from renal hemodynamic alterations; as a result it can enable therapy to be tailored to each patient’s needs. The ability of radiolabeled derivatives of ApoSense to visualize both apoptosis and necrosis, non-invasively and in real-time, may prove useful in reducing the need for routine biopsy. Furthermore, in clinical practice, ApoSense imaging may provide diagnostic information superior to that obtained by means of biopsy, especially as regards the dynamic nature and focal distribution of the disorder; furthermore, it does not suffer from the risks and limitations associated with tissue sampling. Imaging sciences have grown tremendously during recent decades, and many non-invasive techniques for the detection and monitoring of renal hemodynamics, such as advanced ultrasonography techniques, magnetic resonance imaging, and nuclear and X-ray computed tomography [37–39], have become essential clinical tools. Similarly, non-invasive technologies and imaging probes that would enable the detection of evolving renal parenchymal apoptotic and necrotic injury are critically needed. During the past few years, the best-characterized apoptosis marker, annexin V, a calcium-dependent phospholipid binding protein [40, 41], has been extensively investigated in multiple animal models as well as in clinical trials [42]. However, annexin V has a strong affinity for intact renal cortex, which has been attributed to the intrinsic lipid profile of the kidney, where a high concentration of phosphatidylserine exists [43, 44]. The high accumulation of annexin V in the kidneys precludes its use for the study of apoptosis in these organs or in their immediate vicinity [45, 46]. In addition, the biological rationale for engineering DDC, a small non-peptide molecule with a molecular weight of 700 Da (as compared with the 36-kDa annexin V protein), is that such small molecules could benefit from less immunogenicity and the favorable biodistribution kinetics that small compounds in general exhibit over bigger proteins. In summary, this study demonstrates that ApoSense, once successfully radiolabeled with an imaging moiety, may provide a sensitive means for real-time diagnosis and/or monitoring of the extent of renal damage in ATN, utilizing selective targeting, binding, uptake, and accumulation within renal tubular apoptotic and/or necrotic cells. Moreover, as a non-invasive method, ApoSense imaging would allow longitudinal studies in a single individual, rendering important information on the optimal timing and dosing of drugs and on the efficacy of therapeutic interventions.

Psychopharmacologia-3-1-2048539

Acute and constitutive increases in central serotonin levels reduce social play behaviour in peri-adolescent rats

Rationale Serotonin is an important modulator of social behaviour. Individual differences in serotonergic signalling are considered to be a marker of personality that is stable throughout lifetime. While a large body of evidence indicates that central serotonin levels are inversely related to aggression and sexual behaviour in adult rats, the relationship between serotonin and social behaviour during peri-adolescence has hardly been explored. There is a large body of literature that describes the association between serotonin (5-hydroxytryptamine, 5-HT) signalling, social status, aggression and sexual behaviour in humans and animals. For instance, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA; the major metabolite of 5-HT) levels are inversely related to aggression in humans, primates and rodents (Higley and Linnoila 1997; Fairbanks et al. 2001). Selective serotonin reuptake inhibitors (SSRIs), which are well-known anti-depressants, reduce impulsive aggression as well as sexual behaviour in humans (New et al. 2004; Olivier et al. 2006). Furthermore, depending on the social structure of the community, serotonergic drugs are able to interchange the dominant and subordinate status of community members (Edwards and Kravitz 1997; Larson and Summers 2001). It has been proposed that individual differences in 5-HT neurotransmission are an important neural underpinning of personality (Serretti et al. 2006), because central 5-HT levels are relatively stable throughout lifetime (Higley and Linnoila 1997). Studies showing that polymorphisms in 5-HT-related genes are linked to impulsive aggression in humans (Ferrari et al. 2005; Haberstick et al. 2006; Popova 2006) support the idea that the serotonergic modulation of social behaviour is heritable (Higley and Linnoila 1997). An important form of social behaviour is social play behaviour, which is the earliest form of non-mother-directed social behaviour in young mammals. Social play behaviour consists of behaviours found in adult sexual, affiliative and aggressive encounters (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981). However, during social play, these behaviours are displayed in an exaggerated and/or out-of-context fashion (Poole and Fish 1975). In rats, a bout of social play behaviour starts with one rat soliciting another animal, by attempting to nose or rub the nape of its neck. If the animal that is solicited upon rotates to its dorsal surface, ‘pinning’ is the result, i.e. one animal lying with its dorsal surface on the floor with the other animal standing over it. From this position, the supine animal can initiate another bout of play, by trying to gain access to the other animal’s neck. Thus, during social play, pinning, which is considered to be the most obvious posture in social play behaviour in rats, is not an endpoint but rather functions as a releaser of a prolonged play bout (Poole and Fish 1975; Pellis and Pellis 1987; Pellis 1988). The animal that is pounced upon can also respond by evading or by turning around to face the other animal. In the latter situation, a brief period of boxing/wrestling may follow, in which the animals try to push each other away. If the solicited animal evades, the other animal may start to chase it, thus making another attempt to launch a play bout (see Vanderschuren et al. 1995a, for a detailed analysis of the temporal structure of social play behaviour in rats). It is thought that social play sub-serves the facilitation of social and cognitive development because play deprivation, i.e. social isolation during 2 weeks in peri-adolescence when social play is most abundant (i.e. from postnatal day 21 until postnatal day 35), leads to behavioural disturbances, most prominently in the social domain (e.g. Hol et al. 1999; Van den Berg et al. 1999). These behavioural changes are more selective than those of isolation rearing, which entails continuous isolation from weaning throughout further lifetime. This not only affects social behaviour (Ferdman et al. 2007) but also induces behavioural disturbances related to exploratory behaviour (Heidbreder et al. 2000), cognition (Heidbreder et al. 2000; Dalley et al. 2002; Bianchi et al. 2006) and reward sensitivity (Howes et al. 2000), suggesting that social play plays a rather selective role in the development of social skills. There is evidence indicating that 5-HT modulates social play in non-human primates. For instance, low CSF 5-HIAA levels are associated with increased solitary play (Maestripieri et al. 2006; Higley and Linnoila 1997), although these findings are difficult to interpret in terms of central 5-HT levels. The neurobiology of social play has been widely investigated in rats (Vanderschuren et al. 1997; Siviy 1998), but surprisingly, little attention has been paid to the role of the serotonergic system in rat social play itself. While treatment with para-chloro-phenylalanine or a low tryptophan diet, resulting in a decrease in central 5-HT levels, did not affect social play, fenfluramine and the SSRI fluoxetine, agents that increase extra-neuronal 5-HT levels, inhibited play (Panksepp et al. 1987; Knutson et al. 1996). Further, the 5-HT receptor agonist quipazine reduced social play (Normansell and Panksepp 1985). These data suggest that there is an inverse relationship between 5-HT signalling and social play. Furthermore, the findings that 5-HT modulates social behaviour in peri-adolescent animals indicates that 5-HT affects social behaviour throughout development, consistent with the proposed trait-like relationship between 5-HT and social behaviour. The serotonin transporter (SERT) critically regulates extracellular 5-HT levels by its re-uptake into pre-synaptic terminals (Lesch et al. 1996), and SERT knockout models would therefore be very useful to test the hypothesis that central 5-HT levels and social play behaviour are inversely related in a trait-like manner. SERT knockout mice are available (e.g. Bengel et al. 1998), which display reduced aggression (Holmes et al. 2002a) and social interaction (Kalueff et al. 2007). However, because social play behaviour in mice is much less developed than in rats (Poole and Fish 1975; Pellis and Pasztor 1999), mice are not the preferred rodent species to study age-specific patterns of social behaviour during development. As social play behaviour in rats has been well described (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981; Pellis and Pellis 1987; Pellis 1988; Vanderschuren et al. 1997), we measured social play in the recently generated SERT knockout rat (Smits et al. 2006), which displays a ninefold increase in extra-neuronal 5-HT levels (Homberg et al. 2007). In line with the findings that increases in 5-HT levels reduce aggressive and sexual behaviour, we have observed that both aggressive and sexual behaviour (Homberg et al., unpublished observations) are reduced in the SERT knockout rat. The constitutive absence of the SERT in mice causes changes in the functioning of 5-HT receptors (Fabre et al. 2000; Bouali et al. 2003), so that changes in behaviour may be the result of adaptations that have occurred during development. Therefore, we also investigated social play behaviour in wild-type rats after acute treatment with compounds that increase central 5-HT levels, i.e. the SERT-blocker fluoxetine and 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”), which causes 5-HT release by reversing 5-HT transport. Materials and methods Subjects All experiments were conducted with the approval of the animal ethics committees of the VU Medical Center, Amsterdam, The Netherlands, and the University Medical Center Utrecht, The Netherlands. Both commercially supplied and SERT knockout rats were used in this study. Male Wistar rats (Harlan, Horst, the Netherlands) arrived at the age of 3 weeks in the animal facility. SERT knockout rats, which have been generated by ethylnitrosourea-induced mutagenesis in a Wistar background (for detailed description, see Smits et al. 2006), were bred by crosses between outcrossed (six generations) SERT+/− rats. At the age of 2 weeks, ear cuts were taken under anaesthesia and used for genotyping. At the age of 3 weeks, male SERT−/− and SERT+/+ rats (littermates served as controls) were transported from the breeding room to the experimental room. The animals were socially housed (four rats per type IV macrolon cage) under a normal day/light cycle (lights off at 7 p.m.) at controlled room temperature (21 ± 2°C) and relative humidity of 60 ± 15%. Food and water were available ad libitum. Three days after arrival, the rats were weighed and handled daily until testing. Behavioural procedure Testing was performed as described previously (Vanderschuren et al. 1995b). Rats, aged 28–35 days, were tested in an acrylic plastic cage (40 × 40 × 60 cm [l × w × h]) with approximately 2 cm of wood shavings covering the floor. The test cage was illuminated by a 25-W red light bulb mounted 60 cm above the test cage. Background noise, produced by a radio, was used to minimize the influence of extraneous sounds. Two days preceding the test, the animals were habituated to the test cage during 10 min. The animals of a test pair did not differ more than 10 g in body weight and had no previous common social experience. On the test day, test pairs were isolated for 3.5 h before the test to induce a half-maximal increase in the amount of social play behaviour (Niesink and Van Ree 1989). Thirty minutes before the test, pairs of animals in group 1 were treated with 0 or 1 mg/kg fluoxetine (subcutaneous [s.c.]), animals in group 2 were pre-treated with 0 or 10 mg/kg fluoxetine and group 3 received 0, 0.5, 2 or 5 mg/kg MDMA (s.c.). Pairs of similarly treated rats were tested for 15 min in the test cage and the test order was randomized across treatments. Behaviour of the animals was recorded on video tape, and analysis from the video tape recordings was performed afterwards. Coding of the drug solutions ensured that both during experimentation and analysis, the experimenter was unaware of the treatment of the animals. Using Observer 4.0 (Noldus Information Technology B.V., Wageningen, The Netherlands), frequencies and durations of the following behaviours was scored—pinning: one of the animals lying with its dorsal surface on the floor of the test cage with the other animal standing over it; pouncing: play soliciting by nosing the partner’s nape; boxing/wrestling: facing each other in vertical position and struggling using the forepaws; following/chasing: moving in the direction of or pursuing the test partner, who moves away; social grooming and social exploration: sniffing or licking any body part of the test partner. Behaviour was assessed per pair of animals. Animals were used only once. Drugs Fluoxetine was purchased from Sigma-Aldrich (Schnelldorf, Germany), and MDMA was purchased from O.P.G. (Utrecht, The Netherlands). Both drugs were freshly dissolved in saline on the day of the experiment and injected s.c. in the flank in a volume of 2 ml/kg. Statistical analyses Data were analysed using one-way analysis of variance (ANOVA), followed by Student–Newman–Keuls post-hoc tests where appropriate. The level of probability for statistically significant effects was set at p < 0.05 (n.s. = non-significant). Results SERT−/− pairs of rats, as compared to SERT+/+ pairs, showed a strong reduction in pinning frequency (Fig. 1a; F(2, 13) = 5.78, p < 0.05), pouncing frequency (F(2, 13) = 7.08, p < 0.05) and boxing/wresting frequency (F(2, 11) = 9.91, p < 0.01). The duration of following/chasing was significantly increased in SERT−/− rats (Fig. 1b; F(2, 13) = 6.5, p < 0.05), while social grooming and social exploration were not affected by deletion of the SERT gene (social exploration: F(2, 12) = 0.22, n.s.; social grooming: F(2, 12) = 0.76, n.s.). Fig. 1Social play behaviour in peri-adolescent male SERT−/− and SERT+/+ rats. The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a) and mean ± SEM of the duration of social exploration and following/chasing (b) during a 15 min test. Asterisk, p < 0.05 SERT−/− vs SERT+/+ As illustrated in Fig. 2a, 1 mg/kg fluoxetine did not affect pinning (F(2, 13) = 1.33, n.s.), pouncing (F(2, 13) = 2.56, n.s.) and boxing/wrestling (F(2, 13) = 1.75, n.s.). One-way ANOVA did not also reveal effects of 1 mg/kg fluoxetine on social exploration (F(2, 13) = 0.55, n.s.), following/chasing (F(2, 13) = 0.69, n.s.) or social grooming (F(2, 13) = 0.00, n.s.; Fig. 2b). A higher dose of fluoxetine, 10 mg/kg, on the other hand, significantly reduced pinning (F(2, 14) = 15.86, p < 0.005), pouncing (F(2, 14) = 36.18, p < 0.0001), boxing/wrestling (F(2, 14) = 23.82, p < 0.0005; Fig. 2c) and following/chasing (F(2, 14) = 20.17, p < 0.001) but not social exploration (F(2, 14) = 3.03, n.s.) and social grooming (F(2, 14) = 0.03, n.s.; Fig. 2d). Fig. 2Effect of fluoxetine on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 1 (a, b) or 10 mg/kg (c, d) fluoxetine (s.c.). The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a, c) and mean ± SEM of the duration of social exploration, following/chasing and social grooming (b, d). Asterisk, p < 0.05 vs saline MDMA was tested in three different doses: 0.5, 2.0 and 5.0 mg/kg. One-way ANOVA indicated that MDMA treatment dose-dependently suppressed pinning (Fig. 3a; F(2, 30) = 28.8, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05), pouncing (F(2, 29) = 31.77, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05) and boxing (F(2, 29) = 22.07, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05). In addition, MDMA decreased following/chasing (Fig. 3b; F(2, 29) = 8.81, p < 0.0005, post-hoc testing: all MDMA groups vs saline p < 0.05). Social exploration (F(2, 30) = 6.41, p < 0.005, post-hoc testing: 2 and 5 mg/kg MDMA vs saline p < 0.05) and social grooming (F(2, 28) = 24.89, p < 0.0001, post-hoc testing: 2 and 5 mg/kg MDMA groups vs saline p < 0.05; Fig. 3b) were also attenuated by MDMA treatment. Fig. 3Effect of MDMA on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 0, 0.5, 2 or 5 mg/kg MDMA (s.c.). The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a) and mean ± SEM of the duration of social exploration, following/chasing and social grooming (b). Asterisk, p < 0.05 vs saline; double cross, p < 0.05 vs 0.5 mg/kg MDMA Discussion In this study, we show that increasing extra-neuronal 5-HT levels, either constitutively by deletion of the SERT gene or transiently by pharmacological manipulation (i.e. treatment with fluoxetine or MDMA), decreases social play behaviour in peri-adolescent rats. These effects were specific for playful social behaviours, i.e. pinning, pouncing and boxing/wrestling. Mixed effects were observed for following/chasing, social grooming and social exploration. The similarities between the genetic and pharmacological manipulations of the serotonergic system suggest that reduced social play behaviour in SERT−/− rats is the result of increased extracellular 5-HT levels (Homberg et al. 2007). Fluoxetine and MDMA are well known to increase central 5-HT levels, but they affect dopaminergic neurotransmission as well, raising the possibility that reduced social play is due to an effect on the dopaminergic system. Fluoxetine, at 10 mg/kg as used in the present study, increases dopamine release in the frontal cortex (Tanda et al. 1994). MDMA is a potent releaser of 5-HT, but it releases dopamine as well, albeit at higher concentrations (for reviews, see White et al. 1996; Green et al. 2003). Although psychostimulant drugs that increase central dopaminergic neurotransmission have been found to suppress social play behaviour (e.g. Beatty et al. 1982; for review, see Vanderschuren et al. 1997), these effects are most likely mediated through non-dopaminergic mechanisms because the selective dopamine reuptake inhibitor GBR-12909 and the dopamine receptor agonist apomorphine did not suppress play (Vanderschuren et al. 2005). Moreover, the pre-synaptic functioning of dopaminergic, as well as noradrenergic neurons, was not altered in SERT−/− rats (Homberg et al. 2007). Therefore, the present findings are most likely the result of altered function of the serotonergic system, rather than the dopaminergic system. Indirect 5-HT agonists such as MDMA and parachloroamphetamine enhance locomotor activity (Geyer 1996), but the doses required to enhance locomotor activity in peri-adolescent rats are higher than those that suppress play in the present study (see Åberg et al. 2007). Fluoxetine does not alter locomotor activity (Homberg et al. 2004), and locomotor activity does not differ between SERT+/+ and SERT−/− rats under both a novel and habituated conditions (Homberg et al., unpublished observations). In the present study, following/chasing, the social parameter most closely related to locomotor activity, was increased in SERT−/− rats and decreased by fluoxetine and MDMA. Thus, there is no parallel in the effects of fluoxetine, MDMA and SERT knockout on locomotor activity and following/chasing. This indicates that the reduction in social play found in SERT−/− rats and after treatment with fluoxetine and MDMA is not likely to be secondary to effects on locomotor activity. An alternative explanation of the present findings is that the decrease in social play behaviour is due to increased anxiety. Although the animals were habituated to the test cage and tested under red light conditions, reluctance to contact an unfamiliar conspecific may have interfered with our findings. SERT−/− mice display anxiety-related symptoms and reduced social interaction (Holmes et al. 2002b; Kalueff et al. 2007), MDMA and fluoxetine have anxiogenic effects in a variety of tests (Lin et al. 1999; Morley and McGregor 2000; Drapier et al. 2007) and the SSRI citalopram reduces social interaction (Dekeyne et al. 2000). MDMA has also been reported to decrease social investigation and aggression and to increase non-social exploration, which was interpreted as attempts to escape from the test arena, i.e. an anxiogenic-like effect of MDMA (Maldonado and Navarro 2001). On the other hand, pro-social effects of MDMA have also been reported (Morley and McGregor 2000). SERT−/− rats spent more time on following and chasing than SERT+/+ rats. This suggests that while social play was reduced, SERT−/− rats were still interested in their playing partner, which argues against social anxiety as an explanation of reduced play behaviour in SERT−/− rats. Moreover, social exploratory behaviour, which is the parameter used in the social interaction test of anxiety (File and Seth 2003), was not affected in SERT−/− and in fluoxetine-treated rats, whereas MDMA only mildly reduced social exploration at doses that completely eliminated social play. Thus, the observed effects were relatively selective for social play, and increases in extra-neuronal 5-HT levels do not induce a generalized aversion to contact an unfamiliar rat. Long-term SSRI treatment is associated with increased extra-neuronal 5-HT levels and several compensatory adaptations (Dawson et al. 2002) that are strikingly similar to those seen in SERT−/− rats, like desensitization of pre- and post-synaptic 5-HT1A receptors (Homberg et al., unpublished observations). Chronic SSRI treatment decreases aggressive (Fuller 1997) and sexual behaviour (Olivier et al. 2006) in rats, phenotypes we also observed in SERT−/− rats (Homberg et al., unpublished observations). Because acute fluoxetine and MDMA treatment cause an immediate increase in extra-neuronal 5-HT levels without compensatory adaptations, it is likely that reduced social play in SERT−/− rats is due to the increased extra-neuronal 5-HT levels, rather than compensatory adaptations. Ansorge et al. (2004) showed that early chronic SSRI treatment in wild-type mice induces anxiety- and depression-like phenotypes that resembles those of SERT−/− mice. It may follow that SERT−/− rats behave as being chronically treated with SSRIs, but given that acute and chronic SSRI treatment induce opposite effects (Silva and Brandao 2000) and that the effects of anti-depressants on the immature developing brain may differ as opposed to the effects on the mature brain of adults (Ansorge et al. 2004; Taravosh-Lahn et al. 2006), further research is required to support this suggestion. Anti-depressants are frequently prescribed to children with major depressive disorder and anxiety (Cheung et al. 2006), while there is only sparse knowledge of the effects of SSRIs on the developing brain (Spear and Brake 1983; Ansorge et al. 2004). Because social behaviour is reduced in peri-adolescent and adult SERT−/− rats, it is likely that changes in 5-HT homeostasis found at adulthood in SERT−/− rats (Homberg et al. 2007) reflect the situation in the peri-adolescent SERT−/− brain. We thus propose that insight into the function of the serotonergic system in the SERT−/− (peri-adolescent) brain may be helpful to gain insight into the effects of SSRIs on the developing brain. 5-HT is considered to be an important marker of personality traits such as harm avoidance (Gerra et al. 2000), social dominance (Edwards and Kravitz 1997; Larson and Summers 2001), social attachment (Beech and Mitchell 2005) and social impulsivity (Fairbanks et al. 2001), which are relatively stable within individuals. These traits may be the result of changes in SERT function because a polymorphism in the SERT gene of humans and non-human primates, which decreases SERT expression and function (Lesch et al. 1996), has quite consistently been associated with childhood (Beitchman et al. 2006; Haberstick et al. 2006) and adult aggression (Sakai et al. 2006) and childhood (Nobile et al. 2004) and adult depression (Caspi et al. 2003). Furthermore, this polymorphism has been associated with reduced free play behaviour in monkeys (Bethea et al. 2004). Anti-social behaviour in children has been suggested to be related to developmental abnormalities in the serotonergic system, and altered social behaviour during childhood likely extends into adulthood (Van Goozen and Fairchild 2006). Thus, the observations that SERT−/− rats display reduced social play behaviour, reduced aggression and reduced sexual behaviour is likely to be the result of constitutive disruptions in 5-HT function. On the other hand, because social play sub-serves the development of an adequate social repertoire (Vanderschuren et al. 1997; Hol et al. 1999; Van den Berg et al. 1999), the reduction in social play in SERT−/− rats could also itself cause changes in adult social behaviour, independent of 5-HT function. Whichever possibility holds true, we think that because of their lifelong altered 5-HT homeostasis, the SERT knockout rat model may contribute to our understanding of the role of 5-HT during the development of social behaviour.

J_Mol_Med-3-1-2121656

Harnessing hypoxic adaptation to prevent, treat, and repair stroke

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics. Stroke is defined as injury to the brain accruing from a vascular etiology. Strikingly, it has emerged as the third leading cause of death and the leading cause of disability in the USA. Accordingly, the estimated financial costs of stroke are more than 50 billion dollars a year in the USA alone. These financial costs do not begin to tell the story of the personal suffering that amasses from the silent epidemic of stroke disability—over 5 million Americans face the challenges of handicaps from stroke each day. The recognition of stroke as a leading age-associated public health issue has led the government and the pharmaceutical industry to expend enormous resources on developing interventions in the form of drugs that minimize brain damage associated with stroke. Despite their promise, these efforts have been disappointing and have left a nearly indelible sense of frustration on the biomedical research community [1]. In this chapter, we will discuss the pathophysiology of stroke with particular attention to one of its primary mediators, hypoxia, and attempt to revive a sense of optimism and enthusiasm for stroke therapeutics moving forward. Ischemia is a process in which perfusion to tissue is critically reduced creating a deficit in necessary brain fuels. The brain is highly vulnerable to ischemia because the eloquent functions it is assigned to carry out, in the pluralistic society of organ functions, depend integrally on energy—specifically adenosine triphosphate (ATP). The brain comprises only 2% of body weight, but it utilizes nearly 20% of cardiac output to achieve its supply of essential nutrients including oxygen and glucose. Abnormal central nervous system (CNS) symptoms begin to manifest at 40–50% of normal resting hemispheric cerebral blood flow resulting in slowing of the EEG, attenuation of evoked potentials, and reduction in the membrane potential in individual cortical neurons [2]. The resting membrane potential must be maintained to allow proper neuronal function, including synaptic activity and axonal conduction. Once the neuronal membrane potential begins to dissipate, neuronal function ceases. It is important to note that ATP levels at this point may be normal or only slightly reduced [3]. The reason for this inordinate sensitivity is not entirely clear but is likely related to the Km of neurotransmitter systems for ATP or their exquisite sensitivity to tissue acidosis [4]. Gross deterioration and damage requires even more severe reductions in blood flow to 20–30% of normal resting hemispheric cerebral blood flow (Fig. 1). With this level of ischemia, a deterioration of ionic membrane gradients ensues, and the tissue begins to accumulate hydrogen ions (acidosis) [5]. Changes in ionic fluxes likely accrue directly from a reduction of intracellular ATP, a failure of the Na+/K+ATPase activity, and increases in intracellular Na+ and extracellular K+. The failure of ionic homeostasis reflects loss of activity of multiple transporters that normally maintain the electrochemical gradients necessary for normal neuronal signal transduction [6, 7]. Among the ions deregulated, intracellular calcium appears to play a critical role in further ion dyshomeostasis via the calmodulin-dependent activation of neuronal nitric oxide synthase (nNOS) [8–10]. Increased nNOS activity leads to increase in the ambient levels of neuronal nitric oxide (NO) [11]. Changes in NO coupled with ischemia-associated increases in cytosolic and mitochondrially derived superoxide, combine to form toxic peroxynitrite. Peroxynitrite can trigger cell death pathways via DNA damage, poly(ADP-ribose)polymerase (PARP) activation, liberation of apoptosis-inducing factor (AIF) from the mitochondria, and activation of death signaling pathways leading to apoptosis [12, 13]; alternatively, non-selective cation channels such as TRPM2 and TRPM7 can be activated to ensure sustained calcium overload and death [14]. Extracellular acidosis is also postulated to activate acid sensing ion channels, which also contribute to destruction of the normal ionic environment [15]. As one can appreciate from a partial description of the sequence of events after stroke that occur in the neuron alone, targeting a single molecule in the complex parallel and serial pathways of acute hypoxia-ischemia will not maintain neuronal survival (Fig. 2). It is also unlikely to enhance the ability of energy-thirsty neurons to carry out their sophisticated roles in maintaining posture, movement, sequencing language, or making critical executive decisions. Therefore, how might we move forward? One of the most important advances in the treatment of complex medical problems has been the discovery that multimodal therapies can greatly enhance therapeutic efficiency. Treatment of cancer, HIV infection, and tuberculosis with multimodal therapies yields results that are not obtained with the application of single therapeutic agents. Fig. 1Neuronal hypoxia leads to calcium overload and production of free radicals. Stroke is associated with a decrease in cerebral blood flow to the brain. The consequent loss of metabolic fuels leads to failure of sodium pumps leading to an intracellular accumulation of sodium and calcium, depolarization, and activation of voltage sensitive and ligand gated [N-methyl-d-aspartate (NMDA)] ion channels. Increased in calcium in microdomains near the NMDA receptor leads to activation of neuronal nitric oxide synthase. Global dysregulation of calcium in the neuron leads to mitochondrial overload and superoxide production. Nitric oxide and superoxide combine to form peroxynitrite. Peroxynitrite can damage DNA leading to PARP activation and consumption of NAD+. It can also activate TRPM2/7 channels leading to further calcium dysregulation. Hypoxia is sensed by decreased activity of HIF prolyl 4 hydroxylases that can lead to activation genetic responses capable of compensating for the sentinel metabolic stress (decreased cerebral blood flow). Decrease HIF PHD activity can also prevent death via HIF-independent pathways. Acidosis and ROS can also combine with HIF regulated prodeath proteins to trigger cell deathFig. 2Contribution of neurons and astrocytes in mediating excitotoxic neuronal death. 1 Loss of ATP in ischemia leads to inhibition of the Na+/K+ATPase and subsequent collapse of normal ionic gradients. 2 In turn, neuronal membrane depolarization activates voltage sensitive Ca2+ channels, which increase intracellular Ca2+ and stimulate vesicular glutamate release. Severe loss of ionic gradients found in certain ischemic regions may also lead to the reversal of 3 neuronal specific and 4 astrocyte specific glutamate transporters, which in the reverse mode act to release glutamate into the extracellular space. 5 Cell swelling in cerebral ischemia, which is mainly localized to astrocytes, likely activates swelling sensitive anion channels, referred to as volume regulated anion channels (VRACs). VRACs, which are permeable to organic osmolytes, contribute to glutamate release predominantly in the ischemic penumbra. 6 Glutamate regulated NMDA receptors (NMDA-R) are activated by (1) extracellular glutamate and (2) release of Mg2+ from its pore after membrane depolarization (in part due to activation of glutamate regulated AMPA receptors, not shown). 7 NMDA-Rs are permeable to Ca2+ and as such, overabundant NMDA-R activation leads to an intracellular Ca2+ overload. This increase in intracellular Ca2+ then contributes to neuronal death via several mechanisms As stroke does not represent a single homogeneous category of injury, it is also a poor candidate for a single approach to treatment [16]. Challenges involved in promoting recovery from stroke involve reducing the extent of damage that occurs in acute injury. Moreover, many different kinds of damage are found in individuals with stroke ranging from necrotic, apoptotic, or parthanatotic death of neurons [17, 18], demyelination of otherwise functional axons [19], and transection of axons and subsequent loss of critical neuronal populations [20]. Acute injury itself is extremely complex, including waves of cell death, inflammatory responses, edema, and scarring. Indeed, experimental studies have continued to demonstrate that interventions that target single aspects of the complex cascade, including blocking ion gradients, scavenging free radicals, or enhancing growth factors on their own are insufficient to overcome the considerable barriers to protection against hypoxia and ischemia in acute stroke [21]. Instructive data from the experimental paradigm of ischemic preconditioning has pointed the way towards novel strategies that can address the heterogeneity and complexity inherent in stroke pathophysiology [22]. Animals subjected to a non-lethal exposure to hypoxia are found to be more resistant to a host of subsequent lethal stresses, including cerebral ischemia. The mechanism by which a sublethal exposure to hypoxia can render the brain resistant to cerebral ischemia and a host of other insults is a topic of active investigation and debate. However, one point appears irrefutable: The tolerance that develops after a short duration of hypoxia involves not only the activation or inactivation of pre-existing proteins but also de novo gene expression [23, 24]. These transcriptional and post-transcriptional mechanisms reflect a cassette of genes and proteins that work collectively at the cellular, local, and systemic levels to compensate for a discrepancy in oxygen supply and demand. The findings suggest that under conditions where adaptive homeostatic mechanisms are appropriately engaged, damage to the brain can be substantially lessened or even prevented (Fig. 3). Stroke is, almost by definition, a failure of homeostasis. Accordingly, identification of small molecules that augment endogenous adaptive strategies provides a mechanism to tilt the balance away from cell damage and death and toward cell survival and repair (Fig. 3). Fig. 3Drugs that augment endogenous homeostatic mechanisms will more effectively neutralize the heterogeneity inherent in stroke pathophysiology. As these pathways are already used by the body, their activation can occur with decreased threat of toxicity. The term “homeostasis” was coined by Walter Canon in the early twentieth century. It refers to the innate tendency of organisms to mobilize adaptive responses physiological and pathological perturbations that ultimately return the system to a set point that is consistent with survival. a The experimental paradigm of “ischemic preconditioning” has shown that a short, sublethal exposure to hypoxia, or hypoxia-ischemia induces homeostatic responses that make the organism “immune” or “tolerant” to a lethal ischemic insult. Mechanistic studies have revealed that tolerance is the consequence of activation of pre-existing proteins and de novo gene expression. b According to this model, stroke can be conceptualized as a failure of homeostasis. Consequently, neurons die and the brain is permanently damaged. c By extension, small molecules that engage homeostatic mechanisms designed to alleviate hypoxia/ischemia early or enhance their activation should tip the balance away from cell death and toward survival and repair. Such small molecules are currently being developed and represent a new generation of stroke therapies Examination of adaptive responses to hypoxia in the central nervous system has affirmed that the family of transcriptional regulators known as the hypoxia-inducible factors are central players [25–27]. HIF-1 was purified and cloned as a result of a search for proteins that regulate the expression of genes involved in hypoxic adaptation, such as erythropoietin, vascular endothelial growth factor, and glycolytic enzymes [27]. HIF is a heterodimeric transcriptional activator composed of an inducible HIF-1α subunit and a constitutively expressed HIF-1β subunit [28, 29]. HIF-1α stability is regulated via the activity of a class of oxygen, 2-oxoglutarate, and iron dependent enzymes known as the HIF prolyl-4 hydroxylases (HIF PHDs, Fig. 4) [30]. As intracellular oxygen levels drop below a critical threshold, these enzymes fail to hydroxylate HIF-1α. As hydroxylation is required for the recruitment of the constitutively active E3 Ubiquitin Ligase, Von Hippel Lindau protein, HIF-1α becomes stabilized. HIF-1α can partner with its constitutively expressed but induced partner HIF-1β and translocate to the nucleus to regulate the expression of a host of genes involved in hypoxic adaptation [31]. Consistent with this model, several groups have shown that HIF-1α immunoreactivity increases in areas of the cortex that become hypoxic due to stroke [25, 32] An unanswered question has been whether HIF is also upregulated in areas connected to but remote from the site of ischemia. Indeed, neurons projecting to an area of infarction are at risk for cell death due to a loss of trophic support from their damaged targets. Moreover, neurons projecting from an area of damage are at risk for cell death due to a loss of trophic excitatory input from their targets. An elegant recent study in non-human primates demonstrated a dramatic increase in neuronal immunoreactivity for HIF-1α and one of its target genes in the infarct and peri-infarct region [33, 34]. Indeed, the neuronal immunoreactivity for HIF-1α increased from less than 5% to nearly 90% in both regions. Interestingly, areas remote to the area of hypoxia and ischemia also experience increased HIF and vascular endothelial growth factor (VEGF) levels but to a quantitatively much smaller extent. Together, the published rodent and primate histochemical studies support the hypothesis that increased HIF protein levels resulting from direct hypoxia and non-hypoxic mediators such as IGF-1 are a marker for surviving and regenerating neurons after ischemia [32]. Fig. 4HIF prolyl 4 hydroxylases sense hypoxia and transduce a critical insufficiency in oxygen in the brain into transcriptional and post-transcriptional signal changes that mediate protection and repair. Hypoxia regulates the activity of HIF PHDs via direct or indirect mechanisms; production of peroxide via reduction in mitochondrial ATP production and electron transport chain (ETC) impairment (1, 2); accumulation of the tricarboxylic cycle (TCA) intermediates succinate and fumarate (3); or direct inhibition of the activity of PHDs due to lack of oxygen (4). Accumulation of hydrogen peroxide, succinate or fumarate can inhibits the activity of PHDs by competing with 2-oxoglutarate or by oxidizing the active site iron (5). Among its numerous downstream effects, inhibition of HIF PHD activity leads to stabilization of HIF-1α. Stabilized HIF-1α dimerizes with HIF-1β in the nucleus and increases gene transcription (6) Pharmacological and molecular studies have provided additional support for the notion that stabilization of HIF-1 after ischemia is associated with enhanced survival of neurons. Small molecule hypoxia mimics, deferoxamine, and cobalt chloride, were found to stabilize HIF-1α levels, increase DNA binding to a cognate hypoxia response element, and increase the expression of HIF target genes in vitro cultured neurons (Zaman et al. [31]) and in vivo in the intact brain (Fig. 4; [35]). Pretreatment or post-treatment with desferrioxamine or cobalt chloride resulted in reduced cell loss in models of focal or global ischemia in vitro and in vivo [25, 31, 36–39]. Subsequent studies have confirmed that hypoxia, iron chelation, or cobalt chloride appear to confer protective effects on neurons via their ability to inhibit HIF PHDs [35]. Emerging data indicate that inhibition of each of the three of the HIF PHD isoforms (1–3) may lead to cell survival in the nervous system. Of note, inhibition of each isoform may enhance survival via distinct but mutually supportive pathways. Our studies demonstrate that pharmacological inhibition of PHDs, in vitro, leads to inhibition of oxidative stress-induced death, an established mediator of neural injury and death in stroke [35]. More recent studies indicate that molecular suppression of HIF PHD 1 alone can mimic this effect (Siddiq et al., unpublished observations). Interestingly, while HIF-1, HIF-2, and cAMP response element-binding protein are stabilized by PHD inhibition in neurons or astrocytes, the molecular deletion or inhibition of each of these three transcription factors fails to abrogate the protective effects of PHD inhibition suggesting that other, as yet unidentified, pathways are important in protection (Siddiq et al., unpublished observations). Studies are underway to clarify whether molecular deletion of PHD1 selectively in the CNS confers resistance to stroke in a HIF-independent manner. In contrast to PHD1, PHD2 appears to be the isoform most important for tagging HIF for degradation. Deletion of PHD2 but not PHD1 or PHD3 results in increased HIF and VEGF protein and consequent angiogenesis in multiple organs including the brain [40]. Underscoring PHD2’s important role in HIF signaling, the expression level of this isoform is significantly higher as compared to its brethren [41]. Ischemia-induced PHD2 inhibition stabilizes HIF and enhances expression of genes that mediate cellular (e.g., glycolytic enzymes), local (vascular endothelial growth factor), and systemic (erythropoietin, Epo) adaptive responses to hypoxia or hypoxia-ischemia [42]. While Epo is best known as a hematopoietic growth factor that can enhance oxygen carrying capacity to tissue, it also has organ-autonomous roles in the nervous system. Epo is produced in astrocytes in response to hypoxia or ischemia and mediates a number of responses critical to stroke prevention and recovery [43, 44]. Epo can inhibit neuronal death due to excitotoxicity or growth factor deprivation [45–49]. It can also stimulate the proliferation of neural progenitors in the germinal zones of the brain and enhance their migration to sites of injury [50, 51]. The concomitant HIF-dependent increase in VEGF expression provides, among other things, the appropriate angiogenic niche for neural progenitors to survive [52]. New neurons could mediate recovery responses via paracrine effects or due to direct participation in functional circuits. A recent study affirmed the requisite role for HIF transcription factors in mediating some of the salutary effects of low molecular weight PHD inhibitors given after cerebral ischemia [53]. Available evidence suggests that these inhibitors are likely targeting PHD2 to induce HIF and its gene targets in the CNS. Inhibition of the third isoform of PHDs (PHD3) by hypoxia or hypoxia mimetics has also been linked to neuronal survival [54]. Freeman and colleagues first identified PHD3 (then known as SM-20) as a message and protein that is highly upregulated in sympathetic neurons after growth factor deprivation. Subsequent studies have shown that pharmacological or molecular deletion of HIF PHD3 prevents apoptosis associated with trophic loss in neurons and that the protection is HIF independent [55]. As target (post-synaptic) derived trophic factors appear to be lost after stroke and result in cell death remote from the infarct site, HIF PHD3 inhibition may be a rational strategy for maintaining the viability of these neurons in evolving or stable stroke. From the above discussion, a model begins to emerge whereby inhibition of HIF PHD1, 2, or 3 by hypoxia or consequences of hypoxia can mediate HIF-dependent and HIF-independent compensatory responses via distinct but clearly reinforcing mechanisms. How does one reconcile this model with observations from several laboratories, including our own, that HIF PHD inhibition or HIF activation leads to the upregulation of prodeath, Bcl-2 family proteins such as Puma, Bnip3, and NIX [56]? Moreover, constitutive HIF activation has been associated with potentiation and suppression of death [56]. These findings appear to make perfect sense if one considers apoptosis as an adaptive response to stress. After stroke, oxygen may fall below a critical level rendering the prolyl hydroxylases inactive. Accordingly, HIF is stabilized and it translocates to the nucleus to upregulate genes involved in preventing and executing death. The anthropomorphized cell can be visualized stepping to the edge of a steep cliff in response to a potentially lethal stress (Fig. 5). If adequate compensation for hypoxia occurs, prodeath proteins are not activated and the cell survives as a functional component of a complex neural network—the cell moves away from the cliff to survival promoting, terra firma. By contrast, if oxidative stress, hypoxia, and acidosis dominate, indicating that compensatory mechanisms have failed, then proapoptotic proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane and activation of permeabilization transition and release of apoptotic effectors [57–59]. In the latter scenario, the cell jumps off the cliff to preserve limiting resources for its neighbors. The model suggests that state changes in the cell (e.g., redox and pH) that follow the initial ischemic insult will determine the fate of the tissue and potential for recovery (Fig. 5). Of note, low molecular weight or peptide inhibitors of the PHDs, antioxidants, and a small interfering RNA to BNIP3 prevent the prodeath effects of HIF (Amino et al., personal communication). These findings suggest that engaging the adaptive response at an upstream point where stress is detected by the cell (the stress sensor) is going to be more effective in stemming cell loss and facilitating repair than at the point of the transcription factor (HIF). It is our hypothesis that engaging the adaptive response at an upstream point in the pathway results in post-transcriptional changes essential for the homeostatic response. These changes in concert with transcriptional changes optimally alleviate the discrepancy between oxygen supply and demand. Fig. 5Adaptation to hypoxia-cell fate and beyond. Expression of HIF in neurons leads to the constitutive expression of proteins associated with cell death (BNIP3, NIX, and PUMA) and cell survival (VEGF, glycolytic enzymes, Epo, and p21 waf1/cip1). Similar prodeath gene expression is found in neurons exposed to hypoxia or hypoxia mimetics despite the absence of cell death. It appears the oxygen “starved” neurons have stepped to the edge of the cliff. If during the ensuing hours to days the neuron becomes acidotic or oxidized, then prodeath proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane, release of apoptotic effectors, and death. By contrast, if the survival genes are effective in neutralizing the hypoxic stress (e.g., no acidosis or oxidative stress), then the death genes never get activated. Our studies indicate that antioxidants, short interfering RNAs to BNIP3 or inhibitors of the HIF prolyl 4 hydroxylases tip the balance toward survival (away from the cliff) The model has some clear predictions. First, low molecular weight global inhibitors of the HIF prolyl 4 hydroxylases will be more effective at preventing injury and repairing damage after stroke than selective isoform inhibitors. These inhibitors will engage HIF-dependent and HIF-independent pathways at cellular, local, and systemic levels and ultimately alleviate the discrepancy in nutrient supply and demand. They can also (via mechanisms that are only beginning to be defined) divert HIF away from its tendencies as a prodeath transcription factor. The ability of single “drugs” to target an oligopoly of proteins (HIF PHD1–3) to affect a concerted program of neuroprotection involving more than 70 genes and larger number of proteins suggests a strategy for overcoming the heterogeneity inherent in stroke pathophysiology in the short term. While a significant amount of work needs to be done to adequately assess the viability of this strategy for human therapeutics, the notion of augmenting endogenous adaptive programs via HIF PHDs to thwart disease continues to gain currency.

Environ_Health_Perspect-114-2-1367850

Prenatal Methylmercury Exposure and Developmental Outcomes: Review of the Evidence and Discussion of Future Directions

I conducted a review of the published literature to assess the strength of the evidence for an association between prenatal exposure to methylmercury (MeHg) and subsequent child development. I identified 12 studies on this subject published since 1980. Of these, 3 were longitudinal studies—2 conducted in the Seychelle Islands, and 1 in the Faroe Islands. Nine were cross-sectional studies conducted in different countries where seafood, a source of MeHg, constituted a major part of the diet. The ages of the children studied ranged from 2 weeks to 12 years. The results of the longitudinal studies were contradictory. Researchers in the Faroe Islands identified an association between MeHg exposure and developmental effects, whereas those in the Seychelle Islands identified no such association. This inconsistency was mirrored in the results of the cross-sectional studies where there were some positive and some negative findings. It was concluded that it was not possible from currently available data to determine whether there is an association between prenatal MeHg exposure and adverse developmental effects in children. In advance of future research, consideration should be given to resolving the uncertainties surrounding exposure assessment and outcome measurement, as both elements varied between studies. It was suggested that questions of exposure assessment would benefit from the application of an expert review process. Outcome assessment would benefit from the development of theoretically based measures of specific aspects of cognitive functioning to replace the relatively crude measures of attainment and IQ currently employed in most studies. This would assist in the development of classic longitudinal studies by allowing repeated assessment over the full age range and providing data that are more readily interpretable and comparable between studies. Organic compounds of mercury have a variety of industrial uses, and most of the data relating to the neurotoxic effects of mercury exposure have been derived from occupational populations (Chipman et al. 1995). However, in the 1950s a potential risk to the wider community was highlighted when large numbers of people living in the Minamata Bay area of Kyushu Island, Japan, developed symptoms of mercury poisoning. This well-documented incident was traced to a factory using mercuric chloride as a catalyst in the production of vinyl chloride and discharging effluent into the sea (Harada 1995). In aquatic environments, mercury is methylated by the action of common bacteria and methylmercury (MeHg), then passes up the food chain and becomes concentrated in fish and sea mammals. The heavy dependence of the Minamata inhabitants on a fish diet meant that they were subject to high levels of MeHg exposure. Both marine and freshwater fish routinely contain low levels of MeHg as a result of mercury leaching from the earth’s crust. Cases of frank poisoning have not been recorded in fish-eating populations other than those in Minamata, where special circumstances prevailed. However, the Minamata findings raised questions about whether less severe neurologic effects might occur at lower levels of exposure in populations whose diets were rich in seafood. Given the high level of fetal abnormalities observed in Minamata (Harada 1995), particular concern focused on the potentially hazardous effects of prenatal exposure. This concern increased after an outbreak of mercury poisoning in Iraq in 1971–1972, when large numbers of people consumed bread made from grain treated with a mercury-based fungicide. Subsequent neurologic examination of 80 children born to mothers who had eaten the affected bread over a 2- to 3-month period suggested an increase in neurologic abnormalities (Amin-Zaki et al. 1974). Animal and other data have indicated that the developing fetus is more vulnerable both to exposure to neuro-toxicants such as heavy metals and to the effects of such exposure (Hanson 1997). Any adverse effects are likely to be structurally and functionally different from those seen in the exposed adult. Thus, in addition to the obvious concern for the health of those with diets high in seafood, this issue has wider public health implications in terms of the general advisability of consuming seafood during pregnancy. The systematic investigation of potential developmental effects in children after prenatal exposure to MeHg began in the 1980s. Since that time two major longitudinal programs of work and a number of smaller cross-sectional studies have been conducted. These investigations assessed levels of prenatal exposure to MeHg in seafood-eating populations and examined subsequent developmental outcomes in children of varying ages. However, despite considerable research effort over a period of 20 years, there remains a lack of consensus on the central issue of whether exposure to MeHg derived from the routine diets of pregnant women presents a risk to their unborn children. This article contributes to the current debate on this subject with a brief description of the currently available data, some possible explanations for the inconclusive results that have so far emerged, and suggestions for potential ways forward in this contentious area of research. Current Evidence A search of relevant databases [Medline (http://omni.ac.uk/medline), Toxfile (http://www.dialog.com), Embase (http://www.embase.com), Cancerlit (http://cancerweb.ncl.ac.uk/cancernet/cancerlit/), Biosis Previews (http://www.biosis.org.uk), SciSearch (http://www.bids.ac.uk), and the Web of Science (http://wok.mimas.ac.uk)], using keywords such as “methylmercury,” “prenatal exposure,” “child development,” and “neurobehavior,” revealed that 12 investigations have been published thus far on this subject, all of which have been conducted since 1980.These investigations have used either neurologic examinations, developmental rating scales, or psychological tests to evaluate postnatal neurologic effects in children prenatally exposed to MeHg. Nine of the studies were cross-sectional (Table 1), in the sense that a sample of children was tested on one occasion and associations between test results and a measure of prenatal exposure to MeHg were examined. (Cordier et al. 2002; Grandjean et al. 1999b; Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Murata et al. 1999b; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002). Of these cross-sectional studies, however, one consisted of a form of follow-up, in that children were tested at 6–7 years of age and divided into comparison groups on the basis of exposure data collected from their mothers soon after birth (Crump et al. 1998; Kjellstrom 1991). In addition the study of Stewert et al. (2003) comprised a cross-sectional element of a larger longitudinal investigation concerned primarily with the adverse effects of prenatal exposure to polychlorinated biphenyls (PCBs). During the course of this investigation, possible interactions between PCB and MeHg exposure as determinants of adverse health effects were also investigated. Sample sizes in the cross-sectional studies ranged from 43 to 351 and response rates from 64 to 99% (Table 1). Three studies were longitudinal in design (Table 2) and followed children from birth, at intervals, for several years. Of these, the Faroe Islands study (Budtz-Jorgensen et al. 1999, 2000, 2002; Grandjean et al. 1992, 1995, 1997, 1998, 1999a, 2001a, 2001b, 2002a, 2002b, 2003; Murata et al. 1999a, 2002a, 2002b; Weihe et al. 1996) commenced in 1986 with a cohort of newborn infants who were subsequently tested at ages 12 months, 7 years, and 14 years. Two further longitudinal studies were carried out in the Seychelle Islands. The first commenced in 1987 with a sample of newborn infants who were followed up at age 5 weeks, 66 months, and 108 months (Cernichiari et al. 1995a; Davidson et al. 2000; Marsh et al. 1995a; Myers et al. 1995a, 1995b; Shamlaye et al. 1995). This was considered a pilot study by the authors, although a large amount of data was collected of a good standard and was therefore reported. A subsequent study commenced in the Seychelle Islands in 1989 with a sample of newborns who were followed up at ages 6.5 months, 19 months, 29 months, 66 months, and 108 months (Axtell et al. 1998, 2000; Cernichiari et al. 1995b; Cox et al. 1999; Crump et al. 2000; Davidson et al. 1995a, 1995b, 1998, 1999b, 2001; Myers et al. 1995c, 1995d, 1997, 2000, 2003; Palumbo et al. 2000). Sample sizes in the longitudinal studies were larger than those in the cross-sectional studies (Tables 1 and 2), although there was inevitable attrition over the periods of longitudinal investigation (Table 2). Exposure assessment. With the exception of the longitudinal Faroe Islands study and the study of Steuerwald et al. (2000), all studies used maternal hair as the vehicle for measuring prenatal exposure. In the case of the two longitudinal Seychelle Islands studies and six of the cross-sectional studies (Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002), hair samples were taken from the mothers at parturition. However, three of the cross-sectional studies used hair samples taken from the mother and/or the child at the time the child was tested (Cordier et al. 2002; Grandjean et al. 1999b; Murata et al. 1999b). In the study by Grandjean et al. (1999b), for example, exposure was assessed by reference to MeHg concentrations in maternal hair samples and, where this was not available, by reference to concentrations in the child’s hair at the time of testing. Dietary habits and social circumstances had changed little in the community during the previous years. For this reason maternal hair samples were judged likely to be representative of parturition samples. In addition child and maternal hair concentrations were highly correlated. Thus, where it was necessary to use child hair samples, these were also regarded as representing an adequate proxy measure for maternal exposure at parturition The longitudinal Faroe Islands study and the cross-sectional study of Steuerwald et al. (2000) used samples of cord blood as well as maternal hair to assess levels of prenatal exposure. All studies reported details of quality control measures for the analysis of samples. However, the specific form of mercury included in the measurement was not always clear, and a number of different exposure metrics were employed (Tables 1 and 2). Outcome measurements. A variety of outcome measures were used that included neurologic examination, developmental rating scales, neuropsychological tests, and attainment tests. Although this variety was accounted for partly by the differing ages of the children, necessitating different forms of assessment, variation also occurred between studies in terms of the tests used for children of the same age group. All studies used tests or rating scales that were established, published assessment tools. With the exception of the Seychelle Islands studies, information on testing procedures provided in the published reports was rather limited and therefore difficult to evaluate. All studies reported control of some potential effect modifiers such as socioeconomic status, ethnicity, and parental IQ, but the particular factors selected for inclusion varied between studies. All studies used multivariate analysis techniques. Three of the 12 studies, namely, the longitudinal Faroe Islands study and the studies of Kjellstrom (1991) and Grandjean et al. (1999b), reported a statistically significant relationship between prenatal exposure to MeHg and at least one developmental outcome. In the case of the Faroe Islands study, significant associations were observed for the group as a whole, at 7 years of age, between MeHg in cord blood and some, but not all, of the outcome measures on six neurobehavioral tests. Many of these associations were of borderline statistical significance. However, a dose–response relationship between cord blood MeHg and some test outcomes was demonstrated in a subgroup with the highest MeHg levels. Initial analysis in the Kjellstrom study (1991) did not indicate any significant associations between test outcomes and MeHg. However, exclusion of one outlier whose mother had particularly high MeHg levels resulted in the emergence of associations between MeHg and six test outcomes (Crump et al. 2000; Kjellstrom 1991). In the study by Grandjean et al. (1999b), an association between MeHg exposure was observed for three test outcomes. No discernible pattern was evident in these results in terms of effects on particular tests. Three further studies reported some positive associations between MeHg exposure and developmental outcomes, although all qualify their conclusions and express some reservations about the strength of their findings. In two of these studies, children were under 3 years of age, and assessments were in the form of neurologic examination. Steuerwald et al. (2000) reported that examination of children at 2 weeks of age showed that those with higher prenatal exposures had slightly lower overall neurologic scores, although there was no discernible pattern of suboptimal findings. Similarly, McKeown-Eyssen et al. (1983) carried out neurologic examinations on Cree Indian children between 12 and 30 months of age and found an association between MeHg exposure and the prevalence of abnormal muscle tone reflexes in males only. The authors note the mildness of the abnormality, which they consider to be of doubtful clinical significance. Cordier et al. (2002), in a study of children 9 to 12 years of age, found an association between scores on one test and MeHg exposure, but this did not appear in separate analysis of the highest exposure subgroup. Moreover, results from one test showed a positive association with MeHg exposure. This type of counterintuitive result also occurred in a study of children 7–12 years of age (Weihe et al. 2002) and in the main Seychelle Islands study when children were assessed at 66 months (Davidson et al. 1998). Neither of these studies or the first Seychelle Islands study report associations between other test results and MeHg exposure. Similarly, the results of Marsh et al. (1995b) and Murata et al. (1999b) with children between 6 and 7 years of age indicated no associations between psychological test scores and MeHg exposure. Murata et al. (1999b), however, demonstrated an effect of MeHg exposure on two neurophysiologic measures. Finally, the study of Stewert et al. (2003) of younger children 38 and 54 months of age reported an interactive effect of MeHg and PCB exposure as an incidental finding that emerged in the course of a study to investigate the adverse effects of PCB exposure. A finding of this nature, although interesting, should perhaps be regarded as speculative only and requiring further investigation. Our relatively brief review of the literature highlights the current uncertainty in this field of enquiry. The two major longitudinal investigations report contradictory findings, and a number of cross-sectional studies have similarly produced inconsistent results. This data set has already been subjected to much examination [National Research Council (NRC) 2000] and further analysis in an attempt to derive appropriate environmental exposure limits for pre-natal MeHg (Office of Environmental Health 1999; Rice et al. 2003). The generally assumed superiority of longitudinal over cross-sectional designs has tended to focus most attention on the studies conducted in the Faroes and the Seychelle Islands, which are of high methodologic quality (Jacobson 2001). However, it should be noted that, given the complex and challenging nature of this type of research, the majority of the cross-sectional studies are also of relatively good quality and would normally be taken into account in a systematic review. The results of these cross-sectional studies in fact mirror the general inconsistency evident in the results of the longitudinal investigations. One is therefore drawn to the conclusion that it is not possible from currently available data to determine whether prenatal exposure to MeHg, at levels routinely experienced by populations whose diets are rich in seafood, results in adverse effects on the nervous system of the developing child. Discussion A primary objective of the review process is the derivation of conclusions from the available data to guide future policy. In this case, however, the development of such a policy would appear to be hindered by the existence of directly contradictory results obtained from research of equal quality. As noted above, the data from the studies contained in this review have already been the subject of extensive evaluation (NRC 2000; Rice et al. 2003) and continue to excite controversy that is so far unresolved (Davidson et al. 1999a; Grandjean and White 1999; Stern and Gochfeld 1999). A secondary objective of the review process, the identification of data gaps in the literature, appears inappropriate in circumstances where so much research has been carried out to date. Although the contribution of existing published research is unquestioned, it may be time to concede that there is little further that can be drawn from these data or, one suspects, from repeated studies of a similar type. Experience from other fields (Spurgeon 2002) suggests that further cross-sectional studies employing similar neurobehavioral outcomes will serve only to increase rather than reduce the uncertainty surrounding this issue. In the remainder of this article, therefore, I discuss some of the possible reasons for the inconsistency in the existing data and indicate some areas where alternative approaches might be required to achieve some progress in this field. The common objective of the investigations reviewed above was to establish whether there is an association between prenatal exposure to MeHg and developmental effects. Although the various studies had many elements in common, perhaps the most noticeable feature of the studies as a group was the variation in the methods used to assess the two basic elements of the association, namely, the exposure and the effect. It is not surprising that research using different combinations of biological and psychological measures produces inconsistent results. The debate surrounding each of these elements, although undoubtedly complex, merits resolution in advance of any further research. Exposure. In terms of the most appropriate biological marker of prenatal exposure, opinion is divided between maternal hair and cord blood as the biological sample of choice. Studies that have attempted to define the relationship between different biological indices have produced inconsistent and somewhat wide-ranging results, and conversion from one set of values to another appears to involve a number of questionable assumptions (Office of Environmental Health 1999). Other difficulties in the interpretation of the data set arise as a result of the use of different units of measurement and a lack of clarity in some studies about whether the measure is of organic, inorganic, or total mercury concentration. Thus, there is continuing uncertainty about the association between elements of the diet and concentrations in child hair, maternal hair, cord blood, and maternal blood, as well as uncertainty about the strength of any relationship between each of these elements and the relationship between each and the actual exposure of the fetus. Elements of the debate about hair versus blood samples must be linked to a large number of other unanswered questions surrounding prenatal exposure measurement. These relate particularly to the relative importance of exposure at different periods of gestation, the relationship between these and average exposures, and the importance of peak exposures. The development of the central nervous system is time related and unidirectional. The inhibition of one stage of development tends to cause alterations to subsequent processes, with limited capacity for compensation for cell loss (Annau and Eccles 1986; Trask and Kosofsky 2000). Both the dose and timing of any environmental insult are important in terms of the specific nature of any adverse effects. How far do our current methods of prenatal exposure assessment reflect the need to take this into account? The present enthusiasm for evidence-based policy and practice appears to offer an ideal opportunity to address these types of questions, either through the medium of an expert workshop or that of a written systematic review. The important issues in either process include a) definition of the important questions to be addressed to achieve valid and reliable assessment of prenatal exposure, b) identification of available data that could be used to answer these questions, and c) identification of new research required to fill any identified data gaps. In advance of some consensus on these issues, further research is likely to provoke more controversy rather than lead to any resolution of the current uncertainty. Outcomes. The outcomes used in these studies were predominantly psychological tests. Use of such tests in environmental and occupational health research, which began in the early 1980s, has always been controversial, and the apparent inconsistencies in the data produced has provoked much debate in both environmental and occupational health research (Koller et al. 2004; Levy et al. 2004). Results relating to prenatal MeHg exposure represent a particular example of a wider problem and highlight a number of questions related to the more general field of neurobehavioral toxicology. Specifically, two main areas are of concern. The first, and perhaps the more straightforward, relates to the control of variables that either represent potential confounders or may act as modifiers of the effects under investigation (Spurgeon and Gamberale 1997). They are perhaps best considered under the broad headings of situational variables (physical testing conditions and test procedures), tester variables (reliability of the examiners), and subject variables (individual characteristics such as age, gender, and socioeconomic group). In all epidemiologic research involving psychological testing, the list of these variables is potentially very long, and researchers appear divided about which to include. In research on MeHg, the majority of studies consider important subject characteristics such as age, ethnic and socioeconomic group, and aspects of parental lifestyle. However, for a number of other variables (e.g., aspects of the caregiving environment), inclusion is patchy. For many of these variables, useful literature is available on their effects on children’s abilities or on test performance, and it may be possible to reach an evidence-based consensus on their inclusion or exclusion. For other, mainly procedural factors, data appear relatively scarce. A systematic review that encompasses other areas of psychology, for example, that pertaining to human–computer interaction, might reveal relevant information. For example, how much does the size of the screen affect performance on a computer-administered test? How much does the physical location of testing (home, laboratory, hospital) affect test performance? Existing data on the effects of time of day (Smith 1992), for example, indicate that in epidemiologic studies this factor should always be controlled. Intuitively it would seem appropriate that the physical testing situation and procedures should be standardized for all subjects as far as is practically possible, regardless of whether firm evidence exists about the influence of heating, lighting, noise control, or the arrangement of furniture. Less well-researched aspects of the test situation can be explored usefully within the researchers’ data. Is there, for example, a significant difference between test scores obtained at the beginning and at the end of the week or at different times of the year? The effects of the tester, particularly where tests are not computer-administered, may be important, not only because of different interactions with different subjects but also because of the examiner’s variable moods, motivation, levels of fatigue, and tendency to introduce systematic errors into the testing procedure. It cannot be assumed that confining testing to one examiner or using examiners who have undergone a single period of training removes tester variation. In terms of reliability, it may be advantageous to employ more than one tester in some circumstances. Measures such as the videotaping of testing procedures, double scoring, and examination of the test data for trends related to some of these factors have all been used to account for or eliminate this potential source of variation (Harvey et al. 1988). Similarly some estimation, albeit a subjective rating, of the child’s level of co-operation with the testing procedure is important to include. Potentially this is a major source of variation in test performance rarely alluded to in published reports. Ideally, tests should also include parallel forms or practice trials to ensure that maximal performance level is recorded for each subject. All except one of the studies in the field of research under discussion here present detailed accounts of quality control procedures in relation to MeHg assessment. It is relatively rare to find equally detailed discussion of procedures for outcome assessment. This is a situation that occurs frequently in neurobehavioral investigations. Lack of reference to quality control does not necessarily imply that control was limited but may suggest something about the attitude of researchers toward its importance. The implications for further research are 2-fold. First, systematic work is needed on the effects of factors considered likely to affect test performance, including both a review of the available data and, if necessary, further investigative work. Second, consensus must be reached on good practice such as that available in some other areas of toxicology, notably animal experimentation. Although this consensus may exist at an informal level in the field, the considerable methodologic variations between different neurobehavioral studies suggest that many aspects are currently opinion based rather than evidence based. A second and fundamental issue in terms of outcome measures relates to the types of tests used and, by implication, the interpretation of the results they provide and the comparability of these between studies. The tests employed in the studies described above are mainly tests of intellectual functioning. However, those used in different studies, and sometimes within the same study, derive from a number of separate traditions of intellectual assessment, each of which was developed for a different purpose and different client group. Although each has some advantages, none were developed specifically for neurotoxicity research and none is entirely appropriate for this type of application. Attainment tests are attractive in the sense that they offer the opportunity to benchmark the performance of children in basic skills such as literacy and numeracy against that of their peers. However, such tests tend to reflect the use of abilities rather than the underlying abilities themselves. Given the range of social and educational factors interacting with the ability to produce attainment, this effectively introduces additional variables into the equation (Gadzella et al. 1989). In contrast, the neuropsychological approach characterized, for example, by tests such as the Trailmaking test used in the Seychelle Islands study or the Bender Gestalt test used in the Faroes Islands study was developed to provide detailed evaluation of patients with suspected damage to the brain. Such damage might have resulted from head injury or other insult or from a degenerative disease of the nervous system. In these circumstances the purpose of assessment is to provide detailed information about the nature of the problem in functional terms and thus provide a basis for rehabilitation and progress monitoring. Assessment in a clinical setting tends to be a flexible process that draws as much on the qualitative aspects of the interaction between psychologist and client as it does on the numerical test scores. The clinician is interested in the patient as an individual and reaches a professional judgment on the basis of a number of sources of information. There is a risk that tests of this nature lose much of their value when applied in a routine fashion to large groups of people. Many who work in the field of clinical neuropsychology appear to be deeply uneasy about the transfer of these techniques to an epidemiologic setting (British Psychological Society 2001). Particular concern arises when tests designed for administration by a psychologist are adapted for computer presentation. Researchers with a neuropsychological background tend to adopt a clinical approach by administering a very large battery of tests to cover all aspects of functioning (Davidson et al. 1995a; Grandjean et al. 1997; Kjellstrom 1991). In an epidemiologic setting this can be inappropriate, resulting in multiple comparisons and the possibility of chance findings. Moreover, it often leads to confusion from a psychological point of view, where the results appear as a collection of apparently unconnected findings with no discernible meaningful pattern. Where studies use the same tests, it is common for significant associations to appear in both studies but in relation to different outcomes (Grandjean et al. 1997, 1999b). Finally, there are questions about the ability of tests designed for more severely affected patient groups to detect relatively subtle effects in community samples (Spurgeon 1996; Stollery 1985, 1990). Tests derived from a psychometric tradition are concerned with the assessment of intelligence quotients (IQ) in the general population and were originally developed to describe normal distributions of cognitive functioning. The Wechsler Scale (Wechsler 1991) represents the most widely used test battery in this respect. Developmental scales for very young children fit within this tradition, replacing formal testing where this is impractical, although it should be noted that maternal reports of developmental milestones are subject to numerous sources of error such as inaccurate recall, differing definitions of certain behaviors, and presentational bias (Axelson and Rylander 1984). The measurement of IQ is a reassuringly familiar concept supported by a wealth of normative data and experience built up over many years. Unfortunately, IQ tests were originally developed within a theoretical framework of cognitive functioning that prevailed more than a half-century ago. Such tests reflected a contemporary need to categorize individuals on a quantitative scale to predict future performance, an approach now considered somewhat crude and simplistic. Although such tests maintain their predictive validity in some settings (Neisser et al. 1996), they are relatively blunt instruments that combine a number of different abilities within each test (Lezak 1988). This aspect limits the information that can be derived from the assessment and makes interpretation difficult when conflicting results emerge from different studies. When placed in the context of more recent theoretical developments in cognitive psychology, established IQ tests do not provide results that can be easily linked to current models of cognitive processes. A primary objective of neurobehavioral research is the detection of subtle effects on cognitive functioning in community samples after neurotoxicant exposure. For epidemiologic purposes, tests should be quick and easy to administer. The results should be interpretable at group level and comparable between different studies. Given these criteria, none of the tests currently in use appear to be entirely fit for this purpose. Speed and ease of administration do not represent major challenges in an age of advanced information technology. However, improvements in interpretability and comparability are more complex issues likely to require a radical change of approach. In recent years a number of authors have pointed to the overemphasis on empiricism in this field and the lack of a strong theoretical underpinning for the assessment tools employed (Stephens and Barker 1998; Stollery 1990, 1996; Williamson 1990). The development of tests grounded in well-established cognitive theory would allow results to be discussed in terms of the specific aspects of cognitive processing under investigation rather than simply by reference to broad and largely uninformative categories of effect such as “memory” or “attention.” Modern approaches to the study of memory processes, for example, have long distinguished between several elements that contribute to the final outcome (initial registration of information, encoding, transfer to long-term store, loss of information by decay or interference, and use of cues for retrieval) (Baddeley 1987). Each may be differentially susceptible to neurotoxic insult, but effects on one specific process cannot be uncovered by most current tests that provide a simple global outcome score. Moreover, overall scores may mask specific effects where subjects employ compensatory strategies among different processes to achieve maximum performance. The development of tests, for both children and adults, based on techniques currently available to separate and measure these specific processes would provide much more useful information about the nature and size of any observed effect. This type of approach would ultimately pave the way for much greater comparability between the results of different investigations and for the development of comparable assessment techniques for children at different ages during longitudinal investigations. Despite much international effort during the last 25 years, agreement on a universally approved set of tests has proved elusive (World Health Oganization 1989). At the same time, the pursuit of the goal to achieve comparability over time and between studies has tended to inhibit the development of new techniques. It seems unlikely that consensus on appropriate assessment tools will be achieved in advance of a consensus on the theoretical basis for those tools. Fortunately, much of the information required for these new developments is readily available in the existing cognitive, experimental, and developmental psychology literature. Conclusion Reviews of the data relating to the developmental effects of prenatal MeHg exposure have highlighted the inconsistency of the currently available evidence. The size and nature of the risk to children that is associated with seafood consumption by their mothers remains uncertain and a source of considerable controversy. It has been argued here that the present uncertainty derives from the variation between studies in the methods used to measure both the exposure and the effect. Each element would merit further consideration in advance of any future research in this field. Although consensus is required on the appropriate biological marker of exposure, there is also a particular need to address issues of both procedure and content in psychological assessment. Discussion of these issues, particularly those relating to psychological tests, may have implications that go well beyond the immediate needs of this field of inquiry. Investigation of the effects of MeHg provides one particular example of the difficulties in data interpretation that occur repeatedly in neurobehavioral studies and threaten to undermine confidence in this methodology. The increasing international anxiety about potential adverse effects of low-level neurotoxicant exposure in the environment underlines the importance of addressing these concerns, as psychological methods currently represent one of the main tools of research in this field.

J_Gastrointest_Surg-3-1-1852381

Successful Internalization of a Chronic Biliary Cutaneous Fistula After Liver Transplantation: Deepithelializing the Fistula Tract

Biliary cutaneous fistulas are uncommon sequelae after biliary surgery and can be a source of significant morbidity. We describe a liver recipient who developed a biliary cutaneous fistula secondary to hepatic artery thrombosis; this subsequently drained for over 7 years. Through a novel approach, using the transabdominal fistula tract as a conduit, the fistula skin opening was deepithelialized and anastomosed to a jejunal loop, internally draining the tract. For over 7 years postoperatively, this internal drainage procedure has continued to function effectively. This approach may have value in internalizing longstanding biliary cutaneous fistulas in well-selected patients in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal drainage through conventional approaches. Introduction Biliary cutaneous fistulas are uncommon sequelae after biliary surgery and hepatic trauma. Persistent biliary fistulas can be associated with local skin morbidity, malabsorption of fat soluble vitamins, steatorrhea, impaired wound healing, and sepsis as a result of fistula tract obstruction.1 We describe a rare scenario in which a liver transplant recipient developed a biliary cutaneous fistula secondary to hepatic artery thrombosis. Through a novel approach, the fistula skin opening was deepithelialized and, using this transabdominal fistula tract as a conduit, anastomosed to a jejunal loop, thereby internally draining the fistula. This operative procedure, which has provided a successful long-term result, is discussed. Materials and Methods Case Report A 60-year-old man of Chaldean descent underwent orthotopic liver transplant for cirrhosis secondary to chronic alcohol use. His early postoperative course was complicated by hepatic artery thrombosis with necrosis of the extrahepatic biliary system. Because of sepsis and family issues, retransplantation was not a realistic option. Surprisingly, his graft remained viable with good metabolic function despite the absence of normal extrahepatic drainage. For 7 years subsequent to transplantation, his biliary drainage was managed with a U-tube connected to a bulb drain exiting the skin in the right subcostal region (Fig. 1). As a result, he suffered from severe malnutrition, pain, and skin breakdown. The persistent external drainage markedly affected his lifestyle, as the continuously draining bile required dressing changes over the exit site two to four times per day. The U-tube, frequently becoming obstructed with biliary sludge, required bimonthly replacement. Preoperative fistulogram (Fig. 2) demonstrated the continuity of the cutaneous opening with a large channel through the mid portion of the liver, but with no evidence of a true ductal system. Figure 1(a) Longstanding (7-year-old) biliary cutaneous fistula draining through right upper quadrant scar. Functional U-tube in place with copious biliary drainage. (b) Close-up of U-tube exit site.Figure 2Preoperative fistulogram in which contrast was injected through the existing U-tube. The cutaneous opening at the upper left side of the frame is in continuity with the large contrast-filled channel that enters the substance of the liver. Note the complete absence of an extrahepatic biliary system. Surgical Procedure With the patient under general anesthesia, the abdominal cavity was entered through a right subcostal incision along the scar of the previous transplant incision. The incision was fashioned so as to preserve a 1" diameter button of full-thickness skin surrounding the opening to the fistula tract (Fig. 3). Extensive adhesions between the fascia, liver surface, and small intestine were carefully lysed. The fistula tract was dissected retrogradely from surrounding subcutaneous tissue and muscle, preserving a rectus fascial ring. This created a conduit that could be anastomosed to small intestine. Dissection of the tract was stopped proximally at the level of the liver surface to preserve its integrity and vascularity. The fistula opening was lowered into the abdominal cavity. A 60-cm long Roux-en-Y jejunal limb was constructed 50 cm distal to the ligament of Treitz using stapled technique. Figure 3(a) At operation, the U-tube was prepped into the surgical field. (b) Dissection of the epithelialized chronic biliary cutaneous fistula tract. In entering the abdomen through the previous right subcostal incision, a 1" diameter button of full-thickness skin containing the fistula tract was preserved. A 4-mm-wide skin edge was sharply deepithelialized circumferentially around the dissected fistula opening. This conduit was then anastomosed to the Roux limb using two layers of running 4-0 Prolene suture (Fig. 4). The inner layer approximated the deepithelialized skin edge of the fistula tract to full-thickness jejunum. The outer layer approximated the conduit’s scar tissue and the preserved rectus fascial ring to seromuscular jejunum. An omental wrap was fashioned around the anastomosis. The anastomosis was performed over an 8-French feeding tube, which was exteriorized through a previous U-tube exit site and connected to a bile bag. Figure 4(a) Anastomosis of Roux-en-Y jejunal limb to deepithelialized fistula tract. (b) Illustration depicting the anastomosis. The abdominal cavity was irrigated and closed in the standard fashion. The patient tolerated the 3-h procedure well. Blood loss was minimal. He was discharged on postoperative day 8. Result This patient has now been followed for 7 years since the described procedure and is doing well without external drainage of bile. The condition of his periincisional skin improved dramatically shortly after the procedure. At this time his liver function tests and nutritional parameters are normal and his graft function remains adequate on a low dose immunosuppressive regimen. Discussion Chronic biliary cutaneous fistulas are challenging to manage from many standpoints: metabolic, nutritional, hygienic, and quality of life. The patient’s being a liver transplant recipient, added further complexity. First, malignant change was reported in chronic biliary cutaneous fistulas 2; this possibility is of real concern in light of this patient’s immunosuppressed state. Second, this patient had severe malabsorption and resulting difficulty in maintaining acceptable cyclosporin blood levels. Finally, the ongoing presence of his U-tube poses the threats of ascending cholangitis, hemobilia, intrahepatic biloma, and biliary-venous fistula, which are all well-documented complications that have been associated with U-tubes.3 The decision for operative intervention was made out of these concerns and for progressive difficulty in maintaining adequate external biliary drainage. Late retransplantation was considered; however, the patient’s graft function had been excellent over the 7 years subsequent to his transplant, and the patient and his family refused to consider it. In both the posttraumatic and postbiliary surgery settings, existing literature advocates initial nonsurgical management of biliary cutaneous fistulas, as a significant number will close either spontaneously or with nonsurgical intervention.1,4,5 In the series by Zer, four of seven biliary fistulas sealed spontaneously.5 Endoscopic approaches to reducing intrabiliary pressure and thereby encouraging drainage along a path of least resistance include endobiliary stenting, sphincterotomy, and nasobiliary drainage.1,4 Finally, selective biliary embolization, percutaneous transhepatic catheter drainage, sclerosis with tetracycline, and oral nitrates were also described to promote closure.4,6–9 Operative procedures for refractory biliary cutaneous fistula were dictated by the anatomic site of biliary tract disruption. Roux-en-Y hepaticojejunostomy, in which a jejunal loop is sutured directly to the liver capsule, was employed in the context of intrahepatic biliary injury after trauma.11,10,4 Reports in which an actual fistula tract is used as a conduit and sutured to a loop of small bowel, however, are rare. Smith described the anastomosis of a jejunal loop to a divided fistulous tract arising from the lateral surface of the liver in a patient with a penetrating injury to the upper abdomen.12 That same author described the internal drainage of a biliary cutaneous fistula secondary to blunt liver trauma into the gallbladder.12 In both cases the fistula tract was divided and then the proximal end of the divided tract was anastamosed to an intestinal loop over a tube. Deepithelialization of the skin opening and its direct usage in an anastomosis has not, to our knowledge, been previously described. Furthermore, this procedure is unique in that the internalization was done in the case of a functioning liver after hepatic artery thrombosis. The existing anatomy that had been established over 7 years of fistulous drainage dictated the type of procedure that was performed. In contrast to fistulas that arise after biliary tract surgery (for example, after cholecystectomy with common bile duct exploration), there was no remnant of a previous biliary ductal system. The preoperative fistulogram revealed flow of contrast from the skin directly to the liver surface with no evidence of an extrahepatic ductal system. The fistula therefore provided the only source of biliary drainage for the entire liver. An alternative surgical option would have been hepaticojejunostomy, in which the jejunum would have been sutured to a fibrous ring at the origin of the fistula tract from the liver surface. This, however, would have required more extensive dissection with possible disruption of collateral vasculature to the previously ischemic liver and to the tract itself, as well as dissection of the transverse colon, which in part bordered the tract. At operation, the superficial portion of the fistula tract was well established and appeared well vascularized. The potentially harmful deep dissection necessary for a hepaticojejunostomy was therefore avoided. We emphasize the decision to have fashioned a tongue of omentum over the anastomosis. By nature, the tissue comprising a fistula tract is probably somewhat tenuous. This patient’s immunosuppressed and malnourished state compound the risk of anastomotic breakdown. We feel that the well-vascularized, adherent omental tongue was an important aspect of the operation in reinforcing the anastomosis. As of 7 years of follow-up, the patient has not developed signs of biliary obstruction. His immunosuppressant levels are easier to maintain and he does not have evidence of malabsorption. It is unlikely that he will develop cicatricial narrowing of the tract, as it had remained well epithelialized for the 7 years before this operation. There is a theoretical concern about formation of squamous cell carcinoma at the anastomotic site, which we continue to keep in mind during long-term follow-up. Conclusion In summary, through a novel approach this patient’s biliary cutaneous fistula was internalized, which has eliminated his associated skin morbidity and malabsorption. Furthermore, his well-functioning graft was salvaged, thus saving the cost of retransplantation and sparing an additional liver for another patient. Internalization of a long-standing biliary cutaneous fistula through deepithelializing the skin opening and using the tract as a conduit is technically feasible. We believe it to be a valid approach in the patient in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal biliary drainage by conventional approaches.

J_Gastrointest_Surg-3-1-1852388

Middle Segment Pancreatectomy: A Useful Tool in the Management of Pancreatic Neoplasms

Small, benign, or low-grade malignant tumors located in the neck of the pancreas are usually treated with enucleation. However, if enucleation is too risky because of possible damage of the main pancreatic duct, standard pancreatic resections are performed. Such operations can lead to impaired long-term exocrine–endocrine function. Middle segment pancreatectomy consists of a limited resection of the midportion of the pancreas and can be performed in selected patients affected by tumors of the pancreatic neck. Middle segment pancreatectomy is a safe and feasible procedure for treating tumors of the pancreatic neck; in experienced hands it is associated with no mortality but with high morbidity, even if the rate of “clinical” pancreatic fistula is about 20%. Moreover, it allows a surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function. Introduction Whereas neoplastic lesion located in the pancreatic head or body-tail are usually resected by pancreaticoduodenectomy or distal pancreatectomy, tumors in the neck represent a real challenge for a surgeon. In these cases, standard or extended pancreatectomies performed for benign or borderline cases can determine the loss of a great amount of glandular tissue, significantly increasing the risk of diabetes, impaired exocrine function, and splenic loss.1–6 Enucleation would be an adequate alternative for small, benign, and low-grade malignant tumors, such as endocrine and cystic neoplasms of the pancreas. Unfortunately this conservative procedure cannot be always applicable. When the neoplastic lesion measures up to 2 cm or more, or it is encased within the pancreatic gland, enucleation is associated with a high risk of Wirsung’s duct damage; moreover in the case of tumors with uncertain biological behavior this approach should be avoided because of the risk of tumor recurrence1–5. Letton and Wilson7 reported for the first time in the English literature in 1959 two cases of traumatic midpancreatic transection followed by a reconstruction with a Roux-en-Y jejunal loop anastomized to the distal part of the gland. Dagradi and Serio,8 from our own Department of Surgery, were the first in 1984 to propose middle pancreatectomy with an “oncological” indication, treating a pancreatic insulinoma. Subsequently, other authors reported cases of resection of the middle pancreas, of varying extent, using various terms such as “central pancreatectomy,” “middle segment pancreatectomy,” “segmental pancreatectomy,” and “intermediate pancreatectomy.”9–13 The underlying indications for surgery ranged from chronic pancreatitis to benign, uncertain behavior, or low-grade malignant exocrine and endocrine neoplasms1–19. Different techniques were adopted for gastrointestinal reconstruction, including jejunal anastomosis of both the proximal and distal stump, or of only the distal stump, with pancreaticoduodenal or pancreaticogastric anastomosis.1–21 Surgical Technique The abdomen is entered through a midline incision. The gastrocolic ligament is opened, preserving the gastroepiploic vessels, and the pancreatic gland is exposed. The posterior peritoneum along the superior and inferior margin of the pancreas is incised. The superior mesenteric vein and the portal vein must be identified and their surfaces cleared below the gland. The plane between the superior mesenteric and portal vein should be teased apart. The splenic artery and vein are dissected free and separated from the gland. Some venous tributaries to the portomesenteric axis and some minor collaterals of the splenic artery can be ligated. Then, the posterior surface of the pancreatic neck is isolated from the portomesenteric axis and a ribbon is passed behind the gland to elevate it. Sutures are placed along the superior and inferior margins to indicate where the proximal and distal transection should be performed and to ligate those vessels running along the margins. The segment of the pancreas with the tumor is subsequently transected through a knife or a stapler to the left and to the right of the lesion. The cephalic stump is sutured with interrupted stitches after elective ligation of the Wirsung’s duct or by means of a stapler. A small stent is placed in the main pancreatic duct while performing pancreojejunostomy or pancreogastrostomy; the stent can be left in place, even if in our experience we have never done it. Two closed-system suction drains are used to drain the cephalic stump of the gland and the pancreaticojejunostomy/pancreaticogastrostomy. Discussion It has been shown that standard pancreatic resections are nowadays associated with low mortality and morbidity if performed in high-volume centers by experienced surgeons.22–24 It is remarkable that this type of surgery can lead to long-term complications, such as diabetes, exocrine insufficiency, and late postsplenectomy infection25,26. The incidence of postoperative exocrine and endocrine impairment is not predictable in patients with apparently “normal pancreas.” Factors such as fibrosis of the remnant, Wirsung’s duct obstruction, preexisting chronic pancreatitis, benign or malignant disease, and subclinical diabetes may play a role as “risk factors.”1–3 After standard left-sided resection there is an increased incidence of endocrine impairment and onset of diabetes reported from 17 to 85% of patients; it is reasonable that the extent of the resection is strictly related to the incidence of endocrine–exocrine long-term insufficiency27–31. For all these reasons, more conservative surgical techniques have been advocated for small, benign, or low-grade malignant tumors located in the neck of the gland, aimed for sparing, as much as possible, pancreatic parenchyma. Whenever neoplastic lesions are not small and superficial enough to be simply enucleated, middle segment pancreatectomy should be considerated.1–6 Middle segment pancreatectomy accounts for only 3% of the pancreatic resections performed at our institution and about 100 cases have been reported in the English literature1–21,32–34: this means careful selection of patients. In fact, the small number of patients who underwent this type of operation is related to different factors: specific localizations of the neoplasm, well-selected indications (benign or low-grade malignant tumors), and a distal pancreatic stump of at least 5 cm in length. Some authors1,3 have reported that this operation can be performed only in the case of small tumors (<5 cm in diameter); in our experience, although the mean diameter of the resected lesions was 27.4 mm, we have safely performed middle segment resection for tumor measuring more than 5 cm, harboring from the anterior face of the pancreas. Middle segment pancreatectomy was also occasionally used for malignant disease: two islet cell carcinomas, one vipoma who subsequently developed hepatic metastases, one cystadenocarcinoma, and one carcinoma in situ2,3,10–12. In the past we have also performed this operation for malignant tumor but we had pancreatic recurrence of the tumor in two patients (one affected by metastasis and one by intraductal papillary mucinous neoplasms [IPMN] with in situ carcinoma); moreover, two patients with adenoma and borderline main duct IPMN had a tumor recurrence in the pancreatic gland. Thus, we believe that in patients affected by primary or metastatic malignant tumor, a standard resection would be more appropriate. Moreover, middle pancreatectomy in our experience should also be avoided in patients affected by IPMN, especially main duct type because of their potential malignity and the possibility to have different degrees of dysplasia along the Wirsung’s duct in the same patients. The surgeon must be sure to achieve tumor-free proximal and distal resection margins after performing middle segment pancreatectomy and, for this reason, frozen section examination is mandatory. Middle segment pancreatectomy is a meticulous procedure. There is the possibility of leaks from both the closed cut edge of the head and the pancreaticojejunostomy, considering that in most patients we are dealing with a normal soft pancreatic texture with a small Wirsung’s duct. Thus, not only great care must be taken in selecting the patients who will benefit from this operation, but also an experienced pancreatic surgeon working in a high-volume center is required for performing the procedure.1–4,6,32,33 Median pancreatectomy is reported to be associated with no mortality but with a high postoperative morbidity, above all consisting of pancreatic fistula.6 In our experience the “clinical” pancreatic fistula rates after pancreaticoduodenectomy and left pancreatectomy are 10 and 20%, respectively.23,35–37 Between January 1990 and December 2005 61 patients underwent middle segment pancreatectomy at our institution. The incidence of pancreatic fistula—according to the International Study Group on Pancreatic Fistula definition22—was 51%. It is remarkable that most patients complained of Grade A fistula, which is a “biochemical” fistula without any clinical impact, whereas 13 patients (21%) developed a grade B or C fistula, which required prolonged in-hospital stay. In almost all patients the conservative management was successful; no one underwent reoperation and in four cases intraabdominal collections were treated with ultrasound-guided drainage. The mortality rate was zero. The risk of developing a pancreatic fistula must be taken into account in the preoperative decision making; we believe that this risk is acceptable when the procedure is performed in a high-volume center and for patients with a long-life expectancy, such as young or middle-aged people affected by benign or low-grade tumors. The most important advantage of middle segment pancreatectomy is the good endocrine and exocrine long-term function.1–6,10 Iacono et al.1 in a series of 13 patients demonstrated that postoperative oral glucose tolerance, pancreaticolauryl and fecal fat excretion were normal in all cases and they studied six patients pre- and postoperatively with oral glucose tolerance test showing no significant differences before and after surgery. Moreover, Sperti et al.3 showed, in a review of the literature, no case of impaired endocrine function in 59 evaluable patients whereas exocrine function was reported to be normal in 56 out of 59. Another advantage of this procedure is the possibility to preserve the spleen, preventing the risk of postsplenectomy sepsis and hematologic disorders, which is low but exists in an adult.38,39 In conclusion, middle segment pancreatectomy is a safe and technically feasible surgical approach for removing pancreatic neck tumors in well-selected patients; in experienced hands it is associated with no mortality but with high morbidity. Most of the complications do no require reoperation or prolonged in-hospital stay and can be successfully managed conservatively. Moreover, it allows the surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function.

Ann_Surg_Oncol-4-1-2277445

Metinel Node—The First Lymph Node Draining a Metastasis—Contains Tumor-Reactive Lymphocytes

Background We previously identified tumor-reactive lymphocytes in the first lymph nodes that drain the primary tumor. In this study, we performed lymphatic mapping to investigate the possibility of finding the first lymph nodes that drain metastases, and of learning whether these lymph nodes contained tumor-reactive lymphocytes suitable for adoptive immunotherapy. The global cancer registry Globocan shows 10.9 million new incident cases and 6.7 million deaths worldwide due to cancer in 2002.1 A total of 24.6 million persons are estimated to be alive with cancer (within 3 years of diagnosis). Leading causes of death from cancer, in order, are those of the lung (1.18 million), stomach (700,000), and liver (598,000). Most of these deaths were caused by metastatic disease, and they verify the fact that surgery and oncological therapy do not cure most patients with disseminated cancer. The incidence of cancer will continue to increase, especially in developing countries, largely due to smoking, infections, and lifestyles resembling those of people living in industrialized countries.1,2 Thus, intensified efforts are warranted, both to prevent cancer and to find new modalities of treating patients with metastatic cancer. Tumor cells spread by lymphogenous and hematogenous routes into the systemic circulation to disseminate the disease. The importance of tumor lymphangiogenesis has lately been proven in studies where high levels in colorectal cancer specimens of lymphangiogenic peptides, and vascular endothelial growth factor (VEGF)-C and/or VEGF-D promote tumor lymphangiogenesis and metastasis.3 The sentinel node concept implies that the lymphatic drainage from a primary tumor first drains to a certain locoregional lymph node specific for each individual and that its tumor status is representative for the entire lymphatic field. The concept is established in staging for breast cancer4 and malignant melanoma5 and has also been evaluated in several other types of malignancies, such as colon cancer,6 ovarian cancer,7 pancreatic cancer,8 and urinary bladder cancer.9 The technique has been used as a diagnostic tool to improve staging and to tailor the extent of regional lymph node dissections. Probably most solid tumors disseminate first through the lymphatics and thereafter enter the systemic circulation via lymphovenous shunts present in the first draining lymph nodes.10–12 Approximately half of the lymphatic fluid entering a lymph node continues directly into the systemic circulation; therefore, lymphogenic spreading of the tumor cells may be mandatory for hematogenic dissemination.3,13 However, to our knowledge, only one study has previously evaluated lymphatic mapping in metastases where liver metastases were investigated.14 Because it has been proven that both metastases and primary tumors have the capacity to metastasize15–17 and induce lymphangiogenesis,18 we assume that lymphatic drainage from metastases may have an equal importance in metastases as the sentinel node concept in many solid primary tumors for correct staging. We have previously experienced surprises in lymphatic mapping within patients with colon cancer6 and urinary bladder cancer9 regarding primary tumor lymphatic drainage. We believe that a similar technique may be of use in surgery for liver metastases and other solid metastatic tumors for correct staging, and thereby for giving the patients the most appropriate therapy. We previously showed that a sentinel node derived from a primary tumor contains specific tumor-reactive lymphocytes that have immunological reactivity toward the tumor in patients with colon cancer19 and in patients with urinary bladder cancer.20 Through the injection of Patent blue dye or radioactive tracer subserosally during surgery around the colonic tumor or transurethrally around the urinary bladder tumor, the sentinel nodes were found. A biopsy was performed, and the nodes’ immunological role and function were studied. After preparation, the lymphocytes were identified, isolated, and proliferated in vitro by stimulation of interleukins (ILs) and tumor antigen. Lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon (IFN)-γ on stimulation with tumor homogenate. The aim of the present study was to investigate lymphatic drainage from metastases and identify first draining lymph nodes. We also analyzed tumor-reactive immunological properties in lymphocytes derived from these lymph nodes. MATERIALS AND METHODS Patients Nineteen patients (9 men and 10 women, average age 51.5 years) with metastases from solid tumors were included in the study. Patient characteristics are shown in Table 1. The operations were performed during the period of November 2003 to January 2007. The study was approved by the local ethical committee, and each patient provided informed consent. Table 1.Patient characteristicsaPatientAge (y)SexPrimary tumorOrigin metinel nodeTracerNumber of metinel nodesMetinel node pos/neg for metastasesSuccessful expansionAliveFollow-up time (mo)148MColonIntra-abdominal local recurrencePB33 negYesYes38254MRectumLiver metastasisPB22 negYesYes38350MMal melanomaSubcutaneous metastasisPB22 posNoNo–477FColonLiver metastasisPB33 negNoNo–574MColonLiver metastasisPB21 posYesNo–1 neg666MColonLiver metastasisPB1MINoYes29751FOvarian cancerLiver metastasisPB21 posYesbNo–1 neg864FOvarian cancerGroin lymph nodePB/RT55 posNoNo–959FPancreatic cancerIntra-abdominal local recurrencePB4MINoNo–1039FMal melanomaGroin lymph nodePB/RT42 negNoYes111147MMal melanomaTruncal metastasisRT12 posNoNo–1245FBreast cancerCervical lymph nodePB/RT22 negYesYes51333FTongue cancerTruncal lymph nodeRT3MINoYes61464FColonIntra-abdominal local recurrencePB21 posYesYes41 neg1541FBreast cancerLiver metastasisPB22 negNoYes41639MMal melanomaCervical lymph nodePB/RT22 negNoYes21720MCholangiocarcinomaLiver metastasisPB33 negYesNo–1865MColonLiver metastasisPB31 posYesYes12 neg1942FLeiomyosarcomaLiver metastasisPB44 posYesYes1pos, positive; neg, negative; Mal, malignant; PB, Patent blue dye; MI, missing information; RT, radioactive tracer (4 × 10–15 MBq Tc-nanokolloid).a Data regarding the expansions for patients 1, 2, 5, and 6 also exist in unpublished data.b Patient died on the projected day of transfusion. The lymph nodes that drained metastases were identified by injecting approximately 1 mL Patent blue dye (PB) (Guerbet, Paris) subserosally or subcutaneously in four places around one of the metastasis or in the tissues close to the local recurrences with a 27-gauge needle. After the injection of PB, the first draining lymph nodes turned blue within 3 to 10 minutes and were regarded as metastases-draining lymph nodes. In our previous work, a 10-minute cutoff was sufficient to identify sentinel nodes to primary tumors. We have experienced that during this period of time the first draining lymph nodes turn blue. Waiting longer means that a second echelon of draining lymph nodes may be colored blue, which does not represent the first draining lymph nodes. We arbitrarily applied the same 10-minute limit when detecting lymph nodes that drain metastases. These lymph nodes were either immediately removed or marked with sutures. The sources of the liver metastases in our study were from colorectal cancer in five patients and in one patient each from ovarian cancer, breast cancer, cholangiocarcinoma, and leiomyosarcoma. Four patients had subcutaneous lymph node metastases from disseminated malignant melanoma, and one patient each had metastases from ovarian cancer, breast cancer, and squamous cell carcinoma of the tongue. Two patients had intra-abdominal local recurrences after surgery for colon cancer, and one patient had an intra-abdominal local recurrence after surgery for pancreatic cancer. Because of these liver metastases, five patients underwent partial right-sided liver resections (patients 4, 5, 6, 15, and 17), and three patients underwent partial left-sided liver resection (patients 2, 18, and 19). The patient with metastases resulting from ovarian cancer (patient 7) had several large, bulky, cystic tumors, and surgeons performed an almost complete right-sided liver resection. In four cases, surgery was performed for solitary colorectal liver metastases (patients 2, 4, 5 and 6), and in the remaining five cases, tumor-reducing surgery was performed. In six cases, a preoperative lymphoscintigraphy was performed by a subcutaneous injection of 4 × 10 to 15 MBq Tc-nanokolloid radioactive tracer in quadrants around the metastasis. The lymphoscintigraphies were performed to plan the surgical procedure by localizing the lymph node or nodes draining the metastasis. The position of the metastasis-draining lymph node or nodes was marked on the skin with a felt-tip pen. When the lymphoscintigraphy was not performed on the day of surgery, a new peritumoral injection of radioactive tracer was made at start of surgery. The draining nodes were intraoperatively identified by the use of a handheld gamma detection tube. In four of six cases, a simultaneous intraoperative injection of PB was performed. Preparation of Specimens Lymph nodes draining metastases and nondraining lymph nodes (for analytical purposes) were surgically removed and immediately taken to the laboratory on ice. In addition, one 5-mm slice of the whole metastasis (including the invasive margin) was also dissected and sent for analysis. Frequently, most of the lymph node or nodes were used for the expansion procedure. The remaining specimens underwent routine histopathological examinations with hematoxylin and eosin staining. At the laboratory, slices <1 mm were cut from the central and peripheral part of the lymph nodes for analysis by flow cytometry (FACS) and proliferation analyses. The metastasis was also analyzed by FACS, and a preparation of the whole tumor (homogenate) was used. Immunological Evaluation Single cell suspensions were made from the lymph nodes and metastases by gentle pressure with a loose-fitting glass homogenizer as previously described.19 Briefly, 1 × 106 cells were washed in phosphate-buffered saline containing 2% fetal calf serum and .05% NaN3, then stained with fluorophore conjugated antibodies toward the cell surface markers CD4, CD8, CD19, and CD56 (Becton Dickinson). Thereafter the cells were investigated with a FACSCalibur (Becton Dickinson). For cell cultures, single cell suspensions of metastasis-draining lymph node–derived cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum (Sigma), 1% penicillin-streptomycin (Sigma), and 1% glutamine (Sigma). For preparation of the antigen source, the metastasis was homogenized by an Ultra-turrax homogenizer in 5 volumes (w/v) of 2 ×  phosphate-buffered saline, followed by 5 minutes’ denaturation at 95°C. This tumor homogenate was then used for in vitro activation and clonal expansion of lymphocytes. The metastasis-draining lymph node–derived lymphocytes were finally dispersed into cell culture flasks, at 2 × 106 cells/mL of proliferation media, supplemented with 100 IU of IL-2 (Proleukin, Chiron) per milliliter of culture every 3 to 4 days. Tumor homogenate was diluted to 1:10 and 1:100 and added to the cell cultures after 3 to 4 days. After 1 to 3 weeks, the cell cultures were restimulated by irradiated peripheral blood mononuclear cells from the patient. These cells were collected by Ficoll-Hypaque centrifugation19 and used as antigen-presenting cells for restimulation together with further addition of tumor homogenate. To verify clonal expansion of T lymphocytes, Vβ repertoire analyses were performed in a few cases. To test for functionality, single cell suspensions were investigated for Th1 and Th2 cytokine production, IFN-γ, and IL-4. After an average period of 4 weeks, expanded cells were counted, evaluated by FACS, and investigated for the exclusion of malignant cells and bacteria before they were considered ready for transfusion. Cells were washed three times in .9% saline solution (Natriumklorid Baxter Viaflo 9 mg/mL, Baxter) containing 2% human serum albumin (Baxter) and prepared in a sterile erythrocyte transfusion bag at 4 × 106 cells/mL. The transfusions took place at the surgical ward as an intravenous transfusion over the course of 1 hour. Our criterion for adoptive transfer is a transfusion of lymphocytes to a patient, regardless of the number of transferred cells. In our previous studies (M.K., unpublished data) of adoptive transfer originating from sentinel nodes, on average, 71 million clonally expanded autologous tumor-reactive lymphocytes were transfused to each patient, and in that pilot study, the disease of all 16 patients responded to therapy. We do not know the optimal number of cells to be transfused for the best or maximum immune response, but according to our experience, 39.5 million transfused cells (the average number of transfused cells in the metinel node study) may be sufficient. The explanation may be that the CD4 cells proliferate and promote division of effector T cells and memory T cells when they are stimulated by their antigen (which is derived from the tumor). The cell expansion continues in the patient. RESULTS Metinel Nodes Lymph nodes draining liver metastases, intra-abdominal recurrences, and subcutaneous metastases were identified in all 19 cases, with an average number of 2.6 nodes found per patient (range, 1–5) (Table 1). We denoted these metastasis-draining lymph nodes metinel nodes. Seventeen (40%) of the analyzed metinel nodes were positive for metastatic disease. In 4 (25%) of 16 patients where data were available, all metinel nodes were positive for metastatic disease (range, 2–5), and in 8 (50%) of 16 patients, all metinel nodes (range, 2–3) were negative for metastatic disease. Tracer was injected around liver metastases (n = 9), intra-abdominal recurrences (n = 3), or close to subcutaneous metastases (n = 7). Draining metinel nodes appeared visually blue (Fig. 1A) within 3 to 10 minutes after the injection of PB or were found to be disease positive by lymphoscintigraphy (Fig. 1B). No complications due to the lymph node mapping procedure were observed. The method seems valid for detecting the draining metinel node regardless of tumor type because the following were identified by this method: colorectal cancer (n = 7), malignant melanoma (n = 4), ovarian cancer (n = 2), breast cancer (n = 2), pancreatic cancer (n = 1), squamous cell cancer (n = 1), cholangiocarcinoma (n = 1), and leiomyosarcoma (n = 1). Thus, we conclude that lymph nodes draining liver metastases or local recurrences can readily be identified by this simple, safe, and quick procedure. Fig. 1.(A) After intraoperative injection of Patent blue dye around liver metastasis in patient 18, the metinel node in the hepatoduodenal ligament is blue. (B) Preoperative percutaneous injection of 4 × 15 MBq Tc-nanokolloid around ovarian cancer groin local recurrence (patient 8) demonstrates a medially and distally situated metinel node after 10 minutes. In all cases of surgery for liver metastases, lymph nodes draining the area of the metastases were found within the liver hilum or hepatoduodenal ligament. The average number of metinel nodes from liver metastases were 2.4, and 7 (32%) of 22 of the analyzed metinel nodes were positive for metastatic disease. Two patients underwent surgery to treat intra-abdominal local recurrences from colon cancer (patients 1 and 14); resections of the recurrence and reresection of the bowel en bloc were performed. One patient with pancreatic cancer (patient 9) underwent tumor-reducing surgery to treat a local intra-abdominal recurrence after a previous Whipple operation. Four patients underwent surgery, with lymph node biopsies performed, to treat subcutaneous metastases from disseminated malignant melanoma (patients 3, 10, 11, and 16), and one patient each underwent surgery to treat subcutaneous metastases from disseminated breast cancer (patient 12), groin lymph node metastasis due to ovarian cancer (patient 8), and subcutaneous metastases related to squamous cellular carcinoma of the tongue (patient 13) (Table 1). Immunology We previously demonstrated the presence of tumor-reactive T lymphocytes in primary tumor-draining sentinel nodes from patients with colon19 and urinary bladder cancers.20 Here, we investigated whether signs of immune recognition also occurred in metinel nodes draining metastases. Single cell suspensions were collected from identified metinel nodes and investigated by flow cytometry for the presence of CD4+, CD8+ T lymphocytes, CD19+ B lymphocytes, and CD56+ natural killer (NK) cells (Table 2). Most of the metinel nodes were predominated by CD4+ T helper cells, with an average CD4+/CD8+ ratio of 2.3 (range, .1–6.6). However, five patients (patients 3, 6, 11, 12, and 19) displayed an increase in the fraction of CD8+ cytotoxic T cells. Two of these patients had malignant melanoma. The metinel nodes contained an average of 9.5% (range, .1–55) CD19+ B lymphocytes and an average of 2.1% (range, 0–18.9) CD56+ NK cells. Table 2.Immunology of single cell suspensions collected from metinel nodesPatientTime in culture (d)No. of cells at start (millions)No. of cells at end (millions)Ratio of cells start/endStart (% gated), ratio CD4/CD8 and CD19/CD56End, ratio CD4/CD8 and CD19/CD56Ratio CD4/CD8 startRatio CD4/CD8 end13110080.824/11 and 55/−24/16 and −/−1.81.52334240.9551/15 and 13/−24 and .12/−3.41.5328–––8/39 and 1/−–.2–42065.4––37/9 and −/−–4.1–543130–1.347/7.1 and 28/1.373/22 and .09/.176.63.3665––.2/1.6 and .4/.4–.125–765.7.0216.4/6.5 and 24.2/1.6–2.5–85242.6––15.9/7.6 and 9/3.4–2.1–91512––––––1074166.85.0340/7 and .5/.3–5.7–11352091409.312.8/16.3 and 1.9/0–.8–12312852.0116.4/31.8 and .1/0–.5–13243––––––1432301.05––––154836––0/.1 and 0/0–0–164520––28.25/6.95 and 1.15/0–4.1–1720894.045.8/2.6 and 4.2/.234.2/30.4 and 7.6/20.12.21.11829674.069.7/3.8 and 1.3/.1–2.6–194311151.3612.7/15.8 and 3/18.985/3 and .9/8.4.828.3 CD4+ cells are crucial for initializing both the cellular and humoral immune response toward, for example, tumors. CD4+ cells recognize antigens expressed on major histocompatibility complex II and have the capacity to catalyze the immune response in B cells, Antigen presenting cells (APCs), and CD8+ cells. CD4+ cells have, compared with CD8+ cells, a long-term capacity of acting both as effector T cells and as memory cells. CD8+ cells are mainly effector cells and will act during a few weeks, but further recruitment of CD+8 cells is maintained by CD4+ cells. The expansion procedure aims to increase the number of CD4+ cells, and the CD4+/CD8+ ratio can be regarded as a measurement of how well this can be achieved. The average ratio of CD4+/CD8+ cells at the start of the procedure was 2.6 for patients who underwent successful expansions and 2.1 for patients who underwent unsuccessful expansions. Consequently, the CD4+/CD8+ ratios were 24% higher for patients with successful expansions. To test for recognition of tumor antigen, the number of metinel node–acquired lymphocytes from nine patients (patients 1, 2, 5, 7, 12, 14, 17, 18, and 19) were increased successfully by the addition of low dose IL-2 and tumor homogenate. An average of 97 × 106 cells (range, 5–285 × 106) were collected, and the cells were expanded on average for 32 days before transfusion. At the time of transfusion, a mean of 39.5 × 106 tumor-reactive T cells (range, .7–170 × 106) were returned (Table 2). In patient 7, cells were expanded and prepared for transfusion, but the patient suddenly died without receiving any cells. No side effects from transfusion of expanded T lymphocytes were seen, and the patients were discharged from the hospital on the same day of the transfusion. The expansions were successful in five of seven patients who had colorectal primary tumor; one of two in patients with ovarian cancer and breast cancer; and one patient each with cholangiocarcinoma and leiomyosarcoma. The following expansions failed in all patients: malignant melanoma (n = 4), pancreatic cancer (n = 1), and squamous cellular cancer (n = 1). An analysis of the CD4 Vβ repertoire from patient 11 was investigated by flow cytometry of metinel node–acquired lymphocytes (Fig. 2) and revealed an increase and clonal expansion of Vβ families 7.1, 13.2, and 20. In addition, analysis of the Vβ repertoire in two independently cultured metinel nodes derived from one breast cancer metastasis (patient 12) (data not shown) demonstrated clonal expansion after 12 days’ short-term culture of the same T cell receptor families in both nodes (Vβ 4, Vβ 9, Vβ 20, and Vβ 21.3). We conclude that cultures of metinel node–acquired lymphocytes expand in the presence of autologous tumor extract and IL-2. Fig. 2.In patient 13, the T cell receptor Vβ repertoire was investigated by flow cytometry in two sentinel (nodes at the day of operation and after in vitro cell culture (43 days). Clonal expansion of Vβ families 7.1, 13.2, and 20 were detected in the CD4+ T cell population. Cells from patient 8, stimulated at the end of the expansion, demonstrated a high production of IFN-γ of >1000 pg/mL (Fig. 3) and no detectable IL-4 (data not shown), indicating a Th1 response. In an additional patient (patient 5), the stimulated IFN-γ secretion was 155 pg/mL while only a low IL-4 production of 30 pg/mL was found, again demonstrating a Th1-predominant response pattern. Fig. 3.Amount of interferon gamma secreted into the supernatant measured by enzyme-linked immunosorbent assay. Data presented are for patient 7 after 35 days of in vitro cell culture. According to more detailed analyses of some cases, we found that some T cells expressed Fox P3, indicating that these cells in fact were T regulatory cells. DISCUSSION We show that it is possible to find the first lymph nodes draining various types of metastases by lymphatic mapping by means of dye or radioactive tracers. We have named these metastasis-draining lymph nodes “metinel nodes” to show both the analogy and difference to the well-known term “sentinel node,” which means the first node to receive lymphatic drainage from a primary tumor. In 19 studied cases, we demonstrated three different ways of identifying these nodes by injection of tracers subserosally around liver or intra-abdominal metastases, close to subcutaneous metastases, or into local lymph node metastases. The fact that it is possible to expand the metinel node–derived lymphocytes via in vitro expansions during several weeks of time, together with our analysis that they contain lymphocytes that show clonal expansion toward tumor antigens and produce high levels of IFN-γ in the expansions, are evidence that they contain tumor-reactive lymphocytes. These cells are mainly T-helper 1 cells that have developed an immunological response toward the metastatic cells. We suggest that these T cells may be used in future trials of adoptive cellular therapy of disseminated solid cancer. Studies have demonstrated that tumor growth beyond a certain size required angiogenesis21 and that the amount of neovascularization peritumorally was a predictor of metastatic disease.22 Far less is known about the specific factors regulating the lymphangiogenesis around a tumor and its possible importance, much as a result of the difficulty of discriminating between lymphatics and blood vessels. However, during recent years, the molecular pathways signaling for lymphangiogenesis have been described, and these studies have revealed that lymphangiogenesis is a major component of the metastatic process. The lymphatic capillaries consist of thin-walled, low-pressure vessels that drain the interstitium. When the surrounding interstitial fluid pressure increases, the walls of the lymphatics are stretched and open to passage of cells or fluids. The lymphatic capillaries are joined into major lymphatic collecting vessels and filtered through at least one lymph node before entering the venous circulation.23 After entering the node, approximately half of the lymphatic fluid continues directly into the blood before the rest drains into an efferent lymphatic vessel.24 A malignant tumor causes a peritumoral increase of lymphatic vessels mediated through VEGF. Studies have shown that high levels of the cytokine cascade of VEGF-C and VEGF-D promote the tumoral lymphangiogenesis and that inhibition of their joint receptor, VEGFR-3, suppresses the effects. Kawakami et al.25 showed that high levels of VEGF-C and its receptor, VEGFR-3, correlated with increased lymph node metastases and lymphatic invasion in human colorectal cancer. White et al.26 concluded that patients operated on for colon cancer had an overexpression of VEGF-D, but not of its receptor VEGFR-3, which correlated with a decrease in disease-free interval and overall survival. A study based on 110 patients with breast cancer showed that lymph vessels were present more often in metastatic axillary lymph nodes (85%) than in nonmetastatic lymph nodes (25%),18 and that intranodal and perinodal lymphatic endothelial cell proliferation fractions were higher in metastatically involved lymph nodes. Taken together, all these findings support lymphangiogenesis in metastases. Thus, a tumor causes an upregulation of lymphangiogenic factors that leads to an increase in peritumoral lymphatic vessels in both tumors and metastases. It is likely that these newly developed lymph vessels connect to the preexisting lymphatic vessels and lymph nodes. Shed tumor cells may invade these newly formed vessels, and the dissemination of tumor cells is facilitated. The present study shows that this process can be visualized by lymphatic mapping, and the first draining lymph node from the metastasis, the metinel node, may be identified. Animal studies showed that metastases had the capacity to metastasize. Mice inoculated with pulmonary venous blood from mice having pulmonary metastases developed disseminated cancer disease after injection into the tail vein, showing that pulmonary metastases shed viable circulating cells.16 Similarly, Hoover and Ketcham15 demonstrated that mice who had undergone amputation of their primary tumors and now only had pulmonary metastases experienced spread of the tumor cells specifically into the lungs after parabiosis. In surgery for colorectal liver metastases, it is not standard to perform a biopsy of perihepatic lymph nodes. Nakamura et al.27 reported a 4-year survival of 40% in node-positive patients, but the study cohort was small. Most studies identify few 5-year survivors after liver resection for colorectal liver metastases involving positive perihepatic lymph nodes.28 These lymph node metastases are unlikely to be of lymphatic origin from the primary colonic tumor; instead, they may be explained by hematogenous spread to the liver followed by lymphogenic spreading from the liver metastases to the perihepatic lymph nodes.29 Correct perihepatic lymphatic staging is beyond dispute, not only because the increased morbidity in patients having perihepatic lymph node metastases, but also because of the improvements in systemic therapies. In the nine cases of resected liver metastases in our study, all metinel nodes were found among perihepatic nodes within the hepatoduodenal ligament or liver hilum. Most of the liver is drained by deep hepatic lymphatics, the upper inner part of the liver drains to nodes around the end of inferior caval vein, and the lower inner part drains to hepatic nodes through the liver hilum.30 The superficial part of the liver lymphatic drainage is divided into two main directions; the posterior surface drains into pericaval nodes, and the inferior, anterior, and superior surface drains into hepatic nodes through the liver hilum. In all cases of resected liver metastases, the injections of PB were performed subserosally on the inferior, anterior, or superior surface of the liver. Our identified perihepatic metinel nodes were located corresponding to the superficial lymphatic drainage of the liver. Despite the prognostic importance of these perihepatic lymph nodes, we have only found one article in the literature describing lymphatic mapping originating from liver metastases. Kane et al.14 demonstrated in a study of 11 patients with colorectal liver metastases that it was possible to perform lymphatic mapping by the intraoperative injection of isosulfan blue dye in a peritumoral and subcapsular manner. The basic premise was that the occurrence of extrahepatic metastases within the normal lymphatic drainage of the liver was considered to be remetastases from the colorectal metastases. They found blue-colored nodes in seven patients (54%), all within the liver hilum, falciform ligament, or gallbladder fossa. In three of these patients, the nodes were not detected by the surgeon before the mapping procedure. Like our results, they concluded that hepatic lymphatic mapping was safe, rapid, and simple technique to perform in humans. The importance of lymphatic mapping in liver surgery for metastases was underlined by Grobmyer et al.,31 who concluded that routine lymph node sampling had a low yield in patients without suspected perihepatic disease by computed tomographic scan or positron emission tomography, or at surgical exploration. Further, another study demonstrated that microscopic perihepatic positive lymph node disease is associated with the same poor prognosis as in macroscopic positive disease and suggests, therefore, that lymph node dissection should be systematic.32 Lymphoscintigraphy has been used to find sentinel nodes from primary tumors,33 but to our knowledge, it has not been reported as a tool to locate lymph nodes draining subcutaneous metastases. The radioactive tracer facilitated the intraoperative identification of the metinel nodes by a gamma probe used as a complement to PB. In those cases, the lymphoscintigraphy was made days before surgery, and new injections of radioactive tracers were provided on the day of surgery. One to three metinel nodes were detected in our six patients. In the late 1950s, the immunosurveillance hypothesis was introduced34 supporting the fact that tumor cells possessed new antigenic potentials and provoked an immunological reaction that could eradicate a tumor completely. In the last decade, the hypothesis has been proven in mice and humans. The role of the immune system in cancer diseases is demonstrated by immunodeficient patients having both an overrepresentation of virus-induced malignancies and an increased probability of developing cancer of nonviral cause. Cardiac transplant recipients have, for example, been shown to have a 25-fold higher prevalence of lung tumors than the general population.35 Much data also supports the fact that both the cellular and the humoral immune systems develop immune responses against tumors. Bevacizumab, a monoclonal antibody against VEGF, functions as an inhibitor of angiogenesis, inhibits growth of human tumor cells in mice, and prolongs the overall median survival in human metastatic colorectal cancer.36 The presence of tumor-reactive lymphocytes in sentinel nodes19,20 is an example of the function of the cellular immune system and a positive prognostic factor for survival in colorectal and ovarian cancer.37–39 In our earlier study, we made a single cell suspension of the lymphocytes from sentinel nodes. They were expanded in vitro, stimulated by tumor homogenate, IL-2, and antigen-presenting cells. In the present expansions, we proved it was possible to find tumor-reactive lymphocytes in metinel nodes and to perform adoptive immunotherapy based on them. The preliminary results seems promising, with total or partial regression of some liver and lung metastases (M.K., unpublished data). In this study, the expansions were performed in exactly the same way as in our previous studies, except that the source of the lymphocytes was derived from lymph nodes draining metastases—that is, metinel nodes. The fact that we succeeded in nine cases in proliferating the lymphocytes via in vitro expansions over more than a month is evidence for the presence of tumor-reactive lymphocytes. Because the expansions were continuously stimulated with autologous tumor homogenate and antigen-presenting cells together with IL, only lymphocytes that were a priori stimulated by antigens derived from the tumor could proliferate and survive for such a long time. Figure 2 demonstrates the Vβ repertoire in CD4-positive cells in two metinel nodes in patient 11, thus showing which types of T-helper cell receptors were found at the end of expansion. The figure shows clonal expansion of Vβ 7.1, 13.2, and 20, demonstrating the presence of tumor-reactive lymphocytes toward three different types of tumor antigens. These collective facts point out the presence of tumor-reactive type 1 T-helper lymphocytes in metinel nodes. T helper lymphocytes play a central role in initiating the adoptive immune defense. In 9 of 19 patients, the cells survived until the time for transfusion, and 8 patients underwent intravenous adoptive autologous T cell transfusion. Approximately half of all patients who underwent the expansion procedure died as a result of a combination of too few cells at the start and the initial presence of immunosuppressive factors that not could be overcome. Six of eight patients who received transfusions are still alive, although the follow-up time varies (Table 1). Our results are encouraging because the prognosis in these type of cases is usually poor. In this study, we have shown that it is possible to find the draining lymph nodes from different types of metastases. We named these nodes “metinel nodes,” and they contain tumor-reactive lymphocytes, preferentially T-helper cells with Th1 response. We have also demonstrated that it is possible to proliferate these cells in vitro and, if the expansion procedure is successful, to use the cells for adoptive immunotherapy.

Eur_Arch_Otorhinolaryngol-3-1-2099162

Tonsillar metastasis of oesophageal adenocarcinoma

In the literature less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported. Tonsillar metastasis of adenocarcinoma of the oesophagus has not been reported previously. We report a case of a 57-year-old male with a primary adenocarcinoma of the distal esophagus with a metastasis in the right palatine tonsil. Metastatic tumour involving the palatine tonsil is rare. The route of dissemination remains unclear. Hypothetically the dissemination of tumour cells could be lymphogenic or secondary by transportation due to vomiting or at the time of endoscopy, but most likely represents haematogenous spread. Introduction The vast majority of cancers in the head and neck region are of epithelial origin. Lymphoma is the next most common malignancy in this area. Metastases in the head and neck other than lymph node metastases are rare. The occurrence of tonsillar metastases from non-haematological malignant neoplasms is very rare [10]. We present a patient with metastasis in the right tonsil of a primary adenocarcinoma of the distal oesophagus. Case report A 57-year-old man was referred to our department of otolaryngology and head and neck surgery, because of dysphagia, pain and swelling of the right palatine tonsil. He also complained of frequent vomiting. Five months previously, an adenocarcinoma of the distal oesophagus with lymph node and liver metastases had been diagnosed. He received chemotherapy with combined capecitabine and oxaliplatin, which was stopped after two cycles because of disease progression. On physical examination an enlarged and ulcerating right palatine tonsil was seen (Fig. 1). A suspicious ipsilateral cervical lymph node of 2 × 4 cm was found at level II. A biopsy of the tonsillar tumour was performed and revealed intestinal-type adenocarcinoma. The histopathological picture corresponded with that of the oesophageal tumour (Fig. 2). Tonsillectomy was not performed because of severe clotting disorders attributed to liver metastases. The patient died 4 weeks after his first visit to our department. Fig. 1Enlarged right palatine tonsil with ulcerationFig. 2Biopsy right tonsil: adenocarcinoma (intestinal type) Discussion Second primary tumours relatively often involve the head and neck region. These tumours develop in head and neck cancer patients due to their common factors in carcinogenesis, e.g., tobacco, alcohol and, probably, genetic susceptibility [4]. However, this common aetiology applies for squamous cell carcinoma and not for adenocarcinoma. Simultaneous primary adenocarcinomas are not frequently found. In our patient the corresponding histopathology indicates a common origin, suggesting metastatic disease. Primary oesophageal adenocarcinoma is a relatively uncommon tumour in western countries [9]. The pattern of metastasis of oesophageal adenocarcinoma can be lymphogenic, haematogenic and by transportation of tumour cells by regurgitation or endoscopes [2, 10]. Lymphogenic spread of distal oesophageal adenocarcinoma generally involves the regional nodes in the lower posterior mediastinum, the paracardiac region, along the lesser curvature and the left gastric artery [6]. In case of extensive lymphogenic spread, lymph-node metastases can be found in the tracheal bifurcation region, the proximal mediastinum, coeliac axis and supra-clavicular region [6]. Lymphogenic spread of adenocarcinoma of the distal oesophagus and the gastro-oesophageal junction is present in 70% of the patients at the time of diagnosis of the primary tumour [9]. Haematogenous dissemination occurs at a later stage and is less frequent. Liver and lung are the most common organs involved [6]. Metastatic tumour involving the palatine tonsil is distinctly rare. These metastases represent only 0.8% of all tonsillar tumours [5]. Bilateral metastases of gastric carcinoma to the tonsils have been described [7]. Less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported [3, 7]. The most frequent tumours metastasizing to the tonsil are melanoma, lung carcinoma, breast carcinoma and renal cell carcinoma [3]. Eleven cases were associated with gastric adenocarcinoma [2, 7, 8]. Tonsillar metastasis of adenocarcinoma of the esophagus has not been reported previously. The occurrence of a tonsillar metastasis as the first manifestation of an occult neoplasm is very unusual [8]. In the majority of cases the occurrence of a tonsillar metastasis becomes manifest after the diagnosis of the primary tumour and is generally part of a widespread systemic disease [2]. Therefore, mean survival time for patients with tonsillar metastases is short, with a mean of 9 months, irrespective of the histological type of primary tumour [3]. In this case there are four hypothetical pathways for metastatic spread to the tonsils: Haematogenous dissemination of tumour from an esophageal carcinoma may occur via the portal circulation, the liver, the pulmonary circulation and systemic arterial vessels, thus reaching the palatine tonsil. A second haematogenous route may be through the paravertebral plexus of Batson, bypassing the lungs [1]. Retrograde flow via this valveless plexus occurs during elevated intra-abdominal and intrathoracic pressure, as is brought about by straining, coughing or as in this case, vomiting [1]. A third route of metastasis may be through the thoracic duct followed by retrograde cervical lymphatic spread to the tonsil [3]. This route of dissemination is very rare and is mostly seen in cases of seminomas [3]. Finally, some authors believe the possibility of metastasis by direct translumenal implantation, which might be favoured by a previous lesion in the mucosa of the tonsil [2]. The tumour cells would be transported secondary to regurgitation from the oesophagus [8], or as in this case possibly because of frequent vomiting, or at the time of endoscopy [2]. In conclusion, when finding a tumour of the tonsil a metastasis from a distant primary malignancy should be considered, although this is very rare. The route of dissemination eventually remains unclear.

Eur_J_Health_Econ-_-_-1388078

Defining the “Health Benefit Basket” in nine European countries

This article identifies and analyses a framework for “health baskets,” the taxonomy of benefit catalogues for curative services, and the criteria for the in- or exclusion of benefits in nine EU member states (Denmark, England, France, Germany, Hungary, Italy, The Netherlands, Poland and Spain). Focusing on services of curative care, it is found that the explicitness of benefit catalogues varies largely between the countries. In the absence of explicitly defined benefit catalogues, in- and outpatient remuneration schemes have the character of benefit catalogues. The criteria for the in- or exclusion into benefit catalogues are often not transparent and (cost-)effectiveness is applied only for certain sectors. An EU-wide harmonization of benefit baskets does not seem realistic in the short or medium term as the variation in criteria and the taxonomies of benefit catalogues are large but not insurmountable. There may be scope for a European core basket. The Kohll/Decker judgments of the European Court of Justice and more recently the Geraets-Smits/Peerbooms cases, have demonstrated that health services can no longer be regarded as operating in isolation from other EU member states [1]. Increasingly there are flows of patients from one member state to another, sometimes due to individual choice, sometimes induced by sickness funds or even by health ministries. Such movements have the potential to stimulate competition between health care systems of the member states. However, the average volume of imported health care services in the EU has been rather small, with approx. €1.99 per person as yet (1998) [2]. One major reason for this low volume of cross country flows is the lack of accurate information as the basis for competitive behavior. Actors in one health care system, for example, sickness funds, do not have sufficient information on benefit catalogues and prices for benefits in other health care systems in order to induce in- or exports of health services. So far research has mainly focused on health services for selected indications or on the comprehensiveness of services, but information on the benefit catalogues, their taxonomy, and inclusion criteria in each country is widely lacking [3, 4, 5, 6, 7]. As one result of the project “Health Benefits and Service Costs in Europe–Health BASKET” funded by the European Commission, this article identifies and analyses the framework for health baskets, the taxonomy of benefit catalogues for curative services, and the criteria for in- or exclusion of benefits in nine EU member states (Denmark, England, France, Germany, Hungary, Italy, The Netherlands, Poland and Spain). It focuses on services of curative care, although an in-depth analysis of all other health sectors is also available as part of the project results (Reports of the EU Health BASKET project on health benefit baskets in each of the nine countries are available at: http://www.ehma.org/projects/healthbasket.asp). One of the main hypotheses of the study is that in the absence of explicit benefit catalogues, inpatient, and outpatient remuneration schemes have the character of (less explicit) benefit catalogues. After introducing the terminology and methodology of the analysis, different regulatory frameworks for the definition of the overall benefit basket in each country are compared and analyzed. Finally the taxonomy and the inclusion criteria of benefit catalogues and their substitutes for curative services are compared and analyzed. Methodology In general we differentiate between the terms “benefit basket” (also “benefit package”) and “benefit catalogues.” The benefit basket refers to the totality of services, activities, and goods covered by publicly funded statutory/mandatory insurance schemes (social health insurance, SHI) or by National Health Services (NHS). In contrast, we define benefit catalogues as the document(s) in which the different components of the benefit basket are stated in detail, i.e., which enumerate the services, activities, and goods in a more detailed way, listing single interventions (i.e., specific technologies). Thus a benefit basket may be further defined by one or more benefit catalogues. Basically, the coverage of a given population for health services can be characterized in three dimensions: “breadth” as the extent of covered population, “depth” as the number and character of covered services, and “height” as the extent to which costs of the defined services are covered by prepaid financial resources as opposed to cost-sharing requirements. Services or goods which are not covered at all (i.e., with a “copayment” of 100%) are not considered to be part of the benefit basket. Figure 1 summarizes the three dimensions of a benefit basket. This article mainly focuses on the coverage of services (depth of benefit coverage) [8].Fig. 1 Three dimensions of coverage An open questionnaire was developed to explore the different benefit baskets and their underlying benefit catalogues of the participating nine countries, and this served as guide in compiling standardized country reports. To scan the different health systems in search for existing benefit catalogues we followed the framework of functional categories of “health care services and goods” proposed by the OECD in its “System of Health Accounts” (Table 1) [9]. As an initial step the researchers in each country were asked to identify the decision-making processes on the benefit basket as a whole in their country. As a second step they were asked to provide a detailed description of the existing benefit catalogues for each functional category, the actors involved in decision making, and the decision criteria.Table 1 Framework of health care functional categories, system of health accountsHC.1 Services of curative care  HC.1.1 In-patient curative care  HC.1.2 Day cases of curative care  HC.1.3 Out-patient care    HC.1.3.1 Basic medical and diagnostic services (primary health care)    HC.1.3.2 Out-patient dental care    HC.1.3.3 All other specialized care    HC.1.3.9 All other out-patient curative care  HC.1.4 Services of curative home careHC.2 Services of rehabilitative care  HC.2.1 In-patient rehabilitative care  HC.2.2 Day cases of rehabilitative care  HC.2.3 Out-patient rehabilitative care  HC.2.4 Services of rehabilitative home careHC.3 Services of long-term nursing care  HC.3.1 In-patient long-term nursing care  HC.3.2 Day cases of long-term nursing care  HC.3.3 Long-term nursing care at homeHC.4 Ancillary services to health care  HC.4.1 Clinical laboratory  HC.4.2 Diagnostic imaging  HC.4.3 Patient transport and emergency rescue  HC.4.9 All other miscellaneous servicesHC.5 Medical goods dispensed to out-patients  HC.5.1 Pharmaceuticals and other medical non-durables    HC.5.1.1 Prescription medicines    HC.5.1.2 Over-the-counter medicines  HC.5.2 Therapeutic appliances and other medical durables    HC.5.2.1 Glasses and vision products    HC.5.2.2 Orthopedic appliances and other prosthetics    HC.5.2.3 Hearing aids    HC.5.2.4 Medico-technical devices    HC.5.2.9 All other miscellaneous medical durablesHC.6 Prevention and public health services  HC.6.1 Maternal and child health; family planning and counseling  HC.6.2 School health services  HC.6.3 Prevention of communicable diseases  HC.6.4 Prevention of non-communicable diseases  HC.6.5 Occupational health care  HC.6.9 All other miscellaneous public health services Establishing and shaping health baskets A generic pattern of establishing and shaping health baskets is found in most of the countries consisting of two levels. At the higher level, legislation passed by the national parliaments (sometimes even rooted in the country’s constitution, e.g., Poland [10]) establishes the general framework by enumerating the areas of health care, often similar to the OECD health care categories, included in the benefit basket. At the lower level, the benefit basket is further shaped by specifying certain procedures provided within each area of health care as part of the benefit catalogues. These catalogues contain recommendations as well as explicit in- or exclusions of services. The extent to and the way in which this shaping actually takes place varies considerably from country to country and within each country from sector to sector of health care. There are several ways used to establish benefit catalogues such as legislations passed by central or regional parliaments, decrees issued by national or regional governments, directives issued by self-governing bodies or by national and/or local authorities, and other documents considered as “quasilaws” (rules without legal character, e.g., clinical guidelines). In all countries studied a general definition of a benefit basket could be identified at the higher level. Depending on the organization of the system (NHS or SHI), the logic underlying the general definition of the benefit basket differs. In NHS countries the definition of a benefit basket refers to the specification of the duties and obligations of the purchasing organization (regional health authorities), and in SHI countries the issue of the benefit basket is more related to the specification of entitlements of the insured persons. However, it cannot be said that either NHS or SHI countries generally define their benefits more explicitly than the other. In most NHS countries a coherent legislation contains a list of the areas of care to be provided by the respective NHS, including “regional health services” as part of the benefit basket of regions in decentralized NHS systems, for example, Italy and Spain. Denmark represents an exception from this as the legislation consists of separate acts concerning the categories of hospital, primary and long-term care, and pharmaceuticals [11]. The level of explicitness varies considerably from country to country. The vaguest definition of a benefit basket may be that of the NHS Foundation Act (1946) in England and related posterior documents, where the Secretary of State for Health is legally required to provide services “to such extent as he considers necessary to meet all reasonable requirements” [12]. The responsibility for making available general practitioner, dental, ophthalmic, and pharmaceutical services lay until 2003 with health authorities and since then with Primary Care Trusts. In contrast, the framework of the Italian and Spanish benefit baskets, each of which was established in recently implemented legal documents, is structured in more detail [13, 14]. Common to all four, however, is the differentiation between hospital care and primary care, including specialist outpatient services, as well as preventive or health promotion services. The level of explicitness is not only heterogeneous across different countries, but also within the same country. For instance, in Spain some areas of health care may be further shaped by mentioning specific services or in some cases even specific procedures or technologies being in- or excluded from the benefit basket. Thus the definition of the benefit basket within NHS countries does not always follow a systematic approach of going into further detail. It rather addresses shortcomings perceived by decision makers of each health care system, not necessarily being based on evidence. Concerning the origin of more detailed benefit catalogues some similarities are observed across countries with similar organization of the health care system or with similar organizational demands (i.e., the need to reimburse providers in free practice). In the two countries with regionalized NHS, Italy and Spain, the motivation for defining a detailed benefit catalogue is to be seen in the autonomous status of regions. The national benefit basket can be regarded as a minimum basket of health services that must be provided by the regional health authorities. Therefore the regions in both countries are free to offer additional services not included in the national benefit basket. Health baskets in SHI countries stem from two different roots. On the one hand, SHI countries formulate the basket as an entitlement for persons insured under the respective statutory health insurance schemes. The main reason for this lies in the fact that SHI schemes have evolved from fragmented voluntary/statutory health insurance schemes not covering the whole population and only covering certain services, for example, sickness benefits. Statutory health insurance in Germany as well as in The Netherlands does still not cover the whole population [15, 16]. Therefore in SHI countries the health basket is also used to indicate the boundaries between those insured under the statutory health insurance and those insured under other schemes. The second root are fee catalogues which were originally more prevalent in SHI than in NHS countries due to the frequently used fee-for-service reimbursement system, at least in ambulatory specialist care. Another common characteristic of decision making on health baskets in most SHI countries is the role of the self-governing institutions. Within a general framework stipulated by laws, self-governing institutions (e.g., sickness funds, physician associations) specify the rules to explicit benefit catalogues or remuneration schemes with the character of benefit catalogues, limiting the scope of service provision. As observed in NHS countries, the level of explicitness also varies considerably among SHI countries. Poland has by far the most explicit benefit basket, which is even rooted in the constitution. Different legal acts define benefit catalogues specifying detailed procedures or even technologies being provided. At the other extreme, Germany probably has the vaguest legal definition of the benefit basket among the SHI countries, with the Social Code Book as a general framework. For instance, inpatient services in Germany are limited to only a certain extent by the diagnosis-related group (DRG) reimbursement system because they can be provided unless they are explicitly excluded by directives [17]. There is a trend in all SHI countries towards more explicit benefit baskets. In The Netherlands a system of diagnosis-procedure combinations has been introduced in 2005 defining procedures provided in inpatient care and by specialists in outpatient care [18]. In France a similar list is currently being prepared, while Germany and Hungary recently updated their outpatient remuneration schemes specifying certain procedures that are reimbursed by the statutory health insurance [17, 19]. Definition of benefit catalogues for curative services Benefit catalogues for inpatient care As in other sectors of the health care system, the provided inpatient services can either be listed as procedures being part of an explicit benefit catalogue or indirectly determined by grouping systems that serve remuneration purposes, for example, DRGs. France, Poland, and Spain have defined explicit benefit catalogues, grouped according to medical specialties, for inpatient services listing detailed procedures serving as positive lists [10, 14, 20]. While there are clear decision criteria for the inclusion of benefits in the benefit catalogue in France and Spain, no transparent criteria are applied in Poland. In all other countries DRG and other grouping systems serve as a tool for estimating resource consumption supporting budget assignations or providing the basis for remuneration. In general, they classify a single episode of care according to main diagnoses, comorbidities and main surgical interventions into one of a limited number of groups. Thus each classified episode is assumed to require more or less homogeneous resource consumption, independently of whether exactly the same items (e.g., drugs, diagnostics) are used. As, finally, monetary values are attached to the different groups, the use of drugs, diagnostics etc. is confined to the given monetary scope of each group. In our study four countries have introduced DRG systems: Italy [13], Germany [17], Hungary [19], and Denmark [11]. England and The Netherlands have DRG-like grouping systems called, respectively, health care resource groups and Diagnose Behandeling Combinaties. The main features of the DRG and other grouping systems are very similar (Table 2) [12, 18]. In each country the groups have been developed from data collected on resource consumption, clustering in homogeneous resource consumption groups. The observed variability in the number of classified groups in each system may be explained by the different criteria used to classify them (Table 2). Another possible explanation is the creation of additional groups in certain countries to increase the scope for the use of new innovative technologies (devices, procedures, and even drugs theoretically) which are considered to be worth the promotion (i.e., because of higher efficacy). This has been the case in Italy, where the regional health authority of Lombardy added three new DRGs to its system in order to specifically consider the use of drug-eluting stents and to encourage its utilisation [13].Table 2 Inpatient benefit catalogues or substitutesCountryName of taxonomy, year of introductionApplied geographical areaTaxonomy (and grouping criteria)Actors involved in decision makingCriteria for in-/ exclusion of benefitsDenmarkDRG system (Nordic-DRG); 1998National25 MDC (anatomical, etiological, other) with 589 DRG, special category for chemotherapy and radiotherapy. Grouping criteria: main diagnosis, procedures, sex, age, cause of dischargeLegislation at the national level (law, general framework)Need, budgetMinistry for the Interior and Health (approval)National Board of Health (DRG catalogue)County level (budgeting, hospital plan)Clinicians (priority setting in hospital)FranceCommon Classification of Medical Procedures (CCAM); 2005NationalCCAM lists reimbursable and excluded medical procedures thus being a positive and negative list; Grouping criteria: anatomic classification, medical specialtiesNational level (law, general framework)Effectiveness, safetyMinistry of Health (approval)National Union of Health Insurance Funds (in- and exclusion of services)High Health Authority (advisory body on in- and exclusion of services)GermanyG-DRG system (based on AR-DRG 4.1); stepwise 2003–2009National25 MDC (anatomical/ etiological/ other) with 876 DRG, 71 extra remunerations for special services (in 2005). Grouping criteria: main diagnosis, procedures, age, comorbidity, cause of dischargeLegislation at the national level (law, general framework)Services can be provided as long as they are not explicitly excluded. Adequate, expedient and cost-effectiveMinistry of Health (approval)Federal Joint Committee (exclusion of benefits)Institute for Hospital Reimbursement with assistance of the Committee of on HospitalPayment (DRG catalogue)Clinicians (priority setting in hospital)HungaryDRG system (US-DRG); 1993National26 MDC (anatomical, etiological, other) with 786 DRG. Grouping criteria: main diagnosis, procedures, age, comorbidityLegislation at the national level (law, general framework, budgeting)Costs, effectivenessMinistry of Welfare, Health division (DRG catalogue)National Health Insurance FundAdministration, especially (prepares decisions)Clinicians (priority setting in hospitals)ItalyDRG system (HCFA no. 10); 1995National reference list with regional differences23 MDC (anatomical, etiological, other) with 489 to 506 DRG, exceptional DRGs (e.g. liver and bone marrow transplantation). Grouping criteria: main diagnosis, procedures, age, comorbidity, cause of dischargeLegislation at the national level (law, general framework)Effectiveness, costsCentral level (national DRG catalogue)Regional level (redefines DRG catalogue, sets tariffs)Clinicians (priority setting in hospitals)The NetherlandsDiagnose Behandeling Combinaties (DBC) (DRG-like system); Jan. 2005National111,527 procedures regarding diagnosis and therapy (DBCs) are combined to 641 product groups. DBCs are on three different lists determining the status for tariff negotiations or excluding DBCs from the benefit package. Grouping criteria: medical specialty, product groupLegislation at the national level (law, general framework)Costs, effectiveness, Ministry of Health (decrees)DBC Maintenance Organization (DBC system)Clinicians (priority setting in hospitals)PolandGovernmental decrees and catalogue of benefitsNationalCatalogue lists all services covered under social health insurance scheme; services are linked to the respective regulation/law. Grouping criteria: area of care (e.g., hospital care), medical specialtyLegislation at the national level (law, general framework)/Ministry of Health (regulations decrees)National Health Fund (catalogue)SpainRoyal Decree 63/1995 / Law 16/2003 on cohesion and qualtiy of the National Health SystemNational with regional differencesServices are listed explicitly in decree. In some cases services are restricted to specific patient groups. Grouping criteria: area of care, medical specialtyLegislation at the national level (law, general framework)Safety, efficacy, efficiencyFederal Government (decree)Inter-territorial Council and Council of the State (inclusion of new benefits)Clinicians (provision of services relating to entitlements defined by decree)EnglandHealth care resource group (DRG-like system); stepwise 2004–2009NationalIn April 2004 there were only 48 HRGs in use. Grouping criteria: diagnosis, complexity, procedureLegislation at the national level (law, general framework)Costs, budgetMinistry of Health (catalogue)Primary Care Trusts (negotiate with providers on quantity and tariffs) The fact that specific procedures and technologies drive the development of DRG systems confirms our hypothesis that they serve as some kind of benefit catalogues. Technologies specifically mentioned in certain groups may not be used if less resource consuming alternatives are available. In this way a less specific DRG system may act as a hidden negative list of technologies which de facto are not available for beneficiaries of publicly financed care since the monetary value assigned to certain groups does not cover the actual resource consumption associated with its use. Furthermore, in most applied grouping systems certain groups (e.g., in DRG systems so-called surgical DRGs) are even defined by specific procedures or specific technologies, such as drug-eluting stents. These groups can therefore be considered as a kind of explicitly defined benefits, similar to a positive list. Thus it can be assumed that grouping systems are applied as substitutes for benefit catalogues, containing incentives to limit the provision of benefits for those services not being explicitly mentioned. Benefit catalogues for outpatient care In the outpatient sector benefit catalogues are, again, often replaced by grouping systems serving remuneration purposes. Although they generally seem to be more explicit than the inpatient catalogues, the explicitness varies even more than in the inpatient sector (Table 3). These different degrees of explicitness are due mainly to the applied remuneration schemes in each country. If physicians receive fixed budgets or capitations, the benefit “catalogue” (i.e., the procedures that they can offer) is indirectly restricted by the amount of money allocated to them. Therefore in these countries the benefit package for outpatient care is regulated rather implicitly through decrees issued by national or regional health authorities describing the obligation of physicians to provide those benefits that are considered necessary. Examples of these kind of implicit benefit catalogues are the “Health Insurance Treatment and Services Decree” for care provided by general practitioners in The Netherlands and the “General Medical Services Contract” in England. These decrees do not mention specific procedures, although in the case of The Netherlands, the general practitioners’ association, the Landelijke Huisartsen Vereniging, defined a basic general practitioner benefit package in the 1980s [21].Table 3 Outpatient benefit catalogues or substitutesCountryName of taxonomyApplied geographical areaTaxonomy (and grouping criteria)Actors involved in decision makingCriteria for in-/exclusion of benefitsBenefits, procedures explicitly excludedDenmarkHealth Care Reimbursement Scheme Fee ScheduleNationalServices are grouped according to medical specialty and for GPs additionally in basic, supplementary, laboratory and miscellaneous services. Each service has an item number. It is referred to the respective legislation decree specifying the benefit, certain goods, procedures or in rare cases indicationsNational level (law, general framework)NeedAlternative careMinistry for the Interior and Health (approval)Counties (budgeting, health plan)Health care Reimbursement Negotiating Committee and health professional associations (negotiate catalogue)FranceCommon Classification of Medical Procedures (CCAM)NationalLists all medical procedures reimbursable and excluded. Grouping criteria: anatomic classification, medical specialtiesNational level (law, general framework)Effectiveness, safetySpa treatments; cosmetic surgeryMinistry of Health (approval)National Union of Health Insurance Funds (in- and exclusion of services)High Health Authority (advisory body on in- and exclusion of services)GermanySHI-EBM, SHI-BEMA, SHI-BEL-IINationalServices are grouped according to the medical specialty allowed to provide the service. Each service is assigned a numeric code in accordance with the subjection of the catalogueNational level (law, general framework)Diagnostic and therapeutic expedience, medical necessity and cost-effectivenessOrthopedic services after the age of 18 yearsFederal Joint Committee (approval of new benefits)Valuation Committee (negotiates EBM)Dental Valuation Committee (negotiates BEMA, BEL-II)HungaryGovernmental decrees and reimbursement cataloguesNationalSimilar services are listed in groups. Governmental decrees relate to different areas of care (e.g., dental care, specialist services). Items in reimbursement catalogues are listed with the respective ICPM code and a point valueLegislation at the national level (law, general framework, budgeting)Costs, effectiveness–Ministry of Welfare (decrees, approval)National Health Insurance FundAdministration, especially (prepares decisions)Payment Codes Updating Committee (reimbursement catalogues)ItalyNational contract for primary care; decree on specialist outpatient servicesNational benefit package, regions include additional servicesContract for primary care describes obligations of GP. Individual services are not further itemized. Decree on specialist outpatient services lists services in three sections: available, availability restricted to specific indications, excludedGovernment at national level (sets decree, negotiates contract)Effectiveness, costsNonconventional treatments (e.g., acupuncture, phytotherapy); vaccination for traveling purposesRepresentatives of GPs (negotiate contract)Ministry of Health (transfers contract into law)Government at regional level (negotiates additional contracts)The NetherlandsHealth Insurance (Treatment and Services) Decree; Diagnose Behandeling Combinaties (DBC; DRG-like system); Jan. 2005NationalGP services are regulated in generic terms only by decree, DBC catalogue (111,527 DBCs) combine information on diagnosis and treatment for medical specialists. DBCs are on three different lists determining the status for tariff negotiations or excluding DBCs from the benefit package. Grouping criteria: medical specialty, product groupLegislation at the national level (law, general framework)Costs, effectiveness–Ministry of Health (decrees)DBC-Maintenance Organization (DBC-System)Physicians (priority setting)PolandGovernmental decrees and catalogue of benefitsNationalCatalogue lists all services covered under social health insurance scheme. Services are linked to the respective regulation/law. Grouping criteria: area of care, medical specialtyLegislation at the national level (law, general framework)–Vaccination; acupuncture, unless part of chronic pain managementMinistry of Health (regulations)National Health Fund (catalogue)SpainRoyal Decree 63/1995National with regional differencesServices are listed explicitly in decree. In some cases, services are restricted to specific patient groups. Decree lists services in 5 areas of care (e.g., primary care, specialized care, pharmaceutical care) which are further subdivided.Legislation at the national level (law, general framework)Safety, efficacy, efficiencyCosmetic surgery (transplantation of hair and nails); sex changeFederal Government (decree)Inter-territorial Council and Council of the State (inclusion of new benefits)Clinicians (provision of services relating to entitlements defined by decree)EnglandNational Service Framework NationalHealth Resource Groups are linked to procedures. Currently only 48 HRGs are in use. Guidelines recommend services to be used on certain indicationsLegislator at national level (law, general framework)Need, effectivenessCosmetic dental treatmentsGeneral Medical Services ContractNational, with possible variation at PCT-levelNHS Confederation and General Practitioners Committee (negotiate contract)Need, costsClinical GuidelinesNationalPrimary Care Trusts (PCT) (negotiate additional contracts)Need, costs, effectivenessNICE (clinical guidelines) In contrast, the countries remunerating providers on the basis of fee-for-service schemes need detailed lists of procedures or at least of service complexes (aggregated multiple procedures) to be able to negotiate on price and/or volumes. These lists can therefore be interpreted as substitutes for benefit catalogues, as physicians are usually reimbursed only for those items listed. The explicitness of these lists differs largely. Some countries issue detailed lists of all procedures to be performed by physicians (e.g. the “Common Classification of Medical Procedures” in France [20]) while other countries list service complexes making physicians responsible for the priority setting within such a service complex (e.g., SHI-EBM or SHI-BEMA in Germany [17] and the Health Care Reimbursement Scheme Fee Schedule in Denmark [11]). Interestingly, taxonomy and structure are very similar in all countries. For example, in Denmark, France, Germany, Hungary, and The Netherlands services are grouped according to medical specialty. Certain outpatient benefits are also linked to indications or special patient groups in Poland and Spain [10, 14]. The high degree of explicitness regarding the definition of the benefit package is also underlined by the diverse lists of excluded services, common in all countries. Exclusion practices vary from issuing negative lists (e.g., Spain, Poland) and directives of self-governmental institutions with the character of negative lists (e.g., Germany) to excluded services within the framework of the national law (e.g., England). However, most countries exclude similar benefits such as cosmetic surgery (if not closely linked to certain treatments), vaccination for nonstandard diseases (e.g., for traveling purposes), and certain nonconventional treatments (e.g., acupuncture). Benefits of dental care are either restricted to specific treatment methods or age groups (e.g., Germany). Discussion The analysis of benefits defined in the countries under study reveals that there is a clear trend towards a more explicit definition of benefit baskets and their benefit catalogues. Those countries which recently introduced new health care legislations, such as Italy, Poland, and Spain, have more explicitly defined benefit catalogues. Other countries with older health care legislations, for example, the UK’s English-NHS Foundation Act (1946) and Germany’s Social Code Book (1988) have rather implicitly defined benefit catalogues, but increasingly work with negative lists, based on evidence provided by independent institutions such as the Englisch NICE and the German Institute for Quality and Efficiency (IQWiG) [12, 17]. Apart from negative lists remuneration schemes, for example, DRGs and procedures catalogues used for grouping are more and more used as benefit catalogues. These developments indicate that all included countries move towards a more explicit definition of benefit catalogues. Explicitly defined benefit catalogues, however, require clear and transparent decision criteria for the in- or exclusion of benefits. Most countries officially state that (cost)-effectiveness is an important decision criteria. However, further inquiries often demonstrate that there is no rational process of reviewing the available evidence on specific procedures or technologies [22]. In reality the decision-making process is rather guided by lobbying activities of certain actors in the system. Especially those countries with very explicit benefit baskets, e.g. Poland, often lack transparency of decision criteria [10]. In contrast to this, countries with rather implicitly defined benefit baskets, such as England and Germany, define very transparent criteria for benefit exclusion, although lists with excluded services are minor compared to explicitly oriented countries. In addition, criteria such as cost-effectiveness and even effectiveness are often restricted to one or few sectors of the health care system, for example, pharmaceuticals or medical devices, and are not generalizable to all products or services [23]. In general the transparency of decision criteria must be improved in all countries in order to achieve accountability for all actors of the health care systems as well as consumers. This contribution, as well as the overall EU Health BASKET project, provides useful information for health care providers and industrial companies willing to invest in EU countries but do not have the necessary information on benefit baskets and their underlying decision-making processes. However, to improve the environment for investments and to provide confidence for foreign investors, public documents should be regularly prepared by each country giving a transparent overview of the health baskets and the decision-making criteria. The information provided will be beneficial especially to decision makers at all levels of health policy enabling them to compare different approaches of benefit definitions in order to develop their own position. The need for benchmarking will grow in line with the further development of cross boarder flows and the establishment of coherent benchmark criteria as part of the “Open Method of Coordination” initiated by EU policy makers [24, 25]. However, the project results also demonstrate that a harmonization of health baskets of EU countries, which in the view of certain decision makers could be the final stage after identifying best practice in benchmarking, is not realistic in the short or medium term since the definitions of benefit baskets vary substantially. Additionally, as shown in the cases of Italy and Spain, especially in NHS countries there is rather a trend towards more decentralization of decision making on benefits, delegating to regions the autonomy to offer certain benefits in addition to nationally defined health baskets [26, 27]. On the other hand, this could also mean that in future a minimum basket of health benefits may be defined by all countries on the national level, which could be harmonized on the EU level at a certain stage due to systems’ competition as a result of increased cross-border flows. Beyond this minimum basket, there could be regional variations reflecting differences in wealth and of preferences.

J_Fluoresc-3-1-1915606

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages. Introduction Among the biological macromolecules, proteins have become an important source of active substances for medical purposes. Insulin and growth hormone are two prominent examples of the many proteins used in medicine nowadays [1]. Many protein formulations can cause immune reactions in patients, and the presence of protein aggregates, even in minute amounts, has been identified as an important factor leading to the breaking of immune tolerance [2, 3]. Large protein aggregates are regarded as particularly immunogenic [4], and for this reason, analytical procedures for their sensitive and selective detection are important. The most widely used technique for analyzing aggregates in protein formulations is size exclusion chromatography (SEC) [2]. However, large protein aggregates of more than about 5 million Da are out of the separation range of SEC and can even be excluded from separation columns. Moreover, protein-column interactions may cause aberrant elution leading to erroneous interpretations. Field flow fractionation and analytical ultracentrifugation have a greater capacity than SEC for detecting high molecular weight protein aggregates [5, 6], but until now, these techniques are not readily available in every laboratory. Analysis by light scattering techniques [7] and Fourier transform infrared spectroscopy (FTIR) [8–10] is experimentally easy in the sense that these techniques do not rely on physical separation steps. However, the former is prone to artifacts when aggregates are present at low concentrations, and the latter is not very sensitive. Fluorescence spectroscopy with probes covalently labeled to proteins has been used for the direct detection of aggregated species [11], but this approach suffers from the problem that the modified protein may exhibit an altered aggregation behavior. Methods without protein labeling are rarer, but environment sensitive fluorescent dyes can be useful for detecting aggregated structures. For example, Congo red is used for the detection and identification of amyloid fibrils [12, 13]. Another potentially interesting dye is Nile red, which is established for the detection of hydrophobic areas on the surfaces of proteins [14–17]. Recently, this dye has been used for the detection of aggregates of an IgG1 recombinant humanized monoclonal antibody, as well as human calcitonin fibrils by fluorescence microscopy [18]. The fluorescence of Nile red is strongly dependent on the polarity of its environment. Decreasing polarity leads to a substantial blue shift of the absorption and emission maxima, as well as a marked increase of quantum yield and fluorescence lifetime. This has been related to the large dipole moment of the molecules in the excited state and the existence of a non-emissive twisted intramolecular charge transfer (TICT) state, which is more likely to be formed in polar than apolar environments [19, 20]. It has been proposed that the TICT state is non-emissive because of fast, non-radiative transition to the triplet state [19]. Nile red is a hydrophobic molecule with low solubility in water of ∼1 μM [21]. Therefore, it has the tendency to partition into hydrophobic phases [22]. β-Galactosidase from Escherichia coli is used as a model protein in the present study. This tetrameric enzyme (MW = 465 kDa) consists of four equal subunits. It contains four catalytic sites for the hydrolysis of lactose or other β-galactosides to monosaccharides. For maximal enzymatic activity, the presence of Mg2+ or, alternatively, Mn2+ is required [23, 24]. It has been shown that Mg2+ increases the unfolding temperature of β-galactosidase in aqueous solution. In the presence of Mg2+, heat induced unfolding is irreversible. It coincides with activity loss and leads to the formation of denatured monomers, dimers, and aggregates [25]. In this study, steady-state and time-resolved fluorescence spectroscopy experiments with Nile red are used for the sensitive detection of large β-galactosidase aggregates, present in solution at concentrations in the low nanomolar range. Denaturation and aggregation of native protein is induced by incubating aqueous solutions below and above the experimentally determined unfolding temperature of 57.4°C. The presence of aggregates in heat-treated solutions is confirmed by light scattering. Time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy (FCS) are used for analyzing the size of the aggregates detected by Nile red. The results obtained with Nile red are compared with results from SEC analysis. Materials and methods Materials Freeze-dried β-Galactosidase from Escherichia coli (protein content ∼80%) was purchased from Fluka (Buchs, Switzerland) and stored at −20°C. Phosphate buffer (pH 7.4) containing 55 mM sodium-phosphate and 10 mM MgCl2 was prepared with reverse osmosis water, and rendered isotonic by the addition of NaCl. Upon preparation, buffer solution was filtered through a 0.2 μm cellulose acetate filter (Schleicher and Schuell, Dassel, Germany), and stored at 4°C for at most 4 weeks. 0.2 μm HPLC filters were obtained from Alltech (Deerfield, IL). Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) was obtained from Fluka. A stock solution containing 0.25 mM Nile red was prepared with water-free dimethylsulfoxide (DMSO) and stored at −20°C. Methods Preparation of β-galactosidase solution A fresh solution of β-galactosidase in phosphate buffer was prepared before each experiment. The protein was dissolved in the buffer and subsequently, the solution was filtered through a 0.2 μm HPLC filter. Protein concentration after filtration was determined by measuring absorption at 280 nm, assuming an absorption coefficient of 2.09 cm2/mg [26]. Reproducibly, a protein concentration close to 0.10 μM was found after filtration. For incubation at elevated temperatures, 1 ml aliquots of β-galactosidase solution containing 0.10 μM protein were filled into 1.5 ml polypropylene Eppendorf tubes. They were heated for a defined period of time without shaking using an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany), and then cooled at room temperature. Steady-state fluorescence measurements Instrument and measurement settings Steady-state fluorescence measurements were performed with a Fluorolog FL3-21 spectrofluorometer (Jobin Yvon – Horiba, Edison, NJ), equipped with a short-arc xenon lamp. The slit openings of the excitation and emission monochromators were set to a bandwidth of 3 nm. The integration time was 0.05 s, and the signals were corrected for lamp intensity fluctuations by a simultaneously recorded reference signal. Each sample was measured 3–5 times, and the average spectrum was calculated. The sample temperature was controlled by a water bath with a temperature sensor connected to the sample holder. Samples were measured in quartz cuvettes (Hellma GmbH, Muellheim, Germany). Intrinsic tryptophan fluorescence Measurements of intrinsic tryptophan fluorescence of β-galactosidase were performed by exciting 0.10 μM protein solutions at 298 nm and scanning emission between 310 and 450 nm. To establish a protein denaturation curve, the sample temperature was increased from 25 to 70°C in one degree steps. At every temperature, the sample was equilibrated for 5 min and then measured. Nile red fluorescence Before every measurement, a fresh solution of 25 μM dye in phosphate buffer was prepared by dilution of Nile red stock solution. This solution was immediately added to heated 0.10 μM β-galactosidase solution, non-heated 0.10 μM β-galactosidase solution or phosphate buffer in a cuvette, and mixed well. The final concentration of Nile red was 18.75 nM, well below the solubility limit of Nile red in water of 1 μM [21]. Nile red fluorescence was measured by exciting at 550 nm and scanning emission between 565 and 750 nm. All measurements were performed at 25°C. Fluorescence spectra were measured immediately after addition of Nile red. However, Nile red emission in water remained unchanged for at least 25 min upon sample preparation. Time-resolved fluorescence measurements Instrument and measurement settings Time-resolved fluorescence measurements were performed by time-correlated single photon counting, as described in detail elsewhere [27]. Excitation source was a mode-locked titanium:sapphire laser (model Verdi V10, Coherent Inc., Santa Clara, CA), pumped by a CW diode-pumped, frequency doubled Nd:YVO4 laser (model Mira 900-D in fs mode, Coherent Inc., Santa Clara, CA), and tuned at 980 nm. An excitation wavelength of 490 nm was obtained by frequency doubling, using a second harmonic generation system (model 5-050 Ultrafast Harmonic Generation System, Inrad Inc., Northvale, NJ). The repetition rate of the excitation pulses was 3.8 MHz. The emission filters were a Schott 3 mm OG 530 nm cut-off filter (Schott AG, Mainz, Germany), combined with a Schott IL 611.6 nm (Δλ=11.0 nm) interference filter. Measurements were performed at 25°C, and consisted of repeated 10 s sequences of measuring parallel and perpendicularly polarized fluorescence emission until a maximum peak content of at least 50,000 counts in the data files was reached. Samples of β-galactosidase solution without Nile red were measured for background correction. To minimize background luminescence, all solutions, including buffer, were prepared with fluorescence spectroscopy grade water (Fluka, Buchs, Switzerland). For the performance of a deconvolution procedure in data analysis, the dynamic instrumental response of the experimental setup was recorded using the fast and single-exponential fluorescence decay of the reference compound erythrosine B in water. Data analysis was performed using the “TRFA Data Processing Package” of the Scientific Software Technologies Center (Belarusian State University, Minsk, Belarus) [28]. The decay of fluorescence intensity with time, I(t), could be described by an exponential model with three independent fluorescence lifetimes (τi) [29, 30]: where αi are the pre-exponential factors, which were normalized. I(t) itself was calculated from the measured parallel and perpendicular polarized fluorescence emission, III(t) and I⊥(t): G in the above equation represents the g-factor, which had a value of 1 for the used experimental setup. The course of fluorescence anisotropy with time r(t) was calculated from the measured intensities III(t) and I⊥(t): Fluorescence correlation spectroscopy measurements Instrument and measurement settings Fluorescence correlation spectroscopy measurements were performed with a system composed of a krypton-argon laser and an MRC1024 confocal laser-scanning microscope (Biorad, Hercules, CA), a TE300D inverted microscope (Nikon, Tokyo, Japan) with a water immersion objective lens (Plan Apo 60×, NA 1.2, collar rim correction, Nikon), and ALV-5000/E avalanche photodiode detectors (ALV GmbH, Langen, Germany). The 568 nm line of the krypton-argon laser was used for excitation of Nile red samples. Fluorescence emission above 585 nm was detected. Samples were prepared by mixing heated 0.10 μM β-galactosidase solution, non-heated 0.10 μM β-galactosidase solution or phosphate buffer with diluted Nile red stock solution in polypropylene Eppendorf tubes. Heated β-galactosidase solution was mixed with Nile red in the same tubes that were used for heating. The final concentration of Nile red in the samples was 18.75 nM. One hundred μl sample was transferred into a well of a 96 glass bottom well plate (Bio-one, Greiner, Frickenhausen, Germany). For measurements, the focal volume was positioned 100 μm above the bottom of the well. Raw data, i.e. fluctuations of fluorescence intensity with time, were collected during 30 s. The laser power was minimized to prevent photobleaching during this period. Every sample was measured ten times, and ten experimental autocorrelation functions were subsequently obtained from the raw data. Analysis of the experimental autocorrelation functions was performed by non-linear regression, based on a theoretical expression that describes the decay of an autocorrelation function, G(τ), in consequence of diffusion and fast, non-diffusive processes, such as excitation of fluorescent molecules to the triplet state [31–33]: Here, Ei is the relative emission rate of species i, and 〈Ni〉 the average number of molecules of species i in the focal volume. Mi(τ) is the mobility term for free, 3-dimensional diffusion: where τD,i is the diffusion time of fluorescent species i. The coefficients and z0 are defined, respectively, as the distances from and along the optical axis at which the excitation light intensity has dropped by 1/e2. These coefficients were determined from calibration of the focal volume with rhodamine green of known diffusion coefficient (2.8×10−6cm2 s−1). The coefficient F in Eq. (4) represents the fraction of molecules involved in a non-diffusive process, and τf is the decay time of this process. It is justified to assume the existence of a non-diffusive decay process, because the transition of excited state Nile red molecules to a non-emissive TICT state and to the triplet state has been proposed [19]. G(∞) is the value of the autocorrelation function at long time intervals (τ). To reduce the number of fitting parameters, Eq. (4) was simplified. For one diffusing species, it can readily be written as: The assumption of two or more diffusing species gives: where Before data analysis, the experimental autocorrelation functions were truncated at very short time intervals below 0.001 ms and at very long time intervals above 600–1000 ms. The autocorrelation functions were then normalized [34]: where G(0) is the start value of the autocorrelation function and G(∞) is its value at very long time intervals. G(0) and G(∞) were estimated individually for every experimental autocorrelation function by non-linear regression, using Eqs. (6) and (7) [34]. Normalization eliminated possible differences in experimental autocorrelation functions that were related to systematic errors, such as adsorption of dye molecules to the surfaces of the well plate during experiments. The above described normalization changes Eq. (6) to: Here, Gnorm(0) is the start value of the normalized autocorrelation function, which is expected to be one. The value at long time intervals, accounted for by Gnorm(∞), is expected to be zero. Equation (7) changes to: where Equation (12) shows that fi,norm represents an intensity-weighted fraction of diffusing species i. Equations (10) and (11) were used for analyzing the normalized, experimental autocorrelation functions by non-linear regression using the Levenberg-Marquardt algorithm. The calculations were performed with IGOR Pro V.4.04 (WaveMetrics, Inc., Lake Oswego, OR). When applicable, up to ten autocorrelation functions obtained from the same sample were concatenated into one data set and analyzed by a global analysis procedure contained in the software. The standard deviation (σ) of experimental autocorrelation functions, estimated from the measurement repeats, was used as the weighting factor during fitting [34]. Solutions with a heterogeneous distribution of particle sizes showed very complex decays of the experimental autocorrelation functions. These could not be adequately fit by the discrete multi-exponential model using a small number of diffusion times, and increasing the number of diffusion times above three in the model resulted in substantially increased error margins of the determined parameters. For such complex data, calculation of the distribution of diffusion times was performed by the maximum entropy method (MEM) using the MEMFCS software [35]. Translation of diffusion time (τD) to a diffusion coefficient (D) was done by the following relationship: Calculation of the hydrodynamic radius (Rh) was then possible using the Stokes-Einstein equation: Light scattering Detection of β-galactosidase aggregates in solution was done with static light scattering measurements using a fluorometer (Fluorolog FL3-21, Jobin Yvon–Horiba, Edison, NJ). The excitation and emission wavelengths were set to 400 nm, and measurements were performed at 25°C. Furthermore, the hydrodynamic radius of native β-galactosidase was determined by dynamic light scattering measurements of 0.10 μM protein solutions in phosphate buffer. Experiments were performed at 25°C with a CGS-3 goniometer system and an LSE-5003 correlator (ALV, Langen, Germany). The average hydrodynamic radius (Rh) of the β-galactosidase molecules was determined using the method of cumulants. Size exclusion chromatography (SEC) SEC measurements were performed using an Alliance 2695 system with a type 2487 dual wavelength detector and a type 2475 fluorescence detector (Waters, Milford, MA). A Superose 6 column with a separation range of 5 × 103 to 5 × 106 Da, and an exclusion limit of 4 × 107 Da (GE Healthcare Europe GmBH, Roosendaal, The Netherlands) was used. Phosphate buffered saline (pH 7.4) was used as an eluent, and the injection volume was 40 μl. Detection was by UV absorption at 210 nm or by fluorescence emission (λex 280 nm and λem 340 nm for intrinsic protein fluorescence; λex 550 nm and λem 611 nm for Nile red fluorescence). Before injection, samples were centrifuged at 10000 RPM for 5 min. A molecular weight calibration curve using chymotrypsin (25 kDa), human serum albumin (66 kDa and 132 kDa dimer), and thyroglobulin (670 kDa) as molecular weight standards was established. Results and discussion Heat denaturation of β-galactosidase The temperature induced denaturation of β-galactosidase was followed by measuring intrinsic tryptophan fluorescence emission of this protein (not shown). Denaturation started between 50 and 55°C and was completed between 60 and 65°C. An unfolding temperature of 57.4°C was obtained by fitting the denaturation curve with a model for a two-state unfolding process [36]. This is in good agreement with DSC experiments and activity assays performed under comparable conditions [25]. Heating β-galactosidase solutions above the unfolding temperature leads to irreversible aggregation of protein molecules [25]. Accordingly, a 0.10 μM β-galactosidase solution incubated at 62°C for 5 min was heavily aggregated and remained so upon cooling back to room temperature, as detected by light scattering (not shown). The aggregates formed under these conditions were too large to be detected by SEC. This is shown in Fig. 1a, which depicts SEC chromatograms of a non-heated β-galactosidase solution and of a β-galactosidase solutions that was heated at 62°C for 5 min. The former consists of peaks representing native, tetrameric β-galactosidase, oligomers of two and three β-galactosidase molecules, and even some aggregates of higher molecular weight. The latter consists only of a peak representing native, tetrameric β-galactosidase. However, the area under the curve of this peak is reduced to about 10% of the area under the curve of the corresponding peak of non-heated β-galactosidase solution. The apparent loss of protein was caused by the formation of large aggregates that were excluded from the SEC column.Fig. 1(a) SEC chromatograms of non-heated 0.10 μM β-galactosidase solution (solid line) and β-galactosidase solution incubated at 62°C for 5 min (dotted line). The peaks represent native (i.e. tetrameric) β-galactosidase (1) and oligomers of two (2) and three (3) β-galactosidase molecules. (b) Plot of the increase of scattered light intensity at 400 nm against incubation time of 0.10 μM β-galactosidase solution at 49°C. Data points are averages of 5–10 individual samples, and the error bars indicate ±1 SD. (c) SEC chromatograms of non-heated 0.10 μM β-galactosidase solution and solutions heated at 49°C for 15 and 60 min. The peaks represent native, tetrameric β-galactosidase (1) and oligomers of two (2) and three (3) β-galactosidase molecules. The insert shows that after 60 min incubation, high-order aggregates of β-galactosidase appeared, with a retention time of 29 min. There was no detectable difference between non-heated solutions and solutions heated at 49°C for 15 min Static light scattering measurements using a fluorometer indicated that heating β-galactosidase solutions at 49°C also led to the formation of aggregates. Figure 1b shows the intensity of scattered light of 0.10 μM β-galactosidase solutions heated for different times between 15 min and 6 h. The increase of scattered light intensity indicates the formation of protein aggregates. This is clearly an effect of heating, because scattered light intensity did not increase upon storage at 25°C (not shown). Figure 1b also shows that a maximum of scattered light intensity was reached after about 120 min of incubation. This is possibly related to the sedimentation of very large aggregates that had been formed after long incubation times, which would also explain the increasing error margins of the long time-points.Fig. 2(a) Fluorescence emission spectra of Nile red added to phosphate buffer (broken line; λmax = 662 nm), non-heated 0.10 μM β-galactosidase solution (dotted line; λmax = 659 nm), and 0.1 μM β-galactosidase solution that was previously heated at 62°C for 5 min (solid line; λmax = 611 nm). Excitation wavelength was 550 nm. (b) Fit of two Voigt-shaped peaks (broken lines) to an emission spectrum (circles) of Nile red added to β-galactosidase solution that was heated at 49°C for 360 min. The solid black line is the fitted spectrum, and the residuals are indicated. The positions of the fitted peaks are 605.0 ± 2.2 nm and 658.3 ± 1.4 nm. (c) Plot of Nile red fluorescence intensity at 611 nm against incubation time at 49°C of 0.10 μM β-galactosidase solution. Data points are averages of 5 individual samples, and the error bars indicate ±1 SD The SEC chromatograms in Fig. 1c show the formation of large β-galactosidase aggregates by heating solutions at 49°C. Non-heated solution mainly contained the native, tetrameric β-galactosidase, along with some dimer, trimer, and even higher order aggregates. Heating for 15 min at 49°C did not lead to a change of the SEC chromatogram. However, after 60 min incubation an additional peak with a retention time of 29 min appeared, representing large β-galactosidase aggregates. The area of this peak was 1.5% of the total area under the curve of the protein and increased with increasing incubation time at 49°C (not shown). The chromatograms shown in Fig. 1c were obtained by measuring absorbance at 210 nm. The use of an on-line fluorescence detector system (λex 280 nm and λem 340 nm) led to corresponding results, i.e. aggregation was only detected in β-galactosidase solutions that were heated at 49°C for at least 60 min. Nile red steady-state fluorescence Figure 2a depicts the emission spectra of Nile red in buffer, in non-heated 0.10 μM β-galactosidase solution, and in 0.10 μM β-galactosidase solution that was previously heated at 62°C for 5 min. The emission of Nile red in the presence of non-heated protein was almost identical to the dye’s emission in buffer. This indicates that the surface area of native β-galactosidase is highly polar, and that possible apolar sites are not accessible to Nile red. Storage of unheated β-galactosidase solutions containing Nile red for up to 25 min at room temperature did not lead to a change of the dyes fluorescence, showing that the presence of Nile red does not lead to a change of β-galactosidase surface hydrophobicity over time. Interaction of Nile red with heat-denatured β-galactosidase resulted in a shift of emission maximum from 660 to 611 nm and in a substantial increase in fluorescence intensity (Fig. 2a). This is typically observed when Nile red molecules bind to hydrophobic, apolar sites on the surface of a protein [14], and shows that irreversible denaturation and aggregation by heating above the unfolding temperature has increased the surface hydrophobicity of β-galactosidase. Upon 15 min incubation of 0.10 μM β-galactosidase solution at 49°C, the emission spectrum of Nile red reproducibly showed an increased intensity below 640 nm. Upon longer incubation between 1 and 6 h, a shoulder between 605 and 615 nm emerged. Figure 2b shows a two-peak fit of an emission spectrum obtained with Nile red added to β-galactosidase solution that was previously heated at 49°C for 6 h. In Fig. 2b, the λmax of the main peak is 658 nm, whereas the peak responsible for the shoulder is positioned at 605 nm. Emission at 658 nm originates from free Nile red molecules, and emission at 605 nm from Nile red interacting with denatured β-galactosidase. The latter is comparable with the λmax of Nile red in β-galactosidase solution that had been heated above the unfolding temperature (Fig. 2a). This indicates that the surface polarity of protein molecules denatured at different temperatures is similar. The fluorescence of Nile red did not depend on the time that protein solutions were kept at room temperature upon heating at 49°C (not shown), which indicates that irreversibly denatured β-galactosidase species were detected. The effect of increasing incubation time at 49°C on Nile red fluorescence intensity is shown in Fig. 2c. An observation wavelength of 611 nm was chosen, which corresponds to the average emission wavelength of Nile red interacting with irreversibly denatured β-galactosidase. The profile of Fig. 2c is similar to that of Fig. 1b, suggesting that Nile red interacted with aggregated β-galactosidase. A clear decrease of Nile red emission at 611 nm upon centrifugation at 10000 RPM (not shown) supported this notion. However, it was not possible to confirm this by SEC, neither by adding Nile red to the samples before injection and performing on-line fluorescence detection (λex 550 nm and λem 611 nm), nor by staining fractions from SEC runs with Nile red and performing off-line fluorescence detection. For this reason, time-resolved fluorescence spectroscopy and FCS were used to further investigate the interaction between Nile red and aggregated β-galactosidase.Fig. 3Fluorescence intensity decay, I(t), of Nile red in 0.10 μM β-galactosidase solution that was heated at 49°C for 60 min. The fitted decay curve and the weighted residuals are shown. Fitting gave three lifetimes (τ1 = 0.11, τ2 = 1.11, and τ3 = 4.36 ns) and three (normalized) pre-exponential factors (α1,norm = 0.40, α2,norm = 0.24, and α3,norm = 0.36). The value of χ2 was 1.056Table 1Lifetime analysisSampleχ2 totala〈τ〉bbuffer1.0390.20non-heated1.0110.4215–49°C1.0621.3060–49°C1.0561.89120–49°C1.0642.48240–49°C1.0002.105–62°C1.0321.50aχ2 obtained with Eq. (1) using three fluorescence lifetimes.b Time-resolved fluorescence spectroscopy with Nile red Figure 3 shows the analysis of the fluorescence intensity decay, I(t), of Nile red in 0.10 μM β-galactosidase solution that was heated at 49°C for 60 min. The data points could be adequately described using Eq. (1) with three fluorescent lifetimes, as can be seen from the regular distribution of the residuals around zero and the proximity of χ2 to unity. The use of three lifetimes was necessary for the analysis of all intensity decays, and it was not possible to relate particular lifetimes to the free and the protein-bound state of Nile red. In Table 1, the results are summarized by listing 〈τ〉 values, which are proportional to steady state intensities. In agreement with the increase of Nile red steady state intensity at 611 nm, heating of β-galactosidase solution at 49°C for 15 min led to a clear increase of 〈τ〉 as compared to Nile red in buffer or non-heated protein solution. The tendency of 〈τ〉 to further increase with increasing heating time at 49°C corresponds nicely to the increase of fluorescence intensity upon prolonged heating shown in Fig. 2c. Also with β-galactosidase solution that was heated at 62°C for 5 min, 〈τ〉 was substantially increased as compared to non-heated protein solution. However, it was lower than expected from the steady-state fluorescence intensity at 611 nm (Fig. 2a). Possible reasons for this difference are the variation between individual experiments with solutions containing highly aggregated protein and, since there is a lot of light scattering in such samples, different sensitivities of the steady-state and time-resolved instrumentation for scattered light. The course of fluorescence anisotropy with time, r(t), of Nile red in 0.10 μM β-galactosidase solution that was heated at 49°C for 15, 60, 120, and 240 min was measured (not shown). With all samples, anisotropy decreased at short times, then increased again, and finally remained at an almost constant value. The practically constant value of anisotropy at long times indicated rotational diffusion that was too slow to be detected by Nile red fluorescence. Therefore, although indicating that Nile red was in fact bound to large particles such as aggregates of β-galactosidase, it was not possible to estimate the size of these particles by time-resolved fluorescence anisotropy. Fluorescence correlation spectroscopy (FCS) measurements with Nile red Figure 4a depicts an overlay of ten normalized, experimental autocorrelation functions that were obtained with Nile red in buffer. These data were analyzed simultaneously using the one-component model (Eq. (10)). The fitted curve included in Fig. 4a, the regular distribution of the weighted residuals around zero, and the proximity of χ2 to unity show that Eq. (10) was appropriate for describing the experimental data. The determined decay time for non-diffusive process (τf) was 0.022 ms, the fraction of dye molecules involved in this process (F) was 0.45, and the determined diffusion time (τD,1) was 0.309 ms (Table 2). A diffusion time of Nile red around 0.3 ms is in agreement with reported values [37]. It corresponds to a diffusion coefficient of 2.9 × 10−6 cm2 s−1, which is reasonable for a molecule with a molecular weight of 318 Da. Therefore, τD,1 can be associated with the diffusion of free dye molecules. Gnorm(0) deviated slightly from the expected value of unity (Table 2). Forcing it to unity increased χ2 slightly, but had no effect on the determined parameters τD,1, τf, and F. Also Gnorm(∞) deviated somewhat from the expected value of zero (Table 2). This is related to the background subtraction described in the materials and methods section. Leaving out background subtraction did not lead to different values of the coefficients τD,1, τf, and F. Measurement of Nile red in non-heated 0.10 μM β-galactosidase solution gave the same parameters as Nile red in buffer (Table 2), confirming that there was no substantial interaction between the dye and non-heated protein.Table 2Coefficients from autocorrelation function analysisSample withNile redModelτD,1a (ms)f1,normaτD,2a (ms)f2,normaτfa (ms)FaGnorm(0)aGnorm(∞)aPhosphate bufferOne-Component0.309±0.008–––0.022±0.0020.451±0.0100.920±0.016−0.0049±0.00031.03β-Galactosidase non-heatedOne-Component0.291±0.009–––0.018±0.0020.434±0.0150.915±0.021−0.0047±0.00041.08β-Galactosidase 62°C/5 minTwo-Component0.287±0.0170.335±0.00545.7±1.020.690±0.0070.019±0.0010.246±0.008–−0.0368±0.00211.44aEstimates ±1 SD.Fig. 4Analysis of FCS data. (a) Simultaneous fit of 10 normalized autocorrelation functions obtained with Nile red in buffer. Analysis was performed by non-linear regression using Eq. (10). The fitted curve (black) and the weighted residuals for one of the normalized autocorrelation functions are shown. Determined parameters (value±1 SD) were F=0.451±0.010, τf=0.022±0.002 ms, τD,1=0.309±0.008 ms, Gnorm(0)=0.920±0.016, and Gnorm(∞)=−0.0049±0.0003. The value of was 1.03. (b) Simultaneous fit of 9 normalized autocorrelation functions obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62°C for 5 min. Analysis was performed by non-linear regression using Eq. (11) for two diffusing species. The fitted curve (black) and the weighted residuals for one of the normalized autocorrelation functions are shown. Determined parameters (value ±1 SD) were F=0.246±0.008, τf=0.019±0.001 ms, f1,norm=0.335±0.005, τD,1=0.287±0.017 ms, f2,norm=0.690±0.007, τD,2=45.70±1.02 ms, and Gnorm(∞)=−0.037±0.0021. The value of was 1.44Fig. 5Analysis of FCS data. (a) MEM analysis of a single autocorrelation function obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62°C for 5 min. The non-weighted residuals are shown. (b) Distribution of diffusion times (τD) calculated by MEM analysisFig. 6Raw data of two FCS measurements obtained with 0.10 μM β-galactosidase solution that was heated at 62°C for 5 min (upper curve) and at 49°C for 15 min An overlay of nine normalized, experimental autocorrelation functions, obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62°C for 5 min, is shown in Fig. 4b. There is an additional decay of the autocorrelation functions between 20 and 200 ms, which was not observed with Nile red in buffer. Data analysis was therefore performed using a two-component model, i.e. Eq. (11) assuming two diffusing species. The fitted line and the weighted residuals in Fig. 4b show that up to time intervals between 1 and 10 ms, the data could be well described by the two component model. Within this range, the decay of the autocorrelation functions is governed by τf and τD,1. The determined values were 0.019 and 0.287 ms, respectively (Table 2), which is in good agreement with the values found for Nile red in buffer. This shows that also in the heated β-galactosidase solution, not all Nile red was bound to denatured protein. The fraction of dye molecules (F) involved in non-diffusive process was 0.25, which is substantially lower than for Nile red in buffer (Table 2). A possible explanation for this is that in solutions with large amounts of denatured protein, less Nile red molecules undergo transition to the TICT and triplet state, because they can interact with hydrophobic surfaces [19]. The second diffusion time (τD,2) of 46 ms (Table 2) governed the decay of autocorrelation functions between 20 and 200 ms. It corresponds to a diffusion coefficient (D) of 1.9 × 10−8 cm2 s−1 and a hydrodynamic radius (Rh) of 130 nm, which deviates substantially from the values of native β-galactosidase (D = 3.1 × 10−7 cm2 s−1; Rh = 8 nm). This shows that incubation at 62°C for 5 min had led to very large, denatured aggregates of β-galactosidase, which could be detected by Nile red. However, the two-component model (Eq. (11)) was not able to fully describe the very complex autocorrelation function decays shown in Fig. 4b, as reflected by the residuals and the relatively high χ2 value of 1.44 (Table 2). For this reason, the distribution of diffusion times was determined by the maximum entropy method (MEM) [35]. Such an analysis is shown in Fig. 5a for an individual experimental autocorrelation function. The distribution of diffusion times (Fig. 5b) shows 3 peaks, centered at 0.02, 0.36, and 39 ms. The 0.02 and 0.36 ms peaks correspond to τf and τD,1 determined with Eq. (11). The peak around 39 ms is very broad and ranges from 5 to 150 ms, which corresponds to hydrodynamic radii between 14 and 420 nm. The peak maximum of 39 ms corresponds to an Rh of 109 nm, in good agreement with τD,2 obtained with Eq. (11). This is well above the hydrodynamic radius of small products of β-galactosidase denaturation, i.e. denatured monomers, dimers, and tetramers [25], and confirms that interaction with large β-galactosidase aggregates was the reason for the change of Nile red fluorescence. Nile red also detected large aggregates in 0.10 μM β-galactosidase solutions that had been incubated at 49°C. As for protein samples heated at 62°C, these aggregates were detectable by an additional decrease of FCS autocorrelation functions at long times. However, aggregate concentration in samples heated at 49°C was much lower than in samples heated at 62°C. This is shown in Fig. 6, which compares the raw FCS data obtained with Nile red in β-galactosidase solutions heated for 5 min at 62°C and for 15 min at 49°C. The former is full of high-intensity peaks that were caused by large aggregates with bound Nile red crossing the focal volume. The latter contains only one high-intensity peak, representing a single large aggregate crossing the focal volume. As a consequence of the low aggregate concentration in samples heated at 49°C, only 3 out of 100 measurements contained high-intensity peaks. In spite of their rare occurrence, these peaks were not artifacts. No high-intensity peaks were detected in a total of 150 control experiments with Nile red in buffer and in non-heated β-galactosidase solution. The combined results of steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and FCS show that Nile red fluorescence gave complementary information to SEC about the aggregation of β-galactosidase. Native protein molecules and small oligomers could be detected by SEC (Fig. 1a and c), but did not substantially affect Nile red fluorescence. It is possible that both the surface hydrophobicity and the surface area of these protein species were not sufficient for an effective interaction with the dye. However, Nile red was able to interact with large, denatured aggregates of β-galactosidase and could therefore be used as a probe for their specific detection. Although some higher order aggregates could be detected by SEC upon heating at 49°C (Fig. 1c), Nile red fluorescence was sensitive to the presence of even larger aggregates, which were missed by SEC (Fig. 1a). The interaction of Nile red with large aggregates of β-galactosidase makes it very difficult to use the SEC results for estimating the sensitivity of the Nile red method. Figure 1c shows that some large aggregates were first detected by SEC in protein samples that had been heated at 49°C for 60 min. However, the inability of Nile red to stain these aggregates fractionated from SEC runs (see results above) and the possible exclusion from the SEC column of even larger protein aggregates, which are detectable by Nile red, render the use of SEC as a reference method questionable. Furthermore, Nile red fluorescence was clearly more sensitive than SEC, as it already detected irreversible changes in samples that were heated at 49°C for 15 min. Therefore, to estimate the sensitivity of Nile red, non-heated β-galactosidase solutions were doped with defined amounts of β-galactosidase solution that had been irreversibly denatured and aggregated by heating at 62°C for 5 min. The lower limit of detection by Nile red steady-state fluorescence was 1% (v/v) of heated protein solution, corresponding to 1 nM denatured and aggregated β-galactosidase. The fluorescence intensity of this solution at 611 nm corresponded to that of β-galactosidase solution that had been heated at 49°C for 15 min (Fig. 2c). This comparison is justified, because nearly all β-galactosidase molecules had formed large aggregates upon heating at 62°C for 5 min (Fig. 1a), and because the fluorescence intensity of the detected aggregates did not depend on the incubation temperature (Fig. 6), which means that Nile red was equally sensitive to aggregates formed by heating at 49 and 62°C. Conclusions Using the model protein β-galactosidase, it was shown that the presence small amounts of large, denatured protein aggregates in solution can be detected by Nile red fluorescence. Aggregates detected by Nile red had hydrodynamic radii around 130 nm with a broad size distribution. Native protein and small aggregates thereof had no substantial effect on Nile red fluorescence intensity. By steady-state fluorescence measurements, it was possible to detect 1 nM denatured and highly aggregated β-galactosidase in solution. The spectroscopic detection of protein aggregates by Nile red is potentially useful for formulation screening or quality control of protein pharmaceutics. The presence of even minute fractions of aggregates in protein formulations needs to be avoided, because this can cause immune reactions in patients. Since large aggregates are particularly potent for breaking immune tolerance, their analytical detection is very important. SEC, which is the standard method for studying protein aggregation, is not reliable for detecting large aggregates, because they may be excluded from the separation column. In this work we showed that an analytical detection method with Nile red is a possible approach to overcome this shortcoming of SEC. After establishing the method with time-resolved fluorescence spectroscopy and FCS, as was done for β-galactosidase, steady-state fluorescence measurements, which can be performed in most labs, may enable an experimentally simple and sensitive detection of large protein aggregates.

Diabetologia-3-1-1914285

Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression

Aims/hypothesis The thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-γ agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes. Introduction Rosiglitazone (BRL-49653), a thiazolidinedione (TZD) drug, is used in the treatment of type 2 diabetes mellitus [1]. It has been proposed that the glucose-lowering action of TZDs is mainly mediated by activating peroxisome proliferator-activated receptor (PPAR)-γ (PPARG) [2, 3]. PPARG is most abundantly produced in adipose tissue, suggesting that this is the primary site of action of TZDs [4]. Proposed mechanisms underlying the adipocyte-mediated glucose-lowering action of TZDs are the trapping of fatty acids inside adipocytes and away from muscle, the so-called ‘fatty acid steal’ hypothesis and the altering adipokine release [1]. PPARG is a critical transcription factor in adipogenesis [5]. Its production increases strongly during adipocyte differentiation [6]. By activating PPARG, rosiglitazone promotes adipocyte differentiation in vitro [7, 8]. An increase in the number of small adipocytes and fat mass by TZDs in both animal models and human subjects, suggests that adipocyte differentiation also occurs in vivo [9, 10]. Compared with preadipocytes, adipocytes have a much higher capacity for fatty acid uptake and lipid storage and are potentially more insulin-sensitive due to higher numbers of glucose transporters and insulin receptors [6]. By increasing the number of adipocytes, TZDs can lower circulating NEFA and improve lipid exposure of peripheral tissues, thus improving whole-body insulin sensitivity [1]. This hypothesis is clearly attractive, but other effects of TZDs may occur. It has been pointed out that the increased number of small adipocytes caused by TZD treatment is not only a result of the appearance of new adipocytes, but also due to the shrinkage and/or disappearance of existing mature adipocytes [10]. TZD-induced body weight gain in vivo is used as a marker of increased adiposity. However, there is evidence that this may also be due to fluid retention [11]. In fact, the fat mass may be unaffected or even decreased. In ob/ob mouse, whole body weight was increased, but the fat pad weight was not changed after rosiglitazone treatment [12]. In type 2 diabetes patients, the visceral fat area was found to be decreased [13]. ‘Fatty acid steal’ therefore is not just a matter of increased lipid storage. The maturation of adipocytes is positively correlated with the production of adipokines. Several of these profoundly influence insulin sensitivity and glucose metabolism, e.g. leptin, adiponectin and resistin [14]. However, it has been observed that TZDs decrease the circulating level of individual adipokines such as leptin [15], resistin [16] and plasminogen activator inhibitor-1 [17], and also of cytokines [18]. A decreased production of these adipokines by TZDs seems to conflict with the adipogenic properties of these drugs. The above discrepancy could be due to different effects on preadipocytes and mature adipocytes in adipose tissue. It is clear that TZDs stimulate preadipocytes into the process of differentiation. However, their effect on mature adipocytes is less clear. In vivo studies do not allow us to distinguish between the effects on these two cell types. Therefore, to study the effects of TZDs on mature adipocytes, we used in vitro differentiated mature 3T3-L1 adipocytes. In addition to the assessment of lipid accumulation and adipokine secretion, we used transcriptomics to obtain insight into the effects of rosiglitazone. Although high insulin and high glucose levels mimic the situation in vivo when rosiglitazone is administered, we also included low insulin and low glucose levels for comparison. Materials and methods Cell culture and sample preparation Murine 3T3-L1 fibroblasts (CL-173; American Type Culture Collection, Manassas, VA, US) were cultured and differentiated to adipocytes in vitro as described [19]. On day 8, differentiated cells, grown in DMEM/F-12 (1:1) supplemented with 10% fetal calf serum (Perbio Science, Erembodegem, Belgium), were treated with 0.5 μmol/l rosiglitazone maleate (GlaxoSmithKline, Worthing, UK) with low (4.5 mmol/l) glucose and (fetal calf serum level) insulin (Rosi), or with high (15.75 mmol/l) glucose and 1 μmol/l insulin (RosiIG). In parallel, control cells were cultured in the same medium with low (basal) or high glucose and insulin (IG). Each condition was created in quadruplicate. On day 10, two replications were collected separately for total RNA isolation using Trizol as described [19]. Cells of the other two replications were washed and incubated with the corresponding serum-free medium, supplemented with 2.2 mg/l transferrin and 10.6 nmol/l sodium selenite for 6 h. The number of cells was counted and culture medium proteins were collected as described [20]. The duplicated protein samples were pooled for later analysis. Three independent experiments were performed. Oil red O staining An optimised Oil Red O staining method [21] was adapted for 3T3-L1 adipocytes cultured in a six-well plate. We added an additional quick wash with 70% ethanol after fixing and washing with water. After staining and washing, the neutral lipid-bound pigment was dissolved in DMSO and absorbance at 540 nm was measured. The staining for 3T3-L1 preadipocytes was used as background to correct for unspecific binding. The obtained absorbance values were further corrected for cell numbers measured in replicated wells. Five independent experiments were performed in duplicate, but in the first two experiments Basal and Rosi conditions were not measured. Glycerol assay The culture media of cells that had been treated for 48 h were collected per condition, frozen in liquid N2, then stored at −80°C until analysis. The concentration of glycerol in the medium was measured by a quantitative enzymatic assay kit (Sigma, St Louis, MO, USA), according to the manufacturer’s instructions. The concentrations were corrected for cell numbers measured in replicated wells in parallel. Microarray The mouse 10K_A oligo set (MWG, Ebersberg, Germany) and additional oligonucleotides of genes encoding secretory proteins and proteins involved in energy metabolism were printed in-house and further annotated as described [19]. Five total RNA samples of three independent experiments per condition were used for hybridisation against reference RNA, also as described [19]. Microarray data analysis The data were analysed as described [19]. Briefly, spots with an average intensity, over all arrays, of lower than twofold above average background were removed from further analysis, then the intensities of the remaining 4,019 spots were normalised against reference. Fold-change calculations, Student’s t tests and correlation test (Pearson) were performed in Excel (Microsoft). Fold-change equals ratio in the case of increase and equals −1/ratio in the case of decrease. Cut-off criteria for differential expression was set at fold-change >1.3 and p < 0.05. For genes with replicated spots, the average value of individual spots was used.Data were further analysed using Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com). The program recognised 4,005 of 4,019 spot entries, and thus 3,718 genes. Differentially expressed genes that associated with a canonical pathway in the Ingenuity Pathways Knowledge Base were considered for evaluation. The significance of the association between the data set and the canonical pathway was measured in two ways: (1) a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed; (2) Fischer’s exact test was used to calculate a p value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone. A pathway with a significance <0.05 was taken as significantly regulated.Quantitative real-time RT-PCR All six total RNA samples per treatment were used for quantitative real-time RT-PCR (Q-PCR) of 22 genes (primer information in Electronic supplementary material [ESM] Table 1). Q-PCR was performed as described [19]. Ribosomal protein S15 (Rps15) mRNA was used as reference. One- and two-dimensional gel electrophoresis Protein samples were analysed by one-dimensional (1D) and two-dimensional (2D) gel electrophoresis, corrected for cell numbers, as described [19]. Brefeldin-A-treated adipocyte medium protein sample [20] was loaded on the same 1D gel, to verify protein secretion. Differentially expressed secreted protein bands were excised and identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry [22]. Western blotting The secretion level of adiponectin was analysed by 1D western blotting as described [20]. Results Effect of rosiglitazone on the lipid content of mature adipocytes To assess the effect of rosiglitazone on lipid accumulation in mature adipocytes, we measured the lipid content of differentiated 3T3-L1 adipocytes. Visual inspection by microscopy showed no change in morphology and fat cell percentage in the population of 3T3-L1 adipocytes after 2 days of treatment with RosiIG or Rosi (0.5 μmol/l rosiglitazone at either high or low insulin/glucose levels, respectively), as compared with untreated adipocytes. Nevertheless, the lipid content, as detected by Oil Red O staining, was 10% lower (p < 0.001, n = 5) in RosiIG-treated cells, while no difference was observed in Rosi-treated cells (Fig. 1). Fig. 1Oil Red O staining (ORO) for lipid content in 3T3-L1 adipocytes. Data are expressed as mean±SD. n = 5 for high glucose and insulin (control condition) (IG) and rosiglitazone with high insulin and glucose condition (RosiIG); n = 3 for basal and rosiglitazone with low insulin and glucose condition (Rosi). Blank bars: absorbance, relative value; filled bars: absorbance, relative value corrected for cell numbers. ** p < 0.01 compared with IG condition before cell number correction (t test), *** p < 0.001 compared with IG condition after cell number correction (t test) Effect of rosiglitazone on gene expression To reveal the possible mechanisms for the decreased lipid content of rosiglitazone-treated 3T3-L1 adipocytes, we used DNA microarray to profile the gene expression. The complete analysis set was imported into Ingenuity Pathway Analysis for canonical pathway analysis. The significantly changed pathways were involved in metabolism, but not in signalling, such as PPAR signalling or insulin receptor signalling (Fig. 2). The expression of Pparg itself was downregulated 1.8- and 2.2-fold (p < 0.005) by Rosi and RosiIG treatment, respectively. Notably, pathways related to energy metabolism were among those that were most changed. Taking the high redundancy among these pathways in the Ingenuity database into account, we checked the individual pathways for redundancy. This showed that oxidative phosphorylation, fatty acid metabolism, glycolysis and gluconeogenesis, the pentose phosphate pathway, the citric acid (TCA) cycle, glycerolipid metabolism, glutathione metabolism, steroid (androgen, oestrogen and C21 steroids) metabolism and prostaglandin metabolism are independent pathways, while for the others it cannot be ruled out that they were identified as significant just because they contain genes overlapping with other pathways. Fig. 2Regulated canonical pathways by rosiglitazone. Microarray data analysed by Ingenuity Pathway Analysis. Blank bars, with low insulin/glucose; filled bars, with high insulin/glucose. Bold line, thresholdFor these independently regulated pathways, we determined the direction of change by examining the individual genes in the pathways. The regulation of gene expression of rate-limiting or otherwise representative enzymes is presented in Table 1. High insulin/glucose attenuated the effect of rosiglitazone on some genes of complex I and II of the electron transport chain. However, high insulin/glucose treatment itself did not induce a change in the expression of these genes from the basal state. In general, rosiglitazone produced dominant effects, as indicated by the Pearson product moment correlation coefficient of 0.875 for the fold changes of the complete analysis set of Rosi/Basal and RosiIG/IG. The changes in the pathways indicate that rosiglitazone enhanced both energy metabolism at the TCA cycle and oxidative phosphorylation. The generated energy may be dispensed with the help of upregulated uncoupling protein 2 (Ucp2). Enhanced energy metabolism is mainly supported by upregulation of the pathways involved in uptake and activation of fatty acids and in beta-oxidation in both mitochondria and peroxisomes. In contrast, de novo fatty acid synthesis was downregulated, as indicated by fatty acid synthase (Fasn) and stearoyl-coenzyme A desaturase 1 (Scd1). The downregulation of triacylglycerol synthesis enzymes and upregulation of adipocyte lipase (also known as patatin-like phospholipase domain containing 2) together imply a decreased lipogenesis in mature adipocytes (Table 1). Table 1Key enzymes of metabolism pathways regulated by rosiglitazonePathwayChangeGene symbolGene IDNameRosi/BasalRosiIG/IGFold-changep valueFold- changep valueTCA cycleUpIdh3a67834Isocitrate dehydrogenase 3 (NAD+) alpha2.530.0002.550.000Cs12974Citrate synthase1.950.0001.470.000Oxidative phosphorylationUpNdufa568202NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 51.720.0091.200.235Sdhd66925Succinate dehydrogenase complex, subunit D1.830.0011.510.039Uqcrc122273Ubiquinol-cytochrome c reductase core protein I1.470.0071.330.017Cox7a112865Cytochrome c oxidase subunit VIIa polypeptide 11.780.0021.550.003Atp5h71679ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d1.710.0061.490.005Fatty acid uptake and transportUpCd3612491CD36 antigen, fatty acid translocase2.540.0002.270.006Fabp411770Fatty acid bind protein 4, adipocyte1.640.0881.570.142Fatty acid activationUpAcsl114081Acyl-CoA synthetase long-chain family member 12.500.0001.970.000Fatty acid beta oxidationUpCrat12908Carnitine acetyltransferase1.450.0061.470.007Slc25a2057279Solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 201.630.0031.220.092Acox111430Acyl-coenzyme A oxidase 1, palmitoyl2.700.0002.540.000Acaa1113868Acetyl-coenzyme A acyltransferase 1 (peroxisomal)1.670.0021.290.017Acaa252538Acetyl-coenzyme A acyltransferase 2 (mitochondrial)2.700.0001.940.000Fatty acid synthesisDownFasn14104Fatty acid synthase−1.520.000−1.550.001Scd120249Stearoyl-CoA desaturase 1−3.650,000−4.310.000Triacylglycerol synthesisDownPpap2b67916Phosphatidic acid phosphatase type 2B−1.380.001−1.480.000Dgat267800Diacylglycerol O-acyltransferase 2−1.310.003−1.370.006Lipid droplet formationUp/downAdfp11520Adipose differentiation related protein5.970.0006.600.000Cav112389Caveolin, caveolae protein 1−2.200.000−2.300.001Glycerolipid lysisUpPnpla266853Patatin-like phospholipase domain containing 21.520.0061.670.007Glycerol metabolismUpGk14933Glycerol kinase1.860.0071.880.003Gpd114555Glycerol-3-phosphate dehydrogenase 1 (soluble)2.070.0091.680.032Energy expenditureUpUcp222228Uncoupling protein 2 (mitochondrial, proton carrier)1.780.0011.500.003Glutathione metabolismUp/downMgst366447Microsomal glutathione S-transferase 32.110.0011.880.001Gsta414860Glutathione S-transferase A41.360.046−1.010.924Gstm114862Glutathione S-transferase M1−1.880.000−2.010.010Gstt114871Glutathione S-transferase theta 1−1.380.060−1.510.044Gstz114874Glutathione transferase zeta 1−1.440.001−1.570.015Prostaglandin metabolismUp/downCbr3109857Carbonyl reductase 33.040.0003.960.000Ptges296979Prostaglandin E synthase 21.450.0121.610.000Ptgis19223Prostaglandin I2 (prostacyclin) synthase−1.710.002−1.410.004Steroids metabolismUp/downHsd17b715490Hydroxysteroid 17-beta dehydrogenase 71.630.0031.730.002Nsdhl18194NAD(P) dependent steroid dehydrogenase-like1.510.0391.370.017Hsd11b115483Hydroxysteroid 11-beta dehydrogenase 1−2.730.001−2.050.004GlyconeogenesisUpFbp214120Fructose-1,6-bisphosphatase 22.920.0032.820.014Glycogen synthesisUpGyg27357Glycogenin1.520.0011.340.008Pyruvate metabolismUpPdha118597Pyruvate dehydrogenase E1 alpha 11.380.0011.330.001Pdhb68263Pyruvate dehydrogenase (lipoamide) beta1.590.0051.610.004Pentose phosphateUpH6pd100198Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)1.600.0001.470.001Taldo121531Transaldolase 11.570.0011.330.036Leu catabolismDownIvd56357Isovaleryl coenzyme A dehydrogenase−1.420.004−1.720.000Mccc172039Methylcrotonoyl-coenzyme A carboxylase 1 (alpha)−1.260.026−1.350.031Mean value of microarray data (n = 5)Rosi/basal: the effect with low insulin/glucose; RosiIG/IG: the effect with high insulin/glucoseAccompanying fatty acid catabolism, glycerol activation by glycerol kinase and oxidation by glycerol phosphate dehydrogenase to dihydroxyacetone phosphate were upregulated, feeding glycolysis and gluconeogenesis. The enzymes catalysing glycolysis/gluconeogenesis were all upregulated, possibly in favour of gluconeogenesis. In addition, the upregulation of glycogenin, a gene priming glycogen synthesis, implies an increase of glycogen synthesis.Most genes in the amino acid metabolism pathways that were regulated also participate in fatty acid metabolism or in glycolysis. Therefore, we did not take amino acid metabolism pathways as independent rosiglitazone-regulated pathways. However, two key enzymes that catalyse the catabolism of leucine (Table 1) were downregulated. This implies that rosiglitazone saves amino acids from being used as fuel in energy metabolism.Glutathione metabolism was significantly changed by rosiglitazone treatment. Two glutathione S-transferases that detoxify lipid peroxides, microsomal glutathione S-transferase 3 and glutathione S-transferase A4, were upregulated, while other glutathione S-transferases were downregulated by rosiglitazone. This is in line with the upregulation of fatty acid oxidation by rosiglitazone.Microsomal glutathione S-transferase 3 is involved not only in glutathione metabolism, but also in eicosanoid metabolism, which is also regulated by rosiglitazone. The expression of genes encoding enzymes involved in prostaglandin metabolism implies that the conversion from prostaglandin H2 to E2, F2 and D2 may be enhanced, while conversion to I2 may be reduced.Cholesterol biosynthesis and steroid hormone metabolism were also affected by rosiglitazone. Expression of some steroid dehydrogenases were upregulated, while hydroxysteroid 11-beta dehydrogenase 1 was downregulated (Table 1). The latter has been reported as one of the beneficial effects of TZDs, leading to a decrease in the stress hormone cortisol [23]. Effect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteins To assess whether protein secretion by mature adipocytes is altered by rosiglitazone, we checked genes encoding adipocyte-secreted proteins. The list was based on literature [24–27] and our previous work [20]. In total, 61 genes were present on the array. Il1b, Il6, Il10 and leptin showed very low signals (less than twofold of background) and were excluded from the analysis. Among the remaining 57 genes, only apolipoprotein E (Apoe) was upregulated by Rosi treatment, and more than 40% of the genes were significantly downregulated with both Rosi and RosiIG treatment (Table 2). Table 2Effect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteinsFunctionGene symbolGene IDNameRosi/BasalRosiIG/IGFold-changep valueFold-changep valueLipid metabolismAdn11537Complement factor D (adipsin)−3.550.000−4.230.000C312266Complement component 3−2.250.000−2.680.001Lpl16956Lipoprotein lipase1.120.166−1.020.627Apoe11816Apolipoprotein E1.340.0261.180.145Insulin sensitivityRetn57264Resistin−3.070.000−3.170.001Igf116000Insulin-like growth factor 1−1.550.012−1.580.001Pbef159027Pre-B-cell colony-enhancing factor 1−1.240.050−1.280.085Adipoq11450Adiponectin−1.090.462−1.250.108InflammationHp15439Haptoglobin−2.930.000−4.420.000Ptx319288Pentaxin-related gene−1.680.001−1.580.000Tgfb321809Transforming growth factor, beta 3−1.580.008−1.440.056B2m12010Beta-2 microglobulin−1.540.018−1.640.006Ccl220296Chemokine (C-C motif) ligand 2 (previously known as monocyte chemoattractant protein-1)−1.540.012−1.480.023Saa320210Serum amyloid A 3−1.490.047−1.380.001Cxcl1220315Chemokine (C-X-C motif) ligand 12−1.450.006−1.420.046Il6ra16194Interleukin 6 receptor, alpha−1.160.128−1.150.037Tgfb121803Transforming growth factor, beta 1−1.150.1631.020.914Tgfb221808Transforming growth factor, beta 2−1.150.306−1.070.584Il1816173Interleukin 18−1.150.224−1.010.937D17Wsu104e28106DNA segment, Chr 17, Wayne State University 104, expressed (previously known as IL25)−1.030.794−1.160.088Lgals116852Lectin, galactose binding, soluble 11.050.7741.080.590Lcn216819Lipocalin 21.110.2281.200.179Mif17319Macrophage migration inhibitory factor1.180.0761.170.137Vascular functionAdm11535Adrenomedullin−1.410.003−1.500.000Agt11606Angiotensinogen−1.220.161−1.150.150Serpinf120317Serine (or cysteine) peptidase inhibitor, clade F, member 1 (previously known as pigment epithelium derived factor)−1.150.237−1.250.187Vegfa22339Vascular endothelial growth factor A−1.030.724−1.080.644Apln30878Apelin1.010.9691.020.790Serpine118787Serine (or cysteine) peptidase inhibitor, clade E, member 1 (previously known as plasminogen activator inhibitor-1)−1.010.8971.270.013AntioxidantSod320657Superoxide dismutase 3, extracellular−1.090.500−1.390.036Mt117748Metallothionein 1−1.110.577−1.120.417Extracellular matrix componentNid218074Nidogen 2−1.640.002−1.960.003Sparc20692Secreted acidic cysteine rich glycoprotein−1.630.000−1.560.000Col5a212832Procollagen, type V, alpha 2−1.620.000−1.620.003Col5a112831Procollagen, type V, alpha 1−1.590.003−1.480.013Col1a212843Procollagen, type I, alpha 2−1.640.020−1.400.002Col1a112842Procollagen, type I, alpha 1−1.600.065−1.380.020Col3a112825Procollagen, type III, alpha 1−1.520.024−1.590.002Col4a512830Procollagen, type IV, alpha 5−1.440.034−1.370.019Col6a212834Procollagen, type VI, alpha 2−1.320.011−1.190.108Col6a112833Procollagen, type VI, alpha 1−1.310.1171.050.740Col5a353867Procollagen, type V, alpha 3−1.200.046−1.290.076Col4a112826Procollagen, type IV, alpha 1−1.160.114−1.160.069Col4a212827Procollagen, type IV, alpha 2−1.160.153−1.290.003Nid118073Nidogen 1−1.040.7231.040.767Col6a312835Procollagen, type VI, alpha 3−1.030.690−1.010.960Extracellular matrix processingMmp317392Matrix metalloproteinase 3−2.010.003−2.020.013Lox16948Lysyl oxidase−1.870.005−2.170.002Timp221858Tissue inhibitor of metalloproteinase 2−1.810.002−1.920.000Mmp217390Matrix metalloproteinase 2−1.440.054−1.300.100Timp121857Tissue inhibitor of metalloproteinase 1−1.320.095−1.300.055Pcolce18542Procollagen C-endopeptidase enhancer protein−1.310.029−1.480.002Mmp1958223Matrix metalloproteinase 19−1.010.848−1.080.347Mmp1117385Matrix metalloproteinase 111.050.860−1.120.413Timp4110595Tissue inhibitor of metalloproteinase 41.060.6721.250.055MiscellaneousGal14419Galanin−3.040.000−2.890.000Gsn227753Gelsolin−1.630.000−1.520.000Ppic19038Peptidylprolyl isomerase C−1.270.049−1.380.001Mean value of microarray data (n = 5)Rosi/basal: the effect with low insulin/glucose; RosiIG/IG: the effect with high insulin/glucoseInterestingly, the general downregulation of adipokines was also observed for other genes encoding secreted proteins. We used Gene Ontology to cluster the expressed genes on the microarray into secreted protein encoding genes and intracellular/unknown location protein-encoding genes (additional gene expression information is available at www.foodbioactives.nl). In the first cluster, more genes were significantly downregulated (Fig. 3a and c). Of 477 genes, 24.9 and 25.8% were significantly downregulated, while 9.6 and 6.7% were upregulated by Rosi and RosiIG, respectively. Of the upregulated genes, most encode multi-location proteins, which can also reside in the nucleus, mitochondria and/or membranes. If these genes are omitted from the analysis, only seven genes were upregulated (1.5%). In the cellular protein cluster, by contrast, the majority of the regulated genes were upregulated (Fig. 3b and d). Of 3,382 genes, 10.3 and 10.1% were significantly upregulated, while 5.1 and 5.0% were downregulated by Rosi and RosiIG treatment, respectively. Fig. 3Regulation pattern by rosiglitazone of genes encoding extracellular proteins and of genes encoding intracellular proteins or proteins of unknown location. a, c: extracellular pattern; b, d: intracellular pattern. a, b: rosiglitazone with low insulin/glucose; c, d: rosiglitazone with high insulin/glucoseTo complement array data, we used 2D and 1D gel electrophoresis and western blotting to examine the effect of rosiglitazone on secretion. With regard to complement C3, a 30% reduction of secretion by Rosi or RosiIG treatment was observed on the 1D gel (Fig. 4). This is less than the change observed at the gene expression level. Nine spots on the 2D gel were identified as adipsin. We assessed the changes for individual spots, as well as for all spots in total. In both cases, the decrease effected by rosiglitazone at the gene expression level was similar to the decrease found at the secreted protein level with RosiIG treatment. However, this decrease at the protein level was less prominent with Rosi treatment (Fig. 5). The secretion of adiponectin was also analysed by western blotting. A slight increase with rosiglitazone was detected (Fig. 6), although there was no change at the gene expression level. Fig. 4The effect of rosiglitazone on the secretion of complement C3 as measured by 1D gel analysis. The marked bands (arrows) were identified as C3. One representative image is shown. A sample treated by Brefeldin A (+BFA) was used to verify secretionFig. 5The effect of rosiglitazone on the secretion of adipsin as measured by 2D gel analysis. A section of one representative 2D gel image is shown for an individual adipsin spot (squares) from a low glucose and insulin control condition (Basal), b rosiglitazone with low insulin and glucose condition (Rosi), c high glucose and insulin control condition (IG) and d rosiglitazone with high insulin and glucose condition (RosiIG). e Total quantity of adipsin in the culture medium of 3T3-L1 adipocytes expressed in arbitrary units (AU)Fig. 6The effect of rosiglitazone on the secretion of adiponectin as measured by western blotting. Data are expressed as mean ± SD (n = 3) and one representative western blot image is shown. *p < 0.05 compared with Basal condition (t test); **p < 0.01 compared with IG condition (t test). Basal, low glucose and insulin control condition; Rosi, rosiglitazone with low insulin and glucose condition; IG, high glucose and insulin control condition; RosiIG, rosiglitazone with high insulin and glucose condition General confirmation of the microarray data by quantitative real-time RT-PCR analysis To confirm our microarray data, we checked the expression of 22 genes that encode secreted proteins or are related to lipid metabolism, by Q-PCR, using Rps15 as reference. Rps15 was chosen because of its constant expression behaviour on the arrays. A gene was selected as target gene because either itself or its family member showed changed expression based on microarray data. The comparison showed a strong correlation between array data and Q-PCR data (ESM Fig. 1), with Pearson correlation coefficients of 0.987 and 0.986 for Rosi/basal and RosiIG/IG, respectively. The effect of rosiglitazone on lipolysis We assessed the glycerol level in the culture medium as a measure of the lipolysis of adipocytes. RosiIG treatment significantly increased the release of glycerol from 3T3-L1 mature adipocytes, whereas glycerol release was significantly decreased by Rosi treatment (Fig. 7). Fig. 7The effect of rosiglitazone on the culture medium glycerol levels. Data are expressed as mean±SD (n = 3). *p < 0.05 compared with IG condition (t test); **p < 0.01 compared with Basal condition (t test). Basal, low glucose and insulin control condition; Rosi, rosiglitazone with low insulin and glucose condition; IG, high glucose and insulin control condition; RosiIG, rosiglitazone with high insulin and glucose condition Discussion As a PPARG agonist, rosiglitazone stimulates adipogenesis, which is accompanied by an increasing lipid content of the differentiating cells. However, our results showed that rosiglitazone does not induce an increase of the lipid content, but decreases it in mature adipocytes at high insulin/glucose level. The pathway analysis based on transcriptome data points out that an altered energy metabolism may underlie this phenomenon. Although the level of insulin/glucose has some influence, the effect of rosiglitazone on the transcriptome was clearly dominant. The most significantly regulated pathways by rosiglitazone in 3T3-L1 adipocytes were fatty acid oxidation, the TCA cycle and oxidative phosphorylation. Besides an upregulation of fatty acid catabolism pathways, our data indicate that rosiglitazone encourages adipocytes to utilise fatty acids for energy production and spares glucose and amino acids by enhancing glycogen synthesis and inhibiting amino acid catabolism pathways. This is in line with the upregulation of Ucp2 and the downregulation of Scd1 expression. The increase in the level of UCP2 is correlated with a shift in substrate utilisation in favour of lipids as the dominant metabolic fuel [28]. SCD1 is involved in fatty acid synthesis, but it has also been shown that SCD1 deficiency activates metabolic pathways that promote beta-oxidation and decrease lipogenesis [29]. Our conclusions regarding energy metabolism are consistent with recent studies. Mitochondrial changes have been shown in response to TZD treatment in 3T3-L1 adipocytes [30], human adipocytes in vitro [31] and mouse adipocytes in vivo [12]. These studies show that TZDs, including rosiglitazone, upregulate fatty acid oxidation by increasing mitochondrial protein and gene expression, but not in a trans-differentiation manner [12, 30]. This has been confirmed by direct measurement of labelled palmitic acid oxidation [12] or indirect calorimetric calculation [32]. We also found that among the genes involved in protein biosynthesis, more than 80% of those upregulated by rosiglitazone were genes encoding mitochondrial ribosomal proteins. This is consistent with an increase of mitochondrial biogenesis and remodelling [12]. We speculate that the maintenance of increased mitochondrial mass and function utilises (part of) the energy that the cell no longer needs for lipid synthesis under rosiglitazone treatment. A decrease in lipogenesis pathways accompanies the increased fatty acid catabolism pathways caused by rosiglitazone in mature adipocytes, as shown by decreased expression of genes encoding triacylglycerol synthesis enzymes and the genes encoding lipase. The downregulation of Fasn by TZD has also been observed in brown adipocytes [33]. Caveolin-1 (CAV1) is an important protein for efficient lipid droplet formation [34]. The downregulation of its gene expression supports a decrease of lipid accumulation. The upregulation of adipocyte differentiation related protein gene (Adfp), which encodes a lipid droplet-associated protein, seems to conflict with the decreased lipogenesis observed by us (Table 1). However, in mature 3T3-L1 adipocytes, ADFP is spatially associated with lipid droplets subjected to lipolysis and is suggested to play a role in lipolysis [35]. Furthermore, the protein level of ADFP in mature adipocytes is not determined by the mRNA expression, but by the amount of micro-lipid droplets [35]. Therefore the increase in Adfp expression might also be interpreted as being in agreement with upregulated lipolysis. The pentose phosphate pathway provides NADPH to be used in reductive biosynthesis, including fatty acid biosynthesis [36]. Rosiglitazone enhances the pentose phosphate pathway, but inhibits fatty acid biosynthesis, while stimulating other NADPH-dependent pathways, such as glutathione detoxification, cholesterol and steroid biosynthesis, and prostaglandin metabolism (carbonyl reductase 3). The excess of NADPH may be a driving force behind the upregulation of these pathways by rosiglitazone. The present study indicates that in mature 3T3-L1 adipocytes rosiglitazone enhances fatty acid catabolism, increases lipolysis and reduces lipogenesis pathways. This may lead to a net decrease of lipid content of the cells. Indeed, we observed increased lipolysis expressed in terms of an increased glycerol level in the medium and a decreased lipid content of the cells under RosiIG treatment. This is in line with the increased lipolysis effected by rosiglitazone in a recent study on rat white adipose tissue [37]. We did not observe increased lipolysis under Rosi treatment, where in fact the glycerol level was decreased rather than increased. The difference implies that the insulin/glucose level alters the effect of rosiglitazone. It is likely that at low insulin/glucose level, the ‘starved’ adipocytes use glycerol as an energy source, rather than releasing it into the medium. However, it should be noted that low insulin/glucose is not a realistic situation in vivo when rosiglitazone is administered. The decreased lipid content is consistent with the physiological effects of TZDs in both animal models [10, 38] and in humans [9], where TZDs increase the number of small adipocytes. In adipose tissue treated with TZDs, mature adipocytes can become smaller by actively burning fat, in parallel to the emerging of small immature adipocytes due to preadipocyte differentiation. With rosiglitazone, mature adipocytes take fatty acids for burning rather than for storage. This still fits the ‘fatty acid steal’ hypothesis [1] and can explain part of the glucose-lowering effects of rosiglitazone. Rosiglitazone affects the function of mature adipocytes not only with regard to fat metabolism, but also with regard to adipokine secretion. Despite the limitations of the present study, namely that a mid-size array was used and that the expression changes of several genes could not be detected due to sensitivity, our data at both the mRNA level and the protein level confirmed that rosiglitazone represses adipokine expression, except adiponectin and Apoe. Since the majority of adipokines are negatively correlated with insulin sensitivity [39], the general repression may correct the disturbance of insulin sensitivity caused by obesity. Furthermore, our transcriptome analysis suggests that rosiglitazone induces a general decrease in expression of genes encoding secreted proteins. This implies a general mechanism, in which energy redistribution might be involved. We observed that repression at the secretion level was less significant than at the gene expression level. One explanation is that the effect on protein lags behind the effect on mRNA. Another reason might be the influence of insulin. We have recently studied the role of insulin in 3T3-L1 adipocyte secretion [19]. We observed that at a low insulin level, the secretion of proteins is already suppressed. Consequently, the inhibitory effect of rosiglitazone on secretion is less prominent in the low insulin condition. In contrast to most adipokines, adiponectin is reversely correlated with obesity, and its circulating level is upregulated by TZDs [40]. A functional PPAR-responsive element (PPRE) in the adiponectin promoter could be the reason for the stimulation [41]. We observed an upregulation only at the secretion level, but not at gene expression level. This is in line with the conclusion from another study, that although plasma levels of adiponectin were upregulated by TZDs, there was no significant effect on adiponectin gene expression [16]. Rosiglitazone is a potent PPARG agonist. It seems logical to assume that all these effects are mediated through regulation of PPARG. Indeed, we observed that rosiglitazone induced a significant decrease of Pparg expression in mature 3T3-L1 adipocytes. This is in contrast to an overwhelming increase during adipogenesis, but it is in line with several reports [42–44]. With respect to PPARG downstream effects, we found here that two PPRE-bearing genes, Cav1 and Scd1, were downregulated, and two other genes, adiponectin and lipoprotein lipase, remained unchanged, while most PPRE-bearing genes [45], such as CD36 antigen, fatty acid binding protein 4, adipocyte and acyl-coenzyme A oxidase 1, palmitoyl, were upregulated by rosiglitazone (Tables 1 and 2). The effect of TZDs on Scd1 in mature adipocytes is opposite to that in preadipocytes [46], and the effect on Cav1 is in contrast to the reports in other cell types [47, 48]. It has been known that PPARG acts with different transcriptional partners, co-repressors and/or co-activators as a complex, to activate or repress the expression of target genes [49, 50]. Possibly, the PPARG complex is modulated by rosiglitazone in mature adipocytes in a manner that is different from that occurring in preadipocytes and other cell types. In conclusion, we have shown that rosiglitazone, in contrast to the stimulation of adipogenesis in preadipocytes, reduces adiposity of mature adipocytes by increasing fatty acid catabolism pathways. In addition, rosiglitazone represses the expression and secretion of adipokines of mature adipocytes. Our findings suggest that the change of adiposity as seen in vivo [13] may reflect a shift in balance between the effects of TZDs on preadipocytes and those on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocytes. These aspects all contribute to the therapeutic effects of TZDs. Electronic Supplementary Material Below is the link to the electronic supplementary material Table 1 Q-PCR primer sequences (DOC 26 kb) Fig. 1 Comparison between microarray and quantitative real time RT-PCR analysis. The mean of the fold-change is shown. n = 5 for array n = 6 for RT-PCR. The lines at 1.3 and −1.3 fold changes indicate thresholds of differentially expression (DOC 51 kb)

Ann_Biomed_Eng-2-2-1705476

Autonomous Reovirus Strain Classification Using Filament-Coupled Antibodies

We previously described a filament-based antibody recognition assay (FARA) that generates ELISA-like sandwich structures immobilized on a filament. FARA allows the coupling of antibodies to precise locations along a filament, on-line fluorescence detection of captured pathogen, and feedback-directed filament motion. These properties suggest that this approach might be useful as an automated means to rapidly classify unknown pathogens. In this report, we describe validation of the novel decision tree aspect of this technology using mammalian reovirus. Based on available antibodies, we developed a decision tree algorithm to detect virus with increasing specificity at each level of the tree. Using three strains of reovirus and a bacteriophage control, our system correctly classified the reovirus strains at a concentration of 2 × 1012 virions ml−1 and M13K07 phage at 3 × 1011 virions ml−1. Classification of reovirus strain type 3 Dearing (T3D) required three levels of testing: general reovirus classification in level 1, serotype 3 classification in level 2, and final T3D strain classification in level 3. Strain T3SA + also required three levels of testing before a final classification was returned in level 3. Classification of strain type 1 Lang (T1L) required two levels of testing. M13K07 phage detection required only one level of testing for classification. These results indicate that automated pathogen classification using FARA is feasible. Furthermore, the simplicity of the design could be exploited for development of more complex sub-classification networks with additional levels and branches. Introduction Identification of specific pathogens is essential for the selection of pathogen-specific treatments, minimizing the spread of infection, and monitoring for long-term complications. Currently, specific pathogen identification is achieved through available RT-PCR and antibody-based strategies. In the clinic, these tests are usually applied consecutively to evaluate for the presence of the most likely to least likely pathogen based on patient findings. An automated classification strategy that is less dependent on clinical knowledge but achieves rapid accurate identification of a single pathogen from among a group of possible pathogens is currently unavailable. Our previously described filament-based antibody recognition assay (FARA) employs antibodies immobilized at known locations along a filament to detect specific pathogens.9,10 The filament is pulled through a series of small reaction chambers, and pathogens, if present, are captured by filament-bound antibodies. Detection of pathogen binding is achieved by using a fluorescently labeled second antibody specific for the pathogen. FARA was first reported using immobilized anti-M13K07 antibody to detect M13K07 phage.10 This virus and antibody pair provided a well-characterized test system to demonstrate the feasibility of a filament-based, pathogen-detection platform. However, in this first generation approach, the filaments were removed from the system for fluorescence scanning. Subsequent improvements to FARA include an integrated fluorescence detector and a feedback algorithm to control filament position.9 The integrated detector enables adaptive pathogen detection in which regions of interest along the filament can be reincubated in the appropriate reaction chambers to increase filament fluorescence when the initial signal is low. In this report, we describe a small-scale test of the use of a simple classification tree together with feedback-controlled FARA to identify four viruses. The feedback feature of online FARA is used to guide the selection of subsequent tests within the classification tree. Unnecessary tests are not performed, and each subsequent test becomes more specific for a single pathogen. These results establish FARA as a robust platform for classification of diverse pathogens. Materials and Methods Cells and Viruses Murine L929 (L) cells were cultured in suspension in Joklik’s modified Eagle’s medium supplemented to contain 5% fetal bovine serum, 2 mM l-glutamine, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 g mL−1 amphotericin-B. Reovirus strains T1L and T3D are laboratory stocks. T3SA + is a monoreassortant virus isolated from L cells co-infected with T1L and T3C44MA.1 T3SA + contains the S1 gene segment from the type 3 parental strain and all other gene segments from T1L.1 Reovirus particles were purified as previously described.1,3,6 L cells were inoculated with second-passage L-cell lysate stocks of twice plaque-purified reovirus at a multiplicity of infection of 10 plaque-forming units per cell. Virus was purified from infected cells by freon extraction and CsCl-gradient centrifugation. Purified M13K07 virus was obtained from the Vanderbilt Molecular Recognition Core. Antibodies Mouse monoclonal antibodies 4F2, 5C6, 8H6, and 9BG5 specific for reovirus proteins2,12,13 (Table 1) were purified from mouse hybridoma supernatants using Protein A column chromatography. Anti-M13K07 monoclonal antibody was purchased from Amersham Biosciences (Piscataway, NJ). Table 1.Antibody specificity.AntibodyProtein specificityReovirus strainT1LT3DT3SA+5C6T1 σ1+−−9BG5T3 σ1−++8H6T1 and T3 μ1+++4F2T3D σ3−+− Antibodies 4F2 and 8H6 were used for fluorescence detection of virus (step 4, Table 2) and were labeled with Alexa Fluor 555 or Alexa Fluor 647 (AF555 or AF647, Molecular Probes, Eugene, OR), respectively, according to the manufacturer’s instructions (Molecular Probes). Labeled antibodies were purified using PD-10 size-exclusion chromatography (Amersham Biosciences). Antibody concentration and number of fluores per labeled antibody were determined by using absorbance measurements at 280 nm and the peak absorbance wavelength of each label. Aliquots of both labeled and unlabeled antibodies were stored at −20°C, and working solutions were kept at 4°C. Final antibody concentration was adjusted immediately prior to experiments. Table 2.The five reaction chambers in FARA processingChamberDescriptionSolutionIncubation timeChamber ID / Volume (mm / μl)1Block/Wash FilamentPBS-Ta15 min2/2352Virus IncubationUnknown Virus40 min1/60 3WashPBS-T1 min2/2354Detecting Antibody IncubationFluorescently Labeled Detecting Antibody5 min1/60 5WashPBS-T1 min2/235aTween-20 (0.1%) was added to PBS as a blocking agent. Filament Preparation Capture antibodies were passively adsorbed to the filament surface in groups of three by placing the filament across the concave teeth of a PhastGel sample applicator (Amersham Biosciences) (Fig. 1). Three capture-antibody regions, corresponding to the three levels of testing, were prepared along each filament using three applicator combs glued end to end. Antibody solution pipetted onto the filament without the comb spread unimpeded along the filament. Surface tension within the teeth of the comb overcame this tendency and produced a small, distinct circumferential band of immobilized antibody. Antibody was spotted in a volume of 0.75 μL and allowed to passively adsorb to the filament for 45 min in a humidified box. Following incubation at 25°C, filaments were rinsed in phosphate buffered saline with 0.1% Tween 20 (PBS-T) and threaded through the reaction chambers for virus detection experiments. Preliminary experiments were performed to determine the optimal concentration of each capture antibody. Figure 1.Schematic of a device for adsorption of capture antibodies to the filament. The filament is placed within the concave teeth of a PhastGel applicator to localize the spotted antibody to a small circumferential band around the filament. Red fingernail polish (Poisonberry, Noxel Corporartion, Hunt Valley, MO) was used as a visible and fluorescent fiducial marker to identify the leading and trailing edges of capture-antibody regions during experiments and during laser scanning. The polish was applied by pipette between the teeth of the comb flanking the antibody region. A simple bar code system was used to identify each of the three capture-antibody regions. The first and second regions were preceded by a single fiducial marker, and the third region was preceded by two fiducial markers. Micro-reaction Chambers Glass microreaction chambers were fashioned from 0.25 inch stock tubing into 75 mm lengths. The ends were flared outward to facilitate movement of the filament through the chambers. Interior diameters of the chambers were 1 or 2 mm depending on the required reaction volume (Table 2). Chambers were carefully positioned in a straight line on the top of a horizontal aluminum stage using machined aluminum mounts.9 Fine adjustment of chamber position was achieved by using oblong mounting holes on each mount. PBS-T was used in all wash chambers and for virus and labeled antibody solutions. Reovirus was used at a concentration of 2 × 1012 virions mL−1, and phage M13K07 was used at a concentration of 3 × 1011 virions mL−1. Detecting antibodies were present in the detecting-antibody chamber at a concentration of approximately 40 μg mL−1 for each antibody in the three antibody mixture. Solutions were added to appropriate chambers at the initiation of experiments and used for all three levels of testing (Table 2). If testing proceeded to level three, fluid loss from the chambers was monitored and replenished if necessary. Filament Control Movement of the filament and, therefore, the antibody bands through the chambers, was achieved by using a rotary stage to wind or unwind the filament around a spindle. A small weight was attached to the opposite end of the filament to maintain a constant tension. Filament positioning to within several microns was achieved by using a rotary stage encoder from Yaskawa Instruments (Waukegan, IL) and a custom control algorithm written as a LabView Virtual Instrument (VI) (National Instruments, Austin, TX). Parameters including filament speed and residence times within chambers were controlled by using the LabView software interface. Between chambers, the speed of the filament was 1 cm sec−1. Within each chamber, the capture-antibody region undergoing processing was oscillated 2.5 cm back and forth at a speed of 1 cm sec−1 to increase interactions between immobilized molecular structures attached to the filament and the molecular species in solution. Within the detector, filament speed was 4 cm sec−1. Lasers and PMTs Filament fluorescence was measured by passing the filament between two diode lasers. The two laser excitation sources were attached to either side of a detection chamber (Fig. 2). Laser 1 (638 nm, 25 mW diode laser; Coherent, Santa Clara, CA) was used to excite the antibody tag AF647. Laser 2 (532 nm, 20 mW diode-pumped, solid state laser; B&W Tek, Inc., Newark, DE) was used to excite the antibody tag AF555. The effective power of laser 1 was reduced to approximately 5 mW by using a polarizer and an excitation slit. Laser 2 was not attenuated. Two Hamamatsu R928 photomultiplier tubes (PMT) were attached to the top and bottom of the sample chamber and powered by 850 and 800 V signals for the AF647 and AF555 channels, respectively. Current from the PMT was converted to voltage by using transimpedance amplifiers that amplified the signal by a factor of 106 for AF647 and 105 for AF555. Voltage was sampled at a rate of 800 samples sec−1 by using a digital acquisition board from National Instruments (DaqPAD 6020E). Fluorescence signal was acquired as a function of filament position by using LabView. Following fluorescence scanning for most experiments, filament regions were cut and scanned again in a microarray flatbed scanner (GenePix 4000B, Axon Instruments, Union City, CA). Figure 2.Schematic of lasers and photomultipliers (PMT) to detect online fluorescence of the filament. The filament is moved (arrow) through a rectangular sample chamber with a laser excitation source attached to either side. Photomultiplier tubes are attached to the top and bottom of the chamber. Filters Filter sets were placed in the light path between the sample chamber and the photomultipliers (Fig. 2). Long-pass filters with cutoffs at 685 nm (Chroma, Rockingham, VT) and 665 nm (Melles Griot, Rochester, NY) were combined to reduce reflected laser light from the AF647 laser. For the AF555 channel, two long-pass filters (570 nm cutoff, Melles Griot) were combined with a bandpass filter centered at 565 nm (30 nm bandwidth, Chroma) to reduce reflected light. Classification Algorithm The LabView program coordinated all filament movement, filament scanning, and feedback control. Elements of the feedback control are described by the nodes of the decision tree used to classify the viruses shown in Fig. 3. The fluorescent fiducial markers on either side of the capture-antibody region produced characteristically sharp emission peaks. Since the physical location of the immobilized antibody positions between the markers was known, experimental conclusions were based on the distance of the first detected peak from the initial fluorescent marker. Figure 3.Decision-tree algorithm to classify virus strains. The filament control program enters the decision tree from the top at level 1 and, based on the type of virus found at each level, follows different branches of the decision tree. When reovirus is detected at level one, the filament is moved forward to the next antibody region for level 2 testing. If a serotype 3 reovirus strain is detected (T3D or T3SA+ ) at level 2, testing continues to level 3 to distinguish between these strains. The filament-control program was designed to find each peak, calculate its location along the filament, and make a decision about additional tests. Spatially localized fluorescence from the filament was measured as a 0–14 V signal from the transimpedance amplifier. Voltage data from the scan were used as input data by a peak-detection function in LabView. The LabView peak detector fit a quadratic polynomial to sequential sets of points depending on a width parameter entered by the user. Data were then compared to a threshold parameter, also entered by the user, to identify peaks. A binary decision to stop or continue testing was based on the location and number of peaks found. Peak-detection threshold parameters were defined as 0.3 V (approximately three times background) with a width of 30 data points, which corresponded to a width slightly larger than the physical width of the comb tooth. A common set of parameters was used in all experiments based on previous work with M13K07.10 A filament region containing three capture antibodies and two fiducial markers was incubated within each chamber for the times shown in Table 2. Each of the capture-antibody regions contained a PBS negative control in the first position. In level one the second position was a mixture of 9BG5 and 5C6 (0.25 mg mL−1, 0.2 mg mL−1). The third position was anti-M13K07 (0.5 mg mL−1). In levels two and three, the negative control position was followed by 9BG5 (mg mL−1) in the second position and 5C6 (0.2 mg mL−1) in the third position. The logic encoded in the decision tree shown in Fig. 3 was followed. In level 1, if fluorescence was detected from the anti-M13K07 position of the first region of the filament, a classification of M13K07 virus was made, and no further testing was conducted. If fluorescence was detected in the 9BG5/5C6 region, a classification of reovirus was made and the filament was advanced to the appropriate region for level 2 testing, where a more specific test for reovirus was performed. In level 2 testing, fluorescence from the 5C6 region indicated a serotype 1 reovirus, which in this scheme corresponded to reovirus T1L. A classification of T1L represented an endpoint in the decision tree, and testing was discontinued. Fluorescence from the 9BG5 region indicated the presence of a serotype 3 reovirus, and the program advanced the filament for further subtyping in level 3. Level 3 testing used AF555 conjugated 4F2 antibody as the detecting antibody and a second detection channel with a green excitation laser. If fluorescence was found in the 9BG5 region using this channel, a classification of reovirus T3D was made and testing along that branch ended. If no fluorescence was detected from the second channel for that region, the program ended with a classification of reovirus T3SA+ . If no peaks were found between the fiducial markers, a message was generated indicating that no virus was found. Similarly, if a peak was detected in the negative control region of the filament, a warning message was generated. Although theoretically possible, this situation did not arise during testing. Results FARA utilizes a polyester filament with circumferential bands of immobilized antibody that is passed through a series of five glass micro-reaction chambers that are similar to the five major steps of an ELISA (Table 2). In the first reaction chamber of the FARA approach employed in this study, capture antibody coupled to the filament was rehydrated. In this chamber the filament also was blocked to prevent non-specific binding of virus. In the second chamber, the immobilized capture antibody was incubated with virus solution. In this chamber, if virus in solution bound to antibody coupled to the filament, then the virus became coupled to the filament via this interaction. In the third chamber, non-specifically bound virus was removed by washing before incubation with a fluorescently labeled detecting antibody in the fourth chamber. A final wash in the fifth chamber removed non-specifically bound detecting antibody. The capture antibody region was then passed through an integrated fluorescence detector. In these experiments, the test virus was classified with greater specificity at each level of a decision tree (Fig. 3). Representative fluorescence signals in volts obtained during testing for phage M13K07 and reoviruses T1L, T3D, and T3SA+ are shown in Fig. 4. Labels for the PBS control position and the antibodies in each capture antibody position are shown adjacent to the filament. The specificity of each test antibody for its corresponding virus was high, with little or no cross-reactivity. No signal was detected in the PBS negative-control position. Figure 4.Fluorescence intensity in volts as a function of filament position for M13K07 phage (A) and reoviruses T1L (B), T3D (C), and T3SA+ (D). The immobilized capture antibody positions on the filament are labeled within each panel. Captured virus was detected by a fluorescently labeled second antibody (anti-M13K07 [red], 8H6 [red], or 4F2 [green]). Corresponding images of the scanned filaments are shown at the top of each graph. Each panel shows a representative experiment of three performed. Detection of M13K07 was achieved by level 1 testing. Strong fluorescence was observed in the anti-M13K07 position but not in the 9BG5/5C6 or PBS positions (Fig. 4a). This finding indicated capture of the phage M13K07 by the anti-M13K07 capture antibody. For detection of M13K07, AF647 conjugated anti-M13K07 was used. Because a classification of M13K07 was made, the other two capture regions of the filament were not evaluated. Detection of the reovirus strains employed in this study required analysis beyond level 1. Level 1 testing indicated the presence of a reovirus for all three strains with a peak in the second position corresponding to virus capture by the 9BG5/5C6 antibody mixture. In level 2 testing, strain T1L was detected in the 5C6 antibody position but not in the 9BG5 or PBS positions (Fig. 4b). In this case, the capture and detecting antibodies differed. Because a reovirus T1L classification was made, the third capture region of the filament was not evaluated. For reovirus T3D, level 1 testing indicated the presence of a reovirus with a peak in the second position corresponding to virus capture by 9BG5/5C6 (data not shown). Level 2 testing showed a distinct peak in the 9BG5 antibody position (Fig. 4c, red curve). In this case, 8H6 antibody labeled with AF647 was the detecting antibody indicating a type 3 reovirus. Level 3 testing showed a distinct peak in the 9BG5 antibody position (Fig. 4c, green curve). In this case, 4F2 antibody labeled with AF555 was the detecting antibody. Neither curve showed cross reactivity of reovirus T3D with the immobilized 5C6 region or the negative-control PBS region. For reovirus T3SA+ , level 1 testing indicated the presence of a reovirus with a peak in the second position corresponding to virus capture by 9BG5/5C6 (data not shown). Level 2 testing yielded a distinct peak in the 9BG5 antibody position (Fig. 4d, red curve), like the result obtained with T3D. However, level 3 testing with 4F2 did not yield a positive signal in the 9BG5 antibody position, indicating that the captured virus was not T3D. No detectable cross reactivity was found in the 5C6 antibody region or the negative-control PBS region. Thus, the virus was classified as T3SA+. Discussion In this study, we demonstrated the feasibility of a FARA pathogen-classification approach using a well-characterized virus system. Three reovirus strains and one phage were successfully classified by performing sequential antibody-binding assays directed by the decision tree. The structure of the decision tree was based on published characteristics of each virus strain and the known specificity of each antibody. Because the capture antibodies were arrayed at known locations along the filament, and the fluorescence of the detection antibodies was measured as a function of filament location, an increase in filament fluorescence at a particular location indicated antibody interaction with the virus. For each virus strain tested, the expected fluorescence peaks were observed, and the correct classification was made. Moreover, successful transit of all decision tree branches was demonstrated. The biochemical components of FARA are similar to those used in standard ELISAs. Both assays create a dual-antibody sandwich that results in similar detection limits for M13K07 for FARA and ELISA.10 One of the virus-specific antibodies acts as the capture antibody, and the second acts as the detecting antibody. The main difference in the biochemistry of the two assays is that, in the current implementation of FARA, enzyme amplification is not utilized. Like ELISA, FARA utilizes antibodies adsorbed to the surface of a substrate to capture virus from solution. The capacity of bound antibody to retain its antigen-binding activity is essential to the success of both assays. Each of the immobilized test antibodies in our system retained functionality and bound its corresponding virus (Fig. 4). The absence of peaks in the negative-control positions and in positions occupied by antibodies not specific to the test virus demonstrates minimal cross reactivity in the FARA platform employed in this study. We chose reovirus as a test system to show clinical relevance and to avoid potential safety concerns in the laboratory. Reovirus has been used as a model to study mechanisms of viral pathogenesis in mice.14 Although it is a human pathogen, it is rarely associated with human disease.11 Many reovirus field-isolate strains have been characterized,4,7,8 and a broad array of antibodies are available that recognize reovirus with varying specificity.12 We found that not every antibody tested was suitable for use as an immobilized capture antibody. Neither antibody 8H6 nor 4F2 bound virus when immobilized on the filament, even after virus incubation time was increased to greater than 100 min (data not shown). It is possible that these antibodies undergo conformational changes when passively adsorbed to a solid substrate, rendering them inactive. Alternatively, these antibodies may be incapable of antigen binding when immobilized as a consequence of steric hindrance. Antibodies 8H6 and 4F2 recognize the μ1 and σ3 proteins, respectively,12 which form the bulk of the viral outer capsid.5 It is possible that extension of the viral attachment protein σ1 may shield the μ1 and σ3 proteins from binding to the 8H6 and 4F2 antibodies on the filament surface. This conclusion also is consistent with our observation that 9BG5 and 5C6 were both excellent capture antibodies, since they recognize the σ1 protein, which extends farthest from the viral capsid. The failure of the 8H6 and 4F2 antibodies to function as effective capture antibodies complicated our virus detection scheme. Although 8H6 is not serotype-specific and should bind all reovirus strains, we could not use this antibody as the immobilized capture antibody for level 1 testing. Antibodies 5C6 and 9BG5 are specific for serotype 1 and serotype 3 reovirus strains, respectively;12 therefore, we immobilized a mixture of these antibodies for detection of all reovirus isolates in level 1. Although 4F2 recognizes most type 3 strains, T3SA+ is a reassortant virus that contains a σ3-encoding gene segment from T1L.1 As a result, 4F2 does not recognize T3SA+ and is specific only for T3D in our scheme. Since 4F2 could not be used as a capture antibody in level 3, we incorporated a fluorescently labeled 4F2 antibody as a detecting antibody in solution. Although the nature of the biochemical interactions is similar in FARA and ELISA, the capture antibody employed in a standard ELISA is static, and solutions are changed in the well-plate in a fixed sequence. In FARA, the capture antibody is attached to a mobile substrate, and solutions are changed by positioning the filament in different solution chambers. As we have shown previously, this gives FARA a sensitivity limit similar to ELISA on the order of 107 virus particles.10 A major advantage of FARA is that it is dynamic and allows modification of processing in response to results.9 Virus incubation time with the capture antibody can be reduced to as low as one minute depending on the virus concentration and antibody affinity, reducing the overall assay duration. On the other hand, the incubation time can be increased in order to increase sensitivity.9 Moreover, because the filament is mobile, the capture antibody is brought to the virus solution, thus enabling use of capture antibodies with different specificities to interrogate a single aliquot of virus solution. Attachment of the capture antibody to a mobile filament in FARA makes accurate positioning of the filament and correct identification of the capture antibody regions essential for virus strain classification. These parameters are particularly important in multilevel testing of the type used in our study. A simple bar code system based on fluorescent fiducial markers was used to identify the leading edge of each of the capture antibody regions and position the filament in the reaction chambers. The pattern of the fluorescent marks also was used to identify the level of testing. Since our simple test system involved three levels, and testing always began in level 1, only searches for patterns containing one or two fluorescent marks corresponding to levels two and three were required. However, this simple approach allows for the incorporation of more complex bar codes using additional spots or patterns of spots. This study demonstrates the feasibility of an automated system for diagnosing specific virus strains. Although the scheme we report is a relatively simple implementation of this system, more complex designs are possible. For example, each antibody capture region could contain additional antibodies. In addition, there is essentially no limit to the overall length of the filament, which would allow the incorporation of many additional testing regions. The sensitivity of FARA could also be increased by the use of quantum-dot labeled detection antibodies as well as a more sensitive fluorescence detection system. The most important requirement is the availability of a wide range of antibodies to the specific pathogens of interest. Such a strategy may have applications for detecting specific pathogens from complex mixtures. This approach would have both clinical and environmental applications.

Diabetologia-3-1-2063563

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms

Aims/hypothesis Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms. Introduction Genome-wide scans for diabetes susceptibility genes have been performed in various populations. In early 2006, it was reported that variants in the transcription factor 7-like 2 (TCF7L2) gene were strongly associated with an increased risk of type 2 diabetes in an Icelandic, a Danish and a US population [1]. The estimated population attributable risk of type 2 diabetes of this variation lies between 10 and 25%, which is in the range of the peroxisome proliferator-activated receptor γ gene (PPARγ) Pro12Ala (25% [2]) and the potassium inwardly-rectifying channel, subfamily J, member 11 gene (KCNJ11) Glu23Lys (15% [3]) polymorphisms. In the initial report of Grant et al. [1], five single nucleotide polymorphisms (SNPs) within introns 3 and 4 of the TCF7L2 gene (rs12255372, rs7903146, rs7901695, rs11196205, rs7895340) were identified to associate with an increased risk of type 2 diabetes. This finding initiated a series of reports in various populations confirming the effect of these polymorphisms within the TCF7L2 gene on the type 2 diabetes risk [4–12]. Recently, the T allele of rs7903146 was identified as the variant that most strongly determines the risk of type 2 diabetes [13]. In carriers of the risk alleles for SNPs within TCF7L2 several studies additionally showed an impaired insulin secretion estimated from an OGTT or IVGTT [6, 7, 9, 10]. These studies indicate that progressive loss of insulin secretion might be the essential component of the phenotype which predisposes carriers of the TCF7L2 variant to develop type 2 diabetes. The pathogenic mechanism of the impaired insulin secretion due to polymorphisms in the TCF7L2 gene is not yet clear. It could be the consequence of a reduction in beta cell mass or could reflect a dysfunction of pancreatic beta cells. Furthermore, a defect in incretin-induced stimulus secretion coupling could mediate a reduction of insulin secretion in carriers of the TCF7L2 polymorphisms. The latter hypothesis is supported by the molecular mode of action of the transcription factor TCF7L2. TCF7L2 is an essential component of the wingless-type MMTV integration site family, member 1 (WNT) signalling pathway, which is crucial for the regulation of the glucagon gene (GCG) expression and the secretion of its product GLP-1 by the intestinal endocrine L cells [14]. Therefore, an alteration in the WNT signalling pathway through polymorphisms in the TCF7L2 gene might result in an altered GLP-1 response, which in turn could lead to a lower postprandial insulin secretion. To test this hypothesis we analysed the associations of the above-described TCF7L2 polymorphisms with basal GLP-1 secretion and glucose-induced GLP-1 response during an OGTT in non-diabetic participants. We further studied the influence of the TCF7L2 SNPs on insulin secretion kinetics to i.v. administered glucose during an IVGTT and a hyperglycaemic clamp. In addition, we particularly investigated the influence on GLP-1-induced insulin secretion using a combined hyperglycaemic clamp with additional GLP-1 infusion and arginine bolus [15]. Methods Participants We studied 1,110 non-diabetic participants by an OGTT (Table 1). Secretion patterns of GLP-1 were obtained in a subgroup of 155 participants. The anthropometric characteristics of this subgroup are shown in Table 2. All 1,110 participants were also genotyped for the following five SNPs in the TCF7L2 gene: rs7903146, rs12255372, rs7901695, rs11196205 and rs7895340. Table 1Anthropometric and metabolic data from all 1,110 participants who underwent an OGTT rs7903146p value (ANOVA)p value (CC vs XT)GenotypeC/CC/TT/Tn54647490NGT/IGT461/85386/8863/270.0070.03Sex (female/male)346/200315/15950/400.130.64Age (years)39 ± 140 ± 141 ± 10.390.67BMI (kg/m2)29.5 ± 0.428.9 ± 0.428.2 ± 1.00.510.24Plasma glucose (mmol/l) Fasting 5.1 ± 0.025.1 ± 0.025.2 ± 0.070.250.39 2 h 6.1 ± 0.076.2 ± 0.076.7 ± 0.20.060.17Plasma insulin (pmol/l) Fasting62.0 ± 2.153.4 ± 1.749.8 ± 3.10.0040.001 2 h442 ± 19356 ± 17372 ± 340.120.04Insulin sensitivityOGTT (arbitrary units)16.8 ± 0.518.2 ± 0.517.8 ± 1.20.02/0.02a0.005/0.006aInsulin secretionOGTT (pmol/mmol)319 ± 5301 ± 5292 ± 100.003/0.04b0.0009/0.02bInsulin/glucose ratio (pmol/mmol)143 ± 5127 ± 5124 ± 130.003/0.03b0.001/0.01bData are means ± SEMp values were obtained using χ2 test or ANOVAAnalysis for rs12255372 and rs7901695 also revealed a significant difference in insulin secretion (p = 0.003 and p = 0.05), whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected (p = 0.53 and 0.45)ap values are derived from multivariate linear regression models: adjusted for age, sex and BMIbp values are derived from multivariate linear regression models: adjusted for age, sex, BMI and insulin sensitivityTable 2Anthropometric and metabolic data of the subgroup of 155 participants with additional measurements of GLP-1 rs7903146p value (ANOVA)p value (CC vs XT)GenotypeC/CC/TT/Tn736715NGT/IGT64/948/1910/50.030.009Sex (female/male)50/2342/258/70.500.33Age (years)46 ± 147 ± 147 ± 30.990.96BMI (kg/m2)29.8 ± 0.730.3 ± 0.727.1 ± 1.10.150.88Plasma glucose (mmol/l) Fasting5.2 ± 0.055.3 ± 0.075.6 ± 0.20.170.21 2 h6.5 ± 0.17.0 ± 0.27.2 ± 0.50.220.10Insulin sensitivityOGTT (arbitrary units) 12.8 ± 0.713.6 ± 1.015.7 ± 2.60.74/0.92a0.78/0.75aInsulin secretionOGTT (pmol/mmol)304 ± 12287 ± 13235 ± 190.01/0.02b0.02/0.01bGLP-1 (pmol/l) 0 min 16.1 ± 0.917.3 ± 1.317.3 ± 2.00.910.88 30 min34.1 ± 2.138.8 ± 4.038.1 ± 3.50.450.36 120 min28.9 ± 1.529.0 ± 1.728.9 ± 2.40.870.88 Fold increase 0–30 min2.5 ± 0.22.7 ± 0.32.4 ± 0.40.770.84Data are means ± SEMp values were obtained using χ2 test or ANOVAAnalysis for rs7901695 also revealed a significant difference in insulin secretion (p = 0.02), whereas for rs7895340 and rs11196205 no significant differences in insulin secretion were detected (p = 0.73 and 0.76)ap values were derived from multivariate linear regression models: adjusted for age, sex and BMIbp values were derived from multivariate linear regression models: adjusted for age, sex, BMI and insulin sensitivityA subset of 210 participants were studied by an IVGTT combined with a euglycaemic–hyperinsulinaemic clamp to determine insulin secretion capacity and insulin sensitivity in one test [16] (Table 3). Table 3Anthropometric and metabolic data of 210 participants who underwent a combined IVGTT and hyperinsulinaemic–euglycaemic clamp rs7903146p value (CC vs XT)GenotypeC/CX/Tn97113NGT/IGT80/1781/320.06Sex (female/male)59/3864/490.58Age (years)44 ± 145 ± 10.29BMI (kg/m2)29.0 ± 0.528.8 ± 0.50.82Insulin sensitivityclamp (μmol kg−1 min−1 [pmol/l]−1)0.07 ± 0.010.07 ± 0.010.77Insulin secretion indexa IVGTT C-peptide secretion (pmol/l)7,904 ± 3067,679 ± 3230.40 IVGTT insulin secretion (pmol/l)2,059 ± 1311,924 ± 1290.25 OGTT insulin secretion (pmol/mmol)314 ± 8292 ± 80.04Data are means ± SEMp values were obtained using χ2 test or unpaired t test or multivariate linear regression modelsNone of the four other SNPs in TCF7L2 (rs12255372, rs7901695, rs11196205 and rs7895340) affected significantly insulin secretion in the IVGTT (all p > 0.2)aAdjusted for age, sex and BMI, and insulin sensitivityAdditionally, 233 participants were studied by a hyperglycaemic clamp [17]. The participants were selected from three independent studies in the Netherlands and Germany. The German and the Dutch cohort from Utrecht consisted of NGT and IGT participants, the Dutch cohort from Hoorn consisted of IGT participants only (Table 4). All participants in our studies underwent a standard OGTT to define their glucose tolerance status. Details of the study groups have been described previously [17–19]. In the German subgroup (n = 73), the hyperglycaemic clamp was continued with an additional GLP-1 and arginine administration [15, 20–22]. Table 4Anthropometric and metabolic data of 233 participants who underwent a hyperglycaemic clamp and of 73 participants who underwent a modified hyperglycaemic clamp with additional GLP-1 and arginine administration GermanyThe Netherlands (Utrecht)The Netherlands (Hoorn)rs7903146p valuers7903146p valuers7903146p valueGenotypeC/CX/TC/CX/TC/CX/TSex (female/male)15/2024/140.085/3122/11<0.0117/1130/330.27NGT/IGT30/527/110.1331/526/70.530/280/66–Age (years)38 ± 240 ± 20.6647 ± 147 ± 10.9456 ± 158 ± 10.43BMI (kg/m2)26.1 ± 0.925.1 ± 0.60.4125.6 ± 0.726.2 ± 0.70.4929.2 ± 0.827.9 ± 0.40.13Plasma glucose (mmol/l) Fasting5.1 ± 0.15.0 ± 0.10.724.6 ± 0.14.8 ± 0.10.186.6 ± 0.16.6 ± 0.10.75 2 h glucose5.9 ± 0.36.5 ± 0.30.215.7 ± 0.35.6 ± 0.30.749.5 ± 0.19.5 ± 0.10.84Insulin sensitivity (μmol kg−1 min−1 [pmol/l]−1)0.15 ± 0.020.15 ± 0.010.900.17 ± 0.020.18 ± 0.020.600.11 ± 0.010.14 ± 0.010.21Insulin secretion index (pmol/l)a First phase (sum 0–10 min)1,067 ± 158815 ± 1050.18963 ± 75891 ± 960.76699 ± 70679 ± 720.68 Second phase308 ± 47291 ± 410.80389 ± 42376 ± 470.71304 ± 45303 ± 380.18 First phase GLP-12,049 ± 2581,466 ± 1750.03n.a.n.a.n.a.n.a. Second phase GLP-14,567 ± 5682,881 ± 3410.006n.a.n.a.n.a.n.a. Arginine2,680 ± 2262,252 ± 1810.40n.a.n.a.n.a.n.a.Data are means±SEp values were obtained using χ2 test, unpaired t test or multivariate linear regression modelsCarriers of the risk allele for SNP rs12255372 showed reduced first phase (p = 0.02) and second phase (p = 0.005) GLP-1-induced insulin secretionCarriers of the risk allele for SNP rs7901695 showed reduced second phase (p = 0.02) GLP-1-induced insulin secretionn.a. Not availableaAdjusted for age, sex and BMI, and insulin sensitivityThe participants were not related to each other. Informed written consent for all studies was obtained from all participants, and the local ethics committee approved the protocols. Genotyping Detection of the TCF7L2 polymorphisms described by Grant et al. [1] was done using the TaqMan assay (Applied Biosystems, Forster City, CA, USA) in the German population. The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 7000, and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems). As a quality standard, we randomly included six positive (two homozygous wild-type allele carriers, two heterozygous and two homozygous risk allele carriers) and two negative (all components excluding DNA) sequenced controls in each TaqMan reader plate. Because all controls were correctly identified, we assumed that the genotyping error rate of this method did not exceed 0.3% [23].In the Dutch population, only rs7903146 was determined using an ABI PRISM 7900HT sequence detector (Applied Biosystems). OGTT At 08:00 hours, participants ingested a solution containing 75 g glucose. Venous blood samples were obtained at 0, 30, 60, 90 and 120 min for determination of plasma glucose, insulin and C-peptide concentrations and 0, 30 and 120 min for determination of GLP-1 concentrations. The participants did not take any medication known to affect glucose tolerance or insulin sensitivity. Tests were performed after an overnight fast of 12 h. Combined IVGTT and hyperinsulinaemic–euglycaemic clamp After an overnight fast and after baseline samples had been obtained, 0.3 g/kg bodyweight of a 20% (vol./vol.) glucose solution was given at time 0. Blood samples for the measurement of plasma glucose, plasma insulin and C-peptide were obtained at 2, 4, 6, 8, 10, 20, 30, 40, 50 and 60 min. After 60 min, a priming dose of insulin was given followed by an infusion (40 mU/m2) of short-acting human insulin for 120 min. A variable infusion of 20% glucose was started to maintain the plasma glucose concentration at 5.5 mmol/l. Blood samples for the measurement of plasma glucose were obtained at 5 min intervals throughout the clamp. Hyperglycaemic clamp Hyperglycaemic clamps were performed at 10 mmol/l in all participants. The Dutch NGT and IGT participants underwent a 3 h clamp. In the German NGT participants, the clamp lasted for 2 h followed by the GLP-1 and arginine stimulation (see below). After an overnight fast, the participants received an i.v. glucose bolus to acutely raise glucose levels to 10 mmol/l. Plasma glucose levels were measured at the appropriate intervals to maintain a constant plasma glucose during the clamp. Blood samples for insulin were drawn at 2.5 min intervals during the first 10 min of the clamp and at 10–20 min intervals during the remainder. Exact details of the clamping procedures in the different study groups have been described previously [15, 18, 19]. Combined hyperglycaemic clamp This hyperglycaemic clamp combined with GLP-1 and arginine administration was performed as previously described [15, 20–22]. After 120 min of hyperglycaemic clamp at 10 mmol/l, a bolus of GLP-1 (0.6 pmol/kg) was given (human GLP-1(7-36)amide; Poly Peptide, Wolfenbüttel, Germany) followed by a continuous GLP-1 infusion (1.5 pmol kg−1 min−1) during the next 80 min. At 180 min, a bolus of 5 g arginine hydrochloride (Pharmacia & Upjohn, Erlangen, Germany) was injected over 45 s while the GLP-1 infusion was continued. Blood for the measurement of glucose, insulin, proinsulin and C-peptide was obtained during the time-points shown in Fig. 1. This clamp allows measurement of different aspects of stimulus secretion coupling: first and second phases of glucose-induced insulin secretion, GLP-1-induced insulin secretion, and the response to additional arginine administration. Fig. 1Associations between the genotypes of rs7903146 polymorphism in the TCF7L2 gene with insulin secretion during a hyperglycaemic clamp in 73 German participants. Open circles CC; closed circles CT and TT. AIR Acute insulin response. Arrow Administration of 5 g arginine. The p values show the differences for first and second phases of glucose-induced insulin secretion, first and second phases of GLP-1-induced insulin secretion and acute insulin secretory response to arginine (AIR) (for calculation see ‘Methods’; insulin secretion is adjusted for insulin sensitivity) Analytical procedures Plasma glucose was determined as previously described [14, 17, 20, 21]. GLP-1 was determined during the OGTT at baseline, 30 and 120 min. GLP-1 immunoreactivity was determined using an RIA specific for the C-terminus of the peptide [24]. Samples were stored at −80°C. The assay measures the sum of the intact peptide plus the primary metabolite, GLP-1(9–36)amide using the polyclonal anti-GLP-1 antibody 89390 and synthetic GLP-1(7–36)amide as standard.Plasma insulin and C-peptide concentrations in the German cohort were measured by a microparticle enzyme immunoassay (Abbott, Wiesbaden, Germany) and an RIA (Byk-Santec, Dietzenbach, Germany). In the participants from the Netherlands, insulin was measured using an in-house competitive RIA employing a polyclonal anti-insulin antibody (Caris 46), 125I-labelled insulin (IM 166; Amersham, Roosdaal, the Netherlands) as a tracer and regular insulin (Humulin; Eli Lilly, Indianapolis, IN, USA) as a standard. Calculations Insulin secretion in the OGTT was assessed by calculating the AUC for C-peptide divided by the AUC for glucose (AUCCP/AUCGlc). AUCs were determined by the trapezoidal method. Furthermore, insulin secretion was calculated as insulin:glucose ratio by dividing (insulin at 30 min–insulin at 0 min) by (glucose at 30 min–glucose at 0 min). Insulin sensitivity during the OGTT was estimated from glucose and insulin values as proposed by Matsuda and DeFronzo [25].Insulin secretion during the IVGTT was calculated as the sum of C-peptide levels during the first 10 min after glucose administration. Insulin sensitivity during the hyperinsulinaemic–euglycaemic clamp was calculated by dividing the average glucose infusion rate during the last 40 min of the clamp by the average plasma insulin concentration during the same time interval.Insulin secretion during the hyperglycaemic clamp was calculated using insulin levels determined during the clamp. The first phase of insulin secretion was defined as the sum of the insulin levels during the first 10 min of the clamp. The second phase of insulin secretion was defined as the mean of the insulin values during the last 40 min (80–120 min, NGT group, Germany) or the last 30 min (150–180 min, NGT and IGT groups, the Netherlands) of the clamp.In the combined hyperglycaemic clamp with GLP-1 and arginine administration, first-phase GLP-1-induced insulin secretion was defined as the mean of the 125 and 130 min insulin levels, second-phase GLP-induced insulin secretion (plateau) was defined as the mean of the 160–180 min insulin levels. The acute insulin response to arginine was calculated as the mean of 182.5 and 185 min minus 180 min insulin levels [21].The insulin sensitivity index was determined by relating the glucose infusion rate to the plasma insulin concentration during the last 40 min (NGT, Germany) or 30 min of the clamp. Statistical analysis Data are given as means ± SEM. Non-normally distributed parameters were logarithmically transformed to approximate linearity for statistical comparison. Distribution was tested for normality using the Shapiro–Wilk W test. Differences in anthropometrics and metabolic characteristics between genotypes were tested using ANOVA for the three genotype groups. The secretion indices were compared using multivariate regression models. In these models the trait was the dependent variable whereas age, sex, insulin sensitivity and genotype were the independent variables. Due to the relatively low number of participants who were homozygous for the rare alleles in the hyperglycaemic clamp group, a dominant model was used. A p value of <0.05 was considered to be statistically significant. The statistical software package JMP (SAS Institute, Cary, NC, USA) was used. Results Genetic variants in the TCF7L2 gene The SNPs described in the paper of Grant et al. [1] had an allelic frequency of the minor allele of 30% (rs7903146), 28% (rs12255372), 31% (rs7901695), 46% (rs11196205) and 45% (rs7895340) in our population. These polymorphisms were all in Hardy–Weinberg equilibrium and in linkage disequilibrium (Electronic supplementary material Table 1). OGTT: glucose tolerance, insulin secretion, insulin sensitivity and GLP-1 levels The percentage of participants with IGT was significantly higher in carriers of the risk allele for rs7903146 (p = 0.007), rs12255372 (p = 0.02) and rs7901695 (p = 0.02).Insulin secretion assessed as AUCCP/AUCGlc during the OGTT was significantly reduced in participants with the risk alleles for rs7903146 and rs12255372 in the additive and dominant model both unadjusted and adjusted for relevant covariates. Participants with the risk allele for rs7901695 had a significantly decreased insulin secretion in the additive model (p = 0.02) (Table 1).Insulin sensitivity, estimated by the index of Matsuda and DeFronzo [25], was significantly higher in participants carrying the risk alleles for rs7903146 and rs1255372 in TCF7L2 (Table 1).In a subgroup of 155 participants we measured GLP-1 concentrations at baseline, at 30 min and 120 min after oral ingestion of 75 g glucose. The anthropometric characteristics are shown in (Table 1). Neither basal GLP-1 plasma levels nor GLP-1 levels during the OGTT significantly differed between the genotypes in any of the above-described SNPs in the TCF7L2 gene (Table 2). Combined IVGTT and hyperinsulinaemic–euglycaemic clamp: glucose-induced insulin secretion and insulin sensitivity C-peptide and insulin values during the IVGTT were not significantly different between the genotypes in any of the above-described SNPs in the TCF7L2 gene. Table 3 shows the results for rs7903146 in the additive and dominant model adjusted for relevant covariates. Including the glucose tolerance status as an additional co-variate in the model did not affect the results. Interestingly, a lower insulin secretion in this subgroup of participants could be demonstrated when insulin secretion was calculated from the OGTT as described above (Table 3). Insulin sensitivity measured with the clamp technique was not affected by any of the genotypes (all p > 0.4). Hyperglycaemic clamp: glucose-, GLP-1- and arginine-induced insulin secretion and insulin sensitivity First- and second-phase insulin secretion during the hyperglycaemic clamp were not significantly different between carriers and non-carriers of the risk allele for rs7903146 (Table 4) in any of the three populations from the Netherlands and from Germany. The other four SNPs in TCF7L2 which were described above were determined in the German population only and were not associated with glucose-induced insulin secretion (data not shown) during the hyperglycaemic clamp.The first phase of GLP-1-induced insulin secretion was significantly reduced in carriers of the risk alleles for rs7903146 (p = 0.03) and rs12255372 (p = 0.02). In addition, we found significant differences in the second phase of GLP-1-induced insulin secretion for rs7903146 (p = 0.006), rs12255372 (p = 0.005) and rs7901695 (p = 0.02) with carriers of the risk alleles having a significantly lower second phase of GLP-1-induced insulin secretion. Table 4 and Fig. 1 show the results for SNP rs7903146.In contrast to GLP-1-induced insulin secretion, arginine-induced insulin secretion was not affected by any of the analysed SNPs in TCF7L2 (Table 4). Discussion The variants in TCF7L2 described by Grant et al. [1] that mediate an increased risk of type 2 diabetes have been found to be associated with reduced insulin secretion [6, 7, 9, 10]. We could confirm this reduced insulin secretion in our study in a group of 1,110 German non-diabetic participants using C-peptide levels during an OGTT. Unexpectedly, the i.v. application of glucose during an IVGTT did not affect insulin secretion in carriers of the risk alleles for SNPs in TCF7L2. The same results were obtained using i.v. glucose challenge during a hyperglycaemic clamp in subgroups of the German population and a population from the Netherlands. The observed different results between an orally and i.v. administered glucose challenge generate the hypothesis that incretin-induced insulin secretion is affected by the variants in the TCF7L2 gene. Possible mechanisms explaining these findings include impaired incretin production or incretin signalling. To address this issue we first measured basal GLP-1 concentrations and GLP-1 response during an OGTT. None of the tested variants that have been shown in the literature to exert a reproducible effect on type 2 diabetes were associated with basal GLP-1 levels or with GLP-1 response during the OGTT. Furthermore, we combined the hyperglycaemic clamp with additional GLP-1 infusion to measure GLP-1-induced insulin secretion. Both acute (so called first phase) and prolonged (so called second phase) GLP-1-induced insulin secretion during the hyperglycaemic clamp were significantly impaired in carriers of the risk alleles in TCF7L2. These data suggest that participants carrying the risk alleles have an impaired insulin secretion due to an impairment of the GLP-1 signalling chain. The putative molecular mechanism for this finding is not clear. As the transcription factor TCF7L2 plays an essential role in the WNT signalling pathway [14], which on the one hand is required for a normal development of the pancreas and the pancreatic islets [26] and on the other hand is crucial for the regulation of GLP-1 expression and secretion in intestinal L cells [14]. In theory, polymorphisms in the TCF7L2 gene might therefore through alteration of the WNT signalling pathway directly affect beta cell growth, beta cell differentiation and beta cell function. Furthermore insulin secretion might also be affected indirectly via the enteroinsular axis either through an impaired overall GLP-1 secretion or through a defective or dysfunctional GLP-1-induced insulin secretion. An impaired or dysfunctional GLP-1 effect might result first in a reduced postprandial insulin secretion and second, might influence stimulation of beta cell growth and beta cell differentiation. Our data show that basal and glucose-induced GLP-1 secretion during the OGTT were not affected by any of the analysed SNPs, whereas the first and second phases of GLP-1-induced insulin secretion were significantly reduced in carriers of the risk alleles in three of the five above-described SNPs in the TCF7L2 gene, among them rs7903146, which is most strongly associated in the literature with type 2 diabetes [1, 6, 9, 12, 13]. The finding that basal and glucose-stimulated GLP-1 secretion were not influenced by the TCF7L2 polymorphisms indicates that a reduced GLP-1 secretion is not likely to explain the impaired insulin secretion in carriers of the risk genotypes, although it can not fully exclude an effect of these SNPs on GLP-1 levels. First, by measuring total GLP-1 levels, we may have missed a subtle defect in GLP-1 secretion, which may have been detected by measuring the active form of GLP-1. Second, systemic GLP-1 levels may not adequately reflect the level of the active hormone acting in the gut wall on the autonomic nervous system [27]. Third, impaired TCF7L2 activity might tissue-specifically reduce the GLP-1 levels in the brain, which are believed as well to be important for insulin secretion [28]. While we have no evidence for reduced GLP-1 secretion we have strong evidence for an impaired GLP-1-induced insulin secretion in carriers of the above-described polymorphisms in TCF7L2. The data show a reduced efficiency of GLP-1 to stimulate insulin secretion in pancreatic beta cells. Alterations of the GLP-1 signalling pathway in beta cells might thereby play a role. Recently the TCF7L2 gene was also identified in human pancreas [14]. Therefore variations in this transcription factor could specifically impair the transcription of genes involved in the incretin signalling chain, resulting in resistance of the pancreatic beta cells to GLP-1. In contrast to the observed reduction in the first and second phases of GLP-1-induced insulin secretion, the arginine-induced insulin secretion was not significantly affected by the analysed SNPs in the TCF7L2 gene. The arginine bolus in the combined hyperglycaemic clamp produces a maximal challenge for the secretory capacity of the beta cell and can be considered as a surrogate for beta cell mass [15, 20]. The SNPs do not affect this maximal insulin secretion, suggesting that the variants in TCF7L2 do not influence beta cell mass, at least in the prediabetic state. In addition, impaired beta cell function may also include the efficiency of the conversion from proinsulin to insulin [21]. However, there was no evidence for this abnormality related to the variants in the TCF7L2 gene during the hyperglycaemic clamp (data not shown). We also found that insulin sensitivity calculated from the OGTT using an established index [25] was significantly increased in participants carrying the risk alleles for all analysed SNPs in the TCF7L2 gene. This was also described in the study of Florez et al. [9]. When we measured insulin sensitivity with the gold standard, the euglycaemic–hyperinsulinaemic clamp and the hyperglycaemic–hyperinsulinaemic clamp, we found no association of the risk alleles with insulin sensitivity. Taken together, our data suggest that the variants in the TCF7L2 gene do not have a strong effect on insulin sensitivity. In summary, our data show that variations in the TCF7L2 gene are associated with impaired GLP-1-induced insulin secretion. This might be consistent with a state of relative incretin resistance. Increasing the incretin levels by pharmacological tools may thus be a logical therapy to overcome impaired insulin secretion in carriers of the TCF7L2 variants. Electronic supplementary material Below is the link to the electronic supplementary material. ESM 1 Linkage disequilibrium statistics (D′, r2) among the five SNPs reported by Grant et al. [1] in the German population (n=1,110) (PDF 17 KB).

Plant_Mol_Biol-4-1-2295253

Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis

Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought. Introduction Photosynthesis provides plants with sugars that play a central role in the plant life cycle as energy sources, storage molecules, structural components or intermediates for the synthesis of other organic molecules. Next to these metabolic functions, sugars act as signalling molecules with hormone-like properties. Both hexoses and disaccharides are able to induce signalling via different pathways (Jang and Sheen 1997; Loreti et al. 2001; Rolland et al. 2006). In plants glucose has been shown to affect many processes, including germination, early seedling growth, flowering and senescence (Gibson 2000, 2005; Smeekens 2000; Rolland et al. 2006). Moreover, glucose feeding of Arabidopsis seedlings affected the expression of many genes as shown by micro array studies (Price et al. 2004; Villadsen and Smith 2004; Li et al. 2006). Sugar-induced signal transduction has been shown to control gene expression via diverse mechanisms that include transcription, translation, and modification of mRNA and protein stability (Rolland et al. 2006). Genetic analysis showed that sugar signalling in plants is closely associated with plant hormone biosynthesis and signalling, in particular with that of abscisic acid (ABA, for review see Finkelstein and Gibson 2001; Gazzarini and McCourt 2001; León and Sheen 2003; Rook et al. 2006; Dekkers and Smeekens 2007). Four screens for sugar response mutants i.e. sucrose uncoupled (sun), impaired sucrose induction (isi), glucose insensitive (gin) and sugar insensitive (sis) identified ABA deficient mutants (i.e. aba2/isi4/gin1/sis4 and aba3/gin5) and ABA insensitive4 (abi4/sun6/isi3/gin6/sis5) as sugar insensitive (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000; Rook et al. 2001). ABI4 encodes an AP2 domain containing transcription factor that binds a CE1-like element present in many ABA and sugar regulated promoters (Finkelstein et al. 1998; Niu et al. 2002; Acevedo-Hernandez et al. 2005). These observations linked sugar regulation to ABA signalling. However, there are many more examples of co-regulation of sugar and ABA in plants. The expression of many genes is co-regulated by sugar and ABA. A study of Li et al. (2006) showed that 14% of the ABA upregulated genes are induced in response to glucose as well, in total nearly 100 genes. These included genes in stress responses, carbohydrate and nitrogen metabolism and signal transduction. Another group of nearly 40 genes was repressed by both ABA and glucose. In addition the authors identified a group of genes that showed a synergistic upregulation when treated with both ABA and glucose, including the ApL3 (ADP pyrophosphorylase large subunit) gene, which is involved in starch biosynthesis. An earlier study showed that ABA was not able to induce the ApL3 gene but that ABA in combination with sugar boosted transcription levels in comparison to sugar treatment alone (Rook et al. 2001). Interestingly, Akihiro et al. (2005) found a similar ABA/sugar interaction for the regulation of ApL3 expression and starch biosynthesis in rice (Oryza sativa). There are many other examples of processes and genes that are regulated by sugar and ABA. E.g. the ABA, stress and ripening-induced (ASR) protein in grape (Vitis vinifera) that binds to the sugar response boxes in the promoter of the monosaccharide transporter VvHT1. ASR mRNA expression is responsive to sugar and is modulated by ABA (Cakir et al. 2003). Both sugar and ABA signals also regulate the maize (Zea mays) invertase gene, Ivr2 (Trouverie et al. 2004). Thus ABA/sugar interactions were reported for a multitude of processes and in different plant species indicating that these interactions are physiologically relevant. Therefore, a thorough understanding of these sugar and ABA interactions is of importance. An important strategy for the isolation of sugar response mutants makes use of the effect of exogenous supplied sugars on early seedling development in Arabidopsis Although the use of high sugar concentrations (>300 mM) possibly are above the physiological range, it provided an efficient screening method for mutants (reviewed by Rognoni et al. 2007). Such elevated sugar concentrations arrest early seedling development, which is characterized by the absence of cotyledon greening and leaf formation. The difference in phenotype between arrested and non-arrested seedlings was exploited to isolate sugar insensitive mutants that showed proper seedling development on high glucose (gin) and sucrose (sis) media. In addition to the aforementioned role of ABA biosynthesis and signalling such sugar response mutants revealed a role for HEXOKINASE1 (HXK1/GIN2) and ethylene signalling in the GIN response pathway (Zhou et al. 1998; Gibson et al. 2001; Cheng et al. 2002; Moore et al. 2003). ABA deficiency caused by either mutations in ABA1, ABA2 or ABA3 (Koornneef et al. 1998), resulted in a clear gin phenotype but analysis of ABA insensitive mutants resulted in more ambiguous phenotypes. In addition to abi4, only abi5 and abi8 showed a sugar insensitive phenotype, while for abi1-1, abi2-1 and abi3-1 no or only a weak phenotype was reported (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000; Brocard et al. 2002; Brocard-Gifford et al. 2004). Nevertheless, a role for ABI3 in sugar signalling during early seedling development was hinted too by later studies. E.g. ABI3 over expression lines were sugar hypersensitive (Finkelstein et al. 2002; Zeng and Kermode 2004) and abi3 mutants were insensitive to glucose in combination with ABA (Nambara et al. 2002). This insensitivity appeared to be allele specific and the assay used was different from other sugar sensitivity assays. However, in a more recent report WT glucose sensitivity during early seedling development for the abi3 mutant was observed (Yuan and Wysocka-Diller 2006). Thus it is unclear how and to what extend sugar and ABA signalling overlap. ABA by itself blocked post-germination early seedling development, in addition to the role of ABA in the glucose-induced seedling arrest (Lopez-Molina et al. 2001). Similar to glucose-arrested seedlings, ABA-arrested seedlings lacked greening and leaf formation. The ABA-induced seedling arrest is dependent on ABI3 and ABI5 activity and both abi3 and abi5 mutant do not arrest early seedling growth in response to ABA (Lopez-Molina et al. 2002). This ABA sensitivity is confined to a limited time window after the start of germination (Lopez-Molina et al. 2001; Lopez-Molina et al. 2002) Adding ABA after this sensitive phase to seedlings no longer affects vegetative development. Similarly, the glucose-induced seedling arrest could only be triggered in a similar limited time frame after sowing (Gibson et al. 2001). Here a study of the role of ABA biosynthesis and ABA signalling in sugar response pathways is presented. In particular this study was focussed on the glucose-induced early seedling developmental arrest (the GIN pathway). Analysis of transgenic ABA deficient lines with ectopic ABI4 expression (aba2-1/35S::ABI4) suggested that additional ABA-controlled factors are necessary to arrest early seedling development in response to glucose. ABI3 expression and protein stability are controlled by ABA and ABI3 is a key component in the ABA mediated pathway that blocks early development (Lopez-Molina et al. 2002; Zhang et al. 2005). Therefore, we investigated the role of ABI3 in the GIN pathway. ABI3 encodes a B3 domain transcription factor that contains four conserved domains, one acidic activation domain (A1) and three basic domains (B1, B2 and B3, Giraudat et al. 1992; Finkelstein et al. 2002). ABI3 directs seed specific gene expression and is required for induction of desiccation tolerance and seed dormancy and represses genes related to seed germination (Ooms et al. 1993; Hoecker et al. 1995; Nambara et al. 2000; Suzuki et al. 2001; Finkelstein et al. 2002). Other functions for ABI3 include regulation of flowering, resource allocation, lateral root growth in response to auxin and serve as a signal transduction component downstream of PHYB signalling in seeds (Robinson and Hill 1999; Suzuki et al. 2001; Brady et al. 2003; Mazzella et al. 2005). Here we show that multiple abi3 mutants display a gin phenotype. The glucose insensitive phenotype observed for abi3 mutants was comparable to that of other known gin mutants like aba1, abi4 and abi5. Moreover, abi3 mutants showed a reduced expression level of glucose-regulated genes. In addition, physiological and molecular experiments led to the conclusion that the glucose-arrested seedlings mimic ABA-arrested seedlings. This was supported by the observations that two other ABA response mutants, i.e. era1 and abi2-1, showed altered glucose responsiveness as well. The identification of ABI3 and two other ABA response loci as components in GIN signalling suggests that glucose and ABA signalling share more components than originally anticipated. Material and methods Plant material Arabidopsis plants were grown in a climate chamber at 22°C with 70% humidity and a 16 h/8 h light/dark cycle (Sylvania GRO-LUX fluorescent lamps; Technische Unie, Utrecht) for seed production. WT and mutant seeds that were compared in all experiments presented were produced together and stored under identical conditions. Seeds were dry-stored in paper bags at room temperature for at least a month before use in our experiments. The transgenic aba2-1/35S::ABI4 lines were constructed as follows. ABI4 cDNA sequence was PCR amplified and cloned in pDONR vector and sequenced. Using Gateway cloning ABI4 was cloned behind the Cauliflower Mosaic Virus 35S promoter in the pGD625 vector. The pGD625 vector containing 35S::ABI4 was introduced in Agrobacterium which were used to transform aba2-1 plants by the floral dip method (Clough and Bent 1998). Germination assays All germination assays were performed on 0.5 MS: half-strength Murashige and Skoog medium (pH 5.8), including vitamins, solidified with 0.8% plant agar (Duchefa, Haarlem, The Netherlands). Before plating, seeds were surface-sterilized in 20% (v/v) commercial bleach (Glorix) for 15–20 min and rinsed four to five times with sterile water. After a three days stratification period at 4°C in the dark, plates (with or without sugars present as indicated) were incubated in the growth chamber (22°C and 16 h/8 h light/dark cycle). To investigate the effect of stratification on sugar-free (0.5 MS) media seeds were transferred to sugar media after this stratification period. Control experiments showed that transferring of seeds did not affect seed germination. Germination defined as radicle emergence from the seed coat, was scored daily for 3–8 days. Experiments were performed in duplo, each plate containing 50–100 seeds, and every experiment was repeated one to three times. Sugar response assays For gin assay experiments seeds were sterilized, sown on 0.5 MS media and stratified for three days at 4°C in the dark. Hereafter seeds were collected and sown on 0.5 MS media supplemented with 1% sucrose (control) or on control media with the indicated amount of sugar unless otherwise indicated. Different seed batches show slightly different responses and therefore both 7% and 8% glucose media were used. Plates were incubated in the growth chamber (22°C and 16 h/8 h light/dark cycle) for up to two weeks. Seedlings were scored gin when green cotyledons emerged. To determine the isi phenotype of WT Col, aba2-1, L6 and L10 the seeds were stratified first for three days on sugar-free 0.5 MS media. After stratification the seeds were sown on 100 mM sucrose media. After four days plant material was harvested and ApL3 expression levels determined. For the sun phenotype analysis seeds were sterilized, sown on 0.5 MS and stratified for three days at 4°C in darkness. After stratification seeds were transferred to plates containing 0.5 MS or 0.5 MS with the indicated amount of sugar. Seeds were light-treated for at least one hour before they were incubated in the dark (wrapped in three layers of aluminium foil) at 22°C. After two days plant material was harvested for PC expression analysis by qRT-PCR. Gene expression analysis WT Ler-0, abi3-1 and abi3-5 seeds were stratified for three days and germinated on 0.5 MS media. Seed batches were germinated to approximately 70% (t = 0) before the start of the different treatments. At this stage all WT seedlings were developmentally arrested in response to elevated glucose concentrations and ABA treatment. For RNA isolation and qRT-PCR analysis plant material was ground using mortar and pestle in liquid nitrogen and additionally bead-beaten in a mikro-dismembrator S (B. Braun Biotech International, Germany). RNA was isolated according to Schuurmans et al. (2003). Additionally, RNA was purified using RNeasy columns (Qiagen USA, Valencia, CA). RNA samples were DNase treated, checked for the absence of DNA by PCR and used for cDNA synthesis and qRT-PCR analysis as described before by van Dijken et al. (2004). For expression analysis by qRT-PCR either a 5′ FAM/3′ TAMRA labelled probe (with TaqMan® 2× Universal PCR Master Mix, Applied Biosystems, Foster City, CA) or SYBR®Green technology (Power SYBR®Green PCR Master Mix, Applied Biosystems, Foster City, CA) was used. Relative quantitation of gene expression is based on the comparative CT method (User Bulletin No. 2: ABI PRISM 7700 Sequence Detection System, 1997) using the AtACTIN2 (ACT2) as reference gene. The EM1 primers were obtained for the CATMA database (http://www.catma.org, EM1 forward primer ID: 3a44750.5, EM1 reverse primer ID: 3a44750.3). The sequences of primers and probes that were used for gene expression analysis are listed in Table 1. The qRT-PCRs were run on a ABI-prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Table 1Primers and probes used for gene expression analysisGeneForward primerReverse primerProbeACT2gctgagagattcagatgcccaatgggagctgctggaatccacagtcttgttccagccctcgtttgtgcABI3cacagccagagttccttccttttgtggcatgggaccagactcttgaatctccaccgtcatggccacABI4cggtgggttcgagtctatcaacggatccagacccatagaacaacctcatccaccgccgttggttgaABI5ggaggtggcgttgggtttgggcttaacggtccaaccatcccatttgctgtccacccgctEM1agatgggacacaaaggaggagtgttggtgaactttgactcatcgEM6ggtacgggaggcaaaagcttttgcgtcccatctgctgataRAB18gagcaactccacaaggaaaggtagccaccagcatcatatcApL3cgagaagtgccggattgtaaaggaacgttggatgctgcattcccaagaaacatccgtgtgagattaccgPCtctttgaaggatttcggtgtcacatggccatcgcatttccaaaacgatcgaagctgctgttgccact Results Transgenic ABA deficient lines with constitutive expression of ABI4 are glucose insensitive Screens for sugar insensitive mutants revealed the importance of ABA and ABA signal transduction, in particular ABI4 (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000; Rook et al. 2001). To study the interaction between ABA biosynthesis and ABI4 in sugar signalling we generated transgenic lines with constitutive ABI4 expression driven by a CaMV 35S promoter in an ABA deficient (aba2-1) background. ABI4 expression in these transgenic aba2-1 plants is independent from plant sugar status. Two lines homozygous for the 35S::ABI4 construct were selected, i.e. lines L6 and L10. Both lines germinated normally but showed a stunted growth phenotype (Fig. 1a) similar to what has been reported previously for severe ABI4 overexpression lines (Söderman et al. 2000). Expression analysis on control media showed that ABI4 expression is high in L6 and L10 and exceeded glucose-induced ABI4 expression in developmentally arrested WT Col seedlings by approximately 5–8 fold (Fig. 1b). Fig. 1Analysis of transgenic ABA deficient lines with constitutive ABI4 expression. Plant phenotypes of WT Col, aba2-1, and two transgenic lines with constitutive ABI4 expression in aba2-1 (L6 and L10) after 2,5 weeks of growth on soil (a). ABI4 expression was detected by qRT-PCR in Col, aba2-1, L6 and L10 seedlings grown for two weeks on control or 8% glucose media. Values are averages of two experiments ±SD (b). Seedling phenotypes after two weeks of growth on control, 8% sorbitol (sorb), 8% glucose (glc) or 8% glucose + 0,1 μM ABA (glc + ABA). Similar results were obtained in three experiments (c). ApL3 expression was detected by qRT-PCR in Col, aba2-1, L6 and L10 seedlings grown for four days on control half-strength MS (con) or half-strength MS + 100 mM sucrose (suc) media. Values are averages of three experiments ±SD (d) The gin phenotype of Col, aba2-1, L6 and L10 was tested by growing the lines for two weeks on control, 8% sorbitol and 8% glucose media. All lines showed greening on control (con) and sorbitol (sorb, osmotic control) media as expected (Fig. 1c). On glucose the Col seedlings showed an arrested phenotype whereas aba2-1 showed a gin phenotype as reported before (Fig. 1c, León and Sheen 2003). Also ABI4 expression levels in glucose-arrested Col seedlings were enhanced whereas in aba2-1 ABI4 expression was very low. The L6 and L10 aba2-1/35S::ABI4 lines have very high ABI4 levels but, importantly, display a gin phenotype (Fig. 1c). The gin phenotype of aba2-1 and the transgenic lines could be reverted to WT by the addition of 100 nM ABA as was reported before for aba mutants (Fig. 1c, Arenas-Huertero et al. 2000). Thus, in an ABA deficient background ABI4 overexpression does not restore wild type sensitivity to glucose. Sucrose feeding induces ApL3 gene expression in Arabidopsis seedlings. This sugar regulation of the ApL3 promoter has been used as basis for a screen for sugar response mutants. Rook et al. (2001) reported on isi sugar response mutants that were unable to fully activate ApL3 expression in response to sucrose. The identification of aba2/isi4 and abi4/isi3 as mutants with an impaired sucrose induction phenotype revealed a role for ABA biosynthesis and signalling in control of ApL3 expression (Rook et al. 2001). To study the sugar-induced ApL3 expression in WT Col, aba2-1, L6 and L10 stratified seeds were sown on 100 mM sucrose media. After four days plant material was harvested and ApL3 expression levels determined. Sucrose feeding resulted in an approximate six fold induction of ApL3 in Col seedlings (Fig. 1d). The induction of ApL3 is 25% lower in the aba2-1 seedlings, which is in agreement with the isi phenotype of aba2/isi4 (Rook et al. 2001). In the L6 and L10 aba2-1/35S::ABI4 lines the ApL3 expression level on sugar-free control media already exceeds that of sugar-treated WT seedlings. Sugar feeding of L6 and L10 seedlings further boosted ApL3 expression 7–9 fold higher than sugar-treated WT seedlings (Fig. 1d). Glucose and ABA trigger a similar developmental early seedling arrest High sugar concentrations arrest early seedling development in Arabidopsis. After germination vegetative seedling growth is blocked, characterized by the absence of cotyledon greening and leaf formation. Equimolar concentrations of an osmotic control did not block development in this early seedling stage (Fig. 2a; Zhou et al. 1998; Laby et al. 2000; Rognoni et al. 2007). Previously, Lopez-Molina et al. (2001) showed that treatment with ABA arrested this post-germination switch from embryonic to vegetative growth as well. ABA-arrested seedlings showed neither greening nor leaf formation similar to glucose-arrested seedlings (Fig. 2a). In comparison to ABA-arrested seedlings, glucose-arrested seedlings were larger due to the longer hypocotyl and larger cotyledons (Fig. 2b). Moreover, significant root growth was observed in glucose-arrested seedlings, which is absent in ABA-arrested seedlings (Fig. 2a, b). Further, in glucose-arrested seedlings often a pink colouration was observed, indicative for anthocyanin accumulation that was absent in ABA-arrested seedlings (Fig. 2a, b). Fig. 2Glucose and ABA signalling arrest early seedling development and induce a drought tolerant phenotype. Ler-0 seedling phenotypes are shown after growth for twelve days on control (con, half-strength MS + 1% sucrose), or on con medium supplemented with either 7% sorbitol (sorb), 7% glucose (glc) or 10 μM ABA (ABA) (a). A close-up image of a glc-arrested seedling (left) and an ABA-arrested seedling (right) is shown (b). Developmentally arrested seedlings have a drought tolerant phenotype. Seeds were grown on control (con) or on con media containing either 10 μM ABA (ABA) or 7% glucose (glc) for nine days. At day nine the seedlings were subjected to a drought treatment as indicated (c). Hereafter seedlings were allowed to recover on control media for five days after which survival was scored. The survival percentage following drought treatment is presented (c) ABA-arrested seedlings possess an enhanced resistance to drought stress compared to non-arrested seedlings (Lopez-Molina et al. 2001). A comparison was made between the drought resistance phenotype of glucose- and ABA-arrested seedlings. Control (non-arrested) seedlings did not survive a drought treatment of 2.5 h (Fig. 2c). ABA-treated seedlings showed a near 100% survival rate after 6 h of drought, the longest treatment tested in our experiments. Glucose-arrested seedlings showed an intermediate drought resistance. After 2.5 h of drought stress, glucose-treated seedlings showed a nearly 90% survival rate and after 6 h of drought the survival rate was 50% compared to 0% survival observed for control seedlings. Thus, a clear drought resistant phenotype was observed for glucose-arrested seedlings. The ABA-induced early seedling developmental arrest depends on functional ABI3 and ABI5 genes. abi3 and abi5 mutants did not block early seedling development in response to ABA. Moreover, WT seedlings exposed to ABA showed strongly enhanced ABI3 and ABI5 protein levels (Lopez-Molina et al. 2001, 2002). Both genes encode transcriptional regulators important for seed development and LEA gene expression (Koornneef and Karssen 1994; Parcy et al. 1994, Finkelstein and Lynch 2000; Carles et al. 2002). Lopez-Molina et al. (2002) showed that during the early seedling developmental arrest several LEA genes were re-induced as well. This re-induction of seed transcriptional regulators and LEA gene expression suggested that a late embryogenesis program is re-initiated by ABA treatment, inducing a drought tolerant phenotype in such arrested seedlings (Lopez-Molina et al. 2002). It was investigated whether the same transcriptional regulators and LEA genes were expressed by glucose signalling during early seedling development. A role for ABI5 has been proposed in GIN signalling and ABI5 expression is sugar responsive (Arenas-Huertero et al. 2000; Laby et al. 2000; Brocard et al. 2002; Arroyo et al. 2003). ABI5 expression thus is a good positive control in this experiment. Gene expression upon glucose feeding was studied by germinating seeds on control medium until the batch reached 70% germination (time point zero, t = 0). At this time point a sample was collected for RNA isolation while the rest of the seeds were transferred to control, sorbitol, glucose or ABA containing media for an additional 24 h. After 24 h samples were collected for gene expression analysis. Using quantitative RT-PCR (qRT-PCR), the expression of ABI3, ABI5, AtEM1, AtEM6 and RAB18 was studied in response to the different treatments. Gene expression was studied in three independent experiments. In each experiment the gene expression level at t = 0 was set to one (Table 2). Some variation in gene expression levels between the three experiments was observed, likely due to the fact that for each experiment independently generated seed batches were used. Generally, continued growth on control media after t = 0 resulted in a decreased expression of all five genes (Table 2). In contrast, treatments with sorbitol, glucose and ABA enhanced the expression of all genes studied, except for the sorbitol treatment in the second experiment. In this particular experiment gene expression decreased on sorbitol, however, this decrease was less pronounced compared to the control treatment. ABA treatment resulted in the re-induction of ABI3, ABI5 and the LEA genes, which is in line with results reported by Lopez-Molina et al. (2002). Also glucose exposure significantly induced the expression levels of all five genes although the expression levels are in general somewhat lower than in response to ABA. Thus ABI3 is upregulated by glucose as well, which indicated a possible role for ABI3 in glucose responsiveness. The observation that the expression levels of the genes studied are in general lower after sorbitol treatment compared to glucose treatment indicates that the glucose-induced expression is not an osmotic response. Table 2Relative expression levels of ABI3, ABI5, EM1, EM6 and RAB18 after glucose treatmentGeneTreatmentExp. 1Exp. 2Exp. 3ABI3t = 0111con0.140.090.06sorb2.20.191.7glc9.04.57.9ABA178.36ABI5t = 0111con0.480.200.29sorb8.70.4112glc191319ABA404123EM1t = 0111con0.260.340.20sorb740.71164glc7972300ABA749742200EM6t = 0111con0.220.140.15sorb360.3878glc5140173ABA450132237RAB18t = 0111con0.160.120.00sorb770.461.7glc324416.2ABA618633315The relative expression levels of five genes were determined using quantitative RT-PCR. The gene expression levels were measured at t = 0 and after a 24 h treatment on control media (con, half-strength MS + 1% sucrose) and on con media supplemented with either 8% sorbitol (sorb), 8% glucose (glc) or 10 μM ABA (ABA) media. The expression level at t = 0 was set to 1. The experiment was performed three times and the results for each experiment are indicated Expression studies using seedlings grown for five days on control, sorbitol (7%) or glucose (7%) media confirmed the induction of these genes (Fig. 3). In five days old glucose-arrested seedlings enhanced expression levels were detected for ABI3 and ABI5 as well as the LEA genes AtEM1, AtEM6 and RAB18. The induction of these genes by sorbitol was roughly five fold lower (Fig. 3). After 72 h of growth on ABA-free media, seedlings lose the capacity to re-induce ABI3, ABI5 and LEA expression and to arrest early seedling development in response to ABA (Lopez-Molina et al. 2001, 2002). To study whether the induction by glucose is restricted to a similar time frame seeds were germinated and grown for three days on half-strength MS and subsequently switched for two additional days to elevated sorbitol and glucose media. The re-induction by glucose of ABI3, ABI5, EM1, EM6 and RAB18 was largely lost (Fig. 3, glc 72 h). The weaker induction by sorbitol is essentially lost after three days (Fig. 3, sorb 72 h). Thus, the glucose-response that induced ABI3, ABI5 and LEA expression is confined to a similar time frame as observed for ABA. This is in agreement with the finding that glucose regulation of ABI4 and ABI5 expression was dependent on developmental stage (Arroyo et al. 2003) and that sugar arrests early seedling development only in a small 2–3 day time frame after sowing (Gibson et al. 2001). Fig. 3The glucose-regulated expression of ABI3, ABI5 and LEA genes is restricted to a limited time frame after the start of germination. The expression of EM1, EM6, RAB18, ABI3 and ABI5 genes was quantified by qRT-PCR after germination and growth for five days on different media. Ler-0 seeds were plated for five days on control media (con, half-strength MS + 1% sucrose), or on con medium supplemented with either 7% sorbitol (sorb) or 7% glucose (glc). The values are an average of three experiments and in each experiment the expression on glc was set to one. It was investigated whether this gene expression regulation is limited to a small time window after the start of germination as has been shown for ABA regulation (Lopez-Molina et al. 2002). Therefore seeds were germinated on con media for three days and thereafter switched to either sorb (sorb 72 h) or glc media (glc 72 h) In conclusion, the glucose-induced block of early seedling development mimics the ABA-induced arrest of vegetative development in young seedlings. Both glucose- and ABA-arrested seedlings have a drought resistant phenotype and show enhanced expression levels of seed expressed genes. The observation that ABI4 overexpression is unable to restore WT glucose sensitivity in the aba2-1 mutant shows that WT ABA biosynthesis is essential for the GIN response. Apparently, next to ABI4 additional factors which are under control of ABA are important for GIN signalling. Two candidates are ABI3 and ABI5. Both play a role in the ABA-induced early seedling developmental arrest and gene expression and protein stability of both transcription factors is affected by ABA (Lopez-Molina et al. 2001; Lopez-Molina et al. 2002; Lopez-Molina et al. 2003; Zhang et al. 2005; this study). A role for ABI5 in glucose signalling has been established; abi5-1 has a gin phenotype (although somewhat weaker compared to abi4) and ABI5 expression is regulated by sugar (Arenas-Huertero et al. 2000; Laby et al. 2000; Brocard et al. 2002; Arroyo et al. 2003; this study see Table 2). However, a role for ABI3 is controversial (for review Finkelstein and Gibson 2001; Leon and Sheen 2003; Gibson 2004; Rognoni et al. 2007). Since ABI3 is important for the ABA-induced developmental arrest and is induced by glucose, our hypothesis is that ABI3 is involved in GIN signalling as well. Therefore, the gin phenotype and glucose-regulated gene expression were studied in abi3 mutants. abi3 mutants are glucose insensitive The glucose insensitive phenotype of WT Ler-0 and the abi3 mutant was investigated by plating seeds of both genotypes on media with elevated glucose concentrations (6–8% glucose). Initially, two abi3 mutant alleles were tested, i.e. the weak abi3-1 allele (producing normal, drought tolerant seeds) and the strong abi3-5 allele (producing green, drought intolerant seeds, Ooms et al. 1993). As expected WT Ler-0 early seedling development was arrested in response to high glucose concentrations. In contrast, approximately 60–100% (differed between experiments) of both abi3-1 and abi3-5 seedlings showed greening on high glucose media, suggesting that abi3 is a gin mutant, (Fig. 4a). All genotypes showed greening on control and osmotic control media (Fig. 4a, control and 7% sorbitol, respectively). The gin phenotype was observed for abi3 mutants in several experiments using independently grown and harvested seed batches (data not shown). Next, the strength of the gin phenotype of abi3-1 was assessed in comparison to that of the ABA deficient mutant aba1-1, which is in the Ler-0 background as well. The aba1-1 mutant, like other ABA deficient mutants tested, was shown to possess a gin phenotype (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000). A similar percentage of seedling greening was observed for aba1-1 and abi3-1 when grown on glucose media (Fig. 4b) suggesting that both mutants possess a comparable gin phenotype. Fig. 4abi3 mutants have a gin phenotype. Representative Ler-0, abi3-1 and abi3-5 seedlings are shown that were grown for 12 days on control media (half-strength MS + 1% sucrose), or on control medium supplemented with either 7% sorbitol or 7% glucose (a). The gin phenotype of abi3-1 was compared to that of the ABA deficient mutant aba1-1. Seedlings were grown for 12 days on the indicated media before seed germination and seedling greening were scored (b) Six additional mutant alleles (abi3-8, abi3-9, abi3-10, abi3-11, abi3-12, and abi3-13) were obtained (a kind gift of Dr. E. Nambara) and tested for their gin phenotype. These abi3 mutants were tested along with two known gin mutants, abi4-3 and abi5-7 in the gin assay. All these mutants were retrieved from a screen for mutants insensitive to the unnatural (−)-ABA (Nambara et al. 2002). The ABA insensitivity of the mutants was tested by plating the mutants on half-strength MS media containing 1% sucrose + 10 μM ABA. The ABA insensitivity observed was in agreement with that reported by Nambara et al. (2002) (see Table 3). The abi3-8, abi3-9, abi3-10, abi4-3 and abi5-7 mutants had a strong ABA insensitivity, the abi3-12 and abi3-13 showed a weaker phenotype. We did observe 40% greening for abi3-13 on ABA media while originally no abi phenotype was observed for this allele (Nambara et al. 2002). These mutants were isolated on (−)-ABA and the (−)-ABA insensitivity does not necessarily parallel (+)-ABA insensitivity (Nambara et al. 2002). Next, these mutants were plated on control, sorbitol- and glucose-containing media, respectively. On 7% glucose media strong gin phenotypes were observed for abi4/gin6 and abi5 mutants as shown by the high percentages of greening, in agreement with earlier reports (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000). Again a strong gin phenotype was observed for abi3, in agreement with our observation using abi3-1 and abi3-5 mutants. Five out of the six additional abi3 mutants tested show a gin phenotype. For four abi3 mutants the gin phenotype was comparable to that of abi4-3 and abi5-7 (Table 3). The strong ABA insensitive abi3 mutants are strong gin mutants as well whereas the mutants with a weaker phenotype (abi3-11 and abi3-12) show a weaker phenotype on glucose media. However, exceptionally abi3-13 shows a weak abi phenotype but a strong gin phenotype. The abi3-11 mutant lacks an ABI insensitive phenotype and did not show a gin phenotype either. In conclusion, gin phenotype analysis of eight abi3 alleles confirms the glucose insensitive phenotype for abi3. Moreover, the gin phenotype of abi3 is comparable to that of other known gin mutants like aba1, abi4 and abi5. Table 3Glucose insensitive phenotype of six abi3 allelesGenotypeTreatmentcon7% sorb7% glc10 μM ABA3 μM ABAaWT Col10099200abi4-3999894100100abi5-799988996100abi3-81001009791100abi3-9100100100100100abi-310100100100100100abi3-11100100520abi3-1210096338350abi3-1310010098400Seeds of WT Col, abi4-3, abi5-7 and six abi3 mutants were stratified on half-strength MS and after stratification switched to control medium (con, 0.5 MS + 1% sucrose) or con medium containing 7% sorbitol, 7% glucose or 10 μM ABA. Values are the percentage of cotyledon greening scored after 11 days. The experiment was repeated once with similar resultsaThe ABA insensitivity scored for the different mutant alleles based on cotyledon greening by Nambara et al. (2002) Glucose-induced expression of ABI4, ABI5 and RAB18 is reduced in abi3 Glucose-induced early seedling developmental arrest is associated with expression of ABI3, ABI4, ABI5 and a subset of LEA genes (Arenas-Huertero et al. 2000; Cheng et al. 2002; Arroyo et al. 2003; this study). The involvement of ABI3 in glucose-regulated gene expression was tested for three genes ABI4, ABI5 and RAB18 as a representative of the LEA genes. For expression analysis the weak abi3-1 and the strong abi3-5 mutants were used. Seedlings of all three genotypes were grown for 24 h on control media or on control media containing 8% glucose. Both RAB18 and ABI5 expression were sensitive to glucose addition. In WT seedlings, glucose treatment induced these two genes on average 14 times in comparison to t = 0 (Fig. 5a, c). However, on glucose the expression levels of both genes were much reduced in abi3-1 and abi3-5 compared to WT (Fig. 5a, c). Glucose treatment did not result in a clear induction of ABI4, especially when compared to ABI5 and RAB18. Instead ABI4 expression was roughly maintained to the same level observed for t = 0 but was enhanced when compared to the control treatment which displayed a strong reduction of ABI4 expression (Fig. 5b). ABI4 transcripts were 2–8 fold lower in the abi3 mutant backgrounds upon glucose treatment. Thus, the abi3 mutation did not allow full glucose-induced expression of RAB18, ABI4 and ABI5. Interestingly, the reduced expression is most pronounced in the strong abi3-5 mutant background. Fig. 5Altered glucose-regulated gene expression in abi3 mutants. Glucose-regulated gene expression was studied in WT Ler-0 and abi3-1 and abi3-5 mutants. Gene expression was quantified by qRT-PCR at t = 0, the time of glucose application and after 24 h on control medium (con, half-strength MS + 1% sucrose) or con medium supplemented with 8 % glucose. At the t = 0 time point approximately 70% of the seeds had germinated. The values indicate an average of three independent experiments (two experiments for abi3-5). The WT Ler-0 expression on 8% glucose was set to one in each experiment. The glucose-regulated expression in Ler-0 and the two abi3 mutants was studied for RAB18 (a), ABI4 (b) and ABI5 (c). The gene expression ratio of Ler-0 over abi3 was calculated because all three genes were already under expressed in abi3-1 and abi3-5 at t = 0. This ratio was calculated by dividing the average gene expression in Ler-0 by the expression in abi3 (for both mutants) at t = 0 and after 8% glucose treatment for all three genes (d) Already at t = 0 reduced expression levels for all three genes were observed in the abi3 mutant backgrounds. This was expected since the ABI transcription factors were found to affect each others expression in seeds (Söderman et al. 2000). Moreover, ABI3 is an important regulator of LEA gene expression (Parcy et al. 1994; Kermode 2005). To rule out that this reduced expression is responsible for the phenotype observed after glucose treatment the gene expression ratios of WT over mutant were determined for t = 0 and after glucose feeding. For each gene the expression level of WT is divided by the expression level of each mutant. These values show the fold difference of expression between WT and both abi3 mutants (Fig. 5d). E.g. the level of RAB18 expression is 9 fold higher in WT compared to abi3-1 at t = 0. After glucose treatment the fold difference for RAB18 expression was 38. Except for the ABI4 expression ratio of Ler-0 over abi3-1, all WT/mutant expression ratios showed increased values after glucose exposure. This confirms that both mutants have a reduced ability to control glucose-regulated gene expression. Other sugar insensitive phenotypes of abi3 The sugar response phenotype of the abi3-1 mutant was investigated in the sis, sun and glucose-induced delay of germination assays. High sucrose concentrations block early seedling development similar to high glucose concentrations. High sucrose resistant mutants are known as sis mutants (Laby et al. 2000). Several mutants affected in the same genes were retrieved from gin and sis screens, e.g. gin1/sis4/aba2, gin6/sis5/abi4 and gin4/sis1/ctr1. The sis phenotype of WT Ler-0, abi3-1 and abi3-5 was analysed by plating these genotypes on 13% sucrose media. Both abi3 mutants show a sucrose insensitive phenotype (Fig. 6a, b) and can thus be considered as sis as well as gin mutants. Fig. 6The abi3-1 mutant shows additional sugar signalling defects. The sugar response phenotype of abi3-1 was assessed in three additional assays. The sugar insensitive (sis) phenotype was assessed by scoring the early seedling developmental phenotype (by seedling greening) after growth on elevated sucrose concentration. Representative Ler-0, abi3-1 and abi3-5 seedling phenotypes are shown after growth on 13% sucrose media for 13 days (a). The percentage of germination and seedling greening of Ler-0, abi3-1 and abi3-5 was determined after growth for 13 days on control (half-strength MS + 1% sucrose) or on control medium supplemented with either 7% sorbitol or 13% sucrose media (b). The sucrose uncoupled (sun) phenotype was assessed by measuring plastocyanin (PC) expression levels by qRT-PCR in two days old seedlings grown on sucrose containing media in darkness. Sucrose repressed the PC transcript level in dark-grown two days old WT seedlings in comparison to WT seedlings grown on sugar-free media, however in sun mutants PC repression by sucrose is much less pronounced (Dijkwel et al. 1997). Indicated are the relative PC expression on sucrose containing media for WT PC-LUC and sun6/abi4 (assayed on 2% sucrose) and Ler-0 and abi3-1 (assayed on 1% sucrose). Values indicate an average of at least three experiments. PC expression of two days old seedlings grown on sugar-free half-strength MS was set to 1 for each genotype in every experiment (c). Germination is delayed by glucose addition. abi3-1 was tested for glucose-induced delay of germination by plating seeds on half-strength MS (0.5 MS), 0.5 MS + 7% sorbitol (7% sorb) and 0.5 MS + 7% glucose (7% glc). Sugars were already present during stratification (solid lines). In addition, seeds were stratified on sugar-free 0.5 MS media and directly after stratification transferred to 0.5 MS + 7% sorbitol (MS > 7% sorb) and 0.5 MS + 7% glucose (MS > 7% glc, dashed lines). Seed germination after the different treatments was scored daily for eight days for Ler-0 (d) and abi3-1 (e) Dijkwel et al. (1997) previously reported on sugar insensitive mutants that have been isolated using the sun screen. Dark-grown seedlings transiently express photosynthesis genes, including plastocyanin (PC, Dijkwel et al. 1996). This transient increase of photosynthesis gene expression is repressed on sucrose media. In sun mutants sucrose did not repress this transient PC gene expression in dark grown seedlings (Dijkwel et al. 1997). sun6 is such a mutant which was shown to be allelic to abi4 (Huijser et al. 2000). A possible sun phenotype of abi3-1 was investigated by quantifying PC mRNA levels in two days old, dark-grown seedlings using qRT-PCR. Elevated PC expression was observed in sucrose-treated seedlings of sun6/abi4-3 compared to its WT PC-LUC parent confirming its sun phenotype (Fig. 6c). Interestingly, the abi3-1 mutant showed enhanced PC expression as well and, therefore, abi3 has a sun phenotype (Fig. 6c). abi4 mutants displayed a nine fold increased PC expression level in sugar-treated seedlings compared to WT. In the abi3 mutants the PC expression level is only two fold increased, indicating that abi3 is a weak sun mutant. Sugars delay seed germination in Arabidopsis. ABA plays a role in this glucose-induced delay of germination (Ullah et al. 2002; Price et al. 2003; Dekkers et al. 2004; Chen et al. 2006). Despite the involvement of ABA, several abi mutants (abi1-1, abi2-1, abi4 and abi5) are normally sensitive to glucose during germination (Price et al. 2003; Dekkers et al. 2004). Sugar insensitive mutants like gin6/abi4 and gin2/hxk1 that are insensitive to the glucose-induced early seedling developmental arrest are sensitive to glucose during germination (defined as radicle emergence). This indicates that the glucose response during germination (which delays radicle protrusion) and early seedling growth (which arrests seedling development) are two distinct processes (Price et al. 2003; Dekkers et al. 2004). The, abi3-1 and abi3-5 alleles were plated on 2.5% glucose. The presence of 2.5% glucose clearly delayed germination of WT seeds. The osmotic control of 2.5% sorbitol only slightly affected germination compared to control media. Both abi3 mutants showed a clear resistance against glucose as displayed by their reduced germination delay (data not shown), which is in agreement with observations of Yuan and Wysocka-Diller (2006) who reported on the glucose insensitivity of abi3-1 during germination. In our experiments seeds were sterilized, plated on sugar media, and after a three day stratification period moved to the growth chamber and germination was scored the following days. Interestingly, when WT seeds were stratified on sugar-free media (half-strength MS) and after stratification transferred to sugar-containing plates, the delay of germination by 2.5% glucose was completely lost (data not shown). Thus, stratification on sugar-free media suppressed the inhibitory effect of glucose on germination. This effect of stratification was further investigated using higher (7%) glucose and sorbitol concentrations. Ler-0 and abi3-1 seeds were plated either on control, sorbitol or glucose media, stratified for three days and moved to the growth chamber. Alternatively, seeds were stratified on sugar-free control media and shifted to media containing sorbitol or glucose, and then placed in the growth chamber. Continuous treatment with 7% glucose severely delayed germination of WT seeds but affected abi3-1 significantly less as expected (Fig. 6d, e). Also in this experiment, stratification on sugar-free media strongly suppressed the germination response to glucose in WT Ler-0. Even germination of the abi3-1 mutant, which already showed a reduced sensitivity to glucose during germination could be improved by stratification on sugar-free media. The germination speed of the abi3-1 mutant after stratification on sugar-free media was similar to the osmotic control indicating that this combination completely suppressed the germination delay by glucose. The glucose response during germination was affected by stratification but not the osmotic response (sorbitol treatment). This was observed for WT Ler-0 as well as the abi3-1 mutant thereby differentiating glucose from osmotic signalling. abi2-1 and era1-2 mutant are glucose response mutants Our results on the physiological and molecular level indicate that glucose-arrested seedlings mimic ABA-arrested seedlings. The finding that ABI3 is important for glucose signalling in addition to ABI4 and ABI5 led us to test the glucose insensitive phenotype of two other ABA signalling mutants, i.e. abi2-1 and era1. Brady et al. (2003) proposed a genetic pathway for ERA1 and the ABI1-ABI5 genes in seed ABA responsiveness based on double mutant analysis between the era1 mutant and the abi mutants. This analysis suggested that ABI1 and ABI2 act at or upstream of ERA1, while ERA1 acts at or upstream of ABI3, ABI4 and ABI5 (Brady et al. 2003). ABI2 encodes a phosphatase 2C protein that acts as a negative regulator of ABA signalling and the abi2-1 mutation results in an ABA insensitive phenotype (Koornneef et al. 1984; Leung et al. 1997). WT Ler-0 and abi2-1 seeds were plated along with abi3-1 on control, 7% sorbitol and 7% glucose media. In addition to abi3, the abi2-1 mutant displayed a gin phenotype as well, although its phenotype is not as strong as that of abi3-1 (Fig. 7a). On control and sorbitol media all seedlings showed greening. Fig. 7Other ABA signalling mutants have sugar signalling defects as well. Putative glucose signalling defects were determined for the ABA insensitive mutant abi2-1 and the ABA oversensitive mutant era1-2. Ler-0, abi2-1 and abi3-1 were grown on control media (half-strength MS + 1% sucrose), or on control medium supplemented with either 7% sorbitol or 7% glucose. After 12 days germination and seedling greening percentage were scored (a). To investigate whether era1-2 has a glucose oversensitive phenotype both WT Col and mutant were grown on a sugar concentration which did not arrest early development of WT Col. Therefore, seedling greening of Col and the era1-2 mutant was scored daily on half-strength MS (0.5 MS) and on 0.5 MS + 6% glucose. The seedling phenotype was scored for 11 days (b) The ERA1 gene encodes a β subunit of a protein farnesyl transferase (Cutler et al. 1996) and acts as a negative regulator of ABA signalling. The era1-2 mutant has an ABA hypersensitive phenotype. If ERA1 exerts a similar function in glucose signalling, a glucose oversensitive (glo) phenotype is expected for the era1-2 mutant. Seeds of WT Col and era1-2 were plated on half-strength MS + 6% glucose media. This glucose concentration allowed seedling greening of the WT Col seedlings. Greening started for WT seedlings from day four onwards. However, this glucose concentration arrested early seedling development of era1-2 seedlings. Even after eleven days no greening was observed for era1-2 seedlings (Fig. 7b), which indicates that era1-2 indeed has a glo phenotype. The era1-2 mutant has a more dormant phenotype compared to WT (Cutler et al. 1996). Therefore, seed germination was synchronized as much as possible by using after-ripened seeds and employing a three day stratification period on sugar-free media. On control media (half-strength MS) germination was somewhat delayed but that did not affect cotyledon greening. On 6% glucose era1-2 germination reached similar germination levels as WT seeds despite that germination was 1–2 days delayed (data not shown). This slower germination of era1-2 does not explain the much larger difference observed for cotyledon greening on glucose media. Thus, the small difference in germination timing between era1-2 and WT is not responsible for the seedling greening phenotype. Discussion GLUCOSE INSENSITIVE signalling depends on a functional ABI3 gene Our results show that ABI3 is an essential regulator of GIN signalling. This conclusion is based on three observations. Firstly, ABI3 transcript levels accumulate in response to glucose treatment. In our study seedlings were treated for a relatively short period of 24 h and showed a 5–9 fold enhanced ABI3 levels, while ABI3 expression levels dropped after 24 h growth on control media (Table 2). The osmotic control conditions induced ABI3 expression at most two fold showing that the strong induction of ABI3 by glucose is not solely due to osmotic stress. Secondly, glucose-regulated gene expression was altered in the abi3 mutant background. Glucose enhanced the expression of LEA genes and two important regulators of GIN signalling ABI4 and ABI5. In WT, glucose treatment led to a 14 fold induction of RAB18 and ABI5, and a 1.5 fold induction of ABI4. The abi3 mutation caused 2–60 fold under expression of these glucose-regulated genes (Fig. 5). Thirdly, we observed a gin phenotype for seven out of eight abi3 alleles in both Ler-0 and Col accessions. The majority of the abi3 alleles tested showed a gin phenotype that was comparable to well-known gin mutants like aba1, abi4 and abi5 (Fig. 4 and Table 3). This indicates that ABI3 is a key regulator of GIN signalling which is in agreement with previous reports showing that ABI3 overexpression (either AtABI3 or CnABI3) in Arabidopsis results in a glucose oversensitive phenotype (Finkelstein et al. 2002; Zeng and Kermode 2004). Kermode (2005) distinguished at least three functions for ABI3 in seeds. (i) dormancy induction and maintenance (ii) induction of storage proteins and desiccation/stress protectants and (iii) repression of post-germinative gene expression. The observations that abi3 mutants showed a disturbed glucose-regulated induction of RAB18 and a gin phenotype (lack of post-germinative gene repression) are in agreement with the functions defined for ABI3. Although a role for ABI3 in sugar signalling during early seedling development was hinted by ABI3 over expression lines (Finkelstein et al. 2002; Zeng and Kermode 2004) and the observation that abi3 mutants were insensitive to glucose in combination with ABA (Nambara et al. 2002) its role remained controversial. This because studies that investigated the gin phenotype of abi3-1 in the Ler-0 background observed at most a weak phenotype. Importantly however, Ler-0 is a sugar oversensitive accession compared to the Col accession (Laby et al. 2000) and likely, the sugar insensitive phenotype of abi3-1 has been underestimated in comparison with mutants in genetic backgrounds that show more rapid greening. Further, the absence of a clear gin phenotype for abi3-1 in earlier studies could be due to the seed material used. We observed that freshly harvested seeds were particularly sensitive to sugars during germination (data not shown). Low sugar concentrations delay germination while higher levels resulted in severely reduced germination rates. Therefore, we routinely after-ripened seeds for at least a month before these were used in our sugar signalling experiments. Seed quality changes per batch produced, which affects germination and most likely responses to internal and external stimuli as well. Hence, WT and mutant seeds used in the sugar response assays were produced simultaneously under the same growth conditions and seeds were stored under identical conditions. Moreover, we routinely stratified the seeds on sugar-free half-strength MS media before the start of the gin assays, since this procedure dramatically improves germination rates following transfer to glucose-containing media (Fig. 6d; Dekkers and Smeekens 2007). Under these conditions we were able to obtain clear gin phenotypes within 12–14 days for abi3 in the Ler-0 background. In addition, this study identified both abi3-1 and abi3-5 as sis mutants (which are insensitive for the sucrose-induced early seedling developmental arrest, Laby et al. 2000) as well. In addition, abi3 mutants are insensitive to the glucose-induced delay of germination (Yuan and Wysocka-Diller 2006; Fig. 6d, e) and abi3 is a weak sun mutant. These observations imply a broader role for ABI3 in mediating sugar responsiveness. Interestingly, another B3 domain transcription factor, HSI2, was found to regulate the sugar-inducible sporamin promoter (Tsukagoshi et al. 2005). HSI2 is an active transcriptional repressor. A mutation in this gene causes high sporamin promoter activity in both low and high sugar conditions. Glucose and ABA induce a similar early seedling arrest in Arabidopsis Glucose treatment of germinated seeds resulted in an enhanced expression of several transcriptional regulators of late seed maturation (ABI3, ABI4 and ABI5). GIN signalling depends on these glucose-induced factors as shown by mutant analysis (León and Sheen 2003; this study). LEA genes (EM1, EM6 and RAB18) were also induced by glucose treatment. The ABI transcription factors are known to regulate the expression of LEA genes (Parcy et al. 1994; Finkelstein et al. 1998; Finkelstein and Lynch 2000; Carles et al. 2002; Kermode 2005). LEA gene expression is correlated with drought and desiccation tolerance (Hoekstra et al. 2001) and its enhanced expression is in agreement with the drought tolerant phenotype observed for glucose-arrested seedlings. Interestingly, the ABA response during the post-germination developmental arrest depends on the same ABI3, ABI4 and ABI5 transcription factors. ABA similarly induces ABI3, ABI5 and a subset a LEA genes. Lopez-Molina et al. (2001; 2002) suggested that a late embryogenesis program was re-induced by ABA, which resulted in developmentally arrested, drought tolerant seedlings and that this may constitute an important checkpoint during germination and seedling development. The developmentally arrested state could only be induced within a limited time frame and after 60–72 h ABA sensitivity was lost. Beyond this limited time window ABA did not induce ABI3 and ABI5 protein levels, nor, re-induce LEA gene transcription and did not block early seedling development. The sugar-induced seedling arrest is restricted to a similar time window (Gibson et al. 2001). Following this time window glucose failed to re-induce ABI3, ABI5 and LEA genes. Thus far it is not clear whether this time frame specifies a sugar sensitive window. Since ABA insensitivity results in a gin phenotype the loss of ABA sensitivity beyond this time frame may account for the glucose insensitivity observed. In conclusion, both glucose and ABA signalling depend on the same set of genes, induce similar transcription factors and LEA genes, result both in drought tolerant phenotypes and are sensitive for a similar time window. This indicates that both trigger a similar seedling developmental arrest. Interestingly, the induction of desiccation tolerance in germinated radicles is limited to a small time window after germination as well. In germinated seeds with short radicles (<3 mm) desiccation tolerance can be induced while in seeds with longer radicles (>4 mm) this capacity is lost (Buitink et al. 2003). Possibly, this phenomenon depends on the same genetic components and is sensitive in a similar time window observed for glucose- and ABA-induced post-germination developmental block. A possible role was suggested for sugar signalling in desiccation tolerance induction in barrel medic (Medicago truncatula) and cucumber (Cucumis sativa) radicles (Leprince et al. 2004). Non-reducing di- and trisaccharides, e.g. sucrose, protect the structure of membranes and proteins by the formation of a glassy state (Koster 1991; Hoekstra et al. 2001). E.g. elevated sucrose concentrations were measured in desiccation tolerant radicles (Koster and Leopold 1988; Bruggink and van der Toorn 1995). Our study suggests that such elevated sugar concentrations may serve a double role, by protecting cellular structures and by acting as a signal for the induction of protectants such as LEA protein. Interactions between sugar and ABA signalling The early seedling developmental arrest phenotype has been extensively exploited to isolate mutants with sugar signalling defects like gin mutants (Rognoni et al. 2007). The gin2/hxk1 mutant revealed a role for HXK1 in glucose signalling. Such HXK1 signalling does not depend on its metabolic function (Moore et al. 2003) and HXK1 was shown to interact with unusual partners (a vacualor H+-ATPase and a 19S proteasome subunit) to regulate glucose signalling (Cho et al. 2006). Other gin mutants suggested a link between glucose signalling and plant hormone biosynthesis and signalling. The lack of ABA biosynthesis and signalling (gin1/aba2, gin5/aba3 and gin6/abi4) resulted in glucose insensitivity. To study the interaction between ABA biosynthesis, ABI4 and sugar signalling we used ABA deficient lines with constitutive ABI4 expression (aba2-1/35S::ABI4). These strong ABI4 overexpression lines affected ApL3 expression (ISI pathway) and showed a stunted growth phenotype similar to what has been reported previously for severe ABI4 overexpression lines (Söderman et al. 2000), indicating that ABI4 is active in an ABA deficient background. Nevertheless, the gin phenotype of aba2-1 could not be restored by high ABI4 expression levels (Fig. 1c). Thus, ABI4 is necessary but not sufficient for GIN signalling in an ABA deficient background, indicating that other ABA regulated factors are essential for proper GIN signalling as well. Both ISI and GIN signalling rely on ABA biosynthesis and ABI4 but surprisingly, ABI4 overexpression only affected the isi phenotype of aba2-1. Thus, gin and isi sugar signalling depend on the same components but this study shows that the relationship between sugar, ABA and ABI4 is different for both sugar signalling pathways. ABA biosynthesis and ABA signalling are required for the sugar induced seedling arrest. Genetic studies showed that two transcription factors which are involved in ABA signalling, i.e. ABI4 and ABI5, function in the sugar-induced seedling arrest (Arenas-Huertero et al. 2000; Huijser et al. 2000; Laby et al. 2000; Brocard et al. 2002). Here we show that ABI3 is also essential for GIN signalling. Glucose induces ABA biosynthesis genes (Cheng et al. 2002) and increased ABA levels were measured in developmentally arrested seedlings. ABA is able to induce ABI3 transcription (Table 2) and affects ABI3 protein stability (Zhang et al. 2005). Thus glucose may impinge on ABI3 via activation of ABA biosynthesis. ABI3 also functions in seed development and developmental timing (Rohde et al. 1999, 2000). Severe abi3 mutant embryos remain green and are unable to acquire desiccation tolerance and dormancy during seed development (Ooms et al. 1993). Such mature abi3 embryos resemble a developing seedling rather than a dormant embryo (Nambara et al. 1995). The seedling developmental arrest as induced by ABA and glucose is sensitive to developmental signals as well. Arrest of seedling development by ABA and sugars only occurs within a two to three days time window after imbibition (Gibson et al. 2001; Lopez-Molina et al. 2001). Therefore, the phenotype of the severe abi3 mutants is probably not solely due to ABA insensitivity but to seed developmental defects and the heterochronic nature of this mutation as well. Two additional ABA response mutants with altered glucose responsiveness, abi2 and era1, strengthen the link between glucose and ABA signalling. The abi2-1 mutant showed a gin phenotype that is weaker compared to abi3-1. Possibly additional seed developmental phenotypes of abi3 may explain the stronger phenotype of the abi3-1 mutant compared to abi2-1. However, the ABA insensitive phenotype of abi2-1 is somewhat weaker compared to abi3-1 (Koornneef et al. 1984; Finkelstein and Somerville 1990), which could explain the weaker gin phenotype as well. The era1-2 mutant is hypersensitive to ABA and shows a glucose oversensitive phenotype. Interestingly, era3 another ABA hypersensitive mutant shows a similar glo phenotype. Era3 is allelic to ethylene insensitive2 (ein2, Ghassemian et al. 2000) and also other ethylene related mutants show sugar signalling defects (Zhou et al. 1998; Gibson et al. 2001; León and Sheen 2003). The role of ABA and ABA signalling in sugar signalling is well established. Thus far, sugar signalling was thought to rely on only a part of the ABA signalling cascade. The analysis of aba2/35S::ABI4 transgenic lines showed that ABA related factors other than ABI4 are involved in glucose signalling. Furthermore, the glucose-induced early seedling arrest mimics the ABA-induced seedling arrest. Our study indicates that all response loci tested that act in the genetic pathway regulating ABA responsiveness as proposed by Brady et al. (2003) affect glucose signalling. These observations suggest that the genetic pathways regulating glucose and ABA signalling are much more closely linked than assumed thus far. Obviously, both pathways are different as well. Genetic analysis suggested that HXK1 acts upstream of ABA signalling in glucose signalling. Moreover, the gaolaozhuangren2 mutant has a gin phenotype but is not affected in ABA signalling (Chen et al. 2004). Such mutants may provide insight in the mechanism by which glucose affects ABA signalling. Interestingly, glucose delays germination in a process that depends on ABI3 but that does not require ABI1, ABI2ABI4 and ABI5 (Gibson 2005; Yuan and Wysocka-Diller 2006; Fig. 6d, e), showing that the tight interaction between glucose and ABA is not present in all sugar response pathways.

Ann_Biomed_Eng-2-2-1705502

Single-Step 3-D Image Reconstruction in Magnetic Induction Tomography: Theoretical Limits of Spatial Resolution and Contrast to Noise Ratio

Magnetic induction tomography (MIT) is a low-resolution imaging modality for reconstructing the changes of the complex conductivity in an object. MIT is based on determining the perturbation of an alternating magnetic field, which is coupled from several excitation coils to the object. The conductivity distribution is reconstructed from the corresponding voltage changes induced in several receiver coils. Potential medical applications comprise the continuous, non-invasive monitoring of tissue alterations which are reflected in the change of the conductivity, e.g. edema, ventilation disorders, wound healing and ischemic processes. MIT requires the solution of an ill-posed inverse eddy current problem. A linearized version of this problem was solved for 16 excitation coils and 32 receiver coils with a model of two spherical perturbations within a cylindrical phantom. The method was tested with simulated measurement data. Images were reconstructed with a regularized single-step Gauss–Newton approach. Theoretical limits for spatial resolution and contrast/noise ratio were calculated and compared with the empirical results from a Monte-Carlo study. The conductivity perturbations inside a homogeneous cylinder were localized for a SNR between 44 and 64 dB. The results prove the feasibility of difference imaging with MIT and give some quantitative data on the limitations of the method. Introduction Magnetic induction tomography (MIT) is a non-invasive and contact-less imaging modality for reconstructing the changes Δκ of the complex conductivity distribution κ = σ + jω ɛ0ɛr in a target object.8,13–16,22 MIT requires an array of excitation (EXC) and receiving coils. Each EXC couples an alternating magnetic field B0 to the object under investigation (see Fig. 1). Changes Δκ of the complex conductivity cause a field perturbation ΔB due to the induction of eddy currents. The perturbation induces voltage changes ΔV in the receiver coils. It is convenient to normalize ΔV to V0, the voltage which is induced by the unperturbed field B0.Figure 1.Schematic of a possible coil system for MIT with 16 excitation coils and 32 receiver coils. Previous reviews of MIT have been given in8,22,32. The method has been developed for industrial process tomography already more than 10 years ago but is comparatively new in medical imaging. Potential medical applications usually aim at the characterization of biological tissues by means of their complex conductivity. The motivation for measuring the electrical properties is their characteristic dependence on the (patho-) physiological state of tissues, especially hydration and membrane disorders. Medical applications so far suggested are: imaging of limbs,2 imaging of the brain, e.g. for the monitoring of brain edema,14,16,24,26 measurement of human body composition,7 monitoring of wound healing.23 In contrast to electrical impedance tomography (EIT) MIT avoids the ill-defined electrode-skin interface due to its inherently contact-less operation. Figure 1 shows a schematic MIT coil configuration with rectangular coils as receivers and a cylindrical object space. The solenoid excitation coils are distributed on two different rings in order to obtain a true 3-D-arrangement. The reconstruction of the absolute conductivity in a target region Ω requires the solution of a complex inverse eddy current problem. Let be the discretized non-linear forward mapping of the conductivity vector to the vector of induced voltages y. y contains M = a × b entries, a being the number of EXC and b that of receiving coils. The corresponding inverse problem is ill-posed and usually underdetermined. Uniqueness of the solution for this inverse boundary value problem was established in21 provided the angular frequency ω of the AC field is not a resonant frequency. The generic approach for the solution of this type of non-linear problem is the application of an iterative scheme such as the regularized Gauss–Newton approach, including an appropriate regularization scheme. To the knowledge of the authors the full inverse problem of medical MIT in 3-D has not yet been solved satisfactorily, although some approximate solutions, especially for 2-D, have been presented.3,13,33 Some authors9,15 proposed the use of weighted back-projection, similar to EIT. In all published cases the back-projection is done along magnetic flux tubes between excitation and receiving coils, the weights being calculated for the case of conducting perturbations in the empty space. However, own observations17,27,29 suggest that the basic requirements for the applicability of this kind of back-projection are not fulfilled in realistic anatomical structures, so that a more appropriate inverse approach is necessary. This paper is dedicated to demonstrating the feasibility of the 3-D reconstruction of a spherical perturbation within a cylindrical conducting body by means of a regularized one-step Gauss–Newton reconstructor. The conductivities were chosen in the physiological range of human tissues. Methods The solution of (2) requires the target region to be discretized into N voxels. Within each voxel i the assigned component κi of the vector of conductivity is assumed to be constant. A grid of tetrahedral finite elements of second order was employed. In a general setup is then found with the iterative scheme: Define the forward problem with an initial parameter vector . Measure the data vector ym.Solve iteratively for the estimated true parameters whereby means the estimated “true” parameter vector. R and λ are a regularization matrix and a regularization parameter, respectively, which are required to stabilize the iteration. When applying Newton’s method starting from an initial guess the parameter vector is updated by an increment pk in each iteration step k+1 with the update step with . The Jacobian , also called sensitivity matrix, must be recalculated in each iteration step. This procedure is very time consuming, hence a complete iterative identification run requires significant computing power, the bottleneck being the solution of the forward model. However, in EIT it could be shown that in practice most features of the image can already be recognized very satisfactorily after the first iteration. This fact led to the development of the so-called Newton-one-step reconstructor (NOSER4). NOSER is especially appropriate for so-called dynamical imaging where only the change in the conductivity between two different states of the object under investigation (e.g. lung ventilation) are of interest. In this case the first Newton step corresponds to the solution of the linearized forward problem whereby is the change of the conductivity between two states of the observed object, and Δym is the corresponding change of the measured data. In the case of comparatively small changes the inversion of (6) according to (7) yields a fairly correct localization of the perturbed regions. We evaluated the feasibility of this kind of reconstruction in MIT by implementing a NOSER-approach according to (7) under consideration of four different regularization methods. Calculation of the Forward Solution and the Sensitivity Matrix The forward mapping is given by Maxwell’s equations for harmonic excitation: with H: magnetic field intensity, B magnetic flux density, E electric field strength, J: current density, ɛ: dielectric constant, μ: magnetic permeability, σ: real conductivity, κ: complex conductivity, ω: angular frequency. Ω denotes the interior of the object under investigation. This forward problem is solved with a previously published finite element program,12,17 which employs an Ar–V, Ar – formulation with edge elements of second order for the reduced magnetic vector potential Ar and nodal elements of second order for the electric scalar potential V. Boundary conditions on the far boundary (normal component of B vanishes) were prescribed on a spherical surface with a radius sufficiently large such that a change of this radius by 50% resulted in a change of the induced voltages by less than 1 %. Special attention must be paid to the efficient calculation of the Jacobian . A mathematically rigorous treatment of this topic has been given in.31 In our implementation we exploited the integral formulation published by Mortarelli20, which is based on a physical mutual energy concept. With this approach the absolute sensitivity dy/d for a certain pair of coils is calculated according to (9). with Aϕ, AΨ, Vϕ and Vψ denote the total magnetic vector potential and the electric scalar potential in the region Ω due to currents Iϕ and Iψ in the excitation and receiver coils, respectively. The sensitivity matrix is then obtained by evaluating (9) for all individual elements and all coil pairs. The exact numerical implementation of (9) was described in detail in11. The calculation of the sensitivity map for one pair of coils requires only two forward solutions of the eddy current problem for generating Lϕ and LΨ in (10). Regularization In EIT the regularization matrix RTR most frequently used is either the identity matrix I or a discrete spatial derivative operator of first or second order. Such approaches have been discussed extensively in the literature, for a good review see e.g.10 Several regularization matrices can be regarded as simple smoothness criteria for the solution but they have also a more general statistical meaning in the framework of Bayesian estimation theory (see e.g.1). In the case of uncorrelated noise with equal variance for all measurement data the estimator in (7) is a maximum a posteriori (MAP) estimator with RTR being the inverse of the expected covariance matrix of the image E[ppT]. In that sense e.g. the neighbouring matrix accounts for the case that all image values are de-correlated at borders between homogeneous regions with different mean values. According to our own observations good results can be achieved with variance uniformization5 which imposes a special assumption of the prior distribution. The objective here is to uniformize the expected variance of the reconstructed conductivity changes over the region Ω, thus providing approximately equal image noise in the center as in the periphery. The algorithm has been described in detail in5 and requires singular value decomposition of G according to G = UΣVT. Then the regularization term is expressed as λ RTR = VDVT, with D a diagonal matrix with the entries di whereby σi is the Ith singular value. c is a free scalar tuning parameter. Alternatively truncated singular value decomposition (TSVD) has been applied in EIT-reconstruction,19 hence this approach was also implemented for MIT. In this case the inverse solution becomes whereby t denotes the truncation level of the original matrices V, Σ and U, thus removing the contributions of singular values with index > t. In this paper the results obtained with four different regularization schemes were compared: RTR = I. Using the identity matrix is the most simple Tikhonov-regularization method, penalizing high values of the reconstructed conductivity changes. In the following this method will be abbreviated as ‘IM’.RTR = N with N the neighbouring matrix defined as: nn is the number of neighbouring elements for element i, whereby only elements with common facets are considered as neighbours. N is an approximation of the spatial derivative operator of second order. Due to the irregular structure of the grid this filter is not spatially invariant, nevertheless it gives good results and is common practice for this type of inverse problem. In the following this method will be abbreviated as ‘NM’. λ RTR = VDVT according to the variance uniformization approach. In the following this method will be abbreviated as ‘VU’.TSVD while choosing the truncation level t such as to remove all singular values below an appropriately chosen threshold. Methods 1 and 2 require the regularization parameter to be chosen optimally while method 3 in addition implies the choice of the tuning parameter c. In practice it turns out that the value of c is not critical over a very wide range of values because the optimal λ depends on c. That means that c can be fixed at a more or less arbitrary value if a method for the automatic determination of λ is applied. In our case c was set to 0.1. The regularization parameter λ accounts for the degree of smoothness of the reconstructed image and determines the condition number of the term (GTG + λRTR) in (6). Several methods for the optimal choice of this parameter have been published in the past, the most well-known ones being L-curves, Generalized Cross-Validation and the Morozov-criterion.10 Because of its clear physical interpretation we choose the latter method. In this case the optimal λ is the one where the estimated residuals Gp − Δym have the same variance as the measurement noise. The motivation for this criterion is that it is obviously meaningless to make the residuals lower than expected from the statistics of the data. This method provided always stable images independently of the regularization matrix and was considered as a good basis for a fair comparison between the different regularization methods. Modeling Setup The inverse solver was tested with the simple 3-D model comprising a cylindrical conductor with two spherical inhomogeneities placed with their centers at (x =  − 60, y = 0, z = 0) (mm) and (x =  − 30, y = 52 and z = 0) (mm) and the array of 16 excitation coils and 32 receiving coils shown in Fig. 1. The exact geometry is illustrated in Fig. 2. The cylinder had a radius and a height of 100 mm, the perturbing sphere had a radius of 20 mm. The solenoid excitation coils were modeled by cylindrical rings with an inner diameter of 60 mm, a thickness of 1 mm and a width of 21 mm. The exciters were placed in groups of 8 with their centers on two rings with radii of 125 mm in two transversal planes of the cylinder. The lower ring is rotated by 22.5 degrees versus the upper one in order to achieve a lower degree of symmetry. The square receiver coils with an edge length of 40 mm were placed with their centers on two symmetrically arranged parallel rings with a radius of 120 mm, each comprising 16 evenly spaced coils. The orientation of the windings were opposite in both rings so that the 16 vertical pairs can be combined to planar gradiometers as published previously26. The measured data were simulated in terms of induced voltage changes when changing the conductivity of the spherical perturbation from 0.2 S/m (= background conductivity, homogeneous cylinder) to 0.3 S/m. The relative permittivity was kept constant at 80 in all compartments. The excitation frequency was 100 kHz.Figure 2.Schematic of the simulation model. 16 excitation coils and 32 receiver coils are placed on two concentric rings around the tank, respectively. Two spherical perturbations are placed at the shown locations with (x =  − 60, y = 0, z = 0) and (x =  − 30, y = 52, z = 0). All measures are given in (mm). This arrangement was chosen as the model system for our analysis because of two reasons: It represents a true 3-D-arrangement which delivers theoretically 512 independent measuring combinations, i.e. 512 data points for one image reconstruction.It is similar to our experimental system which employs 16 excitation sites in one plane and 14 planar gradiometers which are formed by connecting in counter-phase the coils in the upper and in the lower receiver plane. Two different meshes A and B were used for the generation of an artificial dataset and for the reconstruction. Mesh A comprised 11000 elements within the cylinder and approximately 1200 in the spheres while mesh B comprised 17000 elements for the homogeneous cylinder without perturbation. In both cases the diameter of the surrounding spherical surface which approximated the far boundary was chosen as 1m, requiring approximately 30000 elements (mesh A) and 50000 elements (mesh B), respectively. Uncorrelated Gaussian noise was added to the voltage data in order to simulate the noise of the receiver channels. This type of noise, although common practice in simulations of this kind,, is not entirely valid for real situations. In addition the noise of the excitation coils is propagated to all receiver coils, thus resulting in a certain amount of correlated noise in all receiver channels. This phenomenon has been studied in detail for EIT,6 but should be disregarded here for simplicity. The calculation of the complete sensitivity matrix required 48 forward solutions according to the Mortarelli-approach. Theoretical Limits of Resolution and Contrast/Noise Ratio For EIT the theoretical limits of image quality in terms of contrast/noise ratio (CNR) and resolution have been studied carefully in30 while no such study exists for MIT. A similarly rigorous discussion for MIT is certainly beyond the scope of this paper. However, a simplified analysis on resolution and CNR was carried out for our linear reconstruction scheme so as to have a certain theoretical basis for interpreting our empirical results. There is a fundamental limit for the resolution which depends on the amount of available information in the data. This information depends on the number of data points, i.e. the number of possible sensor–detector-combinations and on the degree of independence between these data points. In the case of EIT and MIT the number of independent data points is usually much lower than the number of voxels, so that the system is under-determined. Moreover the different data are correlated to a certain degree, so that the effective rank is comparatively low. In EIT, e.g. 16 electrodes provide 104 independent data points so that the information is no more than 104 ‘effective pixels’. Including some a priori-information in the form of the regularization terms leads to a defined ‘smearing’ of this information over the imaging plane and provides the typical diffuse images known from EIT. We characterized the resolution of MIT with the Raleigh criterion. Accordingly two point-shaped perturbations are still separable if their point spread functions (PSF) overlap in such a way that the peak of the first one coincides with the first zero crossing of the second one. In the case of a sinc-shaped PSF the lowest separable distance is equivalent to the 64%-width of the PSF. In contrast to e.g. X-ray CT in MIT the PSF depends on the location of the perturbation and on the geometry of the object under investigation. In this paper the object is the model cylinder which is also used for the numerical reconstruction examples and for the phantoms. The PSF is calculated by mapping the true parameter values p* to the reconstructed ones p via the reconstruction equation A means the expression A = (GTG + λ RTR)−1GT for regularized methods 1–3 or A = Vt Σt−1Ut for TSVD. The ith column of M in Eq. 13 is then the shifted PSF for the ith voxel. The theoretical limit was approximated by applying TSVD with the full set of non-zero singular values, i.e. We chose TSVD for the estimation of the theoretical limit because it requires the least explicit assumptions about the a-priori distribution of p. In MIT an additional difficulty is that, in general, the 64%-boundary of the three-dimensional PSF is not spherical and thus the resolution is anisotropic. In our simplified analysis we define as Raleigh-width the largest axis of the ellipsoid which best approximates the 64%-boundary. As CNR at the point x we defined whereby Δp is the reconstructed difference between perturbation and background and std(np) is the standard deviation of the image noise. We recognize that Δp is not a contrast in the classical sense. Usually contrast is the difference between p in the perturbation and background divided by the background value. However, as our method is a differential one, the background value of the image is always zero and the classical contrast definition is meaningless. Moreover the determinant for the detectability of a perturbation is the difference Δp rather than the contrast. The CNR depends on the size and location of the perturbation as well as on the noise level of the measured data. In contrast to resolution there is no theoretical lower limit in the case of noise-free data and a perfect reconstruction method. We estimated std(np(x)) by calculating the Cramer-Rao lower bound of the covariance of the parameters. Given the covariance matrix X of the voltage changes Δym the Cramer-Rao lower bound of the covariance matrix of the image noise vector np is From the diagonal elements of this matrix the expected lower bound of the coefficient of variation (CV) of the reconstructed conductivity in the perturbation can be calculated for any voxel. For the evaluation of Eq. 15 the reconstructed value Δp in voxel i follows from Eq. 13: whereby P is the set of the indices of all voxels inside the true perturbation. This equation clearly shows that the CNR must decrease when the size of the perturbation shrinks. Assuming a small perturbation and an approximately constant PSF inside this region the CNR is approximately inversely proportional to the volume of the perturbation. The detectability limit can then be defined as the one where the CNR equals 1. The shown approach is simple and allows the calculation of detectability limits for perturbations with different radii and locations but it is only valid in the linear case. Alternatively Monte-Carlo studies can be carried out for different perturbations varying in size and contrast. Such a study has been published previously for a spherical perturbation in the center of a brain model.18 For simplicity we assumed the noise to be Gaussian with zero mean, although in the general non-linear case this may not be entirely valid. For characterizing the noise level we defined as SNR the ratio max(|ΔV|)/std(V) whereby std(V) is the standard deviation of the noise voltage. ΔV is the vector of voltage changes in all excitation/sensor combinations when a test object is placed into the empty measurement system. In order to be independent on size and location of the perturbation we chose as the test object the homogeneous background cylinder of our model. Results The PSF was evaluated at 20 equally spaced points along the x-axis between the center and the border of the cylinder. This set includes also the point (0.6R0,0, 0), i.e. the center of one of the two perturbations in our simulation model. To mitigate discretization errors the PSF was calculated by rotating the coordinate system 8 times about 45° and averaging the data assuming radial symmetry of the true model. Then the normalized resolution was calculated from the PSF as the inverse of the ratio between the Raleigh-width and the cylinder radius. This normalized resolution can be interpreted as the number of points which can be resolved per cylinder radius. The resulting data were plotted in Fig. 3 as a function of the normalized x-coordinate in the xy-plane. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64 dB, respectively. The truncation levels were chosen according to the Morozov-criterion and are listed in table 1. The above three SNR levels were chosen because they correspond to the range in which our current measurement system operates.25Figure 3.Dependence of the theoretical normalized resolution on the noise level. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64 dB, respectively.TABLE 1.Tuning parameters for the regularization, chosen according to the Morozov-criterion.Regularization methodTSVD Truncation levelIM λNM λVU λSNR = 64 dB1822.40E − 202.40E − 202.35E − 11SNR = 50 dB1008.40E − 198.80E − 193.20E − 10SNR = 44 dB681.25E − 171.19E − 177.40E − 10The parameter c for VU was always kept at 0.1. A clear increase of the resolution with the distance from the center is observable. The theoretical limit for a point-shaped perturbation in the xy-plane is 3.5 (corresponding to 2.9 cm separation) in the center and a maximum resolution of more than 6 (corresponding to 1.7 cm separation) at the periphery. At the locations of the test spheres the resolution is in the range of 4, i.e. 2.5 cm separation. The relative loss of resolution with the noise level is stronger in the center than in the periphery. At a SNR of 44 dB the resolution does not increase any more continuously but levels off above a normalized x position of about 0.6. In analogy to EIT the PSF depends strongly on the location, showing the broadest distribution in the center of the cylinder. This is reflected by increasing resolution when moving from the center towards the periphery. Increasing the amount of regularization or decreasing the truncation level according to increasing measurement noise the PSF broadens and its center of gravity is shifted towards the borders of the cylinder. Moreover increasingly strong ringing in form of star-like patterns becomes observable close to the border (not shown explicitly in this paper). In Fig. 4 the four regularization methods are compared at a SNR of 50 dB. Except for few points TSVD, performs poorest which is in accordance with the assumption of least explicit a-priori information.Figure 4.Theoretical normalized resolution for the four regularization methods at a SNR of 50 dB. Figure 5a shows the CNR for TSVD as a function of the normalized x-coordinate and in dependence on the noise level while Fig. 5b compares the four methods at a SNR of 50 dB. As expected the theoretical CNR depends strongly on the location of the perturbation increasing from values around 2 in the center up to about 60 at the cylinder border. At the location of the perturbing spheres the CNR drops from about 26 to 13 when decreasing the SNR from 64 dB to 44 dB. In the simulated images the CNR drops from 24 to 9, i.e. remains fairly in the same range. Figure 5b reveals that IM and NM yield the highest CNR, followed by TSVD and VU, whereby VU is characterized by dramatically lower values. In the center VU yields a CNR around 2 which is already very close to the limit of detectability. When comparing the theoretical values with the reconstructed ones (see table 2) the reconstructions always produce a lower CNR than expected, the discrepancy being stronger at high noise levels.Figure 5.Panel A: CNR for TSVD as a function of the normalized x-coordinate (relative to the cylinder radius) and in dependence on the noise level. Panel B: comparison of the four methods at a SNR of 50 dB. Figures 6 and 7 show the reconstructed mean images from a Monte-Carlo study with 50 runs for each of the four methods and an SNR of 64 and 50 dB, respectively. Representative cross-sections in the xy-plane and in the xz-plane were selected. The respective regularization parameters are listed in Table 1.Figure 6.Mean images of the Monte-Carlo study. Reconstructed Δ σ (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 64 dB.Figure 7.Mean images of the Monte-Carlo study. Reconstructed Δ σ (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 50 dB. In all cases the two perturbations can be recognized as diffuse bright disks. The dotted circles in the figures delineate the original position of the perturbing spheres. A number of performance indices were calculated in order to quantitatively assess the results in Figs. 6 and 7. They are summarized in Table 2 and comprise: Mean and CNR of the pixel values in the center of gravity of each reconstructed perturbation. These parameters quantify the correctness of the reconstructed values as well as their uncertainty. The theoretically expected values are listed for comparison. The center of gravity was chosen as evaluation point because the reconstructed perturbations deviate more or less from the spherical shape and show significant outward shift with increasing noise level.Radial outward shift of the spheres in the reconstructed image (fidelity of the location). This shift was determined by localizing the center of gravity for each spot within a wedge-shaped search region with a height of 2.6 times the sphere’s radius and excluding the outermost 2 mm as well as the innermost 40 mm in radial direction from the center. The restriction of the search region to this volume prevented spurious contributions from outliers and negative image values far away from the real perturbing regions. Also for this parameter we present theoretical values as expected from the PSF.TABLE 2.Summary of the performance indices defined in the text.SNR = 64 dBSNR = 50 dBSNR = 44 dBMethodPerformance indexSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundIMMean central0.0210.0160.000540.0060.00430.00350.001NM0.0220.0160.000560.0060.0040.000510.00320.0010.00035VU0.0240.0240.000550.010.00650.000430.00600.00360.00035TSVD0.0330.0250.000540.010.00740.000500.01100.0020.00055IMCNR42.345.016.123.01220.1NM41.144.016.522.510.719.3VU7.98.25.65.23.43.3TSVD23.925.813.718.49.113IMResolution2.842.602.53NM2.812.582.50VU2.942.462.58TSVD2.582.422.24IMNormalized outward shift*0.100.100.210.250.330.35NM0.100.100.210.250.330.35VU0.100.060.110.150.210.15TSVD0.100.100.180.210.350.33Performance measures for the comparison of the reconstruction methods.*Normalized to the cylinder radius. In addition Table 2 lists the theoretical resolution limits for all methods and noise levels at the position of the perturbations. With 64 dB SNR noise the two spheres can be resolved comparatively easily with all four methods. With 50 dB SNR noise the resolution is theoretically still possible for all methods. In the reconstruction the resolution is already somewhat below the limit for IM and VU, the image values in the notch between the two peaks being around 71% of the peak values. TSVD and NM appear to separate the perturbations even worse although theoretically this should not be the case. At higher noise all algorithms tend to shift the objects towards the borders of the cylinder when looking at the parameter ‘normalized outward shift’ in Table 2. Here again the VU performs best by producing the lowest shift. At 64 dB SNR the mean images (not shown here) are in general comparatively poor. IM and NM interestingly still allow a clear separation of two objects, but their localization is very poor, the outward shift being extremely large (see Table 2). VU still provides a much better localization but only at the cost of CNR. TSVD failed to produce a clear image, an observation which was not expected from theory. Depending on the regularization method the central voxel value of the perturbation at (− 0.6R0,0,0) decreases from 0.02–0.03 S/m to 0.002–0.006 S/m, compared to the true value of 0.1 S/m. This means that even under nearly ideal conditions (64 dB SNR) the conductivity changes are strongly underestimated. NM and TSVD yield nearly the same central voxel values as TSVD while VU produces much lower values. Figure 8 shows single reconstructions for IM and VU at all three noise levels. Both methods allow a separation of the perturbations in all cases, but the poor CNR of VU implicates a very noisy image at 44 dB. The pronounced difference in outward shift is clearly visible at 44 dB, where VU still allows a fair localization while IM fails completely to reconstruct the perturbations at the right positions.Figure 8.Comparison of single-shot images for VU and IM at the three different noise levels. IM and NM perform nearly identically, also their optimal regularization parameters are almost identical. VU yields, in general, larger values in the perturbed regions but also a larger STD. Discussion The results demonstrate the feasibility of image reconstruction from MIT-data with the same methods as suggested for EIT. This finding is not self-evident, as the sensitivity distribution is significantly different in EIT and MIT.27,29 In EIT the region of maximum sensitivity is located between the equipotential surfaces which meet the surface at the detection electrodes, i.e. within a tube-shaped region which connects injection and detection sites. As shown in27,29 in MIT the sensitivity is not concentrated within a field tube between excitation and receiver coil but increases with the distance from the tube axis, according to the increase of the eddy current density. This may be the main reason why the reconstructed solution tends to be displaced towards the nearest border of the cylindrical tank, especially at higher noise levels. An extreme case for this effect can be observed if the perturbation is placed exactly in the origin and if the senders and receivers are all in the same plane (image not shown due to space restrictions). Instead of the expected spot in the origin two widely separated spots appear on the cylinder axis close to the top and bottom of the cylinder, respectively. In fact such a coil arrangement cannot distinguish between an object in the center and two objects on the cylinder axis placed symmetrically with respect to the origin, because it is always possible to find two corresponding conductivitiy changes so that the field perturbations in the median plane are the same. Obviously, in this ambiguous situation, the algorithm favors the splitted solution according to the sensitivity distribution. A similar ambiguity occurs when using differential sensors, such as the gradiometers employed in our setup. For getting rid of such artifacts it is very important to use a less symmetric transceiver setup which provides enough spatial information in 3-D. The theoretical resolution limit was calculated from the PSF as derived from the TSVD method. This limit depends on the chosen regularization method, the geometry of the object and on the location within the object. The respective dependences are shown in Fig. 3 and Table 2 for some selected positions inside a cylinder. TSVD was chosen for the calculation of the theoretical limits because it requires no explicit assumptions about any prior distribution of p. In this sense it contains less a-priori information than the other methods and thus describes the worst case, as confirmed by Fig. 3 and Table 2. The calculated PSF shows all basic features of the reconstructed images. The PSF is a 3-D-distribution similar to a 3-D analog of the sinc function. This means that most of its energy is concentrated in a diffuse cloud around the considered point but that there exist three-dimensional ‘side lobes’ which decay with the distance and show some kind of ‘periodicity’. The ‘bean’-shaped artifacts which are visible in most top views of Figs. 6 and 7 are typical features of the PSF as well as the ‘star-artifact’ in the TSVD-images. Therefore these ringing artifacts do not stem from inaccuracies of the reconstruction method or measurement errors, but, instead, are inherent in the PSF. The resolution clearly also depends on the contrast in the presence of noise, because the contrast determines the SNR. Increased noise requires more regularization and hence leads to a broadening of the PSF-distributions. Figure 3 shows the dependence of the resolution on the noise in the case of TSVD at one single contrast of 0.5 only. A more complete analysis similar to that given for EIT in30 should also show the dependence of the resolution on contrast, size and location of the perturbation at a given noise level. However, such a comprehensive analysis requires a separate paper and should not be given here. The CNR depends strongly on the radius and, to a less extent, on the noise level. Obviously IM and NM produce very similar values, followed by TSVD. VU in general yields comparatively small CNR but higher central voxel values. For centrally placed spheres with 4 cm diameter VU yields CNRs close to the detection limit. When comparing the theoretical values with the reconstructed ones (see Table 2) the reconstruction always produces a lower CNR than expected, the discrepancy being stronger at high noise levels. One surprising detail of Fig. 5a is that at higher noise levels the CNR-curves cross the curve for 64 dB. This means that very close to the periphery noisier data yield higher CNR values than less noisy data. The reason for this counter-intuitive effect is not yet entirely clear but may be related to the strong outward shift of the PSF at higher noise. In those cases the evaluation of the CNR at the original position of the perturbation may not be appropriate any more and should be interpreted with caution. In the case of weak perturbation we can assume that the CNR depends approximately linearly on the conductivity difference Δσ. The dependence on the volume of the perturbation is, in general, more complicated because the PSF depends on the location and is therefore not constant throughout the whole perturbation. Only in the case of small spatial extension of the perturbation an approximately linear dependence on the volume can be assumed. The low number of significant singular values even at comparatively low noise (64 dB SNR) suggests that, similar as in EIT, a significant amount of sensor combinations does not provide enough independent information. Intuitively one would expect this finding because there exist pairs of excitation/receiving coils which nearly fulfill the reciprocity condition and hence reduce the amount of useful combinations to about half of the number of possible combinations, i.e. to 256 in our case. Further investigations should determine the maximum ‘useful’ number of sensors in one plane, i.e. that number beyond which additional sensors do not increase the resolution significantly. Adding more sensors off-plane may add more 3-D-information and hence still provide improvement. This possibility should be studied in further research. When comparing the regularization schemes after application of the Morozov-criterion, the IM and the NM approach yield the smoothest visual appearance and the highest CNR. However, they also tend to displace the perturbations towards the border of the tank. The best localization is obtained with VU, probably because the imposed variance counteracts somewhat the lower sensitivity in the center of the object. However, VU yields also the lowest CNR, i.e. the less homogeneous images and more pronounced ghosts. The failure of TSVD at a SNR of 44 dB was not expected theoretically, although, in general, it produces the poorest theoretical resolution. In terms of separability of the two perturbations VU performs best, especially when also taking into account the correct localization. In neither case, however, the single-step solution provides the correct values for Δσ. Even at a SNR of 64 dB the reconstructed differences are too low by a factor of at least 5, thus demonstrating that the method yields the correct search direction but not the correct step size. The highest mean voxel values are provided by VU and TSVD, the drop with the noise levels being lowest. However, on the other hand VU yields the highest standard deviations. Moreover VU tends to produce more pronounced ‘ghost objects’ in the homogeneous region than IM and NM. As expected from the PSF TSVD tends to produce ‘star-artifacts’ at the cylinder border, i.e. a periodic pattern with 16 peaks close to the centers of the receiving coils. This artifact gets worse at increasing noise level. First experiments with smaller models and at least 10 iterations with an iterative solver show that the solution converges towards the correct voxel values. Nevertheless the single-step method may be completely justified in cases where only qualitative changes are sought for or where proportions are to be reconstructed, e.g. in frequency differential spectroscopic imaging. Therefore the area of applicability of a single-step approach has to be analyzed carefully in future work. At least for the shown examples MIT appears relatively robust against Gaussian measurement noise. A SNR of 64 dB allows for a stable and distinct solution. Even 44 dB allow the recognition of the two spheres when applying the correct regularization. This result is very important for the practical implementation because, due to technical reasons, MIT is expected to yield low SNR (around 50 dB) at frequencies as low as 100 kHz, which are interesting for the imaging of pathophysiological processes.28,26 However, our results have only been achieved with two single focal perturbations with a relatively large diameter of 20% of the background object. In a more advanced study the stability and the resolution of the images should be investigated for a series of perturbations with different diameter and spacing. For the monitoring of brain edema which usually do not split up in separate sub-regions our approach may be sufficiently stable. This hypothesis has to be tested both theoretically and empirically for centrally placed perturbations (worst case). As to the detectability of spherical perturbations in a human brain simulation results in18 have shown that a sphere with a diameter of about 40 mm, a background conductivity of 0.1 S/m a contrast of 2 yields a SNR of 24 dB at 100 kHz when applying 1 A to an excitation coil with 45 turns and a receiver coil with 1 turn. The assumed acquisition time was 200 ms, With our present technology single shot images are generated with an acquisition time of 20 ms, an excitation coil with five turns and a current up to 20 A. The receiver coils have 40 turns with otherwise unchanged geometry. This means an overall increase in SNR by 28 dB. Extrapolating the analysis given in,28 an improvement of the SNR by a factor of 5–10 is still technically possible, thus reaching 50–60 dB, which is obviously sufficient for producing fairly acceptable difference images. Another open question is the influence of the mesh quality on the reconstruction results. We used a comparatively coarse non-uniform grid for the reconstruction. Therefore non-negligible numerical errors are to be expected which may explain the discrepancies between theoretically expected and the reconstructed values for CNR and radial displacement. Also the apparently somewhat worse spatial resolution in the reconstructed images than theoretically expected may be due to such numerical problems. The influence of the mesh and the optimization of mesh quality should be a major issue for further developments. The results were obtained at a single frequency only. Future work should concentrate on the exploitation of the frequency dependence of the tissue conductivity and measurements at frequencies up to several MHz. A multi-frequency approach is expected to increase significantly the available information and thus the quality of the images. Possible applications may then in fact be the same as for EIT (lung function monitoring, lung edema monitoring) and hydration monitoring in the brain.

Eur_J_Pediatr-4-1-2413080

Growth charts for Wolf-Hirschhorn syndrome (0–4 years of age)

Wolf-Hirschhorn syndrome is characterized by severe growth and mental retardation, microcephaly, seizures and ‘Greek helmet’ facies, caused by partial deletion of the short arm of chromosome 4. Growth charts are given from 0–4 years of age, based on the study of 101 individuals. Use of these specific growth charts is recommended, because standard growth charts are inapplicable for patients with WHS. Introduction Wolf-Hirschhorn syndrome (WHS) is caused by partial deletion of the short arm of chromosome 4 (4p- syndrome) [3–5, 7]. Cardinal features of this rare syndrome are severe growth and mental retardation, microcephaly, seizures, “Greek helmet” facies and closure defects (Fig. 1) [1, 2, 4, 5, 7]. Growth in children with Wolf-Hirschhorn syndrome differs from that of normal children [1]. Up to date, there are no Wolf-Hirschhorn specific growth charts available. Fig. 1A 7-year-old boy with Wolf-Hirschhorn syndrome Materials and methods The study is based on data from 1,057 examinations of 101 children with WHS, 35 males and 66 females, born between 1975 and 2002. Data from 32 children were collected from records of individuals with WHS in the Netherlands. Another set of data was obtained from 69 children with WHS from Great Britain, North America, Germany and Australia who were contacted through WHS support groups. There were no exclusion criteria except growth hormone treatment. Thus, all children regardless of complicating disease such as heart defects, renal disease or feeding problems, were included. The number of observations in the Dutch group of children was higher than in the group of children from other countries. No difference in parameters related to growth was observed between Dutch and non-Dutch patients. The data used for creation of the growth charts were age at examination, height (cm), weight (kg) and head circumference (cm). The growth charts cover the time period from birth until 4 years of age. The data for each sex were divided into 30 different age groups, with 1-month intervals during the first 2 years of life, 3-month intervals during the 3rd and 4th years of life. Each child contributed only one single set of data for each age group. If data from more than one examination within an interval were available, the data from the first examination were used. The growth data were compared to standard growth charts for healthy children based upon the fourth Dutch growth study of 1997 [6]. As there are no growth charts for children with WHS elsewhere in the world, no comparison could be made with growth charts from children with WHS from other countries. Data for weight and height were transformed into logarithms before statiscal analysis in order to obtain normal distributions. All growth charts are based on means and standard deviations. The software used was SPSS version 10 and Sigmaplot version 8.02 bij SPSS Inc. Results Figures 2 and 3 present growth charts for height for boys and girls. Mean birth length was 43.0 cm in girls and 41.5 cm in boys, corresponding to −3 SDS on growth charts for healthy Dutch children. Mean height at 4 years was 87.2 cm for girls and 85.8 cm for boys, corresponding to −4 and −4.5 SDS, respectively. Fig. 2Growth chart for height for girlsFig. 3Growth chart for height for boys Figures 4 and 5 represent growth charts for weight for boys and girls. Mean birth weight was 1.9 kg for girls and 2.1 kg for boys, corresponding to −3 SDS on growth charts for healthy Dutch children. Mean weight at 4 years was 9.8 kg for girls and 9.7 kg for boys, corresponding to −4 SDS and −4.5 SDS respectively. Figure 6 represents the growth chart for head circumference for boys and girls. Mean head circumference at birth was 30.5 cm for girls and boys, corresponding to −3.5 SDS for both sexes on normal growth charts. Mean head circumference at 4 years was 45.8 cm for girls and boys, corresponding to −3.5 SDS, respectively. Fig. 4Growth chart for weight for girlsFig. 5Growth chart for weight for boysFig. 6Growth chart for head circumference for boys and girls Discussion Growth is used in pediatrics as a marker of health and is an important tool in the medical care for children. Severe growth retardation is one of the cardinal features of WHS [1]. It might be caused by genetic factors, poor feeding status, inherited parental factors and associated diseases. Whether patients with WHS suffer from an additional endocrine disorder is not known and is beyond the scope of this study. Up to date there are no WHS-specific growth charts available in contrast to more common chromosomal disorders like Down’s syndrome, Turner syndrome and Prader-Willi syndrome. Although the ideal manner in constructing growth charts would be a prospective, longitudinal study based on a large study group, this method cannot be implied for rare syndromes such as WHS. To increase the number of observations we combined the available longitudinal data with cross-sectional measurements. We have used data from all children with WHS, including those receiving tube-feeding. Several other associated diseases such as heart or renal disease theoretically could influence growth of the patients. Although 33% of children with WHS have congenital heart defects, these defects are in most cases not complex in nature [1]. Closure defects, hypotonia, gastro-eosphageal reflux, epilepsia, repeated infections and surgical procedures all can have a significant influence on growth. Not all patients with WHS have the same deletion size. Until now there has been no evidence of an influence of the deletion size on growth, but all studies have only involved small numbers of children. Deletion size can be expected to be of influence on growth, and therefore we tried not to include patients with a known microdeletion. However, this study inevitably includes patients with different deletion sizes, but the number of included children was too small to show an effect on growth. Based on the available data from the Dutch cohort, we could not identify a major co-morbidity risk factor aggravating growth retardation. Almost all children with WHS showed a marked intra-uterine growth retardation. Despite adequate feeding none of the children with WHS, demonstrated catch-up growth; all remained short with a profound microcephaly. The developed specific growth charts should be used for children with WHS from birth to 4 years of age for the estimation of their growth and health condition as standard growth charts are inapplicable in these patients. At present the authors are constructing growth charts for older children with WHS.

Pediatr_Nephrol-4-1-2335288

Chronic kidney disease after liver, cardiac, lung, heart–lung, and hematopoietic stem cell transplant

Patient survival after cardiac, liver, and hematopoietic stem cell transplant (HSCT) is improving; however, this survival is limited by substantial pretransplant and treatment-related toxicities. A major cause of morbidity and mortality after transplant is chronic kidney disease (CKD). Although the majority of CKD after transplant is attributed to the use of calcineurin inhibitors, various other conditions such as thrombotic microangiopathy, nephrotic syndrome, and focal segmental glomerulosclerosis have been described. Though the immunosuppression used for each of the transplant types, cardiac, liver and HSCT is similar, the risk factors for developing CKD and the CKD severity described in patients after transplant vary. As the indications for transplant and the long-term survival improves for these children, so will the burden of CKD. Nephrologists should be involved early in the pretransplant workup of these patients. Transplant physicians and nephrologists will need to work together to identify those patients at risk of developing CKD early to prevent its development and progression to end-stage renal disease. Introduction Chronic kidney disease (CKD) is a frequent and increasingly recognized complication of solid-organ and hematopoietic stem cell transplantation (HSCT), increasing the complexity of patient management and impacting survival. The incidence of CKD after cardiac, liver, and HSCT varies from 7–86% in pediatric patients, partly due to the lack of a uniform definition of CKD after transplant (Table 1). Up to 16% of transplant survivors will develop end-stage renal disease (ESRD) [1–19]. Table 1Percentage of patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) based on transplant typeTransplant typeCKDESRDReferenceLiver28–86%0–8%[1–5]Cardiac7–54%2%[6–10]Heart–lung34%7–16%[11–13]Hematopoietic stem cell transplantation18–42%5–8%[14–19] This review focuses on the epidemiology, risk factors, and outcomes of children who develop CKD after liver, cardiac, lung, heart–lung, and stem cell transplant. Liver transplant Epidemiology The majority of studies of renal disease after liver transplantation has been done in adults, and the cumulative incidence of CKD varies from 5% to 30% over 1–5 years after transplant [20]. In a retrospective study of 117 pediatric liver transplant patients who survived 3 years after transplant (median 7.6, range 3–14.6 years), the prevalence of CKD, defined as a glomerular filtration rate (GFR) <70 ml/min per 1.73 m2 at last follow-up was 32% [1]. In a 10-year follow-up study of 12 children undergoing a liver transplant in France, mild to moderate CKD (mild = GFR 60–80 ml/min per 1.73 m2; moderate = GFR 20–60 ml/min per 1.73 m2) developed in six of seven patients. Early deterioration of renal function is usually seen within the first year following transplant, followed by a period of stabilization, then with progression on long-term follow-up [2, 3]. In contrast, in a study of 50 pediatric liver transplant patients in Poland, CKD stages 2 and 3 as defined by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF/KDOQI) (Table 2) developed at 1 year after transplant and remained stable after 3 years of follow-up [4]. In this study, GFR was calculated using both diethylenetriamine pentaacetic acid (DTPA) plasma clearance and estimated using the Schwartz formula. Little correlation was found between the two methods with the Schwartz formula, consistently overestimating GFR [4]. Therefore, the prevalence of CKD is likely underestimated in this patient population if estimated measures of GFR or serum creatinine are used to define CKD. Table 2National Kidney Foundation Kidney Disease Outcomes Quality Initiative definition of chronic kidney disease by stageStageDescriptioneGFR (ml/min per 1.73 m2)1Kidney damage with normal or ↑ GFR≥ 902Kidney damage with mild ↓ GFR60–893Moderate ↓ GFR30–594Severe ↓ GFR15–295Kidney failure<15 or dialysiseGFR estimated glomerular filtration rate Risk factors for developing CKD Many patients will have renal disease at the time of liver transplant secondary to hepatorenal syndrome and/or acute tubular necrosis. In one study, renal histology pretransplant revealed glomerulosclerosis and other mild glomerular changes with mesangial matrix expansion, capillary-wall changes, and mesangial immunoglobulin (Ig) G, IgA, and IgM in the majority of cases despite normal serum creatinines [21]. Ultrasonographic findings included nephromegaly and increased echogenicity, which improved in the majority of cases after transplant [22]. In pediatric patients, there can be renal involvement from the primary liver disease, as in patients with primary hyperoxaluria, autosomal recessive polycystic kidney disease, Alagille’s syndrome, and tyrosinemia (Table 3). Renal dysfunction in patients with these disorders often improves after liver transplant. However, the long-term affects of preexisting renal disease on developing CKD after orthotopic liver transplant has not been studied systematically in pediatric patients. In contrast, in adult liver transplant patients who develop acute renal failure during the peri- and postoperative periods, there is an increased risk for developing CKD after transplant [23] and progression to CKD stage 5 requiring dialysis [24]. Table 3Liver diseases associated with preexisting renal diseaseTyrosinemiaAutosomal recessive polycystic kidney diseaseAlagille’s syndromePrimary hyperoxaluriaHepatitis B- and C-related glomerulonephritis Other identified risk factors (Table 4) for CKD include GFR <70 ml/min per 1.73 m2 at 1 year after transplant and cyclosporine use [1]. In this study, there was an inverse relationship between hypertension at 1 year after transplant and development of renal dysfunction. In addition, in adult patients, preexisting diabetes, pre- and postoperative renal failure, hypertension, age, female gender, and hepatitis C infection increased the risk of CKD and ESRD [20]. Though the majority of renal dysfunction after liver transplant is attributed to the use of cyclosporine and/or tacrolimus, there have been reports of other types of pathology present leading to CKD. Studies have found preexisting renal disease such as focal segmental glomerulosclerosis (FSGS) and hepatitis-C-related injury including membranoproliferative glomerulonephritis, unresolved hepatorenal syndrome, and diabetic nephropathy on renal biopsy after liver transplant [25, 26]. Table 4Risk factors for developing chronic kidney disease by type of transplantRisk factor common to all transplant typesCalcineurin inhibitor useLiverGFR of <70 ml/min per 1.73 m2 at 1 year after transplantPretransplant renal dysfunctionAcute renal failurePreexisting diabetesAgeFemale genderHepatitis CCardiacPretransplant dialysisHypertrophic cardiomyopathyAfrican American racePrevious transplantPretransplant diabetesExtracorporeal membrane oxygenation useHeart–lungHypertension posttransplantElevated serum creatinine at 1 month posttransplantHematopoietic stem cell transplantAcute graft-versus-host disease grades II–IVOlder ageTransplant from an unrelated donorAcute renal failureChronic graft vs. host diseaseTotal body irradiation Calcineurin inhibitors The use of cyclosporine (CSA) and tacrolimus in managing liver transplant patients has greatly improved outcomes. However, the improvement in survival is associated with an increased development of CKD. The manifestations of CSA toxicity range from asymptomatic azotemia and proteinuria to fulminant multiorgan failure [27]. In addition to the commonly described striped fibrosis and arterial and vascular lesions seen in patients on these medications, thrombotic microangiopathy has also been described in as many as 50% of patients after liver transplant [25]. These lesions are typically characterized by mesangiolysis, thrombus formation within glomerular capillaries, and widening of the subendothelial spaces. The nephrotoxic effects of CSA correlate with drug serum levels and therapy duration. CSA is also known to cause arteriolar injury, glomerulosclerosis, and interstitial fibrosis, as well as diffuse expansion of the mesangial matrix [28, 29]. The hypothesized mechanisms behind this injury appears to be vasoconstriction secondary to an imbalance between the vasodilatory hormones such as prostaglandin E1 and vasoconstrictive ones such as thromboxane A2 [30]. Increased synthesis of transforming growth factor (TGF)-β1 by calcineurin inhibitors also contributes to the development of CKD in patients after transplant, and genetic polymorphisms in the TGF-β1 gene have been associated with the development of ESRD after cardiac transplant [31]. Reducing calcineurin inhibitor levels while adding mycophenolate mofetil (MMF) in patients with CKD 5 years after liver transplant resulted in renal function improvements as measured by serum creatinine and creatinine clearance after 24 months of combined therapy, with only minor changes noted in immune function in these patients. Decreases in microalbuminuria were also noted[32]. Some authors have suggested that thrice-daily dosing of cyclosporine leads to decreased nephrotoxicity and only mild histopathologic changes in the kidney after 3 years of follow-up [33]. The authors attribute their findings to more constant trough blood levels and lower, less toxic, peak levels as well as the use of a calcium-channel blockers to manage their patients’ hypertension [33]. In a case series, three children who underwent combined liver and kidney transplant were switched to sirolimus because of prolonged renal failure requiring dialysis and acute calcineurin inhibitor toxicity on biopsy. All three demonstrated improved renal function with cessation of dialysis [34]. These studies suggest that decreasing calcineurin inhibitor exposure by adding MMF or eliminating long-term exposure by switching to sirolimus can have beneficial effects on renal function in children after liver transplantation. Cardiac transplantation Epidemiology Approximately 350 pediatric cardiac transplants are performed annually in the USA [7]. In a 2006 report from the International Society for Heart and Lung Transplantation, the incidence of renal dysfunction (defined as an abnormal serum creatinine) was 10% at 5 years after cardiac transplant [6]. Sixty-three percent of patients had hypertension. After 8 years of follow-up, the percentage of patient with renal dysfunction did not change, but 2% of patients were on dialysis or had received a renal transplant. In reports from single centers, the incidence of CKD at approximately 10 years after cardiac transplant in pediatric patients ranged from 7% to 54%; however, only a small percentage of these patients progress to ESRD requiring dialysis and/or renal transplantation [7–10]. Hypertension is a more common finding, occurring in approximately 70% of patients after transplant [7–10]. Some centers actually report an improvement in kidney function in the first year following cardiac transplant and stabilization thereafter [9]. In adult studies, there is an increased risk of mortality associated with development of CKD [35, 36]. Risk factors for development of CKD after cardiac transplant A recent study of 2,032 pediatric cardiac transplant patients transplanted between 1990 and 1999 identified pretransplant dialysis, hypertrophic cardiomyopathy, African American race, and previous transplant as risk factors for developing CKD (defined in this study as a creatinine >2.5 mg/dl) [37]. Additional risk factors for developing ESRD included pretransplant diabetes and intensive care unit stay or extracorporeal membrane oxygenation [37]. In the adult population, several risk factors for CKD development have been identified and include older age at transplant, pretransplant serum creatinine, preexisting diabetes, abnormal GFR at 1 year after transplant, hypertension after transplant, and cyclosporine immunosuppression within the first 6 months after transplant [12, 35, 36, 38, 39]. Risk factors for progression to ESRD include postoperative development of hypertension and proteinuria of >1 g/24 h [13, 40]. Calcineurin inhibitors Though the nephrotoxic effects of calcineurin inhibitors are well known, data in the pediatric cardiac transplant population are conflicting. After 18 months of triple immunosuppression including cyclosporine, patients’ GFR remained stable, and renal biopsy specimens in four patients did not show signs of cyclosporine toxicity [41]. Minor abnormalities in tubular function resulting in hyperuricemia and hyperkalemia were reported. Similarly, in a study of adult cardiac transplant patients treated with cyclosporine for 5 years, GFR (measured by inulin clearance) remained stable at 66 ml/min per 1.73 m2, as did tubular function. The only abnormalities noted were hypertension and the presence of microalbuminuria. However, other studies of pediatric cardiac transplant patients found an association between cyclosporine use within the first 2 months after transplant and decreases in GFR years after transplant [42]. High cyclosporine levels (>500 μg/L) in the first 6 months after cardiac transplant were associated with developing ESRD at anytime after cardiac transplant [43]. In a pediatric study of 14 patients with progressive decline in renal function over 2–5 years, inulin clearance declined from 84 ml/min per 1.73 m2 at 1 year to 49.8 ml/min per 1.73 m2 at 5 years. Biopsies performed on 13 patients revealed chronic tubulointerstitial lesions of grade II associated with acute changes of vacuolization of the proximal tubules. Arteriolar lesions were also present, along with focal glomerular scarring and fibrosis. Lesion extent correlated with calcineurin therapy duration. After reduction in calcineurin inhibitor dose by 50% and change from azathioprine to MMF, a 67% improvement in GFR (77 ml/min per 1.73 m2) was noted 1 year after the change. The authors suggest that this improvement may be related to a decrease in preglomerular vasoconstriction [44]. In a study of pediatric patients treated with tacrolimus after transplant, 41% of patients had elevations in serum creatinine of 1–2 mg/dl approximately 2–3 years after transplant [45]. Heart–lung and lung transplantation Epidemiology The cumulative incidence of CKD, defined as a doubling of serum creatinine, after lung or heart–lung transplantation varies from 34% at 1 year after transplant to 53% by 5 years after transplant [11]. ESRD occurs in 7.3–16% of patients 5 years after transplant [11–13]. The majority of patients, 51%, developed CKD stage 3 by 1 year posttransplant [12]. Risk factors for CKD after heart–lung and lung transplant Risk factors identified for developing CKD in a study of 219 patients undergoing a lung or a heart–lung transplant include cumulative periods of a diastolic blood pressure >90 mmHg and serum creatinine value at 1 month posttransplant [11]. In this same study, the authors found that tacrolimus use in the first 6 months posttransplant decreased the risk of CKD compared with those who received cyclosporine alone. Other studies found GFR at 1 month to be a predictor of later CKD [12]. Development of postoperative hypertension is associated with an increased risk of ESRD after lung and heart–lung transplants [13]. Hematopoietic stem cell transplant Epidemiology The cumulative incidence of CKD varies from 13% to 60% in adult studies [15–17] to as high as 62% in children [14]. CKD usually becomes apparent 6–12 months after HSCT, although it has been described as early as 2 months and as late as 10 years posttransplant. Though this section discusses injury that occurs after HSCT, baseline or pretransplant renal function can impact the results [46]. Therefore, baseline assessments not only of serum creatinine but also urinalyses and more formal estimations of GFR are warranted. Accurate assessment of baseline renal function can help guide later medication dosing. There are three distinct clinical manifestations of renal disease that can occur in the HSCT patient: thrombotic microangiopathy (TMA), typically hemolytic uremic syndrome (HUS), graft-versus-host-disease (GVHD)-related CKD, and nephrotic syndrome (NS). TMA syndromes represent a spectrum of clinical diseases characterized by systemic or intrarenal platelet aggregation, thrombocytopenia, and microvascular fragmentation of erythrocytes. Platelet aggregation can result in ischemia and organ injury. When renal injury is predominant, a diagnosis of HUS is usually rendered, whereas the presence of extensive extrarenal manifestations leads to a diagnosis of thrombotic thrombocytopenic purpura (TTP) (reviewed in [47]). Histopathology of TMA after HSCT Microscopic examination of kidney biopsy specimens from patients with TMA-associated CKD demonstrates mesangiolysis and loss of endothelial cells with expansion of the subendothelium and occlusion of capillary loops. On electron micrographs, there is extensive widening of the space between the glomerular basement membrane and the subendothelium, with amorphous deposits that are not immune complexes. Risk factors for HUS after HSCT Though no clear relationships have been found to date for the development of TMA after HSCT, a number of risk factors have been examined. In earlier studies, where HUS was the primary diagnosis, risk factors identified were total body irradiation (TBI) [14, 15, 48, 49] and calcineurin inhibitor use [15, 27, 50–54]. However, in more recent studies of TMA, acute graft-versus-host disease (GVHD) grades 2–4, older age, and transplant from an unrelated donor are the primary risk factors identified [55, 56]. Other investigators identified sinusoidal obstruction syndrome, matched unrelated donors or haploidentical donors, and lymphoid malignancy as significant predictors of TMA after HSCT in addition to the above risk factors [17, 57–59]. However, in children who develop HUS after HSCT, the presumptive risk factor in these studies was TBI used as part of the conditioning regimen. For example, Tarbell et al. studied 44 children (aged 3–15 years) with acute lymphocytic leukemia (ALL) or neuroblastoma (NB) who underwent HSCT [14]. Twenty-nine of these patients were alive and in remission 3 months after HSCT and were evaluated in the study. Eleven patients developed increases in blood urea nitrogen (BUN) and creatinine, and ten were anemic and thrombocytopenic with evidence of hemolysis on peripheral blood smear; they also had elevated lactate dehydrogenase (LDH) levels. In every patient except one, the hemolytic process resolved, yet renal insufficiency persisted. The pathologic findings of mesangiolysis with intraglomerular capillary aneurysm formation in conjunction with laboratory abnormalities support the diagnosis of HUS in these children. In another small study, Antignac et al. described seven children referred to their nephrology clinic with CKD approximately 5–10 months after TBI followed by HSCT [48]. All seven children had leukemia, and all received cyclophosphamide alone or with cytosine arabinoside and vepeside, in addition to single-dose TBI as part of their conditioning regimen. Three patients developed CKD without hypertension, and four developed HUS with severe hypertension and microangiopathic hemolytic anemia. Of these, two of the four had normalization of their renal function. Follow-up biopsies, however, showed extensive scarring of the renal parenchyma but almost complete resolution of the mesangiolysis. The glomeruli were globally sclerotic, ischemic, or demonstrated mesangial hypercellularity. Thus, there was evidence of persistent and progressive renal damage in these patients despite normalization of serum creatinine and urinalysis. The occurrence of two different clinical presentations, CKD without hypertension and HUS with hypertension, but similar pathology in these children supports the notion that this is a spectrum of disease rather than distinct pathophysiologic processes. GVHD-related CKD GVHD-related CKD in this patient population is usually defined as an elevated serum creatinine or an abnormal GFR 6–12 months after transplant. The incidence of GVHD-related CKD in children after HSCT varies from 11% to 41% [60–63]. In one recent study, the incidence of CKD (GFR <70 ml/min per 1.73 m2) changed over time, with 41% of children having CKD at 1 year, 31% at 3 years, and only 11% 7 years after transplant [63]. In approximately 19% of patients, hematuria and proteinuria persisted up to 10 years after HSCT. Berg and Bolme followed 44 children with acute lymphocytic leukemia (ALL), acute myeloblastic leukemia (AML), and severe aplastic anemia (SAA) and found a significant decrease in GFR 1–2 years after HSCT when compared with their baseline GFR (ALL and AML groups) or with a healthy control group despite serum creatinines that remained within normal limits. An initial decrease in GFR was followed by stabilization up to 5 years posttransplant [64]. This study supports the contention that serum creatinine is not an accurate measure of kidney function. Serum creatinine level and related estimating equations, routinely used clinical measures to estimate kidney function, are dependent on muscle mass and are influenced by age, race, gender, and weight [65, 66]. Patients undergoing HSCT may have large fluctuations in their nutritional status, muscle mass, and weight that will influence GFR based on estimation equations or serum creatinine levels. Proximal tubular dysfunction has also been described in 14–45% of pediatric patients 1–2 years after HSCT, with initial injury to the proximal tubules being nonspecific as reflected by elevated urinary excretion of alpha-1 microglobulin and beta-N-acetylglucosaminidase (β-NAG) followed by more specific damage manifested by decreases in phosphate reabsorption [67]. Risk factors for GVHD-related CKD The risk factors for GVHD-related CKD in children are similar to those identified in adult studies. Kist-van Holthe et al. also retrospectively identified risk factors for developing both acute and chronic renal insufficiency in a cohort of 142 children undergoing transplant over a period of 5 years in the Netherlands [68]. All children received allogeneic transplants. Ninety-one children received radiation, and 82 of these 91 received TBI. Twenty-five children (18%) had CKD (defined as a GFR <85 ml/min per 1.73 m2). These authors found no correlation between radiation dose used and renal insufficiency at 1 year. In a later study from the same group, only acute renal insufficiency predicted the later development of CKD in patients after HSCT [62]. These studies contradict others in the literature that found TBI to be associated with renal injury [60, 61, 69]. However, the doses used here (5–8 Gy in a single fraction) were much lower than described elsewhere. In a study of 92 pediatric HSCT patients by van Why et al., late renal insufficiency developed in 18 of 64 (28%) patients; in half of these patients, the renal disease persisted for 3 months to 3 years [60]. Amphotericin B use, cyclosporine, and TBI were associated with the later development of CKD. In a large retrospective review of 1,635 children and adults, risk factors for developing CKD after HSCT included acute renal failure and acute and chronic GVHD [17]. In this study, TBI was not associated with development of CKD. Nephrotic syndrome (NS) after HSCT Chronic GVHD may manifest itself in the kidney as NS with or without renal insufficiency (reviewed in [70]). Patients usually present with proteinuria, edema, and hypoalbuminemia. The majority of these case reports demonstrate membranous nephropathy (MN) with subepithelial deposits on biopsy; it is postulated that these deposits are antigen/antibody complexes representing GVHD in the kidney. However, cases of minimal-change disease (MCD), which is thought to be a T-cell-mediated process, have also been described [70]. Comparisons between case reports of MN and MCD after HSCT found that MN occurs in 61% of cases compared with 22% of cases having MCD [71]. The majority of reported patients with MN were slightly older males with a history of acute and chronic GVHD. Both MCD and MN occur later after transplant, at 8 and 14 months, respectively, and tend to occur within 1–5 months of GVHD development and/or the tapering of immunosuppression for their chronic GVHD. MN is more difficult to treat, with only 27% of patients reportedly achieving remission compared with 90% of patients with MCD [71]. Others have reported cases of diffuse proliferative glomerulonephritis, anti-nuclear-cytoplasmic-antibody (ANCA)-related glomerulonephritis, focal segmental glomerulosclerosis, and IgA nephropathy [72–76] occurring after HSCT. The development of each of these diseases seems to be associated with chronic GVHD and/or immunosuppression tapering. Treatment with high-dose prednisone and/or reinstitution of calcineurin inhibitors usually results in resolution of NS. Some physicians have used rituximab successfully in patients with NS after HSCT, typically in cases of MN [77]. Management of CKD after liver, cardiac, lung, and HSCT Patients who develop CKD after liver, cardiac, lung, and hematopoietic stem cell transplants are at increased risk of mortality [18, 26, 78]. Transplant physicians and nephrologists should work together starting from the time prior to transplant to monitor these patients closely. More accurate measures of kidney function are needed, and baseline and follow-up iohexol or iothalamate studies to measure GFR may be indicated to identify patients with underlying CKD prior to transplant and to allow early identification and intervention in patients with mild changes in GFR posttransplant. New markers to estimate GFR, such as cystatin C, may be more informative in certain patient populations than is serum creatinine. Cystatin C is a cysteine protease inhibitor expressed by all nucleated cells and is freely filtered by the glomerulus. Serum cystatin C correlates well with measured GFR and more accurately measures kidney function than does serum creatinine in the elderly, cancer patients, diabetics, and renal transplant recipients [79–83]. Normal ranges for cystatin C have been validated in children. The normal reference range for children older than 1 year is 0.7–1.38 mg/dl [84]. An equation to estimate GFR based on cystatin C levels in children has also been created: log (GFR) = 1.962 + [1.123*log(1/cystatin C)] [85]. Urinalyses including a microalbumin to creatinine ratio should also be part of the pretransplant workup, and urinalyses should be monitored closely following transplant. Animal models of HSCT, and specifically of radiation-induced HUS, offer potential interventions for patients with HUS after HSCT. Angiotensin-converting enzyme inhibitors (ACEI) have been used in rodent models of HSCT-related renal injury. The use of captopril or enalapril at the time of TBI in these animals resulted in less azotemia, lower blood pressures, decreased proteinuria, and long-term preservation of renal function [86]. ACEI and angiotensin receptor blockers (ARBs) also help reduce inflammation and inflammatory markers and reduce circulating levels of TGF-β1 in patients after transplant [87–90]. These agents have also been shown to slow CKD progression and decrease proteinuria in patients with renal disease from various causes [91, 92]. Hypertension management early after transplant is important to prevent CKD development and progression to ESRD in certain transplant populations. ARBs and ACEIs have been shown to be effective and safe in managing hypertension in cardiac transplant patients [93], and ACEIs have been shown to stabilize renal function over 2 years of follow-up [94]. These drugs should be considered as first-line agents to manage hypertension in patients after transplant. In addition, hyperlipidemia management may be important to prevent CKD after transplant. In a study of adult patients undergoing a cardiac transplant, statin use was associated with a decreased risk of development of CKD after transplant [78]. The difficult decision is when to intervene (Fig. 1). Should patients be started on these medications prior to transplant, at the first signs of hypertension and microalbuminuria, or at some set time point after transplant to help protect their kidneys? These decisions need to be made on an individual basis and will vary based on the type of transplant. Frequent discussions between the transplant physician and nephrologist are required to optimize the management of patients with CKD after transplant. Fig. 1Timing of intervention. Bold numbers are estimated glomerular filtration rate based on the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines. Adapted with permission from [102] In addition to the above medications, immunosuppression adjustment is important in managing the CKD that develops after transplant, further supporting the need for close collaboration between transplant physicians and nephrologists. In two reported series of lung transplant recipients, sirolimus was used to decrease and eventually stop calcineurin inhibitors in patients with CKD after transplant. Patients had a sustained improvement in kidney function as reflected by a decrease in serum creatinine for 1–12 months [95, 96]. However, prior to changes in immunosuppression, a kidney biopsy may be indicated to define the etiology of the kidney injury and better tailor therapy to prevent or slow disease progression. Management of patients with ESRD after transplant include the use of peritoneal and/or hemodialysis. Caring for these patients also involves management of complications associated with ESRD, which include anemia, bone disease, hypertension, and metabolic abnormalities. There have been case series of patients undergoing renal transplantation successfully after cardiac, liver or HSCT; renal transplantation is a viable option for patients with ESRD after transplant and can improve outcomes [19, 97–101]. Questions (Answers appear following the reference list) Which of the following are risk factors for developing CKD after cardiac, liver, and HSCT? Calcineurin inhibitorsTotal body irradiationAcute renal failureHypertensionAll of the following are manifestations of CKD seen in patients after HSCT except: Nephrotic syndromeAcute glomerulonephritisThrombotic microangiopathyGVHD-related CKDWhat pretransplant factors increase the risk of CKD in pediatric cardiac and liver transplant patients? Preexisting renal diseaseRacePretransplant diabetesAll of the aboveManagement of pediatric patients who develop CKD after transplant should include all of the following except: Reducing exposure to calcineurin inhibitorsACEI and/or ARBCalcium-channel blockersStatin useWhat is the most common cause for developing TMA after transplant in patients receiving a heart, liver, or stem cell transplant? Graft-versus-host diseaseTotal body irradiationCalcineurin inhibitor useDiabetes Answers: a and c b d c c

World_J_Urol-3-1-1913171

The influence of thermo-chemotherapy on bladder tumours: an immunohistochemical analysis

To study the influence of microwave induced thermo-chemotherapy on high-grade urothelial cell carcinomas. Five groups of each three patients were formed of whom initial biopsies and cystectomy samples were collected. Patients were treated 2 days prior to cystectomy with mitomycin-C (group 1), hyperthermia (group 2) or thermo-chemotherapy (group 3). Group 4 patients had been treated with a cycle of six thermo-chemotherapy treatments prior to cystectomy and group 5 patients served as control (no treatment). Tumour samples were stained with Haematoxylin and Eosin, monoclonal antibody Ki-67 and the monoclonal antibody p53. In six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour was found. Seven out of nine patients treated with hyperthermia showed a decrease in p53 activity. A decrease in proliferation activity and p53 activity illustrate the potential role of thermo-chemotherapy as a promising intravesical treatment. Introduction Bladder cancer is the seventh most common cancer worldwide, accounting for 3.2% of all cancers. In 2000 an estimated 260,000 new cases in men and 76,000 in women were found worldwide [5]. The highest incidence rates of bladder cancer in both sexes are observed in Europe, North America and Australia [12]. Bladder cancer incidence is still rising moderately in most developed countries. Of all these malignant bladder tumours, more than 90% are transitional cell carcinomas of which two third is superficial and one third is muscle invasive. The management of these two types of transitional cell carcinoma differs enormously. The management of superficial bladder cancer consists of transurethral resection in the first place, whereas the primary treatment for muscle invasive bladder cancer consists of cystectomy, a complete different approach. After transurethral resection of a superficial bladder tumour 30–85% of patients develop recurrences, despite the most accurate resection technique. This high risk of recurrences makes bladder cancer one of the most prevalent human tumours. To decrease the number of recurrences after transurethral resection, patients are treated subsequently with chemotherapeutic or immunotherapeutic agents. Immunotherapy, usually BCG treatment, is more effective than any intravesical chemotherapeutic agent, but has more serious and more frequent side effects. So, there is a need for more effective treatment options or treatment options with less frequent and less severe side effects. A new treatment option in patients with intermediate to high risk tumours is the combination of intravesical hyperthermia and intravesical chemotherapy [2, 6, 15]. The endocavitary location and ease of accessibility by the urethra makes thermo-chemotherapy a good therapeutic option for superficial bladder cancer. Local hyperthermia at temperatures of 40–44°C in combination with selected cytostatic agents in several tumours, including transitional cell carcinoma, results in a synergistic anti-tumour effect [10]. In this descriptive study the influence of different treatment modalities on bladder tumours (thermo-chemotherapy, chemotherapy and thermotherapy) was investigated by several immunohistochemical stainings. Methods Subjects The study included 15 patients with high-grade transitional cell carcinoma of the bladder. All patients signed a patient informed consent and all these patients were facing cystectomy. The 15 patients were divided in five groups with each three patients. Group 1 patients were treated 2 days before cystectomy with an intravesical mitomycin-C (MMC) instillation. After the bladder was emptied, 20 mg MMC (Kyowa Hakko Kogyo Co., Tokyo, Japan) in 50 ml saline was instilled. In order to stabilize the MMC concentration in the bladder throughout the entire session, the bladder was emptied after 30 min and with urine diluted solution was replaced by a new solution containing 20 mg MMC. Group 2 patients were treated 2 days before cystectomy with sterile water and local hyperthermia. To standardize treatments the instillation was replaced after 30 min. The local microwave induced hyperthermia was delivered by the SB-TS 101 system as described by Colombo et al. [3]. This system consists of a 915 MHz intravesical microwave applicator that delivers hyperthermia of the bladder walls via direct irradiation. The applicator is part of the specially designed 20F transurethral catheter. The catheter also contains five thermocouples. Two thermocouples measure the temperature in the prostatic urethral tract; the other three are spread out and pushed tangentially against the posterior and lateral walls of the bladder. To avoid urethral overheating, the solution is continuously pumped out of the bladder and re-instilled after being cooled. Hyperthermia was delivered within a temperature range of 41–42°C. Group 3 patients were treated 2 days before cystectomy with two times 30 min intravesical MMC (20 mg in 50 ml sterile water) combined with local microwave induced hyperthermia delivered by the SB-TS 101 system. Group 4 patients were treated with a cycle of six thermo-chemotherapy treatments during the last 3 months. Finally, group 5 patients served as control group. These patients did not receive any intravesical instillations within 3 months prior to cystectomy. Immunohistochemistry All tumour tissue samples were fixed in a 10% buffered formaldehyde solution. The specimens were embedded in paraffin blocks and sections of 4 μm were cut. All specimens were deparaffinized and stained with Haematoxylin and Eosin. A microscopic examination of the samples was performed and the extend of inflammation (1+ to 3+) haemorrhage (1+ to 3+) were semi quantitatively scored. Ki-67, a nuclear protein present during phases G1, S, G2 and M of cycling cells, is accepted as a good indicator of cell proliferation [11]. Sections were deparaffinized and immersed in a 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5 min to enhance antigen retrieval. After washing, the slides were incubated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. After incubation at room temperature for 2 h with the anti-Ki67 monoclonal antibody clone MIB-1 (BioGenex) diluted 1:30, a biotinylated antibody that recognises murine IgG (BioGenex) was applied for 20 min, followed by incubation in streptavidin-peroxidase complex (BioGenex) for 20 min. The peroxidase reaction was developed using 0.5 mg/ml diaminobenzidine tetrahydrochloride (Sigma) in 0.01% H2O2. Haematoxylin was used as a light counter stain. The slides were dehydrated and mounted with a xylene-soluble mounting medium. The mutated p53 tumour suppressor gene product can be detected immunohistochemically and is associated with a lower survival in patients with bladder cancer [7]. Sections were deparaffinized and immersed in a 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5 min to enhance antigen retrieval. After cooling down, 0.6% H2O2 in a 40% methanol solution was used for 30 min to block endogenous peroxidase activity. Slides were pre-treated with 10% normal swine serum for 10 min to block non-specific staining followed by adding primary antibody DO-7 for 1 h at 20°C. After extensive rinsing, sections were incubated for 30 min with biotinylated swine anti mouse antibody (1:200 dilution) and then for 30 min with avidin-biotin complexes (1:50 dilution) at 20°C. Diaminobenzidine (DAB) staining were finally followed by haematoxylin nuclear counter staining. The slides were dehydrated and mounted with a xylene-soluble mounting medium. Immunoreactivity scoring The screening of tumour tissue samples was performed by two independent investigators (CH and AH). The sections were screened for positive cells, defined as cells with nuclear staining. The amount of Ki67 or p53 staining is scored in percentages. The threshold for p53 “positivity” is ≥20% positive staining. The areas with maximal immunohistochemical staining were used for scoring. In total, 300–500 tumour cells were scored. In the visual estimation only definitely brown nuclei were recorded as positive. The results were expressed as percentage of immunoreactive tumour cell nuclei. Results Fifteen patients (11 males, 4 females) signed a patient informed consent and participated in this study. Ages ranged from 42 to 75 years (mean 63.2 years). All patients underwent cystectomy for transitional cell carcinoma of the bladder. Eight patients had a de novo invasive bladder tumour, whereas seven patients had a history of high-risk superficial disease. Patient details are summarized in Table 1. Table 1Patient details containing number of previous occurrences, interval period between initial diagnosis and cystectomy in months, pathology data of biopsy and cystectomy (grading according to WHO 2002)GroupsPatient no/age/sexN previous occurrencesInterval biopsy–cystectomy (in months)Histopathology initial biopsyHistopathology cystectomy1 (MMC)1/75/M02,0≥pT2aGIIIpT3aGIIIN22/46/F04,4pT2GIIpT3bGIIIN13/67/F01,2≥pT2GIII≥pT2GIII 2 (HT)4/64/F01,9pT1GIIIpT2aGIII5/71/M338,7pT1GIII + CISpT1GIII + CIS6/42/M10121,9pTaGIIpTaGII3 (MMC + HT)7/55/M02,6pT1GIII≥pT2GIII8/67/M01,9pT2GIIIpT3aGIIIN19/48/F13,3pT1GIIpT2bGIIIN14 (History of MMC + HT)10/54/M18107,5pTaGIIpTaGII11/73/M222,3CISpT4aGIIIN212/75/M1057,7pTaGIIpTaGII5 (Control)13/71/M9115,5CISpT2GIII14/71/M03,1pT2GIIIpT2GIII15/71/M03,3pT2GIIIpT2GIIIN1HT microwave induced hyperthermia The initial biopsies and tumour tissues obtained with cystectomy were used for histopathological diagnosis and immunohistochemical analysis of Ki-67 and p53. The results from the different treatment groups are summarized in Table 2. In one patient of the MMC group no residual tumour could be retrieved in the cystectomy specimen. The proliferation activity and p53 activity could not be scored in that patient. Table 2The p53 and Ki67 immunoreactivity scorings in percentages of all patients divided in five different treatment groupsGroupsMaterialInflammationProliferation (%)P53 (%)Haemorrhage1 (MMC) aBiopsy+++3020+Cystectomy+++7520++ bBiopsy++2075−Cystectomy+++1575+ cBiopsy+++4075+++CystectomyNANANANA2 (HT) aBiopsy+6080−Cystectomy+++2040++ bBiopsy+3090−Cystectomy++2040− cBiopsy+10-−Cystectomy++10-+3 (MMC + HT) aBiopsy+++40>75−Cystectomy+++2575− bBiopsy+++>75>75+Cystectomy+++6015− cBiopsy+++30>75+Cystectomy+++1575++4 (History of MMC + HT) aBiopsy+1075−Cystectomy+20−−  bBiopsy+++3025−Cystectomy+++3025− cBiopsy++5020−Cystectomy++2010+5 (Control) aBiopsy+15>75−Cystectomy+++40>75− bBiopsy++4020−Cystectomy++4020− cBiopsy++3575+Cystectomy+++3575−The extend of inflammation and haemorrhage is semi quantitatively scored (1+ to 3+)NA not applicable due to absence of residual tumour The intensity of inflammation increased in three out of nine patients treated with hyperthermia. In three out of five patients from the MMC group and control group an increase was seen. The intensity of haemorrhage increased in four out of nine patients treated with hyperthermia, one patient showed a decrease and four patients did not show any difference. In the MMC group and in the control group two patients showed an increase, one patient a decrease and two patients did not show any difference. In six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour tissue sample was found (Fig. 1a). In the MMC group one patient showed a decrease and one showed an increase in proliferation activity. In the control group two out of three patients showed no changes, whereas in one patient an increase in proliferation activity was seen. Fig. 1The p53 and Ki67 positive staining in patient no. 4 (hyperthermia group). a1 shows 60% Ki67 positivity before treatment; a2 shows 20% Ki67 positivity after treatment; b1 shows 80% p53 positivity before treatment and b2 shows 40% p53 positivity after treatment As regards p53, seven out of nine patients treated with hyperthermia showed a decrease in p53 activity (Fig. 1b). In one patient no change was found. The control group and the group treated with MMC did not show any differences concerning p53. Discussion Thermo-chemotherapy has shown to be a promising method for treating several kinds of malignant tumours including superficial bladder cancer [15]. While hyperthermia is important in cancer therapy, it can also damage normal tissues adjacent to the tumour. Fajardo [4] reviewed the effects of hyperthermia from several studies on various tissues of mammals and humans. Hyperthermia with a maximum applied temperature of 44.5°C showed no gross or microscopic alterations of the bladder of dogs and rabbits. Subsequently, Rath-Wolfson et al. [13] studied the effect of hyperthermia, with a maximum applied temperature of 46°C, and simultaneous MMC treatment in sheep. In this study thermo-chemotherapy showed no significant macroscopic or microscopic differences in the bladder wall as compared to a control group with untreated sheep. In the current study the effect of the combination thermo-chemotherapy is compared with solely hyperthermia, solely chemotherapy and no treatment (control group) in human patients with high-grade urothelial cell carcinoma of the bladder. The system SB-TS 101 used to deliver local microwave induced hyperthermia makes it possible to dose the hyperthermia very precisely [1]. In this study hyperthermia was delivered within a temperature range of 41–42°C, the clinical situation. This makes comparison between the different groups more accurate. The sample size of the group studied is small due to the fact that recruitment of patients was difficult. Patients had to agree on an extra treatment session only two days before cystectomy, which did not give them any benefit at all. The degree of inflammation increased in the group treated with solely hyperthermia (N = 3), solely MMC (N = 1) and the control group (N = 2). In the group treated with thermo-chemotherapy 2 days before surgery the degree of inflammation was initially already maximal. In the group treated with a cycle of six thermo-chemotherapy treatments during the last 3 months the degree of inflammation apparently had returned to baseline again. At least one patient per group (except the control group) showed an increase in the degree of haemorrhage. Patients from group 1 (solely MMC) showed the highest degree of haemorrhage. Group 3 patients (treated with thermo-chemotherapy prior to cystectomy) show in one patient an increase, in one patient a decrease and in one patient a stable extend of haemorrhage. This is in line with the results from Rath-Wolfson et al. [13]. Nevertheless, since solely MMC and solely hyperthermia gives an increase in haemorrhage, it would have been logical to find a higher extend of haemorrhage after the combination of both treatments. Possibly, this is not found due to the small sample size. The inhibition of proliferation activity is one of the most important goals in cancer treatment. All groups treated with hyperthermia showed to a different extend a decrease in proliferation activity. Most interesting are the three patients treated with thermo-chemotherapy preceding transurethral resection. All three patients showed a decrease in proliferation activity. The control group on the other hand shows in two patients no difference and in one patient an increase in proliferation activity. In earlier in vitro studies using hyperthermia and bladder cancer cell lines, this decrease in cell proliferation due to hyperthermia combined with chemotherapeutic agents was already shown [14, 16]. However, in these in vitro studies solely hyperthermia did not cause a significant decrease in cell proliferation. Nevertheless, there is a difference between in vitro and in vivo studies. This in vivo study is the first one showing a decreased proliferation activity in humans treated with microwave-induced hyperthermia with or without MMC. The effect of hyperthermia was seen in six out of nine patients treated with hyperthermia preceding cystectomy. Furthermore, the group treated with solely MMC did not show a significant decrease in proliferation activity. This is probably due to the limited penetration properties of this intravesical used drug, especially after one single treatment. p53 is known to be responsible for repair or apoptosis in response to DNA damage. The p53 activity, in other words the expression of mutant p53, decreased exclusively in patients treated with hyperthermia with or without MMC. Previous experiments showed that the p53 pathway is heat sensitive and that the p53 protein is inactivated at temperatures above 41°C [8, 9]. When cells were heated to 42.5°C and returned to normal temperatures, a strong p53 response with an increase in protein levels was observed. In our study we found the opposite, but the time interval between treatment and cystectomy was with 2 days (group 3) and several months (group 4) significantly longer than the time interval used by Guan et al. [8]. Furthermore the results of this study are based on a small number of patients, which makes statistic analysis unreliable. On the other hand, the current results suggest that thermo-chemotherapy could be an effective treatment in patients with a p53 tumour suppressor gene mutated tumour. In all, the results show several trends and encourage carrying out larger experimental studies. Conclusion The degree of inflammation and haemorrhage in bladder tumours did not increase in patients treated with hyperthermia. This, in combination with a decrease in proliferation activity and a decrease in p53 activity, implies that thermo-chemotherapy is a safe and promising treatment.

J_Gastrointest_Surg-3-1-1852393

CD24 Expression is an Independent Prognostic Marker in Cholangiocarcinoma

CD24 has been described as an adverse prognostic marker in several malignancies. This study evaluates CD24 expression in cholangiocarcinoma and correlates the findings with clinicopathologic data and patient survival. Between 1996 and 2002, 22 consecutive patients with cholangiocarcinoma were treated at our institution. Demographic data, SEER stage, pathologic data, treatment, expression of CD24, mitogen-activated protein kinase (MAPK), phosphorylated MAPK, and survival were analyzed. The majority of the tumors demonstrated CD24 (81.8%) and p-MAPK (87%) expression. A negative association was noted between the expression of CD24 and p-MAPK. Median survival for patients with low expression of CD24 was 36 months and high expression was 8 months. Median survival for patients who received chemotherapy with low CD24 expression was 163 months, and for seven patients with high CD24 expression, it was 17 months (p = 0.04). With the addition of radiation therapy, median survival for patients with low expression of CD24 was 52 months and high expression was 17 months (p = 0.08). On multivariate analysis, the use of chemotherapy (p = 0.0014, hazard ratio 0.069) and the CD24 overexpression (p = 0.02, hazard ratio 7.528) were predictive of survival. CD24 is commonly expressed in cholangiocarcinoma, and overexpression is predictive of poor survival and possibly of lack of response to chemotherapy and radiation therapy. These findings may improve selection of patients for the appropriate treatment modality and the development of CD24-targeted therapy. Introduction Malignancies of the biliary tract are uncommon in the Western world. Two-thirds arise in the gallbladder and the remainder in the biliary tree or periampullary region. Cholangiocarcinoma or bile duct cancer is a rare but lethal malignancy with an incidence of 1–2 cases per 100,000 patients in the United States.1 Clinicopathologic factors predictive of survival include curative resection, tumor stage and grade, serum bilirubin level <10 mg/dl, low CA19-9 level, hepatitis viral infection,2–4 lymphovascular or portal vein invasion,5 intrahepatic satellite lesions, inraductal papillary compvonent, tumor angiogenesis,6 and DNA ploidy.7 Reports of molecular markers predictive of survival in cholangiocarcinoma include cluster of differentiation CD24,8 MMP-2, TIMP-2,9 cholinesterase level,10 MUC-4,11 cyclin D1,12 VEGF-C,13 p27,14 p53, and p73.15 Recently, CD24 has been described in a wide variety of malignancies and shown to be a prognostic marker in several solid tumors including colorectal, stomach, lung, prostate, ovarian, and breast.16–21 CD24 is a small, heavily glycosylated, mucin-like, cell-surface protein expressed in developing cells including pre-B cells, keratinocytes, and renal tubular epithelium.22–24 It functions as an alternative ligand of P-selectin, an adhesion receptor expressed on activated endothelial cells and platelets which can enhance the metastatic potential of CD24-expressing tumor cells.25–28 CD24 has apoptotic activity, and its cross-linking induces the sustained activation of p38 MAPK (mitogen-activated protein kinases)—the magnitude of which may determine the survival or death of pre-B cells.29 An improved understanding of the molecular pathways involved in the pathogenesis and progression of cholangiocarcinoma will contribute to the development of targeted therapy. This study correlates CD24 and MAPK expression with patient survival in cholangiocarcinoma with the objective of identifying a subset of patients who may benefit from targeted molecular therapy. Patients and Methods Clinical Data After obtaining approval of the Institutional Review Board, a review of the tumor registry at Roswell Park Cancer Institute identified 31 consecutive patients with histologically proven cholangiocarcinoma between 1996 and 2002. Twenty-two patients had adequate tissue for further histopathologic studies and constitute the basis of this study. Medical records of these patients were reviewed for demographic data including age; gender; surveillance, epidemiology, and end results (SEER) stage at presentation; treatment; and survival from the time of diagnosis. Immunohistochemical Staining For most of the patients, diagnosis was established by examination of conventional hematoxylin and eosin (H&E)-stained slides and, in the remainder diagnosis, was confirmed with ancillary techniques including immunohistochemistry and special histochemistry with mucin and PAS stains. Uniform tissue fixation techniques were used for all patients. For each patient, a representative block containing adequate neoplastic and nonneoplastic tissue was selected. Five-micrometer tissue sections from these blocks were placed on charged slides and dried in a 60°C oven for 1 h. Upon return to room temperature, the slides were deparaffinized in three changes of xylene and rehydrated using graded alcohols. Endogenous peroxidase was quenched with 3% aqueous H2O2 for 15 min and washed with phosphate buffered saline with 0.05% Tween-20 (PBS/T). CD24 primary antibody was obtained from BD Biosciences (clone ML5) and used with the recommended incubation time and antigen retrieval procedures. The primary antibody used for MAPK was obtained from Cell Signaling and for phosphorylated/activated MAPK from Santa Cruz. After a PBS/T wash, 0.03% casein (in PBS/T) was used as a block for 30 min followed by the application of the primary antibody to the slides for an hour or overnight. Another PBS/T wash was followed by exposure to the biotinylated secondary antibody for 30 min. A third PBS/T wash was followed by exposure to the streptavidin–peroxidase complex for 30 min. A PBS/T wash was followed by the application of the chromogen DAB (DAKO, Carpinteria CA, USA) for 5 min. The slides were then counterstained with hematoxylin, rinsed with water, dehydrated, and cleared, and a coverslip was placed. The use of biomarkers, related controls, and interpretation of results using the Histoscore system for quantification of results have been described previously by our group.30 Histoscore was defined as the product of the percentage of positive cells and the intensity of stain. The grade of positive staining depended upon the intensity of staining (0: no staining, 1: weak, 2: moderate, and 3: strong staining) and the percentage of cells stained. The final score was calculated as a sum of each stain intensity multiplied by the percentage of stained cells in the area of interest. For example: if tumor showed 50% weak, 30% moderate, and 20% strong staining, the score assigned was . Histological Grading The cholangiocarcinoma tissue specimens were also stained by routine H&E stains. The specimens were graded based on the degree of tumor differentiation using the World Health Organization (WHO) system. Statistical Analysis Association between biomarker expressions in tumor tissue was investigated using the Kendall’s tau. Biomarker expressions were classified as high and low based on whether their scores were above or below the median value, and survival between low and high expressions was compared using the log-rank test. The Kaplan–Meier method was also used to estimate the survival curves and median survival. The Cox’s proportional hazards survival analysis was used in the multivariate analysis of survival data to explain the effect of biomarker expressions together with other diagnostic parameters. Patient demographics including age, tumor grade, SEER stage, and treatment received were considered as possible parameters for explanatory variables in the model. All statistical tests were two-sided with statistical significance level at 5%. Results Patient Characteristics Of the 22 patients included in the study, 7 were males and 15 females. The median age was 66.5 years (range: 35–77). SEER staging was local in 1 (4.6%), regional in 14 (63.6%), and distant in 7 (31.8%) patients. Differentiation of the tumor was classified as grade 1 in 3 (13.6%), grade 2 in 9 (40.9%), and grade 3 in 10 (45.5%) patients according to the WHO classification. Treatment for cholangiocarcinoma included surgery only (n = 8), surgery and chemotherapy (n = 5), surgery and radiation (n = 1), chemotherapy only (n = 2), and all three treatment modalities (n = 5), and one patient did not receive any treatment. Immunohistochemical Staining Normal bile duct staining was used to set the score intensity. Most of the bile ducts were negative. Occasionally, they demonstrated weak focal and incomplete staining as seen in Fig. 1. The cholangiocarcinoma cells were scored as 1+ when they demonstrated weak expression of CD24, 2+ for moderate expression, and 3+ for strong expression of CD24. Figure 1 depicts cholangiocarcinoma positive for CD24 expression adjacent to normal biliary epithelium. The majority of the tumors demonstrated CD24 (81.8%) and p-MAPK (87%) expression. Immunohistochemical staining for these proteins was higher in malignant tissue in comparison to normal biliary epithelium. The pattern of staining was usually a combination of cytoplasmic and apical, and few specimens demonstrated the apical pattern only. Figure 1Moderately differentiated cholangiocarcinoma with overexpression of CD24. Normal bile duct (right upper corner) demonstrated weak apical staining, whereas neoplastic cells had strong apical and cytoplasmic staining (20×). Relationship Between Biomarkers A negative association was suggested between the expression of CD24 and phosphorylated/activated p-MAPK (Kendall’s τ = −0.32408, p = 0.0501). Survival Median survival was 36 months for nine patients with low expression of CD24 and 8 months for 13 patients with high expression of CD24 as shown in Fig. 2. The median survival for five patients who received chemotherapy with low CD24 expression was 163 months, and for seven patients with high CD24 expression, it was 17 months (Fig. 3, p = 0.04). Median survival for four patients treated with radiation in the presence of low CD24 expression was 52 months, and it was 17 months for two patients with overexpression of CD24 (Fig. 4, p = 0.08). Overexpression of CD24 continued to affect survival adversely despite the overall improvement noted with the addition of radiation therapy. Multivariate analysis using the Cox’s proportional hazards survival analysis demonstrated that overexpression of CD24 (p = 0.02, hazard ratio 7.528) and use of chemotherapy (p = 0.0014, hazard ratio 0.069) were predictive of survival (Table 1). There was no significant association noted between survival and patient’s age, sex, SEER stage, grade of the tumor, surgery, radiation therapy, or expression of MAPK. Figure 2Kaplan–Meier survival curve for patients of cholangiocarcinoma with low and high levels of CD24 expression (n = 22). p = 0.02. Low CD24 (–––––––), high CD24 (- - - - - - - - - - - -).Figure 3Kaplan–Meier survival curve for patients who received chemotherapy with low and high levels of CD24 expression (n = 12). p = 0.04. Low CD24 (–––––––), high CD24 (– - – - – - –).Figure 4Kaplan–Meier survival curve for patients who received radiation therapy with low and high levels of CD24 expression (n = 6). p = 0.08. Low CD24 (–––––––), high CD24 (– - – - – - –).Table 1Prognostic Variables for Survival in 22 Patients with CholangiocarcinomaVariablenMedian survival (months)Survival (p value)Multivariate analysis (p value)Age<681117 (8–36)0.60>681111 (5–52)GenderMale712 (8–163)0.52Female1517 (6–52)Grade1, 21215 (6–52)0.9931011 (8–*)SEER stage1, 21511 (6–52)0.483728 (6–163)ChemotherapyNo106 (4–13)0.00050.0014Yes1252 (17–163)RadiationNo168 (6–28)0.12Yes644 (17–*)SurgeryNo38 (0–28)0.17Yes1917 (7–52)MAPKLow1017 (8–36)0.68High1128 (5–163)P-MAPKLow1013 (8–52)0.34High1136 (4–163)CD24<120936 (13–163)0.100.02>120138 (6–28)*The estimate was not provided because the upper limit of the survival curve had not reached a 50% failure rate.CD denotes cluster of differentiation.p-MAPK denotes phosphorylated form of mitogen-activated protein kinase. Discussion The physiologic function of CD24 is incompletely understood but it has been shown to increase tumor proliferation, cell adhesion, motility, invasion, and apoptosis.22–24,31 Selectins are cell adhesion molecules involved in the rolling adhesion of leukocytes to endothelial cells and platelets under the shear forces of circulation, and P-selectin expressed by thrombin-activated platelets and endothelial cells is a major ligand for CD24 on carcinoma cells.26,27 This suggests that CD24-expressing tumor cells can disseminate more readily due to their capacity to form thrombi with activated platelets or to adhere to endothelial cells. Friederichs et al.28 have demonstrated that the carbohydrate sialylLex abundantly expressed on human cancers is essential for CD24-mediated rolling of tumor cells on P-selectin, and in its absence, human adenocarcinoma cells failed to arrest and colonize the lungs. CD24, a metastasis-associated protein, has been recently identified as a downstream target of Ral signaling.32 Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. Microarray by immunohistochemistry of a human bladder cancer identified CD24 as a novel Ral-regulated target and a prognostic biological marker. In this study, 81.8% of patients with cholangiocarcinoma expressed CD24. Median survival for patients with overexpression of CD24 was significantly shorter, and the addition of chemotherapy improved survival. A negative association was noted between the expression of CD24 and p-MAPK. The use of chemotherapy in patients with low expression of CD24 was associated with a median survival of 163 months compared to 17 months in patients with a high CD24 expression (p = 0.04). The use of radiation therapy in patients with low expression of CD24 was also associated with an improved survival than with overexpression of CD24 although the data did not attain statistical significance possibly due to the small number of patients in this series. It has been reported by Taguchi et al.29 that the cross-linking of CD24 induces apoptosis in Burkitt’s lymphoma enhanced by a B-cell antigen receptor (BCR)-mediated signal. They observed that simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. CD24 cross-linking also induces the sustained activation of p38 MAPK, and whether pre-B cells survive or die may be determined by the magnitude of MAPK activation. Consistent with these observations, the present study suggests an inverse association between CD24 and p-MAPK, and eventual cellular proliferation or apoptosis might be a consequence of the dominant effect in a complex interplay of opposing influences.33 Our data indicate that high expression of CD24 remains an adverse prognosticator despite the use of additional therapy. Chemotherapy and radiation were noted to provide maximal survival benefit to low expressors of CD24 although the data for the use of radiation was statistically insignificant probably due to the small number of patients in this study. Furthermore, correlation between CD24 expression and radiation sensitivity has been noted to vary with the cell type as in human small cell lung cancer, and radiation doses required to induce apoptosis of CD24-negative human ALL (acute lymphoblastic leukemia) cells were higher than those required for CD24-positive cells, suggesting that lack of CD24 surface antigen expression is associated with intrinsic radiation resistance.34,35 Hypoxia is a characteristic feature of tumor cells due to the sustained proliferation which progressively results in an acidic, nutrient-deprived, and hypoxic tumor microenvironment. Tumor oxygenation has been identified as an independent prognostic variable for locoregional control and overall survival following definitive irradiation for squamous cell carcinoma of the head, neck, and uterine cervix.36,37 Recent reports have indicated decreased efficacy of chemotherapy under hypoxic conditions in several tumor types including pancreatic cancer and testicular tumors.38,39 Because treatment failure was a consequence of hypoxia, the authors recommend novel treatment strategies aimed at improving tumor oxygenation or enhancing the treatment sensitivity of hypoxic tumor cells. Aimed at identifying potential oxygen-dependent markers in vascular endothelial cells for therapeutic intervention in tumor angiogenesis, Scheurer et al.40 performed a broad-range transcriptomic analysis of selected extracellular matrix protein gene expression levels in human umbilical cord vein endothelial cells exposed in vitro to hypoxia for different time periods. They noted several genes transcriptionally upregulated including CD24 at late times of exposure to hypoxia, indicating that it was a useful marker of hypoxic activation in vascular endothelial cells. In the present series, low expressors of CD24 demonstrated greater survival benefit from chemotherapy and radiation than the high expressors, suggesting that its expression may be a marker for tumor hypoxia and response to therapy. This finding that shows that patients with low expression of CD24 may benefit from chemotherapy or radiation is of importance because it has been previously reported that adjuvant or palliative radiation had no effect on survival in patients with cholangiocarcinoma.41 However, the small number of patients in the present series limits interpretation of data suggesting that CD24 overexpression may be predictive of lack of response to radiation or chemotherapy. CD24 has been shown to be a prognostic marker for shortened survival and disease progression in several malignancies including colorectal, stomach, lung, prostate, ovarian, and breast cancers.16–21 Weichert et al. report that in colorectal cancer, only the subset of patients with exceptionally strong cytoplasmic CD24 staining comprising 10% of their study group demonstrated a markedly shortened mean survival of 31.5 months compared to 67.5 months for the remaining patients.16 They also reported that cytoplasmic CD24 staining pattern is prognostically more significant than the membranous pattern—the biological significance of which was unclear. Su et al. noted a 51% expression of CD24 by immunohistochemistry in intrahepatic cholangiocarcinoma as compared to the 81.8% in the present series. They reported CD24 expression and tumor stage as independently predictive of survival on multivariate analysis and suggested membrane-bound CD24 protein as a potential target for immunotherapy.8 In conclusion, overexpression of the molecular marker CD24 in cholangiocarcinoma is predictive of poor survival. CD24 overexpressors demonstrated a lack of response to chemotherapy and possibly radiation therapy although these observations were limited by the small sample size. Additional properties of tumor proliferation, invasion, metastasis, and apoptosis make CD24 a potent target for specifically directed molecular therapy and its overexpression a potential criterion in the selection of patients for the appropriate conventional treatment modality.

Knee_Surg_Sports_Traumatol_Arthrosc-4-1-2358931

A cadaveric analysis of contact stress restoration after osteochondral transplantation of a cylindrical cartilage defect

Osteochondral transplantation is a successful treatment for full-thickness cartilage defects, which without treatment would lead to early osteoarthritis. Restoration of surface congruency and stability of the reconstruction may be jeopardized by early mobilization. To investigate the biomechanical effectiveness of osteochondral transplantation, we performed a standardized osteochondral transplantation in eight intact human cadaver knees, using three cylindrical plugs on a full-thickness cartilage defect, bottomed on one condyle, unbottomed on the contralateral condyle. Surface pressure measurements with Tekscan pressure transducers were performed after five conditions. In the presence of a defect the border contact pressure of the articular cartilage defect significantly increased to 192% as compared to the initially intact joint surface. This was partially restored with osteochondral transplantation (mosaicplasty), as the rim stress subsequently decreased to 135% of the preoperative value. Following weight bearing motion two out of eight unbottomed mosaicplasties showed subsidence of the plugs according to Tekscan measurements. This study demonstrates that a three-plug mosaicplasty is effective in restoring the increased border contact pressure of a cartilage defect, which may postpone the development of early osteoarthritis. Unbottomed mosaicplasties may be more susceptible for subsidence below flush level after (unintended) weight bearing motion. Introduction Full-thickness cartilage defects may lead to early osteoarthritis [6,13,14]. Osteochondral transplantation is a successful treatment for these defects, but the outcome is dependent on, among other parameters, stability and restoration of surface congruency [9,15,21]. In our previous study about the effect of perfect depth alignment of a transferred plug [8], we evaluated the difference in stability between bottomed plugs (donor plug length and recipient defect depth accurately matched) and unbottomed plugs (donor plug is left shorter than the recipient defect depth). That study demonstrated that bottoming plugs resulted in a significantly more stable situation than unbottoming plugs. Unbottomed plugs leave a cavity at the bottom of the defect and therefore rely exclusively on frictional forces. When performing only a single-plug transfer the whole plug is, in most cases, nicely surrounded by (subchondral) recipient bone, and stability is fairly good, bottomed or not. However, when performing osteochondral transplantation where more than one plug is needed, stability is probably lower because of gaps between the round plugs and their surrounding bone. Kordas et al. [10] reported in agreement with this that the push-in force below flush level for (unbottomed) multiple grafts was significantly lower compared to a single graft. Theoretically, width differences of the cartilage layer between trochlea (donor area) and condyl (defect area) might have an influence on restoration of surface congruency. Usually a patient is restricted in weight bearing after osteochondral transplantation. Rehabilitation periods of two weeks of non-weight bearing and an additional two to three weeks of partial weight bearing are reported in the literature [6]. This period facilitates good ingrowth of (subchondral) bone [11,15] and thus ensures the preservation of surface congruency as it was intended directly post-operative. Histological research has proven that after transplantation bone resorption takes place on the recipient site and on the graft surface [5,18]. This might be one reason for a gradual loss of the initial press-fit stability postoperatively and a cause of subsiding below flush level of the graft during follow-up. Accordingly, Whiteside et al. [20] reported a reduction in short-term load bearing capacity of a single-plug transplant one week after transplantation. Press-fit mechanisms provide stability up to 4 weeks [6]; subsequently, this is taken over by the integration of the graft and the recipient bone [11]. The objective of the present study was to investigate the biomechanical effectiveness of osteochondral transplantation. More specifically we assessed whether the treatment would decrease peak stresses at the boundaries of the original articular cartilage defect. Subsequently, we analysed if articular stresses were dependent on plug placement (bottomed versus unbottomed) and how this was affected by loads that represented (for instance unintended) early weight bearing. Materials and methods Materials Eight intact human cadaver knees obtained from the anatomical department, from individuals 70–80 years old of unknown gender, were used. Exclusion criteria were severe arthrosis of donor or graft area and malignant processes within the knee. Specimens were freshly frozen and thawed at room temperature overnight before preparation and testing. The K-scan 4000 (Tekscan Inc., Boston, MA) (Fig. 1) was used for measuring surface congruency according to a previously discussed protocol [4]. Before testing, each new Tekscan sensors was pre-loaded and calibrated, as suggested by the Tekscan manual and by Brimacombe et al. [2]. For this loading, a circular plastic plunger, that almost covered the entire width of the sensor, was placed underneath a small loading platform where weights could be placed on top. Each sensor was calibrated separately and calibration files were stored. A new sensor was used for each knee. The sensor measurements were presented as a 22 by 26 pixel matrix with square pixels. For the osteochondral transplantations, a disposable 8 mm Osteochondral Autograft Transfer System (OATS) was used (Arthrex, Somas, Sint Anthonis, The Netherlands). Fig. 1Tekscan K-4000 measuring device with two thin (0.089 mm) and flexible pressure sensors, which were able to conform to the anatomy of the medial and lateral knee compartments. The sensors consist of printed circuits divided into grids of load-sensing regions. Each sensor measured 28 × 33 mm, with a total of 572 pixels with a surface per pixels of 1.27 mm2 Preparations Skin, muscles, excess soft tissues, patella and the anterior part of the knee capsule were removed from each knee, whereas collateral ligaments and intra-articular structures were left intact. In this way the tibio-femoral joint area was uncompromised and yet was accessible from the anterior side. Dorsally two small ‘windows’ were created in the capsule, giving access to the posterior part of both condyles. The femur as well as the tibia and fibula were sectioned at ∼18 cm from the joint space. The knee was then placed in a knee-testing device, which was used in earlier experiments [1,19] (Fig. 2). This device provides five degrees of freedom of motion for the tibial side, whereas the femoral side has one degree of freedom and can additionally be manually displaced for flexing or extending movements of the knee (Fig. 2). Weights could be attached to the femur side such that the force may be directed through the longitudinal axis of the femur. To simulate partial weight bearing, we applied 350 N as a tibio-femoral compressive force. Previous research at our institution has shown that a force of 171 N (unbottomed) and 384 N (bottomed) was necessary to push a single-plug-mosaicplasty below flush-level [8]. Fig. 2a Knee-testing device. b Circular plastic cap for positioning of Tekscan sensor. c Tekscan sensor in situ. d Special mould for OATS. e Three-plug mosaicplasty placed flush in full-thickness osteochondral defect Operation and testing First, donor sites were marked with a permanent marker on the trochleae or the posterior region of the condyles. Three plugs were harvested with a length of ∼15 mm (to be unbottomed) and three plugs of ∼18 mm length (to be bottomed). After harvesting all six donor plugs, Tekscan sensors were placed inside the tibio-femoral joint space. After positioning the knee in approximately 45° flexion, the sensors were guided alongside the cruciate ligaments (one on each side), and placed between each femoral condyle and tibial plateau. Subsequently, a small circular plastic cap (diameter 10 mm, height 1 mm) was pinned on both condyles, at the location where the mosaicplasty was to be performed (Fig. 2). The compression of both markers on the sensors was clearly visible on the computer screen and assisted in positioning the sensors centrally over the area where the mosaicplasty would be executed. To facilitate repositioning of the sensors between different interventions, the sensors were fixated to the joint by four small metal ‘anchors’ on all four corners of the sensor that could be used to re-attach the sensors to the joint (Fig. 2). In order to confirm validity of this method a test was performed where the sensor was repeatedly fixated and completely removed from the knee and the plastic marker was visualized. Reproducibility of the re-attachement of the sensor was confirmed by five independent measurements of the position of a circular cap, each after removing a re-attachment of the Tekscan sensor. Four out of five measurements showed exactly the same position of the cap within the Tekscan matrix, which proves a high accuracy of re-attachment. To investigate the consequences of a cartilage defect and subsequent mosaicplasty on the stress distribution under various conditions, Tekscan recordings were performed under five different conditions: (A) preoperatively, intact condition, (B) cartilage defect without any reconstruction, (C) following mosaicplasty, (D) after mosaicplasty and subsequent non-weight bearing motion of the knee and (E) after mosaicplasty with weight bearing motion (E). All surface measurements were performed with the knee in 0° extension, thereby ensuring loading of the (restored) defective sites, and an axial load of 350 N. The ‘preoperative’ measurement (A) was with intact condyles. After removing the sensors, both on the medial and lateral condyle a standardized subchondral defect, 8 mm deep was created by a circular drill of 16 mm diameter, the center being at exactly the same location as the center of the plastic marker that was measured previously. The ‘defect’ measurement (B) of the condyle surfaces was performed after creating the osteochondral defect. With the use of a special mould (Fig. 2) and the OATS, these defects were prepared to receive three osteochondral plugs. When the mosaicplasty was to be performed as bottomed, three defects were created, approximately 15 mm deep and the bottoms of the defects were tamped. Following measuring of the depths of these defects, three of the 18-mm donor plugs were matched for these depths by carefully removing some subchondral bone with surgical bone-nibbling pliers according to clinical practice. Plugs were orientated such that the most congruent surface would be achieved. These plugs were tampered in place, until flush level of the cartilage surface with the recipient site was achieved (Fig. 2). At the opposite condyle, which was to be unbottomed, three defects of approximately 20 mm depth were created, and the donor plugs of 15 mm were used. The difference of 5 mm in length between the defects was essential for the unbottomed plugs to be sure to have a cavity at the bottom of the defect and therefore rely exclusively on frictional forces. The ‘mosaicplasty’ surface measurement (C) was executed after performing both mosaicplasties. The ‘non-weight bearing motion’ measurement (D) was performed following flexing and extending the knee 20 times, while no weight was attached to the test device. The axial load of 350 N was re-applied before the measurement was performed. The final ‘weight bearing motion’ measurement (E), was performed once the knee had been flexed and extended 20 times with 350 N of axial force applied to the knee while performing the flexion-extension movements. Evaluation of variables Each of the eight knees provided two paired standardized mosaicplasties of three plugs, one bottomed and one unbottomed. Thus, there were 16 mosaicplasties: eight bottomed and eight unbottomed. Three general groups were created for statistical evaluation: the whole group of all mosaicplasties (bottomed and un-bottomed combined), a group with only bottomed and one with only unbottomed mosaicplasties. The five measurements performed were: (A) preoperative, (B) defect, (C) mosaicplasty, (D) non-weight bearing motion, (E) and weight bearing motion. Relative to the reconstructed surface, we defined two specific regions of interest: the mosaicplasty area itself and the border of the reconstructed surface. The first region obviously quantified the amount of stress transferred at the mosaic site, whereas the second region was selected to quantify the amount of stress transferred to the border of the defect. The mosaicplasty area (region 1) was a circle with a diameter of 16 mm, for which the best fitting circle representing the standardized defect was obtained on the Tekscan sensor matrix (Fig. 3). This circle was obtained by calculating the lowest surface pressure of a 16-mm diameter circle in the non-reconstructed (defect) case. The border region (region 2) was selected around the first region and had a width of 3 pixels (3.81 mm). To ensure that the two regions were clearly separated a transition zone with a width of 1 pixel was selected and has not included in the measurements (Fig. 3). The total of the pixel values in regions 1 and 2, respectively, were used for further comparative evaluations. To allow for a direct comparison of the pressure values, the pressures on the border and mosaic areas were normalized to the total force on the sensor. Fig. 3a Typical example of Tekscan report on load distribution for a cartilage defect without any reconstruction. b The mosaicplasty area (region 1) is a circle consisted of square pixels with a diameter of 12 pixels. The border area (region 2) is a ring with a diameter of 3 pixels around the defect circle. A transition zone of 1 pixel around the mosaicplasty area is found between regions 1 and 2 Statistics Contact pressures were calculated at the boundaries of the cartilage defect (region 2) as well as for the defect/mosaicplasty (region 1) itself. This was done for five different conditions as described before (A–E). These values were expressed for three groups, namely bottomed, unbottomed and combined. Differences were examined within and among the groups. For statistical analysis, we used SPSS 12.0 for Windows. The linear mixed model was used to evaluate the influence of bottoming or unbottoming the plugs, with Bonferroni correction. P < 0.05 were considered to be statistically significant. Results The averaged contact-pressures at the border contact surface are shown in Fig. 4. Overall, there were no significant differences between bottomed and unbottomed. All the data showed the same pattern for the three groups: in presence of a defect (B) the border contact surface pressure was significantly higher compared to the intact (preoperative) situation (A), performing a mosaicplasty made the border pressure decline to approach preoperative values (C), and after motion with and without weight the border contact pressure remained stable (C–E). Fig. 4Graph showing the border contact pressure for three groups; bottomed (blue line), unbottomed (green line) and the whole group (grey line). In presence of a defect (b) the border contact surface pressure was significantly higher compared to the intact (preoperative) situation (a), performing a mosaicplasty made the border pressure decline to approach preoperative values (c), and after motion with and without weight the border contact pressure remained stable (c–e). Standardized deviation values are presented in Table 1 The pressure pattern of the mosaic contact surface was inverse to the border contact surface pressure (Fig. 5): the mosaicplasty area had almost no contact pressure after creating a defect (B), in the presence of the mosaicplasty the contact surface pressure was regained to some extent (C vs. A) and the contact pressure remained relatively constant after non-weight bearing and weight bearing motions (C–E). Fig. 5Graph showing the mosaicplasty contact pressure for three groups; bottomed (blue line), unbottomed (green line) and the whole group (grey line). The mosaicplasty area had almost complete lack of contact pressure after creating a defect (b), in presence of the mosaicplasty the contact surface pressure was regained (c vs. a) and the contact pressure remained stable after non-weight bearing and weight bearing motion (c–e). Standardized deviation values are presented in Table 1 Two out of eight unbottomed versus zero bottomed mosaicplasties showed a decreased mosaicplasty pressure and increased rim stress after weight bearing motion on individual Tekscan measurements, indicating that the mosaicplasty subsided below flush level. Five out of eight bottomed plugs were placed on the medial condyle and no significant differences in pressure or rim stress was found. Statistics Mean pressure values measured are given in Table 1. Table 1Results of the surface contact pressure measurements of five conditionsMeasurementWhole group (n = 16)P-valueBottomed (n = 8)Unbottomed (n = 8)BorderA. Preoperative38.7 (7.1)a40.2 (8.3)37.1 (6.0)B. Defect74.3 (5.5)(B vs. A–C–D–E) 0.00075.5 (3.9)73.2 (6.8)C. Mosaicplasty52.1 (9.3)(A vs. C) 0.00154.6 (7.5)49.7 (10.7)D. Motion without weight53.2 (9.8)(A vs. D) 0.00153.9 (8.4)52.4 (11.6)E. Motion with weight53.8 (9.6)(A vs. E) 0.00055.1 (9.0)52.5 (10.6)MosaicA. Preoperative35.7 (7.8)33.6 (9.3)37.9 (5.6)B. Defect4.0 (4.0)(B vs. A–C–D–E) 0.0003.7 (3.8)4.3 (4.4)C. Mosaicplasty23.8 (8.2)(A vs. C) 0.00221.4 (7.4)26.2 (8.9)D. Motion without weight22.8 (8.7)(A vs. D) 0.00122.1 (8.1)23.5 (9.7)E. Motion with weight22.0 (9.1)(A vs. E) 0.00121.0 (8.4)23.0 (10.1)a Values are given as mean (SD), only significant P-values are shown Effect of defect (A–B): Pressure redistribution occurred in the presence of a defect from the mosaic contact surface to the cartilage surrounding it. The border contact pressure increased to 192% (P = 0.000) compared to the preoperative border contact surface pressure, while the pressure on the defect contact surface decreased to 11% (P = 0.000). Effect of mosaicplasty (B–C): The mean stress elevation around the defect was partially restored in the presence of a mosaicplasty, as the border contact pressure decreased by 30% compared to the defect measurement. This resulted in a border contact pressure of 135% compared to intact cartilage. The mosaic contact pressure was restored from 11 to 67% (P = 0.000). Effect of flexion-extension motion (C–D–E): The border contact pressure did not return to preoperative values after performing a mosaicplasty, and did not change after flexion and extension motions. The border contact pressure after non-weight bearing motion’ and ‘weight bearing motion’ remained equal to the mosaicplasty measurement, 138 and 139% (P = 0.001 and 0.000, respectively) compared to intact, preoperative cartilage. For these measurements the mosaic contact pressures remained lower than those of intact cartilage, with pressures 64 and 62%, respectively (respectively P = 0.001 and 0.001). Discussion In this biomechanical human cadaver study we clearly demonstrated that an osteochondral cartilage defect severely affects the contact pressure on the remaining intact joint surface. Obviously, there are some additional limitations to our study. The study had only a limited number of knees. During testing osteoarthritic changes in bone were found in some of these elderly knees. Clearly this is different to the bone quality of the typical patient who is relatively young and active. Another limitation was that during the preparations the knee was positioned in such a way that both condyles would be loaded with approximately the same force, which may be different from in vivo loads. This might have had an influence on the absence of medial/lateral differences. Contrary to other experiments we kept all collateral ligaments and intra-articular structures intact, which resembles the anatomical situation more closely and thus gives a greater translational value of pressure transfer. Clearly, no biological effects were taken into account such as the resorption of the plugs (thereby reducing the stability) or bony ingrowth (thereby enhancing stability). We also measured the contact patterns under static conditions with the knee in extension, whereas in reality shear forces at different flexion angles are also applied to the reconstruction. These limitations should be taken into account when interpreting the results. The results showed that the average border contact pressure almost doubled (increase of 92%) compared to the pressure on an intact congruent joint surface. In the literature, increases in peak pressure between 10 and 30% were found in the presence of a defect with a diameter ranging from 1 to 7 mm [3,9]. Guettler et al. [4] claimed to have found no difference in rim contact pressure for defects below 10 mm diameter, but they found a 64% increase with respect to the healthy situation for all defects above 10 mm. These findings are supported with a recent finite element model, in which it was found that large defects (greater than 0.78 cm2) resulted in significantly increased border contact surface pressures, which may have clinical implications [16]. The relation between increased contact stresses on the joint surface and progressive degenerative changes of the cartilage is well recognized. Lefkoe et al. found a significant decrease in proteoglycan content in the cartilage sampled from the rim of 20-week-old defects [12]. Jackson et al. [7] introduced a ‘zone of influence’, as he found cartilage adjacent to the defect being affected, which may lead to early secondary gonarthrosis. Messner and Maletius [14] reported in a follow-up study radiographic joint space reduction in almost 50% of the patients with severe cartilage damage 14 years earlier. Linden et al. [13] found in a follow-up study of 33 years that 80% of the adults with osteochondritis dissecans developed secondary gonarthrosis. The gonarthrosis seemed to have its onset 10 years earlier (mean age 49 instead of 59 years) in life than primary gonarthrosis. Clearly, the increased potential of degenerative changes in the knee following an articular cartilage defect is of great clinical importance. Especially because this type of cartilage defect frequently occurs in the younger aged population. In our study we were able to reduce the contact pressure at the boundaries of a large articular cartilage defect by 30% with a osteochondral transplantation. In a study by Raimondi et al. [17] a 16% reduction in peak pressure was found in the presence of a fibrin glue graft. One could question as to what extent this reduction lasts since fibrin glue degrades quite rapidly. Koh et al. [9] reported peak contact pressure reduced to normal when plugs were flush. However, they used only one plug, which does not correspond to clinical practice. It may be more difficult to obtain a smooth cartilage surface when using more plugs, which explains why we did not find a complete normalization of the contact stresses. Another objective of this study was to investigate the stability of a mosaicplasty during motion (with or without weight). For the whole group (unbottomed and bottomed together), neither motion protocols significantly changed surface congruency; it remained equal to the mosaicplasty directly after surgery. This can be concluded from the fact that the pressure patterns did not change after weight bearing or non weight bearing motion, as the pressure pattern and rim stress remained equal. When comparing the bottomed and unbottomed groups, two unbottomed mosaicplasties showed a pressure pattern according to subsidence below flush level after weight bearing motion. Since the main focus of this study was to measure peak stresses on the articular surface, we did not assess the actual amount of subsidence. Although no significant differences in contact stresses could be detected between bottomed and unbottomed plugs by comparing the total groups; one can at least have some concern about potential subsidence of unbottomed plugs. Nevertheless, most unbottomed plugs remained stable, which is in agreement with a study of Pearce et al. [15], who reported that unbottomed multiple plug mosaicplasties that were placed flush, continued to stay flush after 3 months of weight bearing motion in sheep. However, human bone is much softer and therefore it may be more difficult to obtain a stable reconstruction with unbottomed plugs. In our previous study with human femora we measured the force required to push a single plug below flush level. For an unbottomed plug of 16 mm, 151 N had to be applied in comparison with 294 N for the bottomed plug [8]. For the comparison with the multiple plug mosaicplasty, Kordas et al. [10] reported that application of a mean force of 54 N was enough to push an unbottomed multiple plug mosaicplasty 3 mm below flush level; unfortunately, there was not a bottomed comparison in that study. The applied force of 350 N during the weight bearing motion in our study may have been too low, to destabilize the reconstruction. This load, which is relatively low, was chosen, as it would simulate partial weight-bearing motion during the direct postoperative rehabilitation period and not level walking. The latter is, according to our protocol, permitted after 2–4 weeks, which should allow for in-growth of the plugs [11,15]. Clinical relevance A cartilage defect results in increased stress levels at the articular cartilage boundaries of the defect. A multiple plug mosaicplasty has a positive effect in reducing these stress elevations, which will reduce the potential of cartilage degeneration, and thus may postpone secondary osteoarthritis. Post-operative non-weight bearing and weight bearing motions did not seem to influence the surface congruency of the mosaicplasty, although the unbottomed mosaicplasties showed a trend of subsidence below flush level after weight bearing motion. It appears feasible to allow limited weight bearing of the knee after osteochondral transplantation, especially when plugs have been bottomed.

Knee_Surg_Sports_Traumatol_Arthrosc-3-1-1779626

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature. Introduction Autologous chondrocyte implantation (ACI) is an established method for the treatment of chondral defects [6]. Some prospective studies have demonstrated subjective and objective improvement in joint function at 12 months after surgery [3, 13]. MRI, second-look surgery and biopsies have shown the formation of nearly normal cartilage in a good number of patients [13, 18, 19]. On the basis of these results, ACI may be considered one of the most effective treatments of articular cartilage lesions. However, this method is not devoid of some problems with regard to, for example, the wide surgical approach, the demanding technique, the presence of periosteal flap and the cell-holding in site [14, 16]. In order to overcome some of these hurdles, new tissue engineering techniques, widely used nowadays, have been developed using chondrocytes seeded on biological matrices such as hyaluronic acid [15] or collagen membranes [7] or atelocollagen gel [17]. Despite the wide diffusion of these methods, there are still some areas that would need better clarification. The presence of numerous, viable and well-differentiated cells at surgery is an essential requisite for the success of all these methods. It has been shown that autologous chondrocytes in suspension before implantation are able to maintain their differentiated phenotype and are capable of proliferating fairly well [9, 20]. On the other hand, little has been reported so far about cell characterization in membrane-seeded ACI techniques. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the moment of the implantation. Materials and methods In 2003, 12 consecutive patients, 8 males and 4 females, mean age of 34 years, suffering from cartilage lesions of the knee (10 cases) and the ankle (2 cases), underwent a collagenic scaffold-based ACI procedure (MACI®-Verigen, D). Autologous chondrocytes were isolated at the Verigen laboratories from cartilage slices obtained from non-bearing areas of the patients’ joints during a preliminary arthroscopic surgery. Cells were propagated in monolayer cultures in autologous serum for 2 or 3 weeks, according to the cellular growth rate, and were passaged on average 3 times to obtain at least 10 × 106 cells. Cells were then seeded on 20 cm2 type I/III collagen membrane of porcine origin. The cell-seeded membranes were implanted in cartilage defects by means of either arthroscopic or mini-open surgery, using fibrin glue to ensure adhesion. At each implantation, the residual part of the membrane was collected and tested for cell viability, and histochemical and ultrastructural analyses were also performed. Cell viability analysis Cell viability was evaluated by MTT (dimethylthiazol-diphenyltetrazol bromide; thiazolil blue) colorimetric assay. MTT (Sigma, Italy) is a water soluble tetrazolium salt that yields a yellowish solution when prepared in medium lacking in phenol red (RTMI 1640, Sigma). Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by the active mitochondrial dehydrogenases of living cells. The MTT solution (5 mg MTT/ml medium) was added to three samples (1 cm2) of each membrane, being assayed to equal 1/10 of the original culture medium volume, and incubated for 3 h. The solution was then removed and acidic isopropanol (0.04–0.1 N HCl in absolute isopropanol) was added to solubilize the stain. The results were evaluated by means of the spectrophotometric assay (570 nm), yielding absorbance as a function of viable cell number. Histochemical and immunohistochemical analysis The samples were fixed by immersion in 4% para-formaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C and then embedded in paraffin. Specimens were stained with safranin-O. For the immunohistochemistry, non-specific binding was blocked with 3% normal goat serum in a phosphate-buffered saline (PBS), pH 7.4, for 30 min at room temperature; slides were then incubated overnight with primary antibodies at 4°C. Sections were incubated with polyclonal antibodies anti S-100 protein (Dako, Italy), a cytoplasmatic marker of chondrocyte phenotype, diluted at 1:3,000, anti-collagen type I (Monosan, The Netherlands) and II (Calbiochem-Oncogene, CA, USA) at 1:150, and monoclonal antibodies anti chondroitin sulphate (chondroitin-S) (Sigma) at 1:200. Rabbit and mouse immunoglobulins, at the same dilutions as the primary antibodies, were used as controls. After three washes with Tris–HCl (0.05 M, pH 7.6), revelation of the reactions was accomplished by DAKO LSAB + kit, HRP. Stainings were viewed and photographed with a Leica Microscope (Leica Cambridge Ltd., UK). Ultrastructural analysis For scanning electron microscopy (SEM), the membranes were fixed in 2% glutaraldeyde in 0.1 M cacodylate buffer (pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in increasing ethanol concentrations and then CPD-dried. They were mounted on stubs and gold-sputtered. Specimens were observed with a Philips 505 microscope. Results Cell viability analysis The presence of numerous viable cells was observed in all samples, with quite a homogeneous stain distribution, even if some of the areas revealed a greater concentration of converted dye (Fig. 1). Fig. 1Macroscopic view of human chondrocytes seeded on type I, III collagen membrane after MTT incubation. The converted blue dye shows the presence of numerous viable cells with quite a homogeneous stain distribution Considerable variability was found in mean cell numbers between the samples from different patients, in relation to the different amounts of cells obtained from cartilage slices and seeded on the membrane. Highest values were observed in samples from the younger patients (Fig. 2, Table 1).Fig. 2The standard curve obtained from each membrane by MTT colorimetric assay (see also Table 1 )Table 1The table shows the mean viable cell number/cm2 obtained from each membrane by MTT colorimetric assayCaseJointAgeSexSeeded cellsViable cells/cm2 (mean ± SD)1 Knee54M19 × 106(7.52 ± 2.79) × 1032 Knee22F17 × 106(12.94 ± 2.54) × 1033Knee50F15.4 × 106(17.82 ± 6.25) × 1034 Knee23M20.3 × 106(100.28 ± 22.14) × 1035Knee29F20.4 × 106(110.07 ± 39.18) × 1036Knee43M18.7 × 106(5.34 ± 3.25) × 1037Knee48M17.5 × 106(4.74 ± 2.37) × 1038Ankle42F18.2 × 106(33.13 ± 25.92) × 1039Knee19M21.6 × 106(99.54 ± 25) × 10310Knee35M20 × 106(103.81 ± 37.26) × 10311Ankle39M19 × 106(79.48 ± 62.57) × 10312Knee13M20.1 × 106(87.88 ± 55.17) × 103 Patients’ characteristics and the number of cells initially seeded on each membrane are also reported (see also Fig. 2) Histochemical and immunohistochemical analysis In all of the samples, chondrocytes that were grown on the membrane were arranged in multi-layered sheets, and sometimes invaded deeper into the matrix. The cells appeared to be quite well differentiated, although some had a flattened morphology. They slightly stained metachromatically for safranin O (Fig. 3), and clearly immunoreacted with anti-S-100 protein (Fig. 4), type II collagen (Fig. 5) and chondroitin-S antibodies. Only a few cells stained for type I collagen. Fig. 3Human chondrocytes seeded on type I, III collagen membrane before the implantation. Numerous cells are located on the membrane and deeper in the matrix forming a multi-layer engineered tissue. (Safranin O stain, ×200)Fig. 4Human chondrocytes seeded on type I, III collagen membrane. A marked cytoplasmic immunoreaction for S-100 protein is evident (×400)Fig. 5Human chondrocytes seeded on type I, III collagen membrane. Cells express a positive immunoreaction for type II collagen, while the membrane remains unstained (×400) Ultrastructural analysis The membranes presented a dual appearance: there was a smooth side with tightly packed fibres and a rougher side, a sparse layer, to which the cells were adhered. Numerous cells firmly adhered to the membrane and showed a round shaped morphology and a rough surface (Fig. 6). Fig. 6Scanning electron micrograph of human chondrocytes seeded on type I, III collagen membrane. Numerous cells (arrows) firmly adhere to the membrane and show a round shaped morphology and a rough surface (×7,500) Discussion Cell phenotype and proliferation analysis should be an essential step in the evaluation of all tissue-engineered products because the implantation of dedifferentiated or not-proliferating cells would not justify the therapeutic employment of these biotechnologies. The chondrocyte culture may undergo a dedifferentiation process, consisting in a fibroblast-like morphology, a reduction of the type II collagen and aggregating proteoglycans synthesis, and an increase of type I collagen expression [1, 4]. Chondrocyte dedifferentiation is common in monolayer cultures, while a long phenotype maintenance is supported by the presence of a tri-dimensional matrix [1, 4, 11]. The chemical and ultrastructural characteristics of the matrix may have great influence on the behaviour of chondrocytes in culture. A Type I collagen membrane appears to be a matrix endowed with such properties which make that ideal and useful for cartilage tissue engineering [8, 10]. MACI is a method for the treatment of articular cartilage defects, that employs autologous chondrocytes expanded in monolayer culture, suspended, and then seeded on type I–III collagen membrane. In a previous study [9], we had observed that autologous chondrocytes in suspension before implantation were of “good cartilaginous quality” and that they possessed an excellent proliferation capacity. Zheng et al. [20] confirmed that cells in ACI procedure maintained chondrocyte phenotype and showed a low apoptotic rate. To our knowledge, there is no previous reference to the analysis of the phenotype and the proliferation activity of human chondrocytes before MACI implantation. Data about cell number and viability, but not about cell phenotype and distribution are provided by Verigen’s laboratory for each implant. It should, however, be remembered that it is quite possible that the presence of a membrane like the one found in the MACI technique could interfere with cell behaviour and modify phenotype expression. The present study has demonstrated that the autologous chondrocytes that were analysed adhered to the membrane and had quite a homogenous distribution. These cells were viable in all the cases analysed, as shown by the spectrophotometric assay. The differences observed among patients in the number of cells seeded on the membrane and those found to be viable are probably due to several factors. In the first place, the amount of tissue collected from patients and sent to the laboratory for culture was variable. The cellularity of cartilage tissue and the proliferation capacity of chondrocytes in identical culture conditions are also quite variable between subjects, and this variability cannot merely be explained with age, but is more likely to stem from a number of characteristics that define tissue “quality” [5, 12]. We did not investigate whether during monolayer culture chondrocytes underwent dedifferentiation, however, other authors [20] demonstrated that cells grown in monolayer with the same culture conditions maintained a differentiated phenotype. Moreover, we felt it was important to establish the phenotypical expression of chondrocytes seeded on the I/III collagen membrane, since those are the cells that are then implanted, independently of what has taken place in the previous steps of the procedure. The positive immunostaining for S-100 protein, chondroitin-S, type II, but not type I collagen, confirms that cells maintained the characteristics of differentiated hyaline chondrocytes at the moment they were implanted. These data reassure that the MACI procedure can indeed provide a suitable engineered tissue for cartilage repair, in line with the mid-term clinical results described in the literature so far [2, 7]. Nevertheless, clinical and histological results of this method to attain stable clinical recovery and a hyaline-like cartilaginous scar are auspicated to substantiate these evidences in the long term.

Cancer_Causes_Control-2-2-1764867

Wine and other alcohol consumption and risk of ovarian cancer in the California Teachers Study cohort

Objective Whether alcohol consumption influences ovarian cancer risk is unclear. Therefore, we investigated the association between alcohol intake at various ages and risk of ovarian cancer. Introduction Consistent evidence that moderate alcohol consumption increases the risk of breast cancer, presumably by elevating estrogen and androgen levels [1], suggests that alcohol intake might also influence the development of other hormone-related malignancies, such as ovarian cancer. From a public health standpoint, this hypothesis is attractive since alcohol consumption could represent a readily modifiable ovarian cancer risk factor, whereas other risk factors, particularly reproductive characteristics [2, 3], are generally less amenable to change. Findings from previous studies examining the association between alcohol and ovarian cancer risk have been discrepant, with published reports of mostly null [3–19], some positive [15, 16, 20, 21], and some inverse associations [14, 17, 22–25], including occasional variation in associations by type of alcohol and/or subgroup of ovarian cancer cases. No systematic differences in findings are apparent between case–control and cohort studies. Although ethanol itself is not a direct ovarian carcinogen, it could influence ovarian cancer risk through effects on steroid hormones, especially estrogens, which are believed to play a primary role in ovarian carcinogenesis [26]. In pre-menopausal women, moderate to high alcohol consumption is associated with elevated levels of total and bioavailable estrogens and androgens [27–29], reduced fertility [30, 31], and decreased menstrual cycle variability and length, resulting in increased cumulative estrogen exposure [32]. In post-menopausal women, moderate alcohol intake markedly increases circulating estrogen levels in hormone therapy (HT) users [33, 34], and it may elevate estrogen and androgen levels in women not using HT [35]. Other mechanisms for alcohol-related carcinogenesis include alteration of gonadotropin levels, promotion of DNA damage, impaired folate metabolism and DNA hypomethylation, inhibition of carcinogen detoxification or clearance, and increased metastatic potential of tumor cells [1]. Given the biologic plausibility of a role of alcohol intake in ovarian cancer etiology, we examined the association between alcohol consumption and risk of ovarian cancer in a prospective cohort in which baseline alcohol consumption was associated with increased breast cancer risk [36, 37]. With data on past and baseline intake of specific alcoholic beverages, as well as information on a variety of demographic and behavioral factors, we were also able to explore the importance of drinking patterns and potential effect modifiers of any association. Methods Study population The California Teachers Study (CTS) cohort includes 133,479 active and retired female public school teachers and administrators who were members of the California State Teachers Retirement System and returned a mailed questionnaire in 1995–1996 [38]. The questionnaire assessed a range of potential cancer risk factors including menstrual and reproductive history, personal and family medical history, physical activity, dietary intake during the previous year (using a food-frequency questionnaire and portion-size assessment [39–42]), alcohol and tobacco use, and other factors. All women provided written informed consent to participate in the study, and the study protocol was approved by the institutional review boards of all participating institutions. For this analysis, we excluded women (in a hierarchical manner) as follows: those who 1.) lived outside of California at baseline (n = 8,867); 2.) did not provide adequate information on personal history of cancer (n = 662); 3.) consented to participate only in analyses of breast cancer (n = 18); 4.) reported having had ovarian cancer before baseline or were identified by the California Cancer Registry as having been previously diagnosed with ovarian cancer (n = 640); 5.) reported having had a bilateral oophorectomy before baseline (n = 14,422); 6.) were aged 85 years or older at baseline (n = 1,874); 7.) reported never having had a first menstrual period (n = 51); 8.) provided multiple invalid, inconsistent, or blank responses to the dietary questionnaire (n = 2,942); 9.) reported food consumption that was judged to be implausibly low (i.e., < 600 calories/day) or high (i.e., > 5,000 calories/day) (n = 1,565 and 69, respectively); or 10.) provided invalid, missing, or inconsistent data with respect to alcohol intake during the previous year (n = 5,094) or the earlier two periods evaluated (n = 6,904). Of the 90,371 remaining women included in this analysis, 227 were diagnosed with invasive epithelial ovarian cancer and 26 were diagnosed with borderline epithelial ovarian cancer (ICD-O-3 site C569, excluding non-epithelial ovarian cancer cases [morphology codes 8240–8245, 8590–8671, and 9060–9989] [43]) after joining the cohort and on or before 31 December 2003. Alcohol assessment Participants reported average weekly consumption of beer, wine/champagne, and cocktails/liquor at ages 18–22 years, at ages 30–35 years, and in the year prior to baseline. Available response categories for average number of drinks per week were none, ≤ 3, 4–10, 11–17, 18–24, and ≥ 25. A typical drink was defined as one bottle, can, or glass of beer; one glass of wine, champagne, or wine cooler; or one cocktail, shot, or mixed drink of liquor. A single drink of beer, wine, or liquor was assumed to contain 13.2, 11.1, or 15.0 grams of alcohol, respectively. Based on these standards, daily intake of grams of alcohol from each type of drink was calculated for each woman during each time period. Alcohol intake in the cohort was reproducible (r = 0.87) and valid compared to multiple 24–hour recalls (r = 0.74) (Pamela Horn-Ross, unpublished data). Daily consumption of alcohol from beer was categorized as 0, < 13.2, or ≥ 13.2 grams/day; alcohol from wine was categorized as 0, < 11.1, or ≥ 11.1 grams/day; alcohol from liquor was categorized as 0, < 15.0, or ≥ 15.0 grams/day; and total alcohol was categorized as 0, < 10.0, 10.0 to < 20.0, or ≥ 20.0 grams/day. These categories were defined based on the grams of alcohol per standard drink, and on findings from previous analyses of alcohol intake and breast cancer risk in the CTS [36, 37]. Based on their drinking patterns in any two time periods (ages 18–22 years and baseline, ages 30–35 years and baseline, or ages 18–22 years and 30–35 years), women were categorized as having been non-drinkers, moderate drinkers (in the middle category or categories of intake), or heavy drinkers (in the highest category of intake) in both time periods; or decreasing or increasing drinkers if their intake changed between the earlier and the later time period. For each type of alcoholic drink and each time period, women also reported how many days per week they usually had at least one drink. Women were categorized as non-drinkers, as drinking alcohol on 1–4 days/week, or as drinking alcohol on ≥ 5 days/week [37]. Residence-based measures Based on residential address at entry, women were geocoded to census block groups. To obtain a measure of each cohort member’s relative socioeconomic status (SES), all 1990 census block groups in the state of California were ranked by three measures, according to deciles based on the statewide adult population: percentage of adults over age 25 years who had completed a college degree or higher; median family income; and percentage of adults employed in managerial/professional occupations [44]. A summary SES metric was created by adding the scores (1 through 10) across these attributes; participants were then categorized into deciles or quartiles of the total score. Women were also categorized by median family income in their census block group, based on deciles in the cohort. Residential census block groups were categorized as rural, town, small city, metropolitan suburban, or metropolitan urban based on population size and density [44]. Women were also classified as residents of the Greater San Francisco Bay Area (Alameda, Contra Costa, Marin, Monterey, San Benito, San Francisco, San Mateo, Santa Clara, and Santa Cruz Counties); the Southern Coastal area (Orange, Los Angeles, and San Diego Counties); or the rest of California [45]. Follow-up Person-time was accrued from the date of completion of the baseline questionnaire until the date of first diagnosis with borderline or invasive ovarian cancer, relocation out of California, death, or 31 December 2003, whichever occurred earliest. Information on incident ovarian cancer and tumor characteristics was obtained through annual linkage of cohort members to the California Cancer Registry (CCR) based on full name, date of birth, address, and social security number, including manual review of possible matches. The CCR is the population-based cancer registry that covers the entire state of California, has agreements with 13 other states for case-sharing purposes, and maintains high-quality data standards as part of the National Cancer Institute’s Surveillance, Epidemiology, and End Results program. Reporting of new cancer diagnoses to the CCR has been mandated by California state law since 1985, and coverage is estimated to be 99% complete [46], such that all members of the CTS cohort are effectively in active follow-up for cancer outcomes as long as they reside in California. Date and cause of death are ascertained through linkages with the California state mortality file and the national Social Security Administration death master file, as well as reports from relatives. Address changes are obtained through annual mailed newsletters, notifications by participants, and record linkages with the California Department of Motor Vehicles, the US Postal Service National Change of Address database, and other sources. Statistical analysis Multivariable Cox proportional hazards regression analysis was performed to evaluate the association between alcohol consumption and risk of ovarian cancer, using ages at the start and end of follow-up (in days) to define the time scale. Models were adjusted for race (White or non-White), total daily caloric intake (continuous), parity (0, 1–2, or ≥ 3 full-term pregnancies), use of oral contraceptives (never, < 5 years, or ≥ 5 years), average strenuous physical exercise ( < 0.5, 0.5–3.99, or ≥ 4 hours/week during lifetime up to age 54 years), menopausal status/use of HT (pre-menopausal, unknown menopausal status, or peri-/post-menopausal and: never used HT, used combination estrogen-progestin HT, used a mixture of combination and estrogen-only (“mixed”) HT, used estrogen-only HT for ≤ 5 years, used estrogen-only HT for > 5 years, used estrogen-only HT for an unknown duration, or unknown HT use), and an interaction between menopausal status/HT use and the time scale (because menopausal status/HT use violated the proportionality assumption), and were stratified by age at baseline (in years). These potential confounders were chosen based on statistically significant (p-value ≤ 0.05) associations with risk of ovarian cancer, and on prior knowledge of ovarian cancer risk factors. Missing values were coded as dummy variables, none of which were found to be associated with ovarian cancer risk. We tested the assumption of proportional hazards for each alcohol variable using significance tests of interactions with the time scale and visual examination of scaled Schoenfeld residual plots [47], and found no violations of the proportionality assumption. Hazard rate ratios, presented as relative risks (RR), and corresponding 95% confidence intervals (CI) were estimated for each type of alcoholic beverage, comparing categories of consumption to non-drinkers of that alcohol type as the reference group. Tests for linear trend across exposure categories were conducted using the median of each category coded as an ordinal variable. The median of the highest category of beer, wine, or liquor intake was equal to the lower boundary because most women in those categories reported consuming one drink per day. Analyses were first performed for all eligible women, and then repeated with restriction to women who were peri- or post-menopausal at baseline. In addition, analyses were performed with restriction to cases of invasive ovarian cancer (excluding borderline cases) or serous ovarian cancer (ICD-O-3 morphology codes 8441–8462 and 9014 [43]; n= 114). Likelihood ratio tests were used to evaluate homogeneity of the estimated RR between strata of women, as well as significant differences in fit between models with and without additional covariates. Tests for non-linearity of trend were based on a likelihood ratio test comparing models with the exposure coded as an ordinal or a categorical variable [48]. Associations with wine drinking were evaluated using multivariate logistic regression, using covariates as defined above. All analyses were performed using SAS Version 9.1 (SAS Institute, Cary, NC). Results Distributions of demographic characteristics and ovarian cancer risk factors in the eligible study cohort are shown in Table 1. The median length of follow-up was 2,959 days (8.1 years) and the median age of participants at baseline was 50 years. As shown in Table 2, there was no overall difference in risk of ovarian cancer by total alcohol consumption in the year before baseline, at ages 30–35 years, or at ages 18–22 years. Similarly, consumption of alcohol from beer or liquor during any of the three time periods was not significantly associated with risk of ovarian cancer. In contrast, intake of alcohol from wine during the year before baseline was associated with statistically significantly elevated risk of ovarian cancer. After adjusting for alcohol intake from beer and liquor, as well as ovarian cancer risk factors, women who drank at least 11.1 grams per day of alcohol from wine—the equivalent of one glass per day—at baseline were at 57% higher risk of ovarian cancer, compared to women who did not drink wine (Ptrend = 0.01). Intake of alcohol from wine at ages 30–35 years or ages 18–22 years was not significantly associated with ovarian cancer risk, although the estimated RRs were consistent with those for baseline wine intake. Table 1Selected baseline characteristics of the California Teachers Study (CTS) cohort included in the present analysis (n= 90,371)Characteristicn(%)Age at baseline (years) <3510,456(11.6%) 35–4418,546(20.5%) 45–5428,275(31.3%) 55–6416,229(18.0%) 65–7411,496(12.7%) 75–845,369(5.9%)Race/ethnicity White78,468(86.8%) Non-white11,266(12.5%) Unknown637(0.7%)Parity (full-term pregnancies) None23,810(26.3%) 1–243,674(48.3%) ≥321,412(23.7%) Unknown1,475(1.6%)Oral contraceptive use (years) None26,643(29.5%) <527,608(30.5%) ≥532,463(35.9%) Unknown3,657(4.0%)Lifetime strenuous physical activity (average hours/week) <0.525,254(27.9%) 0.5–3.949,467(54.7%) ≥4.015,293(16.9%) Unknown357(0.4%)Menopausal status Pre-menopausal42,204(46.7%) Peri-menopausal2,202(2.4%) Post-menopausal39,744(44.0%) Unknown6,221(6.9%)Hormone therapy (HT) use (peri-/post-menopausal women only) None12,968(30.9%) Combination estrogen + progestin HT14,235(33.9%) Estrogen-only HT, ≤5 years4,342(10.4%) Estrogen-only HT, >5 years4,899(11.7%) Estrogen-only HT, unknown duration332(0.8%) Mixed combination and estrogen-only HT4,776(11.4%) Unknown394(0.9%)Region of residence within Californiaa Greater San Francisco Bay Area18,444(20.4%) Southern Coastal/Los Angeles Area35,388(39.2%) Other areas36,483(40.4%) Unknown56(0.1%)Type of residence Rural12,677(14.0%) Town3,136(3.5%) Small city16,135(17.9%) Metropolitan suburban48,278(53.4%) Metropolitan urban9,038(10.0%) Unknown1,107(1.2%)Statewide percentile of socioeconomic status in census block groupa ≤49th18,962(21.0%) 50–59th10,199(11.3%) 60–69th12,620(14.0%) 70–79th14,508(16.1%) 80–89th16,864(18.7%) 90–99th16,076(17.8%) Unknown1,142(1.3%)Smoking history Never60,868(67.4%) Former24,989(27.7%) Current4,428(4.9%) Unknown86(0.1%)Alcohol drinking in the year prior to baseline None31,024(34.3%) Beer only2,222(2.5%) Wine only19,365(21.4%) Liquor only2,868(3.2%) Beer and liquor only1,005(1.1%) Wine and beer/liquor33,887(37.5%)a See Methods for definitionTable 2Relative risks (RRs) and 95% confidence intervals (CIs) for associations between intake of specific types of alcohol at various ages and risk of ovarian cancerAlcohol typeTime periodDaily intake (g/day)Median (g/day)Cases (n)RRa95% CIaRRb95% CIbTotal alcoholYear before baselineNone0.0771.00(reference)—<10.04.5811.04(0.76, 1.42)—10.0– < 20.011.8721.47(1.06, 2.03)—≥20.028.2231.15(0.71, 1.84)—Ptrend = 0.19Ages 30–35 yearscNone0.0671.00(reference)—<10.07.31011.14(0.83, 1.56)—10.0– < 20.011.8471.08(0.74, 1.59)—≥20.029.7160.99(0.56, 1.71)—Ptrend = 0.99Ages 18–22 yearsdNone0.01311.00(reference)—<10.04.5620.76(0.55, 1.03)—10.0– < 20.011.8361.26(0.86, 1.84)—≥20.028.891.00(0.50, 1.99)—Ptrend = 0.63BeerYear before baselineNone0.01991.00(reference)1.00(reference)<13.24.0510.96(0.70, 1.31)0.89(0.64, 1.24)≥13.213.230.58(0.19, 1.84)0.54(0.17, 1.70)Ptrend = 0.40Ptrend = 0.22Ages 30–35 yearscNone0.01801.00(reference)1.00(reference)<13.24.0450.81(0.58, 1.13)0.75(0.53, 1.06)≥13.213.260.72(0.32, 1.64)0.73(0.32, 1.69)Ptrend = 0.20Ptrend = 0.16Ages 18–22 yearsdNone0.01791.00(reference)1.00(reference)<13.24.0460.95(0.68, 1.32)0.93(0.65, 1.33)≥13.213.2131.21(0.68, 2.16)1.30(0.70, 2.39)Ptrend = 0.67Ptrend = 0.57WineYear before baselineNone0.0911.00(reference)1.00(reference)<11.13.3991.08(0.81, 1.43)1.09(0.80, 1.50)≥11.111.1631.50(1.08, 2.09)1.57(1.11, 2.22)Ptrend = 0.01ePtrend = 0.01eAges 30–35 yearscNone0.0901.00(reference)1.00(reference)<11.13.31121.19(0.90, 1.59)1.26(0.92, 1.71)≥11.111.1291.21(0.78, 1.86)1.38(0.87, 2.19)Ptrend = 0.36Ptrend = 0.16Ages 18–22 yearsdNone0.01671.00(reference)1.00(reference)<11.13.3631.11(0.82, 1.50)1.17(0.84, 1.63)≥11.111.181.42(0.69, 2.91)1.63(0.76, 3.50)Ptrend = 0.28Ptrend = 0.15LiquorYear before baselineNone0.01691.00(reference)1.00(reference)<15.04.5681.09(0.82, 1.45)1.06(0.78, 1.44)≥15.015.0160.87(0.52, 1.47)0.82(0.48, 1.39)Ptrend = 0.80Ptrend = 0.56Ages 30–35 yearscNone0.01241.00(reference)1.00(reference)<15.04.5931.07(0.81, 1.41)1.04(0.77, 1.40)≥15.015.0140.77(0.44, 1.35)0.75(0.42, 1.36)Ptrend = 0.52Ptrend = 0.44Ages 18–22 yearsdNone0.01641.00(reference)1.00(reference)<15.04.5690.99(0.74, 1.32)0.94(0.68, 1.29)≥15.015.050.62(0.25, 1.52)0.49(0.19, 1.26)Ptrend = 0.39Ptrend = 0.17a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, and menopausal status/hormone therapy use; stratified by age at baselineb Additionally adjusted for consumption of other alcohol types in the same time periodc Among women over age 35 years at baseline with non-missing data on alcohol consumption at ages 30–35 yearsd Among women over age 22 years at baseline with non-missing data on alcohol consumption at ages 18–22 yearsep for non-linearity of trend > 0.05 Intake of specific alcohol types, beyond total alcohol, was associated with ovarian cancer risk, as assessed by comparing a multivariate model with beer, wine, and alcohol consumption in the year before baseline to a model with alcohol consumption alone (p = 0.05, 2 d.f.). Furthermore, the association of wine consumption with ovarian cancer risk differed from that of beer or liquor consumption, as assessed by comparing a multivariate model with beer/liquor and wine consumption to a model with alcohol consumption (p = 0.02, 1 d.f.). Controlling for total alcohol intake did not attenuate the positive association between wine intake at any time period and risk of ovarian cancer (data not shown). Women who drank wine only in the year before baseline (median daily alcohol intake = 11.1 grams), relative to non-drinkers, had an ovarian cancer RR of 1.40 (95% CI 1.01–1.93). In contrast, risk did not vary between non-drinkers and women who drank beer or liquor only (median daily intake of alcohol = 4.5 grams; RR = 1.03 [95% CI 0.58–1.83]) or beer/liquor and wine (median daily alcohol intake = 11.8 grams; RR = 1.08 [95% CI 0.79–1.48]). The multivariate RR among women who exclusively drank at least one glass of wine per day, compared to non-drinkers, was 1.70 (95% CI 1.10–2.62). After simultaneously adjusting for wine drinking during the year before baseline and at ages 18–22 years and 30–35 years, the RR associated with drinking at least one daily glass of wine at baseline was 1.33 (95% CI 0.91–1.96); at ages 30–35 years, RR = 1.01 (95% CI 0.61–1.68); and at ages 18–22 years, RR = 1.28 (95% CI 0.58–2.87). Women who drank at least one glass per day of wine both in an earlier time period (18–22 years or 30–35 years) and at baseline were at significantly higher risk of ovarian cancer, compared to wine non-drinkers (Table 3). Results were similar when based on intake at ages 18–22 years and 30–35 years (data not shown). Women who were heavy wine drinkers in all three time periods (n= 5 cases) had over four times the risk of ovarian cancer relative to women who never drank wine in any time period (RR = 4.60 [95% CI = 1.76–12.01]), adjusting for beer and liquor drinking patterns. Marginally elevated risk of ovarian cancer was also observed among women who increased or decreased their wine intake between ages 18–22 years and either baseline or ages 30–35 years (latter data not shown). No patterns of total alcohol, beer, or liquor intake were significantly associated with ovarian cancer risk. Furthermore, ovarian cancer risk did not vary according to number of drinking days per week at baseline, whether for total alcohol, beer, wine, or liquor, although the risk of ovarian cancer rose slightly with increasing frequency of wine consumption at baseline (Ptrend = 0.11). Table 3Relative risks (RRs) and 95% confidence intervals (CIs) for associations between patterns of drinking specific types of alcohol and risk of ovarian cancerAlcohol typeTime periodDrinking patternCases (n)RRa95% CIaRRb95% CIbTotal alcoholAge 30–35 years and the year before baselinecNever491.00(reference)—Steady moderate1181.25(0.89, 1.76)—Decreasing291.06(0.67, 1.68)—Increasing271.10(0.68, 1.77)—Steady heavy81.32(0.62, 2.82)—Age 18–22 years and the year before baselinedNever551.00(reference)—Steady moderate701.07(0.74, 1.53)—Decreasing241.06(0.65, 1.72)—Increasing861.30(0.92, 1.84)—Steady heavy31.87(0.58, 6.04)—Year before baseline0 days/week771.00(reference)—1 to 4 days/week981.25(0.92, 1.69)—5 to 7 days/week, ≤ 20 g/day301.36(0.88, 2.08)—5 to 7 days/week, > 20 g/day181.14(0.68, 1.93)—BeerAge 30–35 years and the year before baselinecNever1631.00(reference)1.00(reference)Steady moderate260.80(0.53, 1.21)0.73(0.47, 1.13)Decreasing250.94(0.62, 1.44)0.88(0.57, 1.38)Increasing171.08(0.65, 1.79)0.99(0.59, 1.66)Steady heavy0——Age 18–22 years and the year before baselinedNever1591.00(reference)1.00(reference)Steady moderate241.06(0.68, 1.64)1.02(0.64, 1.62)Decreasing340.95(0.65, 1.38)0.92(0.61, 1.37)Increasing210.77(0.49, 1.22)0.71(0.44, 1.14)Steady heavy0——Year before baselinee0 days/week1991.00(reference)1.00(reference)1 to 4 days/week400.98(0.70, 1.39)0.79(0.53, 1.16)5 to 7 days.week10.33(0.05, 2.37)0.36(0.05, 2.59)Ptrend = 0.30Ptrend = 0.14WineAge 30–35 years and the year before baselinecNever601.00(reference)1.00(reference)Steady moderate651.15(0.80, 1.64)1.19(0.80, 1.77)Decreasing341.25(0.82, 1.92)1.32(0.82, 2.11)Increasing531.25(0.86, 1.82)1.29(0.86, 1.93)Steady heavy191.54(0.91, 2.62)1.75(1.00, 3.04)Age 18–22 years and the year before baselinedNever711.00(reference)1.00(reference)Steady moderate290.98(0.63, 1.52)1.01(0.62, 1.63)Decreasing201.48(0.89, 2.45)1.73(1.00, 2.99)Increasing1131.29(0.95, 1.74)1.38(0.99, 1.92)Steady heavy52.47(0.99, 6.19)2.76(1.09, 7.00)Year before baselinee0 days/week911.00(reference)1.00(reference)1 to 4 days/week971.19(0.89, 1.60)1.23(0.89, 1.71)5 to 7 days.week371.34(0.91, 1.98)1.45(0.96, 2.20)Ptrend = 0.18Ptrend = 0.11LiquorAge 30–35 years and the year before baselinecNever1101.00(reference)1.00(reference)Steady moderate471.13(0.80, 1.60)1.12(0.76, 1.63)Decreasing501.04(0.74, 1.46)0.97(0.67, 1.41)Increasing211.11(0.69, 1.77)1.04(0.63, 1.70)Steady heavy30.49(0.15, 1.54)0.46(0.15, 1.49)Age 18–22 years and the year before baselinedNever1261.00(reference)1.00(reference)Steady moderate321.12(0.76, 1.66)1.10(0.71, 1.69)Decreasing350.84(0.57, 1.23)0.76(0.50, 1.15)Increasing440.98(0.69, 1.38)0.92(0.64, 1.33)Steady heavy10.80(0.11, 5.77)0.69(0.10, 5.04)Year before baselinee0 days/week1691.00(reference)1.00(reference)1 to 4 days/week571.13(0.83, 1.53)1.10(0.78, 1.55)5 to 7 days.week131.00(0.57, 1.78)0.73(0.35, 1.51)Ptrend = 0.92Ptrend = 0.44a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, and menopausal status/hormone therapy use; stratified by age at baselineb Additionally adjusted for drinking patterns of other alcohol types across the same time periodsc Among women over age 35 years at baseline with non-missing data on alcohol consumption at ages 30–35 yearsd Among women over age 22 years at baseline with non-missing data on alcohol consumption at ages 18–22 yearse Excluding women with missing data on number of drinking days per week The lack of an association with total alcohol, beer, or liquor intake, as well as the persistence of the association between wine intake and risk of ovarian cancer after adjustment for alcohol consumption, suggested that determinants of wine drinking, or ingredients of wine other than alcohol, were responsible for the observed positive association. After mutual adjustment, older age (up to ages 65–69 years), White race, higher total caloric intake, nulliparity, ever-use of oral contraceptives, more physical activity, ever-use of HT, higher SES or median family income, residence in the Greater San Francisco Bay Area, lower body mass index, ever-smoking of cigarettes, and higher intake of coffee and/or tea were significantly associated with drinking at least one daily glass of wine, compared to none (data not shown). However, the positive association between wine consumption and risk of ovarian cancer remained statistically significant even after adjustment for these characteristics (data not shown). To further explore the relationship between wine consumption and ovarian cancer risk, we stratified the association by various potential effect modifiers, including demographic characteristics and ovarian cancer risk factors (Table 4). There was no statistically significant heterogeneity in the association between intake of alcohol from wine at baseline and risk of ovarian cancer by any of the factors examined. Among peri-/post-menopausal women, we observed no association with wine intake among those who had never used HT, nor among those who used combined estrogen-progestin HT, adjusting for duration of HT use. In contrast, women who used unopposed estrogen HT were at twice the risk of ovarian cancer if they drank one glass of wine per day at baseline, compared to wine non-drinkers. The association was especially strong among women who used estrogen-only HT for over 5 years (RR = 2.39 [95% CI 0.97–5.89], Ptrend = 0.02), whereas there was no such association among women who used combination HT for over 5 years (RR = 1.34 [95% CI 0.51–3.54], Ptrend = 0.29). We also found that the women in the highest quartile of SES statewide were at double the risk of ovarian cancer if they drank at least a glass per day of wine, compared to none, whereas there was no such association among women in the lower three quartiles of SES. Table 4Stratified relative risks (RRs) and 95% confidence intervals (CIs) for associations between wine intake in the year before baseline and risk of ovarian cancer within participant subgroupsCharacteristic (at baseline)Alcohol consumption from winePtrendPheterogeneity between subgroupsNone < 11.1 g/day≥ 11.1 g/dayCasesRRaCasesRRa(95% CI)aCasesRRa(95% CI)aAge≤Median (50 years)231.00271.07(0.58, 1.99)121.43(0.67, 3.04)0.34>Median681.00721.10(0.76, 1.57)511.62(1.09, 2.39)0.010.95ParityNulliparous181.00261.34(0.69, 2.62)131.56(0.71, 3.40)0.31Parous711.00731.05(0.73, 1.50)481.57(1.06, 2.34)0.020.61Oral contraceptive useNever451.00391.00(0.62, 1.61)291.70(1.02, 2.82)0.03Ever221.00140.76(0.37, 1.58)141.78(0.85, 3.72)0.090.54Lifetime strenuous physical activity≤Median (1.4 hours/week)611.00581.07(0.72, 1.59)401.68(1.09, 2.59)0.01>Median (1.4 hours/week)301.00411.11(0.66, 1.86)231.39(0.77, 2.50)0.260.55Menopausal statusPre-menopausal211.00200.83(0.42, 1.65)101.24(0.55, 2.83)0.53Peri-/Post-menopausal661.00721.16(0.80, 1.66)511.72(1.16, 2.55)0.010.86Hormone therapy (HT) useNoneb211.00221.27(0.64, 2.51)91.20(0.51, 2.78)0.73Combination estrogen + progestin HTb231.00180.69(0.35, 1.37)161.17(0.58, 2.34)0.45Estrogen-only HTb161.00191.27(0.62, 2.61)152.03(0.95, 4.35)0.060.37Region of residenceGreater Bay/Southern Coastal regions451.00621.20(0.79, 1.84)381.69(1.06, 2.71)0.02Other California regions461.00370.94(0.59, 1.52)251.46(0.87, 2.48)0.120.41Type of residenceRural/town/small city351.00341.23(0.74, 2.06)231.77(1.01, 3.11)0.05Metropolitan suburban/urban541.00651.04(0.70, 1.55)401.51(0.97, 2.37)0.050.77Statewide percentile of socioeconomic status in census block groupcLower 75% 571.00501.09(0.72, 1.67)241.35(0.81, 2.27)0.25Upper 25% 321.00491.16(0.72, 1.88)391.96(1.19, 3.24)0.0040.43Body mass index≤Median (23.5 kg/m2)331.00461.31(0.81, 2.11)321.64(0.97, 2.76)0.07>Median531.00520.97(0.63, 1.49)271.48(0.89, 2.45)0.100.69Cigarette smoking statusNever661.00611.05(0.71, 1.56)361.77(1.13, 2.78)0.01Ever251.00381.19(0.69, 2.03)271.42(0.80, 2.50)0.240.57Dietary folate intake≤Median (307.1 μg/day)d231.00210.73(0.37, 1.43)131.15(0.54, 2.44)0.55>Mediand211.00231.16(0.60, 2.24)101.17(0.51, 2.66)0.750.74Total folate intake≤Median (473.0 μg/day)e431.00461.07(0.68, 1.70)251.34(0.78, 2.30)0.27>Mediane411.00481.20(0.77, 1.89)372.07(1.29, 3.35)0.0020.43a Adjusted for race, total energy intake, parity, oral contraceptive use, strenuous exercise, menopausal status/hormone therapy use, and consumption of beer and liquor in the past year; stratified by age at baselineb Including peri-/post-menopausal women onlyc See Methods for definitiond Excluding all multivitamin users (i.e., women consuming supplemental folate)e Excluding short-term multivitamin users (i.e., women consuming supplemental folate for < 2 years) In secondary analyses, we examined the associations between alcohol consumption and risk of ovarian cancer among only women who were peri- or post-menopausal at baseline (46% of the study population, 75% of cases). In this group, we observed the same lack of a significant association with total alcohol, beer, or liquor intake, along with a significant positive association with baseline wine intake. Likewise, when we restricted the case population to invasive ovarian cancer (90% of cases) or to serous ovarian cancer (45% of cases), baseline intake of alcohol from wine, but not from other sources, was associated with significantly increased risk of ovarian cancer. Discussion The lack of association between overall alcohol consumption and risk of ovarian cancer in our study is consistent with most previous studies [3–18], including a pooled analysis of 10 prospective cohorts [19]. Alcohol consumption during ages 18–22 years, ages 30–35 years, or the year before baseline—whether from beer, liquor, or all sources combined—was unrelated to ovarian cancer development. Additionally, changes in beer, liquor, or total alcohol consumption over time, as well as frequency of drinking in the year before baseline, did not affect ovarian cancer risk in this cohort. In contrast, average consumption of at least one glass per day of wine in the year before baseline was associated with elevated risk of ovarian cancer, while wine intake at ages 30–35 years or 18–22 years was associated with nonsignificantly increased risk. The positive association with baseline wine intake persisted after adjustment for total alcohol intake, suggesting that the apparent effect of wine on ovarian cancer risk was independent of alcohol content. The association was also unchanged by further adjustment for characteristics and behaviors associated with wine drinking in this cohort, and was not significantly modified by reproductive characteristics, demographic factors, or folate intake. However, there was a significant positive association between wine consumption and ovarian cancer risk among peri-/post-menopausal women who used unopposed estrogen HT, whereas there was no such association among peri-/post-menopausal women who did not use HT or used combined estrogen-progestin HT. Wine consumption was also associated with increased ovarian cancer risk among women of high SES, but not among women of relatively low SES. To our knowledge, no other study of ovarian cancer has examined alcohol consumption at various ages or drinking patterns over time. A positive association between wine consumption and risk of ovarian cancer has previously been detected in two case–control studies [15, 49], although both of those studies reported a positive association with total alcohol intake as well. In contrast, two case–control studies [14, 24] and one cohort study (among women with high dietary folate intake) [17] found an inverse association between wine consumption and ovarian cancer risk. However, there was no association with wine (or total alcohol) consumption in other studies [13, 18, 19]. Few previous studies have examined modification of the association between alcohol consumption and risk of ovarian cancer. A pooled analysis of 529,638 women, including 2,001 incident ovarian cancer cases, found no interaction between alcohol intake and oral contraceptive or HT use, parity, menopausal status, folate intake, BMI, or smoking [19]. In contrast, a case–control study found that an inverse association between wine consumption and ovarian cancer risk was significantly stronger in women who were more highly educated, had never smoked, or had used oral contraceptives—characteristics that describe the majority of CTS cohort members—but found no heterogeneity by menopausal status, BMI, or HT use [24]. Unlike us, others reported an interaction with folate intake [16, 17, 25] or heterogeneity by histologic subtype of ovarian cancer [14, 15], although others did not [3, 12, 19, 24]. We lacked sufficient cases to perform detailed analyses of ovarian cancer histologic subtypes other than the most common serous type. The restriction of the positive association between wine intake and ovarian cancer risk to peri-/post-menopausal women taking estrogen-only HT suggests a biological mechanism involving estrogen. Likewise, the restriction of the association to women of high SES may reflect reproductive characteristics and/or HT use favoring increased estrogen levels in higher-SES women. A high background level of circulating estrogen, compounded by an upsurge of estrogen resulting from alcohol consumption [1], may promote ovarian carcinogenesis. Furthermore, the lack of an interaction between wine intake and use of estrogen-progestin HT could be explained by a protective effect of progestin against ovarian cancer. Phytochemicals, such as resveratrol, in red wine have been considered as promising cancer preventive agents due to their anti-estrogenic, antioxidant, anti-proliferative, and other anti-carcinogenic effects [50, 51]. However, such phytochemicals have multifarious effects, including pro-estrogenic activity and possible genotoxicity [52–54]; thus, both the potentially beneficial and potentially harmful effects of phytochemicals on cancer development must be considered together. In the present analysis, we were not able to distinguish red from white wine intake. Our findings should be interpreted in light of some limitations. It is possible that the observed positive association between wine consumption and ovarian cancer risk was due to confounding by unmeasured characteristics beyond those for which we attempted to adjust, or by residual confounding or chance. Our efforts to fully evaluate effect modifiers or confounders of the association between wine consumption and ovarian cancer risk were partly hampered by the lack of heterogeneity in some characteristics among CTS participants. The proportion of women who drank beer or liquor was low, although the range of total alcohol intake was adequate to reveal a significant positive association with risk of breast cancer in an earlier analysis [37]. Even though reporting of alcohol intake in the past year using our questionnaire has been validated, we were not able to assess the reliability or validity of self-reported alcohol consumption at earlier ages, which may be difficult to recall, especially among older women. Misclassification of distant past alcohol intake may explain the absence of a significant association between wine drinking at earlier ages and ovarian cancer risk. Nevertheless, it is unlikely that any exposure misclassification differed systematically between ovarian cancer cases and non-cases, since all information was assessed prospectively. In summary, given the lack of association between overall alcohol consumption and ovarian cancer risk in our study, further investigations are necessary to determine whether ingredients of wine, but not beer or liquor, foster ovarian cancer development; whether correlates of wine drinking not measured or imperfectly measured in our study population are associated with ovarian cancer risk; or if an association is largely due to the apparent interaction between wine consumption and unopposed estrogen HT use and/or other characteristics of women of high SES. If the observed association between wine drinking and ovarian cancer risk is due to confounding, then there may exist an as-yet unidentified ovarian cancer risk factor that is also associated with wine drinking. On the other hand, if the interactions with estrogen HT use and SES are confirmed, there may be a biological basis for the increased risk of ovarian cancer among wine drinkers with high endogenous and exogenous estrogen levels. However, if alcohol consumption is indeed unrelated to ovarian cancer development but positively associated with the risk of breast cancer, then differences in the hormonal and non-hormonal triggers between these two malignancies may help us understand the carcinogenic effects of alcohol on hormonally responsive tissue. Further understanding of the complex relationships among steroid hormone levels, metabolism of alcohol and wine, and carcinogenesis will help clarify what role, if any, alcohol and wine play in the development of ovarian cancer.

J_Urban_Health-2-2-1705540

Methods to Recruit Hard-to-Reach Groups: Comparing Two Chain Referral Sampling Methods of Recruiting Injecting Drug Users Across Nine Studies in Russia and Estonia

Evidence suggests rapid diffusion of injecting drug use and associated outbreaks of HIV among injecting drug users (IDUs) in the Russian Federation and Eastern Europe. There remains a need for research among non-treatment and community-recruited samples of IDUs to better estimate the dynamics of HIV transmission and to improve treatment and health services access. We compare two sampling methodologies “respondent-driven sampling” (RDS) and chain referral sampling using “indigenous field workers” (IFS) to investigate the relative effectiveness of RDS to reach more marginal and hard-to-reach groups and perhaps to include those with the riskiest behaviour around HIV transmission. We evaluate the relative efficiency of RDS to recruit a lower cost sample in comparison to IFS. We also provide a theoretical comparison of the two approaches. We draw upon nine community-recruited surveys of IDUs undertaken in the Russian Federation and Estonia between 2001 and 2005 that used either IFS or RDS. Sampling effects on the demographic composition and injecting risk behaviours of the samples generated are compared using multivariate analysis. Our findings suggest that RDS does not appear to recruit more marginalised sections of the IDU community nor those engaging in riskier injecting behaviours in comparison with IFS. RDS appears to have practical advantages over IFS in the implementation of fieldwork in terms of greater recruitment efficiency and safety of field workers, but at a greater cost. Further research is needed to assess how the practicalities of implementing RDS in the field compromises the requirements mandated by the theoretical guidelines of RDS for adjusting the sample estimates to obtain estimates of the wider IDU population. Introduction Evidence suggests recent diffusion of injecting drug use and associated HIV infection in the Russian Federation since 1996.1,2 Approximately 60% of HIV case reports have been associated with injecting drug use,1,3 with recent estimates indicative of increased sexual HIV transmission.4 According to UNAIDS classifications, HIV in much of the Russian Federation and former Soviet Union is a concentrated epidemic, with prevalence consistently above 5% in a single risk group (i.e., IDUs) but less than 1% in the general population.5 Concentrated epidemics require targeted surveillance of the population group most at risk in order to track the spread within that group as well as potential transmission to others. Surveillance among IDUs is problematic, and there has been much discussion on the merits of different methods to recruit marginalized and hidden groups for these purposes.6–8 We know that surveying drug users in treatment settings misses an important segment of the drug using population. Evidence suggests that behaviours, characteristics and HIV prevalence amongst IDUs in treatment often systematically differ to IDUs not in treatment.9–13 Many surveillance studies of IDUs conducted in the 1990s relied on non-probability sampling such as convenience, snowball sampling or chain referral sampling to recruit members of the target group.12,14 These methods work on the assumption that peers are better able to recruit members of a hidden population than researchers.15 Typically studies employed ‘privileged access interviewers’ or ‘indigenous field workers’ to recruit IDUs from community settings. Indigenous field workers are interviewers who are either current or former drug users or individuals who have experience working with drug users and have privileged access to IDU networks. Over the last 15 years, this has become the established sampling method for recruiting hidden populations of IDUs and sex workers both in the UK and internationally.16–21 A refinement of the chain referral methodology called respondent-driven sampling (RDS), has recently been developed.22 RDS is inspired by the insight of “small world theory” that suggests that every person is indirectly associated with every other person through approximately six intermediaries,23 and therefore that everyone in a defined population could be potentially reached through several waves of recruitment in a chain-referral sample.24 This implies that there is a probability greater than zero that everyone in that population will be sampled. The unique selling point of RDS is that the collection of data on participants’ social networks allow for adjustment for non-random recruitment. RDS uses social network data to make inferences about the wider target population from which the sample is drawn to provide proportional population estimates of characteristics and behaviours.24,25 In this paper, we do not attempt to test the statistical superiority of RDS in providing ‘population’ estimates over other sampling strategies but instead focus on RDS as a recruitment strategy examining the unadjusted RDS sample characteristics. This paper compares two sampling methodologies, RDS and chain referral sampling using indigenous field workers (IFS), in terms of cost effectiveness, duration of fieldwork and effects on the demographic composition of the sample. First we offer a theoretical descriptive comparison of the two approaches. Theoretical Comparison of the Sampling Methods Indigenous Field Worker Sampling The IFS recruitment method uses a standard chain referral approach. Indigenous field workers undergo training covering aims of the study, fieldwork protocols, ethics, informed consent, interview skills and safety procedures. Field workers (FWs) identify individuals known to them from IDU networks, recruit; and then interview them in community settings, separate from the rest of the research team. Eligible participants are given an incentive to take part and also asked to introduce their peers to the FW. The use of multiple site and network recruitment ensures a wide coverage of the population, providing as representative a sample as possible. There is some evidence that the use of FWs with direct access to IDU social networks facilitates recruitment and reduces masking (undersampling reclusive respondents), volunteer bias (oversampling cooperative respondents) and underreporting of socially undesirable behaviours.10,26 Respondent-driven Sampling In RDS, a fixed site or “store front” is established where all interviewing takes place, providing the research team with greater control over the fieldwork. Unlike IFS and other chain referral samples, RDS uses a dual incentive system, a primary incentive for participating in the study and a secondary incentive for recruiting others into the study.22 Sampling begins with a set of initial subjects who serve as ‘seeds’ for an expanding chain of referrals, with respondents from each link in the chain or ‘wave’ referring respondents who form subsequent waves. Rather than being asked to identify their peers to interviewers, respondents inform their peers about the study and allow them to decide independently whether they want to participate or not. This theoretically reduces masking since recruiters are part of the target group with direct access to other IDUs, and it reduces volunteer bias since recruitees decide themselves whether to participate. Information on the relationships between recruiters and recruited and their estimated network size is collected during the interview to allow for the calculation of selection probabilities.27 This information is used to assess homophily, the extent to which recruiters are likely to recruit individuals similar to themselves, and to weight the sample to compensate or control for differences in network size, homophily and recruitment success.24 Materials and Methods Data Collection Between 2001 and 2005, we undertook nine community surveys of IDUs in the Russian Federation and Estonia (Table 1). Four studies used IFS to recruit IDUs, and five used RDS. All IDUs were recruited from community settings. Seven of the studies had an epidemiological focus and measured the prevalence of HIV, HCV and associated injecting and sexual risk behaviours in IDUs.13,28,29 Two of the studies collected data on the social and economic characteristics of IDUs.30 All studies collected some standardised indicators and defined current IDUs as individuals who injected drugs for non-medical purposes in the last 4 weeks. Table 1.Research amongst hard to reach populations recruited via respondent-driven sampling (RDS) and indigenous field workers (IFS) in Estonia and the Russian Federation, 2001–2005 PlacePopulation sizeDates of field work (days)Average number of interviews conducted per daySample sizeAim of studyRecruitment methodReference1Togliatti,Russia740,6361/10/2001–18/10/2001 (18)23426HIV prevalence and risk behaviourIFS292Moscow, Russia13,251,40127/09/2003–21/10/2003 (27)18514HIV prevalence and risk behaviourIFS133Volgograd, Russia∼1,012,00026/09/2003–23/10/2003 (21)21597HIV prevalence and risk behaviourIFS134Barnaul, Russia∼750,00026/09/2003–30/10/2003 (21)24512HIV prevalence and risk behaviourIFS135Volgograd, Russia∼1,012,00019/8/2004–7/9/2004 (20)20400Social and economic studyRDS306Barnaul, Russia ∼750,00026/8/2004–16/9/2004 (22)18400Social and economic studyRDS307Togliatti, Russia740,63621/05/2004–09/06/2004 (20)24472HIV prevalence and risk behaviourRDS348Tallinn, Estonia 396,37502/05/2005 (34)10350HIV prevalence and risk behaviourRDS289Kohtla Jarve, Estonia46,34602/05/2005 (16)6100HIV prevalence and risk behaviourRDS28 For each of the IFS studies, IDUs were recruited using a team of 10–12 FWs at each site. Settings included street locations and respondents’ homes but excluded drug treatment centres and STI clinics. Volunteers and outreach workers at local harm reduction non-governmental organizations (NGOs) were employed as FWs, as well as two researchers at a local university in each site. In all IFS studies, two experienced supervisors from Moscow and a researcher from the UK provided technical expertise and management for all studies. Measures to ensure data quality and to minimise network bias included limiting the number of interviews per FW, random spot-checks in the field, and follow-up validation interviews with 10% of participants. Primary incentives included HIV prevention materials (including needles/syringes), chocolates and cigarettes. In each RDS study, recruitment was undertaken by a team of seven to eight FWs at each site. The interview team comprised trained research staff from a local university, two FWs recruited from local harm reduction NGOs, two to three trained research staff from a local university, and two supervisors from Moscow. A researcher from the UK was also present at the studies, with the exception of the two socio-economic studies in Volgograd and Barnaul, Russia. In each study, a pre-fieldwork focus group was held with outreach workers from the local harm reduction NGOs to obtain information about the drug scene and to identify seeds to begin recruitment. Respondents received the same primary incentives for participating in the RDS study as in the IFS study and also an additional secondary incentive for each respondent they recruited into the study.22,24 In all studies FWs recorded their observations on the drug scene, progress of the fieldwork and any difficulties arising from the research in detailed notes. These observations provide a useful additional comparison between the two sampling methods. Duration and Cost of Fieldwork We compared the duration of fieldwork for the IFS and RDS methods by calculating the mean number of days of fieldwork for each method and the proportion of the sample recruited on each day as the studies progressed. Means were compared using t-tests. Costs were estimated for five of the seven surveys conducted in Russia and analysis focused on examining the cost effectiveness of recruiting a given sample for each of the sampling methodologies from a programmatic point of view as opposed to examining societal or health system costs. The IFS studies in Moscow, Barnaul and Volgograd were conducted in 2003 and the RDS studies in Togliatti, Barnaul and Volgograd were conducted in 2004. Costs were calculated as: (1) ‘outside’ costs including salary, accommodation and travel of field work consultants; (2) local salary costs of FWs and researchers; (3) recruitment costs including the packages of goods valued at 140 roubles and 300 roubles, respectively, for primary and secondary incentives; and (4) other costs including local transport, telephone calls and logistical costs of training FWs. For the RDS study the cost of the fixed site used for interviews is not included as an explicit cost, rather it is subsumed into the local salary costs since local staff contracted to undertake the work were employed from syringe exchange programmes. Costs are presented assuming that there are elements of fixed and variable costs at each sample size and that an extra 20 respondents will require keeping the entire survey team in the field for one extra day. Demographic and Injecting Risk Behaviours of Sample Demographic and injecting risk behaviours of IDUs recruited through IFS and RDS were compared in the two sites (Volgograd and Barnaul) where both survey methodologies were used to ensure a cleaner comparison between survey methods. Demographic and injecting characteristics were used as the outcome variables with recruitment method included as an independent variable. In the univariate analysis, chi-squared tests were used to compare outcomes for categorical variables and Bartlett’s test for equal variance to compare continuous variables. For the multivariate analysis, logistic regression models were used to explore associations between explanatory variables and a binary outcome, multinomial logit models were used for categorical variables with multiple values and ordinary least squares for continuous variables. The multivariate analysis includes all common independent variables and a categorical variable indicating survey method used. This allows outcomes to be compared controlling for all independent variables and to identify impacts associated with only survey methodology. All statistical analyses were conducted using Stata 7 with significance set at 5% (Stata Corporation, College Station, Texas). Results The Surveys A total of 3,771 IDUs were recruited into nine surveys across four cities in the Russian Federation and two cities in Estonia (Table 1). A total of 2,049 (54%) participants were recruited through IFS and 1,722 (46%) through RDS. Only IDUs are included in the analyses we present here. Duration of Field Work The mean (standard deviation) duration of fieldwork for IFS surveys was 23.8 (4.1) days and for RDS 20.6 (0.9) days (t  =  27.9, p < 0.001). Figure 1 depicts the number of respondents recruited by each successive day of fieldwork by city and recruitment method. The RDS studies appear to follow a pattern of recruitment that we might expect: the number of respondents increases steadily as the number of waves increase and then declines towards the end of the study as completion of the target sample size approaches and respondents are asked to refer fewer contacts to the study. Kohtla Jarve, Estonia, appears to be an exception to this as recruitment peaks more sharply then abruptly finishes. The recruitment for the IFS studies does not appear to follow any set pattern across the cities. In Moscow the highest number of respondents in any 1 day occurs at the start of the study. In Barnaul, and to a less extent Volgograd, the number of participants recruited per day is more even across the duration of the study. Figure 1Frequency of recruitment per day amongst studies of injecting drug users in Russia and Estonia (2001–2005), by city and recruitment method. Sample Characteristics Table 2 summarizes the characteristics of IDUs by recruitment method from the four surveys conducted in Volgograd and Barnaul. In both cities, RDS participants were younger, more likely to be male, to have attended higher education and to have official residency permits for the city. RDS participants were more likely to report injecting heroin in both sites and less likely to report injecting vint or mak1 than IFS participants. Frequency of injecting did not differ by recruitment method in either city. Regarding injecting risk behaviour, there was no difference between recruitment methods in the proportion of IDUs reporting injecting with a used needle/syringe in the last 4 weeks in Volgograd but in Barnaul a higher proportion of RDS respondents reported this behaviour (21 vs. 15%, p  =  0.02). In Volgograd IFS respondents were more likely to report ever having injected with a used needle/syringe than RDS respondents (61 vs. 40%, p  <  0.001). The opposite was found in Barnaul (53 vs. 63%, respectively, p  <  0.003). In both cities and with both methods, the main source of new needles/syringes was pharmacies. A higher proportion of IFS respondents reported using needle/syringe exchanges or treatment centres in both cities and in Barnaul a higher proportion of RDS respondents reported using a source other than needle/syringe exchange (defined as friend, dealer, family or on the street) as their main source of needles/syringes. Table 2.Comparison of characteristics and injecting risk behaviours among injecting drug users in Volgograd and Barnaul (2003–2004) by recruitment method CharacteristicBarnaulVolgogradIFRDSaIFRDSan/total%/mean (SD)n/total%/mean (SD)χ2P valuen/total%/mean (SD)n/total%/mean (SD)χ2P valueTotal† age (years)25.8 (8.2)24.8 (6.1)2.20.0324.5 (3.9)23.8 (5.4)2.30.022Male348/50469%293/36580%13.8<0.001388/51276%317/34692%35.3<0.001Attended higher education 67/50413%136/35339%73.1<0.001140/50928%150/33445%27.1<0.001Duration of injection (years)7.1 (6.9)6.7 (4.3)1.10.295.4 (3.4)6.1 (4.3)−2.60.009No official residency permit34/4957%7/3562%11.20.00119/4714%8/3382%1.90.174Injecting risk behaviours in the last 4 weeksMain drug injected  Heroin280/50456%263/35774%424/50883%328/34595%  Vint135/50427%38/35711%37/5087%7/3452%  Mak84/50417%51/35714%21/5084%6/3452%  Other5/5041%5/3571%39<0.00126/5085%4/3451%27<0.001 Daily injection 100/50020%69/35619%0.050.82386/51217%63/34118%0.40.53 Injected with used needles/syringes 75/50015%75/35321%5.60.01863/51012%37/33611%0.350.55Main source of new needles/syringes  Pharmacy466/49894%325/36589%463/50891%327/34196%  Treatment21/4984%12/3653%33/5086%6/3412%  Other**11/4982%28/3658%14.90.00112/5082%8/3412%10.50.005 Ever injected with used needles/syringes255/48153%225/35663%8.70.003301/49361%137/34540%37.1<0.001aThe RDS data are not adjusted for homophily and network effects.†Numbers do not always add up to total because not all respondents answered each question.**Other refers to friends, family, found on the street, drug dealers. Multivariate Analysis Table 3 summarizes the multivariate analysis for the categorical and continuous variables for Barnaul and Volgograd. After controlling for all independent variables, our findings indicate that RDS recruited a population 0.07 years younger in both cities. In both cities RDS participants had been injecting slightly longer than IFS participants. In Barnaul, RDS participants were less likely to report obtaining their new needles/syringes from pharmacies (−5%) but there was no evidence to suggest a difference in Volgograd. In Volgograd RDS participants were less likely to report using needle/syringe exchanges (−3%) but there was no difference in Barnaul. RDS participants in Barnaul were 4% more likely to report using another source for their new needles/syringes (Table 3). Table 3.Effect of recruitment method on demographic indicators and injecting risk behaviours amongst injecting drug users in Barnaul and Volgograd (2003–2004)OutcomesRDSaType of analysisBarnaulVolgogradCoefficient95% CIStd. err.Coefficient95% CIStd. err.log AGE−0.07−0.1–(−0.04)0.01−0.07−.0.9–(−0.05)0.01Ordinary least squaresYears of injecting0.1 0.04–0.20.040.20.1–0.20.04Negative binomial regressionIncrease in probability of using pharmacies−5%−8–(−1%)0.023%0.1%–5%)0.1Multinomial logitIncrease in probability of using needle/syringes exchanges0.5%−1–3%0.01−3%−5%–(−0.2%)0.01Multinomial logitIncrease in probability of using another source for needles/syringes4%1–7%0.01n/an/an/aMultinomial logitaThe RDS data are not adjusted for homophily and network effects.n/a = not applicable. In the logistic regression analysis (Table 4), RDS was more likely to result in a higher proportion of male IDUs (Barnaul OR  =  2.0, Volgograd OR  =  3.8) and participants who had attended higher education (Barnaul OR  =  5.2, Volgograd OR  =  3.0). In Barnaul RDS participants were more likely to have official residency permits (OR  =  4.6) but not in Volgograd. In both cities, RDS participants were more likely to inject heroin over mak or vint than IFS participants (Barnaul OR  =  2.5 and Volgograd OR  =  3.4). In Volgograd RDS participants had almost twice the odds of reporting daily injection (OR  =  1.7) than IFS participants and had reduced odds of ever injecting with used needles/syringes (OR  =  0.3). In Barnaul, RDS participants were more likely to report injecting with used needles/syringes in the last 4 weeks and ever (OR  =  1.6 and OR  =  1.4, respectively). Table 4.Effect of recruitment method on demographic indicators and injecting risk behaviours amongst injecting drug users in Barnaul and Volgograd (2003–2004)OutcomesRDSaType of analysisBarnaulVolgogradOdds ratio95% CIStd. err.Odds ratio95% CIStd. err.Male2.01.3–3.10.43.82.1–6.91.2Logistic regressionAttended higher education5.23.5–7.61.03.02.1–4.30.5Logistic regressionOfficial residency permit 4.61.8–12.12.31.40.5–4.30.8Logistic regression Injected heroin as primary drug in the last 4 weeks versus mak/vint2.51.7–3.50.43.41.7–7.01.2Logistic regressionDaily injected0.80.5–1.20.21.71.04–2.60.4Logistic regression Injected with used needles/syringes in the last 4 weeks1.61.0–2.10.41.00.6–1.80.3Logistic regression Ever injected with used needles/syringes1.41.0–1.90.20.30.2–0.50.1Logistic regression Sold sex in the last 4 weeks1.40.6–3.00.51.80.6–5.41.0Logistic regressionaThe RDS data are not adjusted homophily and network effects. Costs The total cost of conducting an IFS survey recruiting 400 respondents was estimated to be $14,651 (USD) but $16,100 for the RDS survey (Table 5). This translates to $43 per respondent using RDS and $37 using the IFS method. Increasing the sample from 400 to 500 reduced the average cost per respondent by $1 for the RDS method and by $3 for the IFS method. Reducing the sample from 400 to 300 respondents increased the cost per respondent by $2 for the RDS method and $5 for the IFS method. These results are presented in Table 5. Table 5.Analysis of costs of recruiting IDUs and sex workers from five surveys in Moscow, Volgograd and Barnaul, Russian Federation (2003–2004) by recruitment method and sample size Recruitment methodIndigenous field workersRespondent-driven samplingSample size300400500300400500Outside costsa−15.40$20.3011.40−0.18$19.620.18Local costsb−13.20$7.9310.40−0.25$5.500.25Recruitment of respondentsc−33.30$7.0020.00−0.25$9.950.25Other0.00$1.400.00−0.25$5.250.25Total−17.20$14,651.0012.60−0.21$16,1000.21Cost per respondent$42.00$37.00$34.00$43.00$41.00$40.00At the time of writing 28 rubles was equivalent to one US Dollar. The 2003 costs are adjusted for inflation that occurred between August 2003 and 2004 based on price indices taken from the Bank of Russia.aCosts are presented assuming that there are elements of fixed and variable costs in each sample size and that an extra 20 respondents will take one extra day necessitating employing the entire fieldwork team for that extra day.bIn order to protect the confidentiality of staff we report only the total amount of all salaries and fees paid to project staff.cThe costs for the RDS surveys are also based on providing a package of goods valued at 140 rubles for each survey participant and a secondary reward of 300 rubles for each participant recruited. Costs for the IFS surveys are based on providing a package of goods valued 140 rubles for each participant and no secondary reward. Discussion Our findings suggest that RDS does appear to be a faster recruitment method and that there are significant differences in the demographic characteristics of IDUs recruited via RDS in comparison with those recruited via IFS. However, evidence from the two cities is conflicting with regard to whether RDS recruits IDUs who engage in riskier injecting practices. One of the suggested benefits of RDS is its apparent ability to recruit the hardest to reach sections of hidden populations.22 We found some differences in measures of marginalization and risk behaviours between the two recruitment methods, RDS participants tend to be slightly younger and are less likely to use needle/syringe exchange programs. Some evidence in Russia suggests that IDUs whose primary source of needles/syringes is informal are at more risk of engaging in high-risk behaviours.31 However, IFS participants were less likely to have attended higher education and have official residency permits to live in the city and more likely to be female. Lack of residency permit is an indicator of marginalization as it will affect an individual’s ability to use health services or obtain employment.32,33 As no consistent trend emerges from the analysis of the effect of recruitment method on sample characteristics, then choice of method might be made on the basis of methodology and cost. Inclusion Criteria and Data Validity With IFS the responsibility for selecting the right target group is placed with the FWs, and its success depends on establishing a trusting relationship between the researchers and the fieldwork team. With RDS, issues of trust are less important, as researchers undertake the interviews themselves. The problems of establishing whether respondents are genuine members of the target group remain. Although measures can be put in place which might reduce this from happening, such as using indigenous field workers to screen participants or recording biometric measurements to avoid the same respondent being interviewed twice, it is very difficult to measure to what extent fabricated data may enter a survey. A disadvantage of both methods is that study participants who are not members of the target group may lie about their membership in order to receive a reward. This was the case in two of the IFS sites where 9 and 14% of questionnaires were subsequently found to be fakes. This was discovered because strict validation processes had been set up and there was a good relationship between the FW supervisor and indigenous field workers. In the Togliatti RDS study 15 people were refused entry into the study, as they were suspected of not being current injectors. However these may be considered a minimum estimate as one cannot rule out that additional fabrication might have occurred and gone undetected. Having a modest primary and secondary incentive can minimize the chances that participants who are not members of the target population will be recruited. Determining the best incentive size is difficult and has many implications for the study, especially for RDS studies where the secondary incentive is so crucial to recruitment success. The networks recruited through RDS are largely artificial, created as a result of the study and since their composition is dependent on the incentive, changes to the amount of incentive offered would change the composition of the network. This is illustrated with the case of sex workers in Eastern Europe, who have been found to be harder to recruit through RDS in part due to the small incentive and social network properties; this is discussed in more detail in a paper in this issue by Simic et al. Adjusting the RDS sample to obtain ‘population’ estimates depends on the ability to recruit a random population within a subject’s social networks and a positive probability of recruiting everyone in that network. The possibility that the network is highly dependant on the incentive raises the question whether the latter condition obtains. This is particularly relevant when the definition of the population of study is fluid or artificially constructed by the research as with IDUs and sex workers. It should also be noted that the collection of information describing network characteristics which allows RDS analysis to produce ‘population’ estimates requires the respondent to recall detailed information on the composition of their network, including its size and each member’s relationship with the recruiter. This process carries a large potential for error. Personal Safety and Capacity Building There are safety considerations that favour RDS as respondents attend a fixed site for an interview in which a minimum number of staff is always present. In the IFS method, interviewers may find themselves travelling to an area in which they are unfamiliar, and unintentionally put themselves in danger, especially if it becomes known that they are carrying financial rewards or gift packs. Limitations Whilst we have tried to limit confounding in our analysis by comparing RDS and IFS studies conducted in the same cities, the studies were conducted in different years, and the findings may be confounded by time. Time may be important in relation to behaviour, but is likely to be less important in relation to socio demographic characteristics of the target group. None of the studies were set up specifically with the aim of comparing sampling methodologies, and this limited the number of characteristics that could be compared between study methodologies. A study set up specifically with the aim of comparing the methodologies might produce different results and facilitate more detailed comparison of characteristics. Additionally the starting point for both the IFS and RDS studies in all sites was the local outreach team. This may have led to more similarities in the sample composition than would have occurred if seeds had been selected through other methods. However, according to the principles of RDS, the selection of seeds does not ultimately influence the composition of the sample, since after several waves of recruitment the sample should be independent of the non-randomly selected seeds.24 Conclusion The HIV epidemic is driven by populations engaging in high-risk behaviours mixing with those engaging in lower risk behaviours. It is important to identify the parameters of risk behaviour in order to model these epidemics and to design appropriate interventions. If we assume that, after adjusting for network sizes and homophily, RDS is more successful at estimating risk behaviours across a more representative population than IFS, then it could lead to more effective modelling and prediction of such epidemics; however to date there is no evidence to suggest that this is the case. Our findings indicate that as a recruitment strategy, RDS is no better than IFS in identifying populations with highest risk behaviours. It does have practical advantages in terms of safety of the FW team, with faster recruitment at only additional costs. In the meantime, until the statistical superiority of RDS can be proven, a preferred approach may be to adopt the best aspects of both methodologies, depending on the resources available. A combination could include the use of coupons for recruitment, but also training indigenous field workers to work alongside researchers to undertake interviews, serving to increase their capacity in research skills whilst ensuring that the correct target group is being reached.

Skeletal_Radiol-3-1-2042032

Are radiographs needed when MR imaging is performed for non-acute knee symptoms in patients younger than 45 years of age?

Objective The objective was to determine the value of radiographs in young adults with non-acute knee symptoms who are scheduled for magnetic resonance imaging (MRI). Introduction Radiographs in patients with acute knee problems that are obtained at, or close to, the time of trauma serve mainly to exclude fractures and most of these radiographs are normal. To reduce the total number, and especially the number of normal radiographs, clinical decision rules that identify patients with a high risk of fracture have been developed and validated [2–4]. In patients with non-acute knee symptoms radiographs are the mandatory minimal initial exam in patients with non-traumatic knee pain, according to ACR appropriateness criteria [5]. Magnetic resonance imaging (MRI) is frequently obtained if additional imaging is clinically needed to search for suspected intra-articular abnormalities. The value of radiographs relative to MRI in a heterogeneous group of patients was recently reported [6]. Radiographs were rated “essential” or “very important” in 46–58% of cases and added “not needed information” in 14–29% of cases. However, the value of radiographs relative to MRI in young adults presenting with non-acute knee symptoms has, to our knowledge, not been established. If in these patients radiographs could be omitted, or obtained only when needed when reading MRI, this could possibly lead to a significant reduction of the volume of knee radiographs with ensuing reduction in radiation dose and costs. The purpose of our prospective study was to determine the value of radiographs in young patients with non-acute knee symptoms and suspected intra-articular pathology who are scheduled for MRI based on the results of a physical exam. We therefore determined the prevalence of osseous abnormalities visible on radiographs and MRI and the yield of MRI compared with that of radiographs. In addition, we measured the radiation dose of standard radiographs of the knee. Material and methods Over 3 years, 961 patients referred from general practitioners to the outpatient surgery or orthopedic surgery clinics of three hospitals (two general hospitals and one university hospital) because of non-acute knee symptoms agreed to participate in our prospective study. The symptoms had to have been present for at least 4 weeks. The institutional review board of each hospital approved the study. We obtained written informed consent from all patients. At study entry a standardized history, including inventory of traumatic events and other knee-related events such as morbidity and medical procedures was taken. Physical exam was performed by one of 15 (orthopedic) surgeons or by residents under their supervision. All the patients included underwent MRI and radiographs of the knee. Recent radiographs taken for the same complaints that were obtained prior to study inclusion were not repeated. We included patients who met the following inclusion criteria: knee symptoms (pain, swelling of the joint, feeling of instability or giving way, history of locking) of at least 4 weeks’ duration and age between 16 and 45 years. To obtain a homogenous study population without co-morbidity we did not include patients who had had previous surgery of the affected knee (including arthroscopy) or previous MRI, a history of rheumatoid arthritis, and clinical diagnosis of retropatellar chondromalacia, contra-indication for MRI, or an incomplete data set. One hundred and sixty-three patients (17.0%) were eventually excluded (Table 1), leaving 798 patients as our study group. Of these excluded patients a substantial number should not have been included by the orthopedic surgeons in the first place according to our study protocol, for instance, because they were either too young, too old or had knee complaints of less than 4 weeks’ duration. Table 1Excluded patients. Of the 961 patients referred, 163 (17.0%) were excludedExclusion criterianSymptoms of less than 4 weeks’ duration65Aged under 16 years1Aged over 45 years6Previous surgery8Retropatellar chondromalacia14Previous MRI1Radiographs unavailable or not obtained65MRI or arthroscopy prohibited2Immediate arthroscopy required30Criteria are not mutually exclusive. One out of six musculoskeletal radiologists (all with at least 5 years’ experience) evaluated the radiographs without knowledge of the MR images and recorded their findings on a Case Record Form (CRF). The radiation dose of standard antero-posterior and lateral radiographs was determined in two of the three hospitals using flat ionization chambers. We used PCXMC software (STUK, Finland) to convert the measured radiation doses into effective doses. The radiologist evaluated the radiographs for articular surface lesions, using the classification developed by Bohndorf [7]. Osteoarthritis was scored using the scoring system developed by Kellgren et al. [1]. In the analysis, grades 1 to 3 were considered as one group, with osteoarthritis being present. Radiographs were also evaluated for fractures [8–10], loose bodies, and incidental findings. A loose body was defined as a radiodense circumscriptive lesion located in the femorotibial, patellofemoral jointline or suprapatellar pouch. Incidental findings, such as non-ossifying fibromas and enchondromas were diagnosed using established criteria [11, 12]. On radiographs a lesion was considered a non-ossifying fibroma if it met the following criteria: asymptomatic, cortical metaphyseal location, and radiographically non-aggressive. A lesion was considered an enchondroma if it met the following criteria: central metaphyseal location, chondroid matrix, geographic pattern of destruction with or without sclerotic margin. We performed the MR studies in all three hospitals on an identical 0.5-T system (Gyroscan T5, Philips Medical Systems, Best, the Netherlands), with the same software release using a dedicated transmit–receive knee coil. The standardized scanning protocol consisted of three sequences: a sagittal and a coronal dual spin-echo (SE) sequence and a sagittal T1-weighted 3D gradient-echo (GE) sequence with frequency selective fat suppression. The following parameters were identical for both SE sequences: field of view 140–160 mm, echo time (TE) 20/80 ms. The coronal dual SE had a repetition time (TR) of 2,100 ms, a 256 × 205 matrix and a slice thickness of 5 mm with a 0.5-mm interslice gap. The sagittal dual SE had a TR of 2,350 ms, a 256 × 179 matrix and a slice thickness of 4 mm with a 0.4-mm interslice gap. The parameters for the sagittal frequency selective fat-suppressed T1-weighted 3D GE were: TR 70 ms, TE 13 ms, 45° flip angle, field of view 160 mm, 256 × 205 matrix, 4 mm slice thickness with a 2-mm overlap. The total imaging time of the standard protocol (including the initial survey sequence) was 26 min. After reading the radiographs one out of six radiologists evaluated MR images and reported his findings on a CRF. In accordance with usual management the observer had access to the radiographs, but not to the reports, when evaluating MR images. The CRF was similar to the one used for radiographs [8–12], except that bone marrow edema was added as an item [13]. For articular surface lesions we again used the classification developed by Bohndorf [7]. For osteoarthritis we used the scoring system developed by Recht et al. [14]. For the purpose of this study, grades 1 to 4 were considered as one group, with osteoarthritis being present. To overcome bias in the reading of MR studies, introduced by knowledge of the results of conventional radiographs, one radiologist (JLB) re-evaluated MR studies of patients with abnormalities on conventional radiographs with the exception of osteoarthritis of Kellgren [1] grades 1–2. We used the same CRF format. At this second evaluation, MR studies of patients with osseous abnormalities on conventional radiographs were mixed with age- and gender-matched MR studies of patients without abnormalities on conventional radiographs. The radiographs and the findings on the CRF were not available at that time. We divided the completed CRFs over two groups: group A had no history of trauma, and group B had a history of trauma of more than 4 weeks before presentation at the hospital. We used the Chi-squared test to identify significant differences between the yield of radiographic and MR diagnoses, and between the number of findings in group A and that in group B. To look for statistically significant differences between the yield of radiographs and MRI within groups A, B and the study population as a whole, we used McNemar’s Chi-squared test. Results The median age of the 798 patients included was 31 years (range 16–45 years), and 261 patients (32.7%) were female. Minimal and median duration of symptoms were 4 and 20 weeks respectively (range 4–1,490 weeks). Three hundred and thirty-two patients reported no history of trauma (group A), and 466 patients reported an old (more than 4 weeks prior to consultation) history of trauma (group B). Magnetic resonance imaging depicted arthroscopically treatable intra-articular pathology in 341 patients (42.7%). Medial and lateral meniscal tears were found in 225 (28.2%) and 111 patients (13.9%) respectively. The average radiation dose of supine antero-posterior and lateral radiographs was 0.2 and 0.3 mSv respectively. The median duration of the interval between radiographs and MRI was 8 days (95 percentile, 85 days). In 196 patients (24.6%) radiographs were obtained prior to inclusion in the study. These radiographs were not repeated since the knee complaints had not changed between the moment they were obtained and inclusion in this study. In group A, radiographs showed 36 osseous abnormalities in 332 patients (10.8%, Table 2). All these abnormalities, except 13 Kellgren [1] grade 1 osteoarthritis cases, were also depicted on MRI. There were 23 concordant radiographic and MRI diagnoses (Fig. 3; Table 2). One incidental finding (enchondroma) was demonstrated on radiographs and MRI. The majority of the 72 diagnoses made only with MRI were osteoarthritis and bone bruising (Table 2). Most osseous lesions (osteoarthritis, articular surface lesions, loose bodies and bone marrow edema) with the exception of incidental findings (one case) and old fractures (not present in this group) were significantly more often diagnosed with MRI than with radiographs (Table 2). Table 2Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 332 patients with non-acute non-traumatic knee complaintsOsseous abnormalityR (%)R and MR (%)MRI (%)Total (%)McNemar test, p valueOA13 (3.9)18 (5.4)39 (11.7)70 (21.1)0.00ASL0 (0)3 (0.9)7 (2.1)10 (3.0)0.02LB0 (0)1 (0.3)6 (1.8)7 (2.1)0.03Fractures0 (0)0 (0)0 (0)0 (0)NAIncidental finding0 (0)1 (0.3)0 (0.0)1 (0.3)1.00Bone marrow edema0 (0)0 (0)20 (6.0)20 (6.0)NA Total13 (3.9)23 (6.9)72 (21.7)108 (32.5)0.00OA: osteoarthritis grade 1 or higher according to Kellgren [1] on radiographs and according to Recht [14] on MRI ; ASL: articular surface lesion with or without disrupted cartilage; LB: loose body; R: abnormality detected with radiographs only; R and MR: abnormality detected with both radiograph and MRI; MRI: abnormality detected with MRI only; NA: not applicable. Categories are not mutually exclusive. In group B, radiographs showed 40 osseous abnormalities in 466 patients (8.6%, Table 3). All these abnormalities, except 15 Kellgren [1] grade 1 osteoarthritis cases, were also depicted on MRI. There were 25 concordant radiographic and MRI diagnoses (Figs. 1, 4, 5 and 6; Table 3). Four of the six incidental findings were diagnosed on radiographs and MRI (1 non-ossifying fibroma and 3 enchondromas, Fig. 7). Two enchondromas were depicted on MRI only (Fig. 8). The majority of the 192 diagnoses made only with MRI were bone marrow edema (Fig. 2), osteoarthritis and articular surface lesions (Table 3). These three diagnoses were significantly more often diagnosed with MRI than with radiographs (Table 3). There was no significant difference between MRI and radiographs in the diagnosis of loose bodies, old fractures and incidental findings. Table 3Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 466 patients with non-acute traumatic knee complaintsOsseous abnormalityR (%)R and MR (%)MRI (%)Total (%)McNemar test, p valueOA15 (3.2)13 (2.8)65 (13.9)93 (20.0)0.00ASL0 (0)3 (0.6)22 (4.7)25 (5.4)0.00LB0 (0)1 (0.2)4 (0.9)5 (1.1)0.13Fractures0 (0)4 (0.9)3 (0.6)7 (1.5)0.25Incidental finding0 (0)4 (0.9)2 (0.4)6 (1.3)0.5Bone marrow edema0 (0)0 (0)96 (20.6)96 (20.6)NA Total15 (3.2)25 (5.4)192 (41.2)232 (49.8)0.00Categories are not mutually exclusive.Fig. 1Severe osteoarthritis on both radiographs and MRI. a, b Lateral and antero-posterior radiographs showing deformation of the femoral condyles and tibia plateau with marked subchondral cyst formation and osteophyte formation in a 44-year-old female patient with a history of knee trauma (group B). c–e Coronal proton density and T2-weighted spin-echo (SE) images and of the same lesion showing, in addition to the deformities and changes already noted on the radiographs, the osteochondral defect to a better extent. e Sagittal T2-weighted SE image showing the same features, but also focal full-thickness cartilage lossFig. 2Three-month-old transchondral fracture on both radiographs and MRI. a, b Antero-posterior and lateral radiograph showing irregularity and deformation of the lateral femoral condyle consistent with a transchondral fracture of the lateral femoral condyle in a 40-year-old male with a history of knee trauma (group B). c–d Sagittal T2-weighted SE images and T1-weighted 3D gradient-echo (GE) with fat suppression showing subtle contour deformity of the lateral femoral condyle with kissing bone bruises in the lateral femoral condyle and tibial plateau Osseous abnormalities were more frequently encountered in group B than in group A (p < 0.001). This difference is explained by a significant difference in the number of old fractures (p < 0.05, no old fractures were seen in group A), and patients with bone marrow edema (p < 0.001). Bone marrow edema was demonstrated with MRI in 116 patients (14.5%, Table 4); 96 of these were found in group B (82.8% of patients with bone marrow edema, Table 3). Table 4Osseous abnormalities detected on radiographs only, on radiographs and MRI, and on MRI only in 798 patients with non-acute knee complaintsOsseous abnormalityR (%)R and MRI (%)MRI (%)Total (%)McNemar test, p valueOA28 (3.5)31 (3.9)104 (13.0)163 (20.4)0.00ASL0 (0)6 (0.8)29 (3.6)35 (4.4)0.00LB0 (0)2 0.3)10 (1.3)12 (1.5)0.00Fractures0 (0)4 (0.53 (0.4)7 (0.9)0.25Incidental finding0 (0)5(0.6)2 (0.3)7 (0.9)0.5Bone marrow edema0 (0)0 (0)96 (12.0)96 (12.0)NATotal28 (3.5)48 (6.0)264 (33.1)340 (42.6)0.00Categories are not mutually exclusive. In 6 (30%) of the 20 group A patients with bone marrow edema, it was either associated with intra-articular damage of non-osseous origin or it was an isolated finding. The associated osseous abnormalities in the other 14 patients were as follows: bone marrow edema was found in 4 of the 10 patients with articular surface lesions, in 1 of the 7 patients with a loose body, and in 9 of the 70 patients with osteoarthritis. In 89 (92.7%) of the 96 group B patients with bone marrow edema, it was either associated with intra-articular damage of non-osseous origin or it was an isolated finding. The associated osseous abnormalities in the other 7 patients were as follows: bone marrow edema was found in 4 of the 25 patients with articular surface lesions (Fig. 2), in 1 of the 5 patients with a loose body and in 2 of the 93 patients with osteoarthritis. In our population we did not find any other osseous lesions like osteopenia or femoropatellar disease. Also, we did not find chondrocalcinosis on radiographs. Subsequently, all patients with radiographically detected abnormalities with the exception of Kellgren [1] grade 1 and 2 osteoarthritis were identified. MRI studies of the 17 patients with 18 radiographically detected osseous abnormalities were mixed with 17 gender- and age-matched MRI studies of patients without abnormalities on radiographs. There were 6 females in each group, and the mean age in each group was 33.1 years, SD 7.9 years). At revision, the reader (JLB) was blinded to all information and had no access to the radiographs. All 18 osseous abnormalities (1 caae of osteoarthritis Kellgren grade 3 [Fig. 1], 6 articular surface lesions [Figs. 2, 3], 2 loose bodies [Fig. 4], 4 old healed fractures [Figs. 5, 6] and 5 incidental findings [4 enchondromas and 1 non-ossifying fibroma, Figs. 7, 8]) were all diagnosed on MRI. Fig. 3Twenty-six-month-old transchondral fracture on both radiographs and MRI. a Antero-posterior radiograph showing abnormal density, subtle irregularity and deformation of the medial femoral condyle in a 35-year-old male patient with no history of knee trauma (group A). b–d Sagittal T1-weighted 3D GE with fat suppression and coronal proton density and T2-weighted SE images of this knee showing the transchondral fractureFig. 4Loose body on both radiography and MRI. a Lateral radiograph showing a ventrally located loose body in the left femorotibial joint in an 18-year-old male professional skater with a history of knee trauma (group B). b–c Sagittal T1-weighted 3D GE with fat suppression and coronal proton density SE images of the same patient, also showing the loose body that is ventrally located in the lateral compartment of the femorotibial joint. At subsequent arthroscopy this loose body was removedFig. 5Eight-month-old healed fracture on both radiograph and MRI. a Lateral radiograph showing a healed fracture of the inferior patellar pole in an 18-year-old male patient with a history of knee trauma (group B). b Sagittal proton density SE image of the same patient, also delineating the healed fracture of the inferior pole of the patellaFig. 6Two-month-old healing fracture on both radiograph and MRI. a Lateral radiograph showing a healing fracture of the left inferior patellar pole in a 40-year-old male patient with a history of knee trauma (group B). b, c Sagittal T1-weighted 3D GE with fat suppression and T2-weighted SE images of the same patient, also delineating the healing fracture of the inferior pole of the patella without cartilaginous irregularities. Around the fracture line bone marrow edema can be appreciatedFig. 7Incidental finding depicted on both radiographs and MRI. a, b Lateral and antero-posterior radiographs showing a central, metaphyseally located lesion with chondroid matrix consistent with enchondroma in the femur in a 44-year-old female patient with a history of knee trauma (group B). c, d Sagittal proton density and T2-weighted SE images of the same lesionFig. 8Incidental finding depicted on MRI only. a Lateral radiograph showing no femoral osseous lesion in a 40-year-old female patient with a history of knee trauma (group B). b, c Sagittal proton density and T2-weighted SE images showing a metaphyseal lesion consistent with an enchondroma Discussion Key characteristics of our population such as age, gender, clinical suspicion of intra-articular abnormalities, intra-articular abnormalities detected with MRI, and history suggest that our conclusions can be applied to typical populations scheduled for MRI to analyze non-acute knee problems. The yield of radiographs in patients with subacute or chronic knee symptoms who are otherwise healthy and who are scheduled for MRI based on the results of a physical exam suggesting the presence of intra-articular damage is only 9.5% (76 patients). The majority of these patients (77.6%) have osteoarthritis. We failed to confirm with MRI the radiographic diagnosis of Kellgren [1] grade 1 osteoarthritis in 28 patients, but none of the other abnormalities were diagnosed only with radiographs. This was confirmed in a second reading of MRI blinded to clinical and radiographic information. Radiographs do not, in the setting of this study, contribute to clinical decision-making or clinical management of the patient. Although the dose administered when obtaining radiographs of the knee is small, the size of the population with knee complaints make the reduction of an administered dose a relevant issue. Radiographs can therefore be omitted in this specific population when the decision to perform MRI is taken based on clinical assessment. The disadvantage of occasionally missing Kellgren [1] grade 1 osteoarthritis is limited when we realize that because of the lack of a gold standard these radiographic diagnoses may also be false-positive. Taljanovic et al. [6] rated radiographs as “essential” or “very important” in 46–58% of cases and radiographs added “not needed information” in 14–29% of cases. They conclude in their study that radiographs are an important, and sometimes essential initial, complementary study for reading musculoskeletal MR examinations. This apparently different conclusion can be explained by the differences in study populations. Taljanovic et al. [6] analyzed a large variety of diagnosis (trauma, infection, tumors, degenerative or miscellaneous) in all anatomical locations in patients of all ages. We only analyzed patients between 16 and 45 years of age with non-acute knee symptoms lasting at least 4 weeks, excluding amongst others patients with acute knee symptoms (i.e., less than 4 weeks), history of rheumatoid arthritis, and clinical diagnosis of retropatellar chondromalacia. Other major methodological differences are mainly related to this difference. The concordance between radiographs and MRI is not high because of the large number of diagnoses (77.6%) made only with MRI. In groups A and B osteoarthritis, articular surface lesions, and bone marrow edema were significantly more often diagnosed with MRI than with radiographs. Loose bodies were significantly more frequently diagnosed with MRI in group A only. This higher yield of MRI is no surprise, since abnormalities such as articular surface lesions and bone marrow edema are known to be better, or even exclusively appreciated on MRI. These findings have, in addition to the intra-articular abnormalities detected with MRI, potential clinical significance in view of the patients’ presenting symptoms. The advantage and comprehensiveness of MRI is illustrated by its ability to demonstrate bone marrow edema that is associated with other abnormalities. In the patients with a history of old trauma, bone marrow edema was mainly (92.7%) associated with intra-articular damage. In the group with no history of trauma, however, bone marrow edema was mainly (70%) seen in association with osseous abnormalities, thus facilitating the diagnosis of articular surface lesions and osteoarthritis. This prospective study has several limitations that are mainly related to the decision to follow usual care. This means that radiographs were available at the time of MRI. To overcome this limitation, one radiologist (JLB) re-evaluated MR studies of patients with abnormalities on conventional radiographs without knowledge of the radiographs, as described. Another limitation is that we could not determine accuracy because of the absence of a reference standard. If we look at the discordant cases, there are no potentially false-negative MRI diagnoses (with the exception of Kellgren [1] grade 1 osteoarthritis), but the number of potential false-positive MRI diagnoses (abnormal finding on MRI in combination with a normal radiograph) is substantial, mainly because of the high prevalence of bone marrow edema. Another possible limitation of this study is the selection bias introduced by including only patients between 16 and 45 years of age and excluding patients with co-morbidity. The study design was not tailored toward assessment of the value of conventional radiographs only. The overall goal of this multicenter study was to evaluate the efficacy of MRI compared with direct arthroscopy in patients with subacute knee complaints, based on clinical outcome. To this end we tried to obtain a homogenous study population without co-morbidity. This is most likely the reason we encountered only 1 patient with high-grade osteoarthritis (Kellgren [1] grade 3). However, because the majority of MR examinations performed in our institutions will be of patients in this age range without known co-morbidity, we do not think this selection bias will alter our overall conclusions. We tried to mimic usual care as much as possible in this study. However, in our study MRI was performed within 2 weeks of inclusion in the study. Due to the limited availability of MRI compared with conventional radiographs in most hospitals, MRI often cannot be performed at such short notice. This can lead to a considerable waiting time for MR examinations and this may direct physicians to prior conventional radiographs to rule out gross pathology and reassure the patient. To prevent these unnecessary radiographs, the waiting time for MRI must be as short as possible. In the Netherlands waiting times for MRI in most institutions are down from several months a couple of years ago to several weeks nowadays. We conclude that radiographs should not be obtained routinely when MRI is being performed in non-acute young patients with knee complaints because the number of clinically relevant lesions detected on radiographs is low. In addition, the small number of abnormalities found on radiographs is, with the exception of an occasional case of Kellgren [1] grade 1 osteoarthritis, also depicted on MRI. Reduction of costs and radiation dose is small in the individual patient, but, because of its size, substantial in the entire population with subacute and chronic complaints suspected to be knee injury.

Int_Arch_Occup_Environ_Health-3-1-1915641

Work-related physical and psychosocial risk factors for sick leave in patients with neck or upper extremity complaints

Objectives To study work-related physical and psychosocial risk factors for sick leave among patients who have visited their general practitioner for neck or upper extremity complaints. Introduction Many people suffer from work-related neck and upper extremity complaints. In a population-based study in the Netherlands, the 12-month prevalence of work-related neck and upper extremity symptoms was found to be 31% (Blatter and Bongers 1999). These complaints have an important impact on loss of time from work. Sick leave has enormous cost implications, considering costs to employers, insurers, health care providers, and patients themselves. In the Netherlands, 8% of the employed population reported lost days from work in the past year because of complaints at the neck, shoulder, arm, elbow, wrist or hand (Blatter et al. 2001). The results of a British national survey in 1995 showed that musculoskeletal disorders of the neck and upper extremity were responsible for the loss of 4.2 million working days in a 12-month period (Jones et al. 1998). Borghouts et al. (1999) estimated the total number of sick days due to neck pain in 1996 in the Netherlands to be 1.4 million, with a total cost of US$ 185.4 million. It is well recognized that neck and upper extremity complaints have a multi-factorial aetiology. Several reviews have identified individual characteristics, and (work-related) physical and psychosocial factors as risk factors for the onset of neck and upper extremity complaints (Bernard 1997; Bongers et al. 2002a, b; Malchaire et al. 2001; Muggleton et al. 1999; van der Windt et al. 2000; Zakaria et al 2002). Furthermore, these factors have also been recognized as potential prognostic factors for chronic pain and disability in patients with neck or upper extremity complaints (Ariëns et al. 2000, 2001; Cole and Hudak 1996; Eriksen et al. 1999; Feuerstein et al. 2000). However, relatively little is known about the association of work-related factors with sick leave in patients with neck or upper extremity complaints. Risk factors for the onset of neck and upper extremity complaints may differ from risk factors for sick leave due to these complaints (IJzelenberg et al. 2004). Ekberg and Wildhagen (1996) studied the impact of physical workload, work organization, psychosocial conditions and individual characteristics on days of sick leave after rehabilitation in patients with neck and shoulder disorders. They found two work-related factors (i.e., work content and uncomfortable sitting) to be independently related to sick leave days. A prospective cohort study on work-related determinants of sick leave in employees without neck pain at baseline suggested that work-related neck flexion and rotation, sitting, decision authority and skill discretion were independently related to sick leave due to neck pain (Ariëns et al. 2002). In contrast to these findings, a recent cross-sectional study on risk factors for musculoskeletal sick leave showed that physical workload nor psychosocial workload were significantly related to sick leave due to neck or upper extremity pain (IJzelenberg et al. 2004). Hansson and Jensen (2004) reviewed the literature on the causes for back and neck disorders and found that heavy physical workload, bent or twisted working position and low work satisfaction increased the risk of short-term and long-term sick leave, and self-reported pain and functional impairments were associated with a high risk for long-term sick leave. Exposure to work-related physical risk factors may lead to pain and disability, which in turn may lead to sick leave. Psychosocial exposures at work may lead to psychological distress that may cause physiologic changes in the neck and upper extremity (e.g., increased muscle tension (Hagg and Astrom 1997; Lundberg 2002; Rissen et al. 2000), causing pain and disability, which in turn may lead to sick leave. Furthermore, psychosocial exposures at work may modify the effect of physical workload on sick leave. The objective of our study was to determine the influence of work-related physical and psychosocial risk factors on sick leave due to neck or upper extremity complaints in patients who have visited their general practitioner (GP) for these complaints. Methods Design and study population This study is part of a large observational cohort study on musculoskeletal disorders conducted in 61 general practices (97 GPs) (van der Waal et al. 2003). Part of the GPs participated in the second Dutch National Survey of General Practice (NS2), carried out by the Netherlands Institute for Health Services Research in co-operation with the National Information Network of General Practice in 2001 (Schellevis et al. 2003). The Medical Ethics Committee of the VU University Medical Center approved our study. Written informed consent was obtained from all patients. Details of the study design are described elsewhere (van der Waal et al 2003). Over a period of 1 year the GPs recruited 727 patients with a new complaint or new episode of a complaint at the neck, shoulder, elbow, arm, wrist or hand. An episode was considered to be ‘new’ if patients had not visited their GP for the same complaint during the preceding 3 months. Patients were eligible for participation if they were 18 years or older and capable of filling in Dutch questionnaires. Patients were excluded from the study if the presented symptoms were presumably caused by a fracture, malignancy, prosthesis, amputation or congenital defect or if the patient was pregnant. At baseline a self-report questionnaire was sent to collect data on a broad range of factors (including work-related factors) that may be predictive of sick leave. In total, 643 patients (88%) returned the baseline questionnaire, of whom five patients were excluded (two were too young; three were pregnant). For our study, patients who reported to have paid work for more than 8 h per week were included (8–16 h: n = 41; 17–24 h: n = 69; 25–36 h: n = 101; >36 h: n = 172). Outcome (sick leave due to neck and/or upper extremity complaints) was assessed after 3 months using a self-report questionnaire. Outcome measure Sick leave was measured at 3 months after baseline by the question: “how long were you absent from work because of your complaint since the baseline questionnaire”. Response categories were (1) no sick leave; (2) less than 1 week; (3) between 1 and 2 weeks; (4) between 2 weeks and 1 month; (5) between 1 and 3 months; (6) more than 3 months. Sick leave was dichotomised into no sick leave (response category 1) and sick leave (response categories 2–6). Determinants At baseline, work-related physical risk factors were assessed by a questionnaire consisting of two scales “heavy physical workload” [12 items; e.g., does your work involve (a) standing for long periods of time, (b) walking for long periods of time, (c) moving loads, (d) physical hard work] and “long-lasting postures and repetitive movements” [six items; e.g., does your work involve (a) making the same movement for long periods of time; (b) bending or twisting your neck often; (c) working in the same position for long periods of time; (d) doing repetitive tasks with arms, hands or fingers many times per minute) (Bot et al. 2004). Response options varied from “seldom or never”, “sometimes”, “often”, and “(almost) always”. Scoring was done by simply adding up the response to each item, which produced a raw score from 0 to 36 for the first subscale, and 0 to 18 for the second subscale. The final scores were calculated by dividing the raw score by the maximum score, multiplied by 100, resulting in a final score ranging between 0 (no workload) and 100 (highest workload) for both subscales. The internal consistency and validity of this questionnaire were considered to be good in a population with upper extremity and lower extremity musculoskeletal disorders (Bot et al. 2004). Furthermore, two questions about “prolonged sitting” and “ working with visual display units (VDU) for long periods of time” (yes/no) were asked. Work-related psychosocial risk factors were assessed according to the Demands–Control model of Karasek (1979). We used three subscales of the Job Content Questionnaire (JCQ): “job demands”, “decision authority” and “co-worker support” and one single item on “job security” (Karasek et al. 1998). The hypothesis of the Demands–Control model is that the most adverse reactions of psychological strain occur when demands are high and the worker’s decision latitude is low (Karasek et al. 1998). Therefore, we decided to combine the subscales high job demands and low decision authority into an additional determinant “job strain”. Both subscales were dichotomized by their median score and four categories were created: (1) high decision authority and low job demands (reference category); (2) high decision authority and high job demands; (3) low decision authority and low job demands; (4) low decision authority and high job demands. Statistical analysis Descriptive statistics (e.g., means and standard deviations) were used to present baseline characteristics of the study population. Differences between patients reporting sick leave and patients without sick leave were tested with the unpaired Student T-test (continuous variables) and the Chi square (χ2) test (categorical and dichotomous variables). In case a continuous variable was not normally distributed the Mann–Whitney test was used. Instead of using logistic regression analysis, Cox regression models with equal survival time were used to investigate the association between work-related risk factors (determinants) and sick leave (outcome). Hazard ratios are more easy to interpret and provide a better estimate of relative risks than odds ratios (Barros and Hirakata 2003; Lee and Chia 1994; Skov et al. 1998; Thompson et al. 1998) Associations were expressed as hazard ratios (HR) and corresponding 95% confidence intervals (95% CI) per unit increase of the risk factor involved. For continuous factors the linearity of the relation between the risk factor and sick leave was examined. Factors that were found to be non-linearly related to sick leave were either dichotomized or divided into tertiles with equally sized groups (Table 1). The “low-category” served as the reference category in all analyses. Table 1Potential confounders of the association between work-related risk factors and sick leave in patients with neck or upper extremity complaintsPotential confoundersCategorizationIndividual factors AgeContinuous (per year) SexMale versus female SmokingPresent versus previous smoker EducationPrimary versus secondary; college/university Pain copinga: retreating (seven items)Continuous (scale 7–28) Pain copinga: worrying (nine items)Continuous (scale 9–36) Distress (six items)Continuous (scale 1–9)General health ACSM position standcNorm not met versus norm met Norm healthy activityNorm not met versus norm met Perceived healthdContinuous (scale 1–5) Vitalityd (four items)Continuous (scale 0–100) Quality of lifeContinuous (scale 1–5)Characteristics of the complaint Kind of complaintLocalized versus generalized Duration of the complaintCategorical (<1, 1–4 weeks; 1–6 , >6 months) History of neck/upper limb complaintsHistory versus no history Musculoskeletal co-morbidityYes versus no Other co-morbidityYes versus noWork-related psychosocial factors (%) Decision authorityf (three items)Tertiles (low, medium, high) Job demandsf (five items)Tertiles (low, medium, high) Co-worker supportf (four items)Tertiles (low, medium, high) Job-securityfLow security versus high security Job as perceived cause of complaintYes versus noaPain Coping Inventory (Kraaimaat and Evers 2003; Kraaimaat et al. 1997)bFour Dimensional Symptom Questionnaire (Terluin 1998)cAmerican College of Sports Medicine Position Stand (American College of Sports Medicine Position Stand 1990)dMedical Outcomes Study 36-item Short Form Health Survey (Ware and Sherbourne 1992)ePhysical workload questionnaire (Bot et al. 2004)fJob Content Questionnaire (Karasek et al. 1998); %work-related psychosocial factors were considered as potential confounders in the relation between work-related physical factors and sick leave To prevent multicollinearity we checked whether the psychosocial risk factors, the physical risk factors, and potential confounders were highly correlated (P > 0.5 or P < −0.5). The factor with the highest association with sick leave was retained and the other factor was removed from further analyses. This was the case for prolonged sitting and prolonged VDU work (P = 0.69), distress and worrying (P = 0.52); prolonged VDU-work and heavy physical work (P = −0.56), and prolonged sitting and heavy physical work (P = −0.65). We performed the analyses in several stages. First, the unadjusted relationships between the work-related risk factors and sick leave were examined. Second, these relationships were adjusted for each of the potential confounders individually. Potential confounders of the relationship between work-related risk factors and sick leave included individual factors, general health, characteristics of the complaint, and other work-related risk factors (Table 1). Factors were considered as potential confounders if they had been reported as predictive of sick leave or poor prognosis in previously published scientific papers. Only those factors that led to a considerable change in the regression coefficient of the risk factor (>10%) were considered as potential confounders in the multiple regression models. Next, a manual forward selection procedure was used to sequentially include potential confounders that induced the most change in the regression coefficient of the risk factor (i.e., in order of highest change of the regression coefficient). Confounders that changed the regression coefficient of sick leave by more than 10% were retained in the model. If the unadjusted relationship between the work-related risk factor and sick leave was not statistically significant (P > 0.05) and remained not significant after controlling for all potential confounders individually, the relationship was adjusted for sick leave at baseline and sex only. Individual factors may interact in the relation between work-related physical and psychosocial risk factors and sick leave. We considered possible effect modification by sick leave at baseline, sex, coping style worrying (dichotomized) and musculoskeletal co-morbidity. Product terms of the potential effect modifier and the work-related risk factor were added to the model (e.g., sex × risk factor). Furthermore, possible effect modification by psychosocial risk factors was investigated in the relation between the four physical risk factors and sick leave. Factors significantly interacting with determinants in the model were retained in the models. In case of significant effect modification stratified analyses were carried out presenting the effect of the work-related risk factor for relevant subgroups of workers. All analyses were performed with the use of SPSS for Windows version 10.1 (SPSS Inc., Chicago, IL, USA). Results In total, 342 of the 383 included patients (89%) completed the 3 months follow-up questionnaire. Fifty-eight of the patients were excluded, because data on sick leave at follow-up were missing. There were no significant differences between responders and dropouts by age, sex, and any of the determinants (P > 0.05). Baseline characteristics of patients reporting sick leave in the 3 months following baseline (n = 73) and patients without sick leave (n = 252) are shown in Table 2. The mean age of the total study population was 43 years (SD 10.3) and 54% was female. At baseline 80 patients reported sick leave in the 3 months preceding baseline due to their neck or upper extremity complaint. Half of these patients reported sick leave in the 3 months following baseline. With reference to physical and psychosocial risk factors, there was a statistically significant difference between patients reporting sick leave and patients without sick leave for decision authority, heavy physical work, static postures and repetitive movements, prolonged sitting and prolonged VDU-work. Table 2Baseline characteristics and values of patients with neck or upper extremity complaints reporting sick leave (n = 73) and of patients reporting no sick leave (n = 252) due to their complaints in the 3 months following baseline assessmentCharacteristicSick leaveaNo sick leaveaTotalIndividual factors Age, years [mean (SD)]42.0 (11.5)43.6 (9.8)43.0 (10.3) Sex (female)75%49%**54% Sick leave in 3 months before baseline59%16%**25% Education level  Primary48%30%*34%  Secondary40%49%48%  College/university12%21%18% Number of hours work (h)  8–1611%12%11%  17–2418%25%20%  25–3626%30%27%  >3645%33%42%Married/living together63%83%*78%Smoking (now, ever)77%67%70%Pain coping: retreating [scale 7–28; median (IR)]11.0 (5.0)9.0 (4.8)**10.0 (4.5)Pain coping: worrying (scale 9–36; median (IR))18.0 (7.0)14.0 (5.0)**15.0 (6.0)Psychological distress (scale 0–12; median (IR))7.0 (5.0)4.0 (5.0)**5.0 (6.0)General health Meeting ACSM position stand4%16%*13% Meeting Norm Healthy Activity41%42%43% Perceived health [scale 1–5; mean (SD)]3.1 (0.8)3.4 (0.9)*3.3 (0.8) Quality of life [scale 1–5; mean (SD)]3.2 (0.7)3.5 (0.8)*3.4 (0.8) Vitality [scale 0–100; mean (SD)]54.3 (20.9)62.4 (17.2)**60.0 (18.5)Characteristics complaint Localized complaint40%53%*49% Complaint at both arms10%15%13% Complaint at dominant arm50%44%47%Duration of current episode <1 week5%7%7% 1week–1 month36%32%33% 1–6 months29%37%34% >6 months30%24%26%History of elbow complaints51%43%45%Musculoskeletal co-morbidity56%47%50%Pain intensity [scale 0–10; mean, (SD)]6.0 (1.9)4.4 (2.1)**4.8 (2.2)Functional disability [scale 0–100; median (IR)]36.3 (28.8)16.5 (21.3)**20.0 (25.3)Work-related factors Heavy physical work [scale 0–100; mean (SD)]36.1 (40.2)16.7 (36.1)**22.2 (42.8) Static postures and repetitive movements (scale 0–100)55.6 (47.2)44.4 (44.4)**44.4 (38.9) Sitting for a long period of time22%48%**42%VDU-work for a long period of time21%36%*31% Decision authority [scale 3–12; mean (SD)]9.0 (3.0)9.0 (3.0)*9.0 (3.0) Job demands [scale 5–20; mean (SD)]13.4 (3.6)12.8 (2.9)12.9 (3.1) Co-worker support [scale 4–16; mean (SD)]12.0 (1.0)12.0 (2.0)12.0 (2.0) Job security78%85%83% Job as perceived cause of complaint62%49%56%VDU visual display unit, SD standard deviation, IR interquartile range* P < 0.05; ** P < 0.001 (independent T-test, Mann–Whitney test, or χ2-test)avalues are percentages of patients unless indicated Table 3 shows the effect of work-related physical and psychosocial risk factors on sick leave. After adjustment for confounders, a statistically significant effect was found for heavy physical work and prolonged sitting, but only in patients with a high score on pain coping strategy “worrying”. In patients who scored low on the pain coping subscale “worrying” the hazard ratio of sick leave was 1.05 (0.93–1.18, P = 0.46) per 10% increase in heavy physical work. In patients scoring high on “worrying” the relative risk of sick leave was 1.32 (95% CI 1.07–1.62) per 10% increase in heavy physical work. Patients who were sitting for long periods of time had a reduced risk of sick leave as compared to patients who did not spent a lot of time sitting, again only in patients who scored high on “worrying” (HR = 0.17, 95% CI 0.04–0.72). In patients who scored low on “worrying”, sitting for long periods of time did not have an effect on sick leave (P = 0.43). Sick leave at baseline, sex and musculoskeletal co-morbidity did not modify the relationship between the physical and psychosocial risk factors and sick leave. The psychosocial risk factors neither modified the relationship between heavy physical work and sick leave. Table 3Relationship between work-related risk factors and sick leave in patients with neck or upper extremity complaints: results from the multiple Cox regression analysesDeterminantNCrude HR95% CIAdjusted HR95% CIHeavy physical work (per 10% increase)69/3201.23[1.12; 1.36]* For patients with low scores on worrying d21/1401.05[0.93; 1.18]a For patients with high scores on worryingd48/1801.32[1.07; 1.62]a,*Static postures, repetitive movements (per 10% increase)69/3201.16[1.06; 1.27]*1.04[0.94; 1.14]bSitting for long periods of time (vs. never/now and then)16/3220.40[0.23; 0.70]* For patients with low scores on worryingd2/1800.81[0.43; 1.52]a For patients with high scores on worryingd14/1420.17[0.04; 0.72]a,*VDU-work long periods of time (vs. never/now and then)15/3220.56[0.32; 1.00]*0.70[0.36; 1.36]cDecision authority Low1.00 Medium0.80[0.47; 1.35]0.94[0.55; 1.59]a High0.59[0.31; 1.13]0.87[0.45; 1.68]aJob demands Low23/1151.001.00 Medium20/1110.90[0.49; 1.64]0.91[0.50; 1.66]a High27/951.42[0.81; 2.48]1.13[0.64; 1.97]aCo-worker support Low16/861.001.00 Medium31/1401.19[0.65; 2.18]1.22[0.67; 2.23]a High23/881.40[0.74; 2.66]1.14[0.59; 2.17]aLow job security (vs high)70/3191.43[0.78; 2.60]1.47[0.80; 2.69]aJob strain High decision authority, low job demands10/771.001.00 High decision authority, high job demands11/521.63[0.69; 3.84]1.29[0.54; 3.07]b Low decision authority, low job demands25/1041.85[0.89; 3.85]1.30[0.62; 2.73]b Low decision authority, high job demands 24/862.15[1.03; 4.49]*1.15[0.54; 2.47]bN Number of patients reporting sick leave/total, HR hazard ratio* P < 0.05aAdjusted for sick leave at baseline and sexbAdjusted for sick leave at baseline, sex and worryingcAdjusted for sick leave at baseline, sex, education level, quality of life, working in static postures and with repetitive movementsdEffect modification by worrying Discussion We studied a population of working employees who consulted their GP with a complaint at the neck or upper extremity. The results show that heavy physical work and prolonged sitting in patients that worry a lot predicted sick leave in the three successive months after baseline. Other physical and psychosocial risk factors appeared not to be related to sick leave after adjustment for confounding variables. Individual factors and stress may have a modifying effect on the relation between work related factors and outcome in musculoskeletal complaints. The pain coping style “worrying” appeared to be an effect modifier in the relationship between heavy physical work and sick leave. An increase of 10% of heavy physical work in patients who worried a lot increased the probability on sick leave with 1.3. A passive coping style like worrying (e.g., “focus on pain all the time”; “I think the pain will get worse”) is thought to generate a preoccupation with bodily symptoms, which may increase the perception of pain and disability (Covic et al. 2000; Evers et al. 2003) or generate a different appraisal of the work situation and musculoskeletal symptoms (Bongers et al. 2002b), which in turn may lead to sick leave. Sitting for long periods of time reduced the risk of sick leave, only in patients who scored high on the pain subscale “worrying” In the study of Ariëns et al. (2002) sitting was also related to a reduced risk of sick leave. However, uncomfortable sitting has been found to increase the number of days on sick leave due to neck and shoulder disorders (Ekberg and Wildhagen 1996). Sitting was highly negatively correlated with heavy physical work. Perhaps, people who sit for long periods of time at their work do not experience heavy physical work, and thus the absence of heavy physical work results in a reduced risk. This may explain why sitting had only a protective effect in patients that worry a lot. In the unadjusted analyses, all the physical risk factors and two of the psychosocial risk factors (i.e., decision authority and job strain) had a statistically significant effect on sick leave. It is partly due to the underlying distribution of the exposure which physical factors are most strongly associated with sick leave in the multivariate analyses. Adjustment for sick leave at baseline had the strongest effect on the presented associations, diminishing the strength of the relations between work-related risk factors and sick leave. Sick leave may have been the result of exposure to these risk factors, but once patients were on sick leave work-related risk factors no longer seem to have an impact on continued sick leave. Half of the patients who reported sick leave within the 3 months before baseline also reported sick leave in the 3 months following baseline. The effect of sick leave in the months preceding baseline apparently outweighed the impact of work-related risk factors in the analyses. The proportion of patients reporting sickness during the 3 months follow-up was too small to be able to exclude patients reporting sick leave at baseline. Cox regression produces large estimates of the standard errors, which results in conservative estimates of the confidence intervals (Skov et al. 1998). Therefore, it may be possible that relevant associations did not reach statistical significance. Sick leave data were collected by a self-reported questionnaire. Sick leave data collected by an objective method (i.e., sick leave records) may be more accurate than self-reported sick leave. However, in large epidemiological studies the use of questionnaires is more feasible and can be a source of reliable data on sick leave. Burdorf et al. (1996) investigated the reliability of self-reported sick leave with company records as reference. A good agreement was found between prevalence, frequency and duration of self reported sick leave and company records in patients with back pain. In our study we only examined the prevalence of sick leave and consider the reported sick leave data to be fairly reliable. Our study population differs from an occupational cohort, consisting of workers from a wide variety of occupational settings who had visited the GP due to neck or upper extremity complaints. This makes our results more widely generalisable than a selective sample of workers from a specific company or industry. However, due to the fact that our population already had symptoms at baseline we may not be able to discriminate between factors that are a consequence of sick leave at baseline and factors that may increase the risk of sick leave. For example, patients may worry more about their pain problem because they are no longer able to work, or worrying about their pain problem may be the reason for sick leave after their visit to the doctor. In the Netherlands the GP is often confronted with patients who are on sick leave due to musculoskeletal complaints, as patients who seek medical care usually first consult their GP. The GP acts as a gatekeeper in the health care system. At the time of our study, referrals to the second or third level of care could, in principle, only be made by the GP. This is comparable to, for instance, the British and the Canadian health care systems. As a result of their position in the Dutch health care system, GPs could play an important role in the prevention of aggravation of complaints. In this study the copying style “worrying” seemed to be an important factor in patients who have jobs involving heavy physical work. Reassuring patients might help to reduce aggravation of complaints, which might improve the chances of return to work in this group of patients with neck or upper extremity complaints. It may be interesting to investigate whether early intervention aimed at promoting particular coping styles can prevent or reduce sick leave in patients with neck or upper extremity complaints in a primary care setting. In conclusion, heavy physical work increased the risk of sick leave and prolonged sitting reduced the risk of sick leave in a subgroup of patients who worry much. Other work-related physical risk factors and work-related psychosocial risk factors were not significantly related to sick leave. Additional large longitudinal studies of sufficiently large size among employees with neck or upper extremity complaints are needed to confirm our results.

Curr_Eye_Res-1-1-2430176

Branch Retinal Vein Occlusion: Pathogenesis, Visual Prognosis, and Treatment Modalities

In branch retinal vein occlusion (BRVO), abnormal arteriovenous crossing with vein compression, degenerative changes of the vessel wall and abnormal hematological factors constitute the primary mechanism of vessel occlusion. In general, BRVO has a good prognosis: 50–60% of eyes are reported to have a final visual acuity (VA) of 20/40 or better even without treatment. One important prognostic factor for final VA appears to be the initial VA. Grid laser photocoagulation is an established treatment for macular edema in a particular group of patients with BRVO, while promising results for this condition are shown by intravitreal application of steroids or new vascular endothelial growth factor inhibitors. Vitrectomy with or without arteriovenous sheathotomy combined with removal of the internal limiting membrane may improve vision in eyes with macular edema which are unresponsive to or ineligible for laser treatment. BACKGROUND Method of Literature Search Eligible studies were identified through a comprehensive literature search of electronic databases (Medline, 1966–September 2007 and Science Direct, all years). Additional articles were selected from review of the reference lists of the articles generated from the above search. The following keywords and combinations of these words were used in compiling the search: branch retinal vein occlusion, retinal circulatory disorders, pathogenesis, hematological disorders, risk factors, therapy methods, visual prognosis. In total, 150 of these were used for this mini-review. Epidemiology, Classification Retinal vein occlusion (RVO) is the second most common retinal vascular disorder after diabetic retinopathy and is a significant cause of visual handicap. Its prevalence has been shown to vary from 0.7% to 1.6%.1,2 In a population-based study,3 an overall incidence of symptomatic RVO was found in 0.21% of patients aged 40 or older. Hayreh et al.4 investigated the demographic characteristics of various types of RVO in 1108 patients (1229 eyes). In this study, a male:female ratio of 1.2:1 was noted in a group of patients with RVO. Of the two main types of RVO, central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO), the latter is more common. A further group is hemi-vein occlusion, a distinct clinical entity presenting as occlusion of only one trunk of the central retinal vein in the area of the anterior part of the optic nerve.4 Hayreh et al.4 postulated that its pathogenesis is closely related to CRVO. The first case of BRVO was reported by Leber in 1877.5 Some studies showed a higher proportion of BRVO patients older than 65 at the onset of the disease compared to CRVO,4,6 but others found no significance of age in the distribution of CRVO and BRVO.7,8 BRVO is divided into two distinct entities: major BRVO, when one of the major branch retinal veins is occluded, and macular BRVO, when one of the macular venules is occluded. In 66% of eyes with BRVO, there is occlusion of the major branch in the superotemporal quadrant followed by 22–43% of eyes with occlusion of the major branch in the inferotemporal quadrant.9 Owing to absent subjective BRVO symptoms in nasal quadrants, the diagnosis of occlusion in this localization is mostly accidental and therefore rare.10 Very often BRVO in nasal quadrants is diagnosed only when its complication as bleeding from neovascularizations into the vitreous cavity occurs. The cumulative probability of developing a second episode of occlusion in the other eye within 4 years is about 7% in patients with BRVO.4 Pathogenesis The pathogenesis of RVO is multifactorial while BRVO may be due to a combination of three primary mechanisms: compression of the vein at the arteriovenous (A/V) crossing, degenerative changes of the vessel wall, and abnormal hematological factors. In the following sections these factors are discussed. Arteriovenous Crossing Koyanagi in 192811 first reported the association between BRVO and A/V crossing, and now it is established that mechanical narrowing of the venous lumen at these intersections plays a role in the pathogenesis of BRVO. Anatomic features of A/V crossings and secondary effects of arteriolar sclerosis may explain the apparent vulnerability of the crossing site to venous occlusion. In the majority of A/V crossings, the thin-walled vein lies between the more rigid thick-walled artery and the highly cellular retina. The sharing by artery and vein of the common adventitial sheath and the narrowing of the venous lumen that normally occurs at the A/V crossing provide the setting for BRVO.12 The risk of occlusion may be accentuated when arteriolar sclerosis results in increased rigidity of the crossing artery. Duker and Brown13 provided further support for a mechanical basis of BRVO development when they examined the relative anatomic position of the crossing artery and vein at the site of occlusion in 26 eyes with BRVO. They found in all 26 eyes the artery anterior to the vein (towards the vitreous cavity). Zhao et al.12 evaluated the anatomic position of the crossing vessels in 106 eyes with BRVO and found the artery anterior to the vein at the obstructed site in 99% of affected eyes. However, other mentioned risk factors must play a role, too, because in approximately 60% of normal A/V crossings without BRVO the artery lies anterior to vein.12 Degenerative Changes of Vessel Wall A number of studies have investigated the histological changes of vessel wall at the A/V crossing.14,15 An investigation by Jefferies et al.14 showed that the expected venous compression at the crossing in histological view does not exist. He described the bending of the vein into the nerve fiber layer at this point without its compression. Histological investigation of the venous lumen at the A/V crossing in patients with a number of months to several years duration of BRVO showed organized thrombus with varied extent of recanalization in this part. Seitz15 described the clinical histological correlation in one eye with BRVO of a few hours after onset. There was no blood thrombus obliterating the venous lumen at the A/V crossing and even the fundoscopic examination showed strong dilated and tortuous vein distal to the crossing. In the area of the A/V crossing, alteration of the endothelium and intima media was present. Seitz suggests that the trophic changes of venous endothelium and intima media, as they follow the compression from overlaying artery, is the root of the pathogenesis of BRVO.15 The formation of the thrombus follows as a secondary process. The findings of Frangieh et al.16 support this hypothesis; 90% of the patients in their study had evidence of intima media layer hypertrophy, and all had evidence of intravenous thrombosis. Systemic hypertension, diabetes mellitus, atherosclerosis, and smoking are reported to be more common in patients with RVO.1,2,10 Sclerosis of the retinal artery which is associated with these systemic disorders may result in further compression of the vein, when the increased rigidity of arterial wall and contraction of the adventitial sheath shared by artery and vein occur. Mechanical obstruction of the vein through the rigid artery in the A/V crossing may result in turbulent blood flow producing damage to venous endothelium and intima media and the sequence of events leading to occlusion of the vein.12,14 The turbulent blood flow was confirmed by Christoffersen and Larsen in an investigation which analyzed the fluorescein angiograms of 250 patients with BRVO.17 Hematological Disorders Some studies have revealed an association between BRVO and hyperviscosity due to high hemotocrit.18,19 Higher blood viscosity increases under conditions of low blood flow and erythrocyte aggregation.18 Viscosity is mainly dependent upon the hematocrit (the greater the number of erythrocytes, the larger they aggregate) and plasma fibrinogen (required for aggregation to occur).20 Another discussed hematological disorder in the pathogenesis of BRVO is dysregulation of the thrombosis-fibrinolysis balance.21 The coagulation cascade including different blood factors results in the production of thrombin which converts circulating fibrinogen to fibrin. The coagulation sequence is held in check and inhibited by specific anticoagulants including protein C, protein S, and antithrombin. Table 1 shows the major disorders studied in patients with RVO. The results of published studies, however, are inconsistent, and the role of coagulation factors in the development of RVO remains unclear. TABLE 1 Most discussed coagulation and anticoagulation disorders in the etiology of BRVO Resistance to activated protein C (especially factor V Leiden mutation) Protein C or protein S deficiency Deficiency of antithrombin III Genetic mutation in the prothrombin (factor II) gene Anti-phospholipid antibodies Hyperhomocysteinemia Resistance to Activated Protein C and Deficiency of Protein C or Protein S Protein C is serine proteinase whose activated form is a potent inhibitor of coagulation factors V and VIII.22 Factors V and VIII are a part of the coagulation cascade leading to conversion of fibrinogen to fibrin. Patients with protein C deficiency frequently manifest superficial and deep venous thrombosis and pulmonary embolism. Protein S and phospholipids are co-factors in the inactivation of factors V and VIII by activated protein C.22 An absolute deficiency of protein C or S is relatively rare. Tekeli23 and several other authors have reported normal levels in patients with RVO.24–26 The concept of resistance to activated protein C (so-called APC resistance) was first introduced by Dahlbäck et al. in 1993.27 APC resistance was subsequently shown to be a risk factor for venous thrombosis.28 More than 90% of patients with APC resistance have been shown to have a single point mutation in factor V gene.29 This mutation hinders the degradation of factor V normally occurring through protein C. Several investigators have reported an increased frequency of APC resistance in a cohort of patients with RVO,30–34 but this association has not been confirmed in other studies.35,36 Moreover, some results are inconclusive due to small patient samples or the lack of control groups. The meta-analysis of Janssen et al.21 showed the odds ratios for factor V Leiden mutation in patients with RVO 1.5 (95% CI 0.8–3.2). Despite the evidence of the significance of Leiden mutation, the effect of this hematological disorder in the etiology of RVO is only marginal.21 Deficiency of Antithrombin and Mutation in the Prothrombin Gene In recent studies of patients with RVO, no significant association with a deficiency of antithrombin or with prothrombin mutation was found.21,26,34,37–40 Anti-Phospholipid Antibodies and Hyperhomocysteinemia Antiphospholipid antibodies (APA) consist of a heterogeneous group of immunoglobulins, mainly anti-cardiolipin antibodies (ACA) and lupus anticoagulants (LA). Circulating APA leads to a hypercoagulable state and recurrent thrombosis through thrombocyte activation and inhibition of the natural anticoagulant pathways by binding of membrane phospholipids. Both the presence of LA and increased level of ACA are associated with a 3- to 10-fold increased risk of venous thrombosis.41 An elevated level of the amino acid, homocysteine is now generally accepted to be a risk factor for systemic vascular disease.42 Homocysteine appears to have a deleterious effect on vascular endothelium and may induce increased platelet aggregation and thrombosis. Levels of homocysteine may be increased by dietary habits, prescription medicines, or enzymatic mutations affecting homocysteine metabolism.43 The results of meta-analyses confirm total homocysteine to be an independent risk factor for RVO.21,44 Loewenstein et al.45 investigated the prevalence of genetic mutation in the enzyme methylentetrahydrofolate reductase (MTHFR) whose impaired activity may lead to hyperhomocysteinemia. The prevalence of this mutation was significantly higher in patients with RVO compared with the incidence of MTHFR in a control population. However, these results were not confirmed in other studies.46 The meta-analysis of Cahill et al.44 showed an association between retinal vascular occlusion and hyperhomocysteinemia but not with the mutation in the gene for MTHFR. Pathogenesis of Macular Edema in BRVO The development of macular edema (ME) followed by BRVO has been hypothesized to be caused by fluid flux from vessels to tissue according to Starling's law,47,48 which is based on the breakdown of the blood-retinal barrier (BRB) as a result of damage to the tight junctions of capillary endothelial cells,49 vitreoretinal adhesion,50 and secretion into the vitreous of vasopermeability factors produced in the retina.51,52 Observations by Noma et al.52 suggest that in patients with BRVO, vascular occlusion induces the expression of vascular endothelial growth factor (VEGF) and Interleukin-6 (IL-6), resulting in BRB breakdown and increased vascular permeability. Thus, VEGF and IL-6 may contribute to the development and progression of vasogenic ME in BRVO. ME is closely associated with retinal hypoxia, and the degree of hypoxia in the center of the macula corresponds to the decrease in visual acuity (VA). If marked hypoxia persists, irreversible structural changes in the macula occur, and the disturbed VA is almost always lasting. It is generally known that ME and intraretinal hemorrhage occurring in BRVO usually disappear within 6 to 12 months.53 In these cases, collateral systems often develop. The main purpose of the treatment is to decrease the duration of edema to prevent photoreceptor damage, if no spontaneous improvement occurs. Clinical Signs and Diagnosis In general, diagnosis of BRVO is not a problem owing to its classical features. Major BRVO can be asymptomatic or with visual blurring usually involving the sector of visual field corresponding to the area of the retina involved. In macular BRVO, there is always a central visual disturbance with normal peripheral vision. Acute BRVO presents characteristic clinical features with flame-shaped, dot and blot hemorrhage, soft and hard exudates, retinal edema, and dilated, tortuous vein in a segmental distribution. Signs of old occlusion are vascular sheathing and venous collaterals. The diagnosis is based on clinical examination under slit lamp and fundoscopy in artificial mydriasis. VA is of great importance for future visual prognosis. BRVO often leads to retinal non-perfusion zones in the occlusion area. Fluorescein angiography is particularly useful in determining the extent of ME and ischemia, although the ischemic areas are often obscured by the presence of intraretinal hemorrhage. Retinal neovascularization occurs in 36% of eyes with an area of non-perfusion greater than 5 disc diameter.54 RVO is associated with an increase in vascular causes of death (both cerebral and cardiac) in large prospective follow-up studies.55 In all patients with RVO, the systemic risk factors (hypertension, diabetes mellitus, blood lipid disorders) should be investigated and managed by appropriate specialists. Natural Course and Visual Prognosis The visual outcome following the natural course of BRVO is well documented.56–58 In general, BRVO has a good prognosis: 50–60% of eyes have been reported to have a final VA of 20/40 or better even without any treatment.56–59 The natural course of BRVO is determined by the site and degree of occlusion, the integrity of arterial perfusion to the affected sector, and the efficiency of the developing collateral circulation.59 Chronic ME and bleeding into the vitreous from neovascularizations account most frequently for a poor final VA.54,58,60 Retinal neovascularization and persistent ME develop in 25% and 60% of eyes, respectively.57,61 Gutman et al.60 found that in the natural course of BRVO, only 14% of eyes with chronic ME retained a VA of 20/40 or better, while 86% had a final VA of 20/50 or worse. He concluded that chronic ME has a poor prognosis in terms of final VA.60 Schilling et al.62 observed a worse visual prognosis in cases of ischemic ME compared to perfused ME. However, findings by Finkelstein63 showed that 91% of 23 eyes with macular ischemia recovered vision within one year with a VA of 20/40 or better. The conflicting reports and small number of studied eyes make it difficult to reach definitive conclusions on visual prognosis in patients with BRVO. VA is a very sensitive indicator of the oxygen situation in the macula. For this reason, pre-treatment VA may be an important prognostic factor. Six studies analyzing the relation between initial and final VA were found.53,56,58,64–66 Five were used in an analysis of the data of eyes with unsatisfactory final VA (20/200 or worse) in relation to initial VA (Table 2). There were 2 groups; the first consisted of eyes with an initial VA of 20/50 or better and the second group of eyes with an initial VA of 20/200 or worse. In the second group were found a considerably higher percentage of eyes with a final VA of 20/200 or worse, regardless whether the eyes had undergone laser treatment or not. Since there were differently divided subgroups for final VA, the study of Subramanian et al.65 was not included in our analysis. Magargal et al.58 investigated the visual prognosis in 246 eyes with BRVO divided into two groups: with and without laser treatment. The obtained analysis illustrates that in the group of eyes with an initial VA 20/50 or better, no eye (not receiving laser treatment) and only 13% eyes (had undergone laser treatment) had a final VA of 20/200 or worse, whereas in the group of eyes with an initial VA 20/200 or worse, 83% of eyes (not receiving laser treatment) and 50% of eyes (had undergone laser treatment) had this unsatisfactory final VA. In an analogous way, the data for the final VA 20/50 or better in relation to the initial VA were analyzed (Table 3). We can see that in the group of eyes with an initial VA 20/50 or better, 89% of eyes (not receiving laser treatment), and 75% of eyes (had undergone laser treatment) retained this good VA, whereas in the group of eyes with an initial VA 20/200 or worse, only 14% of eyes (not receiving laser treatment) and only 22% of eyes (had undergone laser treatment) had a final VA 20/50 or better.58 Similar data are reported in the other studies (Tables 2 and 3). A chi-squared test with Yates correction was used to analyze the data. In 4 cases, in Table 2, and in 5 cases, in Table 3, respectively, the results were statistically significant (p < 0.05). Our analysis shows that in eyes with an initial VA 20/50 or better, the visual prognosis is good even without treatment. It could also be concluded that the cases of BRVO with an initial VA of 20/200 or worse have a statistically significantly poorer visual prognosis than those with an initial VA of 20/50 or better. Subramanian et al.65 showed that in patients with BRVO who underwent laser treatment of ME, the level of preoperative VA can be a useful predictor of visual outcome. TABLE 2 Final visual acuity of 20/200 or worse in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05) Initial visual acuity 20/50 or better Initial visual acuity 20/200 or worse Chi-squared test p < 0.05 Natural course—without laser treatment Gutman56 5% (1/20) 50% (6/12) Significant Magargal58 0% (0/35) 83% (24/29) Significant Had undergone laser treatment Wetzig53 25% (2/8) 67% (10/15) No Jalkh64 0% (0/9) 33% (3/12) No Magargal58 13% (5/40) 50% (32/64) Significant Lang66 8% (1/13) 50% (8/16) Significant TABLE 3 Final visual acuity 20/50 or better in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05) Initial visual acuity 20/50 or better Initial visual acuity 20/200 or worse Chi-squared test p < 0.05 Natural course—without laser treatment Gutman56 90% (18/20) 33% (4/12) Significant Magargal58 89% (31/35) 14% (4/29) Significant Had undergone laser treatment Wetzig53 63% (5/8) 20% (3/15) No Jalkh64 56% (5/9) 9% (1/12) Significant Magargal58 75% (30/40) 22% (14/64) Significant Lang66 77% (10/13) 13% (2/16) Significant Treatment Current treatment options focus on the sequelae of the occluded venous branch, such as ME, retinal neovascularization, vitreous hemorrhage, and traction retinal detachment. There have been a number of treatment modalities advocated for the management of BRVO (Table 4). Many studies that examine interventions for BRVO suffer from methodological limitations, including insufficient power resulting from small sample sizes, short follow-up periods, absence of a control group or inappropriate control group (absence of placebo or best practice intervention as control groups), and lack of distinction between clinical entities. A number of such investigations have therefore produced conflicting data. Hence, the results of randomized clinical trials are the most important. The complex pathogenesis of this disease requires investigation and treatment of all risk factors (hypertension, diabetes mellitus, blood lipid disorders, hematological disorders). TABLE 4 Treatment modalities for BRVO Anti-aggregative therapy and firbrinolysis Isovolemic hemodilution Laser treatment Intravitreal and periocular application of steroids Intravitreal injection of VEGF inhibitors Sheathotomy and vitrectomy Anti-Aggregative Therapy and Fibrinolysis Systemic treatment with oral acetylsalicylic acid, subcutaneous heparin, or intravenous thrombolysis have not been shown to be effective treatments for CRVO, while for BRVO no randomized clinical trials have been published as of the date of this review. Thrombolysis using administration of tissue plasminogen activator intravitreally or directly into the retinal vein (mostly upper temporal branch close to the optic disc) has been demonstrated to improve VA in patients with CRVO,67 but the results were based only on noncomparative interventional series. There is no general current acceptance of this treatment. Houtsmuller et al.,68 in a double-blind study, examined the platelet aggregation inhibiting effect of ticlopidine in 54 patients with BRVO less than 3 weeks from the onset of symptoms. Compared with placebo therapy a significant improvement in VA was observed with ticlopidine therapy for six months. In the treated group, 69% of patients experienced an improvement in VA, whereas 52% of placebo group reported improvement. Troxerutin has been suggested to inhibit erythrocyte and platelet aggregation and to improve erythrocyte deformability, thus reducing blood viscosity and the retinal microcirculation.69 A double-blind randomized study of 26 patients with BRVO compared troxerutin with placebo.69 At 4 months follow-up, more of the patients receiving troxerutin treatment had a mean VA of 20/40 or better than the control group, although this difference was not found to be statistically significant. After 4 months, all patients were treated with troxerutin for 2 years. At the completion of this follow-up period for those patients initially treated with troxerutin, a significant improvement in VA and improvement of ME was demonstrated. The limitation of this study is that there is no separation in the analysis of results for patients with BRVO and CRVO who were included in the study, too. Both studies mentioned that investigated the medical treatment of BRVO are limited by a small sample size and short follow-up period (6 and 4 months). Isovolaemic Hemodilution Chen et al.20 demonstrated positive results for isovolemic hemodilution given up to 3 months after the on-set of the symptoms of BRVO in patients with a hematocrit of 35% or more. In this randomized controlled study, 18 patients were treated for 6 weeks with venesection and volume replacement using hydroxyethylstarch and compared to 16 untreated control patients. After a one year follow-up, the final VAs were 20/40 and 20/80 for treated and untreated patients, respectively (p = 0.03). Patients with ME and a VA 20/40 or worse underwent 3 months after including into the study macular grid laser photocoagulation (MLG). Sector photocoagulation was applied if ocular neovascularization developed or if, at 3 months, the fluorescein angiogram showed an area of capillary non-perfusion greater than 5 disc areas. 28% of the hemodiluted patients required MLG compared to 44% of the control group; this difference was not statistically significant (p = 0.2). Sector photocoagulation was required by 50% of both groups of patients.20 Hydroxyethylstarch has a capacity to expand the plasma volume by up to 172% of the volume infused and has a duration of action of approximately 36 hours.70 It is non-antigenic and has a low incidence of allergic reactions.71 Poupard et al.72 randomized 25 patients to either hemodilution with dextran for 21 days (n = 10), hemodilution combined with heparin for 21 days (n = 10), or heparin treatment for 21 days followed by anti-vitamin K drugs for a further 30 days (n = 5). The study showed that, for those receiving heparin followed by anti-vitamin K drugs, mean VA remained unchanged to baseline values by 60 days. For those treated with hemodilution and heparin, a statistically significant increase in VA was found by 60 days. For those treated with hemodilution alone, a significant improvement in VA was found by day 14. In a randomized study by Hansen et al.73 of 35 patients with BRVO, 18 patients were treated by hemodilution for a period of 5 to 6 weeks (targeted hematocrit 30–35%). A control group of 17 patients were only observed. At follow-up 12 months later, 25 patients had completed the therapy. Seven of the 13 who received hemodilution demonstrated a VA increase of 2 lines or more compared with none of the 12 patients who did not receive hemodilution (p < 0.005). Reported complications of hemodilution include headache, exertional dyspnea, tiredness, deep vein thrombosis, and hypotension. The treatment was noted to be generally well-tolerated even in elderly patients.20,73,74 The use of hemodilution to treat BRVO is currently not generally accepted. Interpretation of the above-mentioned studies is difficult because most of them incorporated other treatments in combination with the hemodilution. Further prospective randomized trials with adequate controls and sufficient follow-up are required for any definitive conclusions and recommendations. Arteriovenous Crossing Sheathotomy and Vitrectomy Osterloh and Charles75 first reported improvement in VA in patients with BRVO after treatment using the technique of surgical sheathotomy. The principle steps of this procedure are a pars plana vitrectomy followed by separation of the retinal artery from the vein by creating an incision in the adventitial sheath adjacent to the A/V crossing and then separation of the adhesions. Several studies have shown significantly better functional outcomes in patients treated by sheathotomy compared to controls (Table 5).75–96 Reported complications are few but include cataract, hemorrhage, retinal tears, postoperative gliosis, and retinal detachment.75–96 Garcia-Arumi76 described a combination of A/V sheathotomy and injection of thrombolytic into the occluded vein which resulted in thrombus release in 28% cases and significant correlation with early surgery and better final VA. The role of the sheathotomy alone in visual improvement is insufficiently clear. Some authors suggest that vitrectomy is the most important part of the sheathotomy surgery, leading to reduction of ME.77,80,96 Yamamoto et al.77 compared the effect of sheathotomy combined with vitrectomy to the effect of vitrectomy alone and found no advantage of sheathotomy. Eyes with pre-existing posterior vitreous detachment were not studied. For this reason, the benefit of vitrectomy of these eyes is unknown. Surgical detachment of posterior hyaloid could be more important than the sheathotomy itself .78 The vitreous is postulated to have a role in the pathogenesis of neovascularization and ME, which may complicate BRVO and its removal may help in the management of these sight threatening complications.78 Vitrectomy and removal of the posterior hyaloid with peeling of the internal limiting membrane (ILM) appears to improve oxygenation of the retina, which may lead to visual improvement.97,98 Peeling of the ILM improves the surgical outcome during A/V adventitial sheathotomy, too.84 To date, no randomized clinical trials on the surgical treatment of BRVO have been published. Any evidence supporting these procedures is based on non-ramdomized case series only. TABLE 5 Summary of studies evaluating the treatment of macular edema in BRVO by sheathotomy (VA = visual acuity, ME = macular edema, ILM = internal limiting membrane) Author Study type Patients Follow-up (mean) Outcomes Comments Osterloh and Charles75 Case report. 1 eye. 8 months. VA improved from 20/200 to 20/25. First report of sheathotomy. Garcia-Arumi et al.76 Prospective interventional nonrandomized study. 40 eyes—all underwent vitrectomy, sheathotomy and injection of 25 mg of tissue plasminogen activator into occluded vein. 13 months. Thrombus release in 11 eyes (27.5%)—correlated with early surgery. VA increased from 20/100 to 20/40 (p = 0.016). Yamamoto et al.77 Retrospective interventional comparative case series. 20 eyes—sheathotomy 16 control eyes (posterior vitreous detachment via vitrectomy). 12 months. VA: significantly better in both groups (p = 0.008 and p = 0.001, respectively). VA and foveal thickness were not significantly different between the groups. Charbonnel et al.78 Prospective nonrandomized, interventional case series. 13 eyes—sheathotomy. 7 months. Improvement in VA ≥ 2 ETDRS lines in 9 eyes (69%). Absence of previous posterior vitreous detachment correlated with improvement in VA. Sohn et al.79 Retrospective interventional case series. 22 eyes—sheathotomy + ILM peeling in all eyes. 3 months. Improvement in VA (log MAR) from 0.79 ± 0.29 to 0.57 ± 0.33 (p < 0.01). All eyes pretreated with grid laser or triamcinolone. Kumagai et al.80 Prospective, randomized, comparative, interventional study. Group 1: 18 eyes—sheathotomy. Group 2: 18 controls (vitrectomy without sheathotomy). 31 months. VA (log MAR) in group 1: 0.52 → 0.08. In group 2: 0.53 → 0.014. Differences between group 1 and 2 was not significant. Avci et al.81 Retrospective interventional comparative case series. 11 eyes—sheathotomy. 10 control eyes—grid laser photocoagulation. 9 months. VA (log MAR): sheathotomy: 0.84 → 0.36. Grid laser: 1.06 → 0.82. Difference was significant. Horio et al.82 Interventional case series. 7 eyes. 6 months. Significant improvement in retinal blood flow (p < 0.01) and reduced macular thickness (p = 0.03). Lakhanpal et al.83 Retrospective interventional case series. 12 eyes. 49.9 weeks. VA (logMAR) improved from 1.00 ± 0.32 to 0.56 ± 0.28 (p = 0.0003). 25-gauge transvitreal limited arteriovenous crossing manipulation without vitrectomy. Mester et al.84 Prospective interventional nonrandomized case-control study. 43 eyes—sheathotomy. 16 eyes additionally + ILM peeling. 25 control eyes. 6 weeks. 26 patients (60%) gained ≥ 2 lines of VA. Better result in patients with ILM peeling. ME and intraretinal hemorrhage resorbed in all patients. All patients had isovolaemic hemodilution for 10 days. Opremcak et al.85 Prospective interventional case series. 15 eyes. 6.5 years. Snellen VA improved in 10 patients (67%) by an average of 4 lines vision (range 1–9 lines). In 3 patients resolution of ME but no improvement of VA. Retinal vascular bleeding in 2 patients. Asensio Sanchez et al.86 Prospective interventional nonrandomized study. 13 eyes—sheathotomy, 5 eyes underwent additionally ILM peeling. 12 months. VA improved in 12 patients (92%). Better results in patients with ILM peeling. Lerche et al.87 Prospective nonrandomized intervention case series. 12 eyes – sheathotomy. 3 months. VA (logMAR) improved from 0.74 to 0.56. Mason et al.88 Prospective, nonrandomized, comparative interventional study with concurrent control group. 20 eyes—sheathotomy. 20 control eyes (10 of them without intervention and another 10 underwent grid laser). 14 months (sheathotomy). 19 months (controls). VA improvement: Sheathotomy: from 20/250 to 20/63. Controls: from 20/180 to 20/125 (p = 0.02). 45% of the surgical group had final VA ≥ 20/40 compared with 15% of the controls. Data only for whole control group together. Cahil et al.89 Retrospective non-controlled case series. 27 eyes—sheathotomy. 12 months. Resolution of ME in 8 (29.6%) patients, reduction in 14 (51.8%) and persistence in 5 (18.5.%). Becquet et al.90 Prospective nonrandomized interventional case series. 6 eyes (sheathotomy + ILM peeling. 6 controls (ILM peeling only). 6 months. Significant improvement of VA in both groups. No difference in VA or foveolar thickness between the groups (p = 0.5; p = 0.6 respectively). Martinez-Soroa et al.91 Retrospective interventional case series. 17 eyes—sheathotomy. 6 months. Improvement in VA from 0.26 to 0.4. 53% patients improved ≥ 4 lines (Snellen). Le Rouic92 Retrospective interventional case series. 3 eyes—sheathotomy. 10 months. No improvement in VA observed. All patients with initial VA < 20/40. Dotrelova et al.93 Retrospective interventional case series. 3 eyes—sheathotomy. 12 months. VA improved in 2 patients to 20/40, in 1 patient stabilized (20/180). Shah et al.94 Retrospective interventional case series. 5 eyes—sheathotomy. 6.5 years. VA preoperative in all patients ≤ 20/200. Improved in 4 eyes from 20/30 to 20/70. 1 eye with counting fingers remained unchanged. Crafoord et al.95 Retrospective interventional case series. 12 eyes—sheathotomy. 20 months. VA improved in 9 eyes (75%), in 1 eye (8.3%) remained unchanged and deteriored in 2 eyes (16.7%). 2 patients received additionally 25 mg triamcinolone acetonide at the end of the surgery. Han et al.96 Retrospective interventional case series. 20 eyes—pars plana vitrectomy and dissection of the arteriovenous crossing without separation of the vessels. 10.5 months. In 16 eyes (80%) improved VA ≥ 2 lines. Mean improvement of VA (logMAR) was = 0.44 ± 0.14 (p = 0.016). Steroids Intravitreal Corticosteroids In several nonrandomized comparative studies, intravitreal corticosteroids were successfully used for the treatment of BRVO. Currently published randomized studies are very rare and limited by virtue of evaluating patients with ME of different etiology, making comparisons difficult. In various studied doses from 4 to 25 mg, triamcinolone acetonide (TA) has been reported to be effective99–117 (Table 6). In a randomized, interventional, three-arm clinical trial, Avitable et al.99 compared the results of treating diabetic patients and a small group of BVRO patients with cystoid ME by TA and MLG. From a total of 63 patients, 22 were treated by TA (4 mg), 21 underwent MLG, and in 20 patients these methods were combined (TA + MLG). The greatest improvement in VA was found in patients treated by TA combined with MLG. VA (log MAR) in this group increased significantly from 0.83 at baseline to 0.20 at the end of follow-up 9 months later (p = 0.003). In patients treated by TA, VA improved significantly, from 0.82 at baseline to 0.23 at 9 months after injection (p = 0.04). VA in the group of patients treated by MLG remained the same. The results of this study are limited, however, owing to the different ME etiologies in evaluated patients; only 6 patients had ME secondary to BRVO. Oh et al.100 used a retrospective interventional case series to compare VA after single TA injection (4 mg) in 10 patients with mean duration of ME ≤ 3 months after onset of BRVO versus 10 patients with ME > 3 months after onset. In patients with a disease duration ≤3 months, VA significantly improved from baseline over 6 months of follow-up. However in those with a duration of >3 months, improved VA, though apparent at one month, was not maintained at 3 or 6 months after TA injection. This study is limited by its retrospective design and short follow-up period. Ozkiris et al.101 evaluated the effect of TA injection on persistent ME in BRVO that failed to respond to previous laser photocoagulation. During a mean follow-up time of 6.2 months, best corrected VA (log MAR) improved significantly (p < 0.001) from 1.01 at baseline to 0.55 at one month after the injection. VA after 3 months was 0.56, and at the end of follow-up was 0.62. The authors concluded that intravitreal application of TA may be helpful in patients who do not respond to laser photocoagulation. However, in published studies, the resulting reduced macular thickness and improved VA, is only temporary and requires repeated treatment. One to four times re-application has been reported. Cekic et al.103 performed a retrospective chart review of 13 patients who underwent intravitreal injections with 4 mg TA. Six eyes received a single injection. Repeated injections were performed in 1 eye twice, 4 eyes three times, and 2 eyes four times. During a mean follow-up of 13 months, central foveal thickness decreased by more than 50%. Final VA improved in 7 eyes (range 2–6 Snellen lines), remained the same in 4 eyes (range 0–1 Snellen lines), and worsened in 2 eyes (range 1–4 Snellen lines) compared to baseline. Retinal thickness decreased in all cases, while vision improved in most cases. One of the most common side effects of TA was steroid-induced elevation of intraocular pressure.118 Other complications were infectious endophthalmitis, post-injection steroid-induced cataract, and retinal detachment.119,120 Reported risk of infectious endophthalmitis per injection range was from 0.1% to 1.6%.120 The most recent report by Bhavsar et al.121 found in two large studies-Diabetic Retinopathy Clinical Research Network (DRCR.net) and SCORE (Standard Care versus Corticosteroid for Retinal Vein Occlusion), an endophthalmitis prevalence of 0.05% (one case in the 2009 injections). TABLE 6 Summary of studies evaluating the treatment of macular edema in BRVO by intravitreal application of triamcinolone acetonide (TA = triamcinolone acetonide, VA = visual acuity, ME = macular edema, MLG = macular laser grid photocoagulation) Author Study type Patients Follow-up (mean) Outcomes Comments Avitabile et al.99 Randomized interventional, parallel, three-arm clinical trial. Intravitreal TA (4mg): 22 eyes. MLG: 21 eyes. TA+MLG: 20 eyes. 9 months. TA group: VA improved from 0.82 to 0.23 log MAR (p = 0.04). MLG-group: VA unchanged. TA+MLG group, VA improved from 0.83 to 0.20. logMAR (p = 0.003). Different etiology of ME, only 6 eyes with BRVO. Oh et al.100 Retrospective interventional comparative case series. 20 eyes with ME (4 mg TA) Disease duration: 10 eyes ≤ 3 months; 10 eyes > 3 months. 6 months. Group ≤ 3 months: VA (logMAR) improved from 1.07to 0.63 in 1 month (p = 0.012) and to 0.34 in 6 months (p = 0.005). Ozkiris et al.101 Retrospective, non-controlled case series. 19 treated eyes (8 mg TA). 6.2 months. VA (logMAR) improved from 1.01 ± 0.16 to 0.62 ± 0.22. VA improved in 17 eyes and remained unchanged in 2 eyes. Jonas et al.102 Prospective nonrandomized comparative study. 10 treated eyes (20 mg TA). 20 untreated controls. TA patients: 10.1 months. Controls: 6 months. TA patients: VA increased from 0.27 ± 0.11 to 0.45 ± 0.27 (p = 0.02). Controls: VA decreased significantly (p = 0.007). VA increased higher in non-ischemic group. Significant increase of intraocular pressure in treated group. Cekic et al.103 Retrospective non-controlled case series. 13 eyes (4 mg TA). 13 months. VA: improved in 7 eyes, remained the same in 4 eyes, worsened in 2 eyes. Foveolar thickness decreased in 56% of patients (p < 0.001). VA improvement significantly correlated with patient age (p = 0.026). Lee et al.104 Retrospective, non-controlled case series. 6 eyes (4 mg TA). 149.5 days. Improvement in VA ≥ 2 lines in 5 eyes (83.3%). VA from 20/166 to final 20/106. 3 eyes treated with re-application of TA. Ozkiris et al.105 Retrospective interventional comparative case series. 15 eyes (8 mg TA). 19 eyes MLG. 6.3 months. VA (logMAR) improved in TA group from 0.98 to 0.24 and in MLG group from 1.02 to 0.5 (in both groups p < 0.001). Improvement in TA group was significantly higher than in MLG (p < 0.001). Yepremyan et al.106 Retrospective, non-controlled case series. 12 eyes (4 mg TA). 15.3 months. VA improved >3 lines in 50% of eyes after 1 month and in 42% of eyes at last follow up. 8 eyes developed recurrent ME at an average of 5.5 months after initial TA injection. Cheng et al.107 Prospective nonrandomized interventional comparative study. 16 eyes (4 mg TA). 11 controls (without TA). 103 days in TA-group. 94.5 days in controls. VA (logMAR). In TA-group: improvement from 0.77 ± 0.43 to 0.44 ± 0.43 (p < 0.001). No significant change of VA in controls. Significant reduction of ME in TA-group (P < 0.001). Chen et al.108 Case report. 1 eye (4 mg TA). 3 months. Improvement in VA from counting fingers to 20/80. Eye with macular ischaemia. Chen et al.109 Prospective interventional non-controlled case series. 18 eyes (4 mg TA). All patients completed 9 months, 12 eyes completed 12 months. VA (logMAR) improved from 0.81 ± 0.36 to 0.65 ± 0.3 (p = 0.03) after 1 months, no significant difference in VA after 3, 6, 9 and 12 months. Eye with macular ischaemia. All eyes with macular ischaemia. Tsujikawa et al.110 Prospective interventional non-controlled case series. 17 eyes (vitrectomy + 10 mg TA intravitreal), 12 eyes of them with recurrent ME received sub-tenon 20 mg TA. 12.1 months. 82% of eyes rapid resolution of ME within 2 months (p = 0.041). 12 eyes (70.5%) received sub-tenon TA because of recurrent ME. Final VA (logMAR) improved from 0.74 ± 0.40 to 0.40 ± 0.34 (p = 0.010). 14 eyes with vitrectomy underwent additional phacoemulsification with lens implantation. Karacorlu et al.111 Prospective interventional non-controlled case series. 8 eyes (4 mg TA). All eyes with serous macular detachment. 6 months. After TA regression of ME and serous macular detachment in all eyes. After 6 months recurrence in 2 eyes (25%) re-treatment occurred. Final VA improved in 7 eyes (87.5%). Krepler et al.112 Prospective interventional non-controlled case series. 9 eyes (4 mg TA). 6 months. Significant improvement in reading VA only after 1 month (p = 0.02). No significant improvement in VA for distance. No significant reduction in macular thickness. 5 eyes non-ischaemic BRVO. 4 eyes ischaemic BRVO. Degenring et al.113 Case report. 2 patients: 1 eye BRVO 1 eye CRVO (25 mg TA). 5 weeks. Patient with BRVO improved VA from 0.25 to 0.5. Patient with CRVO from 0.4 to 0.5. Wakabayshi et al.114 Prospective interventional non-controlled case series. 5 eyes with CRVO. 11 eyes with BRVO. All eyes received sub-tenon injection of 20 mg TA. 7 months. 8 eyes (50%) improved VA and 2 eyes (12.5%) had worsening of VA at the time of final examination. Reduction of ME >30% of initial thickness in 13 eyes (81.3%). Because of recurrent ME – in 7 eyes repeated sub-tenon application of TA. 1 eye with BRVO pretreated with laser photocoagulation because of retinal ischemia. Salinas-Alaman et al.115 Retrospective interventional case series. 5 eyes (4 mg TA). 6 months Improvement of VA in 4 eyes. 1 eye underwent re-injection after 3 months because of recurrent ME. Hirano et al.116 Retrospective interventional comparative case-control study. 8 eyes TA-injected group (simultaneous intravitreal and sub-tenon TA injection). 7 eyes vitrectomy with TA group (treated by vitrectomy and intravitreal or sub-tenon TA). 12 months. VA improved significantly from baseline in both the TA-injected (p = 0.0069) and vitrectomy with TA groups (p = 0.0145). There was no significant difference in VA and macular thickness between the two groups. Kuppermann et al.117 Randomized interventional clinical trial. 105 (20 with venous occlusion) eyes in each group: I. 700 μg Posurdex II. 350 μg Posurdex III. controls. 3 months. Improvement of VA of ≥10 lines (ETDRS) 35% eyes in group 700 μg Posurdex, 24% in 350 μg Posurdex and 13% in control group (p < 0.001 versus 700 μg group; p = 0.04 versus 350 μg group). Preliminary report 60 eyes with retinal venous occlusion include eyes with CRVO and BRVO. Most published studies on intravitreal TA for BRVO, however, suffer from two serious flaws: either the designs are not randomized or they often do not clearly differentiate between nonischemic types and ischemic types of occlusion. To compare the effectiveness and safety of standard care versus TA injection in the treatment of ME in patients with CRVO and BRVO, the multicenter randomized study SCORE is ongoing (https://web.emmes.com/study/score). In each of the two disease areas, 630 participants will be randomized in a 1:1:1 ratio to one of three groups: standard care, intravitreal 4 mg of TA, or 1 mg of TA. The follow-up is planned for 3 years. The results are not published as yet. Biodegradable intravitreal implants may allow steroid delivery over a more sustained period, permitting longer duration of action. A multicenter randomized clinical trial which evaluates implantation of dexamethasone 350 μg or 700 μg (Posurdex) versus observation (no therapy) for ME secondary to a variety of retinal disorders (including BRVO) has been reported.117 The preliminary 90-day results of all 315 evaluated patients showed that an improvement in VA of 10 letters or more (in ETDRS) was achieved by a greater proportion of patients treated with dexamethasone 700 μg (35%) or 350 μg (24%), than untreated patients (13%; p < 0.001 versus 700 μg group; p = 0.04 versus 350 μg group). The results were similar for patients with diabetic retinopathy, retinal vein occlusion, or uveitis or Irvine-Gass syndrome. In total, 60 patients with BRVO were randomized 1:1:1 to receive 350 μg or 700 μg dexamethasone or observation (no therapy). In the case of RVO, the effect of the treatment was evaluated only in a common group (CRVO and BRVO patients together): an improvement in VA of 10 letters or more was achieved in 15% of untreated patients versus 31% of patients treated with dexamethasone 700 μg. The number of patients with an increase in intraocular pressure of more than 10 mmHg from baseline anytime during the study was 12% for 350 μg, 17% for 700 μg, and 3% for the untreated controls.117 Periocular Application of Triamcinolone Acetonide Kawaji et al.122 evaluated in 20 patients the effectiveness and safety of trans-tenon retrobulbar injection of 40 mg of TA for ME associated with BRVO after vitrectomy. Improvement in VA was seen in 14 (70%) eyes. Hayashi et al.123 compared in a randomized clinical trial, the short-term effect of intravitreal versus retrobulbar injection of TA for the treatment of ME caused by BRVO. Sixty patients received either a single intravitreal injection (4 mg) or repeated retrobulbar injections (40 mg, three times) of TA. The first injection in the retrobulbar group was given approximately one week after focal laser photocoagulation. Foveal thickness, macular volume, and improvement in VA were significantly better after intravitreal injection than after repeated retrobulbar injections. The need for re-injections was significantly greater in the retrobulbar group than in the intravitreal group. Intravitreal Injection of VEGF Inhibitors VEGF inhibitors are a treatment option for ME associated with RVO that target the disease at the causal molecular level. Randomized studies evaluating the results of treatment of all available VEGF inhibitors (bevacizumab, ranibizumab, and pegaptanib) are ongoing. Case reports, small retrospective or prospective non-controlled studies of VEGF inhibitors in the treatment of ME and retinal neovascularizations secondary to BRVO, have been published.124–140 Rosenfeld et al.124 first reported improved VA and reduced ME measured by optical coherent tomography (OCT) following intravitreal injection of bevacizumab for recurrent ME secondary to CRVO in an eye previously treated by intravitreal TA injection. In a short-term study, Iturralde et al.125 treated 16 eyes of CRVO with ME that had failed intravitreal corticosteroid therapy, and nearly every eye showed some anatomic or VA improvement following bevacizumab injection. In various reports, doses from 1.25 to 2.5 mg bevacizumab have been intravitreally administrated.125–134 The most recently published studies evaluated the results in a group of patients with BRVO combined with patients with CRVO. In all of these studies, bevacizumab injection improved VA and reduced macular thickness measured by OCT within the first 3 to 9 weeks. Few studies are available for BRVO patients alone.126,127 Rabena et al.126 reported a significantly increased VA and reduced macular thickness after treatment with 1.25 mg bevacizumab in a retrospective study of 27 patients with BRVO. Recurrent ME was observed in 6 (22%) patients an average of 2.1 months after the initial injection. These patients were reinjected and all showed moderate to complete reduction in ME. The limitations of this retrospective study are short follow-up and lack of control group. Additionally, most of the eyes in the study were previously treated and thus failed standard treatment, and perhaps represent a group unlikely to benefit from any treatment. All published reports provide evidence that this treatment is well tolerated. The most common adverse events were conjunctival hyperemia and subconjunctival hemorrhage at the injection site. However, the duration of reduced ME after bevacizumab administration is currently unknown. Frequent repeated injections are required to prevent a rebound effect with no clearly defined endpoint.128 Campochiaro et al.129 presented preliminary results of a randomized study in the treatment of BRVO with intravitreal injection of ranibizumab at the 2007 Annual Meeting of Association on Research and Vision in Ophthalmology (ARVO). Patients with ME due to CRVO or BRVO were randomized 1:1 to receive 3 monthly injections of 0.5 or 0.3 mg of ranibizumab. Interim results without regard to treatment assignment, which is unknown, showed that 12 randomized patients with BRVO gained an improvement in VA (in ETDRS) from 21 to 37 letters and a reduction in ME from 508 to 208 μm after 3 months of treatment. The endpoint results are expected to clarify any differences between the treatment groups. Another indication for anti-VEGF drugs are retinal neovascularizations, rubeosis iridis, and neovascular glaucoma. Rapid regression of neovascularizations and compensation of intraocular pressure have been described in several studies.136–139 Intracameral application of bevacizumab as successful treatment of rubeosis iridis and neovascular glaucoma has also been reported.140 Prospective, controlled studies are mandatory to develop standardized treatment protocols that allow safe and effective application of anti-VEGF drugs. Laser Treatment Laser treatment is an established method for use in patients with BRVO. A large number of publications concerning the role of photocoagulation in the management of BRVO have appeared. Various laser techniques can be used: macula grid photocoagulation and the method of arterial crimping for treatment of ME, and peripheral scatter photocoagulation for treatment of retinal and/or disc neovascularization. Macular Grid Laser Photocoagulation The Branch vein occlusion study group remains the largest randomized prospective trial that has evaluated the efficacy of grid-pattern laser photocoagulation for the treatment of ME in BRVO.141 In this study, only eyes with recent BRVO, perfused ME, resolved foveal hemorrhage, VA 20/40 or worse, and no other ocular comorbidities were included. After a 3-year follow-up period, 65% of treated eyes gained improvement of 2 or more lines from baseline, as opposed to 37% of untreated eyes. The number of eyes that lost 2 or more lines was not significantly different in the two groups.141 Parodi et al. published two randomized controlled studies, in which no significant benefit of MLG on VA was found.142,143 MLG is recommended as an effective treatment to reduce the ME in BRVO after a period of 3 to 6 months after onset and following absorption of the majority of hemorrhage if VA is 20/40 or worse.61,62,141 If the fluorescein angiogram reveals macular nonperfusion, laser therapy is not warranted.141 Subramanian et al.65 recommended laser treatment in patients with poor VA (20/200 or worse) secondary to ME due to BRVO, before more aggressive approaches (as intravitreal TA). Argon MLG is usually used for this purpose. However, diode laser (810 nm) and krypton red laser (647 nm) also can be used.63,141,144 Scatter Photocoagulation The randomized controlled study by Branch vein occlusion study group54 reported that peripheral scatter laser photocoagulation significantly reduced the development of retinal neovascularization and vitreous hemorrhage. This study also demonstrated that, if all eyes with large retinal nonperfusion were treated, 64% of these patients would never develop neovascularization. If only the eyes that develop neovascularization were treated, the events of vitreous hemorrhage would decrease from 61% to 29%. Since loss in the lower part of the visual field can produce marked disability and BRVO involving the superior retina is common, a significant worsening of visual fields with laser treatment becomes a very important, clinically relevant finding.145 Therefore, waiting is generally advocated until neovascularization actually develops before scatter photocoagulation is considered.54 Arteriolar Constriction An alternative type of laser treatment involves arteriolar constriction (called also “crimping technique”) and may be considered in order to reduce the inflow into the affected area if the ME is excessive. This procedure was first described by L'Esperance146 in 1975. It may lead to a decrease in arterial pressure in the occluded region resulting in better drainage of the ME due to reduced blood inflow. The technique is employed by placing coagulations at approximate intervals of ½ disc diameter (using the green beam of argon laser) through the afferent arteriole in the region of venous blockage. In 1984 Jalkh et al.64 proposed their own modification of this method and published the results obtained in 41 eyes. In this study, arterial constriction was applied in the treatment of the chronic stage of BRVO. Rehak et al. published several studies describing the modified arteriolar constriction in patients with BRVO.147–149 This technique consists of the application of coalescent coagulation spots through the afferent arteriole that supports the occluded venous region. 83% of patients treated by this method within the first 2 months after the onset of occlusion achieved a final VA 20/40 or better.149 In a study by Erdol and Akyol,150 the improvement in VA was higher in a group of patients receiving the MLG combined with arteriolar constriction than in a group treated by MLG only. However, the difference in the resolution of ME between the groups was not statistically significant. The authors suggest that arteriolar constriction in addition to grid pattern laser photocoagulation is more effective for resolving ME in patients with BRVO. CONCLUSIONS The pathogenesis of BRVO is multifactorial. Its resulting visual loss is due primarily to ME, macular nonperfusion, and retinal neovascularization. A large number of treatments have been advocated in its management. Unfortunately, almost all of these lack sufficient evidence for their effectiveness. Randomized prospective trials are essential. The only one established treatment for ME is macular grid photocoagulation in patients with BRVO longer than 3 months and a VA of 20/40 or worse. Additionally, the initial VA may play a crucial role in the prognosis of BRVO and determinates the final VA.

Curr_Genet-4-1-2413079

A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein–protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas. Introduction The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model organism for plant biology (Harris 2001; Gutman and Niyogi 2004; Pröschold et al. 2005). It represents one of the simplest photosynthetic eukaryotes, can be easily grown at large scale either photoautotrophically, mixotrophically or heterotrophically, and can be propagated sexually or asexually. Moreover, Chlamydomonas combines a powerful genetics with the availability of unique genetic and genomic resources: all three genomes are fully sequenced (nuclear, plastid, and mitochondrial; Merchant et al. 2007), large mutant collections have been established, and all three genomes are amenable to genetic manipulation by transformation (Hippler et al. 1998; Remacle et al. 2006). One of the few drawbacks of Chlamydomonas is that it has been notoriously difficult to express transgenes to reasonably high levels from the nuclear genome. Use of specialized promoters (Schroda et al. 2000; Fischer and Rochaix 2001) and adjustment of the transgene’s codon usage to that of the highly GC-rich nuclear genome of the alga (Fuhrmann et al. 1999, 2004) helped in some cases, but no general solution to the problem has been found to date. This is highly unfortunate, because a number of valuable tools available in higher plants currently cannot be used routinely in Chlamydomonas. These include all applications of in vivo reporters of gene expression, such as, promoter-YFP/GFP fusions for gene expression analyses and subcellular localization studies, as well as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) for monitoring protein–protein interactions. Recently, codon-optimized reporter genes have been developed for nuclear and chloroplast expression in C. reinhardtii. Two such genes have been developed for nuclear expression: a gfp gene encoding the green fluorescent protein from the jellyfish Aequorea victoria (Fuhrmann et al. 1999) and a luciferase gene (R-Luc) from the sea pansy Renilla reniformis (Fuhrmann et al. 2004). Synthetic luciferase and gfp genes were also designed for chloroplast transformation and successfully used to measure plastid gene expression (Minko et al. 1999; Mayfield and Schultz 2004; Barnes et al. 2005). However, the use of these reporter genes for nuclear transformation is still far from being routine. While both reporters allowed detection or quantitation of the expression of some (fusion) genes (Fuhrmann et al. 1999; Shao et al. 2007), their generally low sensitivity has precluded the universal use of these reporters. To overcome these limitations, we have explored a recently discovered new luciferase for its suitability as a more sensitive reporter of gene expression in Chlamydomonas. The luciferase from the copepod marine organism Gaussia princeps represents one of the smallest and brightest bioluminescent proteins known to date (Tannous et al. 2005; Remy and Michnick 2006). In an ATP-independent reaction, it catalyzes the oxidation of the substrate coelenterazine resulting in light emission at a wavelength of 480 nm. The Gaussia luciferase gene (G-Luc) has been successfully used as a reporter of gene expression in mammalian cells (Tannous et al. 2005) and could also be split and used for the detection of protein–protein interactions by protein fragment complementation assays (Remy and Michnick 2006). It encodes a non-toxic, monomeric protein of only 185 amino acids and this small size may make it particularly suitable for expression in recalcitrant species. We report here the development of the luciferase gene from G. princeps as a sensitive reporter gene for the in vivo monitoring of gene expression in Chlamydomonas. A codon-optimized version of the gene fused to Chlamydomonas expression signals generated more than 7-fold higher bioluminescence activity than the R-Luc gene from R. reniformis. Furthermore, G-Luc displayed drastically higher signal intensity than R-Luc in luminescence imaging (about 40-fold), thus facilitating the in vivo monitoring of responses to environmental stress stimuli in C. reinhardtii. Materials and methods Algal strains and culture conditions Chlamydomonas reinhardtii strain 325 (CW15,mt+, arg7–8) was used in this study (kindly provided by Dr. Christoph F. Beck, University of Freiburg, Germany). Cultures were grown mixotrophically in Tris–acetate phosphate (TAP) medium (Harris 1989) on a rotary shaker at 23°C under continuous irradiation with white light (55 μE m−2 s−1). The TAP medium was supplemented with 100 mg l−1 of arginine when required. Nucleic acid manipulations The coding region of the G. princeps luciferase gene (G-Luc) was synthesized de novo according to the nuclear codon usage of C. reinhardtii (GenScript, Piscataway, NJ). The synthetic G-Luc gene (GenBank accession number EU372000) was ligated as NdeI/EcoRI fragment into the similarly digested PsaD expression cassette (Fischer and Rochaix 2001). Analogously, a codon-optimized R. reniformis luciferase gene (R-Luc; Fuhrmann et al. 2004) was cloned as NdeI/EcoRI fragment into the PsaD cassette. For inducible expression, the G-Luc coding region was excised by digestion with HincII and BamHI and inserted into an inducible expression cassette driven by the Hsp70A promoter (Shao et al. 2007; Fig. 1b).Fig. 1Codon usage optimization of the G. princes luciferase and construction of expression cassettes for the transformation of Chlamydomonas reinhardtii. a Adaptation of the G-Luc gene to the codon usage in the nuclear genome of Chlamydomonas reinhardtii. The relative frequencies of the individual codons of the native G-Luc gene (GenBank accession number AY015993) in the Chlamydomonas nuclear genome are indicated by grey bars with the most frequently used triplet in Chlamydomonas set to 100%. The synthetic G-Luc gene (diamonds) was optimized by changing all codons to the most frequently used ones in Chlamydomonas reinhardtii. bG-Luc and R-Luc expression cassettes. To comparatively assess constitutive expression levels, the G-Luc (dark grey box) and R-Luc (hatched box) coding regions were inserted into the PsaD expression cassette (PsaD promoter and 5′ UTR shown as open box; Fischer and Rochaix 2001). Both the R-Luc (Fuhrmann et al. 2004) and the G-Luc genes are optimized with regard to the codon usage in the Chlamydomonas nuclear genome. For inducible expression, the Hsp70A promoter (from position −23 to −285 with respect to the translation initiation codon of Hsp70A) was fused to a gene fragment containing the 5′ UTR and the first three exons of the Hsp70B gene (Shao et al. 2007). The positions of the three heat shock elements (HSE, black boxes) within the promoter region are indicated. Light grey bars represent exons, introns are depicted as broken lines. The third exon of Hsp70B was fused to the coding region of R-Luc and G-Luc, respectively. The 3′ UTR of the reporter gene cassettes is derived from RBCS2, a nuclear gene for the small subunit of Rubisco (Shao et al. 2007) Nuclear transformation of Chlamydomonas Chlamydomonas nuclear co-transformation was carried out using the glass bead method (Kindle 1990). Plasmid DNA used for transformation was purified by PEG precipitation. Prior to transformation, plasmid pCB412 containing the C. reinhardtiiARG7 gene as selectable marker was linearized by digestion with EcoRI; all Luc constructs were linearized with ScaI. Co-transformation and selection for arginine prototrophy were employed to introduce the Luc constructs into the Chlamydomonas nuclear genome. Arginine prototrophic clones were selected on TAP medium. Transformants harboring the Luc constructs were identified by luciferase assays. Bioluminescence assays Coelenterazine (P.J.K. GmbH; Kleinblittersdorf, Germany), the substrate for G-Luc and R-Luc, was dissolved in ethanol (1 mM stock solution). To assay luciferase activity, C. reinhardtii cultures were grown in liquid TAP medium under constant illumination (55 μE m−2 s−1) to a final cell density of 3–6 × 106 cells ml−1. After sampling, cells were spun down, resuspended in the same volume of sample buffer [1.5 mM Tris–HCl (pH 7.8), 1 mM EDTA], and frozen at −20°C for at least 20 min. After thawing, 20 μl samples were transferred to 96-well, white microtiter plates and 125 μl of the assay buffer [0.1 M K2HPO4 (pH 7.6), 0.5 M NaCl, 1 mM EDTA] was added to each well. Following incubation at room temperature for 15 min in the dark, bioluminescence was assayed using a luminometer (MicroBeta TriLux; PerkinElmer) by auto-injecting the substrate (coelenterazine 0.01 mM; 50 μl per well). The luminescence units are presented as luminescence counts per second (LCPS). The background was normalized by measuring wells containing only buffer or buffer with cells lacking the Luc gene. Inducible expression of the Luc fusion genes was normalized to the chlorophyll content of the cultures (Porra et al. 1989). The induction factor was calculated by comparison with untreated cells. For in vivo luminescence imaging, C. reinhardtii cultures were grown in TAP medium under constant illumination (55 μE m−2 s−1) to a final density of 1–2 × 106 cells ml−1. A volume of 3 μl cell suspension were spotted onto TAP agar plates and incubated under constant illumination (55 μE m−2 s−1) for 4 days. Luminescence of the cells was visualized in the presence of the substrate (coelenterazine 0.05 mM) using an ultra sensitive Photon Counting Camera (C2400-30H; Hamamatsu). Samples imaged in the absence of the substrate served as control. The luminescence images were acquired and processed with the HPD-LIS software (Hamamatsu) using an integration time of 20 min and linear signal intensity. RNA gel blot analyses Total cellular RNA was extracted according to published protocols (von Gromoff et al. 1989). RNA samples (15 μg total RNA) were electrophoresed in formaldehyde-containing 1% agarose gels and blotted onto Hybond XL membranes (GE Healthcare). To produce a hybridization probe for detection of G-Luc transcripts, the coding region of the gene was excised from a plasmid clone. A Hsp70A-specific probe was prepared from a plasmid clone originally described as hsp70-2 (von Gromoff et al. 1989). The probes were purified by agarose gel electrophoresis following extraction of the DNA fragments of interest from excised gel slices using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and then radiolabeled with 32P-dCTP using the MegaPrime kit (GE Healthcare). Hybridizations were performed at 65°C in Church buffer (Church and Gilbert 1984). Results Design of the Gaussia luciferase as reporter gene for Chlamydomonas Bioluminescent proteins are widely used as reporter genes to measure gene expression, determine subcellular protein localization, and study protein–protein interactions. Luciferases are nontoxic, bioluminescent reporter proteins suitable to monitor gene expression quantitatively. Unfortunately, the previously constructed Chlamydomonas-specific Renilla luciferase (Fuhrmann et al. 2004) suffers from low sensitivity, presumably due to low expression levels and/or low protein stability. As in mammalian cells, the luciferase from the marine copepod G. princeps proved to be a much more sensitive reporter than firefly and Renilla luciferases (Tannous et al. 2005); we set out to test this luciferase as a reporter gene in C. reinhardtii. Previous work had shown that, in Chlamydomonas, adaptation of the codon usage of trangenes significantly improves expression levels (Fuhrmann et al. 1999, 2004). As the codon usage in the native luciferase from G. princeps deviated strongly from that in nuclear genes of C. reinhardtii, we adjusted all codons to the most preferred triplets in Chlamydomonas (according to the codon usage table for Chlamydomonas: http://www.kazusa.org.jp/codon; Fig. 1a). Following this codon optimization in silico, the gene was resynthesized and will be subsequently referred to as G-Luc standing for Gaussia luciferase gene (GenBank accession number EU372000). In all subsequent experiments, G-Luc was compared side-by-side to the Renilla luciferases (Fuhrmann et al. 2004), referred to as R-Luc. Both luciferase genes were cloned into two different expression cassettes: (1) the PsaD cassette (Fischer and Rochaix 2001), whose promoter is constitutively active at least under photosynthetic conditions and (2) an inducible expression cassette driven by the Hsp70A promoter fused to the 5′ region of the Hsp70B gene (Shao et al. 2007; Fig. 1b). This heat shock gene promoter was shown previously to positively respond to a variety of inducing signals, including heat stress, light, retrograde signals from the plastid, and reactive oxygen species (von Gromoff et al. 1989; Kropat et al. 1997; Kropat and Beck 1998; Schroda et al. 2000; Shao et al. 2007). All gene constructs were introduced into arginine-auxotroph Chlamydomonas cells by glass bead-mediated co-transformation followed by selection for arginine prototrophy. Expression and heat inducibility of the Gaussia luciferase reporter To compare the sensitivity of the Chlamydomonas-specific G-Luc as a reporter gene with that of the previously designed R-Luc, 24 transgenic clones from each construct with the constitutive PsaD promoter were randomly chosen and the six best-expressing ones were assayed for their luciferase activities. While only three out of six best R-Luc clones had significant luciferase activity, all six G-Luc clones showed high activity (Fig. 2a). Moreover, when the activities were compared quantitatively, the G-Luc clones displayed, on average, more than 7-fold higher bioluminescence signal intensity than the R-Luc clones (Fig. 2a), indicating that the new G-Luc is more sensitive and more efficient than previously established reporter genes for Chlamydomonas. The very high luciferase activities measured indicate that G-Luc will also be suitable for reporting expression from promoters that are considerably weaker than the PsaD promoter.Fig. 2Comparison of G-Luc and R-Luc activity in transgenic Chlamydomonas reinhardtii strains. a Expression of G-Luc and R-Luc under the control of the constitutive PsaD promoter. For each construct, bioluminescence assays with six independent transformants were performed using a luminescence counter. The bars represent the mean of three independent experiments. The standard deviation is indicated. b Heat induction of the G-Luc and R-Luc reporters under the control of the Hsp70A promoter. Expression of luciferase in transformants harboring the PHsp70A-Luc constructs was induced by a temperature shift from 23 to 40°C for 1 h. After a 1 h recovery phase at room temperature, luciferase activity was assayed and the induction factors were calculated by comparison with untreated samples. Four arbitrarily chosen transformants were assayed for each luciferase construct. The bars represent the mean of three independent experiments, the standard deviation is indicated. The basal expression levels of the transformants under non-inducing conditions were 105, 5, 133 and 2 LCPS μg−1 chlorophyll for the four R-Luc clones and 4, 5, 37 and 18 LCPS μg−1 chlorophyll for the four G-Luc clones Next we wanted to compare the two luciferase genes when expressed under the control of the inducible Hsp70A promoter. To this end, inducibility was determined by measuring luciferase activities in the uninduced and induced states for the four best-expressing co-transformants from each construct (identified among 32 randomly picked clones). Transgene expression was induced by shifting the growth temperature of the algal culture from 23 to 40°C for 1 h. While background expression under non-inducing conditions was comparably low in R-Luc and G-Luc transformants, the G-Luc transformants showed much higher bioluminescence under inducing conditions (on average more than 7-fold; Fig. 2b). This confirms the higher sensitivity of the G-Luc reporter for another expression cassette (Hsp70A promoter + RbcS terminator) and, moreover, indicates that G-Luc can be used as a highly sensitive reporter gene for measuring inducible gene expression in C. reinhardtii. Luciferase imaging and assessment of protein stability One of the most powerful applications of luminescent reporter proteins is their use in genetic screens for mutants in cellular signal transduction pathways. This usually requires detection of the reporter gene activity by imaging techniques to facilitate high-throughput screening of mutagenized organisms. Unfortunately, due to the lack of sufficiently sensitive reporters, this has not been possible in Chlamydomonas to date. We, therefore, were interested in testing whether the sensitivity of our new G-Luc reporter gene was sufficiently high to allow visualization of gene expression by luciferase imaging. To this end, we assayed luciferase activity from both the constitutive and the inducible expression constructs in vivo using a photon-counting camera. Even the best-expressing clone with R-Luc controlled by the PsaD expression cassette (Fig. 2a) did not show enough luminescence to be detectable by luciferase imaging (Fig. 3a). In contrast, G-Luc activity was sufficiently strong to be readily detectable (Fig. 3a). Similar results were obtained for inducible expression from the Hsp70A promoter. While R-Luc activity was barely above the detection limit, inducibility of G-Luc expression was detected with high sensitivity (Fig. 3b).Fig. 3In vivo assay of Gaussia luciferase and Renilla luciferase activities in Chlamydomonas reinhardtii by visualizing luminescence with a photon-counting camera. Left panels show photographs of the algal colonies prior to luminescence imaging, right panels show the luminescence images. The clones with the highest luciferase expression levels in Fig. 2 were used. a For detection of luciferase activity in living algae, wild-type cells (WT) and transformants harboring the PpsaD R-Luc or PpsaD G-Luc constructs (strains R1 and G9 from Fig. 2a) were spotted in three replicas onto agar-solidified medium and grown under normal light conditions (55 μE m−2 s−1) for 4 days. The ratio of luciferase signal intensities of PpsaD G-Luc:PpsaD R-Luc was approximately 40 (40.4 ± 2.5); the signal intensity of PpsaD R-Luc was only slightly above background (PpsaD R-Luc: WT = 3.4 ± 1.5). b Luminescence of the PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken with a photon-counting camera. Luminescence intensities are color-coded with the maximum set to 1.3 × 105 Different possible explanations can account for the much better performance of G-Luc compared to R-Luc: higher expression rates, higher enzymatic activity or higher stability of the Gaussia enzyme. To distinguish between these possibilities, we performed stability assays by measuring luciferase activities in dependence on the temperature. To this end, algal cultures were subjected to 30 min of high temperature incubation followed by a 30 min recovery phase at room temperature prior to measurement of luciferase activity. If the Gaussia enzyme were indeed more stable than the Renilla enzyme, its activity should decline less sharply with temperature. This was indeed the case (Fig. 4a): while the Renilla luciferase suffered a strong temperature-dependent decline in activity, the Gaussia enzyme was much less affected, suggesting that higher enzyme stability contributes substantially to the superior performance of G-Luc.Fig. 4Monitoring the heat stress response in Chlamydomonas strains expressing luciferase constructs driven by the heat-inducible Hsp70A promoter. a Assessment of the thermostability of the Gaussia and Renilla luciferases. PpsaD-Luc transformants were subjected to 30 min of high temperature treatment as indicated followed by a 30 min recovery phase at room temperature. Luciferase activities were measured with a luminescence counter. Note the high temperature sensitivity of the Renilla enzyme with all activity being lost at temperatures above 46°C. In contrast, the Gaussia enzyme appears to be much more stable, facilitating its use as a sensitive reporter of heat stress-induced gene expression. b Visualization of the heat stress response with G-Luc. For luciferase imaging, wild-type cells (WT) and transformants harboring PHsp70A R-Luc, PHsp70A G-Luc or P∆HSEG-Luc (G-Luc fusion to a Hsp70A promoter lacking the HSE region; Shao et al. 2007) were spotted in three replicas on agar plates, grown under normal light conditions (55 μE m−2 s−1) for 4 days, photographed (left picture), and then shifted from 23 to 47°C for 15 min. After 3.5 h at room temperature (RT), the luminescence of the transformants upon substrate addition was recorded with a photon-counting camera (right picture). c Analysis of the kinetics of luciferase induction in a PHsp70A G-Luc transformant exposed to heat stress. Colonies of the PHsp70A G-Luc strain growing on a TAP agar plate were exposed to 40 or 47°C for 15 min. After 1, 3 or 5 h recovery at RT, the luminescence of the colonies was visualized using a photon-counting camera. d Comparison of the induction of G-Luc expression by heat stress with induction of the endogenous Hsp70A gene at the mRNA level. Expression from the Hsp70A promoter was induced by a 15-min incubation at 40 or 47°C, and then followed over time by incubation at room temperature (RT) for the time spans indicated. The induction kinetics of luciferase expression from the Hsp70A promoter parallels that of the endogenous Hsp70A gene To explore the heat inducibility of G-Luc under the control of the Hsp70A promoter in somewhat greater detail, we sought to identify optimum experimental conditions for conducting genetic screens for signaling mutants. We, therefore, tested different combinations of temperatures of the heat shock and recovery times and also included a control construct, in which the heat-shock elements (HSE) were deleted from the Hsp70A promoter (Shao et al. 2007). As expected, this deletion completely abolished heat inducibility under all conditions tested (Fig. 4b and data not shown). Efficient heat induction of the G-Luc reporter was achieved in a wide temperature range, from 40 to 47°C (cp. Figs. 3b, 4b). However, heat shock at higher temperatures required longer recovery times at room temperature before luciferase activity could be visualized by imaging. Whereas following heat shock at 40°C, maximum bioluminescence was measured after 1 h recovery, a recovery phase of 3 h was required to obtain similarly high bioluminescence after a heat shock at 47°C (Fig. 4c). Next we wanted to confirm that heat induction of luciferase activity parallels G-Luc mRNA accumulation. This was clearly the case upon both induction at 40°C and induction at 47°C (Fig. 4d). At both temperatures, mRNA levels peaked at about the same time as enzyme activities (cp. Fig. 4c, d). Moreover, the kinetics of G-Luc mRNA accumulation correlated, by and large, with heat induction of the endogenous Hsp70A gene (Fig. 4d), ultimately confirming that the luciferase reporter faithfully mirrors promoter activity. Having established that G-Luc expression can be readily monitored by luminescence imaging, we finally wanted to provide a quantitative assessment of the superior performance of the G-Luc reporter by direct luciferase imaging of primary transformants. To this end, Petri dishes with transformed Chlamydomonas colonies were exposed to the substrate and analyzed by luminescence imaging (Fig. 5). While transformation with G-Luc produced a high number of brightly luminescing colonies, R-Luc luminescence was much lower and barely detectable (Fig. 5). These data ultimately confirm the much higher sensitivity of the G-Luc reporter and its suitability for luminescence imaging.Fig. 5In vivo assay of Gaussia luciferase and Renilla luciferase activities in primary Chlamydomonas transformants by visualizing luminescence with a photon-counting camera. Co-transformation experiments were conducted using identical amounts of linearized DNA (500 ng Luc-containing plasmid + 100 ng ARG7-containing plasmid) and identical amounts of algal cells. Upper panels show photographs of the algal colonies prior to luminescence imaging, lower panels show the luminescence images. Luminescence intensities are color-coded with the maximum set to 1.3 × 105. a Luminescence of PpsaD-Luc transformants. The ratio of total-plate luminescence of PpsaD G-Luc:PpsaD R-Luc was approximately 36. b Luminescence of PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken. The ratio of total-plate luminescence of PHsp70A G-Luc:PHsp70A R-Luc was approximately 16. Note that a large fraction of the non-luminescing colonies is not co-transformed (i.e., harbors the selectable marker gene but not the luciferase reporter) Discussion In this work, we have established the luciferase from the marine copepod G. princeps as a novel and highly sensitive bioluminescent reporter in the model alga C. reinhardtii. The G-Luc reporter outperforms previously developed reporter genes for Chlamydomonas, improves the monitoring of gene expression and, most importantly, represents the first nuclear reporter gene that is sufficiently sensitive to facilitate in vivo imaging in Chlamydomonas. This expands the toolbox available for Chlamydomonas genetics and cell biology and will make possible experimental approaches that theretofore could not be taken in Chlamydomonas. First and foremost, the possibility to conduct large-scale mutant screens by bioluminescence imaging of live algal colonies will facilitate powerful genetic strategies for the isolation of novel components of all those signal transduction cascades that modify gene expression by targeting specific promoters. For example, the inducible promoter used in this study (Hsp70A) is the target of several distinct signal transduction pathways in response to heat, retrograde signals from the chloroplast, and reactive oxygen species (Shao et al. 2007). Mutagenesis of our algal strains expressing G-Luc from this promoter (Fig. 4b, c), followed by selection for mutants incapable of inducing the luciferase gene in response to a specific stress stimulus, should allow the genetic dissection of the underlying signal transduction pathways. Our preliminary results indicate that reactive oxygen species, which are much weaker inducers of the promoter than heat stress (Shao et al. 2007), induce G-Luc expression sufficiently strongly to facilitate such a screen in a microtiter plate format, although screening on agar plates will require further optimization and improvement of the assay sensitivity. In mammalian cells, a split version of the Gaussia luciferase was successfully used for the detection of protein–protein interactions in vivo by protein fragment complementation assays (Remy and Michnick 2006). As currently no method is available to identify protein–protein interactions in Chlamydomonas cells, the development of screens for protein interaction partners seems to be a particularly promising future application of the G-Luc reporter. It should be noted that thus far, the G-luc reporter gene has been tested only in cell wall-deficient Chlamydomonas strains, which are easily transformable. It remains to be tested whether substrate uptake or luminescence imaging are influenced by the more rigid walls present in strains with wild type-like cell wall structure. Although the Chlamydomonas-specific G-Luc described here provides a workable reporter of gene expression that is significantly more sensitive than previously established reporter genes, our data indicate that the superior performance of the Gaussia luciferase gene is not due to its better expression in C. reinhardtii, but rather due to its very high enzyme stability. Most probably, the adaptation of other widely used reporter genes to C. reinhardtii (like the genes for the fluorescent proteins GFP and YFP) will require a general solution to the transgene expression problem in Chlamydomonas. This could be achieved by either developing novel expression tools or generating dedicated expression strains in which the suspected epigenetic transgene silencing mechanism is inactivated.

Osteoporos_Int-3-1-1820756

Bone loss in elderly men: increased endosteal bone loss and stable periosteal apposition. The prospective MINOS study

Introduction Longitudinal studies on the age-related bone loss in men concerns the decrease in areal bone mineral density (aBMD), which can be qualified as “apparent bone loss” because it does not reflect the change in bone mineral content (BMC). Loss of BMC can be referred to as “net bone loss” because it does not take into account the morphological basis of the bone loss (decreased periosteal apposition; endosteal bone loss, i.e. bone loss on the trabecular, endocortical and intracortical surfaces). The aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and age-related changes during a prospective follow-up in a large cohort of elderly men. Introduction Osteoporosis in elderly men is a major problem of public health. Fracture incidence increases exponentially with age, thus, total number of fragility fractures in men increases rapidly, mainly due to the increasing life expectancy [1]. Currently, 25–30% of fragility fractures occur in men [1, 2]. Moreover, postfracture morbidity and mortality are higher in men than in women [3, 4]. Bone loss is defined usually as the rate of decrease in areal bone mineral density (aBMD) measured by dual X-ray absorptiometry (DXA) [5–7]. However, aBMD of a given region of interest (ROI) is determined by its bone mineral content (BMC) and by its projected area. For tubular bones, ROI length is kept constant, and the projected area depends on bone width. Bone width increases with ageing due to the periosteal apposition [8, 9]. Thus, if the amount of bone deposited on the periosteal surface and that resorbed on the endosteal surfaces (i.e. trabecular, endocortical and intracortical) are equal, BMC remains constant, but aBMD decreases because bone width and its projected area have increased. Therefore, it is more appropriate to refer the decrease in aBMD assessed longitudinally as “apparent bone loss”. Changes in BMC of a given ROI are determined not only by the quantity of bone resorbed on the endosteal surfaces but also by the quantity of bone deposited on the periosteal surface. Thus, change in BMC does not reflect the quantity of bone really lost on the endosteal surfaces and, therefore, the decrease in BMC is better qualified as “net bone loss”. Estimation of bone gain due to periosteal apposition would allow assessment of the quantity of bone really lost on the endosteal surfaces. This quantity of bone could be referred as to “endosteal bone loss”. In elderly men, apparent bone loss accelerates with age [5–7]. However, the morphological basis underlying this acceleration has not been studied, and it is unclear whether it is related to faster outward displacement of a constant amount of bone, to higher endosteal bone loss and/or to a lower periosteal apposition. Moreover, only a few studies have compared the rate of apparent bone loss at different sites of measurement in elderly men [10–12], although it appears that the morphological basis underlying apprent bone loss may vary according to ROI [13]. Therefore, the aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and the age-related changes during a long-term prospective follow-up in a large cohort of elderly men (the MINOS study). Subjects and methods Cohort The MINOS study is a prospective study of osteoporosis and its determinants in men, which was initiated in 1995 [14]. It is the result of a collaboration between the National Institute of Health and Medical Research (INSERM) and Société de Secours Minière de Bourgogne (SSBM) in Montceau les Mines, a town located 130 km northwest of Lyon in the Department (District) of Saône et Loire. The town has a population of 21,000 inhabitants, including 7,150 men > 19 years if age. SSMB is one of the largest health insurance companies in this town. The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983. The MINOS cohort consists of 1,040 men aged 19–85 recruited between 1995 and 1998. All men responded to an epidemiological questionnaire covering demographic and behavioural information as well as detailed medical history. Men aged 19–50 were examined once, whereas 790 men aged 50–85 were followed up prospectively for 90 months. Every 18 months, they were invited to attend the follow-up examination composed of an epidemiological questionnaire and DXA measurement. Radiograph of the spine were performed at baseline, 36 and 90 months. Measurements aBMD and BMC were measured at the lumbar spine (L2–L4), hip and whole body using pencil-beam DXA (QDR 1500, Hologic Inc., Waltham, MA, USA) and at the distal nondominant forearm using single energy X-ray absorptiometry (Osteometer, DTX-100, Denmark). The OsteoDyne Hip Positioner System (HPS) was used to minimise hip positioning error. ROI of the femoral neck was positioned perpendicularly to the axis of the femoral neck to cover its narrowest part. When necessary, the femoral neck edges were adjusted manually. The QDR 1500 device was calibrated daily using a lumbar spine phantom, yielding a CV for aBMD of 0.33 %. Twice a month, the Hologic hip phantom was measured, yielding a long-term CV of 0.94 % for femoral neck aBMD and 1.05 % for the femoral neck projected area. Also twice a month, a human lumbar spine embedded in methyl methacrylate was measured. Its long-term CV was 1.07 % for BMC of L2–L4, 1.07 % for the projected area of L2–L4, and 0.62 % for aBMD of L2–L4. At the distal forearm, the distal site includes 20 mm of radius situated proximally to the site where the spacing between the medial edge of the radius and the lateral edge of the ulna is 8 mm. Scans with evident error of positioning were excluded. The densitometer was calibrated daily using a calibration standard for DTX 100; its long-term CV was 0.47 % for aBMD and 0.15 % for the projected area. Dimensions of the vertebral body of the third lumbar vertebra (L3) were measured on the anteroposterior and lateral radiographs of the lumbar spine performed at baseline and at 90 months. The cross-sectional area of L3 was calculated based on the anteroposterior and frontal diameters measured in the narrowest site of the vertebral body. Osteoarthritis of the lumbar spine was assessed, as described previously [14]. The external diameter of bone was calculated as the projected area of ROI divided by its length. The rate of age-related periosteal expansion was calculated as the average annual increase in the external diameter (femoral neck, distal radius and distal ulna), projected area (total hip) or cross-sectional area (L3). Age-related periosteal apposition (ΔBMCPA) was estimated as the mass of bone deposited on the outer surface of bone since the first measurement (Fig. 1). The volume of the ellipsoid cylinder was calculated assuming that the short axis was 0.75 of the long axis (external diameter). Then, the mass of the deposited bone was calculated as the product of the cylinder volume and the volumetric density of bone mineral (1.15 g/cm3) [15]. We assumed that during the follow-up, BMC was determined by baseline BMC, bone mass deposited on the outer surface (ΔBMCPA) and endosteal bone loss (ΔBMCEBL). Thus, ΔBMCEBL can be calculated using BMC at baseline and during the follow-up and the calculated value of ΔBMCPA. The concept of endosteal bone loss does not make any assumption as to its underlying morphological basis (cortical thinning, increased cortical porosity, trabecular bone loss) nor on the proportion of cortical to trabecular bone. It only reflects the loss of bone mineral “inside” bone, which is represented in Fig. 1 by the change of the colour from black to grey. Fig. 1Calculation of bone mass deposited by periosteal apposition (ΔBMCPA) and of endosteal bone loss (ΔBMCEBL) between baseline (t0) and follow-up (t1). Long semi-axes of the ellipse at baseline (R0) and during the followup (R1) are equal to half the external diameter of the region of interest (ROI). Bone mass deposited by periosteal apposition (ΔBMCPA) is the product of the ellipsoid volume and the vBMD of cortical bone (d = 1.15 g/cm3). Coefficient 0.75 reflects bone flattening in the anteroposterior projection in comparison with its diameter in the frontal projection. Endosteal bone loss (ΔBMCEBL) is presented graphically as the brighter colour of the entire cross-sectional area of bone (black at baseline and light grey at follow-up) in order to indicate that the “endosteal bone loss” makes no distinction as to the morphological basis (cortical thninning, increase in cortical porosity, trabecular bone loss) Statistical methods All calculations were performed by using SAS version 8.2 software (SAS Institute Inc., Cary, NC, USA). Correlation between continuous variables was assessed by Pearson’s simple correlation coefficient. Individual slopes were calculated by using simple linear regression. Comparisons of the individual slopes between age groups were performed by analysis of variance (ANOVA) and adjusted for multiple comparisons by Dunnett-Hsu test. Participants attended two to six exams; those who had few measurements, e.g. two, had them over different periods of follow-up (18–90 months). Individual slopes were calculated by using two to six points distributed over 18–90 months, which could influence accuracy of the calculation. We adjusted for the duration of follow-up or the number of measurements to check whether duration of follow-up and number of measurements influenced the results. Each of them entered significant in the majority of models, although they influenced the results only to a limited degree. We present data adjusted for the duration of follow-up because this variable attained higher level of significance in the models and contributed more to the final model. Results Characteristics of the investigated cohort Sixty-five men who abandoned the study after the first examination were, at baseline, older and had lower physical activity but higher prevalence of diabetes mellitus, rheumatoid arthritis and parkinsonism than the 725 men who participated in the follow-up (Table 1). They had lower aBMD and BMC at all sites of measurement except for the lumbar spine. The difference between the groups became weaker after the adjustment for age; however, it remained significant for certain sites. Table 1Comparison of 725 men participating in the prospective study and 65 men lost to follow-up after recruitmentParameterFollow-up (n = 725)No follow-up (n = 65)p value*p value**Age (years)65 ± 770 ± 8< 0.0001Body weight (kg)80 ± 1379 ± 150.29Body height (cm)169 ± 6168 ± 70.21BMI (kg/m2)27.98 ± 3.6427.75 ± 4.510.63Fat mass (kg)22.02 ± 7.4823.23 ± 9.110.28Lean mass (kg)54.54 ± 6.6652.16 ± 7.56< 0.01NSTobacco smoking (%)11.811.60.98Physical activity (h/week)21.8 ± 12.717.2 ± 11.3< 0.005NSPrevalent fractures (%)13.819.70.18Diabetes (%)6.515.7< 0.005Rhumatoid arthritis (%)1.45.7< 0.01Parkinsonism (%)1.55.7< 0.02Lumbar spine BMD (g/cm2)1.031 ± 0.1841.052 ± 0.2130.21Femoral neck BMD (g/cm2)0.845 ± 0.1210.803 ± 0.127< 0.01NSFemoral neck BMC (g)5.111 ± 0.8494.883 ± 0.934< 0.04NSFemoral neck width (cm)4.082 ± 0.3164.123 ± 0.3560.31Trochanter BMD (g/cm2)0.740 ± 0.1090.691 ± 0.121< 0.001< 0.03Total hip BMD (g/cm2)0.966 ± 0.1270.910 ± 0.157< 0.001NSWhole-body BMC (g)2706.6 ± 410.42550.3 ± 472.7< 0.005NSWhole-body BMD (g/cm2)1.210 ± 0.1081.167 ± 0.121< 0.003< 0.05Distal forearm BMD0.524 ± 0.0650.483 ± 0.070< 0.0001< 0.01Ultradistal radius BMD0.430 ± 0.0640.399 ± 0.072< 0.001< 0.05Radius BMD (g/cm2)0.556 ± 0.0680.513 ± 0.075< 0.0001< 0.01Radius BMC (g)2.743 ± 0.4032.527 ± 0.422< 0.0001< 0.01Radius width (cm)2.471 ± 0.2072.468 ± 0.2160.94Ulna BMD0.476 ± 0.0660.438 ± 0.070< 0.0001< 0.01Ulna BMC (g)1.502 ± 0.2441.401 ± 0.246< 0.0001< 0.01Ulna width (cm)1.659 ± 0.1361.684 ± 0.1520.16BMD bone mineral density, BMC bone mineral content, NS not significantp* difference between the groupsp** age-adjusted difference between the groups Characteristics of bone loss BMC and aBMD of the lumbar spine increased significantly (Table 2) faster in men with severe arthritis (5.95 ± 16.15 vs 1.74 ± 10.96 mg/cm2/year; p < 0.0001). At the femoral neck, aBMD decreased, whereas BMC increased. For other sites of measurement (hip, whole body, distal forearm), both aBMD and BMC decreased. The fractional apparent and net bone loss (percentage of initial value) were fastest at the distal forearm and slowest at the whole body. All the above absolute and fractional changes in aBMD and BMC were significantly different from 0 for all ROIs. Table 2Average rate of apparent bone loss [change in areal bone mineral density (aBMD)], net bone loss [change in bone mineral content (BMC)] and of periosteal expansion (increase in bone width or area) as well as the simple correlation coefficients of these variables with age in 725 men aged 50–85 at baseline followed up prospectively for 90 months (the prospective MINOS study)Site of measurementYearly changeCorrelation with ageBone mineral density(mg/cm2/year)(%/year)rp Lumbar spine4.205 ± 14.210.495 ± 2.910−0.0690.07 Femoral neck−2.463 ± 8.305−0.282 ± 1.019−0.154< 0.0001 Trochanter−1.963 ± 8.031−0.276 ± 1.123−0.196< 0.0001 Total hip−4.714 ± 8.451−0.496 ± 0.930−0.213< 0.0001 Whole body−2.081 ± 8.712−0.177 ± 0.723−0.0450.22 Distal forearm−2.937 ± 4.178−0.580 ± 0.870−0.202< 0.0001 Distal radius−2.986 ± 5.340−0.561 ± 1.041−0.180< 0.0001 Distal ulna−3.353 ± 5.131−0.730 ± 1.206−0.119< 0.002 Ultradistal radius−1.823 ± 5.334−0.426 ± 1.285−0.128< 0.001Bone mineral content(mg/year) (%/year) −0.0410.27 L3118.17 ± 552.900.658 ± 2.954 Femoral neck12.67 ± 50.380.263 ± 1.010−0.0510.18 Total hip−226.1 ± 0.687−0.504 ± 1.551−0.120< 0.002 Whole body−7565.9 ± 22500.9−0.294 ± 0.877−0.192< 0.0001 Distal radius−11.26 ± 25.90−0.426 ± 0.978−0.179< 0.0001 Distal ulna−8.64 ± 16.82−0.559 ± 1.164−0.123< 0.001Bone size L3 cross-section (mm2/year)35.64 ± 225.400.167 ± 1.184−0.0560.10 Femoral neck (μm/year)133.1 ± 217.20.321 ± 0.5030.0700.06 Total hip (mm2/year)4.82 ± 42.230.112 ± 0.9150.0070.85 Distal radius (μm/year)60.48 ± 362.660.257 ± 1.4970.0340.36 Distal ulna (μm/year)58.27 ± 156.610.355 ± 0.9370.0130.73 External diameter of the femoral neck, distal radius and distal ulna as well as the cross-sectional area of L3 and the projected area of total hip increased significantly during the follow-up. Fractional increase in bone size varied from 0.17% to 0.36% per year across the sites. Characteristics of the rate of bone loss according to age at baseline At the hip and distal forearm, rate of apparent bone loss (change in aBMD) was negatively correlated with age, indicating and age-related acceleration of apparent bone loss (Table 2). After the age of 70, the apparent bone loss was two (for distal forearm) to 23 (for trochanter) times faster than in men aged younger than 60 at baseline (Fig. 2). A similar trend was found when apparent bone loss was expressed as the percentage of the baseline value of aBMD. Fig. 2Comparison of the absolute values of apparent bone loss [change in areal bone mineral density (aBMD)] according to age group (black bars 50–60 years, pointed bars 61–70 years, white bars > 70 years at baseline). The slopes are significantly different from 0 (p < 0.005–0.0001) for all regions of interest (ROIs) and for all age groups, except for the trochanter in the youngest group Net bone loss (change in BMC) also accelerated with age at the total hip, whole body and bones of distal forearm. In men aged older than 70, net bone loss was two to four times as fast in comparison with men aged younger than 60 at baseline, both for the absolute values of net bone loss and for data expressed as percentage of baseline BMC (Fig. 3). Fig. 3Comparison of the absolute values of net bone loss [(change in bone mineral content (BMC)] according to age group (black bars 50–60 years, pointed bars 61–70 years, white bars >70 years old). The slopes are different from 0 (p < 0.005–0.0001) for all regions of interest (ROIs) and for all age groups In 183 men aged 50–60, rates of apparent and net bone loss were significantly different from 0 for all ROIs, except the trochanter. In 387 men aged 61–70 and in 155 men aged older than 70 at baseline, rates of apparent and net bone loss were significantly different from 0 for all ROIs. The rate of periosteal expansion (expressed as the change in the projected area of bone) was significantly different from 0 for all ROIs and for all three age groups. However, it did not vary across age groups for any site of measurement. Similarly, at distal forearm bones, periosteal apposition estimated as bone mass deposited annually did not change across the age groups (Fig. 4). By contrast, estimated endosteal bone loss was about twice as high after the age of 70 compared with men aged younger than 60 at baseline. Fig. 4Upper panel: comparison of absolute values of annual rates of increase in external diameter at the distal radius and ulna according to age group. Lower panel: comparison of absolute values of rates of deposition of bone mass by periosteal apposition (positive hatched bars) and rate of endosteal bone loss (negative pointed bars) at the distal radius and ulna according to age group. The slopes are significantly different from 0 (< 0.005–0.0001) for periosteal apposition and for endosteal bone loss for both bones and for all age groups. Please note, in the lower panel, scales are different for radius and ulna Discussion In men, aBMD increased at the spine and decreased at the hip, distal forearm and whole body. At these sites, apparent bone loss (change in aBMD) was associated with net bone loss (change in BMC) except for the femoral neck. Apparent and net bone loss accelerated with age, whereas periosteal expansion rate remained stable. At the distal radius and ulna, endosteal bone loss accelerated with age, whereas the rate of periosteal expansion remained stable. Apparent bone loss at the hip and distal forearm was associated with a parallel net bone loss. At these ROIs, endosteal bone loss exceeded the periosteal apposition, and apparent bone loss was determined by outward displacement of lower quantity of bone mineral. Longitudinally assessed age-related apparent bone loss at the hip and distal forearm has been described in several cohorts of Caucasian men [5–7, 10, 16], but their morphological basis has not been investigated. In elderly men, apparent bone loss was associated with net bone loss at the intertrochanter ROI but with a gain in BMC at the total hip and femoral neck (narrow neck ROI) [13]. We found the same trend for the femoral neck but not for the total hip. This discrepancy is surprising because our cohort is younger. As the net bone loss accelerates with age, faster net bone loss (and not bone gain) could be expected in the older British cohort. We confirm longitudinal and cross-sectional data on the gain in aBMD of lumbar spine in elderly men [7, 12, 14, 16–18]. Increase in the cross-sectional area of L3 at the midheight of the vertebral body is determined mainly by the periosteal expansion. This part is less involved in arthritis. Measurements performed on the X-rays allow for exclusion of the pathological calcifications. We do not speculate on the morphological basis underlying the increase in spine aBMD. Vertebral BMC and aBMD in the posteroanterior projection are determined by changes in the vertebral body and the posterior arch. Posterior arch and its processes are built mainly of cortical bone. As their periosteal surface is relatively high, its age-related gain of BMC due to periosteal apposition may be considerable. At the vertebral body, changes in BMC are determined by loss of trabecular bone inside and by the increase in BMC due to the periosteal apposition and the aggravation of arthritis. At the hip and distal forearm, both aBMD and BMC decreased, in agreement with previous studies [6, 16, 17]. Both apparent and net bone loss accelerated with ageing; by contrast, periosteal apposition remained constant. Thus, age-related acceleration of the apparent and net bone loss is determined by the acceleration of bone loss on endosteal surfaces. Apparent and net bone loss were significant in all age groups. In women, menopause results in the acceleration of endocortical resorption and deceleration of periosteal apposition [19]. Thus, after menopause, periosteal apposition continues, as reported previously by Ahlborg et al. [20]. However, it does not increase sufficiently to offset endocortical bone loss. On the contrary, it decreases, which may contribute to the deterioration of bone strength and increased fracture risk. We postulated that postmenopausal osteoporosis is a disease of failed adaptation of bone [19]. However, in men also, age-related increase in endosteal bone loss is not accompanied by an increase in periosteal apposition that would offset the possible loss of strength. Periosteal apposition does not adapt to the endosteal bone loss and remains stable. This imbalance may result in a decrease of bone strength. Thus, in men also, osteoporosis seems to be a disease of failed adaptation. Our data are consistent with the cross-sectional data obtained in elderly men by using the high-resolution peripheral quantitative computerised tomography (hr-pQCT) showing the increase in bone size at the axial and peripheral skeleton, as well as endosteal bone loss characterised by decrease in the trabecular volumetric BMD (vBMD) determined mainly by the decrease in the trabecular number, increase in total marrow area and cortical thinning, indicating endocortical resorption, and decrease in cortical vBMD [21, 22]. The decrease in cortical vBMD is determined mainly by the increase in the midcortical and subendocortical porosity, whereas subperiosteal porosity and real volumetric density of the mineral of cortical bone remain relatively stable during ageing [15, 23]. Our data help in understanding the difference in the age-related increase in fracture incidence. Young men have bigger bones than women, even after adjustment for age, weight and height [24, 25]. Then, aBMD and BMC decrease. Age-related acceleration of apparent and net bone loss is consistent with the exponential increase in fracture incidence. However, in contrast to women, periosteal apposition rate remains stable in men and may partly offset the endosteal bone loss and associated loss of bone strength. In elderly men, bones are larger than in women (because they were larger in youth and age-related periosteal apposition was greater), net bone loss is less (because periosteal apposition continues to deposit bone on the outer surface) and loss of bone strength starts later (because continuing periosteal expansion compensates partly for the loss of bone strength due to endosteal bone loss). These data are consistent with epidemiological observations that in men, fracture incidence increases later and, for a given age, is lower than in women [1, 26, 27]. Our study has limitations. Montceau les Mines is a small town, and its inhabitants may not be representative of the French population. The response rate for the invitation was 23%. Men who abandoned the study after the first examination were older and sicker, although they represent only 8% of the initial cohort. Men who were followed up may have been healthier than the general population, especially in the oldest group. However, this difference would have underestimated the age-related bone loss and its age-related acceleration. A number of men had lower number of DXA scans because they did not attend examinations regularly or abandoned the study before the end of the follow-up. A low number of DXA values and shorter follow-up could influence the accuracy of estimation of slopes. However, adjustment for the follow-up duration or the number of scans did not influence the results. DXA presents limitations in the evaluation of bone width. In very old men, subperiosteal bone mass can be low and not recognised by the edge-detection system. This artefact can underestimate the bone width in elderly men and the age-related increase in bone width mainly in the cross-sectional studies (where the age range is large) but less so in the longitudinal study (where the follow-up period is shorter). The projected area of femoral neck may be overestimated because of calcifications in fibrous tissue. The measured radius site is established by the device. According to the individual anatomy, this site may be more distal (larger and more trabecular) or more proximal (narrower and cortical). Again, this artefact may introduce a bias mainly in cross-sectional studies. Calculation of endosteal bone loss is indirect and based on the assumptions such as uniform bone flattening, constant subperiosteal bone vBMD and proportional periosteal expansion in all axes. By contrast, the advantage of this concept is that we do not make any assumption on the morphological basis underlying the endosteal bone loss (cortical thinning or trabecular bone loss, proportion of cortical to trabecular bone, similar or different rates of trabecular and cortical bone loss, etc.). Finally, our calculation of endosteal bone loss was carried out for the predominantly cortical sites and, although globally consistent with the cross-sectional data obtained by hr-pQCT, may not necessarily apply for the predominantly trabecular sites. In conclusion, in a large cohort of elderly men, age-related apparent bone loss (aBMD) at the hip, distal forearm and whole body was determined by the net bone loss (BMC), except for the femoral neck. Apparent and net bone loss accelerated with age, whereas the periosteal expansion rate (widening of ROI) remained constant. At the distal forearm, age-related acceleration of the apparent bone loss was determined by the higher endosteal bone loss, whereas the periosteal apposition rate (estimated mass of deposited bone) remained constant.

Eur_J_Pediatr-4-1-2292481

Symptomatic asymmetry in the first six months of life: differential diagnosis

Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. The prevalence of an asymmetric positional preference is 12% of all newborns during the first six months of life. The asymmetry is either idiopathic or symptomatic. Pediatricians and physiotherapists have to distinguish symptomatic asymmetry (SA) from idiopathic asymmetry (IA) when examining young infants with a positional preference to determine the prognosis and the intervention strategy. The majority of cases will be idiopathic, but the initial presentation of a positional preference might be a symptom of a more serious underlying disorder. The purpose of this review is to synthesize the current information on the incidence of SA, as well as the possible causes and the accompanying signs that differentiate SA from IA. This review presents an overview of the nine most prevalent disorders in infants in their first six months of life leading to SA. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. We recommend to design a valid and useful screening instrument. Introduction The objective of this descriptive review is to determine and classify the possible causes of asymmetry seen in young infants who have an asymmetric head and/or body posture, as well as to present an overview of the nine most prevalent disorders in infants in the first six months of life leading to the diagnosis of symptomatic asymmetry (SA). Asymmetric infants form an increasing and complicated group of children seen by professionals from various clinical specialties, such as well baby clinic physicians, pediatricians, pediatric physiotherapists, orthopedic surgeons, and plastic surgeons [4, 6, 19, 37, 50, 61]. Asymmetry in infancy is a mostly benign symptom, but in this early phase of life, the differential diagnostics are extensive. The background of the professional influences the way in which associated clinical problems are evaluated. A screening instrument would be helpful. The first step in this process is to synthesize the current information in the literature about differential diagnostics. Twelve percent of all Dutch newborns develop a positional preference in the first few months of life, different from the physiological asymmetry [6]. A “positional preference” is defined as a condition in which the infant’s head is turned toward one side most of the time and active movement to the other side is restricted [4, 6]. About 25% of these infants (approximately 5,000 a year in the Netherlands) are referred to pediatric physical therapists [6]. Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. From the referred infants, the asymmetry is either idiopathic or symptomatic, and originates ante- and/or postpartum [37, 48, 50, 51, 61]. In case of an idiopathic asymmetry (IA), the etiology is uncertain; environmental factors play a major role in the development of the asymmetry [6, 34, 35, 48, 62]. In SA, an underlying disorder, disease, or dysfunction causes the asymmetry. The majority of cases will be idiopathic, but an initial presentation of positional preference might be a symptom of a serious underlying problem. In the last decade, many studies on the appearances of IA have been published. If the focus in diagnostics and pattern recognition is on IA, there is a chance that an SA will be missed [3, 20]. When examining young infants with a positional preference, differentiating SA from IA is necessary to determine the prognosis and to choose appropriate intervention strategies. This review will address the following question: which diagnoses, incidence rates, signs, and symptoms are described in the literature and are thought to cause a symptomatic asymmetrical posture or movement pattern in infants during their first six months of life? Methods Search strategy This review is based on a comprehensive literature search on SA. The following strategy is used: peer-reviewed literature on this topic in journals with a science citation index was searched, as well as clinical textbooks from the various clinical specialties. Computerized bibliographic databases were searched (PubMed, Pedro, Cinahl, and Cochrane Controlled Trials Register), and related papers and their references. General keywords used were: asymmetry, plagiocephaly, torticollis, posture, scoliosis, (differential) diagnosis, and screening. The search then focused on specific diagnoses that might cause asymmetry in infancy condition with the keywords: etiology, tumors, disorders (related to) vision, hearing, central nervous, or musculoskeletal system, obstetric complications, brachial plexus palsy/lesion, clinical syndromes (Grisel, Sandifer), congenital anomalies and syndromes, gastroesophageal reflux, developmental dysplasia of the hip, paroxysmal torticollis, (birth) trauma, and clavicle fracture. Finally, we focused on the incidence and prevalence. The search was limited to citations that included: “all infants, birth–23 months,” had an abstract, were written in English, and the search terms were in the title or abstract. The year of publication was not restricted. When more papers on the same subject were found, the most current studies were chosen. Only diagnoses that could be observed in infants in the first six months of life were included. Unique case reports and innocuous abnormalities that require no specific treatment were not included. Results The prevalence and/or incidence of the various medical diagnoses leading to SA was not always documented. Some disorders had no consistency in their reported incidence rates. The majority of children with a positional preference or asymmetry during the first six months of life are diagnosed with an IA [4, 6, 34, 35, 37, 48, 50, 51]. Table 1 shows a selection of the most frequently detected disorders causing an SA. Table 1Disorders related to symptomatic asymmetry (SA) from the literature searchDisorders with known incidenceIncidence/1,0001. Developmental dysplasia of the hip (DDH)402. Perinatal fracture of the clavicle353. Congenital muscular torticollis (CMT)204. Obstetric brachial plexus palsy45. Central nervous system disorders26. Craniosynostosis/lambdoid suture0.03Remaining groups of disorders7. Congenital abnormalities or malformations Musculoskeletal Chromosomal8. Sensory systems Ocular disorders Hearing disorders9. Acquired asymmetry postpartum in one of the remaining systems (non musculoskeletal) In the last decade, discussion on positional preference leading to deformational plagiocephaly (DP) has increased substantially. A relatively high number of hits found during a search in January 2008 within PubMed resulted in the following: asymmetry (811), plagiocephaly (206), torticollis (225), posture (405), and scoliosis (623). However, when combined with “differential diagnosis” or “screening,” the result decreased to less than 20 each. Differential diagnosis from craniosynostosis was often described [24, 33, 36, 51]. The main designs were retrospective or prospective descriptive studies and reviews. In diagnoses with a low incidence, the studies were predominantly case reports. All diagnoses were classified according to the International Classification of Diseases [1]. The results of the literature search are presented below, starting with the diagnosis with the highest incidence rate. Developmental dysplasia of the hip Developmental dysplasia of the hip (DDH) has a high rate of co-morbidity with congenital muscular torticollis (8–20%) [11] and, to a lesser extent, with postural torticollis or scoliosis [50]. The reported incidence rate in the Netherlands ranged from 3 to 4% of all newborns [4, 11, 48], with 80% being unilateral [5, 56]. The clinical signs which are described include asymmetry in hip abduction and leg length and/or asymmetrical skin folds in the inguinal and upper thigh region. The strong association with other asymmetries warrants a thorough screening on the signs of developmental dysplasia of the hip in infants with an asymmetry. Perinatal fracture of the clavicle A fracture of the clavicle during birth may induce a positional preference in the first weeks of life and, as such, may cause an asymmetry. A perinatal fracture can be an option in the differential diagnostics of asymmetry during the first weeks of life. A co-incidence with other perinatal injuries (like brachial plexus injury) was described by Perlow et al. [49]. The obstetric brachial plexus lesion is described separately. The incidence varies between 0.1 and 3.5% [28, 40, 49, 54], and these fractures usually consolidate within 3 weeks without complications. The clinical presentation can be asymptomatic. When symptomatic, signs include: decreased or absent movement and pain, or tenderness on movement of the arm on the affected side and palpable irregularity along the clavicle [54]. Congenital muscular torticollis Congenital muscular torticollis (CMT) is frequently described in the literature. Unilateral fibrosis or thickening of and tightness in the sternocleidomastoid muscle can cause a characteristic posture of the head and restricted neck movements. The etiology of the pseudo-tumor or mass is unclear [10, 11, 19, 58]. A compartment syndrome due to intra-uterine malposition is the most frequently mentioned etiological hypothesis [10, 12, 19, 39]. An association with birth trauma and breech presentation is mentioned, but the evidence is weak, since CMT is also seen in infants born via a cesarean section [11, 32]. A pseudo-tumor can be palpated in the second or third week after birth. Incidence rates of CMT vary between 0.3 and 2% [10–12, 19, 58]. Ultrasound screening soon after birth has indicated that the incidence rate could be 3.9%. This screening method tends to be especially sensitive in detecting occult cases of fibrosis [10]. Fibroids of the uterus and other intrauterine tumors are described as a possible etiology to an atrophy of the sternocleidomastoid muscle of the child [18]. This phenomenon can cause a unilateral muscular dysfunction and a strong imbalance between both muscles. The distinction between CMT and postural torticollis is not always clear [15]. A CMT is primarily a condition with a structural fibroid shortening of the sternocleidomastoid muscle, visible and palpable in the first weeks of life, as opposed to a postural torticollis that occurs secondary to a positional preference and a DP [6, 37, 48, 51, 61]. Obstetric brachial plexus palsy Palsy of the brachial plexus during delivery is caused by traction or compression of the plexus during labor. In most cases, the upper brachial plexus is affected; in 15% of the patients, hand function is also impaired. The described incidence is 0.1–0.4% [31, 49, 52]. The extent of the neural damage becomes evident during the first six months of life [52], although in severe cases, the inactivity of the extremity is observed from birth onwards. Timely recognition of severe cases is important, since neurosurgical intervention can enhance future capacities. Between 20 and 25% of the infants experience persistent functional impairments [31]. Central nervous system disorders Cerebral palsy (CP) syndromes, in particular, spastic unilateral CP, are neurological disorders that can cause asymmetry [3, 7]. Serious disorders of the central nervous system are generally easy to recognize, but a CP may also be discrete with subtle features. Early diagnosis, before the age of six months, might be difficult [45, 57]. The neurodevelopmental (motor) behavior is an important issue in early recognition: persistent infantile reflexes and abnormal muscle tone, motor delay, abnormal spontaneous movement patterns (especially “general movements”), and poor postural control are more or less predictors of CP [25, 45, 46]. In a review on the epidemiology of CP, the world-wide prevalence of all types of CP is estimated at 0.2% [43]. Hypotonia and developmental delay were also mentioned as causes for developing positional preference and DP. A neurological disorder might be the underlying problem, but it is not always diagnosed at this young age [3, 7, 51]. Craniosynostosis Craniosynostosis, the premature fusion of one or more cranial sutures, is most frequently described in relation to asymmetry and plagiocephaly, possibly as a result of the over-referral of infants with deformational non-synostotic DP to craniofacial or plastic surgery clinics. Primary craniosynostosis is either simple or compound and part of a genetic syndrome [36]. Premature closing of one lambdoid suture is the most frequently mentioned differential diagnosis of DP [24, 33, 36]. The incidence of this single suture craniosynostosis is rare (1–3 cases to 100,000 newborns) [37, 42] and can be clinically differentiated from DP by four major signs: from the vertex view, a trapezoid head shape can be observed, a palpable unilateral ridge, bulging of the unilateral mastoid, and an asymmetric skull base with tilt to the ipsilateral side [24, 33, 42]. The impact of the premature closure of cranial sutures in complex craniosynostosis is impressive: strong progressive deformation of the skull, risk of increasing intracranial pressure, and developmental problems. When evident, timely surgical intervention is warranted [24, 33, 36, 50, 51]. The following three categories are groups of disorders. Clear incidence rates could not be found. Congenital abnormalities or malformations Musculoskeletal congenital malformations must be considered in an asymmetry that is present immediately after birth [3, 7]. Well-known malformations are those of the spine, such as a Klippel-Feil syndrome, hemi-vertebrae, and a hemi-atlas [22, 38, 64]. Exceptional phenomena are hypoplasia or aplasia of the face, neck, or trunk muscles [2]. Patients may show defects in other systems as well, such as syndactyly, deafness, or a congenital heart disease. The co-incidence of defects may be an important sign of a syndrome. An asymmetric development or posture can be an associated finding in a variety of syndromes and abnormalities. These features are often present immediately after birth, but will not always be discovered until a second stage [64]. Local abnormalities, such as a vascular ring (around the trachea) or tracheomalacia, are occasionally an indirect cause of an asymmetric posture [60]. Disorders in sensory systems In the screening of infants with asymmetry, eye movement and/or vision and hearing disorders must be considered. Infants with congenital nystagmus and restrictive or paralytic strabismus may use anomalous head positions to maximize visual function [26, 47, 63]. No clear incidence rates were found. A predictive factor for an ocular origin of torticollis is the family history of ocular problems, in particular, congenital nystagmus. The ocular pathology may be subtle. In case of doubt, infants must be referred to an ophthalmologist [63]. Theoretically, a unilateral hearing disorder can induce a positional preference in young infants. In the literature search, no match was found for hearing loss and torticollis, except in syndromes such as Klippel-Feil or Moebius. A connection between ear malformation and hearing loss is mentioned [23]. Acquired asymmetry, non-musculoskeletal A number of disorders in systems other than the musculoskeletal system can cause a postpartum asymmetry, but the asymmetry is not the only symptom. The disorders have in common that their symptoms are not stable and occur some time after birth. The signs and symptoms can be seen as so-called “red flags” and require immediate medical evaluation. It may be secondary to a trauma [27] or to inflammatory conditions, such as pharyngitis [8, 13, 29, 55]. Grisel syndrome (a non-traumatic atlanto-axial rotatory subluxation following infections of the upper respiratory tract) is often described, but never under the age of six months [7, 19, 29]. Another cause can be related to the cardio-respiratory or the digestive system, such as Sandifer syndrome (fluctuating asymmetry with abnormal body movements and contortions of the neck, associated with gastroesophageal reflux) [9, 17, 21]. The most alarming causes of asymmetry are related to neurological syndromes, such as syringomyelia, epilepsy, high intracranial pressure, postencephalitic syndromes, or life-threatening tumors of the central nervous system [3, 7, 12, 19, 20, 30]. These disorders are mainly described in case studies, without proven incidence rates. The signs and symptoms of these non-musculoskeletal causes are described in Table 2. Table 2Signs and symptoms of acquired symptomatic asymmetry disorders with a low incidenceSigns and symptomsHints for disordersGeneral historyHeavy painRetro-pharyngeal abscess [13]Vomiting/drowsinessIncreased intracranial pressure [30]Lethargy/irritabilityTumor [12, 30], intracranial injury [27]TraumaIntracranial injury [27]Seizures/convulsionsEpilepsy; increased intracranial pressure; Sandifer syndrome [17]Acute onsetInfection, abscess [13]; Grisel syndrome (>6 months) [29, 55]Stridor, dyspneaVascular ring [60]RefluxSandifer syndrome; pathological gastroesophageal reflux [17]FeverInfection, abscess [13]Specific examinationSunset phenomenonIncreased intracranial pressureBulging anterior fontanelIncreased intracranial pressure, intracranial injury [27]Abnormal courseIncreasing head tiltInfection [13]; tumor [12, 30]Recurrent episodesBenign paroxysmal torticollis [9, 21] Discussion Asymmetry in infancy is a condition with a high prevalence in infants in the first six months of life. In the majority of cases, the origin is idiopathic and is often related to environmental factors [6, 15, 34, 35, 37, 62]. This review addresses the possible causes, incidence rates, and symptoms of symptomatic asymmetries due to an underlying disorder, dysfunction, or disease. Not all of the incidence rates could be found, while some inconsistencies were observed in the current literature. The disorders with a high prevalence are well described in epidemiologic studies. The rarer diseases were, most of the time, documented in case reports without incidence rates. The incidence rates mentioned in the studies are inconsistent because of different opinions regarding the operationalization and assessment of the SA. Frequently, psychometric properties of instruments and concepts have not been described or evaluated. Variations in incidence rates (e.g. CMT) are inevitable, considering the variety in inclusion criteria and diagnostic tests used in the studies. The sequence in estimated incidence rates, as proposed in Table 1, is open to debate. A clear description of signs and symptoms was not always presented in the literature. The variety in the etiology of asymmetry is considerable. The level of evidence of the included studies varies. Literature of more than 10 years ago mainly described underlying causes of SA, in particular, non-muscular torticollis [3, 7]. They still turned out to be useful in establishing criteria for differential diagnostic screening and are widely cited in current studies. However, an update regarding new developments in studies on infant asymmetry is needed. The exponential increase of plagiocephaly in the last decade, related to the recommendations to put babies on their back to sleep, is reflected in the objectives of recent studies [4, 6, 37, 41]. They mainly focused on IA and its predispositions, with little attention to SA. A number of recent papers described features to distinguish craniosynostosis from DP. Although craniosynostosis has a very low incidence, craniofacial clinics are deluged with infants with DP [14, 15, 37]. One of the positive effects of this situation is that authors from this background described useful clinical diagnostic criteria for craniosynostosis. van Vlimmeren et al. [61] stated in their review on diagnostic strategies, that asymmetry in infancy is a diagnosis with a large spectrum of features and a multifactorial etiology without consensus on definition, nomenclature, or classification. In the present review, a classification by virtue of etiology is proposed. The dichotomy, symptomatic versus idiopathic, is often used in medicine [16, 44, 53, 59] and fits well with this health problem, since a large number of children have unexplained asymmetry. Although flow diagrams for diagnostic strategies are presented in some reviews [3, 7, 15, 19, 61], clear clinical diagnostic criteria that could be used were not mentioned. The criteria found in the present review might be considered in a future study. An expert validation, such as a Delphi study with clinical experts, could be a next step towards establishing clinical diagnostic criteria as warning flags in young infants with IA or SA. This review presents an overview of the most common disorders underlying SA in infants less than six months of age. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. The endpoint of this review may be a starting document for the creation of a protocol, but it needs additional studies in order for it to become a valid and useful screening instrument.

J_Urban_Health-2-2-1705478

Assessment of Respondent Driven Sampling for Recruiting Female Sex Workers in Two Vietnamese Cities: Reaching the Unseen Sex Worker

Respondent driven sampling (RDS) is a relatively new method to sample hard-to-reach populations. Until this study, female sex workers (FSWs) in Vietnam were sampled using a variety of methods, including time location sampling (TLS), which may not access the more hidden types of FSWs. This paper presents an analysis from an HIV biological and behavioral surveillance survey to assess the feasibility and effectiveness of RDS to sample FSWs, to determine if RDS can reach otherwise inaccessible FSWs in Vietnam and to compare RDS findings of HIV risk factors with a theoretical TLS. Through face-to-face interviews with FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), data were collected about the venues where they most often solicit their clients. These data were used to create three variables to assess whether FSWs solicit their clients in locations that are visible, semi-visible and non-visible. For this analysis, the visible group simulates a sample captured using TLS. Survey results in HIV prevalence and related risk factors and service utilization, adjusted for sampling methodology, were compared across each of the three FSW visibility groups to assess potential bias in TLS relative to RDS. The number of self-reported visible FSWs (HCMC: n=311; HP: n=162) was much larger than those of the semi-visible (HCMC: n=65; HP: n=43) and non-visible (HCMC: n=37; HP: n=10) FSWs in HCMC and HP. Non-visible FSWs in both cities were just as likely as visible and semi-visible FSWs to be HIV positive (HCMC: visible 14.5%, semi-visible 13.8%, non-visible 13.5%, p value = 0.982; HP: visible 35.2%, semi-visible 30.2%, non-visible 30.0%, p value = 0.801), to practice behaviors that put them at risk for contracting and transmitting HIV (injecting drug use—HCMC: visible 13.8%, semi-visible 12.3%, non-visible 5.4%, p value = 0.347; HP: visible 38.9%, semi-visible 23.3%, non-visible 30.0%, p value = 0.378, to have no condom use in the past month —HCMC only: visible 52.7%, semi-visible 63.1%, non-visible 48.6%, p value = 0.249) and to have symptoms of a sexually transmitted infection (STI) in the past year (HCMC: visible 16.1%, semi-visible 12.3%, non-visible 16.2%, p value = 0.742; HP: visible 13.6%, semi-visible 18.6%, non-visible 20.0%, p value = 0.640). There was a difference found among the visible, semi-visible and non-visible groups in HP for no past month condom use (visible 53.1%, semi-visible 79.1%, non-visible 60.0%, p value = 0.009). This study found that RDS was successful at recruiting hidden types of FSWs in Vietnam. Past reports of FSWs in Vietnam have assessed the more visible FSWs as being the most vulnerable and at risk for HIV. Although the number of visible FSWs is much higher than those of the semi and non-visible groups, this study found that the non-visible FSWs are very vulnerable to HIV infection. If prevention programs are targeting and responding to those who are most likely to be assessed (e.g., more visible types of FSWs) then this analysis indicates that a significant proportion of the FSW population at risk for HIV may not be receiving optimal HIV information and services. Introduction and Background Vietnam is in the midst of an HIV epidemic concentrated among injection drug users (IDUs) and, increasingly, female sex workers (FSWs). In recent years, the epidemic in Vietnam appears to be moving rapidly from IDU populations to FSWs through sexual contact with injecting male clients and the adoption of injection drug use behaviors by FSWs. Sexual transmission of HIV from FSWs to their non-injecting clients, who in turn have non-commercial sex partners, greatly increases the probability that HIV will spread to the general population. In 2002, the Department of Social Evils in the Ministry of Labor, Invalids, and Social Affairs estimated that 130,000 women work as sex workers in Vietnam (other estimates put the number much higher).1–3 To understand and respond to HIV transmission patterns in Vietnam, numerous surveys have measured HIV prevalence and related risk factors among FSWs. However, the lack of sampling frames, relative rarity of this population, and their stigmatized and/or illegal behaviors, hinder investigators from obtaining representative samples of FSWs. Further, it is important to recognize the heterogeneity of the sex worker population. Broadly, FSW can be dichotomized into direct and indirect sex workers. Indirect sex workers typically have a primary job or source of income other than sex work, though that job may lead to contacts with potential clients (e.g., bar maids or masseuses). Direct sex workers typically do not have another job. Direct sex workers can be further broken down into street-based, brothel-based, or gatekeeper-based. The latter may involve a formal “pimp”/agent relationship or informal referrals from hotel concierges or taxi drivers. Use of the Internet to find clients is increasing also. There is a concerted effort by the Government of Vietnam to reduce prostitution by placing FSWs in administrative detention in government rehabilitation centers where they can spend anywhere from 1 month to several years. Fear of detention leads FSWs to exercise greater caution and more subtlety on the streets. Recently, FSWs have become even more difficult to reach because the Vietnamese government is imposing penalties (fines, jail sentences and possible closure) on the owners of entertainment establishments where sex is allegedly sold.4 As a result, FSWs will now wait off-site for an establishment owner or some other gatekeeper to contact them at a client’s request. FSWs are also hard to reach because they are highly mobile and move regularly from bar to bar, between districts and towns, and within and to other provinces.5–9 Some FSWs are difficult to locate because they have primary jobs and engage in sex work part-time. For instance, women from rural areas may travel to urban areas to sell agricultural goods and only sell sex as a way to earn extra income.5 Higher paid FSWs, such as those who solicit clients from scooters, the Internet, through agents and over the telephone, have the financial capacity to remain hidden.5,10 Past efforts to survey FSWs in Vietnam, including annual sentinel HIV surveillance, have relied on convenience samples from rehabilitation centers and street-based FSWs. These samples comprise the type of FSWs who are reportedly the most vulnerable, and therefore most at risk, and visible, (i.e., visible enough to be captured by the police or found by public health staff) and do not represent the entire sex work community.5 Further efforts to assess HIV related risk factors among FSWs have used other non-probability sampling methods, such as institutional sampling, targeted and chain referral sampling.11–16 FSWs in Vietnam have also been sampled using a probability sampling method called time location sampling (TLS), also known as time–space or venue–day–time sampling.17,18 However, TLS can only be representative of those FSWs who are easily located at visible sites, thereby missing potentially important information from other types of FSWs. The Vietnamese Ministry of Health, with technical assistance from Centers for Disease Control and Prevention—Global AIDS Program, conducted a survey using a relatively new sampling method to assess HIV prevalence, risk factors and service utilization among FSWs. The goal of the survey was to provide baseline information before increasing numbers of HIV prevention programs targeting these populations were initiated. This sampling method, known as respondent driven sampling (RDS), is a variant of chain-referral sampling that utilizes an incentive for being interviewed and another incentive for recruiting peers (other FSWs) to be interviewed.19–22 Sampling begins by selecting a small number of non-randomly selected members of the target population, known as seeds, to take part in the study. Seeds initiate the chain referral by recruiting a fixed number of peers who, in turn, recruit other peers. The process continues until the target sample size is attained. Links among peers are recorded using anonymous identification numbers. Although RDS is relatively new to public health researchers, it is starting to be used widely in place of other well known types of sampling methods for hard to reach populations. Researchers are considering the advantages of using RDS in place of TLS, the only other probability method for hard to reach populations, to conduct future HIV biological and behavioral surveillance surveys. TLS has been used to sample FSWs who gather and are accessible at public locations (or venues) that can be listed in a sampling frame.23–25 TLS entails identifying days and times when the target population gathers at specific locations (e.g., brothels, city blocks, bars), constructing a sampling frame of time and location units, randomly selecting these units and systematically intercepting consenting members of the target population for interviews.24–26 When implemented correctly, TLS allows researchers to construct a sample with known probabilities, make statistical inferences to the larger population of venue visitors, and theorize about the introduction of biases that may limit generalization of results to the target population.23 Two TLS surveys of FSWs in Vietnam have been reported. In 2000 Family Health International (FHI) conducted a behavioral survey with karaoke (n=2,302) and street-based (n=1,300) FSWs in five Vietnamese cities using TLS.18 During 2003, the Vietnamese National Institute of Hygiene and Epidemiology collected data from karaoke (n=1,367) and street-based (n=976) FSWs from five provinces.17 Although TLS can produce a probability sample by ensuring that time–location units are enumerated and selected randomly, it allows researchers to reach only those segments of the population who are visible and identifiable. Depending on the population of interest, this may introduce a bias by differential sampling of the portion of the population which is most at-risk. TLS can be expected to miss those segments that are less visible (e.g., FSWs who solicit clients through agents, by telephone or Internet). The “visible” portion of a population does not represent the whole population any more than an institution or clinic-based population does; “hidden” population members have a zero probability of selection, reducing representativeness of the sample. The objectives of this paper are to present findings from a survey on HIV prevalence, risk behaviors and service utilization to (1) assess the effectiveness of RDS to sample FSWs in Vietnam; and (2) to compare RDS findings with theoretical TLS. This is especially important given that evaluations of RDS used for HIV surveillance have only come from IDUs and males who have sex with males populations in the United States.20,22,27–29 Until this study, RDS has not been evaluated in Asia among FSWs. This methodology warrants investigation to assess its ability to sample hard to reach populations in international settings. Materials and Methods Data to evaluate RDS were collected from FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), Vietnam. Eligible participants were over the age of 18 and had exchanged sex for money in the previous month. FSWs were recruited by their peers (other FSWs) and provided a small monetary incentive for completing a questionnaire and providing a saliva sample to test for HIV (US \$3 in HP and US \$4 in HCMC). FSWs were provided another incentive for recruiting up to three other FSWs to participate (approximately US \$1 in HP and US \$1.50 in HCMC for each eligible recruit). Data were gathered from FSWs in HP from April to June and in HCMC from May to July, 2004. Recruitment began with 20 FSWs, non-randomly selected by survey staff, in each city. Outreach workers selected these initial recruits, or “seeds,” based on where they most often solicited their clients. A majority of the seeds were from the visible groups, street-based and karaoke-based FSWs. A few seeds from hotels and guesthouses were recruited. Once seeds consented, they completed a one-hour face-to-face interview with a trained Vietnamese interviewer. The interview instrument consisted of questions about the participant’s demographic background, current and past drug use, sexual behaviors, HIV knowledge, risk perceptions and service utilization, and about where they usually solicit their clients. After the interview, survey staff provided participants with three coupons with which to recruit up to three other FSWs who were co-workers, friends, acquaintances or family members. Each coupon contained a unique identification number and provided basic information about the study and the addresses of the interview sites. Respondent/recruiters were asked to return to collect their incentives for successful recruits. A brief follow-up questionnaire was administered during this visit to ascertain the recruiter’s relationship to their recruits and the number and type of refusals they received. This permitted the assessment of participation bias due to refusals or no-shows. Oraquick™ HIV antibody rapid test kits for oral fluids were used for HIV testing. No one refused to provide an oral sample. Participants did not learn their test results at the interview site but were provided a voucher to receive a free HIV test with counseling at a nearby voluntary counseling and testing (VCT) center. For the assessment of bias in a theoretical TLS-drawn sample in comparison to an actual RDS-drawn sample, respondents were classified as accessible, i.e., visible to TLS, if they reached their clients in a public venue. Categories were established from the response to the question, “Where do you usually reach your clients?” Responses were aggregated into three groups: visible (street, park, karaoke bar); semi-visible (brothel, guesthouse, hotel); non-visible (agent, telephone, Internet). Our assumption is that the visible group could potentially be captured by TLS. The semi visible group may or may not be captured with TLS, and the non-visible group would be missed with TLS.1 Statistical Analysis Adjusted proportions and 95% confidence intervals (ci) for FSWs’ level of visibility were analyzed using the RDS analysis tool (RDSAT).30 RDSAT adjusts proportion estimates for participants’ network sizes and recruitment patterns.20,31 RDSAT adjustments use principles of social network theory to account for biases found in conventional chain referral sampling.31,32 Chi-square proportions and p-values for comparisons of HIV risk factors and service utilization of RDS and theoretical TLS were analyzed using SPSS 11.0. Five of the 20 non-randomly selected seeds in the HP sample, and four seeds in the HCMC sample were not used in the final analysis since they did not recruit any peers. It is not known why these seeds did not recruit anyone. The sample size was calculated based on 15% increase in use and knowledge of CDC sponsored VCT HIV services and outreach. We estimated low baseline knowledge and use of services (no actual data available) with high HIV prevalence. HP sample size was 200 based on higher knowledge from existing health education programs. The sample size in HCMC was 400, assuming lower baseline knowledge. Design effect was set at 2. This study was reviewed by the Associated Director for Science of the National Center for HIV, STD and TB prevention at CDC who classified this as a surveillance activity. The protocol for this study was reviewed and approved by the LIFE-GAP Office of the Vietnam Ministry of Health. Findings A total of 628 individuals, including seeds, were interviewed in fixed interview sites in HCMC (n=413) and HP (n=215). The majority of the FSWs in the HCMC (54.6%) and HP (51.3%) sample were under the age of 29 years old. More than 50% of FSWs in HCMC and HP had no occupation other than selling sex. During the follow-up visits to collect recruitment incentives, most participants reported giving out all of their coupons. According to the 174 recruiting participants in HCMC and 90 in HP, each of whom received three coupons, a total of 75 (15.3%) individuals in HCMC and 24 (10%) in HP refused to accept a coupon; 147 recruiters in HCMC and 83 in HP reported that they were successful in distributing their coupons and that no one to whom they tried to give a coupon rejected one; so refusals were clustered among a handful of recruiters, 27 in HCMC and 7 in HP. According to recruiters, most refusers did so because of “fear of identification,” (57% in HCMC; 75% in HP), others “felt uncomfortable” (27% in HCMC; 8.3% in HP), still others thought they were “too busy” (10.7% in HCMC), others thought the “incentive not worth the time” (17% HP) and a small number of FSWs in HCMC responded that they were “not interested.” Overall, the recruited FSWs were diverse with respect to where they solicited their clients. Figure 1 depicts the diversity of sex work types among the links within a single recruitment chain of FSWs in HCMC (n=59 recruits). In this example, the seed recruited three FSWs who were not street-based: two based in guesthouses and one based in karaoke.2 The selected karaoke-based FSW recruited two other karaoke-based and one guesthouse-based FSW. Near the outer waves of this recruitment chain, there are some more hidden types of FSWs such as agent-based and telephone-based FSWs. Figure 1RDS recruitment network for one seed for type of female sex worker in HCMC, Vietnam. The distribution by visibility was visible (HCMC, n=311; HP, n=161), semi-visible (HCMC, n=65; HP, n=43) and non-visible (HCMC, n=37; HP, n=12). Figure 2 shows that the largest adjusted proportions of FSWs in both HCMC and HP reached by RDS were visible [HCMC, 73.7%; 95% ci(67.0%, 79.5%) and HP, 75.9%; ci(67.1%, 85.3%)]. RDS reached a small proportion of non-visible [HCMC, 9.3%; ci(6.0%, 13.1%) and HP, 9.7%; ci(1.3%, 18.2%)] and semi-visible FSWs [HCMC, 16.9%; ci(11.5%, 23.4%) and HP, 14.2%; ci(9.1%, 21.2%)], who theoretically could have been missed by TLS. Figure 2Distribution of visible, semi-visible and non-visible FSWs in HCMC and Hai Phong, Vietnam; adjusted proportions (number). Visible FSWs are assessed to be the most visible of the FSWs in Vietnam; semi-visible FSWs are assessed as not always being easily identifiable; and non-visible FSWs are assessed as the most hidden types of FSWs in Vietnam. HIV Risk Factors There were minor differences among FSW visibility groups with regard to HIV status and related risk behaviors (see Table 1). Table 1HIV prevalence, risk factors and drug use among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value Ho Chi Minh City (n=413)Hai Phong(n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueHIV positive status14.5 (45)13.8 (9)13.5 (5)0.98235.2 (57)30.2 (13)30.0 (3)0.801No condom use with a partner in the past month52.7 (164)63.1 (41)48.6 (18)0.24953.1 (86)79.1 (34)60.0 (6)0.009Ever used non-injection illicit drugs20.9 (65)18.5 (12)29.7 (11)0.38546.9 (76)37.2 (16)60.0 (6)0.338Ever injected illicit drugs13.8 (43)12.3 (8)5.4 (2)0.34738.9 (63)23.3 (10)30.0 (3)0.378Symptoms of a sexually transmitted infection in past year16.1 (50)12.3 (8)16.2 (6)0.74213.6 (22)18.6 (8)20.0 (2)0.640 HIV positive test status did not differ by visibility group in HCMC or HP. In HCMC and HP, a higher proportion of visible FSWs tested positive for HIV (14.5 and 35.2%, respectively) than semi-visible (13.8%, 30.2%) and non-visible (13.5 and 30.0%) FSWs. The only significant difference among the groups for any variable was in HP, for “no past month condom use with a partner” (p value = 0.009). Visibility groups in both HCMC and HP did not differ with respect to reported use of non-injection illicit drugs ever and injected illicit drugs ever or reporting a sexually transmitted infection (STI) during the previous year. Service Utilization Contact with a peer educator (PE), source of HIV information and HIV testing did not vary significantly by visibility group in either city. Among all three visibility groups in both cities, fewer than 50% had ever made contact with a PE. More than 50% of FSWs in both cities (with the exception of semi-visible FSWs in HP) reported receiving their HIV information from television, radio, newspapers or magazines. More HCMC FSWs reported receiving HIV information from friends than from health workers and PEs; this pattern was reversed for HP FSWs (Table 2). Table 2HIV service utilization among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value Ho Chi Minh City (n = 413)Hai Phong (n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueEver met with a peer educator33.8 (105)49.2 (32)40.5 (15)0.31031.5 (51)27.9 (12)40.0 (4)0.720Most common source of HIV information0.0880.106 TV, radio, newspaper or magazine56.1 (171)70.8 (46)52.9 (18)57.5 (92)45.2 (19)70.0 (7) Health worker/peer educator17.0 (52)9.2 (6)20.6 (7)26.3 (42)38.1 (16)30.0 (3) Friends20.7 (63)13.8 (9)26.5 (9)11.3 (18)16.7 (7)0 Other6.2 (19)6.2 (4)05.0 (8)00Ever had an HIV test45.3 (141)46.2 (30)40.5 (15)0.84134.0 (55)27.9 (12)50.0 (5)0.398Type of facility providing last HIV test0.9760.285 Public clinic35.6 (51)43.4 (13)33.3 (5)46.3 (25)16.7 (2)40.0 (2) Hospital43.7 (62)36.7 (11)33.3 (5)29.6 (16)25.0 (3)40.0 (2) Private clinic4.2 (6)3.3 (1)6.7 (1)1.9 (2)16.7 (2)0 VCT center6.3 (9)6.7 (2)13.3 (2)9.3 (5)16.7 (2)2.0 (2) Rehab center9.9 (14)10.0 (3)13.3 (2)13.0 (7)25.0 (3)0 Fifty percent or fewer FSWs in each visibility group reported ever having an HIV test. Among those HCMC and HP FSWs who ever had an HIV test, the majority received their test at either a public clinic (district or community public health center or STI clinic) or hospital, with the exception of HP semi-visible FSWs who reported receiving their HIV test from either a hospital or a drug user/sex worker rehabilitation center. Twice as many non-visible (13.3%), in comparison to visible and semi-visible (6.3% each) FSWs in HCMC, reported receiving their HIV test at a VCT center. Discussion The analysis of RDS coverage demonstrates that this method may provide modestly better estimates for FSW populations than TLS because it reaches individuals ostensibly inaccessible by TLS. The level of risk and HIV prevalence in the less visible groups of FSWs was similar to the semi-visible and visible FSWs. This would indicate that less visible FSWs merit reasonable efforts to assure that these groups are targeted within all interventions for FSWs. The differences might have differed if two groups emerged rather than three. However, the semi-visible group could not be clearly dichotomized. The three visibility groups in HCMC and HP did not differ significantly, which may contradict previous reports that higher paid, indirect FSWs, in general, have less HIV risk than direct FSWs.5,11,33 High proportions of respondents among all visibility groups in HCMC and HP reported inconsistent condom use with a sexual partner in the past month, possibly indicating that all three groups of FSWs are not being appropriately educated about using condoms or that they cannot easily access them. Other possible barriers to increased condom use might include poor negotiating skills with their clients or a false perception that a well-dressed client with an occupation may not be HIV-infected. Understanding the perceptions, negotiation skills and the reasons why some FSWs use condoms and others do not is essential to addressing the low proportion of condom use among FSWs in these cities. The high proportion of non-visible FSWs in HCMC and HP who ever met with a PE indicates that the more hidden FSWs may be receiving more exposure to HIV information than previously believed. HCMC and HP FSWs reported similar sources for HIV information, indicating that PEs may be equally disseminated among the visibility groups. Furthermore the high proportion of non-visible FSWs who ever had an HIV test and their access to HIV testing at public clinics, hospitals and VCT centers indicate that this group has some awareness of and interest in their risk. It would be useful to gather qualitative data to determine how they obtained information and services. Another key finding was the nature of social networks formed by FSWs in Vietnam. Networks were dense and diverse enough to provide a large sample in a short time that crossed the various types, direct and indirect, of FSWs working in the respective cities. These network properties are essential to the successful use of RDS. RDS had an added advantage over TLS by providing some measure of selection bias by questioning respondents on the characteristics of peers who demurred. TLS cannot determine reasons for not using a venue to solicit clients, only for respondents’ refusal to speak with interviewers. There were limitations to our study. Categories of visibility were based on the authors’ perceptions of the sex work industry in the respective cities. We did not conduct extensive fieldwork so there was limited knowledge about the types of FSWs in Vietnam. There was too little formal research about characteristics of hotel and guesthouse-based FSWs to confidently dichotomize them as visible or non-visible. With a better understanding of these types of FSWs, reclassification and reanalysis of the data could better demonstrate the capacity of RDS to reach hidden FSWs. Furthermore, the question “Where do you usually reach your clients?” does not signify where a FSW always reaches her clients. The findings here may have non-differentially misclassified some respondents. Currently RDS data analysis is limited to estimates of proportions. Stratified analysis of the differences in risk factors among visibility groups might yield more information when RDSAT is updated. The best scenario for testing whether RDS captures more hidden elements of the FSW population would be to conduct nearly contemporaneous surveys using TLS and RDS in the same population. Using RDS data to simulate TLS for comparisons of the two sampling methods is subject to any biases unique to RDS that might not be found in TLS. Although non-visible FSWs make up a small proportion of the FSW sample in HCMC, their high HIV prevalence and associated risk factors is alarming, especially when past efforts to sample FSWs, using TLS and convenience sampling, and sentinel surveillance in STI clinics, are likely to have missed these groups. Missing the most inaccessible portions of the FSW population could result in a biased understanding of HIV sero-prevalence and risk factors and, consequently, could lead to inefficient planning for HIV prevention and intervention programs for the entire FSW population. Recurrent round-ups of FSWs drive FSWs off the street, making them increasingly difficult to find in public venues. If current prevention programs are targeting and responding to those who are most likely to be assessed (more visible FSWs) then it is possible that an important segment of the FSW population at risk for HIV is not receiving optimal HIV information and services. However, this study found that segments were accessing services at about the same low rates, indicating a need to increase service utilization among all groups of FSWs. Based on these findings, we recommend using RDS for future surveys of FSWs in Vietnam to prevent the omission of the less accessible members of this population.

Purinergic_Signal-4-2-2377319

Purinergic signaling microenvironments: An introduction

The common theme of this introductory article and the minireviews that follow in this special issue is the concept of microenvironments within tissues and surrounding cells that would be ideal signaling venues for a biologically active purinergic ligand. Collectively, the editors/authors and the other contributing authors agree that nucleotides and nucleosides would be most potent within a confined system. A talented cadre of purinergics has been solicited to discuss purinergic signaling in his or her favorite microenvironment within a given organ or tissue. We are gratified by the large number of original articles that also have successfully navigated the peer review process and are part of this special issue. These concepts are not simply purinergic, but the idea of maximal potency in a tissue microenvironment and surrounding specialized cells within a tissue pertains to any autacoid or paracrine agonist. Introduction Adenosine 5′ triphosphate (ATP) and adenosine are extracellular autocrine and paracrine signaling molecules. The most potent effects of ATP and adenosine are likely limited to extracellular microenvironments within tissues or immediately surrounding a cell or group of cells. These extracellular microenvironments may have a limited area of diffusion, have a small volume, have an ideal ionic composition and pH, represent an unstirred layer near a membrane surface, represent a trapped interstitium, or any and all of the above. If P1 or P2 nucleotide receptors are present on a membrane that is exposed to released nucleotide or nucleoside in this microenvironment, these ligands can engage P1 G-protein-coupled receptors, P2Y G-protein-coupled receptors or P2X receptor channels and affect the function of the cell on which it binds. Besides ATP and adenosine, other forms of nucleotides and nucleosides may also be active extracellular signaling ligands and may have unappreciated receptors. Inosine, thymidine, and even cyclic nucleotides are released by and may have effects on mammalian cells. The self-aggregation signal for the slime mold Dictyostelium discoideum, is cyclic adenosine monophosphate (AMP). Receptors for cyclic nucleotides have only been defined until now in the lower biological systems. Receptors for cyclic nucleotides as well as inosine or thymidine may also exist on mammalian cells and receive this autocrine signal within a microenvironment. Ideal microenvironments for purinergic signaling For simplicity, we focus on ATP and its metabolites. Many different microenvironments have been studied in great detail and are described and included below. One classical microenvironment that has emerged in importance in the cystic fibrosis (CF) research community is in an open system but is a stirred liquid layer trapped between the luminal membrane of the airways epithelium and the humidified air that fills the lung and airways. The airway surface liquid [ASL, also termed airway surface fluid (ASF), the periciliary liquid layer (PCL), and the epithelial lining fluid (ELF)] bathes motile respiratory cilia [1–4]. The humidified air above this layer and the ciliated apical or luminal membrane beneath this layer help close this microenvironment. Submucosal gland secretions may also contribute importantly to its composition. Ric Boucher and his many coworkers have presented many recent seminal contributions on the purinergic signaling that may occur in the ASL—among its many other properties [3, 4]. There are similar microenvironments of this type that are also discussed in review articles within this special issue. Daniel Marcus and colleagues and Claire Mitchell and coworkers discuss liquid microenvironments in the ear and the eye, respectively [5, 6], which provide a medium for purinergic signaling. A second classical microenvironment is a closed and open system that is compromised of a secretory gland or acinus and a collecting or draining duct. Iva Novak and colleagues have done seminal work in the pancreas in this regard [7, 8]. Geoff Burnstock has often discussed the rich purinergic signaling that occurs in tubes or ducts [9, 10]. In this microenvironment, glandular and acinar secretions may convey the purinergic ligands so that they may act in an autocrine manner at the acinus or in a paracrine manner within the duct. However, it is an open system in that these secretions eventually exit this tissue environment. The airway submucosal gland beneath the airway surface epithelium may be one such example that conveys purinergic signals to the surface where they may drive ciliary beat and other functions. The pancreatic acinus and duct, the sweat gland and duct, and the bile secretory apparatus and the bile duct are all important examples. A third microenvironment is a closed system in which the purinergic ligands concentrate within a small volume and within a tissue. Here, the purinergic ligands bind and activate receptors until they are either degraded or reabsorbed by the cells that secreted them or by neighboring cells. The endolymph of the ear and the humors of the eye are ideal examples of such closed systems [11, 12]. Marcus and colleagues and Mitchell and coworkers discuss these systems in minireviews alluded to above [5, 6]. Original articles and reviews in this issue and future issues of Purinergic Signalling speak to such systems in pancreatic islets and in the many renal tubules that comprise the nephron of the kidney [13]. In polycystic kidney and liver disease, tubules remodel into abnormal fluid-filled cysts. Multiple laboratories have argued that the fluid-filled lumen of the encapsulated cyst is a rich yet pathophysiological microenvironment for purinergic signaling [14–20]. The minireviews that follow this introductory article focus on specific microenvironments, cells and tissues that create and/or come in contact with such microenvironments, and all possible modes of purinergic signaling within these microenvironments. An ideal microenvironment paradigm for extracellular purinergic signaling: the synapse Many reviews in the past [21–23] and in the initial issues of this journal have documented well the roles of the P2X receptor channel at the synapse between neurons. Indeed, P2Y receptors may play roles in synaptic transmission as well. This is analogous to the interplay between cholinergic and muscarinic receptor systems in cholinergic neurons and synapses in many different nuclei and regions of the brain. In fact, the purinergic field was born from the classical studies of extracellular ATP on the heart by Drury and Szent-Gyorgyi [24], in the definition of purinergic nerves in the enteric nervous system by Burnstock and colleagues [25], and on the release and effects of purinergic ligands such as ATP on skeletal muscle contractility by Tom Forrester (see “Historical Perspective” by T. Forrester in this issue, [26]). The authors lead this series of minireviews with the idea that the synapse is the ideal and, perhaps, the first or founding microenvironment for autocrine and paracrine purinergic biology. The synapse is designed to have a limited diffusion distance between pre- and postsynaptic membranes where the principal ligand or neurotransmitter is reabsorbed by the presynapse or degraded in the synapse. For the sake of simplicity, our narrative here focused first on a purinergic synapse where released ATP acts as the principal ligand to evoke an action potential in the postsynaptic membrane. ATP as the lead neurotransmitter is released in response to an action potential in the presynaptic bulb that opens voltage-dependent calcium (Ca2+) channels. This voltage-dependent Ca2+ entry signal triggers the fusion or exocytosis of ATP-filled synaptic vesicles so that labile ATP is secreted into the synaptic cleft. Extracellular ATP-gated P2X receptor channels on the postsynaptic membrane have a large extracellular domain poised to bind that ATP immediately upon release within a synaptic microenvironment where there is a short diffusion distance and a small volume. In other words, the synapse is an ideal microenvironment for paracrine purinergic signaling. In this paradigm, the ATP is likely degraded to adenosine diphosphate (ADP), 5′ AMP and adenosine, where adenosine may be recycled back into the presynapse by nucleoside transporters only to be a substrate to synthesize new ATP for metabolism or neurotransmission. Several additional functions for P2X receptor channels have been proposed in neurotransmission driven by other neurotransmitters. For example, at the glutamate synapse, it is well known that ATP is released as a cotransmitter. A classical example of this is the hair-cell sound transduction process, where glutamate and ATP are coreleased to stimulate auditory afferents [11]. How may ATP affect responses to glutamate at glutamate, N-methyl-D-aspartate (NMDA), and kainate receptor channels in the postsynaptic membrane and/or at the presynapse in terms of release of ligand? Many possible cotransmitter effects have also been entertained and studied. We do not wish to debate the relative roles of P2X receptor channels in neurotransmission. The possible explanations of ATP effects at an excitatory glutamatergic synapse are given in simplistic terms. We only wish to show the synapse as a lead microenvironment to illustrate how and why local ATP signaling may be important. First, the presence of a postsynaptic P2X receptor channel or channels would potentiate the excitation of glutamate postsynapse. Not only do NMDA, kainate, and glutamate itself potentiate Ca2+ and sodium (Na+) entry as Ca2+ permeable nonselective cation channels, but extracellular ATP-gated P2X receptor channels open a separate pathway for Ca2+ and Na+. Flow of Ca2+ and Na+ through parallel pathways driven by these cotransmitters would lead to a much quicker development of a postsynaptic potential. Moreover, with Ca2+ entering with Na+, it is likely that the action potential may be more long-lived and possibly more robust (i.e., having a shoulder or plateau due to Ca2+ entry beyond the Na+-driven spike). Second, ATP could potentiate glutamate release at the presynaptic neuron by potentiating Ca2+ entry driven by the preceding action potential. A more robust Ca2+ entry would cause more or secondary glutamate release and would keep the postsynaptic neuron firing. As for acetylcholine, ATP also has G-protein-coupled receptors, the P2Y receptors, which could modulate presynaptic and/or postsynaptic neuron function. Third, could metabolites of ATP limit activation of the postsynaptic and presynaptic neuron? One does not wish for the postsynaptic neuron to continue firing ad infinitum. One must have a limiting or negative feedback signal. Could this signal be adenosine? Not only does adenosine need to be recycled back into the neuron as a substrate for new ATP synthesis, but adenosine may also bind to P1 G-protein-coupled receptors to have limiting effects or neuronal function. Of course, ATP release and signaling at an inhibitory synapse driven by γ-aminobutyric acid (GABA) or glycine would have different and modulating effects on inhibitory synapse. We do not discuss or illustrate these possible mechanisms because that is not the intent of this introductory minireview. Synaptic purinergic neurotransmission has been reviewed by Burnstock and others in many seminal reviews [9, 10, 21–23]. Conclusions and future directions The founding concepts of purinergic signaling within tissues and tissue microenvironments were found in the heart, the enteric system (later to be duplicated in urinary bladder afferents, nociceptive neurons at the dorsal root ganglia of the spinal cord, etc.), and skeletal muscle by Forrester and coworkers [26]. This special issue of Purinergic Signalling focusing on the “Physiology of Nucleotide Release” is dedicated to these and other pioneers who would not give up the fight. All purinergics would agree that we owe the purinergic pioneers a great debt. Through their efforts, establishment of this journal, and the increasing volume of research in purinergic signaling, it is now accepted that ATP and its metabolites have two principal biological roles—as an extracellular signaling ligand and as an intracellular fuel.

Matern_Child_Health_J-2-2-1592154

Women with Depression—“You Can't Tell by Looking”™

Introduction Perinatal depression, a mood disorder that occurs during pregnancy or the first year postpartum, affects 10–15% of women and up to 28% of women living in poverty. A previous history of depression, either preconceptionally or during the interconception period, is the strongest predictor that a woman will suffer from perinatal depression. Perinatal depression can adversely affect not only the woman, but also her fetus or infant, and has been linked with prematurity, difficulties with maternal-infant bonding and infant behavior problems later in life. If a woman has a known history of depression, she can be counseled preconceptionally about her increased risk of experiencing a subsequent episode during the perinatal years and thus take an active role in recognizing signs and symptoms of depression and accessing treatment as early as possible should an episode occur. The earlier treatment is sought for perinatal depression, the greater the chance for improved outcomes. Background Depression is currently the leading cause of disease-related disability among women worldwide [1]. Depression can cause moodiness, irritability, changes in sleep patterns, sexual interest or appetite, and an inability to carry on with daily activities. In the most severe cases depression can cause thoughts of harming others or oneself or suicide. Prevalence rates of perinatal depression, which occurs during pregnancy or at some point within the first postpartum year, are similar to those in the general population, affecting 10–15% of women and up to 28% of women living in poverty [2, 3]. Some studies suggest that a woman's risk of becoming depressed during the early postpartum period is approximately three times that of women in the general population [4]. Depression may affect how a woman is able to relate and respond to her infant and if untreated, may lead to thoughts of harming the infant or infanticide. In addition, depression can have significant effects on prenatal care-seeking behavior [5], substance abuse [6], and infant/child mental health [7–9] and nutrition [10]. Depression has also been associated with low birthweight [11] and premature delivery [12]. Approximately 50% of women with perinatal depression remain undiagnosed when they are screened based on clinical observations alone [13]. Perhaps the strongest argument for routine screening of all pregnant and postpartum women is the fact that you can't tell simply by looking that a woman is depressed. However, there are some women who may be at increased risk for perinatal depression, including women living in poverty, those in abusive relationships, and women with a prior history of depression [14]. In fact, it is thought that having a history of depression is the strongest predictor that a woman will suffer from an episode of major depression during pregnancy or the postpartum period [15, 16]. Women with other mental disorders are at risk as well. For example, women with bipolar disorder also have an increased risk of experiencing postpartum depression, and some studies suggest an increased risk for a much more serious illness, postpartum psychosis [17]. Due to the predictive value of a history of depression, it is vital that women with a history of depression be counseled about the relationship between pregnancy and depression prior to conception. One method to incorporate depression screening & counseling into preconception care Counseling about depression should become a routine part of preconception care. Health care providers who have contact with women during the preconception period can assist women in becoming aware of how pregnancy and depression can affect one another. By controlling depression prior to conception, just as one would with an illness such as diabetes or epilepsy, a woman's chances for healthy perinatal outcomes may improve [18]. The Wisconsin Association for Perinatal Care (WAPC) is a 35-year-old statewide, non-profit organization dedicated to the improvement of healthy outcomes for women, infants and their families. Since 1989, WAPC has been working toward infusing depression screening and counseling into preconception care by encouraging health care providers to ask women and their partners about their histories with depression and other mental illnesses prior to conception. One tool that has been developed by WAPC is an information sheet entitled Women with Depression that answers four basic questions: How does depression affect pregnancy? How does pregnancy affect depression? How could medications for depression affect the pregnancy? What can you do before pregnancy? Case examples The following are two case examples illustrating the impact of depression on pregnancy and the positive effects of being educated prior to a subsequent depressive episode. Although these women were not educated about perinatal depression prior to their first pregnancies, these examples illustrate the importance of depression counseling in the interconception period as well. These cases demonstrate that addressing the questions in Women with Depression can provide an anticipatory contextual framework on which to develop a plan of care. Case example #1 Sara began experiencing symptoms of depression six months after giving birth to her first child. She had no prior history of depression and had received no counseling prior to conception about the symptoms and presentation of perinatal depression. Sara waited approximately six months before seeking help, but encountered numerous barriers to accessing care. She did eventually get into treatment and attended 15 sessions of interpersonal therapy. Through both her therapy and additional self-education, Sara became quite informed about the effects and consequences of postpartum depression. Approximately six months after giving birth to her second child, Sara began experiencing depression again. This time, Sara sought treatment within one week of the presentation of symptoms. In addition to being able to recognize the signs of depression earlier, as reported by both her and her husband, Sara also remarked on how much easier it was to access treatment, as she knew how to “navigate the system.” She knew what type of therapy worked for her and was able to resolve her depression in only seven sessions. Case example #2 Melanie had a history of anxiety and depression prior to conception, but was never diagnosed or treated. Melanie experienced extreme continuous sleep disturbances almost immediately after the birth of her first child. When this problem had not resolved itself within two months, Melanie sought treatment. She was initially reticent about taking medications because she was breastfeeding her infant, but at nine months postpartum, Melanie began a regimen of paroxetine, which finally resolved the depression. After Melanie's second birth, she felt slight anxiety about the possibility of suffering from postpartum depression again, but this time had a plan of action in place. When she began having trouble sleeping at two months postpartum, she immediately contacted her therapist and began treatment. She was once again hesitant about taking medication, but at nine months postpartum, began taking paroxetine on the recommendation of her care provider. Although she waited to begin medications with the second depressive episode, Melanie reported having “a kind of comfort knowing I was on paroxetine before and it didn't hurt my baby.” Benefits of preconception counseling for women with a history of depression The benefits of counseling women about depression prior to conception are numerous. By treating depression as a chronic condition and by actively managing depression prior to conception, a woman's chances of being prepared for a depressive episode during the perinatal period greatly increase. In a recent meta-analysis of psychosocial and psychological interventions to reduce the risk of postpartum depression there was no statistically significant effect on the prevention of postpartum depression [19]. In the absence of preventive treatment, then, understanding postpartum depression could allow families to anticipate possible consequences of maternal depression so they could take steps to help assure healthy outcomes [20]. By fully disclosing the increased risk of depression if she chooses to become pregnant, a woman can make informed decisions about planning for a family [21]. If the woman is taking antidepressant medication, she can discuss the impact of those medications on her future pregnancy and breastfeeding. By working with her health care provider, steps can be taken to change the medication or dose if necessary. One of the most devastating effects of perinatal depression is that it occurs at a time when most women feel they should be experiencing joy. By educating women prior to pregnancy or birth about the high prevalence of perinatal depression, they may be better prepared for an episode, should it occur. Women with a history of depression especially should have a plan of action in place in the event of perinatal depression. Women who have suffered from depression once may be more likely to recognize the signs and symptoms of a subsequent episode whether or not the first episode occurred preconceptionally or postpartum. This may be especially true if they are counseled about the possibility of a subsequent episode occurring. Women are not the only ones who can be counseled about the interaction between pregnancy and depression. Loved ones often do not know how to help women suffering from perinatal depression and cannot understand why the woman is depressed. By counseling significant others and family members, they, too, can become better prepared and in addition, can help recognize signs of severe depression and assist with accessing treatment. Seeking treatment may present fewer challenges to women who have been successfully treated and counseled about depression as well. Women with a history of depression can be counseled about seeking treatment at the earliest possible sign of depression, rather than waiting, as so often happens in primary episodes. The earlier treatment can be sought, the better the outcomes [22]. Stigma may be reduced as well if women are counseled ahead of time, making them more open to seeking treatment and more comfortable with their treatment decisions. Conclusions Testimonials from affected women suggest that counseling women with a history of depression leads to improved outcomes should they suffer from a subsequent episode during the perinatal period. Women may be more likely to recognize the signs of depression, more likely to seek treatment earlier and will know what treatment options work for them. Due to an earlier recognition of depression, treatment may have a shorter duration as well. In addition, by counseling women about depression prior to conception, they and their families will have time to develop a plan of action should a depressive episode occur and will be equipped to make informed decisions about family planning. Tools such as Women with Depression may be beneficial in both educating consumers and assisting providers with consultations about depression prior to pregnancy or during the interconception period. The major challenge currently lies in convincing health care providers to address women's mental health and to screen and counsel for depression during the preconception period. Intervention strategies using medication or counseling have not been significantly effective in preventing postpartum depression. Anticipating the potential interplay between depression and pregnancy may be an intervention that can lead to better mental health and perinatal outcomes. Future research should address the content and timing of preconception counseling, including what information is needed and when and how it should be disseminated. Treatment algorithms and strategies could then be developed for practitioners and families to help prepare for the postpartum period. Finally, well-controlled intervention studies utilizing the materials developed could focus on maternal mental health, perinatal, and infant outcomes to answer questions about efficacy and effectiveness.

Addiction-1-_-2253702

Social cognitive determinants of ecstasy use to target in evidence-based interventions: a meta-analytical review

Aims The health hazards and prevalence of ecstasy use have been documented in two decades of research, but no review reporting on potentially modifiable antecedents of use is available. The aim of this study was to integrate systematically research identifying cognitive correlates of ecstasy use. Such research has the potential to identify targets for evidence-based interventions designed to discourage use. INTRODUCTION Ecstasy use is potentially damaging to health [1–3] yet prevalent [4,5]. Legislative changes have not been effective in discouraging ecstasy use, and the development of theory-based behavioural interventions is warranted because these have been successful in generating behaviour change in other areas [6,7]. Behaviour change interventions are more likely to be effective if they target modifiable antecedents of the target behaviour. For example, if expected positive outcomes of a target behaviour differentiate between those who do and do not engage in that behaviour, it is prudent to target outcome expectancies in behaviour change interventions [8]. Which potentially modifiable cognitive antecedents of ecstasy use should interventions target? Unfortunately, although ecstasy was synthesized in 1912 [9], and ecstasy use has been studied for 20 years (e.g. [10,11]), there is no systematic review of this research identifying potentially modifiable cognitive antecedents of use. We aimed to summarize research to date, synthesizing quantitatively all published, quantitative studies of psychological determinants of ecstasy use among young people living in western society. METHOD The search strategy comprised three iterative steps. First, the databases PsycINFO, MedLine and ERIC (Education Resources Information Center) were searched using several combinations of keywords (see Appendix I). The results of the final query were then scanned manually for relevant entries by examining the paper titles and abstracts (see Appendix II). Four inclusion criteria were used. Firstly, a study should investigate the target population of young recreative ecstasy users in western society, as factors influencing behaviour can be population-specific [12–14]. Secondly, a study should measure one or more potentially modifiable determinants of ecstasy use-related behaviour; that is, an antecedent that could be influenced potentially by health promotional interventions (excluding for example demographics, personality, etc.); for a list, see chapter 7 of Bartholomew et al. [8]. Thirdly, the study should measure either actual behaviour or intention. Finally, it should assess quantitatively the relationship between determinants and behaviour or intention. Publications selected by this process were examined in detail. Second, reference lists of these papers were scanned for relevant publications (the ancestry approach). Third, texts citing the relevant papers were located using the Web of Science database (the descendancy approach). Potentially modifiable determinants were extracted from the studies using a recommended published list [8], on the basis of which two authors selected determinants from each paper and perfect agreement was observed. Associations between determinants and behaviour or intention across studies were integrated by converting all statistics to the correlation coefficient r. These coefficients were then transformed to Fisher's Z and weighed by sample size −3 (cf. [15]). The mean Fisher's Z was then transformed back to the correlation coefficient r+. When a study tested a variable several times (e.g. frequency of use and intensity of use), the resulting effect sizes were averaged before being included in the calculations. RESULTS The search yielded 367 hits, from which 15 publications were included (see Appendix II). Many excluded studies had a biological focus (e.g. [3]), examined determinants not feasibly changed by health promoting interventions (e.g. [16]) or used qualitative methods (e.g. [17]). All included publications studied the behaviour ‘using ecstasy’ (or the intention to use); none examined determinants of trying out ecstasy, ceasing use, changing use patterns or applying harm reduction practices. Of these 15 publications, six were discarded after thorough examination, as they were then discovered to yield no quantitative information on the relevance of potentially modifiable determinants of behaviour in the target population ([18–23]; see also Appendix II). Application of the ancestry approach yielded no additional publications (365 citations scanned), but the descendancy approach yielded one additional publication (of 85 unique citing publications; [24]). Of the final set of 10 publications [24–33], one publication described two studies ([26]; 26a and 26b refer to studies 1 and 2, respectively). Table 1 describes the 11 included studies, listing the sample details, the extracted potentially modifiable antecedents and how they were measured in the original studies. Table 1 Quantitative studies into the determinants of using ecstasy and measures used. No. Sample details n Age % ♀ Relevant variables I R [24] UK, before July 2002 Polydrug users 364 19 44% Negative mood function scale 3 5 Social function scale 5 5 Negative effects 4 5 Other functions (9 functions) 1 5 Extent of peer use 1 4 Partner/best friend use 1 2 Intensity of use 1 – [25] UK, before March 1998 Alcohol and drug users 100 19 45% Mood function scale 3 5 Social/contextual function scale 5 5 Negative effects/events scale 3 5 Extent of peer use 1 5 Intention 1 7 Intensity of use 1 – Frequency of use 1 – [26a] UK, March 1992 Students 186 19–25 58% Attitudes 6 7 Subjective norms 1 7 Perceived behavioural control 6 7 Behavioural beliefs (17) 2 19 Normative beliefs (5) 2 19 Control beliefs (8) 2 19 Intention to use ecstasy 4 7 [26b]* UK, mid-1996 Club members t1: 203 t2: 123 23 41% Attitudes 8 7 Normative influences 13 7 Perceived behavioural control 1 7 Self-efficacy 1 7 Behavioural beliefs 13 7 Control beliefs 6 7 Intention 3 7 Behaviour (longitudinal) 4 – [27] Netherlands, 2000, 2001 Party attendants 844 22 33% Negative outcome expectancies 11 2 Enhancement outcome expectancies 3 2 Euphoria outcome expectancies 4 2 Sex outcome expectancies 4 2 Dancing outcome expectancies 3 2 Insight outcome expectancies 4 2 Communication outcome expectancies 4 2 Whether ecstasy was currently used 1 2 [28] * UK t1: October 1994–1995 t2: May1995–1996 Students t1: 461 t2: 136 19–22 19–22 55% 65% Attitude 2 7 Injunctive norms 6 7 Perceived behavioural control 8 7 Descriptive norms 2 6 Moral norm 1 7 Intention 1 8 Behaviour (longitudinal) 1 8 [29] UK, before 2003 College students 657 19 55% Frequency of past use 1 7 Intentions to use 1 9 Normative influence (friends’ use) 1 6 Beliefs about ecstasy use (7 beliefs) 1 5 [30] * UK t1: 84 t2: 32 20 74% Attitude 10 8 Subjective norm 2 8 Perceived behavioural control over obtaining ecstasy 3 8 Perceived behavioural control over taking ecstasy 4 8 Intention 5 8 Habit 2 8 Specific attitudinal beliefs (13 beliefs) 1 5 Behaviour (longitudinal) 1 2 [31] the Netherlands, 2001–2002 490 22 34% Energy motives 4 5 Euphoria motives 3 5 Self-insight motives 2 5 Sociability/flirtatiousness motives 8 5 Sexiness motives 4 5 Coping motives 3 5 Conformism motives 4 5 Perceived positive effects 24 2 Perceived negative effects 11 2 Perceived friends’ use 1 5 Frequency of ecstasy use 1 5 [32] UK 200 21 66% Attitude 5 ? Subjective norm 5 5 Perceived behavioural control over obtaining ecstasy 3 7 Perceived behavioural control over taking ecstasy 11 ? Intention 6 7 Habit 2 7 [33] USA Club rave attendees 70 20 47% Risk associated with using ecstasy once or twice 1 4 Risk associated with using ecstasy regularly 1 4 Harmful short-term physical effects 1 4 Harmful long-term physical effects 1 4 Harmful short-term psychological effects 1 4 Harmful long-term psychological effects 1 4 Positive physical effects 1 4 Positive psychological effects 1 4 Ecstasy use within the past 12 months 1 2 No. = number in reference list, I = number of items used to measure variable, R = number of scale points on response scale of each item. * Longitudinal design. All 11 studies can be viewed as tests of two theoretical frameworks which are applied frequently in drug use research [34]. Six studies [26a,26b,28–30,32] tested the theory of planned behaviour (TPB; [35]). The TPB proposes that the most proximal cognitive determinant of behaviour is intention which, in turn, is predicted by attitude (i.e. evaluation of probable consequences of that behaviour), subjective norm (i.e. perception of others' approval of the behaviour) and perceived behavioural control (PBC; i.e. perception of control based on perception of skills and external obstacles/facilitators). Each of these constructs is based on underlying beliefs. Several extensions of the TPB have been proposed [36]; for example, personal norm (personal moral evaluation of the behaviour), descriptive norm (perception of others' performance of the behaviour [37]), habit [38] and anticipated regret (the regret one experiences when prospectively imagining having—or not having—performed a behaviour [39,40]). Five studies [24,25,27,31,33] tested expectancy models (e.g. [41]), which propose that behaviour is determined by expectations people have of the behaviours' consequences. Two studies [24,25] assessed how often participants used ecstasy for particular reasons (e.g. ‘in the past year, how often have you used ecstasy to help you to let go of inhibitions?’), implying germane expectations (such as ‘taking ecstasy helps me let go of inhibitions’). Three studies [27,31,33] used more traditional measures (e.g. ‘I have experienced/would expect that ecstasy makes it easier to communicate’). Note that these expectations are viewed as underlying beliefs in the TPB, particularly in relation to the structure of attitudes [12,35]. In both the TPB and outcome expectancy models, higher-level constructs, such as attitudes, are based on lower-level beliefs. Most studies tested models involving this hierarchical cognitive structure. For the purposes of this review, higher-level constructs are referred to as ‘compound constructs’ and lower-order cognitions as ‘expectancies’ (e.g. beliefs about probable consequences of ecstasy use). Details of the particular theoretical models explored in the original studies are not provided here. Only bivariate analyses were synthesized because multivariate analyses were incommensurable over studies as they tested different models. [In order to resolve this by conducting uniform regression analyses, all authors were asked to provide matrices of covariance. However, due to lost data sets, missing data and non-response, not enough data could be retrieved to render this feasible. The authors are grateful to M. Conner and T. ter Bogt, who did supply additional data.] It is worth noting none the less that in the two TPB-based studiesin which ecstasy use was regressed onto compound constructs, the average (weighed) R2 was 0.51 [26b,28], and in the five TPB-based studies in which intention to use was regressed onto compound constructs the average R2 was 0.67 [26a,26b,28,30,32]. The average R2 for the three expectancy studies in which ecstasy use was regressed onto expectancies was 0.35 [24,27,31], and in the expectancy study where intention to use was regressed onto expectancies was 0.64 [25]. In three prospective studies ecstasy use was found to be related strongly to prior intention to use with an average (weighed) r of 0.71 [26b,28,30]. Table 2 shows the strength of association between predictor variables and measures of ecstasy use and intention to use. The strongest predictor of intention and use was the TPB-specified attitude measure. Attitudes are thought to be based on more specific expectancies [12,35] and these are shown separately in Table 2, grouped into ‘positive’ and ‘negative’ expectancies. In addition to these perceived advantages and disadvantages of ecstasy use, normative measures have been used, especially subjective and descriptive norms, as well as perceived behavioural control over ecstasy use. Variables proposed as extensions to the TPB have been categorized as ‘miscellaneous’. Table 2 Effect sizes of predictors of ecstasy use and intention to use. Association with behaviour Association with intention Variable type Compound or expectancy k r+ k r+ Attitude Attitude [C] 5 0.53 (L) 5 0.63 (L) Positive expectancies Social function [C] 4 0.09 2 0.30 (M) Produces a positive mood state 3 0.12 (S) 2 0.28 (M) Mood function [C] 4 0.20 (S) 1 0.41 (L) Helps lose weight 2 0.09 1 0.08 Enhances sex 3 0.11 (S) Helps stay awake 3 0.08 Helps keep fit 1 0.14 (S) 1 0.22 (S) Enhances insight/openness 2 0.18 (S) Helps to relax/helps coping 2 0.08 Produces excitement 1 0.31 (M) Produces intoxication 1 0.19 (S) Eases after-effects 1 0.15 (S) Improves other drugs' effects 1 0.11 (S) Is conforming to peers 1 0.05 Helps work 1 0.05 Negative expectancies Short-term negative effects [C] 4 −0.47 (L) Produces mood swings 1 −0.34 (M) 2 −0.31 (M) Leads on to more frequent use 1 −0.30 (M) 2 −0.39 (L) Produces mental side-effects 1 −0.24 (M) 2 −0.40 (L) Leads on to ‘worse’ drugs 1 −0.23 (S) 2 −0.31 (M) Produces physical side-effects 1 −0.18 (S) 2 −0.39 (L) Makes one unhealthy 1 −0.18 (S) 2 −0.30 (M) Produces depression 1 −0.18 (S) 2 −0.28 (M) Produces lethargy 1 −0.05 2 −0.20 (S) Produces addiction 1 −0.32 (M) 1 −0.12 (S) Leads to death 1 −0.30 (M) 1 −0.32 (M) Produces paranoia 1 −0.27 (M) 1 −0.26 (M) Makes one feel run down 1 −0.17 (S) 1 −0.25 (M) Subjective injunctive norm Subjective norms (approval) [C] 5 0.39 (L) 5 0.52 (L) Best friends' approval 1 0.36 (M) 1 0.25 (M) Partners approval 1 0.15 (S) 1 0.30 (M) Parents' approval 1 0.10 (S) 1 0.01 Health experts' approval 1 0.09 1 0.00 Other ecstasy users' approval 1 0.07 1 0.08 Subjective descriptive norm Subjective norms (descriptive) [C] 1 0.52 (L) 1 0.63 (L) Perceived peer use 2 0.39 (L) 1 0.43 (L) Perceived use in close relatives 2 0.21 (S) Perceived best friend/partner use 1 0.37 (L) Perceived behavioural control PBC [C] 3 0.33 (M) 3 0.52 (L) PBC (over obtaining ecstasy) 2 0.20 (M) 2 0.25 (M) PBC (over taking ecstasy) 2 0.01 2 −0.03 Being with friends who use 1 0.45 (L) 2 0.62 (L) Going out dancing 1 0.41 (L) 2 0.67 (L) Being offered ecstasy 1 0.40 (L) 2 0.59 (L) Ecstasy being available 1 0.40 (L) 2 0.57 (L) Cheap ecstasy 1 0.19 (M) 2 0.54 (L) Having alcohol 1 −0.02 2 0.10 (S) PBC (over not taking ecstasy) 1 −0.06 1 0.07 Needing to lose weight 1 −0.05 1 0.00 Needing to exercise 1 −0.01 1 0.05 Miscellaneous Habit [C] 2 0.45 (L) 2 0.46 (L) Moral norm [C] 2 −0.28 (M) 2 −0.31 (M) Denial of negative consequences 1 0.17 (S) 1 0.18 (S) Anticipated regret 1 −0.11 (S) 1 −0.22 (S) [C] = compound construct, k = no. of samples, r+ = weighed average correlation, (S) = small, (M) = medium, (L) = large effect size magnitude according to Cohen [42]. PBC: perceived behavioural control. Table 2 is based on the Pearson correlations reported in five studies [24,25,28,30,32] and correlation matrices received from the authors of two publications [26,31]. One study [27] reported t-values, which were converted into effect size correlations using r = √[t2/(t2 + df)]. The results from two studies [29,33] could not be converted and will be provided later. Only associations found in at least two samples, of magnitudes corresponding to a medium (Cohen's d = 0.5 [42], r = 0.24) or large effect size (d = 0.8 and r = 0.37), are considered. All significant associations were in the expected direction and some large effect sizes were observed, suggesting that both the theory of planned behaviour and the expectancy approach can help to explain ecstasy use and intentions to use. In the TPB studies, intention and behaviour are associated most strongly with TPB's attitude, with a large effect size (r+ = 0.53 with behaviour, r+ = 0.63 with intention). Specifically, this compound construct appears to be associated mainly with positive outcomes regarding mood control and social facilitation, and negative expectations regarding short-term negative effects, escalating use and physical and mental side effects. Normative influences are also important covariates of use and intention to use with stronger associations observed for descriptive (r+ = 0.52 with behaviour, r+ = 0.63 with intention) than injunctive norms (r+ = 0.39 with behaviour, r+ = 0.52 with intention). The strongest effect sizes for expectancies underpinning these compound constructs were observed for perceived ecstasy use by peers and approval of use by one's best friend and partner. The results also indicate that whether parents, health experts and ‘other ecstasy users’ approve is inconsequential. Perceived behavioural control was related to use with a medium (near large) effect size (r+ = 0.33) and also related strongly to intention (r+ = 0.52). Underlying beliefs showed a medium effect regarding control over obtaining ecstasy and large effects of control in relation to being with friends who use, going out dancing, being offered ecstasy and ecstasy being available. Two proposed extensions to TPB appear relevant: habit, with a large effect size (r+ = 0.45 with behaviour, r+ = 0.46 with intention) and moral norm, with a medium effect size (r+ = −0.28 with behaviour, r+ = −0.31 with intention). Two studies reported results using statistics that could not be transformed to an effect size measure. One study [29] conducted a polynomial logistic regression predicting user group (six levels: rejectors, at-risk non-users, ex-users, and light, moderate and heavy users; for details, see [29]), testing whether a number of variables contributed significantly to model fit when predicting user group. Another study [33] conducted t-tests on beliefs (also predicting user group, with two levels: non-user and user), but did not report t-values, exact P-values, or variance information. The variables tested in these studies are shown in Table 3. Effect sizes were estimated (conservatively) on the basis of sample size and significance. Table 3 Significant and non-significant predictors of user group (user group had six levels in study [29] and two levels in study [33]). Variable Study Significance Association Effect size Perceived use by friends [29] < 0.001 Positive M Anticipated regret (‘use would induce guilt’) [29] < 0.001 Negative M Is hard to resist [29] < 0.001 Positive M Moral norm (‘ecstasy use is immoral’) [29] < 0.05 Negative S Perceived availability of ecstasy [29] < 0.05 Positive S Is bad for one's physical health [29] None – – Is bad for one's mental health [29] None – – Subjective norm (approval) [29] None – – Harmful long-term physical effects [33] < 0.01 Negative M Risk associated with using regularly [33] < 0.01 Negative M Risk association with using once or twice [33] < 0.05 Negative M Harmful long-term psychological effects [33] < 0.05 Negative M Harmful short-term physical effects [33] None – – Harmful short-term psychological effects [33] None – – Positive physical effects [33] None – – Positive psychological effects [33] None – – S = small, M = medium, effect size magnitude according to Cohen [42]. These additional results confirm the relevance of descriptive norm, negative expectancies (particularly long-term effects) and perceived control, and add anticipated regret, with at least a medium effect size, to the list. DISCUSSION Synthesis of the included studies shows the main predictors of intention to use and actual ecstasy use to be attitude (specifically positive outcomes regarding mood control and social facilitation and negative outcomes regarding escalating use and physical and mental side-effects); subjective and descriptive norms regarding one's friends, partner and peers; perceived control regarding obtaining ecstasy and control in relation to being with friends who use, going out dancing, being offered ecstasy and ecstasy being available; and habit, moral norm and anticipated regret. In addition to lending support to both the TPB and expectancy models, these findings show that some expectancies underlying attitude are irrelevant (e.g. ecstasy enhances sex), as are norms relating to some social referents (e.g. parents), and perceived control regarding some ecstasy-related behaviours (e.g. taking ecstasy). As it is now clear which determinants best predict intention to use and ecstasy use according to the research so far, these determinants seem advisable intervention targets. However, not all determinants are equally easy to modify. As attitude encompasses several specific expectancies, it may be easier to target these more concrete expectancies than the abstract attitude construct. Also, not all expectancies are influenced equally easily. Because most users have experienced positive mood shifts, it may be difficult to develop persuasive messages that undermine this expectancy among users [43]. Negative variables are also associated strongly, and changed more easily. However, simply presenting information about negative outcomes (‘fear appeals’) has been shown not to work, or even work inversely, unless a number of critical conditions are met, such as efficacy enhancement (see [6,44]; also see [45]). Similarly, it may be difficult to change a subjective norm if it reflects reality. As ecstasy use is social [46] and most users take ecstasy at a dance event [47] where ecstasy use is high (in the Netherlands, about two-thirds of the visitors use ecstasy; [48]), it might be hard to reduce descriptive norms, especially if participants' friends use ecstasy. Similarly, it may be challenging to establish a disapproving norm (see [49]). Similarly, it may be difficult to reduce perceived control over obtaining ecstasy among users, as they probably have repeatedly performed this behaviour successfully. However, the large effects of specific control beliefs suggest that users who wish to stop may well be aided by adopting a stimulus control strategy avoiding the social contexts of use. In addition, enhancing refusal skills would influence perceived behavioural control, while also diminishing the effect of undesirable subjective norms (by decreasing users' motivation to comply, see [35]). Thus, among those with intentions not to use, refusal skills training and stimulus control are recommended [50]. Finally, habit and moral norm are useful predictors but may be difficult to change ([8], but also see [51]). Although anticipated regret has a smaller association with use and intention to use, it can be changed more easily (e.g. [39]). A list of methods and strategies to change these determinants, and the theoretical parameters prerequisite to success, is provided in chapter 7 of Bartholemew et al. [8]. This review is limited mainly by the fact that only bivariate associations could be synthesized and by two consequences of the paucity of research into ecstasy use thus far. First, the small number of studies into determinants of ecstasy use limit the robustness of the current findings. Second, many theories and models have not yet been studied and are therefore not included in this review. Only social cognitive theories have been tested; no studies have investigated, for example, the predictive value of implicit cognitions. However, recent research implies that implicit processes may be changeable [52]. Moreover, within social cognitive research, recently developed constructs such as consideration of future consequences [53] have not yet been applied to ecstasy use, but may aid in intervention development. Regarding the theories that have been studied, in order to gain a more comprehensive understanding of motives for ecstasy use future research should combine the two theoretical perspectives studied thus far, so that relative overlap can be determined. It would be interesting to see whether, and to what degree, particular expectancies account for the predictive utility of the TPB attitude measures. Also, the list of expectancies that has been studied so far may omit a number of consequences (such as ‘suicide Tuesday’, a term for a period following use when low serotonin levels can induce depressive feelings). Another important gap in the literature concerns behaviours other than ‘using ecstasy’. Not only can the determinants of trying ecstasy out, starting use, ceasing use and maintaining cessation differ from the determinants of using ecstasy [12], little is known about the determinants of harm reduction practices, such as having one's ecstasy tested by a test service, ensuring sufficient hydration and maintaining a low body temperature (although studies such as [54] are a step in the right direction). Intervening to promote these behaviours could prove to be more beneficial to the health of party visitors, given the difficulty of intervening on most variables determining ecstasy use. In conclusion, this review suggests that there is sufficient evidence to guide intervention development so that evidence-based practice is established. These interventions could then be evaluated to test the utility of particular theoretical frameworks. The priorities for interventions should be negative expectancies, perceived behavioural control and anticipated regret. Tailored interventions can offer refusal skills training and strategies to avoid risky situations to participants not intending to use ecstasy. Appendix I Search terms used in PsycINFO (equivalent terms used in corresponding fields in MedLine and ERIC) [query in words, as corresponding to ‘concepts’ column, in brackets].No.ConceptOperationalizationFields1Language(English) or (Dutch)Language2Publication type(journal*) or (peer-reviewed-journal)Publication type3Publication date> 1980Publication year4Ecstasy(clubdrug*) or (club near drug*) or (dance near drug*) or (dancedrug*) or (party near drug*) or (partydrug*) or (xtc) or (mdma) or (methylenedioxymethamphetamine) or (‘3,4-methylenedioxymethamphetamine’) or (ecstasy)Title, abstract, keywords5Theoretical(theor*) or (attitud*) or (motivat* near functio*) or (mode*) or (norm*) or (perceived near control) or (pbc) or ('social cognitive’) or (self adj efficacy) or (stages near change) or (perceived adj (harm or risk or functions)) or (functional) or (outcome adj (expectancies or expectations)) or (sct) or (tpb) or (patter*) or (psychosoc*) or (health adj belief adj model) or (hbm)Title, abstract, keywords6Determinants(determin*) or (facto*) or (variabl*) or (parameter*) or (reason*) or (caus*) or (motiv*) or (incentive*) or (correlat*) or (antecedent*) or (character*)Title, abstract, keywords7Initiation(start*) or (commenc*) or (originat*) or (onset) or (initiat*) or (instigat*) or ((use) not (user)) or (using) or (usage) or (establish*)Title, abstract, keywords8Maintenance(maint*) or (sustain*) or (continu*) or (uphold*) or (persist*) or (further*) or (prolong*)Title, abstract, keywords9Cessation(end*) or (stop*) or (discontinu*) or (terminat*) or (ceas*) or (cessat*) or (abstain*) or (abstin*) or (quit*) or (remiss*) or (resolut*) or (recover*)Title, abstract, keywords10Harm reduction(harm or risk or damage or casualt*) and (reduc* or manag* or limit* or minimi*)Title, abstract, keywords11Excluded((treatment not (‘not in treatment’ or ‘non-treatment’ or ‘non- treatment’ or ‘no treatment’)) or rat or rats or mouse or mice or animal or monkey* or pigeon* or spectro* or cardio* or seroton* or dopamin* or neurotransm* or receptor* or psychiatr* or psychopath* or cell* or diagnos*)Anywhere12Inclusion#1 and #2 and #3 [Language and Publication Type and Publication Date]–13Behaviour#7 or #8 or #9 or #10 [Initiation or Maintenance or Cessation or Harm reduction]–14Empirical*#6 near #13 [Determinants near Behaviour]–15Final query*#12 and #4 near (#5 or #14) not #11 [Inclusion and Ecstasy near (Theoretical or Empirical) not Excluded]–When executed, the query consisted of one command; therefore the use of the ‘near’-operator was valid here. Appendix II Search procedure, number of resulting hits, and results of each step. Step Activity Number of resulting publications 1 Input of query at 20 August 2007 in PsycINFO (162), MedLine (194) and ERIC (11) 367 2 Removal of duplicate records (83) 284 3 Removal of records about publications that (entries were removed in this order): 3.1 studied biological variables (e.g. sequelae of ecstasy use; 75) 209 3.2 did not study ecstasy use or a related behaviour (such as trying out ecstasy, ceasing use, changing use patterns, or applying harm reduction practices; 32) 177 3.3 studied variables that cannot be changed using a health promotion intervention (e.g. sex, ethnicity or religion; 68) 109 3.4 studied ecstasy use as an independent variable in a multivariate or longitudinal analysis (22) 87 3.5 did not employ quantitative methods (e.g. qualitative studies; 30) 57 3.6 did not study behaviour or cognitions (25) 32 3.7 studied a ‘non-normal’ subpopulation or gathering data from samples inseparably encompassing these subpopulations (e.g. dependent participants, patients or delinquents)*, studying generic drug categories (e.g. ‘hard drugs’)†, or not explicitly stating which drugs were studied (14) 18 3.8 were not published in a peer-reviewed journal (3) 15 * inclusion of these subpopulations would restrict generalization of the results to the target population of the current study (i.e. the average adolescent; see [12,55]). † this demand of drug specificity is necessary because previous research has shown that beliefs about drugs can vary between different drugs [56], rendering aggregation questionable.

Eur_J_Pediatr-3-1-2151775

Incidence of late vitamin K deficiency bleeding in newborns in the Netherlands in 2005: evaluation of the current guideline

Vitamin K prophylaxis is recommended to prevent the hazard of haemorrhage caused by vitamin K deficiency in newborns. The present Dutch guideline recommends 1 mg of vitamin K1 orally at birth, followed by a daily dose of 25 μg of vitamin K1 from 1 to 13 weeks of age for breastfed infants. Since the introduction of this prophylaxis, the incidence of vitamin K deficiency bleeding (VKDB) has decreased; however, late VKDB is still reported. From 1 January to 31 December 2005, a nationwide active surveillance was performed by the Netherlands Paediatric Surveillance Unit (NSCK) to study the current incidence and aetiology of late VKDB in infants. Six cases could be validated as late VKDB: all were breastfed, one fatal idiopathic intracranial haemorrhage at the age of 5 weeks and five bleedings secondary to an underlying cholestatic liver disease between the age of 3 and 7 weeks. The total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 (95% CI: 1.2–6.9) and 0.5 (95% CI: 0–2.9) per 100,000 live births, respectively. With the current Dutch guideline, idiopathic late VKDB is rare but late VKDB secondary to cholestasis still occurs in breastfed infants. Doubling the daily dose of vitamin K1 to 50 μg, as is comparable to formula-feeding, may possibly prevent VKDB in this group. Further research, however, is needed to prove this hypothesis. Introduction Vitamin K deficiency can cause severe haemorrhage in the newborn. The term “haemorrhagic disease of the newborn” (HDN) was coined by Charles Townsend in 1894 to describe an acquired bleeding disorder in the newborn due to vitamin K deficiency [26]. Nowadays, the term HDN is replaced by vitamin K deficiency bleeding (VKDB), as neonatal bleeding is often not due to vitamin K deficiency and VKDB often occurs after the 4-week neonatal period [23]. VKDB can be classified according to the time of presentation after birth into early (0–24 h), classical (1–7 days) and late (2–12 weeks) VKDB. Late VKDB occurs primarily in exclusively breastfed infants who have received no or inadequate vitamin K prophylaxis. In addition, intestinal malabsorption defects (cholestatic jaundice, cystic fibrosis, α-1-antitrypsin deficiency etc.) are a well known frequent cause of late VKDB [13]. In some children, “warning bleeds” such as mild bruises, nose bleeds or umbilical oozing are the first manifestation of VKDB. In about 50% of the cases in late VKDB, intracranial haemorrhage occurs, accounting for death and serious neurological and mental handicaps [22]. The efficacy of neonatal vitamin K prophylaxis (oral or parenteral) in the prevention of classic VKDB is firmly established [16]. It has been the standard of care since the Committee on Nutrition of the American Academy of Paediatrics recommended to administer vitamin K prophylaxis to all newborn infants in 1961 [1]. Since then, many countries adopted this recommendation, although controversies concerning the best dose, route and frequency of administration still exist. In 1990, the Dutch Paediatric Association recommended the administration of a single oral dose of 1 mg of vitamin K1 at birth to all healthy infants. Parenteral administration of vitamin K1 at birth was recommended for newborns especially at risk (preterm babies, babies born with instrumental help or caesarean section, asphyxia or if maternal medication was interfering with coagulation). For infants who are largely or fully breastfed, a daily dose of 25 μg of vitamin K1 orally was recommended from 1 to 13 weeks of age. In case of additional risk factors like a known malabsorption, this dose should be increased to 50 μg per day [29]. After the introduction of this guideline, the incidence of late VKDB, which was studied by a nationwide active surveillance performed by the Netherlands Paediatric Surveillance Unit (NSCK) in 1992 to 1994, decreased from about 7.0 to 1.1/100,000 live births [2]. However, still, some cases of late VKDB have been reported in the Netherlands, merely due to an underlying cholestatic liver disease [7, 10, 11]. Therefore, a one-year surveillance was started again to study the current incidence and the aetiology of late VKDB in the Netherlands. Discussion will rise about the efficacy of the current guideline in order to completely prevent late VKDB. Methods From 1 January to 31 December 2005, an active surveillance programme for VKDB was in operation in the Netherlands, using methods developed by the British Paediatric Association Surveillance Unit [8]. Surveillance was based on monthly report cards sent to paediatricians responsible for in- and outpatient care in all hospitals by the NSCK. A “nothing to report” option was included. The return rate of these report cards was 94.7% in 2004 [17]. The paediatricians were asked to report all infants in whom bleeding may have resulted from VKDB. The reported cases were validated with a questionnaire asking for information about the infant, type of feeding, clinical presentation, dose of vitamin K prophylaxis, route of vitamin K prophylaxis, associated diseases and laboratory data. For all reported cases, we asked for an anonymous hospital discharge letter to verify the diagnosis of late-onset VKDB. The case definition of late-onset VKDB was as internationally defined: any infant between 8 days to 26 weeks of age, with spontaneous bruising, bleeding or intracranial haemorrhage associated with a grossly prolonged prothrombin assay, not due to an inherited coagulopathy or disseminated intravascular coagulation [23]. Confirmed VKDB was diagnosed when the prothrombin assay results were grossly abnormal compared with the standards for age: international normalized ratio (INR) ≥4 control value, prothrombin time (PT) ≥4 control value and at least one of the following was present: Platelet count normal or raised and normal fibrinogenProthrombin assay returned to normal after vitamin K administrationConcentration of proteins induced by vitamin K absence (PIVKA) exceeded normal controls PIVKA is a sensitive indicator of vitamin K status, allowing the detection of subclinical deficiency states which would not be detected by conventional coagulation assays [20]. Idiopathic cases were defined as cases in whom no factor predisposing to vitamin K deficiency was identified. Secondary cases were those in whom an underlying condition such as cholestasis or other causes of malabsorption of vitamin K could be identified. Vitamin K prophylaxis was considered to be “as recommended” if the Dutch guideline was followed at the time of bleeding: 1 mg of vitamin K1 (oral, intravenous, intramuscular) directly after birth, followed by a daily dose of 25 μg of vitamin K1 from 1 to 13 weeks of age for all breastfed babies. The Konakion mixed micellar preparate (Phytomenadion 10 mg/ml, Hoffmann-La Roche, Basel, Switzerland), which can be used for both oral, intramuscular and intravenous administration, an oral preparate called Davitamon K (marketed by Chefaro, Rotterdam, the Netherlands, 25 μg/5gtt) and an oral preparate, Phytomenadion, which is made by the Dutch pharmacist Mixtura (Phytomenadion 10 mg/ml FNA) were the only vitamin K preparations licensed in the Netherlands. The 95% confidence intervals (CI) were calculated according to a binomial distribution and the chi-squared test was used for the comparison of incidences between different periods, with a value of P < 0.05 being considered as significant. Results The response rate to the surveillance amounted to 93.4% and the response to our questionnaires was 100%. In total, seven cases of late VKDB were reported: four reports from academic paediatric centres and three from general district hospitals. A hospital discharge letter was obtained in six cases. In all cases, the diagnosis could be verified by the completed questionnaire or the additional discharge letter. One case did not fulfil the criteria of the case definition because there were no clinical signs of bleeding, only biochemical clotting disorders due to a vitamin K deficiency. Six cases fulfilled the criteria of the case definition and could be validated as late VKDB. The surveillance data are shown in Table 1. The mean age of presentation was 5 weeks and 2 days, and all of the cases were exclusively breastfed. In all cases, the PT time or INR was at least four times greater than normal and vitamin K deficiency was confirmed by a documented PT or INR normalisation after the administration of vitamin K. Five of the cases also received fresh frozen plasma. Table 1Confirmed cases of late vitamin K deficiency bleeding (VKDB) in the Netherlands from 1 January to 31 December 2005Patient; sex; age (weeks)PresentationFeedingVitamin K prophylaxisProthrombin assay (after vitamin K administration) Additional morbidityOutcome of bleedingA; female; 5 + 1/7Intracranial bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayPT>120 s (13.3); APTT>120 s (37)NoDiedB; female; 6 + 6/7Intracranial bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayINR>8 (INR<2); APTT>240 s (49)Biliary atresiaHemipareses rightC; male; 4 + 4/7Intracranial bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayPT>90 s (30); APTT>120 s (38); Vitamin K: 0.1 nmol/lBiliary atresiaHemipareses rightD; male; 5 + 2/7Nasal bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayPT>75 s (normal); APTT: 118 s (normal)Biliary atresiaNo sequelaeE; male; 6 + 2/7Nasal bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayPT>165 s (normal); APTT>240 s (normal)Biliary atresiaNo sequelaeF; male; 3 + 6/7Nasal bleedingBreastfeedingBirth: 1 mg p.o.→25 μg/dayPT>71 s (12.7); APTT: 128 s (39.1)CholestasisNo sequelae Concerning the cause of VKDB, there was one case of idiopathic VKDB, who presented with signs of bleeding at the age of 5 weeks. She was breastfed and died due to the consequence of an intracranial bleeding. She received exactly the recommended prophylaxis. Unfortunately, no post-mortem obduction was performed but, biochemically, there were no signs of cholestasis. Five out of six cases could be validated as secondary late VKDB. The age of presentation in this group was between 3 and 7 weeks of age. All of these bleedings were due to an underlying cholestasis which was diagnosed after the first presentation of bleeding. In two cases, an intracranial bleeding was the first presenting sign. Four cases were diagnosed as having a bile duct atresia. All infants had been exclusively breastfed and all had received exactly the recommended prophylaxis. In the study period, the number of live births amounted to 187,910 [21]. Therefore, the total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 (95% CI: 1.2–6.9) and 0.5 (95% CI: 0–2.9) per 100,000 live births, respectively. Discussion Data from the United Kingdom, Germany and Switzerland prove that intramuscular vitamin K1 prophylaxis (1 mg) is highly effective in the prevention of late VKDB. This is supported by data from Australia, which demonstrate a higher incidence of late VKDB in babies who received oral vitamin K compared to those with parenteral vitamin K at birth. However, most of these failures of oral vitamin K were in cases reported with an underlying cholestatic liver disease [3, 12, 14, 25, 32, 33]. In 1992, Golding et al. [5] reported an increased risk of developing childhood cancer after parenteral vitamin K1 prophylaxis. Recent studies, however, show that solid tumours are no commoner in children who had parenteral vitamin K. Although no convincing evidence between parenteral vitamin K and childhood leukaemia was found in these studies, it cannot be fully excluded that there is no risk [4, 18]. Since the report of Golding et al., there has been an increasing trend towards oral vitamin K administration and many oral prophylaxis regimes have been developed. In 1997, we summarised the results of surveillance studies of over 2 million infants in four different countries using different multiple oral dose schedules and parenteral administration of vitamin K. A daily low oral dose of 25 μg of vitamin K1 following an initial oral dose of 1 mg vitamin K1 directly after birth for exclusively breastfed infants, as was used in the Netherlands since 1990, seemed to be as effective as parenteral vitamin K1 prophylaxis [3]. The present surveillance study shows that the current Dutch guideline still seems quite effective in preventing idiopathic late VKDB, but it is insufficient against secondary late VKDB. In our surveillance, the incidence of all late VKDB and idiopathic late VKDB was 3.2 (95% CI: 1.2–6.9) and 0.5 (95% CI: 0–2.9) per 100,000 live births, respectively. The incidence of the latter is comparable to 0.5 (95% CI: 0.1–1.6) per 100,000 live births reported in our surveillance study performed by the same Netherlands Paediatric Surveillance Unit (NSCK) in 1992–1994 [2]. The incidence of late VKDB secondary to an underlying cholestatic liver disease is, however, significantly higher (P < 0.05); 2.7 (95% CI: 0.9–6.2) compared to 0.7 (95% CI: 0.1–2.0) in 1992–1994. A higher incidence of cholestatic liver diseases in general or a delayed recognition of symptoms can be explanations for this difference. Unfortunately, data about the incidence or recognition of cholestatic liver diseases in the Netherlands are not available. In this surveillance, there were no reports of late VKDB after parenteral administration of vitamin K1. This can be explained by the assumption of the higher efficacy of parenteral vitamin K1 or the low use of intramuscular and intravenous vitamin K1 these days. Since 1994, a new preparate, Konakion mixed micellar, has been available. This mixed micellar formulation is prepared with the phospholipids lecithin and the bile salt glycocholic acid, and it was presumed that this preparate would reduce the incidence of late VKDB due to a better absorption of oral vitamin K. However, a recent randomised controlled trial shows that the mixed micellar vitamin K gives unreliable and still severely impaired intestinal absorption in infants with cholestasis [15]. Furthermore, von Kries et al. [31] reported no significant reduction in the incidence of late VKDB in healthy infants and infants with an underlying cholestatic liver disease with this preparate compared to the use of the original Konakion (Cremophor EL) and Kanavit (Polysorbat 80). In view of the fact that vitamin K has limited tissue reserves and is rapidly catabolised, with 60–70% of a single dose being excreted via the urine and bile in about 3 days, it seems reasonable that repeated doses rather than a higher single dose is more effective in preventing late VKDB [15]. An important study from Denmark has established that 1 mg of oral vitamin K1 given weekly during the first 3 months of life, following an initial oral dose of 2 mg of vitamin K1 directly after birth, is as effective as 1 mg of vitamin K1 given intramuscularly at birth [9]. Therefore, we hypothesise that the dosage of 25 μg of vitamin K1 daily is not enough to prevent the secondary cases of late VKDB, as we encountered in this study. In most co-operative studies, the upper age limit for inclusion was set arbitrarily at the end of 12 weeks of age [32]. In our study, we included infants presenting with VKDB up to the age of 26 weeks of age, as suggested by Tripp et al. [27]. However, no infant between 12 and 26 weeks of age was reported. The mean age of all infants with late VKDB was 5 weeks, which is comparable to other reports [22]. In our study, the boy:girl ratio was 2.5:1. Why late VKDB occurs more in boys than girls is unknown, but this is consistent with other European reports [22, 24]. Unfortunately, this study has some limitations. First, this surveillance was based on one data source only: the Netherlands Paediatric Surveillance Unit. In spite of the high return rate of the report cards (93.4%), it is plausible that dramatic conditions, such as intracranial bleedings, are more likely to be reported, as also noted by von Kries et al. [31]. For this reason, our data could give an under-reporting of the true incidence of late VKDB in the Netherlands. However, the fact that 50% of late VKDB cases were intracranial bleedings is comparable to other, larger reports [3, 13, 14, 19, 22, 30, 31, 33]. Secondly, the surveillance lasted only 1 year. In all probability, the confidence interval would be smaller when the length of the surveillance was prolonged. Third, the proportion of infants that is breastfed is unknown, and, additionally, we lack data about compliance to the prophylaxis. It is likely, however, that less than 100% of the eligible cases received the recommended prophylaxis. Prophylaxis failure, therefore, is likely to be underestimated due to a greater denominator than the true numbers exposed. We would suggest to double the daily dose of vitamin K1 to 50 μg for all breastfed babies from 1 to 13 weeks of age to prevent bleedings in infants with an underlying cholestatic liver disease as well. This amount is comparable to the ingested dose of vitamin K1 by formula-feeding [6]. Formula is supplemented with vitamin K1 to a minimal concentration of 50 μg/l. VKDB are not reported in formula-fed babies and there is no evidence that they are at an increased risk of childhood cancer [28]. Studies with such a concentration of vitamin K1 have never been performed and further research is necessary to study the efficacy of this prophylaxis in infants with underlying cholestatic liver disease. Due to the low incidence of late VKDB, it would be difficult to perform a randomised trial of 25 μg versus 50 μg of vitamin K1 daily. Alternatively, a new surveillance for 1 or 2 years after implementing 50 μg of vitamin K1 as the prophylaxis could give an indication of the efficacy of this dose. Furthermore, an earlier recognition of symptoms associated with cholestatic liver disease and immediate investigation and treatment of “warning bleeds” helps to prevent the severe consequences of secondary late VKDB. In conclusion, with the current guideline of vitamin K prophylaxis in the Netherlands, idiopathic late VKDB is rare, but late VKDB secondary to an underlying cholestatic liver disease still occurs. We suggest to double the daily maintenance dose of vitamin K1 to 50 μg per day for all breastfed infants from 1 to 13 weeks of age in order to prevent bleedings in children with an underlying cholestatic liver disease as well. Further research, however, is recommended.

Pediatr_Nephrol-3-1-2064946

Molecular mechanism of edema formation in nephrotic syndrome: therapeutic implications

Sodium retention and edema are common features of nephrotic syndrome that are classically attributed to hypovolemia and activation of the renin–angiotensin–aldosterone system. However, numbers of clinical and experimental findings argue against this underfill theory. In this review we analyze data from the literature in both nephrotic patients and experimental models of nephrotic syndrome that converge to demonstrate that sodium retention is not related to the renin–angiotensin–aldosterone status and that fluid leakage from capillary to the interstitium does not result from an imbalance of Starling forces, but from changes of the intrinsic properties of the capillary endothelial filtration barrier. We also discuss how most recent findings on the cellular and molecular mechanisms of sodium retention has allowed the development of an efficient treatment of edema in nephrotic patients. Introduction Interstitial edema is a common clinical feature of nephrotic syndrome (NS). It is often massive (up to 30% of body weight) and constitutes a functional constraint, owing to locomotive restriction and eyelid shutting. Expansion of the interstitial compartment volume results from the combination of abnormal renal sodium retention and alterations of fluid transfer across capillary walls. Renal retention of sodium in nephrotic syndrome Site of sodium retention Most of our current knowledge on the site and mechanism of renal sodium retention in NS comes from experimental models of the disease, in particular the puromycin aminonucleoside (PAN) rat model. Following a single injection of PAN, rats develop massive proteinuria within 4–5 days and sodium retention within 2–3 days. Sodium excretion resumes after 9–10 days, while proteinuria lasts for 2–3 weeks [1, 2]. Unilateral NS can be induced by the injection of PAN into one of the renal arteries [3]. Early in vivo micropuncture studies in rats with unilateral PAN nephrosis demonstrated that sodium retention in the nephrotic kidney originates beyond the last nephron segment accessible to micropuncture, i.e., between the late distal convoluted tubule and the tip of the collecting duct [3]. This part of the nephron encompasses the connecting tubule and the cortical and outer medullary collecting ducts, which reabsorb sodium and are the major sites of the adjustment of sodium balance under the homeostatic control of aldosterone, and the inner medullary collecting duct, which is able to secrete an overload of sodium in response to atrial natriuretic peptide [4]. Methods allowing in vitro analysis of isolated sub-segments of the distal nephron demonstrated a marked stimulation of sodium reabsorption in the cortical collecting duct (CCD) of PAN nephrotic rats [1, 5]. This stimulation of sodium transport likely extends upstream to the connecting duct but not downstream to the outer medullary collecting duct. In addition, in NS, the inner medullary collecting duct becomes insensitive to the natriuretic action of atrial natriuretic peptide [6–8], thereby preventing any compensation of sodium retention in upstream nephron segments. Cellular mechanism of sodium retention in the collecting duct In vitro microperfusion of isolated CCDs confirmed that the rate of sodium reabsorption and the trans-epithelial voltage are negligible in CCDs from control rats, whereas both are high in CCDs from PAN nephrotic rats [5]. CCDs are made of principal and intercalated cells, which account for sodium and water reabsorption and potassium secretion for principal cells, and proton, bicarbonate and likely chloride transport for intercalated cells [4]. In principal cells, sodium reabsorption proceeds along a two-step mechanism (Fig. 1): Na,K-ATPase, exclusively present in the basolateral membrane, energizes the active extrusion of sodium at the expense of ATP hydrolysis. The electrochemical gradient for sodium generated by this primary process drives passive entry of sodium through the apical membrane, which contains selective sodium channels (epithelial sodium channels, ENaCs). Both Na,K-ATPase and ENaCs are targets for multiple, and often coordinated, regulations in CCDs [4].Increased sodium reabsorption along the CCDs of PAN nephrotic rats, as well as sodium retention and edema formation, are associated with stimulation of both basolateral Na,K-ATPase [1, 9, 10] and apical ENaC [11–13] (Fig. 1), but the latter is dispensable (see below). Stimulation of Na,KATPase is fully accounted for by transcriptional induction of its α and β subunits and targeting of newly synthesized pumps to the basolateral membrane of principal cells [14]. In contrast, stimulation of ENaC mainly results from the targeting of a pre-existing pool of latent intracellular channels to the apical membrane of principal cells and slightly from transcriptional induction of the α and β subunits [11–13]. The patch-clamp technique shows that the intrinsic properties of ENaC (open probability and unitary conductance) are not altered in the CCD of nephrotic animals [13]. Fig. 1Cellular mechanism of sodium reabsorption in principal cells of collecting ducts from normal rats and nephrotic rats. Sodium reabsorption in principal cells proceeds along a two-step mechanism that includes active extrusion of intracellular sodium ions by the basolateral Na,K-ATPase and passive apical entry of sodium via the amiloride-sensitive epithelial sodium channel (ENaC). In CCDs from normal rats (top panels), most ENaCs are sequestered in the intracellular compartment of principal cells (left panel), and basolateral expression of Na,K-ATPase in collecting ducts (asterisk) principal cells is very weak, in comparison with that in thick ascending limbs (T) and even proximal tubules (P) (right panel). Accordingly, the rate of sodium reabsorption is very low. In CCDs from PAN nephrotic rats (bottom panels), ENaC is expressed at the apical border of principal cells (left panel), and expression of basolateral Na,K-ATPase is drastically increased in collecting ducts (asterisk). Polarized increases in expression of ENaC and Na,K-ATPase in principal cells account for increased sodium reabsorption in CCDs. In both normal and nephrotic rats expression of Na,K-ATPase is undetectable in the glomerulus (G); in CCDs, unlabeled cells for both ENaC and Na,K-ATPase are intercalated cells (redrawn from [13, 14]) Aldosterone and activation of mineralo-corticoid receptors are not involved in sodium retention in nephrotic syndrome Historically, it is acknowledged that sodium retention and edema formation result from hypovolemia-induced stimulation of the renin–angiotensin–aldosterone system. Hypovolemia is supposed to proceed as follows: proteinuria induces hypoalbuminemia and reduces plasma oncotic pressure, which generates an imbalance of Starling’s forces across capillary walls, leading to interstitial leakage of fluid and decreased efficient volume [15]. Although this mechanism is consistent with the renal site of sodium retention, with the activation of ENaC and the induction of Na,K-ATPase in the collecting duct, and with the hyperaldosteronemia observed in PAN nephrotic rats [13], a large body of clinical evidence argues against this theory. Analbuminemic patients display no sodium retention and no, or only modest, edema, despite low plasma oncotic pressure [16].In children with steroid-sensitive minimal change disease, natriuresis resumes at the same time as proteinuria stops, before normalization of albuminemia [17].Blood volume is not correlated with plasma oncotic pressure in nephrotic patients [18].Among children with relapse idiopathic nephrotic syndrome, (a) 21% display hypertension but only 4% exhibit collapse [19], and (b) only 1% display low blood volume, whereas 17% present hypervolemia [20].Intravenous injection of albumin induces volume expansion but promotes only mild natriuresis (reviewed in [21]).Blockade of mineralo-corticoid receptor or inhibition of angiotensin-converting enzyme has no effect on natriuresis in most patients [22, 23].The lack of a role for hyperaldosteronemia in sodium retention in NS was directly demonstrated in PAN nephrotic rats. In this model it is possible to blunt PAN-induced hyperaldosteronemia through bilateral adrenalectomy and corticosteroid replacement through implanted mini-pumps delivering constant physiological level of aldosterone and glucocorticoids. Administration of PAN to these corticosteroid-clamped rats reduces sodium excretion, establishes sodium balance and promotes ascites formation with similar time course and intensity as in adrenal-intact rats [11, 13].These findings also exclude a possible role in sodium retention of promiscuous activation of the mineralo-corticoid receptor (MR) by glucocorticoid brought about by decreased 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Indeed, use of dexamethasone instead of corticosterone for glucocorticoid replacement prevents MR activation, even in the presence of 11β-HSD2 inhibition. The lack of involvement of MR activation in sodium retention in nephrotic syndrome is consistent with the fact that nephrotic syndrome does not induce potassium secretion. None of the known factors that control sodium reabsorption in the collecting duct accounts for sodium retention in PAN-nephrotic rats Since aldosterone, the major factor that controls sodium reabsorption in collecting ducts, is not involved in sodium retention, several hormones or paracrine factors that modulate this process have been considered as putative candidates: Vasopressin (AVP), through activation of its V2 receptors coupled to the cAMP pathway, increases sodium reabsorption in the collecting duct synergistically with aldosterone [24, 25].Angiotensin II (AII) increases sodium reabsorption directly through activation of AT1 receptors coupled to phospholipase C, independently of the induction of aldosterone release [26].Insulin-like growth factor I (IGF-1) used in the treatment of insulin resistance in type 2 diabetes can induce sodium retention and edema [27], likely through its stimulatory action on sodium transport along the collecting duct [28].In mouse collecting duct cells, tumor necrosis factor alpha (TNFα) increases membrane expression of Na,K-ATPase [29], and administration of etanercept, a TNFα receptor antagonist, to diabetic rats induces natriuresis [30].More recently, the thiazolidinedione agonists of peroxisome proliferator-activated receptor γ (PPARγ) were reported to induce sodium retention [31] through a stimulatory effect on the collecting duct [32].Quite unexpectedly, inhibition of nitric oxide synthase promotes sodium excretion in cirrhotic rats with ascites [33].Except for AII, all maneuvers aimed at blocking these different pathways had no effect on sodium retention in nephrotic syndrome (Fig. 2). Only the AT1 blocker irbesartan was able to improve sodium excretion, although not up to normal level. However, this effect was limited to the initial sodium retention observed 2–4 days after PAN administration and did not alter the main effect observed at days 5 and 6. This suggests that AII, independently of aldosterone, may be involved in the early retention of sodium observed after the peak of sodium excretion at day 1. Fig. 2Profile of sodium excretion in PAN nephrotic rats. Daily urinary sodium excretion, expressed as a function of urinary creatinine excretion, following administration of puromycin aminonucleoside (150 mg/kg body wt, intravenously) in normal rats (dotted lines) or genetically modified or pharmacologically treated rats (solid lines). Arrows or grey boxes show the time of treatment. Brattleboro rats genetically lack vasopressin secretion. JB1, an inhibitor of IGF-1 receptors, was continuously administered via subcutaneous mini-pumps at a dose of 12 μg/100 g body wt per day, starting on day 3. The antagonist of AT1 receptor, irbesartan, was administered per os at a dose of 2 mg/100g body wt per day. Etanercept, a chimeric antibody directed against TNF receptor, was administered twice (days −1 and 2) at a dose of 0.2 mg/100g body wt. The inhibitor of nitric oxide synthase, L-NAME, was administered twice daily by gavage (0.5 mg/100 g body wt per 12 h) throughout the study. The antagonist of PPARγ, SR202, was given per os at a dose of 20 mg/100 g body wt. All controls were treated in parallel with the vehicle. Values are means ± SE from 4–5 ratsAs a matter of fact, sodium retention in PAN nephrosis unlikely results from the presence of an abnormal blood concentration of any factor, since only the treated kidney displays increased sodium reabsorption in unilateral PAN-nephrotic rats. Rather, alterations in collecting duct function may be accounted for either by a direct effect of PAN on CCD or by the abnormal presence of an unidentified stimulatory factor in the luminal fluid that reaches the distal nephron. The presence of this factor may result from its abnormal filtration secondarily to the alteration of the glomerular filtration barrier (e.g., a high molecular weight protein), or from its generation in the proximal tubule secondarily to alteration of protein/peptide handling. Na,K-ATPase is the primary target of sodium retention in PAN nephrosis Analysis of corticosteroid-clamped rats not only demonstrated that hyperaldosteronemia is not involved in the etiology of edemas in NS, but it also provided insights into the primary molecular target of sodium retention in the CCD. Indeed, in CCDs from corticosteroid-clamped PAN rats, Na,K-ATPase is induced as in adrenal-intact nephrotic rats [13]. In contrast, targeting of ENaC to the apical membrane is fully blunted [11, 13], and the residual channels expressed at the apical membrane are drastically inhibited, as their open probability is reduced >10-fold [13].Thus, induction of Na,K-ATPase is primarily responsible for sodium retention in PAN nephrotic syndrome. This conclusion likely applies to all experimental models of nephrotic syndrome and, possibly, to the disease in humans, since there is a significant reverse correlation between Na,K-ATPase activity in collecting duct and urinary sodium excretion during the phase of sodium retention in rats with nephrotic syndrome induced by PAN, Adriamycin or mercury chloride [1]. Edema formation in nephrotic syndrome Most sodium-retaining states are associated with high blood pressure but not with development of edema or ascites. Edema formation in NS results from the asymmetry of extracellular volume expansion brought about by sodium retention: the vascular volume is not, or only slightly, modified, whereas water and solutes accumulate in the interstitium. Distribution of fluid between vascular and interstitium compartments is governed by fluid exchanges across the capillary wall and by lymphatic draining. Capillary filtration capacity is increased almost twofold in nephrotic patients [34]. This fluid leakage is governed by several parameters according to Starling’s law: where Jv is the trans-capillary flux of fluid, Lp the hydraulic conductivity of capillaries, S the exchange surface, Pc and Pi the hydrostatic pressure of the capillary and interstitium, respectively, σ the reflection coefficient of proteins across the capillary wall, and Πc and Πi the oncotic pressure in the capillaries and interstitium, respectively. Unexpectedly, data from the literature indicate that the increase in Jv in NS is not accounted for by change in the oncotic pressure gradients, as intuitively thought, but by changes in the intrinsic properties of the capillary walls that govern their hydraulic conductance and reflexion coefficient for proteins. This means that the capillary wall is a direct target in NS, to the same right as the glomerular filtration barrier. The trans-capillary gradient of oncotic pressure (Πc–Πi) is unchanged in NS The lack of edema and ascites in analbuminemic rats and patients [16, 35] has questioned the importance of low plasma oncotic pressure in the genesis of edema in NS. As a matter of fact, the trans-capillary gradient of oncotic pressure is unchanged in analbuminemic rats, owing to a parallel decrease in plasma and interstitium oncotic pressure [35]. Experiments in dogs, in which plasma oncotic pressure was progressively decreased by 50% through plasmapheresis, showed that the interstitium oncotic pressure decreased in parallel and that the trans-capillary gradient remained unaffected. Extracellular volume increased transiently during the phase of variation of plasma oncotic pressure, in response to hyperaldosteronemia and renal sodium retention, but returned to normal level during the period of stabilized low plasma oncotic pressure [36].Essentially similar observations were made in humans. During NS, it was also observed that the oncotic pressure of the interstitium decreases in parallel with that of the vascular compartment, so that the trans-capillary gradient is only slightly reduced [37]. In addition, diuretic treatments [37] or extracorporeal ultrafiltration [38] allow the withdrawal of significant amounts of edema without significant change in the trans-capillary gradient of oncotic pressure.Thus, decrease in plasma oncotic pressure in animal models as well as in nephrotic patients does not alter significantly the trans-capillary gradient of oncotic pressure and is neither a determinant parameter in the genesis of edema nor a resistance factor to edema withdrawal. The trans-capillary gradient of hydrostatic pressure (Pc–Pi) is unchanged in NS Capillary pressure is unchanged in nephrotic patients [34]. Because soft tissues display an almost infinite compliance, their interstitium pressure increases by only 2 mmHg with their fluid filling [39]. In nephrotic patients interstitial pressure in edematous and non-edematous sectors differs by <4 mmHg [40]. Thus, it can be admitted that the trans-capillary gradient of hydrostatic pressure is not significantly altered in soft edematous tissues during NS. The capillary hydraulic conductivity Lp is increased in NS The threshold of venous pressure triggering fluid transfer across capillaries is significantly reduced in NS [34], suggesting that endothelial Lp is decreased. The main determinants of the hydraulic conductivity of capillaries are the occlusive junctions (constituted by occludin, claudins and proteins ZO) and adhesive junctions (made of cadherins, catenins and actinin) between endothelial cells. These proteins are direct targets for intracellular signaling cascades, in particular protein kinase C (PKC), which phosphorylates occludin [41] and alters the endothelial permeability [42]. In NS this pathway may be activated through two mechanisms. Firstly, hypoalbuminemia increases Lp via an increase in intracellular calcium [43]. Secondly, high plasma levels of TNFα observed in patients with minimal change disease [44] activate PKC and increase Lp [45]. The reflexion coefficient of proteins (σ) is increased in NS Increased coefficient of reflexion of macromolecules in NS is evidenced by the higher rate of leakage of technetium-labeled albumin towards the interstitium in nephrotic patients than in controls [46]. Because this change in reflexion coefficient is observed in patients with NS of different origin, it is likely not accounted for by the circulating permeability factor of lymphocyte origin responsible for glomerular hyperfiltration in minimal change disease. Increased σ was also reported in blood–peritoneal barrier permeability in PAN-nephrotic rats [47]. Therapeutic implications Nephrotic edema results from the combination of renal sodium retention and increased capillary permeability. Although treatment of either of these two alterations would prevent edema, treatment of capillary permeability alone would lead to hypertension. Thus, treatments of edema must primarily target renal sodium retention. Different therapeutic strategies are needed to prevent edema formation in children with chronic proteinuria and to treat massive edema. In the first case it is sufficient to prevent sodium accumulation, either through limiting dietary sodium uptake or by natriuretic drugs that target the collecting duct, e.g., amiloride. In clinical practice the daily uptake of sodium is usually limited to 0.5 mmol/kg. In PAN-nephrotic rats administration of amiloride starting before the onset of sodium retention fully prevents sodium retention and formation of ascites [5]. More classically, clinicians are confronted with patients who already display massive edema. Treatment aims both at limiting further sodium retention and at promoting the excretion of the mass of sodium and water sequestered in edema. While amiloride is well suited to block further sodium retention, it is inefficient in promoting massive sodium excretion, because, under normal conditions, sodium reabsorption along the collecting duct is quantitatively low. Recourse to more potent diuretics, such as loop diuretics, is restricted by the functional resistance of nephrotic patients to the natriuretic effect of furosemide. Several explanations have been proposed to account for this resistance, but none has been confirmed: The pharmacokinetics of furosemide urinary elimination is not significantly altered in nephrotic children [48]Binding of furosemide to albumin in the tubular fluid does not account for furosemide resistance, since, despite controversial data [49, 50], inhibitors of furosemide–albumin binding do not improve sodium excretion significantly.The intrinsic sensitivity of the Na/K/2Cl transporter of the loop of Henle, the molecular target of furosemide, is not altered in nephrotic rats [5]. Based on our present knowledge of the site and cellular mechanism of sodium retention in NS, another explanation of furosemide resistance, and a therapeutic strategy to circumvent it, can be proposed. In non-nephrotic patients, furosemide decreases sodium reabsorption along the thick ascending limb of the loop of Henle, which increases sodium delivery to the distal nephron. Because the sodium reabsorption capacity of the distal nephron is rather limited, only a small fraction of the overload of sodium is reabsorbed, and the major fraction is excreted in the urine, accounting for the natriuretic effect of loop diuretics. Note that following long-term treatment with furosemide, there are adaptations of the distal nephron which increase its sodium reabsorption capacity and, thereby, reduce the natriuretic effect of furosemide. Because nephrotic patients display a huge sodium reabsorption capacity along their connecting and cortical collecting tubules, most of the overload of sodium brought about by furosemide-induced inhibition of transport in the thick ascending limb is reabsorbed, thereby blunting the natriuretic effect. Thus, the apparent resistance of nephrotic patients to loop diuretics can be circumvented by the inhibition of distal sodium reabsorption with amiloride. As a matter of fact, co-administration of furosemide and amiloride to nephrotic children increases urinary sodium excretion, induces a negative sodium balance and promotes complete edema withdrawal within 1 week [21]. Conclusion Whatever their etiology, nephrotic syndromes are always associated with renal retention of sodium. Renal sodium retention results from enhanced sodium reabsorption along the connecting and cortical collecting ducts and from blunted responsiveness of medullary collecting ducts to the natriuretic response to atrial natriuretic peptide. Induction of de novo synthesis of Na,K-ATPase is the primary effector of increased sodium reabsorption. It is not accounted for by any circulating factor, in particular aldosterone, known to stimulate sodium reabsorption along the distal nephron. New research strategies will be required to identify the unknown regulatory pathway that is dysregulated in NS. Sodium retention in NS does not lead to high blood pressure but leads to an asymmetric expansion of the interstitium, while the vascular volume remains unchanged in most patients. This asymmetry of extracellular volume expansion is accounted for by changes in the intrinsic properties of the endothelial capillary barriers, i.e., an increase in its hydraulic conductivity and permeability to proteins, rather than to an imbalance of Starling’s forces. Thus, the pathophysiology of nephrotic syndrome relies on at least three disorders: a major alteration of the glomerular filtration barrier responsible for proteinuria and hypoalbuminemia, an induction of distal nephron Na,K-ATPase responsible for sodium retention, and alterations in the capillary permeability accounting for the asymmetry of volume expansion. Although causal relationships between these three events have not been formally established, it is assumed that the glomerular defect engenders both the tubular and the capillary alterations. This conclusion is based on the facts that: (a) whatever its origin, alteration of the glomerular filtration barrier always leads to sodium retention and edema formation, i.e., to the tubular and capillary defects, and (b) loss of function mutations of nephrin, which is expressed in the glomerular slit diaphragm but in neither the collecting duct nor capillary endothelial cells, is sufficient to promote proteinuria, sodium retention and edema [51]. Curiously, in PAN-induced nephrosis, sodium retention precedes proteinuria, suggesting that it is not secondary to the glomerular dysfunction. It should be stressed, however, that microproteinuria may appear sooner and concomitantly with sodium retention.

Anal_Bioanal_Chem-3-1-1797063

The effect of the chemical, biological, and physical environment on quorum sensing in structured microbial communities

As researchers attempt to study quorum sensing in relevant clinical or environmental settings, it is apparent that many factors have the potential to affect signaling. These factors span a range of physical, chemical, and biological variables that can impact signal production, stability and distribution. Optimizing experimental systems to natural or clinical environments may be crucial for defining when and where quorum sensing occurs. These points are illustrated in our case study of S. aureus signaling in biofilms, where signal stability may be affected by the host environment. The basic signaling schemes have been worked out at the molecular level for a few of the major quorum-sensing systems. As these studies continue to refine our understanding of these mechanisms, an emerging challenge is to identify if and when the local environment can affect signaling. Introduction As illustrated by many articles in this special issue, the diversity of mechanisms bacteria use to communicate with one another is remarkable. Quorum sensing is widespread among different bacterial species, emphasizing the importance of coordinating behavior as a group. When considering the relevant context of quorum sensing, it is clear that the environment can be profoundly important. Many bacteria exist in spatially structured, multi-species communities, such as biofilms on submerged surfaces or flocs in the water column of aquatic environments [1, 2]. Within these communities bacteria may achieve the high cell numbers capable of generating the local signal concentrations required to produce a quorum sensing response. Quorum sensing undoubtedly occurs in situations where bacteria are growing planktonically, for example Vibrio harveyi quorum sensing-controlled bioluminescence in the ocean, thought to produce the “milky seas” phenomenon [3, 4]. An argument could, however, be made that quorum sensing is more likely to occur in structured communities. If the reader accepts this point, further considerations lead to some interesting questions for example: Are certain signals better suited for signaling in different types of environments?What are the important parameters influencing signaling in different environments? Take, as an example, the well-studied, environmentally ubiquitous Pseudomonas aeruginosa. This bacterium has been isolated from a range of soil and aquatic samples [5–7]. The nature of signaling in these environments could be quite different, because signal production and diffusion dynamics may change. This review will discuss factors that may affect signaling in structured microbial communities. In addition, quorum sensing in Staphylococcus aureus, a peptide-based signaling system, will be briefly reviewed and its role in biofilm formation will be discussed. Factors affecting signal diffusion, stability, and distribution The traditional way of studying quorum sensing in the laboratory is in shaken liquid-batch cultures. The quorum sensing response occurs at a specific point in the growth curve, coinciding with a threshold concentration of signal. Volume is fixed in a culture flask and signal molecules produced by bacteria usually increase in concentration with time (unless they are degraded). Batch cultures represent a closed system in which only gaseous exchange occurs. Most structured communities are in open systems in which exchange occurs with the surrounding liquid [1]. In an open, flowing system, signals may be “washed” away by the overlying fluid. In open systems the concentration of signal molecules within structured communities is primarily a function of: signal-production rate;the degradation rate or half-life of the signal;the diffusion properties of the signal; andthe external hydrodynamic or mass-transfer conditions. The prevailing environmental conditions and resident biology are, to different degrees, important for each of these processes. The effects of signal and environmental chemistry on quorum sensing Some of the best-studied quorum-sensing signals include the acyl-homoserine lactones (AHL) used by many Gram-negative bacterial species, small peptides used by some Gram-positive species, and the furans of the phylogenetically widely spread AI-2 signaling (reviews are available elsewhere [8–10]). Signal chemistry varies widely in different quorum-sensing systems and is important for determining its stability and diffusion characteristics in different environments. All AHL signals have a characteristic homoserine lactone moiety. The homoserine lactone is derived from S-adenosylmethionine, one of the substrates for AHL synthesis [11–13]. Although stable at neutral and slightly acidic pH, the lactone ring is subject to chemical hydrolysis under basic conditions [14, 15]. The resulting product (e.g. butyryl-homoserine lactone would become butyryl-homoserine) lacks biological signaling activity and is capable of spontaneously re-lactonizing when the pH is lowered. The formula describing the relationship between pH and the lactone ring stability is 1/(1×107×[OH−]) [14], which gives the half life in days. For example, at pH 7 the homoserine lactone ring would be stable for hours whereas at pH 8.5 the stability would be of the order of minutes (Fig. 1). In many environments this might be insignificant. In some alkaline environments, however, like the photosynthetic mats at Octopus Springs in Yellowstone National Park, the pH is high enough to potentially affect signal gradients. Kaufmann et al. recently demonstrated that in addition to lactone hydrolysis, the 3-oxo-C12 AHL of P. aeruginosa is capable of spontaneously undergoing a Claisen-like condensation reaction in aqueous environments, forming a tetramic acid product, 3-hydroxydecylidene 5-(2-hydroxyethyl) pyrrolidine-2,4-dione (Fig. 2) [16]. This compound was shown to have significant antimicrobial activity against other species and a high affinity for iron. It was proposed this iron-binding activity constituted an alternative means of acquiring iron by P. aeruginosa. Fig. 1Predicted AHL half-lives in different alkaline environmentsFig. 2Structures of quorum-sensing signals and their derivatives. Letter designations for the Gram-positive peptide signals indicate amino acids. For the L. lactis signal nisin, the structural abbreviations were: Bu, dehydrobutirine with a lanthionine bridge; Ha, dehydroalanine; Hb, dehydrobutirine Structured communities can create highly heterogeneous localized niches where chemistry can vary drastically over very small distances. For example, consumption of dissolved oxygen by laboratory biofilm cells can result in completely anaerobic regions within the biofilm only 100 μm from the fully oxygen saturated overlying liquid [17]. Similarly, pH can vary dramatically, as demonstrated by pH microelectrodes positioned in biofilms formed from the dental pathogen Streptococcus mutans. On addition of the growth substrate sucrose, the pH dropped from 7 outside the biofilm to 4.6 approximately 100 μm deep within the biofilm (Gieseke A, Nguyen D, von Ohle C, Stoodley P; unpublished results). Clearly, if the half-life of signaling molecules is a function of local chemistry it is reasonable to expect that degradation rates will vary in the community. The acyl side-chains of AHLs range in length from 4 to 18 carbon atoms [18, 19]. The substitution chemistry of the acyl side-chain is also highly variable. Although most AHLs have a ketone group at the C1 position, some AHLs also have either a ketone or hydroxyl group at the C3 position. In addition, the acyl side-chain can vary in saturation. Although most AHLs have fully saturated acyl side-chains, some, for example the 3-hydroxy,7-cis-tetradecanoyl homoserine lactone of Rhizobium leguminosarum, are unsaturated [19, 20]. All these features of AHL acyl side-chain chemistry, in particular acyl chain length, affect the relative hydrophobicity of the signal. Pearson et al. observed this for P. aeruginosa, which produces two major AHL signals, a C4 (i.e. four-carbon side-chain) and 3-oxo-C12 AHL (Fig. 2) [21]. They found that the C4 signal diffused freely across the cell membrane whereas the 3-oxo-C12 signal partitioned to the cell membrane, where its extrusion was aided by the activity of the MexAB-OprM efflux pump. The hydrophobicity of the local environment could have a large effect on AHL diffusion properties. For example, several AHL-utilizing bacterial species are also capable of producing highly hydrophobic, secreted polysaccharides (e.g. the pel/psl polysaccharides of P. aeruginosa) [22]. In a structured community, such as a biofilm, secreted polysaccharides can serve as the scaffolding holding the community together [23]. Under such conditions these polysaccharides might also act as an AHL-sequestering matrix. Charlton et al. provided evidence of this, indicating that the 3-oxo-C12 signal of P. aeruginosa partitioned into the extracellular biofilm matrix [24]. Perhaps the C4 signal has reduced reactivity with the biofilm matrix, making it a more effective signal in a biofilm and explaining why it is produced in greater amounts than 3-oxo-C12 by P. aeruginosa growing as a biofilm [25]. Another factor affecting AHL stability in the environment is biologically-driven signal degradation. Emphasizing this point, Wang et al. demonstrated that radiolabled AHLs fed to soil microbial communities were almost instantly mineralized [26]! Several bacterial species have the ability to use AHLs as carbon and nitrogen sources. These include species that produce their own AHLs and those known not to make them. Why some organisms both make and break down their own AHLs is a mystery. AHL degradation occurs as a result of two primary types of enzymatic activity, lactonases, which break the homoserine lactone ring, and acylases, which cleave the amide bond linking the acyl side-chain to the homoserine lactone ring [27–31]. Biological AHL degradation is a key consideration in multi-species environments. Leadbetter proposed the concept of “insulation” in which AHL-degrading organisms could prevent AHLs produced by bacteria spatially fixed in one location from reaching other bacteria of the same species [26]. Environmental chemistry also plays a key role in AI-2 signaling. The paradigm for this type of quorum sensing is the luxS system of Vibrio harveyi [8]. One of the key steps in AI-2 signal synthesis is the LuxS-catalyzed conversion of S-ribosyl homocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD) [32]. DPD is believed to be the key precursor to all AI-2 signals. DPD undergoes non-enzymatic cyclization and a number of spontaneous, reversible rearrangements to form the active signal-a furanosyl borate diester and other furans [33, 34]. The AI-2 signal for Salmonella typhimurium was recently solved and found to be, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran, a molecule related to, but structurally distinct from, the V. harveyi AI-2 (Fig. 2) [35]. Spontaneous extracellular chemical rearrangements of S. typhimurium AI-2 can produce the V. harveyi AI-2 signal; the reverse is also true [35]. Current thinking is that the local chemical environment can affect the ratio of the different furanosyl esters. In particular, the presence of borate may shift the equilibrium toward formation of the V. harveyi AI-2 [35]. The chemical structures of the Gram-positive quorum-sensing peptides vary greatly in the number of residues and the types of modification (Fig. 2). Because of the post-translational modifications on these peptides and their inability to diffuse across membranes, their biosynthetic pathways are more complex than those of the AHLs and dedicated signal-export systems are usually required. Many also induce their own biosynthesis, and are thus frequently termed “autoinducing peptides”. Several signals are simple linear peptides, for example the 17-residue competence-stimulating peptide (CSP) which regulates competence in Streptococcus pneumoniae [36]. The corresponding linear competence pheromone in Bacillus subtilis, called ComX, is more unusual in that an internal tryptophan residue has been cyclized and isoprenylated with a geranyl moiety [37], a modification that is catalyzed by ComQ [38]. Perhaps the largest and most complex peptide signals are the lantibiotics, which also have antimicrobial activity [39]. These molecules, for example nisin produced by Lactococcus lactis, are known to positively regulate their own biosynthesis in a density-dependent manner. They also have extensive post-translation modifications, including dehydrated residues, such as dehydroalanine and dehydrobutyrine, and thioether bridges called lanthionines (Fig. 2 depicts the base structure of the lantibiotic, nisin, of L. lactis). An emerging class of peptide signals are the cyclic lactones and thiolactones [40]. The first of this class to be discovered was the type I autoinducing peptide (AIP) of Staphylococcus aureus, an eight-residue thiolactone-containing peptide with the C-terminus constrained through linkage with a cysteine side-chain [41]. Since the discovery of AIPs, related signals have been identified in Enterococcus faecalis [42], Listeria monocytogenes [43], and other Staphylococci [44]. Environmental conditions can have a significant effect on peptide signal stability. The linear peptides are likely to have short half-lives as they are rapidly metabolized by the action of secreted proteases. Indeed, this issue has led some to question their role as a means of communication, raising the possibility they serve as a general strategy for a single cell to time regulatory events [45]. In contrast, the lantibiotics have multiple lanthionine bridges that block the activity of many proteases [46], greatly improving the longevity of these molecules. Like AHLs, lantibiotics are pH-sensitive, with solubility and stability dropping rapidly at higher pH; those with dehydrated residues are also reactive with thiols [47]. Because of these chemical constraints, the lantibiotic nisin has a half-life of only 0.9 h in mouse serum [48], the pH of which is approximately 7.3. For L. lactis in the environment, however, growth and production of nisin are optimum at lower pH, at which stability is greater, facilitating autoinduction. In a similar fashion, the thiolactone-containing peptides are resistant to proteolysis and sensitive to high pH and reactive thiols (chemistry of the S. aureus AIP is discussed in more detail below), and some have increased sensitivity to oxidative damage [49]. Although it remains to be demonstrated, protease resistance may be one of the most significant environmental constraints faced by bacteria using peptide communication signals and extensive post-translational modifications could have evolved to overcome this problem. The effect of the hydrodynamic environment on quorum sensing Mass transfer is the physical process by which molecules are transported in a system, and it has the potential to affect cell-to-cell signaling in many ways. Delivery of nutrients to the active biomass in the structured community is one example. This could affect the relative metabolic activity of the community, which in turn has the potential to affect signal-production rates. Mass transfer is affected by the hydrodynamics of the bulk fluid and the geometry of the structured community. These two factors affect each other, because a microbial community both shapes, and is shaped by, its external environment. If liquid flow is a feature of the environment, it will wash signal away, diluting its concentration within the community. In these circumstances the system can be divided into three zones (Fig. 3). First, the biomass of the community which is producing signaling molecules, second, the viscous sublayer (δ) located at the biomass-bulk fluid interface, and, third, the well mixed turbulent core of the overlying fluid. In laminar flow there is no turbulent core and δ essentially extends to either the center of the channel if flow is in a closed conduit (e.g. pipe or catheter) or to the fluid surface if flow is in an open channel (e.g. river). Using this simplified system we have constructed a one-dimensional model based on a flat biofilm to illustrate how different values of δ may result in different concentration profiles while the thickness of the biofilm, the cell density in the biofilm, and the rate of signal-production in the biofilm remain constant. The model is based on coupling the diffusion of signal through the viscous sublayer with the simultaneous diffusion and production of signal that occurs within the biofilm itself. In the fluid outside the biofilm, the concentration of signal changes linearly: where Cz is the signal concentration (mg L−1) at depth z, C0 is the signal concentration outside the viscous sublayer (0 mg L−1), k0 is the signal production rate (2.96×10−17 mg cell−1 s−1), Xb is the active cell density in the biofilm (1×1010 CFU cm−3), Lf is the biofilm thickness (50 μm), δ is the thickness of the viscous sublayer (0, 25, or 50 μm), Daq is the diffusion coefficient of the signal in water (4.9×10−6 cm2 s−1), and δz is the proportional depth within δ (μm). Within the biofilm, the concentration of the signal changes quadratically with depth: where De is the effective diffusion coefficient of signal in the biofilm (1.23×10−6 cm2 s−1) and Lz is the proportional depth within the biofilm (μm). Fig. 3Schematic diagram illustrating how external flow may affect the concentration of a cell signal produced by biofilm cells. The biofilm is 50 μm thick (so there is no oxygen limitation and we can assume equal signal production throughout the depth of the biofilm) and the relative concentration profiles are for three flow conditions: (a) Very high shear, negligible diffusion boundary layer (δ). (b) Lower flow with a diffusion boundary layer of 25 μm (δB). (c) Lower flow, again, with a diffusion boundary layer of 50 μ (δC). The model is steady-state Fickian diffusion for flat-slab geometry with a uniform density of bacteria producing signals at a constant rate with no degradation. There is no convection within the biofilm and no flux at the substratum. The concentration of signal in the turbulent core of the flow is zero. The effective diffusion coefficient in the biofilm is assumed to be 0.25 times that of the bulk fluid based on the molecular weight of 3-oxo-C12 AHL [50]. For simplification the viscous sublayer is shown as the diffusion boundary layer. In reality the transition (the “buffer region” of the boundary layer) from the viscous sublayer to the turbulent core is not a sharp line, as depicted, but gradual. If we assume an inducing 3-oxo-C12 AHL concentration of 3 mg L−1 (10 μmol), depicted by the vertical dashed line, biofilm “A” is not induced at all, in biofilm “B” the bottom 50% is induced, and in biofilm “C” the bottom 75% is induced, as shown by the horizontal dashed lines where the inducing concentration intersects the concentration profile Where known we used data as best we could, based on the signal 3-oxo-C12 AHL, unknown model data were hypothetical. All data were based on those used for illustrative purposes by Stewart [50]. These considerations led us to hypothesize that external flow conditions can affect quorum sensing in many ways. For example, in a somewhat closed, static system (e.g. a biofilm in a mud puddle or in the well of a microtitre plate), signals produced by a large, biofilm cell aggregate may induce a quorum-sensing response in neighboring bacteria that are not part of the aggregate. In contrast, in an open system subject to convective flow (e.g. a biofilm in a river, or the channel of a flow cell biofilm reactor), signals might be continuously removed from the system. Under these conditions mass transfer may prevent signals produced by a large biofilm cell aggregate from inducing cells in the vicinity of the aggregate. Experimental results on interspecies communications in dental plaque biofilms by Egland et al. [51] support this idea, and suggest that diffusible signals are adapted to function over short distances. At higher flows the higher flux of signal molecules from structured communities may reduce signal concentration in the interior or core of the community. A subsequent consequence is that more biomass would be necessary to produce an inducing concentration of signal. Few studies have directly addressed the effect of hydrodynamics on quorum sensing in structured communities. Purevdorj et al. [52] found that at relatively high flow rates flow velocity was a stronger determinant of P. aeruginosa biofilm structure than quorum-sensing-required functions. Yarwood et al. [54] grew wild type (WT) and accessory gene regulator (agr) mutant biofilms of S. aureus by batch culture under static conditions, batch culture on spinning disks, and in flow cells. The QS mutation had the greatest effect under static conditions, resulting in an increase in biofilm formation, consistent with the study of Vuong et al. [55], who hypothesized that agr was a repressor of biofilm formation. Interestingly, there was no difference between WT and agr (quorum sensing) mutant biofilms when grown in flow cells. Care must, however, be taken in correlating growth system with phenotype, because media type and concentration were also varied in this study. Nevertheless, Fux et al. [56] obtained similar results for flow cell-grown biofilms. After 24 h the WT S. aureus biofilms had less biomass than a TRAP mutant but were significantly rougher. After 4 days, however, there was little difference between the biofilms as measured by COMSTAT confocal image-analysis software [57]. In the same study the opposite trend, as measured by viable plate counts, was observed for “filter colony biofilms” [58] grown under static conditions on nutrient agar. The WT biofilms contained an order of magnitude more cells than the TRAP mutant. Interestingly, a regulatory network mathematical model has predicted that at intermediate concentrations of 3-oxo-C12, bacteria can suddenly switch between induced and noninduced states [59]. This effect may help explain experimental observation of different biofilm phenotypes under what are assumed to be similar or even replicate growth conditions. Quorum sensing in the gas phase with volatile signals Quorum sensing is usually discussed and studied in the context of aqueous environments. Some environmental conditions, however, for example those found in soil, may not always be ideal for signaling in the liquid phase. Soil is subject to wetting and drying cycles, and even somewhat dry soils are teeming with metabolically active bacteria [60, 61]. Under these conditions it may be advantageous to some bacterial species to engage in quorum sensing. Local cell numbers may be quite high, but not linked to one another through the liquid phase. The production of signals able to act through the gas phase would circumvent these issues. There is precedence for volatile signaling in the microbial world. An interesting example is the signaling molecule, 3-OH palmitic acid methyl ester (3-OH PAME) of Ralstonia solanacearum (Fig. 2) [62]. The signal of this quorum-sensing system can act in the gas phase. This plant pathogen regulates the production of secreted polysaccharides (an important virulence factor) by use of 3-OH PAME [63, 64], the synthesis of which is catalyzed by the phcB gene product [63]. When 3-OH PAME concentrations reach ∼5 nmol L−1, 3-OH PAME-regulated genes are induced, presumably because of interaction of the signal with the membrane-associated PhcS sensor kinase. Although homologs of phcB are not found on many of the microbial genomes yet sequenced, signaling in the gas phase may be one of the next important frontiers in quorum sensing. A case study of signaling in structured communities-Quorum-sensing in Staphylococcus aureus biofilms Continuing with the themes of this review, a case study will be presented with a discussion on environmental, chemical, and biological factors discussed above that have the potential to affect quorum-sensing in S. aureus biofilms. S. aureus is a human commensal that resides in a non-pathogenic state in the nasal airways. When there is a breach in the host defenses enabling access, S. aureus can convert to a pathogenic state and secrete an impressive array of toxins, hemolysins, and degratory enzymes [40], causing damage to host tissues. This lifestyle switch is mediated, in a cell-density-dependent manner, by the action of the AIP molecule. Early in the growth phase surface adhesins and antigens are produced, and when AIP reaches a critical concentration the quorum-sensing cascade is activated and these surface proteins are down-regulated and invasive factors are secreted. Microarray studies have revealed that 104 genes are up-regulated and 34 are down-regulated by the action of this quorum-sensing molecule [65]; this represents almost five percent of the genome. Intriguingly, there are four specific classes of AIP molecule that approximately correlate with the type of disease caused by the producing S. aureus strain. In a fascinating mechanism of bacterial cross-talk these different classes of AIP cross-inhibit quorum-sensing in other S. aureus groups and other staphylococcal species [41]; this may serve to isolate specific sub-populations for cooperative action. The locus responsible for the quorum-sensing regulation in S. aureus is the accessory gene regulator or Agr locus and is known to contain two divergent transcripts, named RNAII and RNAIII [40]. The RNAII transcript is an operon of four genes, agrBDCA, that encode factors required to synthesize AIP and activate the regulatory cascade (Fig. 4). The biosynthetic pathway leading to functional AIP is not clear, but it is known that AgrD is the peptide precursor of AIP and AgrB is a membrane protease involved in its processing [66]. AgrC and AgrA form a typical two-component regulatory pair, and the binding of AIP to a surface receptor on AgrC activates this phosphoryl-transfer cascade [67]. When phosphorylated, AgrA is known to bind and induce expression of the RNAIII transcript [68], which encodes a regulatory RNA molecule that acts as the primary effector of S. aureus quorum-sensing [69]. AgrA also induces expression of the Agr proteins through the RNAII transcript, triggering the autoinduction phenomenon. Fig. 4Schematic diagram of quorum-sensing systems of S. aureus. The gene locus for the agr system is shown in black and contains two divergent transcripts, RNAII and RNAIII, driven by the P2 and P3 promoters, respectively. The RNAII transcript encodes the agrBDCA operon, which encodes the signal, processing, and detection components for quorum-sensing in S. aureus (see text). The RNAIII transcript is a regulatory RNA that up-regulates and down-regulates all genes related to quorum-sensing. This transcript also encodes the amphipathic peptide δ-hemolysin. RAP and TRAP are part of a second quorum-sensing system thought to be a precursor of the agr system. RAP is secreted and induces TRAP phosphorylation, which in turn induces expression of the RNAII transcript. RIP is a heptapeptide known to inhibit the ability of RAP to induce TRAP phosphorylation A second quorum-sensing cascade is thought to serve as a precursor to the Agr system [70], setting the stage for AIP regulation. As S. aureus cells multiply, the RNAIII-activating protein (RAP) is secreted and accumulates outside the cell, and at a threshold concentration, RAP triggers the phosphorylation of the cytoplasmic protein TRAP [71], which induces expression of the RNAII transcript (Fig. 3). Intriguingly, a linear heptapeptide called RIP is known to block the activity of RAP, enabling small-molecule control over S. aureus pathogenicity [72]. Chemistry of the S. aureus AIP signal The reasons for evolving a cyclic thiolactone structure as a quorum-sensing signal are still not clear. The thioester bond and the presence of other labile amino acids reduce stability, because of the potential for oxidative damage, base-catalyzed hydrolysis, and thioester exchange. Indeed, the methionine residue in AIP is rapidly oxidized in vitro to a methionyl sulfoxide, converting the signal to an inactive byproduct [49]. Similarly, oxidants produced in vivo by the phagocyte NADPH oxidase are known to accelerate this inactivation [73]. Despite these issues, the AIP lifetime in host tissues is reported to be 3 h [74], which is more than adequate for regulation, bearing in mind that S. aureus doubles every 60 min in the host [75]. Structure-function studies have shown that the thiolactone cannot be replaced with a more stabile lactone, suggesting that the signal receptor might require acylation to activate the cascade [76], although a lactam substitution does activate at high concentrations [49]. As discussed above, constraining the peptide will improve metabolic stability to proteases [77], by protecting the C-terminus and impeding access to endoproteases, which probably increases the half-life compared with that of the linear AIP counterpart. Clearly, further study is necessary to determine the physiological benefit of this thiolactone structure. The relationship between S. aureus quorum-sensing and biofilm communities Biofilm formation is increasingly being recognized as an important virulence factor in S. aureus pathogenesis. Several biofilm-associated diseases, including osteomyelitis [78], endocarditis [79], medical device infections [80], and potentially even skin infections [81], have much clinical relevance. The formation of S. aureus biofilms progresses in a similar fashion to the Gram-negative biofilms, with attachment followed by development into a highly-structured cell community. Several attachment factors, for example the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the surface-attached Atl protein, have been implicated in this initial stage of biofilm formation (reviewed elsewhere [82, 83]). Secretion of a polysaccharide adhesin is thought to be critical for development of a structured biofilm [84], although some biofilm-forming S. aureus strains cannot produce this polymer [85]. Unlike some well-studied Gram-negative bacterial species, S. aureus are non-motile, which leads to significantly reduced biofilm architecture in flowing systems. The significance of this difference is not clear, because simple alterations of growth conditions for P. aeruginosa are known to eliminate higher-ordered biofilm structure [86]. Because the development of a robust P. aeruginosa biofilm under some conditions requires an active quorum-sensing system [53], one might assume the behavior of S. aureus is similar; the opposite seems to be true, however, because inactivation of the Agr system tends to enhance attachment [87]. Agr is known to down-regulate surface attachment factors, providing an explanation of this observation. The Agr cascade also up-regulates both the secretion of proteases that can degrade these attachment factors and the secretion of amphipathic peptides (phenol-soluble modulins) that facilitate detachment [83, 88], suggesting quorum-sensing may play a role in biofilm turnover. Despite these intriguing observations, deciphering the literature on this topic has been challenging, because of non-uniform biofilm culturing methods and strain-to-strain differences [89]. To emphasize this point, enhanced attachment by Agr mutants in static biofilm systems is not observed in flowing biofilm reactor systems [87, 90]. An elegant study by Yarwood et al. addressed some of the discrepancies in the literature and suggested an alterative role for S. aureus quorum-sensing in biofilms [87]. Under static conditions attachment of Agr mutants was markedly better than that of wild-type; as shear force increased with increased flow rate, however, this advantage was lost and under some experimental conditions became a disadvantage. As in P. aeruginosa biofilm studies, the method and type of experiment seemed to dictate the requirement for the Agr system. By following a quorum-sensing promoter in a flow cell biofilm, Yarwood et al. observed that only patches of surface cells activated the Agr cascade. Surprisingly, these cells detached from the biofilm in periodic waves, suggesting an alternative role for the Agr system in the detachment and recycling phase of biofilm development. In a biofilm infection of the host it seems probable that S. aureus will be subjected to increased flow and/or shear force, in addition to many other factors, which will affect the quorum-sensing phenomenon. Increased flow will probably dictate the local AIP concentration, potentially deactivating quorum-sensing by washing away the signal, whereas increased shear force could also perform this function, while also accelerating cell detachment. We can only speculate on how this interplay will affect S. aureus in the context of an infection, because studies in this area are limited, but given the medical importance of these biofilms, it warrants further exploration. Complicating these biofilm studies are the frequent occurrence of quorum-sensing negative isolates in clinical models. In one study, 36% of S. epidermidis isolates from joint prostheses infections were Agr mutants [80]; these mutants can also be isolated from chronic infections or through extended passage in vitro [91, 92]. It has been proposed that the metabolic burden of the Agr cascade leads to the mutations in this locus [40], with parallels to the in vitro conversion of mucoid P. aeruginosa strains to non-mucoid [93]. The frequent occurrence of Agr mutants suggests it may be advantageous for some portion of an S. aureus population to dispense with the quorum-sensing system. By inactivating Agr, a more heterogeneous, robust biofilm is likely to form, with attachment of the mutants improved by the higher level of surface factor expression. Although it remains to be investigated, this heterogeneity could mirror findings for P. aeruginosa biofilms, in which variants arise at a greater frequency than in planktonic cultures [94]. As studies on S. aureus progress, a plausible picture of the role of quorum-sensing in biofilm development is beginning to emerge. As S. aureus attaches and develops an initial biofilm, heterogeneity arising as a result of generation of Agr mutants may lead to a more robust structure with improved adherence properties. Communication between cells will be dictated by the secretion and sensing of AIP molecules, which will enable cross-activation or inhibition of quorum-sensing, depending on the staphylococcal subspecies present in close proximity. When a quorum is reached, a portion of the biofilm will slough off by down-regulation of adherence factors, which could potentially be dictated by the action of secreted proteases or amphipathic peptides. The detached S. aureus cells will have an activated quorum-sensing system that leads to the secretion of numerous invasive factors, enabling spread through the host tissues and the development of an infection. This model is in direct contrast to the well-studied P. aeruginosa paradigm, for which quorum sensing-regulated functions are important for maintaining the structural integrity of the biofilm. Clearly more studies are required to confirm or alter this hypothetical view of the S. aureus lifecycle, but as this model is refined, it may play a significant role in the development of treatment for staphylococcal diseases.

Dev_Biol-2-1-2279743

Redundancy and evolution of GATA factor requirements in development of the myocardium

The transcription factors, GATA4, 5 and 6, recognize the same DNA sequence and are all expressed in the developing myocardium. However, knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium. Here we present evidence for redundancy in this family for the first time. Using morpholinos in both Xenopus and zebrafish embryos, we show that GATA4 knockdown, for example, only affects cardiac marker expression in the absence of either GATA5 or GATA6. A similar situation pertains for GATA5 in Xenopus whereas, in zebrafish, GATA5 (faust) plays a major role in driving the myocardial programme. This requirement for GATA5 in zebrafish is for induction of the myocardium, in contrast to the GATA6 requirement in both species, which is for differentiation. This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling, suggesting that a mutual maintenance loop for GATA, BMP and Nkx expression is the evolutionarily conserved entity. Introduction The GATA factors are zinc finger transcriptional activators that bind to the consensus DNA sequence (A/T)GATA(A/G). They have been identified throughout eukaryotes and been shown to play critical roles in both haematopoiesis and cardiogenesis in vertebrates and Drosophila (Fossett and Schulz, 2001; Nemer and Nemer, 2001). Of the six evolutionarily conserved GATA genes in vertebrates, GATA4, 5 and 6 are expressed in the heart as it develops. Loss and gain of function studies in P19 embryonal carcinoma cells indicated a requirement for GATA4 in the differentiation of cardiac restricted cells to beating cardiomyocytes (Grepin et al., 1997, 1995). In addition, overexpression of GATA4 in Xenopus embryos and explants resulted in expression of cardiac differentiation markers and in some cases spontaneously beating tissue (Jiang and Evans, 1996; Latinkic et al., 2003). However, in the GATA4 null mouse, normal amounts of myocardial tissue appeared to be formed (Holtzinger and Evans, 2005; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996). Thus, even though cardia bifida and defects in looping morphogenesis were observed in the null mouse embryos, specification of the myocardium appeared to take place normally. A suggested explanation for this was the elevated expression of GATA6 (Holtzinger and Evans, 2005; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Pu et al., 2004). Consistent with this proposed redundancy of function within the family, GATA5 and 6 are also active in the P19 cell line and Xenopus explant assays described above. Thus, it appears that each of these three GATA family members possesses the capability of inducing cardiac differentiation in gain-of-function assays, however demonstration that they exhibit such redundancy in vivo awaits combinatorial loss-of-function assays. The GATA5 null mouse shows no cardiac phenotype, however it may not be a true knockout due to the potential formation of a truncated protein containing the DNA binding domain (Nemer and Nemer, 2002). In the zebrafish, a critical role for GATA5 in specification of the myocardium has been demonstrated by loss and gain of function assays (Reiter et al., 1999). The fausttm236 mutant shows a severe reduction in expression of cardiac markers and injection of GATA5 RNA induces ectopic expression of the same markers. However, GATA4 expression in the zebrafish fausttm23 mutant is significantly reduced and overexpression of GATA5 results in ectopic expression of GATA4. Thus, these studies raise the possibility that the GATA5 knockdown phenotype is due to the combined loss of GATA4 and 5. The GATA6 null mouse is an embryonic lethal due to an extra-embryonic defect and chimeras have indicated that GATA6 is not required for specification of the myocardium (Kabrun et al., 1997; Koutsourakis et al., 1999; Kuo et al., 1997; Molkentin, 2000; Molkentin et al., 1997; Morrisey et al., 1997). However, we have presented evidence that GATA6 is required for the maintenance and differentiation of cardiac progenitors in zebrafish and Xenopus embryos (Peterkin et al., 2003). The likely resolution of these apparently contradictory data is that the major consequence of lost GATA6 function is non-cell autonomous and can therefore be rescued by surrounding wild type cells in mouse chimeras. The likely non-cell autonomous target for GATA6 is BMP (Peterkin et al., 2003). However, this requirement for GATA6 is for differentiation of cardiac progenitors and not for their initial specification. In this study we use antisense morpholinos in Xenopus and zebrafish embryos to deplete combinations of GATA4, 5 and 6 for the first time. This has allowed us to provide the first experimental support for redundancy in vivo. In addition, we show that the strong dependence of zebrafish on GATA5 is not mirrored in Xenopus, where this GATA factor plays only a redundant role, like GATA4 in both species. The requirement for GATA5 in the zebrafish is for the induction of the myocardial programme, whereas in Xenopus, GATA activity is only required for differentiation. We propose that the primary function for GATA factors in development of the myocardium is in creating a sub-circuit of the regulatory network, involving another critical transcription factor, Nkx, and a crucial signalling pathway, BMP. This mutually supportive sub-network is evolutionarily stable, even though where the network is initiated appears to be more flexible. Materials and methods In situ hybridisation of whole-mounted and sectioned embryos Xenopus and zebrafish were maintained and embryos were raised and staged using standard conditions (Nieuwkoop and Faber, 1967; Westerfield, 1993). In situ hybridisations on whole-mounted and sectioned embryos were carried out as previously described (Ciau-Uitz et al., 2000; Jowett, 2001; Walmsley et al., 1994). All RNA probes used were labelled with digoxigenin (DIG) except for MyoD and Krox20 which were used in double in situ hybridisations and labelled with fluorescein. Detection of the antibody–alkaline phosphatase was done using BM purple (Roche) or Fast red (Sigma). After in situ hybridisation, embryos were re-fixed in 4% paraformaldehyde, zebrafish embryos were transferred into 75% glycerol to be photographed. Cryostat sections were performed after in situ hybridisation, embryos were fixed as above and washed in 30% sucrose. Embryos were transferred into embedding chambers in O.C.T Compound (Tissue-Tek) and 30 μm sections were cut on a Leica CM3050S. Morpholino (MO) injection The GATA4/5/6 antisense morpholinos were designed and manufactured by Genetools. Morpholino sequences: Xenopus GATA4 MO 5′ctggcaactcaatccacaaaatcca3′ (data shown here), a second morpholino designed against the same pseudo-allele as described by Afouda et al. (2005) (data not shown, 5′agctatactctgatacatcctgatc3′), and a third GATA4 MO designed to block both pseudo-alleles (a kind gift from Todd Evans) (data not shown) gave the same results. Zebrafish GATA4 MO 5′gccatcgttacaccttgatacatat3′ or a second splice morpholino as described by Holtzinger and Evans (2005). For Xenopus GATA5 MO 5′gctacaaacctcacagctcc3′ see Afouda et al. (2005). Zebrafish splice GATA5 MO 5′tgttaagatttttacctatactgga3′. For Xenopus and zebrafish GATA6 MOs see Peterkin et al. (2003). MOs were diluted in deionised water and injected as described (Peterkin et al., 2003). For zebrafish, 25 ng GATA4 MO, 25 ng GATA5 MO and/or 5 ng GATA6 MO were injected into single-cell embryos individually and in combination. For Xenopus embryos, a total of 20 ng of GATA4 MO or GATA5 MO and 10 ng of GATA6 MO were injected individually or in combination. Results GATA6 is the only essential GATA activity in Xenopus myocardium We have reported previously that GATA6 is required for the maintenance and maturation of cardiomyocytes in Xenopus (Peterkin et al., 2003). The phenotypes consequential upon depletion of GATA4 or 5 in Xenopus, however, have not been previously reported. In the case of GATA5, the need to know is made greater because of its major contribution in zebrafish, and the inability to determine if this is a general requirement in vertebrates by comparison with the mouse knockout, because the reported mutation in the mouse appears not to be a null (Nemer et al., 1999). Therefore, before examining depletion of combinations of GATA factors, we examined the individual loss of GATA4 and 5 in comparison to the already known GATA6 phenotype. The design and quality control of MOs against Xenopus GATA4, 5 and 6 have been reported previously (Peterkin et al., 2003; Afouda et al., 2005). For GATA4, as well as the MO reported previously, two other MOs, one against both pseudo-alleles (Todd Evans, personal communication), were tested and gave the same results. For GATA5 and 6, MOs were designed to target both pseudo-alleles of the Xenopus laevis genes. The optimal amount of each MO injected was determined by titration to ensure that the maximum dose without non-specific effects was used. The extent of knockdown by these ATG MOs was determined by co-injection of tagged reporter RNAs followed by Western blotting (Afouda et al., 2005; Peterkin et al., 2003). Very little residual protein was detected in each case. When MOs against individual GATA factors were injected separately into the presumptive heart field, the dorsolateral marginal zone, of 4-cell Xenopus embryos, cardia bifida was induced in each case (Fig. 1A, visualised by staining the cells for expression of Myosin Light Chain 2 (MLC)). Cardia bifida has been reported previously for the GATA4 knockout mouse (Kuo et al., 1997; Molkentin et al., 1997), the GATA5 mutant zebrafish, faust (Reiter et al., 1999), and GATA6 morphant Xenopus and zebrafish embryos (Peterkin et al., 2003), but this is the first direct comparison in a single species showing that all three GATA factors are required for the timely migration of cardiac precursors to the midline for fusion of the heart tube. This is in contrast to requirements in the differentiation of the myocardium (see below), where only one member of the family is essential. It seems likely that the requirement for GATA4, 5 and 6 in midline migration of myocardial precursors is actually in the underlying endoderm, where they are all expressed and which has been shown to be essential in mouse and zebrafish for heart tube fusion (Afouda et al., 2005; Alexander et al., 1999; Molkentin et al., 1997; Narita et al., 1997; Reiter et al., 1999; Weber et al., 2000). To determine the effects of the GATA MOs on programming of the myocardial cells, as opposed to their morphological movements, the levels of expression of the transcription factors, Nkx2.5 and Tbx5, and of the contractile machinery genes, cardiac actin (CA), and MLC, were monitored by whole mount in situ hybridisation. In contrast to the GATA6 MO, which causes a profound reduction in the expression of these genes (Peterkin et al., 2003) (Fig. 1D), GATA4 and GATA5 MOs had minimal effects (Figs. 1B, C; for all three MOs and for each marker n was 30–50). Despite the cardia bifida at tailbud stages (stages 28–32) (Fig. 1A), the gross morphology of the hearts at later stages (stage 43) in GATA4 and GATA5 morphants looked similar to those in wild type embryos, i.e. the cardia bifida was only transient (data not shown). In contrast, as previously described (Peterkin et al., 2003), little or no cardiac tissue was observed in embryos depleted of GATA6 (data not shown). Thus, it would appear that, apart from the transient bifida, the loss of GATA4 or GATA5 has little effect on cardiogenesis in Xenopus. To ensure that the GATA4 and 5 morpholinos were properly functional, they were injected vegetally at the single-cell stage, and were shown to reduce the expression of Sox17α during gastrulation (data not shown) (Afouda et al., 2005). Furthermore, the gut of GATA5 morphants failed to coil properly, as previously reported (Afouda et al., 2005). In addition, for these and several of the combination experiments described below, all three GATA4 MOs gave the same results. We therefore conclude that for development of the myocardium in Xenopus embryos, GATA6 is the only essential GATA factor. GATA factor redundancy in Xenopus myocardium On the basis of slightly increased expression of GATA6 in GATA4 knockout mice, redundant roles for the GATA factors in the myocardium have been suggested (Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Watt et al., 2004). In Xenopus, expression of neither GATA5 nor GATA6 was significantly increased in GATA4 MO injected embryos (data not shown). Similarly, in GATA5 and GATA6 MO injected embryos: in neither case was an increase in expression of the other two GATA factors observed (data not shown). However, redundancy does not necessarily depend on an increase in expression of the redundant family member: continued expression could suffice, and that is what we see in all three cases. Therefore to formally test redundancy within the GATA family, we injected combinations of MOs into the presumptive heart field of 4-cell Xenopus embryos, and monitored MLC and Nkx2.5 expression by whole mount in situ hybridisation (Fig. 2). Embryos were classified as unaffected (wild type, +), mildly (−) or strongly (− −) down regulated, or displaying no expression at all (− − −) (Fig. 2A). Numbers of embryos in each category were scored and the results displayed in histograms (n = 31–94) (Figs. 2B, C). The greater effects of the GATA6 MO are immediately apparent, with clear increases in the affected categories at the expense of the wild type category compared to both control embryos and also to GATA4 or GATA5 MO injected embryos. When combinations of two MOs were injected, evidence for redundancy was revealed (Figs. 2B, C). Despite having little effect on their own, both GATA4 and GATA5 MOs made the phenotype of GATA6 MO injected embryos more severe when injected with it. Furthermore, the phenotype observed when GATA4 and 5 MOs were injected in combination was significantly worse than either alone, suggesting that the minimal phenotype for the single injections relied on the continued presence of the other GATA factor. When all three MOs were injected together, the phenotype was the most extreme of all with the vast majority of embryos having no expression of MLC at all. We therefore conclude that, while GATA6 is the only individually essential player in driving the myocardial programme in Xenopus, the other two GATA factors are responsible for the residual expression of cardiac genes. Furthermore, in the absence of GATA6, their roles are increased. This is evident from their significantly greater effects on embryo phenotypes when combined with GATA6 MO compared to on their own. GATA activity is required for differentiation but not induction of the myocardium in Xenopus We have shown previously that GATA6 is required for the maintenance/maturation of the myocardium rather than its induction in both Xenopus and zebrafish embryos (Peterkin et al., 2003). As expected, based on the absence of a late phenotype, embryos injected with GATA4 or GATA5 MOs had no effect on early Nkx2.5 expression, as seen for GATA6 MO (n = 60, 72, and 89, respectively) (Fig. 3A). In order to determine if the lack of an early effect, even for GATA6 which has a strong late phenotype, was the result of redundancy within the GATA family, we examined Nkx2.5 expression at neurula stages in embryos injected with all three MOs (n = 102) (Fig. 3A). Expression was unaffected, as seen with each of the MOs on their own. Although Nkx2.5 is also expressed in the underlying endoderm at this time, we showed by examining sections that the signal in the cardiac mesoderm is unaffected (Fig. 3B, territory delineated by dashed lines). Furthermore, a similar result was obtained for Nkx2.3 (n = 55) (Fig. 3C), which is not expressed or is very weak in the endoderm at this time (Fig. 3D). The expression of eHAND was also unaffected at this stage (Fig. 3E, territory delineated by dashed lines). We therefore conclude that, despite their earlier expression, GATA factors are not required for induction of the myocardial programme in Xenopus, as seen by the continued expression of the other early regulators, Nkx2.5, Nkx2.3 and eHAND, and their own continued expression, but rather for its maintenance/maturation. GATA4 is not essential for induction or differentiation of zebrafish myocardium GATA5 (faust) mutant zebrafish have profound defects in the myocardium, displaying reduced expression of several myocardial genes (Reiter et al., 1999). In addition, GATA6 has been shown to be required for maintenance/maturation of the myocardial programme in zebrafish as seen in Xenopus (Peterkin et al., 2003). In order to determine the relative effects of these two GATA factors, and to determine the contribution of GATA4, we separately injected into zebrafish embryos MOs against each of these GATA factors. The GATA4 MO was shown to specifically block translation of a co-injected GATA4 RNA and not GATA5 or GATA6 RNAs (Supplementary Figs. 1A, B, C). The GATA5 MO was designed to block splicing between exons 1 and 2 of the GATA5 gene, which was confirmed in injected embryos by RT–PCR (Supplementary Figs. 1D, E). This splice blocking morpholino was designed upstream of the exons encoding the zinc fingers to prevent any protein produced binding DNA. However, the creation of a dominant negative GATA5 via splicing from an upstream cryptic site is formally possible (see Supplementary Fig. 1D) but the ability of GATA4 and 6 morpholinos to enhance the cardiac phenotype in combinations (see below) makes this unlikely. Furthermore, the GATA5 morphant heart phenotype was indistinguishable from that seen in the faust mutant, both in single and combination experiments (Supplementary Fig. 1F and see below). The GATA6 MO has been reported previously (Peterkin et al., 2003). The effects of the three MOs injected separately into zebrafish embryos were determined by monitoring expression of the transcription factor, nkx2.5, and the contractile machinery genes, ventricular myosin heavy chain (vmhc) and cardiac myosin light chain 2 (cmlc2) (Fig. 4). GATA5 and 6 MOs induced cardia bifida as described previously for the faust mutant and the GATA6 MO (Peterkin et al., 2003; Reiter et al., 1999). In contrast, in GATA4 MO injected embryos, the myocardial cells appeared to have migrated and fused normally at the midline. We therefore conclude that, in zebrafish, only GATA5 and 6 are required for the proper migration of cardiac precursors. In contrast to mice and Xenopus, GATA4 appears to be uninvolved in this process. The previously reported effects on myocardial gene expression of GATA5 or GATA6 knockdown (Peterkin et al., 2003; Reiter et al., 1999) were immediately evident in these MO injected embryos (Fig. 4). GATA5 MO injection led to substantially reduced expression of nkx2.5 (19/19), vmhc (42/43) and cmlc2 (40/41), as seen for the faust mutant. GATA6 MO injection also resulted in reduced expression of these markers (6/6, 60/60 and 42/44) but to a lesser extent. In contrast, GATA4 MO injection had little or no effect on cardiac marker gene expression levels (n = 28, 69 and 53). Spatially the expression of the markers in the GATA4 morphants looks altered compared with the controls due to defects in late cardiac morphogenesis, consistent with those described by Holtzinger and Evans (2005). We therefore conclude that for laying down the myocardial programme in zebrafish, GATA5 has the greatest effect with a significant contribution from GATA6. In contrast, GATA4 makes little or no contribution, at least to the expression of the markers tested. GATA factor redundancy in zebrafish myocardium To determine if, as in Xenopus, there is redundancy within the GATA family in zebrafish, we injected the MOs in combinations (Fig. 4). Morphant embryos were classified into three types, unaffected (type +), down regulated (type −) or absent (type −−). Both the GATA5 and the GATA6 MO phenotypes were made worse by the co-injection of the GATA4 MO (Figs. 4B, D and F), as seen in Xenopus, and despite the fact that the GATA4 MO had little or no effect when injected on its own (n = 18–39). We therefore conclude that a significant proportion of the residual cardiac gene expression in GATA5 or GATA6 MO injected embryos is driven by GATA4, even though the consequences of its loss in the presence of GATA5 or GATA6 are minimal. Thus, redundancy within the GATA family is apparent in the zebrafish myocardium as in Xenopus. The level of residual cardiac marker expression in the GATA4 and 5 MO combination or the GATA4 and 6 MO combination at 26 hpf was very low (Fig. 4). The level for the GATA5 and GATA6 MO combination was undetectable with 100% of the embryos losing expression, suggesting that, while GATA4 can cover for the absence of either GATA5 or GATA6, it cannot cover for the absence of both, which seems unlikely. We therefore monitored the expression of GATA4 in flat-mounted (Fig. 5A) MO injected 10-somite embryos to determine if it was still expressed (Fig. 5C). We found that GATA5 MO on its own caused a reduction in GATA4 expression (22/36 embryos), and residual expression was removed completely by the addition of the GATA6 MO (n = 35) (Fig. 5C). The GATA4 expression seen in GATA4 morphants reflects the use of a translation-blocking morpholino rather than a splice-blocker. In the same experiment the expression of nkx2.5 was affected in the same way as already described (Fig. 5B). We therefore conclude that the complete absence of cardiac marker expression in GATA5 plus GATA6 MO injected embryos results from the simultaneous absence of GATA4 expression. Thus, as seen for Xenopus embryos, the absence of all three GATA factors completely abolished cardiac marker expression. GATA activity is required for induction of the myocardial programme in zebrafish We have shown that GATA activity is only required for the maintenance/maturation of the myocardial programme in Xenopus. While we have shown that the GATA6 requirement in zebrafish is also late (Peterkin et al., 2003), nkx2.5 expression at 6 somites has been shown to be affected in zebrafish faust mutants (Reiter et al., 1999), suggesting that an additional difference between the species might be the timing of requirements for GATA activity. We therefore tested this earlier requirement with more markers and to determine if it is subject to redundancy. Firstly, we examined nkx2.5 expression in MO injected embryos at 5 somites when it is first expressed (Fig. 6A). For the GATA5 MO, we found a major reduction in expression totally consistent with the reductions seen later and with those reported for the faust mutant (Reiter et al., 1999). We also observed very little effect for the GATA4 or 6 MOs on their own, but both made the GATA5 MO phenotype more severe, consistent with their back-up roles being active at this early stage. Similar observations were made for GATA4 and hrt expression in 5-somite embryos and for tbx5 expression in 10-somite embryos (tbx5 expression is first detected at ∼ 7 somites) (Figs. 6B, C, D). In contrast, nkx2.7 expression was unaffected even by triple knockdown (Fig. 6E). We therefore conclude that, for the markers studied and in contrast to Xenopus, establishing the full early myocardial programme in zebrafish depends on GATA activity. The continued presence of cells expressing nkx2.7 suggested that apoptosis had not yet occurred, and this was confirmed by TUNEL and acridine orange assays (data not shown). Furthermore, re-specification to more anterior or more posterior mesodermal fates was not observed, as judged by the domains of expression of anterior lateral plate and pronephric markers (data not shown). We therefore conclude that in the absence of GATA activity, the cells remain undifferentiated at least up to the 10-somite stage. Discussion Redundancy GATA4, 5 and 6 are an example of a gene family co-expressed in a specific tissue, in this case the myocardium. Although some differences in their binding site preferences have been detected (Sakai et al., 1998), all three bind to canonical GATA sites with high affinity. Because of this and the relatively mild phenotypes generated in loss of function experiments, they have been suggested to act redundantly (Jiang et al., 1998; Kuo et al., 1997; Molkentin et al., 1997; Narita et al., 1996; Watt et al., 2004). Here for the first time we present evidence in support of this with respect to laying down the genetic programme of the myocardium. The redundancy is particularly striking for GATA4, whose individual loss has essentially no effect on induction or maturation of the myocardium in either zebrafish or Xenopus, in contrast to assumptions of its importance in much of the literature. For this member of the family, its contribution is only revealed in the absence of GATA5 or 6, thereby constituting a formal demonstration of redundancy. Similar demonstrations are evident for both Xenopus GATA5 and in early heart induction for zebrafish GATA6, where they are not the essential players. These redundant GATA activities thus most likely account for the residual expression of cardiac markers in the absence of the essential GATA factor. Indeed little change was observed in expression of the remaining GATA factor in double morphant embryos compared to wild type siblings in either zebrafish or Xenopus (data not shown). The one exception was GATA5 and 6 double morphant zebrafish embryos where the complete loss of GATA4 was used to effect a triple knockout (Fig. 5C). GATA factors are an ancient family and in vertebrates have existed with three family members in the heart at least since fish (Patient and McGhee, 2002). Thus, the redundancy reported here would appear to be evolutionarily very stable. Maynard Smith and colleagues have developed simple genetic models to analyse selection pressures on redundant genes and have concluded that evolutionary stability can be achieved if the two (or more) genes perform the same function, but with slightly different efficacies, as seen here (Nowak et al., 1997). The less efficient family member comes into its own when paired with a mutant form of the more efficient family member. Another evolutionarily stable model can be achieved where two (or more) genes perform more than one function: the redundancy occurring only with respect to one specific function. GATA4, 5 and 6 have an ever-growing list of functions in other tissues, so this scenario is more than adequately satisfied as well (Afouda et al., 2005; Capo-Chichi et al., 2005; Ketola et al., 2004; Molkentin, 2000; Yang et al., 2002). The evolutionary stability of this model depends on random mutations being more likely to render the genes inactive for all functions rather than just for one of their functions. Finally, yet another model suggests that redundancy should be more common in genes displaying specific spatio-temporal expression patterns during development, as is the case for GATA4, 5 and 6. For this model, the developmental error rates applicable to these genes need to be higher than their germ line mutation rates: a requirement that is currently unknown. The primary GATA factor An unexpected finding was that the member of the family whose loss has the biggest effect differs between Xenopus and zebrafish. For single knock downs, GATA6 has the strongest effect on myocardial gene expression in Xenopus whereas GATA5 does so in zebrafish. Although at first glance this might suggest a switch in roles for GATA5 and 6, a consideration of the timing of their actions suggests an alternative view. The action of GATA6 in Xenopus is after the initial expression of other early markers such as Nkx2.5, suggesting a role in differentiation of the myocardium (Peterkin et al., 2003). GATA6 knockdown in zebrafish has a very similar effect. Thus, in both organisms, knockdown of GATA6 leaves early marker expression initially intact but decaying with time, whereas when GATA5 was knocked down in zebrafish, expression of Nkx2.5 and other early markers was compromised from the outset (Reiter et al., 1999; this study). The difference between the two organisms therefore can be characterised as the gain or loss of an early function for GATA5. The early role for GATA activity in zebrafish appears not to be masked by redundancy in Xenopus because even triple knockdown of GATA4, 5 and 6 leaves early expression of myocardial markers intact. The role of GATA5 in myocardial induction in mouse and chick embryos is currently unclear. Although in P19 embryonal carcinoma cells induced to differentiate into cardiomyocytes, GATA5 up-regulation occurs after Nkx2.5, precluding an early function during induction (Alexandrovich et al., 2006), the mouse knockout of GATA5 retained the capacity to synthesise a truncated form of the protein containing both zinc fingers, which would likely have significant activity, preventing a definitive conclusion (Nemer and Nemer, 2002). Likewise, the attempts to date to knock down GATA5 activity in the chick were only partial and, in addition, attempted after induction of the myocardium (Jiang et al., 1998). It is therefore not yet clear if the early role for GATA5 has been acquired by zebrafish or lost by Xenopus. In Drosophila, the GATA factor, pannier, is required both upstream and downstream of the Nkx2.5 homologue, tinman (Gajewski et al., 2001; Klinedinst and Bodmer, 2003). In the nematode, the GATA factors, Med1 and 2, are expressed in the mesendodermal precursor to the mesoderm giving rise to part of the pharynx, an organ that has homologies to the heart, and upstream of the Nkx2.5 homologue, ceh22, suggesting that an early role for GATA factors may be ancestral (Broitman-Maduro et al., 2006; Maduro et al., 2001; Rodaway and Patient, 2001). Mesendodermal expression is seen for both GATA5 and GATA6 in zebrafish, while in Xenopus, mesendodermal expression is seen for GATA4 and 6 (Fletcher et al., in press; Rodaway et al., 1999; J. Broadbent, A. Gibson and R. Patient, unpublished observations). Thus, the early role for GATA5 in zebrafish may reflect its early expression in the lineage of cells leading to the myocardium whereas, in Xenopus, early expression of GATA5 is restricted to the endoderm, appearing in the cardiac mesoderm at a later stage (Weber et al., 2000). That GATA6 is also expressed early in this lineage in both species, and GATA4 likewise in Xenopus, and yet neither plays a role in induction of the myocardium, suggests that GATA5, at least in zebrafish, is alone in containing the requisite amino acid sequence for this function. All three GATA factors recognise the same DNA sequence therefore, in the absence of known binding site preferences for GATA5, activities specific to GATA5 are likely to include protein–protein interactions. Thus, GATA5 may be more suited to the necessary interactions in the early mesendoderm than either GATA4 or 6. Feedback loops and timing Genetic regulatory networks (GRNs) consist of functionally linked regulatory genes encoding transcription factors and their controlling extra-cellular signals (Davidson et al., 2002). They contain motifs, or recurring wiring patterns, which occur with frequencies far greater than in randomised networks (Lee et al., 2002; Milo et al., 2002; Shen-Orr et al., 2002). One such motif is a feedback loop. Mathematical modelling of positive feedback loops indicate that they promote the persistence of signals and have the potential to store information, such that, for example, signalling can readily flip the system from one state to another (Bhalla and Iyengar, 1999). The observation in Drosophila that pannier is both upstream and downstream of tinman, raises the possibility that the establishment of a mutual regulatory loop for these two key regulators is critical evolutionarily (Gajewski et al., 2001; Klinedinst and Bodmer, 2003). Evidence for a similar feedback loop exists in mice, where a cardiac GATA gene has been shown to be Nkx dependent and vice versa (Brewer et al., 2005; Davis et al., 2000; Lien et al., 1999; Molkentin et al., 2000). Davidson and Erwin have recently proposed that regulatory motifs of this type are evolutionarily stable components of GRNs, which they called kernels (Davidson and Erwin, 2006). They highlight a heart-field specification kernel, which is conserved from Drosophila to vertebrates. Strikingly the Nkx2.5/tinman GATA/pannier feedback loop is central to this kernel. Our work supports this hypothesis and further suggests that the establishment of this kernel is more critical in evolution than where the loop is initiated. Thus, GATA activity is required to initiate Nkx2.5 expression in zebrafish but not in Xenopus, nevertheless both establish the loop (Fig. 7A). Interestingly, as seen for GATA activity, the requirement for BMP signalling differs between zebrafish and Xenopus. Thus, in zebrafish, BMP signalling is required to initiate expression of cardiac markers including GATA5 (Reiter et al., 2001), whereas in Xenopus, it is only required for their maintenance (Walters et al., 2001). In view of the links between BMP and GATA factors in several different tissues, including the myocardium, it seems likely that the early requirement for GATA activity in zebrafish is linked to the early requirement for BMP, and likewise the later requirement in Xenopus (Fig. 7B) (Peterkin, 2003). The cascade of events in Drosophila predicts that the Drosophila BMP signal, Decapentaplegic (Dpp), activates tinman, and they then act in concert to initiate the expression of pannier. Subsequently tinman and pannier maintain each other's expression, whilst pannier (in the ectoderm) maintains Dpp expression. Dpp signalling feeds back to maintain expression of tinman and pannier thus completing the loop (Fig. 7C; for review see Sorrentino et al., 2005). Thus, in summary, the data imply that the initiating factor and the direction in which the loop flows are not important. Ultimately it is the establishment of the loop that is essential and failure to do so leads to the loss of differentiated myocardium.

Bioinformation-2-1-2139994

Hypo, hype and ‘hyp’ human proteins

Genes with unknown function are called orphan genes while their transcripts and peptides are called hypothetical proteins. There are many genes and their associated proteins that remain uncharacterized in the human genome. A database of human hypothetical proteins with ascribed functions could be helpful for biologists to search for potential proteins of interest. In recent years, the rapid completion of genome sequences has created essential information to link genes to gene products. In order to better explain functions for un-annotated proteins we designed BioinformaTRICKS (an open source project) and used it to develop a database called HYPO. Background As the amount of genome sequence data now available is enormous with more than 750 genomes being either finished or in progress, a biologist is thrown into using several databases with increasing attention to find any novel genes or proteins or function. However, various analyses based on sequence, structure, function and “Omic” data have revealed different annotation criteria leading to different sets of predicted genes. However, more than 50% of proteins in the proteome zone remain un-annotated and un-identified for function (Table 1 in supplementary material). The human genome contains many different regulatory sequences that have roles in controlling gene expression. The protein-coding sequences is only less than 1.5 percent of the genome and then rest remains as non-coding inter and intra-genic regions with undetermined role. [1] The annotation of various human chromosomes is well supported by computational predictions where there is no similarity to known proteins or EST sequences. The genes that have unknown function called as orphan genes code for proteins annotated as “hypothetical proteins”. Hence, there is a need to begin constructing and analyzing protein families clustered as “hypothetical proteins” with an aim to elucidate function and protein subunit interactions. After several drafts of Human Genome Project, there are many proteins that remain to be characterized. Tools have been developed to utilize evolutionary relationships towards understanding uncharacterized proteins despite the need to generate functional interaction networks. [2,3] In particular, these approaches are being used to annotate functions for hypothetical proteins. Although several databases explore protein functions through data-mining, there is a requirement to list all hypothetical proteins. There are reports that address the problem of orphan genes. [4] However, there is no adequate information to necessitate function of genes that cannot be based on homology alone, except connected to other known gene family. There are several hypothetical proteins such as the KIAA that have remained hype for some time now. [5] Systems biology integrated with protein-protein interaction (PPI) studies could identify the role of these unknown proteins (Figure 1a). Systems biology is a science of constructing networks of genes and proteins thereby providing a framework for predicting models. [6] The proteins connected through the networks could perhaps throw light on the plausible function of the hypothetical proteins through the bona fide interactions they are involved with. In the context of PPI networks, we could consider if a model is to be developed from the network or a network is to be generated with an already established model. Precisely, the putative function of a protein could be better known from a PPI network to develop a model from it. We show here an example in finding the putative function of a hypothetical protein (figure 1b, NP_438169 - B3BP Homo sapiens Nedd4-binding protein 2) using a PPI network. The nearest interacting partners of the protein B3BP were mapped using the STRING that could show the probable function of the query [7]. Information on “known” or “unknown” protein-protein interactions is still mostly limited but integrating tools such as these could generalize a way to find the function of hypothetical proteins. While we started mining the proteins, it seemed that there are a few hypothetical proteins that have amino acid residues HYP (histidine, tyrosine and proline) in succession. These might have been long-established through the mutations that are introduced into the proteins at one or more predicted non-essential residues. While a few KIAA proteins are conserved and have been known to be identified as novel [8], functional analysis of the proteins encoded by these KIAA cDNAs could be established from our database of hypothetical proteins. [5] A “conservative amino acid substitution” is the one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are distinct and include conserved amino acids such as histidine (aromatic/basic side chains), tyrosine (polar side chain) and proline (non polar side chains). On the other hand, these might have appeared during annotation through the mutations introduced randomly along all or part of a coding sequence. Our database has over 6362 hypothetical proteins that could be searched for different functions. Observations and challenges We observe in tandem that few hypothetical proteins present on different chromosomal loci are known to have the same putative function. Categorizing several approaches beyond traditional sequence similarity that utilize tremendously large amounts of data that is available for computational prediction of functions is the need of the hour these days. Having said this, one could use a subset of proteins that match from several of the experimental approaches and be used as a predictor to circumvent the use of wet laboratory experiments in the near future. Another concern now is specifically on hypothetical protein domains in intrinsically disordered proteins (IDPs). [7] With Protein Data Bank (PDB), not accepting theoretical structures, there is an emphasis for experimental approach by researchers to determine the structure and functional relationship of a protein. Nevertheless, carefully considering the functional annotation methods as discussed above could definitely devise selection for proteins that could be experimented. This could be an interesting approach to pursue further. Yet another issue to be noted is the appraisal to understand if any of the hypothetical proteins have proper functional annotations’ been attributed to sequence: structure: function relationship in case of ordered proteins while sequence: un-structure: function in case of intrinsically disordered proteins. In conclusion, the current methods could play an important role in establishing functions for proteins annotated as hypothetical in the genome. Note The title of the article contains hypo abbreviated for hypothetical proteins. Supplementary material Data 1

Cancer_Immunol_Immunother-3-1-1914259

No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies

Aim In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+ T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+ T cells in a large cohort of Hu-PNS patients and controls. Introduction Paraneoplastic neurological syndromes (PNS) are considered as naturally occurring, successful anti-tumour immune responses in humans [6]. However, this tumour immunity goes along with autoaggression against the nervous system, resulting in severe neurological dysfunction [9]. The mechanisms responsible for the anti-tumour response and neuronal damage are poorly understood. Antigens expressed by the tumour that are normally restricted to neurons (so-called onconeuronal antigens) hypothetically trigger an immune response that cross-reacts with the same antigens in the nervous system [7]. One of the most frequently involved tumours is small cell lung cancer (SCLC) and approximately 50% of patients with PNS and SCLC have high-titre Hu antibodies (Hu-PNS). Hu antibodies are directed against a family of neuron specific, mRNA binding proteins, of which HuD is the best-documented member. Consistent expression of HuD in all SCLC suggests that HuD plays a central role in triggering the immune response [15, 17]. High titres of Hu antibodies in serum and cerebrospinal fluid suggested a pathogenic role for these antibodies, that could, however, never be proven in animal models [3, 22]. Furthermore, expression of Hu antigens is exclusively intracellular, and it is therefore difficult to understand how such antibodies could target tumours or neurons [5]. In addition, pathological examination of PNS neuronal tissue demonstrated localized inflammatory cell infiltrates, containing B cells, CD4+ and CD8+ T cells, in the proximity of overt neuronal cell damage [2, 13, 18, 25]. The presence of an oligoclonal CD8+ T cell infiltrate in nervous tissues and tumours of Hu-PNS patients further suggests an immunopathogenic role for such cells [18, 25]. Some authors report the presence of HuD peptide-specific CD8+ T cells in the blood of these patients, but also in that of apparently healthy controls (HC) [19, 20, 24]. In addition, the presence of circulating HuD-specific CD4+ T cells has been suggested [1]. Here, we have investigated the presence of circulating HuD-specific T cells in a large cohort of Hu-PNS patients and controls. Despite a multifaceted approach mainly geared towards the detection of HuD-reactive CD8+ T cells and, to a somewhat lesser extent, CD4+ T cells, no such cells were detected in the blood of Hu-PNS patients and controls. Materials and methods Patients Forty-three patients with high-titred Hu antibodies and a definite clinical diagnosis of PNS [12], 31 Hu antibody negative SCLC patients without neurological symptoms or signs (SCLC) and 54 apparently HC were tested. The Erasmus MC Institutional Review Board approved the study and all individuals provided written informed consent. The individuals’ class I HLA alleles were typed by standard diagnostic PCR at the two-digit resolution level. Patient characteristics are shown in the Table 1. Anti-Hu IgG titres were determined as described previously [21] and in Hu-PNS the dependence in activities of daily living was scored using the modified Rankin scale [21]. Twenty-five HC were male and 29 female, their median age was 46 years (range 17–89) and all were Hu antibody negative. No HC had received previous chemotherapy or immunosuppressive treatment and 30 HC (56%) were CMV-seropositive. Table 1Patient characteristics at the time of study entryHu-PNS SCLC N4331Age (median, range)64 (4–81)a61 (40–83)Gender (M/F)15/2821/10Hu-Ab titre (median, range)12,800 (400–204,800)NegativeCMV serostatus (pos/neg)29/1417/14Paraneoplastic neurological syndromeNA PSN27 PEM5 PCD4 PLE/ BE3 Pseudo-obstruction2 Motor neuron disease2Tumour No tumour8b0 SCLC3131  Limited2817  Extended314 NSCLC20 Prostate10 Neuroblastoma10Prior treatment None2727 Chemotherapy ± immunosuppression164Neurological symptomsNA Interval onset symptoms (study entry)5 months (2–15)c Interval onset symptoms (diagnosis)4 months (1–12)c Progressive at study entryd34 (79%)Modified Rankin scoreNA MRS = 27 MRS = 322 MRS = 410 MRS = 54Hu-Ab Hu antibody, CMV cytomegalovirus, pos positive, neg negative, NA not applicable, PSN paraneoplastic sensory neuronopathy, PEM paraneoplastic encephalomyelitis, PCD paraneoplastic cerebellar degeneration, PLE paraneoplastic limbic encephalitis, BE brainstem encephalitis, SCLC small cell lung cancer, NSCLC non-small cell lung cancer, MRS modified Rankin scorea One Hu-PNS patient was a 4-year-old boy with an underlying neuroblastoma. The remaining Hu-PNS patients were aged between 49 and 81 yearsb No tumour mass visible on CT-scan or FDG-PET scanc Median (ranges) of intervals are shownd Progression of neurological symptoms was defined by the increase of at least one point on the modified Rankin scale during 2 months prior to study entry Reagents Ninety-three HuD protein-spanning synthetic peptides, 15-mers with 11 amino acids overlap, were pooled to constitute the HuD peptide mix (HuDmix) and smaller peptide pools [Jerini Peptide Technologies (JPT), Berlin, Germany]. For interferon-γ enzyme-linked immunosorbent spot-forming (IFN-γ ELISPOT) assays and the construction of HLA class-I multimers, HuD-derived 9- and 10-mers were selected based on previous studies [19, 20]. The phycoerythrin (PE)-labeled multimers and corresponding peptides used were: HLA-A*0101-147ELEQLFSQY155, HLA-A*0101-245RLDNLLNMAY254, HLA-A*0201-86SLGYGFVNYI95, HLA-A*0201-248NLLNMAYGV256, HLA-A*0201-315QLFGPFGAV323, HLA-A*0201-362RLGDRVLQV370, and HLA-A*2402-154QYGRIITSRI163 (ProImmune, Oxford, UK). As positive and negative controls, HLA-A*0201-495NLVPMVATV503 [CMV phosphoprotein-65 (CMV-pp65); Beckman Coulter, San Diego, CA] and HLA-A*0201 presenting an irrelevant peptide (ProImmune), respectively, were included. To measure general T-cell responsiveness, we used phorbol myristate acetate (PMA) plus ionomycin, or phytohemagglutinin (PHA). A peptide pool containing 15-mers spanning CMV-pp65 (JPT) was used as positive- and negative antigen-specific control in CMV seropositive and seronegative individuals, respectively. Cytokine flow cytometry Peripheral blood mononuclear cells (PBMC) were isolated within 12 h after venipuncture and stimulated in duplicate as described elsewhere [14]. Briefly, 2 × 106 PBMC were incubated at 37°C in a CO2 incubator for 18 h with 1 μg/ml HuDmix, 1 μg/ml CMV-pp65, 1 μg/ml ionomycin plus 25 ng/ml PMA, or without antigen. After 2 h of stimulation, brefeldin A was added to one of the duplicate tubes allowing for intracellular accumulation of cytokines in activated T cells. Brefeldin A was not added to the second tube to allow detection of secreted cytokines in the supernatant. In some individuals, additional stimulation was performed in the presence of co-stimulatory monoclonal antibodies (mAb) directed against CD28 and CD49d (BD Biosciences, San Jose, CA). Stimulated PBMC were stained and analysed using anti-CD3 conjugated with peridinyl chlorophyllin (PerCP), anti-CD8 conjugated with allophycocyanin (APC), anti-interferon (IFN)-γ conjugated with fluorescein isothiocyanate (FITC), anti-tumour necrosis factor (TNF)-α conjugated with PE, or appropriate isotype control mAb (all from BD Biosciences) [14]. CD4+ T cells were defined as CD3+, 8−. Positive responses were defined by (1) percentage of cytokine-positive CD4+ or CD8+ T cells >2 times the negative control (i.e., no antigen) and (2) ≥0.1% of the total number of CD4+ or CD8+ T cells, each after subtraction of isotype control results. Detection of secreted cytokines The secretion levels of IFN-γ, TNF-α, interleukin (IL)-2, IL-4, IL-5 and IL-10 were measured in supernatants using a cytometric bead array (BD Biosciences). Based on CMV data (not shown), a positive result was defined as cytokine concentration >2 times the background (no antigen) and a minimum level of 50 pg/ml. IFN-γ ELISPOT PBMC (2 × 105/well) were pre-stimulated in duplicate in 96-well plates with culture medium containing 3 μg/ml HuD 9-mers, HuDmix, CMV-pp65, PHA, or no antigen for 1.5 h at 37°C and 5% CO2 [10]. The PBMC were subsequently transferred to anti-IFN-γ-coated ELISPOT plates (Nalge Nunc, Rochester, NY) for a further 18-h incubation. The ELISPOT assay was performed using standard protocols and automated reading (AELVIS GmbH, Hanover, Germany) [10]. The mean number of spot-forming cells (SFC) in duplicate wells was used as assay outcome. Positive results were defined by numbers of SFC/well >3 times background (no antigen) and a minimum of 15 SFC/well. Detection of HuD-specific CD8+ T cells using Class-I HLA multimers Thawed PBMC were stained as described previously [11]. Following acquisition of 1 × 105 viable (i.e., 7-amino-actinomycin-D [7AAD] negative) CD8+ T cells, a positive result required a percentage of ≥0.1% of viable CD8+ T cells binding the HuD multimer and a brightly staining HuD multimer-binding CD8+ T-cell population that did not overlap with the dimly staining irrelevant multimer-binding T-cell population. Results Cytokine flow cytometry of PBMC after stimulation with HuD-derived peptides Stimulation with PMA and ionomycin induced IFN-γ production in both CD4+ and CD8+ T cells of all individuals (not shown). Whilst all CMV-seropositive individuals specifically responded to CMV-pp65, no HuD-specific T-cell reactivity was observed in any of the Hu-PNS patients, SCLC patients or HC (Fig. 1a, b). Similar results were obtained when intracellular TNF-α (not shown) or secreted cytokines (Fig. 1c) were measured. The use of co-stimulatory antibodies in combination with HuDmix did not result in the detection of HuD-specific T-cell reactivity (not shown). Fig. 1Cytokine production in response to HuDmix and HLA-matched HuD 9-mers. Proportions of CD8+ (panel a) and CD4+ (panel b) T cells expressing intracellular IFN-γ after stimulation with HuDmix in Hu-PNS patients, SCLC and healthy controls. Each dot represents the result observed in a single individual (panel c). After stimulation of PBMC with HuDmix and control antigens, the indicated cytokines were measured in assay supernatants. The results are shown for Hu-PNS patients only. Horizontal lines indicate median values of each group (panel d). ELISPOT assay showing the number of IFN-γ SFC after stimulation of 2 × 105 PBMC with HuDmix or HuD 9-mers. Each dot represents the mean result of duplicates for each stimulus in each individual (panels a–d). Responses to CMV antigens are shown for CMV seropositive individuals only, as they were consistently negative in CMV seronegative individuals (not shown). The numbers of individuals tested are given in between brackets. NEG negative control (incubation without antigen), HuDmix HuD protein-spanning peptide pool, PP65 CMV pp-65 protein-spanning peptide pool, IL interleukin TNF tumour necrosis factor, IFN interferon, SFC spot-forming cell, PHA phytohemagglutinin. ELE, RLD, SLG, NLL, QLF, RLG and QYG designate individual HuD-based peptides IFNγ-ELISPOT assay on PBMC stimulated with HuD-derived 9-mers All individuals responded to PHA as determined by IFNγ-ELISPOT assay. In addition, all CMV-seropositive individuals responded to CMV-pp65. However, no T cell reactivity towards the previously described class-I HLA-binding HuD peptides [20] was detected in individuals with the appropriate HLA types in any of the study groups (Fig. 1d). Analysis of HuD-specific CD8+ T cells using Class-I HLA multimers Finally, we investigated the presence of HuD-specific CD8+ T cells in PBMC using Class-I HLA multimers containing HuD-derived peptides. Whilst CMV-seropositive individuals with the appropriate HLA types showed distinct populations of pp65 multimer-binding CD8+ T cells, no HuD-specific CD8+ T cells were observed in individuals with the appropriate HLA types in any of the study groups (Fig. 2). Fig. 2Analysis of HuD peptide-loaded, Class-I HLA multimer-binding CD8+ T cells. PBMC from a CMV-seropositive Hu-PNS patient were stained with HLA-A*0201 multimers loaded with the HuD peptide SLGYGFVNYI (panel a), an irrelevant peptide (panel b) or with the CMV-pp65 peptide NLVPMVATV (panel c). All data shown are obtained after selection of T cells (i.e., CD3+, low sideward scatter signals) [12]. Binding of Class-I HLA multimers (panels a–c, horizontal axes) was analysed in relation to CD8 expression. In this example, the proportion of SLGYGFVNYI multimer-binding CD8+ T cells (0.36%) was similar to that of irrelevant multimer-binding CD8+ T cells (0.46%); binding resulted in low-intensity fluorescence signals only. In contrast, 2.46% of CD8+ T cells bound the NLVPMVATV multimer resulting in high-intensity fluorescence signals from most CD8+ T cells Discussion We set out to detect HuD-specific T cells in the blood of Hu-PNS patients as they are postulated to play a pivotal role in the immunopathology of this disease. Although we applied three different approaches we could not detect circulating HuD-specific T cells. First, to induce cytokine responses in T cells, we used 15-mer protein-spanning peptide pools that have the advantage of covering the full protein sequence and of eliciting both CD8+ and CD4+ T-cell responses [14], as demonstrated by the CD8+ and CD4+ CMV-pp65-specific T-cell responses. However, no CD8+ or CD4+ HuD-specific T-cell responses were observed. These results are at variance with the detection of HuD-specific CD4+ T-cell proliferative responses in PBMC of Hu-PNS patients by Benyahia et al. [1]. This discrepancy may be explained by differences in read-out (i.e., 3-day lymphocyte proliferation in Benyahia’s study [1] vs. overnight cytokine production in ours) and the use of recombinant HuD protein [1] versus a protein-spanning 15-mer peptide pool (this manuscript). We then studied responses to HLA class-I binding HuD peptides that were previously selected [20]. Using the same experimental setup and HuD 9-mer peptides, Rousseau et al. [20] reported HuD-specific T-cell reactivity in 7/10 Hu-PNS patients and in 3/10 HC [20]. In that small study a positive response was defined as an experimental value ≥2 times above background. With that criterion, 3 PNS, 2 SCLC and 4 HC would have been classified as HuD T-cell responders in our study. However, using that cut-off we would also have detected T-cell reactivity in individuals whose Class-I HLA molecules did not have the appropriate binding motifs (data not shown). Therefore, we used more stringent cut-off levels resulting in a negative outcome. As most patients in both studies had progressive neurological disease and were tested shortly after start of symptoms and prior to therapy, differences between the study populations do not explain this discrepancy. Finally, we could not detect HuD-specific circulating CD8+ T cells using Class-I HLA multimers with the same fine specificities as defined by Rousseau et al. [20]. The absence of detectable circulating HuD-specific CD8+ T cells may not be surprising. In a PCR-based study, Plonquet et al. [18] detected the same T-cell clone in neoplastic and nervous tissues, but not in blood. This finding suggests that T cells involved in the pathogenesis of Hu-PNS circulate in concentrations below detection level. An immune response taking place in the central nervous system parenchyma may deplete the circulating pool of CD8+ T cells with that specificity [23]. Furthermore, vaccination studies in melanoma patients demonstrate that clinically effective anti-tumour immune responses may occur despite low levels of melanoma-specific cytotoxic T cells, i.e., below the detection limit of multimer-based assays [4, 16]. In conclusion, we were unable to detect HuD-specific T cells in a large cohort of Hu-PNS patients and controls. However, two of our three assays were designed for the detection of CD8+ T cells only. The IgG1 isotype predominance of serum Hu antibodies in Hu-PNS indicates a T-helper response to the Hu antigen [13]. Therefore, further studies are warranted that focus on the detection of circulating HuD-specific CD4+ T cells. In this context, regulatory CD4+ T cells—which down regulate immune responses towards auto-antigens and tumour-antigens—are of interest. Although the numbers of regulatory T cells are increased in cancer patients [26] and in PNS patients [8] (de Beukelaar et al. unpublished data) their (possibly impaired) function in PNS remains to be studied. Finally, examination of the antigen-specificity of T cells in affected tissues may shed further light on the role of HuD-specific T cells in the pathogenesis of Hu-PNS.

Rev_Endocr_Metab_Disord-3-1-1894828

Noonan syndrome and related disorders: Alterations in growth and puberty

Noonan syndrome is a relatively common multiple malformation syndrome with characteristic facies, short stature and congenital heart disease, most commonly pulmonary stenosis (Noonan, Clin Pediatr, 33:548–555, 1994). Recently, a mutation in the PTPN11 gene (Tartaglia, Mehler, Goldberg, Zampino, Brunner, Kremer et al., Nat Genet, 29:465–468, 2001) was found to be present in about 50% of individuals with Noonan syndrome. The phenotype noted in Noonan syndrome is also found in a number of other syndromes which include LEOPARD (Gorlin, Anderson, Blaw, Am J Dis Child, 17:652–662, 1969), Cardio-facio-cutaneous syndrome (Reynolds, Neri, Hermann, Blumberg, Coldwell, Miles et al., Am J Med Genet, 28:413–427, 1986) and Costello syndrome (Hennekam, Am J Med Genet, 117C(1):42–48, 2003). All three of these syndromes share similar cardiac defects and all have postnatal short stature. Very recently, HRAS mutations (Aoki, Niihori, Kawame, Kurosawa, Ohashi, Tanaka et al., Nat Genet, 37:1038–1040, 2005) have been found in the Costello syndrome and germline mutations in KRAS and BRAF genes (Rodriguez-Viciana, Tetsu, Tidyman, Estep, Conger, Santa Cruz et al., Nat Genet,2006; Niihori, Aoki, Narumi, Neri, Cave, Verloes et al., Nat Genet, 38:294–296, 2006) in the Cardio-facio-cutaneous syndrome. Phenotypic overlap between these genetic disorders can now be explained since each is caused by germline mutations that are major components of the RAS-MAPK pathway. This pathway plays an important role in growth factor and cytokine signaling as well as cancer pathogenesis. Introduction Noonan Syndrome (NS) has been recognized for about 40 years [1] but the genetic cause was not found until 2003 when mutations in the PTPN11 gene were reported [2]. Shortly afterwards LEOPARD syndrome (LS), a rare alleic variant of NS was found to have specific mutations in the PTPN11 gene [25]. Cardio-facio-cutaneous (CFC) syndrome and Costello syndrome (CS), both rare syndromes, share significant phenotypic overlap with NS especially in infancy. The common features of facial dysmorphism, short stature and similar cardiac findings in all four conditions suggest a similar underlying pathogenesis. In 2005 [6], mutations in the HRAS oncogene were found to be the cause of CS and in 2006 mutations in KRAS, BRAF, MEK1 and MEK2 genes [7, 8] were identified in CFC. All of these germline mutations are components of the RAS-MAPK pathway which plays an important role in growth factors and cytokine signaling. In this review, the clinical findings of the four syndromes will be reviewed. NS will be discussed in more detail since it is common and more clinical studies are available. Noonan syndrome NS is one of the more common non-chromosomal syndromes seen in children with congenital heart disease with an estimated incidence of 1 in 1,500 [1]. Although there is wide phenotypic variation in NS, distinctive facial features include hypertelorism, down-slanting palpebral fissures, a high arched palate, low set posteriorly rotated ears, malar hypoplasia, ptosis and often a short neck. The phenotype changes significantly with time. In the newborn, there is excessive nuchal skin which is the result of prenatal cystic hygroma. During infancy, the head is relatively large, the eyes are often prominent and round, there is a high nasal bridge which may be flat, and the neck appears short. At 3 to 4 years of age, the body becomes more stocky and the chest more prominent. The chest deformities often become significant. In later childhood, the facial appearance begins to show coarse features and becomes more triangular as the chin lengthens. The eyes become less prominent and the ptosis may be more apparent. In the teenager and young adult, as the neck lengthens webbing may become more apparent, the facial features are more triangular and become sharper, the nose has a pinched root and a thin high bridge. An older adult has prominent nasal labial folds, a high anterior hairline and the skin often appears rather transparent and wrinkled. In most, the prenatal history is unremarkable but polyhydramnios is frequent. Height and weight are within normal limits at birth but height begins to drop off within a few months and over 70% of patients with NS have significantly short stature. Some patients with NS have significant feeding difficulties with resulting failure-to-thrive and require tube feedings. Although this may contribute to the poor gain in weight, short stature occurs equally in children who have no feeding problems in infancy. Other important findings include a chest deformity which may be in the form of a pectus carinatum or pectus excavatum, apparent widely spaced nipples and a relatively broad chest. Scoliosis and kyphosis occur in about 15% of patients. Muscle hypotonia is frequent and may account for some of the motor delay. Significant mental retardation is uncommon but some degree of learning disability is frequent and may require special help in school. Eye findings, especially strabismus and refractive errors are common and an occasional patient will have a coloboma. All children with NS should have a complete eye examination. Since conductive hearing loss is rather frequent, children should have a hearing evaluation. Over half of the males with NS have either one or both testes undescended and delay in puberty is common for both males and females. Easy bruising is common in NS and a variety of bleeding problems have been reported [9]. These include deficiency of Factor XI, Von Willebrand’s disease, thrombocytopenia and platelet function defects. In addition, low levels of Factor VIII and XII have also been noted. Hepatosplenomegaly, usually unexplained, is present in about 25% of patients particularly in infancy. Lymphatic abnormalities occur in less than 20% of patients but may present serious problems. Over 80% of patients with NS have a cardiac finding. Pulmonary stenosis is, by far, the most common but nearly every cardiac lesion has been reported. In addition, hypertrophic cardiomyopathy may occur. It was recognized early on that NS could be transmitted in an autosomal dominant manner. In 1994, Jamieson et al [10] was able to map the gene for NS to the distal part of chromosome 12q. Not all families with NS studied showed this linkage suggesting that more than one gene was likely to be involved in the etiology. In 2002, Tartaglia [2] found a mutation in the PTPN11 gene to be present in about 50% of patients with NS. This gene regulates the production of a protein called SHP-2 which is essential in several intracellular signal transduction pathways and controls a number of developmental processes including cardiac semilunar valvulogenesis. The protein is expressed throughout the body and it is an important player in cellular response to growth factors, hormones, cytokines and cell adhesion molecules. The PTPN11 mutations in NS are clustered in interacting portions of the N-SH2 in PTTP domains. This mutation results in a gain of function for SHP-2. Children with NS often present to the endocrinologists because of the short stature, delayed puberty or undescended testes in males. Although height and weight are usually in the normal range at birth, height drops off within the first few months. In general, there is at least a 2-year delay between bone age and chronological age. Continued growth may occur until the early 20s. In both males and females, there is a delay in puberty. Females seem to possess normal fertility. Males, as expected due to undescended testes, appear to have decreased fertility but male transmission is well described and not uncommon. The cause of short stature in NS is really not clear. After pharmacological stimuli, growth hormone secretion was usually normal in patients studied but a small number showed a subnormal response. Others have shown a neurosecretory growth hormone dysfunction to be present in some patients but this did not appear to have any effect on the response to growth hormone [11]. There have been previous studies looking at the IgF-1 levels which have been below normal for the majority reported. A considerable number of children have undergone treatment with human biosynthetic growth hormone. The majority of studies have shown similar results. There is a significant increase in growth velocity in the first and second year of growth hormone treatment [12–16]. The velocity in growth tends to diminish in succeeding years. Several authors claim that the predicted adult height has been increased in patients treated with growth hormone but there have been no real controlled studies to document the long-term effect of growth hormone on adult height. In those studies where bone age as well as actual height were measured, the increase in bone age was equal or slightly greater than the overall increase in height. If the bone age acceleration exceeds the acceleration in height, it is even possible that growth hormone therapy could actually decrease the adult height. Since there was little data available regarding adult height in NS, we recently [17] published a study of 73 adults who were all over 21 years of age at the time of their last measurement. Thirty-one percent of males and 32% of females achieved an adult height that was at the tenth percentile or greater which would be considered within a normal range. The remainder all fell below the tenth percentile but importantly 38% of males and 54.5% of females had an adult height below the third percentile. Adult height was unrelated to the presence or severity of cardiac disease and none of the adults achieving a normal height had been treated with growth hormone. Dr. Municchi et al [16] suggests that long-term serial height measurement over years comparing treated and untreated patients is needed to clearly show the benefit of growth hormone therapy on final adult height. There is no question that adult height can be achieved at an earlier age with the use of growth hormone. With the discovery of the PTPN11 mutation, it is now possible to study patients with NS positive for a mutation and compare them with NS patients who do not carry a mutation of the PTPN11 gene. The large cohort reported by Ranke [18] suggested that weight and length were normal in 119 newborns with NS. With the availability of genetic testing, several recent studies have shown that mean birth length for NS patients with a mutation is slightly below normal and is less than that of the non-mutated NS newborns [19, 20]. In a study by Zenker [20], 88% of PTPN11 positive mutated children older than 3 years of age had a height less than two standard deviations and were significantly shorter than non-mutated children. All recent studies [21] suggest that there is a more severe mechanism acting on growth retardation in NS patients carrying a PTPN11 mutation. A recent study from France [19] evaluated 35 patients with NS, 20 of whom had a PTPN11 mutation. There was a trend to a shorter birth length in mutated versus non-mutated newborns and small for gestational age tended to be more frequent in mutated versus non-mutated patients. By 6 years of age, patients with mutations were significantly shorter than patients without mutations. The results of hormonal studies showed a normal growth hormone secretion after pharmacological stimuli and a low serum IgF-1 and ALS concentrations which is in contrast with a normal IgFBP-3 level. Since the PTPN11 gene has a negative effect on intracellular signaling downstream from several growth factor receptors, a growth hormone post receptor signaling resistance could represent the mechanism of stunted growth in NS. They felt that the lower growth response to growth hormone treatment observed in mutated versus non-mutated patients suggests some degree of resistance to growth hormone. Another recent study by Binder et al [22] showed a similar pattern. Data from these two studies would be in favor of growth hormone resistance by a late post receptor signaling defect specific for IgF-1 and ALS that does not effect IgFBP-3 stimulation. Changes in height during the first 2 years of growth hormone therapy in the pre-pubertal group show catch-up growth was less pronounced in patients with a mutation compared to those without a mutation. Fortunately, in all the studies using growth hormone, no adverse results have been observed. Many patients have undergone serial echocardiograms and none have shown an increase in their left ventricular mass index during growth hormone treatment [23]. These recent studies suggesting that there may be a primary IgF-1 deficiency in NS has stimulated and been incorporated into a phase II clinical study which will investigate the use of IPLEXtm (mecasermin rinfabate) (rDNA origin) which will be given by injection to treat growth failure due to insulin-like growth factor IgF-1 deficiency. Although we still do not understand how the mutation in the PTPN11 gene affects SHP-2, the result apparently is a disruption in the growth hormone IgF-1 axis and IgF-1 deficiency which could be the cause of growth failure. LEOPARD syndrome (LS) Gorlin [3], in 1969, introduced the acronym LEOPARD (LS) to describe a rare syndrome that shares many features similar to NS. These include autosomal dominant inheritance, similar facial dysmorphism and similar cardiac defects with an overabundance of hypertrophic cardiomyopathy compared to pulmonary stenosis. The characteristic cutaneous finding of lentigines is the main distinguishing characteristic. In addition, unilateral or bilateral hearing loss is frequent. Sarkozy et al [24, 25] recently reported clinical and molecular studies in a consecutive study of 30 patients with LS and found mutations in the PTPN11 gene in 27 of the 30 patients studied. Mutations in LS have all occurred in exons 7, 12 and 13 while the more typical NS have the great majority of mutations occur in exons 3, 8 and 13. It is of interest that the mutations in patients with LS show a loss of function rather than gain a function as is found in the more typical NS patients. Zenker [20] noted a specific mutation T468M in exon 12 which was also reported by Sarkozy in seven of their patients with LS to have less adverse effects on body growth. Only two of the ten patients with the T468M mutation have short stature.A cardiac abnormality was present in 71% of the patients with LS, with 80% of those showing hypertrophic cardiomyopathy. Pulmonary stenosis was present in two patients and a partial AV canal in another. Cardio-facio-cutaneous syndrome (CFC) It is often difficult to distinguish an infant with CFC from NS although, with time, the phenotype becomes more distinctive. Patients with CFC [4] have a high forehead, a relatively large head and bitemporal constriction. Like NS, they have a downward slant of the palpebral fissures, posteriorly rotated ears and a flat nasal bridge. The hair is usually sparse, curly and friable and absent eyebrows are frequent. The skin changes are variable but include keratosis pilaris with patchy hyperkeratosis [26]. In time, the phenotype for CFC becomes more distinctive and less Noonan-like. These patients are significantly delayed in both motor and mental skills and, like NS, they are hypotonic, often have failure-to-thrive with frequent gastrointestinal complaints and often require tube feedings. Cardiovascular abnormalities are similar to NS although hypertrophic cardiomyopathy is more common than in the typical NS patient. Although these children appear to be of normal height and weight at birth, they soon fail-to-thrive and short stature is found in 78% [27]. Bone age is significantly delayed and osteopenia is occasionally observed. Very recently, mutations in four separate genes have been found to be associated with CFC [3, 4]. These four genes include BRAF, KRAS, MEK1 and MEK2. All of these genes belong to the same RAS-ERK pathway that regulates cell differentiation, proliferation and apoptosis. It is likely that the mechanism causing short stature in NS may be similar to that causing short stature in CFC. This is a very rare disease and there is little information regarding endocrine studies in patients with CFC. Costello syndrome Costello Syndrome (CS) [28] is a rare condition with a distinctive facial appearance which may be difficult to distinguish from NS and CFC in infancy. Although height and weight are normal or above normal at birth, severe growth retardation is the rule postnatally. Like NS, they have a large head, short wide nose and short neck. Unlike NS or CFC, these patients usually have thick and relatively prominent lips and tongue. They also have loose skin on the hands and feet and deep palmar and plantar creases. There is significant mental and motor delay. Cardiovascular abnormalities are found in about 60% and they are remarkably similar to that found in NS and CFC. Pulmonary stenosis, atrial septal defect and hypertrophic cardiomyopathy are the most common lesions [29]. Of particular interest is the high incidence of cardiac arrhythmias noted particularly in infancy [30] that is not characteristic of either CFC or NS. Recently Aoki et al [4] reported mutations in HRAS a proto-oncogene to cause CS. Gripp et al [31] confirmed this finding and reported 33 of 40 patients with a clinical diagnosis of CS to have a HRAS mutation. All the mutations were de novo. Patients with CS are at a significantly increased risk for the development of malignancy, particularly rhabdomyosarcoma, neuroblastoma, ganglioneuroblastoma, and transitional cell carcinoma of the bladder. Although few studies of growth hormone have been carried out in Costello patients, the findings are very similar to that of NS. Response to growth hormone treatment has been variable. Kerr et al [32] have suggested that in CS, hormone treatment may be harmful because of the propensity to tumor formation and the presence of hypertrophic cardiomyopathy. He described two patients, one who had mild left ventricular hypertrophy with normal function before growth hormone was started. After 3 months of treatment, the cardiomyopathy progressed with significant left ventricular outflow tract obstruction with a gradient of 60 mmHg. He was treated with Propranolol. There was no further progression of the cardiomyopathy in spite of continuation of growth hormone. In the second case, growth hormone was started at 12 months and continued until age 26 months when a large pelvic mass was discovered which was proven to be an embryonal rhabdomyosarcoma. Growth hormone was stopped after the tumor was recognized. In spite of extensive chemotherapy, the patient died. Since CS is associated with both subaortic hypertrophic cardiomyopathy and tumors, the role of growth hormone in these two patients is unknown. At the present time, it is unclear whether growth hormone is beneficial or harmful in CS. Conclusion NS, LS, CFC and CS all have significant phenotypic overlap. Although they may be difficult to distinguish early on in life, in time they can usually be distinguished clinically. Recent studies show that each of these syndromes is caused by a germline mutation in a key component of the highly conserved RAS, RAF-ERK-MAP kinase cascade which is better known for its roles in growth factor and cytokines signaling in cancer pathogenesis. We still do not understand how these specific mutations cause the disease and why there are such distinct phenotypic differences in mutations within the same signaling pathway. Tartaglia et al [33] recently provided evidence that specificity in amino acid substitution is relevant to the functional deregulation of SHP-2 and disease pathogenesis. They showed NS mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated mutations and that Y279C and T468M amino acid substitutions noted in LS engender a loss of SHP-2 catalytic activity. It is not surprising that a mutation in the HRAS gene associated with CS has an increased incidence of tumors since these mutations are identical to the human tumor associated mutations. So far, CFC syndrome has not been associated with malignancy but the number of reported cases is still quite small with long-term follow-up limited. For the endocrinologist, it is interesting that all of the syndromes have a high incidence of short stature. LS, on the other hand, with loss of function appears to have a lower incidence of short stature but the higher incidence of hypertrophic cardiomyopathy. It will be of interest to see how effective IPLEXtm will be in treating the growth failure in NS and perhaps these related syndromes as well. Since the growth failure starts very soon after birth and may indeed start before birth, it is certainly possible that treatment may be necessary very early in life to achieve anywhere near a normal growth stature.

Eur_J_Clin_Pharmacol-3-1-1914304

Hypothermia following antipsychotic drug use

Objective Hypothermia is an adverse drug reaction (ADR) of antipsychotic drug (APD) use. Risk factors for hypothermia in ADP users are unknown. We studied which risk factors for hypothermia can be identified based on case reports. Introduction Antipsychotic drugs (APDs) can influence thermoregulation. Even before its psychotropic properties were clear in the early 1950s, the first manufactured APD, chloropromazine, was used to suppress compensatory responses to body cooling in surgery (artificial hibernation) [1]. The hypothermic effects of APDs seem less well known than the hyperthermic effects (e.g., malignant neuroleptic syndrome). Besides occasional case reports, little emphasis has been placed in scientific literature on hypothermia as adverse drug reaction (ADR). To our knowledge, a review of all reported and published cases of antipsychotic associated hypothermia has not yet been published. Methods The WHO international database for Adverse Drug Reactions was searched for reports of hypothermia and APD use (ATC-code N05A, with exclusion of lithium). The data in the database are collected from 77 countries participating in the WHO Program for International Drug Monitoring. This database comprises more than 3.5 million case reports, to which around 50,000 new reports are added quarterly. The relationship between the APD and hypothermia is evaluated by calculating the Reporting Odds Ratios (RORs) and 95% confidence intervals (95% CI) in a case/non-case design. The ROR compares the frequency of the reported ADR for a certain drug with the frequency of reports of that adverse drug reaction for all other drugs in the database. Reports concerning hypothermia were considered as cases, all other reports as non-cases. Index reports included all reports on an APD (ATC code beginning with N05A, with exclusion of lithium), all other reports were controls. When the number of reports of hypothermia in association with the APD is high and the number of reports of hypothermia in association with other drugs is low, ROR will be high. This also happens when the number of reports of other ADRs in association with the APD is low and the number of reports of other ADRs in association with other drugs is low. Since the vast majority of cases in the WHO database do not contain any details on indication of drug use, start and/or end dates and outcome, these factors cannot be analyzed with data from the WHO database. To get more information regarding characteristics of patients developing hypothermia during APD use, we performed a literature search in Medline and Embase for case reports with search terms “(antipsychotic OR neuroleptic) AND (hypothermia OR body temperature regulation), with no selection on date or language. From these articles, we searched the references for missing articles. Two reviewers judged all case reports. All relevant case reports were studied for patient, drug and environmental characteristics. Results In the WHO database, in January 2007, 480 reports were registered of patients developing hypothermia during the use of APDs. Characteristics of these reports are presented in Table 1. In the same period, 524 reports of hyperthermia associated with antipsychotic drug use were registered. Based on the reports, no specific pharmacological subgroup can be associated with an increased risk for hypothermia. Atypical antipsychotics are responsible for 55% of the reports, but this is mainly attributable to risperidone. Risperidone alone was responsible for 27% of all reports. A remarkable high association is found for pipamperone (ROR:24.62; 95% CI 13.16–46.07), an antipsychotic drug mainly used in Europe. Table 1Antipsychotic drugs and hypothermia: reports from the WHO databaseAntipsychotic drugNumber of reports for hypothermiaNumber of reports for any ADR with this drugReported OR (CI)a for ADRs with ≥3 reportsTioxanthenes Zuclopenthixol13109415.88 (9.18–27.47) Flupenthixol616774.73 (2.12–10.55) Chlorprothixen450210.58 (3.95–28.31) Tiotixene39044.38 (1.41–13.62) Clopenthixol2293-Phenothiazines Thioridazine2344366.90 (4.57–10.41) Chlorpromazine1661823.43 (2.09–5.60) Levomepromazine1123486.21 (3.43–11.24) Cyamemazine911979.99 (5.18–19.27) Periciazine843025,00 (12.41–50.36) Pipothiazine319720.36 (6.50–63.71) Fluphenazine326561.49 (0.48–4.62) Trifluoperazine318952.09 (0.67–6.48) Perphenazine21522- Prochlorperazine24451- Promazine2307- Mesoridazine1215-Butyrofenones Haloperidol32105436.21 (3.43–11.24) Pipamperone1054624.62 (13.16–46.07) Droperidol21178- Benperidol1146-Benzamides Tiapride559611.14 (4.62–26.89) Sulpiride418282.89 (1.08–7.70) Amisulpiride11514- Sultopride172-Others Loxapine49285.70 (2.13–15.23) Pimozide36286.31 (2.03–19.65) Zotepine2260- Prothipendyl193- Penfluridol158-Atypical Risperidone129184319.65 (8.09–11.52) Clozapine68442552.05 (1.61–2.61) Olanzapine44160903.65 (2.71–4.91) Quetiapine2153745.19 (3.38–7.98) Aripiprazole1145663.18 (1.76–5.76) Ziprasidone829633.57 (1.78–7.15)aROR= (a/c) / (b/d); (a = no. of reports of adverse drug reaction with suspected drug; b = no. of reports of adverse drug reaction in total database; c = no. of reports regarding the suspected drug in database; d = total no. of reports in database). ROR is only calculated for APDs with three or more reports The literature search resulted in 32 articles containing 43 case reports (December 2006) [3–34] from which characteristics are summarized in Table 2. Hypothermia following antipsychotic drug use is not associated with a specific age group. Reported ages vary from 0 to 90 years. In most cases, hypothermia is detected shortly following the start or dose increase of an antipsychotic drug. Most patients suffered from schizophrenia. Table 2Characteristics of cases with hypothermia following antipsychotic drug use in literature (43 case reports, 46 episodes)CharacteristicsDataMale41%Age: mean (SD)49 (23.0)Range 0–90 yearsReported body temperature: mean (SD)32,6°C (2.7)Range 20.0–36.1°CDiagnosis known (n = 35)Schizophrenia51%Mental retardation11% Bipolar disorder11%Dementia11%Drug change Start or dose increase80% No change16%Interval drug change detection hypothermia <2 days57% 2–7 days16%Outcome death4% ICU admission24%Hospitalization (incl. prolonged)69% Discussion Hypothermia in patients using an APD is a serious, unpredictable, type B adverse event frequently leading to hospital and ICU admission and sometimes even to death. Some authors have even suggested that a substantial proportion of unexplained deaths should be attributed to antipsychotic-induced hypothermia [3, 35]. No single sufficient cause for hypothermia can be found in case reports. First, drug-receptor profile may play a role. Serotonin is associated with thermoregulation and APDs with a stronger affinity for the 5-HT2a receptor than for the D2-receptor (pipamperone, the atypical APDs) seem to be associated with hypothermia. The high association for relatively new drugs, like the atypical APDs, can partially be explained by reporting bias (reporting incidence for adverse drug effects is higher for new drugs and tends to decline in time; the so-called Weber-effect), but the high number of reports for risperidone should keep clinicians alert. Blocking alpha2-adrenergic receptors (e.g., chloropromazine, risperidone, clozapine, thioridazine) may also increase the hypothermic effect, by inhibiting peripheral responses to cooling (vasoconstriction and shivering). Next to this receptor profile, many patient-bound factors must be considered. Patients with pre-existing brain damage may be more susceptible to hypothermic effects. The pre-optic anterior hypothalamic region regulates body temperature. Animal studies show that lesions of this region give a hypothermic response following administration of an APD [36, 37]. In patients with multiple sclerosis, hypothermia is also associated with thalamic lesions [38]. Studies in schizophrenic patients show that core temperature decreases following the administration of APDs [39, 40]. Our search shows a predominance of case reports for schizophrenic patients and little for other frequent APD user groups like demented or delirious elderly. In schizophrenia, thermal regulation is altered. This may be explained by changes in neurotensine levels in schizophrenia. Neurotensine (NT) is one of the most important thermoregulatory peptides that also plays a role in the antipsychotic actions of APDs. In schizophrenic patients, NT concentration in cerebro-spinal fluid (CSF) is low and will be normalized following antipsychotic drug use [41]. The hypothermic reaction is also dependent on ambient temperature. In animal studies, APD administration at ambient temperatures below 22°C led to hypothermia, whereas APD administration in a room temperature of 29°C gave no thermal response and at 32°C an increase in rectal temperature [41, 42]. Normally, a cold environment will result in behavior aimed at protection against the cold (taking extra blankets or clothes). APDs, however, will induce apathy and indifference, resulting in unawareness of developing hypothermia. Since some case reports also mention the co-existence of infections at the time of development of hypothermia, this may also play a role in the dysregulation of thermal homeostasis. There is an ongoing discussion concerning the value of case reports and spontaneous reports in the field of drug safety. There can be no discussion that pharmacodiligence requires prospective studies, access to regulatory filings for controlled and monitored use of drugs, and some sense of relative potency for comparison between different drug entities. However, large cohorts are needed for the detection and analysis of type B reactions. Since these data are lacking, case and spontaneous reports by alert clinicians must be analyzed [43]. The outcome of such an analysis cannot be seen as solid evidence, but can help to get more insight in the adverse drug reaction (ADR). The use of measures like ROR and disproportionality can help to detect drugs with an increased association with an ADR. A statistically significant ROR may be indicative of a higher risk for that particular event during the use of a specific medication, but is never conclusive for the actual existence of a causal relation [2]. The results of this study should alert physicians of the risk of hypothermia in psychiatric patients using APDs. There seems to be no direct relation between stable drug dose and the ADR; the period shortly after starting the APD or dose increase seems to be the high risk period. Often, drug changes are indicated by behavioral problems also leading to separation or isolation of the patient. In the case of separation, patients will be dressed lightly and, even at normal room temperature, can cool down easily. In these cases, the patient’s body temperature should be monitored daily (with a thermometer that can measure low body temperatures). Also, every change in behaviour or co-morbidity (e.g., infections) should be a warning sign to look for hypothermia.

Virchows_Arch-3-1-1888718

Frequency and diagnostic patterns of lymphomas in liver biopsies with respect to the WHO classification

The recent World Health Organization (WHO) classification of hematopoietic and lymphoid tissue tumors represents the first worldwide consensus classification of these malignancies. However, the applicability of this classification to a representative number of hepatic lymphomas in liver biopsy specimens has not yet been investigated. The frequency and infiltration pattern of a series of 205 liver biopsies with lymphoma manifestations was analyzed with the aid of immunohistochemical and molecular pathological analyses. Diffuse large B-cell lymphoma (DLBCL) was by far the most frequent entity, comprising 45% of the cases analyzed. Using a previously published immunohistochemical algorithm, 35% of 80 DLBCL were assigned to a germinal center B-cell-like (GCB) and 65% to a non-GCB group. Most B-cell lymphoma entities involving the liver revealed a characteristic infiltration pattern. Diagnostically challenging entities were T-cell-rich B-cell lymphomas, anaplastic large cell lymphomas and peripheral T-cell lymphomas, which frequently required additional molecular clonality assessment. Overall, the percentage of T-cell lymphomas in the liver (12%) was higher as compared to other extranodal sites except for the skin and the small intestine. This study provides relevant data on the distribution of hepatic lymphomas and demonstrates the applicability of the WHO classification proposing a diagnostic algorithm for liver biopsies. Introduction The liver is most commonly involved in non-Hodgkin lymphomas (NHL) next to lymph nodes, spleen, and bone marrow. In the vast majority of cases hepatic involvement reflects secondary dissemination in advanced disease [15, 18] rather than a primary site according to the definition of Caccamo and coworkers [5]. Histopathological analysis of liver biopsy may be required in patients with an established diagnosis of malignant lymphoma to differentiate lymphoma manifestation from other causes of hepatic dysfunction or to clarify elevated transaminases such as toxic damage because of chemotherapy or other medications. On the other hand, a biopsy may reveal a previously unknown lymphoma during the exploration of a solid hepatic mass or after measuring elevated liver enzyme serology [12]. Occasionally, a malignant lymphoma can be detectable in the setting of other liver diseases such as chronic hepatitis B [20, 22], chronic hepatitis C [2, 3, 7, 21, 25], or primary biliary cirrhosis (PBC) [24]. The goals of the present study were to test the feasibility of lymphoma subtyping according to the World Health Organisation (WHO) classification of tumors of hematopoietic and lymphoid tissues [16] in liver biopsy specimens and to describe the frequencies by which the different entities occur. For this purpose, a series of 205 liver biopsies with a diagnosis of malignant lymphoma were analyzed retrospectively with a specific focus on the histopathology, especially infiltration patterns, which may ultimately allow the use of diagnostic algorithms for subtyping of lymphomas in liver biopsies. This is an important addition in relation to previous studies, which have mainly described the frequency of liver involvement in autopsy material of patients with leukemia or lymphoma [8, 13, 26, 28, 29] differentiating only between low-grade and high-grade NHL [26] or having performed a categorization according to the outdated Kiel classification [29]. Overall, this study represents the largest series on hepatic lymphomas to date and due to its restriction to bioptic material reflects the primary diagnostic situation. Materials and methods All cases of hepatic lymphoma involvement diagnosed by liver biopsy during the years 1994–2003 were retrieved from the archives of the Institute of Pathology, Campus Benjamin Franklin, Charité-University Medicine Berlin and the Institute of Pathology, University of Cologne, Germany. The series comprised a total of 205 cases with 135 cases from Berlin and 70 cases from Cologne. All cases were reevaluated and reclassified independently by three pathologists (C.L., T.L., H.S.) according to the WHO classification [16]. For cases in which the diagnosis was not unanimous, a final consensus diagnosis was reached after further immunophenotyping, molecular analyses, and final consultation. In six cases initially considered “suspicious” for lymphoma, a final diagnosis of lymphoma was made after reevaluation and additional analyses. Nine liver biopsies with insufficient material for additional immunohistochemical or molecular analyses were excluded beforehand, as well as five cases where T-cell receptor (TCR) polymerase chain reaction (PCR) had revealed a polyclonal or oligoclonal rearrangement pattern without a reproducible dominant PCR product. Overall, 32/205 (16%) cases were reclassified, including eight “low-grade” [5× B-CLL, 2× follicular lymphomas (FL), 1× marginal zone lymphoma (MZL)] and 20 “high-grade” [19× DLBCL, 1× Burkitt lymphoma (BL)] B-cell NHL that were assigned to a specific WHO lymphoma category as well as four cases with a change in the final diagnosis [BL, classical Hodgkin lymphoma (cHL), T-cell-rich B-cell lymphoma (TCRBCL), lymphoplasmacytic lymphoma (LPL) to DLBCL, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (pTCL) and B-CLL, respectively]. The analyses of the infiltration pattern were based on the assessment of the hepatic architecture. Three main patterns were distinguished: Infiltrates for which an exclusive or predominant association to portal tracts was evident were recorded as portal infiltrates. The second pattern consisted of lymphoma infiltrates which showed a coherent growth pattern thus predominantly resulting in the replacement of the acinar structures. These infiltrates were designated as a nodular growth pattern. Finally, infiltrates that showed prominent intrasinusoidal dispersion of the lymphoma cells were recorded as a sinusoidal growth pattern. Additionally, the density of the infiltrate was semiquantitatively assessed. The presence of scattered neoplastic cells in a background rich in reactive bystander cells (e.g., nonneoplastic T-cells and/or macrophages in T-cell rich B-cell lymphoma) was defined as a loose infiltration pattern, whereas the appearance of coherently appearing neoplastic B- or T-cells was referred to as a dense infiltrate. According to clinical information available, in 76/205 (37%) of the cases the diagnosis of a lymphoma had been previously established from extrahepatic biopsies or peripheral blood indicating secondary involvement of the liver. In four of these cases with known low-grade lymphoma (2× B-CLL, 2× FL) liver biopsy revealed transformation to a high-grade lymphoma (DLBCL). In the remaining cases, the biopsy obtained from the liver represented the site of primary diagnosis. In 26 (13%) patients, additional biopsies from extrahepatic sites were available (13 bone marrow biopsies, 10 lymph node biopsies, and three spleen biopsies). Furthermore, in a few cases clinical data regarding a potential predisposing condition for a primary hepatic lymphoma was available: chronic hepatitis C was reported in four patients (3× DLBCL, 1× B-CLL), HIV infection was present in three patients (2× DLBCL, 1× BL), and one marginal zone lymphoma occurred in a patient after liver transplantation. The frequency of the various lymphoma entities diagnosed in liver biopsy specimen was compared to their frequency and distribution in other extranodal sites (cases from the Consultation and Reference Center for Lymph Node Pathology and Hematopathology, Berlin). Immunohistochemistry For immunostaining, 4 μm thick sections were cut, deparaffinized, and subjected to heat-induced epitope retrieval before incubation with antibodies. For this purpose, sections were immersed either in sodium citrate buffer at pH 6.0 or, alternatively, in ethylenediamintetraacetic acid (EDTA) at pH 8.0 and heated in a high-pressure cooker. After cooking, the slides were rinsed in running water, washed with Tris-buffered saline, pH 7.4, and incubated with the respective primary antibodies. All primary antibodies employed are listed in Table 1. With the exception of immunoglobulin detection, in which the streptavidin–biotin peroxidase method was applied [14], labeling was carried out using the alkaline–phosphatase/anti-alkaline–phosphatase complex method [6]. Alkaline phosphatase was developed using Fast Red as the chromogen, whereas peroxidase was visualized with diaminobenzidine chromogen as the substrate. Table 1Antibodies used in this studyAntibodyCloneAntigen retrievalDilutionSourceALK1ALK1Citrate1:20DakoBCL2124Citrate1:25DakoBCL6594Citrate1:25DakoCD2AB75Citrate1:50NovocastraCD3F7.2.38Citrate1:100DakoCD41F6Citrate1:25NovocastraCD54C7Citrate1:25NovocastraCD7CD7-272EDTA1:50NovocastraCD8C8/144BCitrate1:100DakoCD1056C6Citrate1:25NovocastraCD15C3D1Citrate1:20DakoCD20L26Citrate1:50DakoCD211F8Protease1:50DakoCD231B12Citrate1:20NovocastraCD27137B4EDTA1:100NovocastraCD30BerH2Citrate1:50DakoCD43DF-T1Citrate1:50DakoCD68PG-M1Citrate1:50DakoCD79aJCB117Citrate1:100DakoCD138B-B4Citrate1:10SerotecCyclin D1P2D11F11Citrate1:50NovocastraEBV-LMPCS1-4Citrate1:100Dakoα-heavychainRabbit polyclonalCitrate1:40 000Dakoγ-heavy chainRabbit polyclonalCitrate1:30 000Dakoδ-heavy chainRabbit polyclonalCitrate1:2000Dakoμ-heavy chainRabbit polyclonalCitrate1:2000DakoKi-67MIB-1Citrate1:2000Dakoκ-light chainRabbit polyclonalCitrate1:100 000Dakoλ-light chainHP6054Citrate1:16 000DakoMUM1/IRF4MUM1pCitrate1:20Generously provided by Prof. B. Falini, Perugia, ItalyPax-524Citrate1:10Transduction LaboratoriesTIA-12G9Citrate1:500Coulter Molecular pathology analyses Additional molecular pathology analyses were selectively performed for those cases in which immunoprofiling alone was not sufficient to establish a definite diagnosis of a malignant lymphoma. For this purpose, DNA was extracted after dewaxing 20-μm-thick paraffin sections with QIAEX (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. TCR-gamma rearrangements were analyzed using four different primer combinations (JGT1/2, JGT3, BioMed-2 Set A and Set B). The primer combinations JGT1/2 und JGT3 suitable to amplify the most frequent TCR-gamma rearrangements were used in a two-step seminested PCR. The BioMed-2 primer sets A and B able to detect all possible TCR-gamma rearrangements were performed as single round PCRs. The detailed cycling conditions (50 rounds of amplification for BioMed-2 set A and set B; two-time 35 cycles for primer combinations JGT1/2 and JGT3) for all PCRs have been described in detail elsewhere [9, 27]. Amplification of rearranged IgH genes was independently performed at least twice per case employing three different framework primer sets (BioMed-2 FR1, FR2, and FR3) separately in conjunction with a JH primer (JH22). PCR conditions consisted of 50 cycles of denaturation (95°C, 15 s), primer annealing (60°C, 40 s), and elongation (72°C, 45 s), and the reaction mixture contained 1.5 mM MgCl2, 0.8 mM deoxyribonucleotide triphosphates (dNTPs), 70 pmol VH primers, 30 pmol JH22 primer, and 2 U of AmpliTaq Gold polymerase (Applied Biosystems, Weiterstadt, Germany). Results Characteristics of hepatic lymphomas Almost 90% (182/205) of the hepatic lymphomas belonged to the group of NHL, whereas only 23 of the 205 cases were cHL. Regarding NHL, 86% (157/182) were of B-cell and 14% (25/182) of T-cell origin. Overall, diffuse large B-cell lymphoma (DLBCL) was the most common type [51% (93/182) of all B-NHL analyzed] with TCRBCL—a morphologic variant of DLBCL—comprising 14% (13/93) of all DLBCL. According to the algorithm of Hans et al. [11] a predominance of DLBCL of non-GCB (germinal center B-cell-like) type (52/80; 65%) compared to the DLBCL of GCB type (28/80; 35%) was found (Fig. 1a–d,f). Only 4 out of 80 cases (5%) corresponded to the group of CD5-positive DLBCL (Fig. 1e). Fig. 1Nodular pattern: DLBCL of GCB group positive for CD10 (a) and BCL6 (b) and negative for IRF4/MUM1 (few scattered plasma cells serve as positive intrinsic control; c). CD5 positive DLBCL of ABC type negative for CD10 (positive bile canaliculi serve as intrinsic control; d), positive for CD5 (e) and IRF4/MUM1 (f). TCRBCL with less than 10% large CD20 positive neoplastic B-cells (g) associated with numerous CD3 positive T-cells (h) and CD68 positive histiocytes (i) In all lymphoma subtypes, men were affected more often, except for FL, which occurred with equal frequencies in both sexes. The male predominance was most striking in lymphomas of T-cell origin, in which ALCL and hepatosplenic T-cell lymphoma (HSTCL) were encountered exclusively in men. Additional molecular analyses were essential to confirm the suspected diagnosis of a lymphoma in 5 out of 19 T-cell lymphomas (26%) and 3 out of 116 (3%) B-NHLs (Fig. 2). Fig. 2Size fragment analyses (GeneScan) of the amplificates after TCR-gamma PCR (primer combination JGT1/2). Fluorescence-labelled PCR products (blue lines) were separated on capillary electrophoresis system (Applied Biosystems, Model 310A) in parallel to a size standard (red line). The dominant PCR product (arrow) indicating the presence of a clonal T-cell population was reproducibly detectable in addition to a moderate oligo-/polyclonal T-cell background The relative frequencies of the lymphoma subtypes, the available patient data and the infiltration patterns are summarized in Table 2. Table 2Frequency, age distribution and pattern of infiltration in the different lymphoma entities presenting in the liver (Berlin/Cologne 1994–2003) BerlinCologneBerlin + Cologne, n (%)Age (range)Age (mean)Male/femalePatternDensity of infiltrateSinusoidalPortalNodularDenseLooseDiffuse large B-cell lymphoma (DLBCL)a522880(39)16–876450/3061972755T-cell rich B-cell lymphoma (TCRBCL)9413(6)35–85599/40115013B-cell chronic lymphocytic leukemia (B-CLL)131326(13)44–826616/107252251Classical Hodgkin lymphoma (cHL)101323(11)21–915414/912110320Follicular lymphoma (FL)11314(7)38–83627/70114140Marginal zone lymphoma (MZL)617(3)49–80706/127061Leukemic Plasmacytoma (PL)426(3)61–88704/241251Burkitt lymphoma (BL)5–5(2)30–70473/200550Mantle cell lymphoma (MCL)3–3(1)60–66632/113130B-lymphoblastic leukaemia (B-ALL)2–2(1)18–28221/120020Hairy cell leukaemia (HCL)1–142–0/110010Peripheral T-cell lymphoma, unspecified (pTCL)13518(9)38–846214/4668117Anaplastic large cell lymphoma (ALCL)b5–5(2)24–76585/033223Hepatosplenic T-cell lymphoma (HSTCL)112(1)38–51452/020020Total13570205aIncluding centroblastic (n = 69), immunoblastic (n = 3) and anaplastic variant (n = 8)bIncluding one anaplastic large cell lymphoma kinase (ALK) protein positive case Infiltration patterns of the various lymphoma types Most of the cases showed the predominance of a certain growth pattern, although some cases exhibited more than one growth pattern. Almost all DLBCL and BL showed a nodular infiltration pattern (91%) composed of a dense infiltrate (94%; Table 3), except for the TCRBCL variant, which was located predominantly within the portal tracts (85%) and exhibited the characteristic scattered infiltrate of fewer than 10% large neoplastic B-cells admixed with many reactive T-cells and histiocytes (Fig. 1g–i). Lymphomas showing a dense portal infiltration pattern were chronic lymphocytic leukemia/small lymphocytic lymphoma (96%; Fig. 3a–c), FL (79%; Fig. 3d–f), MZL (100%; Fig. 3g–i), and mantle cell lymphoma (100%). In contrast, in precursor B lymphoblastic leukemia/lymphoblastic lymphoma (B-ALL/B-LBL; Fig. 4a–c), HSTCL (Fig. 4d–f), leukemic plasmacytoma (Fig. 4g–i), and hairy cell leukemia, a sinusoidal infiltration pattern was observed in the majority of cases. The cHL displayed a predominantly portal infiltration pattern (Fig. 5a–c). Peripheral T-cell lymphoma and ALCL showed a spectrum of portal, sinusoidal, nodular, or mixed patterns with a varying density of infiltration, and thus did not allow for pattern-based selection of additional analyses (Fig. 5d–f). The infiltration patterns, differential diagnoses, and discriminative immunohistochemical markers are summarized in Table 3. Table 3Infiltration pattern, differential diagnosis and characteristic immunohistochemical markersFig. 3Portal pattern: chronic lymphocytic leukemia (B-CLL) with a predominantly portal infiltrate of small lymphocytes (a) with expression of CD23 (b) and CD5 (c). FL (d) with expression of CD10 (e) and BCL2 (f). Marginal zone B-cell lymphoma with portal involvement (g), expression of memory B-cell marker CD27 (h) and a lymphoepithelial lesion of a CK7 positive bile duct (i)Fig. 4Sinusoidal pattern: precursor B lymphoblastic lymphoma (B-LBL) with a sinusoidal infiltrate of small blasts (a) with cytoplasmic expression of CD79a (b) and nuclear expression of terminal deoxynucleotidyl transferase (TdT; c). HSTCL with a sinusoidal infiltrate of monotonous neoplastic cells (d) with expression of CD3 (e) and the cytotoxic granule associated protein TIA-1 (f). Peripheral blood involvement (plasma cell leukemia) in a plasmacytoma with a sinusoidal infiltrate of plasma cells (g) with expression of CD138 (h) and IgG (i)Fig. 5Classical Hodgkin lymphoma vs. anaplastic large cell lymphoma: cHL with portal infiltrates of Hodgkin and Reed-Sternberg HRS cells (arrows) in a background rich in eosinophils (a). The HRS cells strongly express CD30 (b) and the EBV encoded latent membrane protein 1 (LMP1) (c). Anaplastic large cell lymphoma (ALCL) with portal infiltrates of pleomorphic large cells resembling HRS cells (arrows) (d) and strong positivity for CD30 (e) and the cytotoxic molecule perforin (f) Comparison of hepatic lymphomas with other extranodal lymphomas The percentage of T-cell lymphomas was considerably higher in the liver (12%, 25/205) when compared to other extranodal sites (5%, 165/3252) except for the skin (50%, mainly mycosis fungoides/Sézary syndrome) and small intestine (34%, mainly enteropathy-type T-cell lymphomas). The high proportion of DLBCL in lymphomas of the liver (45%) was only surpassed by the small intestine (64%), the brain (83%), and the testis (97%). Of note, TCRBCL was extremely rare in other extranodal sites (0,3%) with only a few cases occurring in the bone marrow and spleen. Interestingly, one case fulfilled the criteria of TCRBCL in the liver, and an additional lymph node biopsy showed the picture of a typical DLBCL, whereas in another case, both liver and spleen biopsy showed the characteristic features of TCRBCL. The frequencies of other extranodal lymphomas compared to hepatic lymphomas are summarized in Table 4. Table 4Frequency of extranodal lymphoma at different sites (Berlin 1994–2003) Brain (n = 31)Testis (n = 61)Lung (n = 79)Bone marrow (n = 1996)Stomach (n = 855)Small intestine (n = 71)Large intestine (n = 102)Spleen (n = 128)Skin (n = 427)Liver (n = 135)DLBCL255722662383032217852TCRBCL–––7–––3–9FL1–21681633209111B-CLL1–265612–410713PL3––473–––124MZL––401757741218*126MCL––310777411963LPL––3122––2135–HCL–––112–––6–1B-ALL–1–78–––1–2BL–1–4–32–25cHL––352–––31010B-NHL total (%)30/31 (97%)59/61 (97%)75/79 (95%)1862/1996 (93%)850/855 (99%)47/71 (66%)96/102 (94%)115/128 (90%)213/427 (50%)116/135 (86%)pTCL1–393466129413NK/T–1–––2––2–T-ALL–1–29––––9–ALCL––1816––265Enteropathy-type TCL–––––10––––HSTCL–––5–––1–1MF––––––––83–T-NHL total (%)1/31 (3%)2/61 (3%)4/79 (5%)134/1996 (7%)5/855 (1%)24/71 (34%)6/102 (6%)13/128 (10%)214/427 (50%)19/135 (14%)*Including nine cases of splenic marginal zone lymphoma (SMZL) Discussion To the best of our knowledge, this study represents the largest series focusing on the histopathology of hepatic lymphoma involvement and the first application of the WHO classification of tumors of hematopoietic and lymphoid tissues for this purpose [16]. Our data demonstrate the feasibility of in vivo lymphoma subtyping in liver biopsy with the aid of infiltration pattern analysis, the selection of differentiating immunophenotypic markers and additional molecular methods. Many B-cell-derived lymphomas involving the liver revealed a characteristic infiltration pattern, which facilitated the use of a restricted panel of immunohistochemical markers to reach a final diagnosis. This approach is critical in those liver biopsies in which a lymphoma diagnosis has previously not been established, as accurate subtyping of lymphomas is fundamental for the initiation of adequate treatment. Diffuse large B-cell lymphomas (DLBCL) accounted for 51% of all B-NHL cases. DLBCL was recently subdivided into different prognostic groups designated GCB, activated B-cell-like (ABC or non-GCB) and Type 3, according to their gene expression profiles [1]. The GCB group is described to have a better survival than the ABC group and the heterogeneous Type 3 group. Hans et al. [11] have proposed a simple immunohistochemical algorithm to assign cases to a GCB and a non-GCB group. In the present study, the frequency of either group in 80 liver biopsies with DLBCL was investigated, excluding the T-cell/histiocyte rich variant (n = 13). DLBCL of non-GCB type (65%) were more frequent in the liver than the GCB type (35%), whereas these two groups are described in other sites at about equal frequency [23]. As liver involvement with malignant lymphomas occurs secondary in advanced disease [15, 18], this observation may reflect the biological behavior of ABC-group DLBCLs through the selection of more aggressive cases by liver biopsy. Approximately 10% of the de novo DLBCL express CD5 [4] and may have a poor outcome [31]. They tend to be associated with extranodal sites, especially bone marrow and spleen [19]. In the present series of hepatic lymphoma involvement, the number of CD5 positive DLBCL (4/80 or 5%) was not increased. The TCRBCL, a variant of DLBCL in which the majority of cells are nonneoplastic T-cells and histiocytes, are often misdiagnosed as a reactive inflammatory condition or as a T-cell lymphoma infiltration of the liver [8, 17]. TCRBCL is relatively rare in lymph node biopsies [(23/976) or 2% of the DLBCL in the Berlin Reference Centre for Lymph Node Pathology from 1994–2003]. However, in the present study, 13/93 (14%) of the DLBCL in the liver were found to fulfill the WHO criteria for TCRBCL thus confirming previous data from Dargent and coworkers in their series of 62 liver specimens [8]. Additionally, one case with TCRBCL characteristics in the liver showed the typical morphology of a conventional DLBCL in an additional lymph node biopsy. This finding indicates that the composition of the reactive coinfiltrate may be influenced by the tissue-specific microenvironment. Therefore, the specific environment of a certain organ may influence the intensity of the T-cell response and subsequently explain the higher frequency of the TCRBCL variant of DLBLC presenting in the liver [10]. Limitations were encountered in the grading of follicular lymphoma and the subtyping of the cHL, which is generally not possible with sufficient certainty in needle biopsies. In contrast to B-cell lymphomas, T-cell lymphomas infiltrating the liver generally lack a typical infiltration pattern, making their diagnosis more challenging. In particular, ALCL may mimic cHL due to the possible resemblance of Hodgkin and Reed-Sternberg cells and expression of CD30. Thus, additional markers against pan-T and pan-B cell antigens (including pax-5), CD15, ALK-1, cytotoxic molecules, e.g., TIA-1, granzyme B, and perforin) are required when confronted with this differential diagnosis (Fig. 5). Similarly, the distinction between a T-cell lymphoma and a drug-induced or viral hepatitis (e.g., due to Epstein-Barr virus) can be very difficult in biopsies containing an increased number of sinusoidal T-cells. Therefore, additional clonality analyses by TCR or IgH PCR were necessary in 5 out of 19 (26%) T-cell lymphomas compared to only 3 out of 116 (3%) B-cell lymphomas to establish a diagnosis. The reason for the relatively high frequency of liver involvement by T-cell lymphomas is not known. As T-cells play an important role in the regulation of the hepatic immune responses, especially in chronic viral and autoimmune hepatitis [30], it is tempting to speculate whether this may influence the relatively high incidence of T-cell lymphomas in the liver compared to other extranodal sites. In the present study, 25 of the 205 (12%) cases were found to represent lymphomas derived from T-cells, whereas lymphomas of the testis, brain, lung, and stomach were B-cell lymphomas in nearly all instances (Table 4). In summary, the present study demonstrates the feasibility of subtyping lymphoma infiltrates in liver biopsies according to the WHO classification. The large number of 205 cases provides reliable information regarding the relative frequencies of the different lymphoma entities encountered in the liver and demonstrates the usefulness of infiltration pattern analysis for diagnostic purposes.

Int_Urogynecol_J_Pelvic_Floor_Dysfunct-3-1-2062491

Is there an association between depressive and urinary symptoms during and after pregnancy?

Depressive symptoms and urinary symptoms are both highly prevalent in pregnancy. In the general population, an association is reported between urinary symptoms and depressive symptoms. The association of depressive and urinary symptoms has not yet been assessed in pregnancy. In this study, we assessed (1) the prevalence of depressive symptoms, over-active bladder (OAB) syndrome, urge urinary incontinence (UUI) and stress urinary incontinence (SUI) during and after pregnancy using the Center for Epidemiologic Studies Depression Scale (CES-D) and the Urogenital Distress Inventory (UDI) and (2) the association of depressive symptoms with urinary incontinence and over-active bladder syndrome during and after pregnancy, controlling for confounding socioeconomic, psychosocial, behavioural and biomedical factors in a cohort of healthy nulliparous women. Our data show a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in prevalence of depressive symptoms, SUI and OAB after childbirth. UUI prevalence did not significantly decrease after childbirth. In univariate analysis, urinary incontinence and the OAB syndrome were significantly associated with a CES-D score indicative of a possible clinical depression at 36 weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated (OR 4.4 [1.8–10.5]). No association was found between depressive and urinary symptoms at 1 year post-partum. Only OAB was independently associated with depressive symptoms during pregnancy. Possible explanations for this association are discussed. Introduction The lifetime risk of depressive symptoms in women (5.9–21.3%) is about twice that in men and symptoms often start in the childbearing years [1–3]. Prevalence rates of 10–25% during pregnancy [4–9] and of 6–16% post-partum are reported [5, 6, 8, 10–12]. Prevalence rates vary because of the diversity of populations and diagnostic methodology. Most researchers found higher rates during pregnancy than after childbirth [5, 6, 8]. Urogenital symptoms are also more prevalent in pregnancy than in the general population. Prevalence rates of 9–50% have been reported for urinary incontinence (UI) during first pregnancy and 34–95% for frequency and urgency symptoms (over-active bladder [OAB] syndrome) [13, 14]. Strong associations are found between depressive symptoms, urinary incontinence and over-active bladder syndrome in non-pregnant women [15–19]. Whilst the relationship of depressive symptoms and urinary symptoms has not yet been explored in pregnancy, several factors have been found to be associated with depressive symptoms in a general, pregnant and post-partum population. These factors include biomedical factors such as obesity, age, chronic pain (like back pain), previous depressive symptoms [12, 20, 21], behavioural factors like excessive use of alcohol, smoking, lack of leisure time physical exercise and socioeconomic factors like unemployment and low job satisfaction [5, 7, 22]. Psychosocial factors, such as poorer social support, stressful life events and personality features have also been found related to depressive symptoms [11, 22–25]. The aim of this study was to (1) analyse the prevalence of depressive symptoms and urinary symptoms during and after pregnancy and (2) assess the association of urinary symptoms with depressive symptoms, controlling for psychosocial, behavioural, socioeconomic and biomedical factors during and after pregnancy. Materials and methods Study population Between January 2002 and July 2003, 1,366 nulliparous pregnant women from 10 urban midwifery practices in the center of The Netherlands were approached to take part in a prospective longitudinal cohort study assessing pelvic floor problems, sexuality and back pain during first pregnancy until 1 year after delivery. All nulliparous pregnant women received information about the study from the midwives. After 1 week, the women were approached by phone and asked if they wanted to participate in the study. Inclusion criteria were a singleton low risk pregnancy and sufficient knowledge of the Dutch language. One hundred and twenty-two women were excluded due to having a twin pregnancy (n = 2), miscarriage (n = 13) or insufficient knowledge of the Dutch language (107). Thus, 1,244 women met the inclusion criteria. Of these, 672 (54%) decided to participate in the study. The most common reasons for refusal were lack of time and the intensity and intrusiveness of the questions. The present study is a separate analysis of data collected from the larger study. The Medical Ethics Committee of the University Medical Center Utrecht approved the study. All participants signed an informed consent form. Data collection To assess the prevalence rates at different points in time of depressive symptoms, stress urinary incontinence (SUI), urge urinary incontinence (UUI) and over-active bladder (OAB) syndrome, we used data obtained from all respondents of questionnaires sent at 12 and 36 weeks gestation and 3 and 12 months post-partum. In addition, we analysed data obtained from the questionnaires sent at 36 weeks gestation and 12 months after delivery to assess the possible association between urinary symptoms and depression. We did so because the prevalence of urinary symptoms peak in the third trimester and because pelvic floor symptoms, occurring in pregnancy and persisting 1 year after delivery, may be associated with depression at this time [26]. Depressive symptoms were investigated using the Center for Epidemiologic Studies Depression Scale (CES-D). This scale is developed for use in non-psychiatric populations and gives an impression of depressive symptoms [27, 28]. The total score ranges from 0 to 60; a higher score corresponds with more depressive symptoms. A cut-off score of 16 is frequently used as an indication of a possible clinical depression. We refer in this study to women who scored B16 on the CES-D as having depressive symptoms. Urinary symptoms were assessed with the Urogenital Distress Inventory (UDI) [29, 30]. The UDI is a validated, standardised questionnaire, translated in Dutch. This questionnaire consists of 19 questions about urogenital symptoms and the experienced discomfort of these symptoms. We looked specifically at self-reported urge and stress urinary incontinence and over-active bladder syndrome. Although the UDI also consists of questions on prolapse, obstructive micturition and pain and heaviness in the pelvic area, these symptoms have not been linked to depression in current literature. Therefore, these questions were not used in the present study. We followed the definitions of the International Continence Society (ICS) [31]. Urge incontinence was determined by a positive answer to the question: “Do you experience urine leakage related to the feeling of urgency?” Stress incontinence was determined by a positive answer to the question: “Do you experience urine leakage related to physical activity, coughing or sneezing?” Over-active bladder syndrome was determined when both of the following questions were answered positively: “Do you experience frequent urination?” and “Do you experience a strong feeling of urgency to empty your bladder?” Validated questionnaires were used to assess potential confounding variables. The Dutch Personality Questionnaire (DPQ) contains 133 statements, which are divided into 7 domains: inadequacy, social inadequacy, rigidity, hostility, egoism, dominance and self-esteem [32]. The higher the score, the more these characteristics are part of the subject’s personality. Personality traits are stable and this questionnaire was completed at 24 weeks gestation for logistic reasons. The Maudsley Marital Questionnaire (MMQ) was used to measure the subjective emotional and sexual relationship of the woman with her partner [33]. The MMQ consists of 15 questions, of which 10 concern emotional aspects (range 0–80) and 5 concern sexual aspects (range 0–40) of the relationship. The higher the score, the worse this specific aspect of the relationship is perceived. In addition, the questionnaires addressed biomedical and socioeconomic variables. Biomedical factors included length and weight at 36 weeks gestation and 1 year after delivery, age at time of delivery, the presence of a chronic illness or back pain and the use of medication. Data on mode of delivery and pregnancy-related complications (hypertensive disorders, premature delivery, growth retardation and vaginal blood loss) were obtained from the case files of obstetricians and midwifes. Data on length and weight were transformed into a body mass index (BMI = weight/(height)2). Socioeconomic factors consisted of level of education, marital state, employment and job satisfaction. For practical reasons, the education level was dichotomised into high school or less and more than high school. Marital state was dichotomised into married and unmarried/divorced. Behavioural factors we studied were whether participants smoked, used alcohol or were involved in leisure time physical activity in early pregnancy. In addition, women were able to add comments to the standard questions. Statistical analyses Changes in prevalence of depressive symptoms, UUI, SUI and OAB between 6 and 12 weeks gestation and between 36 weeks gestation and 3 and 12 months after delivery was determined using the McNemar tests. Possible variables associated with depressive symptoms, including urinary symptoms, were analysed in women that responded to the questionnaire at 36 weeks gestation and 1 year after delivery. Women with and without CES-D B16 were compared in univariate analyses using the independent samples t-test for continuous variables and chi-squared test for categorical variables. The relationship between depressive symptoms (CES-D _16) (dependent variable), urinary symptoms and potential confounders was assessed using multivariate logistic regression analysis (stepwise forward method) to assess whether urinary symptoms remained associated with depressive symptoms. Only significantly associated variables in univariate analyses (p < 0.05) were put in the model. Odds ratios (Exp (B)) for continuous variables were calculated when appropriate. The Hosmer and Lemeshow test was used to establish the goodness of fit of the model. A p value of >0.05 indicates that the model provides a valid representation of data. All analyses were performed with the SPSS for Windows 11.5. Results Of the 672 women who started in the study at 12 weeks gestation, 642 (95%) women responded to the personality questionnaire sent at 24 weeks gestation. The questionnaires at 36 weeks gestation and 3 and 12 months after delivery were answered by 511 (76%), 503 (75%) and 509 (76%) women, respectively. The average age at delivery was 30.0 years (standard deviation (SD) 3.6 years). Average BMI at 36 weeks gestation was 27.9 kg/m2 (SD 4.1 kg/m2). The percentage of women whose education stopped after high school was 52.7%. None of the women were breast-feeding 1 year after delivery. The percentage of women who did not have a spontaneous vaginal delivery was 33.9%. Pregnancy-related complications were reported in 18.3%. In our series, urinary tract infection was not mentioned by any of the women. The prevalence rates of depressive symptoms, UUI, SUI and OAB at different points in time during and after pregnancy are shown in Table 1. The increase in prevalence during pregnancy (from 12 to 36 weeks gestation) and decrease in prevalence after pregnancy (from 36 weeks gestation to 12 months after delivery) of all symptoms, except urge urinary incontinence, was statistically significant in the McNemar tests (p < 0.05). The increase in prevalence of urge incontinence during pregnancy was significant; however, the seemingly decrease after childbirth was not significant. Factors significantly associated with depressive symptoms at 36 weeks gestation in univariate analyses and in multivariate logistic regression model are shown in Table 2. Many factors were significantly associated in univariate analysis; however, in logistic regression only (social) inadequacy (personality traits), worse sexual relationship with the partner, not being involved in leisure time physical activity, pregnancy-related complications and OAB syndrome remained independently associated with depressive symptoms. The Hosmer and Lemeshow test provided a p value of 0.214, which indicates a proper goodness of fit for this model. Explained variance in this model (R2) is 40.9%. At 1 year post-partum, no significant association was found between depressive and urinary symptoms in the chi-squared tests; therefore, no logistic regression analysis was performed. Table 1Incidence of depressive symptoms (CES-D ≥ 16), incontinence and over-active bladder syndrome 12 weeks gestation (%)36 weeks gestation (%)3 months post-partum (%)12 months post-partum (%)Depressive symptoms18.220.716.712.2Urge incontinence7.319.116.115.6Stress incontinence20.142.226.534.3Over-active bladder syndrome54.260.17.814.4Table 2Univariate and multivariate associated factors with depressive symptoms at 36 weeks gestation CES-D < 16 (n = 405)CES-D ≥ 16 (n = 106)Crude OR (95% CI)Adjusted OR (95% CI)UDIUrge incontinence17.4%26.7%1.73 (1.05–2.86)Stress incontinence39.2%52.4%1.71 (1.11–2.63)Over-active bladder syndrome57.0%73.3%2.08 (1.29–3.34)4.40 (1.84–10.48)MMQEmotionality6.62 (6.21)13.74 (11.33)1.10 (1.07–1.13)Sexuality11.52 (6.95)16.53 (8.12)1.09 (1.06–1.13)1.12 (1.06–1.17)NPVInadequacy8.08 (5.09)14.50 (7.79)1.17 (1.13–1.22)1.21 (1.13–1.30)Social inadequacy6.89 (6.19)9.84 (6.96)1.07 (1.04–1.10)1.08 (1.01–1.16)Rigidity24.65 (6.81)26.47 (5.91)1.04 (1.01–1.08)Hostility12.36 (5.36)17.12 (6.71)1.14 (1.10–1.19)Egoism9.03 (4.23)10.56 (4.90)1.08 (1.03–1.13)Self-esteem30.30 (4.83)27.09 (4.56)0.88 (0.84–0.92)BMI (kg/m2)27.59 (3.65)29.22 (5.24)1.10 (1.04–1.15)Age (years)30.50 (3.67)29.66 (4.02)0.94 (0.89–1.00)Education high school/below46.9%63.2%1.95 (1.25–3.02)Unemployed4.5%12.3%3.00 (1.42–6.34)Low job satisfaction5.2%15.1%3.23 (1.57–6.68)Smoking7.1%16.2%2.53 (1.33–4.83)Use of alcohol16.0%6.7%0.38 (0.17–0.85)No leisure time physical activity43.2%65.1%2.46 (1.57–3.83)2.83 (1.35–5.92)Back pain51.9%67.0%1.90 (1.21–2.98)Pregnancy related complications8.8%20.8%2.71 (1.38–5.34)3.22 (1.12–8.87)Shown are significantly associated factors at the p < 0.05 level with depressive symptoms. Non-significantly associated factors are left out in this table.CES-D: Center for Epidemiologic Studies Depression Scale, UDI: Urogenital Distress Inventory, MMQ: Maudsley Marital Questionnaire, DPQ: Dutch Personality Questionnaire, BMI: body mass index, OR: odds ratio, 95% CI: 95% confidence interval. Discussion In our study, we set out to analyse the prevalence of depressive and urinary symptoms during and after pregnancy and to assess the possible association of urinary symptoms with depression, whilst controlling for psychosocial, behavioural, socioeconomic and biomedical factors. We showed a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in the prevalence of depressive symptoms, SUI and OAB after childbirth (p < 0.05). The prevalence of UUI did not significantly decrease after childbirth. In univariate analysis, UUI, SUI and the OAB syndrome was significantly associated with a CES-D score of B16 at 36 weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated. At 1 year after delivery, no association was found between urinary incontinence, OAB syndrome and depressive symptoms. In non-pregnant women, an association is reported between depression and urinary incontinence, especially urge incontinence, and over-active bladder syndrome with and without urge incontinence [15–19]. These studies mainly used univariate statistics and did not account for potential confounding factors. Our univariate analysis also showed that urinary incontinence and over-active bladder syndrome were significantly associated with depressive symptoms. However, in multivariate analysis, we did not find an association between depressive symptoms and stress or urge urinary incontinence during or after pregnancy. On the other hand, we did find an independent significant relation between depressive symptoms and over-active bladder syndrome at 36 weeks gestation, though not 1 year after delivery. In a research in a non-pregnant population, several explanations are suggested. Firstly, urinary incontinence as a chronic disorder may lead to depressive symptoms. The finding that depression at 1 year after delivery was not associated with urinary symptoms does not support this explanation. Secondly, it is suggested that psychological factors might influence urgency and detrusor instability [34]. Thirdly, a mutual pathologic origin of urinary incontinence and depression is proposed: both are suggested to be caused by the reduction of serotonin [15, 35]. This suggestion is at the least dubious because serotonergic depletion as the suggested aetiology in depression has been seriously questioned [36]. Furthermore, the therapeutic effect of selective serotonin re-uptake inhibitors (SSRIs) in the treatment of depression is only slightly better than placebo [37]. In a pregnant population, several other explanations can be considered. First, during the third trimester of pregnancy, the increase in cardiac output, increasing size of the uterus with compression of the bladder and sleep disturbances may all contribute to an increased voiding frequency. Secondly, the definition used for over-active bladder syndrome (combination of urgency and frequency) has been developed for use in a non-pregnant population. Because the majority of women will have frequency and urgency symptoms as part of their normal third trimester pregnancy, it is questionable if the diagnosis of OAB in pregnancy has the same meaning and impact as OAB in non-pregnant women. The strength of this study is that we used a prospective, longitudinal design with validated questionnaires in a large number of healthy nulliparous women. By measuring not only urogenital symptoms and depressive symptoms, but also psychosocial, behavioural, socioeconomic and biomedical factors, we were able to perform solid multivariate analysis techniques on the associations between bladder symptoms and depression. Our study has also some potential drawbacks that need to be discussed. First, because of the observational, epidemiological design of the study, all information gathered through the questionnaires is subjective. We used a symptom-based definition of urinary symptoms recommended by the ICS [31]. Although we could rule out, to a certain extend, urinary tract infection as potential confounding factor for urinary symptoms from the records of the midwife or gynecologist, we were unable to confirm the reported symptoms by means of a clinical diagnosis. However, the sensitivity of clinical tests like the stress test and also urodynamic investigation in relation to urogenital symptoms is still under debate [38, 39]. Our data have to be interpreted from a symptom-based point of view, which is most often the best there is to get out of epidemiological studies. It would be worthwhile to test our findings in a clinical setting, although the use of urodynamics during pregnancy in healthy women may turn out to be a difficult ethical question. Secondly, we had a moderate participation rate. Due to the quantity and intimate nature of the questions on sexuality and pelvic floor discomfort, the response rate was 54%. However, the prevalence rates of pelvic floor problems and back pain found in this cohort are in concordance with prevalence rates in other studies and the obstetrical outcome (mode of delivery, birth weight, etc.) of the study population was similar to that of comparable women registered in The Netherlands Perinatal Registry 2001 [13, 40, 41]. In addition, the percentage of women scoring above the cut-off score of 16 on the CES-D is similar to that found in a large American study among pregnant women [7]. Finally, the associations we found between psychosocial, behavioural, socioeconomic and biomedical factors at 36 weeks gestation have all been reported before in ante-natal and post-natal study populations [5, 7, 21, 22, 42–45]. So, the chance that our results are biased by the moderate participation rate is low. A third limitation is that we did not have information on urogenital and depressive symptoms before pregnancy. We decided not to ask about previous symptoms because of the risk of recall bias. Longstanding pre-pregnancy depressive disorders or urogenital symptoms may affect the prevalence rate recorded in pregnancy, but it is questionable if it would affect the associations we studied. Our main conclusion that there is no association between UUI, SUI and depressive symptoms during and after pregnancy was reached after multivariate adjustment in which many factors were accounted for. Finally, the prevalence of depressive symptoms was measured with the CES-D and diagnosis of depression could therefore differ from diagnosis obtained through interview following the DSM-IV criteria. However, the CES-D is a widely used questionnaire with adequate sensitivity and specificity [46], gives a good indication of depressive symptoms and is very suitable for large cohort studies. Conclusion We found significantly higher rates of depressive symptoms, SUI and OAB syndrome during pregnancy than after childbirth. After controlling for other associated factors, we found an independent association between depressive symptoms and the OAB syndrome in pregnancy but not with urinary incontinence. This association between OAB syndrome and depressive symptoms is lost after pregnancy, so it is likely that pregnancy-related factors have confounded this association. In general practice, this information on the natural course of the OAB syndrome, and its lack in causing major depressive symptoms after childbirth, can be used to counsel women who are confronted with these problems during pregnancy.

Matern_Child_Health_J-4-1-2335294

Preconception Brief: Occupational/Environmental Exposures

In the last decade, more than half of U.S. children were born to working mothers and 65% of working men and women were of reproductive age. In 2004 more than 28 million women age 18–44 were employed full time. This implies the need for clinicians to possess an awareness about the impact of work on the health of their patients and their future offspring. Most chemicals in the workplace have not been evaluated for reproductive toxicity, and where exposure limits do exist, they were generally not designed to mitigate reproductive risk. Therefore, many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use and thus present exposure potential to workers. Examples of these include heavy metals, (lead, cadmium), organic solvents (glycol ethers, percholoroethylene), pesticides and herbicides (ethylene dibromide) and sterilants, anesthetic gases and anti-cancer drugs used in healthcare. Surprisingly, many of these reproductive toxicants are well represented in traditional employment sectors of women, such as healthcare and cosmetology. Environmental exposures also figure prominently in evaluating a woman’s health risk and that to a pregnancy. Food and water quality and pesticide and solvent usage are increasingly topics raised by women and men contemplating pregnancy. The microenvironment of a woman, such as her choices of hobbies and leisure time activities also come into play. Caregivers must be aware of their patients’ potential environmental and workplace exposures and weigh any risk of exposure in the context of the time-dependent window of reproductive susceptibility. This will allow informed decision-making about the need for changes in behavior, diet, hobbies or the need for added protections on the job or alternative duty assignment. Examples of such environmental and occupational history elements will be presented together with counseling strategies for the clinician. Introduction The influence of environmental exposures on the general status of health has been increasingly acknowledged for numerous disease outcomes. The connection between air pollution and acute respiratory disease, for example and, more recently, the observation linking poor indoor air quality to increases in the incidence of childhood asthma has been widely publicized. Such epidemiologic observations are often reported in the media and, when combined with the growing public interest in a clean and healthy environment, have translated into an increasingly sophisticated patient population that expects healthcare providers to be conversant with the environmental contribution to disease risk. Indeed, the lay public often has shown more interest in– and occasionally knowledge of– the relationship between health and the environment than has the medical community. Medical educators and the Institute of Medicine (IOM) both have addressed this dilemma by promoting enhanced medical student and physician training in environmental medicine in order to develop ‘environmentally literate’ physicians. In this brief, the term ‘environmental medicine’ will refer to “diagnosing and caring for people exposed to chemical and physical hazards in their homes, communities and workplaces through such media as contaminated soil, water and air” [1, 2]. This definition was taken from a 1992 IOM Committee report on Curriculum Development in Environmental Medicine. The preconception clinical visit already includes environmental history queries regarding smoking and alcohol use, [3] but must be enlarged to address the broader concept of environmental exposures occurring in the woman’s home, community and workplace. Sources of environmental exposure Organizing the environmental exposure history can be facilitated by using the definition referred to above and by eliciting information on aspects of the patient’s larger environment; this includes the specific community locale (workplace and her home environment) and infrastructure which relates to air quality and soil or water pollution, all of which the patient deals with on a regular basis. Community Surprisingly, a clinician would likely not know if a given patient in his/her practice was living in an area near by an environmentally polluted location, such as a National Priority Listed (NPL) waste site or a local toxic waste dump. In large metropolitan areas, many such sites are present and unknown to most citizens. Although clinically significant exposure to a toxic hazard present on the site would be unlikely to threaten the wider community, a risk may exist for some residents living very close to the site, possibly allowing for soil or drinking water contamination. In specific instances often covered by the news media, patients would likely be able to report knowledge of living in the vicinity of such a place. Such a ‘self-report’ response could be elicited by simply asking the question, “Do you live near or have contact with a waste site?” Probably the most potentially important exposure opportunity is via contaminated drinking water. Here the biggest risk is usually from a small private water source such as a well, not subject to municipal water treatment standards and testing. According to the EPA approximately 15% of the U.S. population has a private drinking water source. Workplace An intimate relationship exists between occupational and environmental health, because often the source of environmental contamination is a former (or present day) work site. The workplace is an important ‘special case’ in environmental history taking, because populations exposed at work tend to be exposed at higher concentrations than the larger community, regardless whether contamination is via air, water or soil. Thus, though the working population is smaller than the public at large, workers are exposed at higher ‘doses’. It is thus sometimes observed that workers are the ‘canaries’ or ‘sentinels’, exhibiting first the health effects which might be expected in a wider community exposure from an environmental pathway. It is fair to say that the principal exposure opportunity to an environmental reproductive or developmental toxicant in patients will be from their work place. Many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use, and thus pose a continued potential risk in the occupational environment. Several employment sectors where such toxicants commonly are found employ women workers primarily and include: laboratory and clinical medicine, printing and dry cleaning [4] though this is clearly not always the case and the gender mix varies by sector. (See Table 1). Whereas some of these chemical toxicants are regulated by public health agencies, the majority of chemicals considered for regulation are not evaluated for reproductive endpoints [5] but rather, for other toxic effects. This gap in the regulatory safety net allows reproductive toxicants to be encountered in both environmental and work settings by men and women.Table 1Employment sectors and associated reproductive/develop-mental toxicantsSectorToxicantExamplesAgriculturePesticides/HerbicidesEthylene DibromideManufacturingOrganic SolventsGlycol ethers, lead,Heavy MetalsCadmiumDry CleaningSolventsPerchloroethylenePrintingSolvents/inksPharmaceutical Compounding/ManufactureHazardous DrugsAntineoplastics, hormones, immunologic modifiersHealth CareBiologicsRubella, CMV, Hepatitis virusPhysical AgentsIonizing Radiation/HeatChemicalsAntineoplastics/Hazardous Drugs Anesthetic Gases SterilantsPhysical ExertionLifting/Prolonged Standing Shift WorkNote. Ref: Summarized from GAO, 1988; Stellman, 1994. Home Here, one must consider the exposure opportunities posed by the woman’s residential environment, such as those involved with household tasks, those related to her pursuit of hobbies and those related to her ‘micro-environment,’ including diet. Diet Beyond the current recommendations related to healthy eating, the uses of non-prescription herbal or alternative medicines or supplements are crucial parts of the medical history for women planning a pregnancy. Also, some ethnic home remedies must be queried for, as some contain hazardous contaminants such as lead and mercury [6]. There are also several dietary cautions, which should be reviewed with the woman contemplating pregnancy. One is not new and relates to the warning regarding exposure to food borne listeria infections. Although listeriosis may cause only mild flu-like symptoms in the pregnant mother, serious outcomes in the fetus may result. These include premature delivery, stillbirth or neonatal infection [7–8]. General advice relies upon avoidance of soft cheese such as feta, Brie, Camembert, blue-veined and Mexican-style cheeses and unpasteurized milk or milk products [9]. Strict adherence to food preparation safety, such as vigilant washing of raw fruits and vegetables, avoidance of undercooked and raw meat and careful separation of stored raw food from uncooked meats serves to enhance safety for the entire family as most food borne illness is preventable. Consumer advisory on methylmercury in fish In 2004, the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) issued a joint consumer advisory on methylmercury in fish and shellfish. The warning specifically targets women who may become pregnant, are pregnant or are nursing mothers, and also includes young children. The document emphasizes the health benefits of fish in the diet generally as a good source of protein, and also warns of several types of fish which contain comparatively higher concentrations of mercury and should be avoided. These include: shark, swordfish, King mackerel and Tilefish. Other types of fish may be consumed in up to two meals (6 oz each) per week, including: shrimp, canned light tuna (but not albacore, which has more mercury), salmon, pollock and catfish. The Advisory also warns about local fish advisories, which are generally posted for specific populations which supplement their diets with locally, caught fish. Appropriate discussion of relevant advisories should take place in the preconception visit and is especially important for non-meat eating patients whose total dietary intake of fish may be relatively greater than is the case for meat consumers. Also of note, the U.S. Department of Health’s Women’s Infants, and Children Program (WIC) sometimes gives canned tuna as a diet supplement. This practice should be weighed in light of the above concerns, however, at least in terms of the relative amount of tuna in a weekly diet. The complete consumer advisory can be found at: www.epa.gov/ost/fish. Hobbies and home-based work The hobbies of concern generally would include those involving similar types of chemical toxicants discussed in the occupational section above, including heavy metals (lead, cadmium, arsenic) and solvents (paints (other than latex based), furniture stripper, metal cleaners etc.) Hobbies to be discouraged include: painting, ceramics, stained-glass window making (lead solder), furniture re-finishing and the like. Leisure activities to be avoided include use of saunas and hot tubs. A number of home-based work activities, which resulted in contamination of the home and family members, have been documented in the literature. Although uncommon, they deserve mention here. At one end of the spectrum most involve the use of metals, either in metal parts reclamation (lead batteries being melted down) or jewelry making (heating and soldering) [10]. At the other, the home environment may be unwittingly contaminated by family members who bring work contaminants home on their shoes or dirty work clothes. If the work clothes are laundered at home, there not only is opportunity for exposure to the launderer but also other household members. For this reason, some OSHA standards require changing rooms so work clothes will not secondarily expose family members, in the case of lead and asbestos work, for example [11]. Household exposures Pesticides, herbicides and rodenticides are likely the most common chemical toxicants in the average home. Certainly, a pregnant woman or one actively attempting to conceive, should not apply any of these [12]. Because the concept of ‘second hand’ exposure as described above applies here also, care needs be taken so that the chemicals are not ‘tracked’ into or throughout the house, no matter who performs the chemical applications. Of far greater importance on a population basis is second hand smoke in the home and/or workplace. Numerous polycyclic aromatic hydrocarbons present in cigarette smoke are carcinogenic and have been shown to affect reproductive outcomes in animal studies. They cross and affect the placenta, and enter breast milk [13]. Laundering of contaminated work clothes is also a concern, as discussed above. A hiatus from caring for the family cat and litter box should be mentioned due to the toxoplasmosis risk. Fig. 1Example checklist for initial qualitative evaluation of reproductive hazards, (modified from Grajewski, 2005)Fig. 2Example of checklist for initial quantitative evaluation of reproductive hazards, (modified from Grajewski, 2005) Many non-latex paints are solvent based and contain small amounts of metals to enhance wear, and as preservatives. This is especially true of exterior paint, such as that used on porches or building exteriors or even in the interiors of older buildings. Rehabbing older homes, which often involve paint stripping, either with a heat gun or a chemical stripper is particularly hazardous. Inhalation is a very efficient means of producing a clinically significant exposure to lead which was commonly used years ago in interiors of homes. Many commercial paint strippers contain methylene chloride (dichloromethane), which metabolizes to carbon monoxide, and is particularly toxic to the fetus. Guidance for clinician Certainly, awareness that occupational and environmental hazards encountered by patients may play a clinically significant role in a pregnancy outcome is the first step in effective patient management. Therefore, enlarging the standard health history form, completed by the patient on or before the first visit to obtain a more detailed environmental history is a necessary first step and is also often very informative. Whereas a number of preconception checklists exist, only some of which address fish consumption or residence near a waste site, most poorly capture, or even fail to query about occupation, despite this being the likely greatest ‘environmental’ risk the patient faces. A ‘qualitative’ evaluation of a patient’s potential exposure to reproductive and developmental toxicants can be obtained with a screening questionnaire as seen in Fig. 1. This involves examining occupation by industry sector of employment and then by chemical, physical and biological agents of concern. If a reproductive hazard is present in the patient’s work environment, an initial quantitative assessment can be made regarding the exposure intensity (Fig. 2). However, at this point, the clinician may want to consult an occupational medicine colleague to elicit a more detailed history and assist in the preconception recommendations. Preconception management strategies, should be based on the occupational history and include a decision on the safety of continued employment during the preconception period and pregnancy. If the patient works with bona fide reproductive and development toxicants, continued employment and under what circumstances must be determined. This may involve identifying additional protections, such as a respirator, gloves or other personal protective apparel or equipment that may make the job safer for the patient. There are some jobs, however, where it is recommended by professional organizations as well as governmental safety and health agencies, that pregnant persons, or those actively trying to conceive be provided with alternative duty [14]. A good example here is nurses who handle cancer chemotherapy [15]. However, a related issue involves crafting elements of an existing job which the pregnant patient, such as the oncology nurse may safely continue, such as patient education, telephone triage and the like. This arrangement is termed ‘alternative duty’. Also involved in preconception counseling is the potential impact on the quality of breast milk if the mother is planning to breastfeed. The majority of organic solvents and pharmaceuticals absorbed by the mother in the workplace invariably make their way into milk. Thus, both work during pregnancy and return to work while breastfeeding need to be considered. We have thus far focused on the working patient’s current job, which is generally considered the most influential on pregnancy outcome, as most reproductive toxicants are thought to act via an acute toxicity mechanism during the three to four months prior to conception and pregnancy [16–18]. However, there are also several examples of remote past exposure that are important to elicit, though likely less commonly encountered. An example includes the patient who has high bone lead stores from remote past (childhood) exposure that can be mobilized during pregnancy and expose the fetus [19–20]. Medical management at the preconception visit would include a blood lead level so this can be tracked during pregnancy. Also, maternal stores of fat-soluble organic chemicals, such as dioxins and PCBs may also expose the fetus [21, 22]. Positive responses to exposure to occupational or environmental agents by the preconception patient may require consultation. The Association of Occupational and Environmental Clinics can supply referrals to clinicians in various locales nationwide at (www.aoec.org). Conclusion The preconception office clinic visit presents a strategic opportunity to minimize the environmental and occupational sources of reproductive risk facing the preconception patient. This requires the two-way exchange of information between the patient and her clinician, clarifying misunderstandings and implementing reasonable strategies to minimize exposures from the wider community, in the workplace and at home. Taking an environmental and occupational history and tailoring recommendations based on that, enlarges the likelihood that preventable, adverse pregnancy outcomes can be avoided.

Cancer_Immunol_Immunother-3-1-2150627

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (“CIMT”) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the “CIMT monitoring panel”. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such “two-step” inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring. Introduction In the last two decades, more than 200 clinical trials of different anti-tumor vaccines aiming to induce tumor-specific immunity in cancer patients have been described [1]. Most of these trials primarily assessed safety and immunogenicity while reporting partial or complete clinical responses in a minority of patients [2, 3]. Despite the fact that the low fraction of clinical responders still precludes the establishment of a direct correlation between clinical efficacy and T-cell reactivity, it has become clear from animal models and clinical observations that naturally-occurring or vaccine-induced CD8+ or CD4+ T-cells play an important role in the control and regression of tumors [4–9]. Therefore, the number of subjects that mount a vaccine-induced T-cell response as well as the strength of a detected T-cell response represent important surrogate markers for vaccine efficacy. The enzyme-linked immunospot (ELISPOT) assay [10, 11], staining with HLA-peptide multimers [12] and intracellular cytokine staining (ICS) [13, 14] are technologies used commonly for the monitoring of antigen-specific immune responses. For these three assays, a huge variety of different protocols are available worldwide. This heterogeneity, together with the fact that the sensitivity of the individual protocols can vary significantly, makes a comparison of the results obtained in different trials a difficult task. Moreover, an increasing number of new technologies are constantly being introduced to the field, which makes interpretations even more complex [15–25]. Current data and opinion support the use of a functional assay like the ELISPOT or ICS in combination with a phenotyping assay like HLA-multimers [26, 27], but recognized international standards for all these methodologies are still lacking. The main aim of the “CIMT monitoring panel” is to harmonize and optimize the monitoring of antigen-specific T-cells among the participating laboratories, based on objective rationales with respect to the testing procedure, the analysis and the interpretation of results. Important requirements for an immunological test are sensitivity, applicability to large amounts of clinical material and feasibility at reasonable cost. The results generated by the tests should be reproducible and sensitive, independently of the place where they have been performed. After the first meeting of the working group, a series of inter-laboratory testing projects was initiated, in which individual laboratories could compare their performance, express their needs and exchange experience in order to improve their local assays. Here we report the results of the first two phases of the CIMT monitoring panel, with 13 participating centers from six European countries. Materials and methods Preparation and screening of PBMC samples Buffy coats from HLA-typed healthy volunteers were kindly provided from the Blood Bank of the University Mainz. HCMV sero-status was known. PBMC were isolated by Ficoll density gradient separation (Pharmacia, Uppsala, Sweden), washed two times in RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) containing 10 mM Hepes buffer, l-arginine (116 mg/ml), l-glutamine (216 mg/ml), penicillin (10 IU/ml), streptomycin (100 mg/ml) and 10% FCS (GIBCO BRL), counted and frozen at 10 to 20 × 106 cells per cryovial in 1 ml of FCS 90% + DMSO 10% at −80°C in freezing-boxes filled with iso-propanol. After 20 h, all cryovials were transferred to liquid nitrogen and stored until distribution to the participating laboratories. Pre-screening and selection of the PBMC donors for influenza- and CMV- T-cell reactivities were performed by a central lab using the IFNγ ELISPOT assay following a local protocol as described previously [38]. Five donors were selected for the first phase of the panel and eight for the second phase. One HLA-A*0201-negative donor was included in each phase (negative control), all other samples were HLA-A*0201-positive. Synthetic peptides and HLA-tetramers Peptides were synthesized using standard Fmoc chemistry, dissolved at 10 mg/ml in DMSO, aliquotted and stored at −80°C. The purity was checked by reverse-phase HPLC and was found to be >80%. Two known HLA-A*0201 T-cell epitopes were used: influenza MP 58–66 GILGFVFTL and HCMV pp65 495–503 NLVPMVATV (http://www.syfpeithi.de). Biotinylated recombinant HLA-A*0201 monomers folded with the influenza MP 58–66 or the HMCV pp65 peptides were produced essentially as described, purified by gel filtration and stored as aliquots at −80°C [12]. Fluorescent multimers were obtained by incubation with streptavidin-PE (Molecular Probes, Leiden, The Netherlands), then frozen as aliquots after addition of 0.5% BSA and 16% glycerol. HLA-concentrations of influenza-tetramer and HCMV-tetramers were 700 and 350 μg/ml, respectively. Both tetramers were checked by HPLC and/or validated by staining of a specific CD8+ T-cell line (Influenza) or PBMC from HLA-A2-negative and HLA-A2-positive CMV seronegative donors (CMV). Such tetramers are stable at 4°C for at least 1 month (personal observation) and participants were asked to perform all tests within this time period. Participating centers Twelve centers from five European countries participated in the first phase of the monitoring panel. As one of the investigators moved to another institution during the study a 13th center from a 6th European country was added to the group in the second phase of the panel. Participation in the panel was open to all interested laboratories with a focus on T-cell monitoring, independently of membership in the Association for Immunotherapy of Cancer. Reagent distribution and assay guidelines Coded PBMC samples, synthetic peptides and HLA-A*0201 tetramers were shipped on dry ice to the participants. Additionally, guidelines for the two T-cell assays were distributed for each phase: Phase I/2005. A protocol for tetramer staining was included. Briefly, 1 × 106 PBMC per test were transferred directly after thawing into one well of a 96 well u-bottom plate and washed in FACS buffer consisting of PBS, 2% FCS, 2 mM EDTA, 0.02% azide. Incubation with 5 μg/ml HLA-tetramer was then performed in FACS buffer with 50% FCS for 30 min at room temperature in the dark. After one wash in FACS buffer, mAb for T-cell staining were added for 20 min at 4°C. Finally, cells were washed twice before fixing in FACS buffer containing 1% formaldehyde solution. Three mAb combinations were proposed, CD8 alone, CD3 plus CD8, or CD4 plus CD8. Each lab could choose here the antibody clones, fluorescent dye and concentrations used. Stainings were performed in duplicate. For the functional assays, synthetic peptides were diluted at 1 mg/ml in PBS as a stock solution. Concentrations in further tests were 1–10 μg/ml, left to the choice of the participants. There were no recommendation which functional test should be performed, so that each group could choose the test either routinely used, or to be implemented for its own needs. In this first phase, 11/12 laboratories chose the IFNγ ELISPOT assay, one lab (Z10) a FACS-based intracellular IFNγ staining and one lab performed both assays (Z7). Spot counting was performed locally. Phase II/2006. Following the results obtained in the first testing phase, requirements were introduced and participants were asked to apply exactly these new criteria (two for the tetramer staining, and four for the ELISPOT, see “Results” section). The assay guidelines were modified accordingly. However, in order to reduce the variability in the FACS analysis of the 13 laboratories, a figure showing exemplary dot-plots, settings of gates and quadrants, and statistical analysis was provided. All laboratories were now required to perform an IFNγ ELISPOT as the functional test, with a fixed peptide concentration of 1 μg/ml. Participants were encouraged to use a distributed model protocol but were allowed to use their local protocol, provided that they applied the four new requirements introduced in the second phase. Collection and analysis of results After performing the required tests in each phase, participants returned a completed report form containing all relevant information. Number of cells recovered after thawing was included to assess viability after transport. For the tetramer staining experiments, mAb clone, manufacturer, amount, cytometer type and number of lymphocytes and/or CD8+ cells analyzed were noted. Results were expressed as percentage of tetramer-positive cells among CD8+, CD3+CD8+, or CD4− lymphocytes, depending on which mAb combination was used for the staining. Additionally, FACS dot-plots containing all gates, quadrants and deduced statistical analysis were collected. For the functional test, medium and thawing procedure (e.g. addition of DNAse, of a resting phase, etc.) had to be described, as well as the number of cells per test, the antibodies used (clone, manufacturer, concentration), the final peptide concentration and the incubation times. For the ELISPOT assay, the type of plate, the enzymatic visualization system and the spot reader were also noted. Absolute spot numbers were given by each participant, and filter plates were kept for possible second analysis. All results from both phases were collected and centrally analyzed. For the tetramer stainings, the number of lymphocytes, number of CD8+ T-cells and frequencies of tetramer-positive cells were calculated on the basis of the stainings and statistics provided by the participants. Apart from these calculated frequencies, a “visual evaluation” was necessary (see “Results”). For the ELISPOT, analysis was performed based on the spot numbers reported by the participants, followed by a student t test. Results were accepted as positive reaction only when the numbers of antigen-specific spots exceeded the number of spots in the background wells by atleast a factor two. The coefficients of variation (CV) were calculated for all results (CV = SD/mean × 100) and are shown in supplementary Tables S1a, b. The raw data from both panel phases will be provided to interested readers upon request. Results Phase I/2005 of the interlaboratory testing project—general aspects Coded PBMC samples from four HLA-A*0201-positive and one HLA-A*0201-negative healthy donor (D1–D5) were included in this first testing phase. The thawing procedure for PBMC samples in the test centers was not standardized and the recovery of viable cells varied greatly between 45 and 102% (mean 73%) in the 12 labs. However, the number of cells recovered was in all cases sufficient to perform the required analyses. When all the data from the tetramer staining and functional tests were combined it became clear that subjects D1 and D5 had responded to the HLA-A*0201 restricted CMV-derived peptide, consistent with their CMV seropositive-status, and that subjects D1, D2, D3, and D5 had responded to influenza. In total, each laboratory should in theory have been able to measure six positive (2× CMV and 4× influenza) responses. Detection of antigen-specific T-cells by tetramer staining and IFNγ ELISPOT The protocol required that all PBMC samples should be analyzed by the 12 participants for the presence of HLA-A*0201-restricted CMV-specific and influenza-specific CD8+ T-cells using centrally-prepared tetramers. The indicated frequencies of antigen-specific CD8+ cells generally represent the mean of two separate stainings with CD3 Ab/CD8 Ab/tetramer, except for centers Z1 (CD8/tetramer), Z7 (CD3/CD4/CD8/tetramer), Z5 and Z10 (one staining CD3/CD8/tetramer and one staining CD3/CD4/tetramer) and are based on the analysis and dot plots provided by each participant. As illustrated in Fig. 1, the absolute numbers of tetramer-positive T-cells were influenced by the individual decision of where to set the gates and quadrant markers for the analysis. For example, the inclusion of the subset of T lymphocytes expressing CD8 at a low density influenced the number of CD8+ and consequently the frequency of tetramer+ cells. Moreover, non-specific binding of the tetramer (as seen on the CD8-negative subset) also varied between the different laboratories. For these reasons, not only the frequencies, but also the appearance of the tetramer-positive populations was carefully examined. Two parameters were chosen for validation of “positive” results: (1) a clustered, but not diffuse, tetramer binding-population, and (2) strong intensity of tetramer staining, especially marked for the CMV-tetramer-binding population (Fig. 1). Table 1 shows: (I) the minimum, mean and maximum frequencies of antigen-specific CD8+ T-cells, (II) the results obtained from the individual centers Z1–Z12, and (III) the number and percentage of centers that detected a response. The high frequencies of CMV-specific CD8+ T-cells in donors D1 and D5 were readily detected by all participants (mean of 1 per 141 CD8 ± 113 in D1 and mean of 1 per 80 CD8 ± 24 in D5, respectively). For influenza-specific CD8+ T-cells, the results were more variable. Influenza-tetramer+ cells in donor D3 were detected by all participants with a mean frequency of one cell in 1014 CD8+ T-cells ± 355. In Donor D5, 11 of 12 laboratories detected a mean of one tetramer binding cell per 1106 CD8+ T-cells ± 508. Influenza-specific cells were less numerous in healthy subjects D1 and D2 and were only detected by five and eight laboratories, respectively. No false positive reactivity was reported by any of the participants. Fig. 1Example of tetramer staining results as provided by four selected participating centers Z5, Z12, Z8 and Z1. All stainings were performed on donor D1 from phase I/2005 who showed reactivity with both of the tested tetramers. Cells were gated either on the lymphocyte population (Z1), or the subsets of CD3+CD8+ (Z5) or CD3+ (Z8, Z12), according to the Ab combination used by each lab. The upper panel shows results for tests with the CMV-tetramer, the lower panel shows results for tests with the influenza-tetramer. In all dot-plots, the tetramer staining is displayed on the y-axis and anti-CD8-staining on the x-axis. Number of counted CD8+ T-cells and percentage of tetramer-positive cells among the CD8 subset are indicatedTable 1Overview of the tetramer results from phase I/2005 of the CIMT monitoring panelD1 CMVD1 FluD2 FluD3 FluD5 CMVD5 FluMin44820,0006,6671,8181061,786(I)Mean1418,0951,9091,014801,106Max353,77452659526588Z11293,774635870106758(II)Z2112––1,818701,770Z3n.d––701n.d–Z497––8541061,342Z5161––90988606Z61396,8961,3161,00091651Z744820,0006,6671,266821,724Z81005,12854559587877Z9n.d–1,3161,274n.d1,786Z1094–2,5971,360861,439Z11994,6751,66787758588Z1235–52664526625Detected by10/105/128/1212/1210/1011/12(III)Detected %100426710010092(I) Minimum, mean and maximum frequencies of antigen-specific T-cells(II) Results obtained from the individual centers Z1–Z12. All frequencies are indicated as 1 per x counted CD8+ T-cells(III) Number and percentage of centers which detected a given reactivity in donors D1, D2, D3 and D5 Eleven laboratories analyzed the five PBMC samples for the presence of HLA-A*0201-restricted CMV-specific and influenza-specific IFNγ-producing T-cells by ELISPOT assay. Only one group (Z10) used an intracellular cytokine staining as a functional test (data not shown because no comparison with other groups possible). Table 2 shows (I) the minimum, mean and maximum frequencies of antigen-specific cells, (II) the results obtained from the individual centers Z1–Z12, and (III) the number and percentage of centers that detected each reactivity. As described in the “Materials and methods”, results of spot-forming cells per seeded PBMC were accepted as a positive reaction only when passing statistical testing and when the number of antigen-specific spots exceeded the number of spots in the background wells by at least a factor of two. IFNγ-producing cells reactive against CMV were detected by 10 of the 11 laboratories in donor D1 (mean reactivity was 1 per 1,855 PBMC ± 825) but only by 8 of 11 in donor D5 (mean reactivity was 1 per 4,405 PBMC ± 3,762). The influenza-specific T-cells present in subject D3 were detected by six laboratories, while the responses in the healthy subjects with markedly lower numbers of peripheral specific T-cells (D1, D2 and D5) were detected by three laboratories only. Table 2Overview of the IFNγ ELISPOT results from phase I/2005 of the CIMT monitoring panelD1 CMVD1 FluD2 FluD3 FluD5 CMVD5 FluMin3,06162,50055,55533,33311,42850,000(I)Mean1,85538,14143,58917,5474,40530,811Max88810,25630,7698,5711,03914,705Z11,006––8,571––(II)Z22,439–––2,816–Z31,29562,500–22,7271,41227,727Z41,312––10,9091,980–Z5––––––Z6233*––1,960*253*–Z71,89510,25644,44433,33311,42850,000Z83,061–––6,896–Z988841,66655,55512,1951,03914,705Z10NDNDNDNDNDNDZ111,769–30,769–––Z123,030–––5,263–Detected by10/113/113/116/118/113/11(III)Detected %912727557327(I) Minimum, mean and maximum frequencies of antigen-specific cells(II) Results obtained from the individual centers Z1–Z12. All frequencies are indicated as 1 per x seeded PBMC except for Z6* where it is indicated as 1 per x seeded CD8+ T-cellsResults from Z6 were not included for calculation of the mean frequency of antigen-specific T-cells in D1, D3 and D5(III) Number and percentage of centers that detected a given response in donors D1, D2, D3 and D5ND not determined Subgroup analysis reveals that the number of CD8+ T-lymphocytes analyzed affects the sensitivity of the tetramer staining Although the tetramer stainings were performed with centrally prepared reagents following set guidelines, centers were left free to select several parameters according to their own protocols, and this could have influenced the test results (see “Materials and methods”). Most of the participants used monoclonal antibodies specific for CD3 and CD8 to co-stain the cells. There were no obvious differences in the performance of the centers depending on which antibody clones, antibody combinations or cytometer were used (data not shown). There was a high degree of variability in the number of CD8+ cells which were analyzed per staining, ranging from only 0.5 × 104 to about 19 × 104 (inter-center variation). In addition, a non-negligible intra-center variation was observed for the number of counted CD8+. We therefore analyzed each individual staining independently of the center that performed it and focused on the number of CD8+ T-cells that had been counted. For the six different antigen-specific populations detectable, a total of 68 tests was performed by the group (see Table 1). Overall, antigen-specific T-cell reactivities were reported in 82% of the tests (56/68, mean of duplicate stainings). When less than 30,000 CD8+ T-cells were counted, only 70% of all responses were found. In contrast, 89% of all responses were manifest when more than 30,000 CD8+ T-cells were counted (Fig. 2a). When antigen-specific T-cells were present at high frequency, the number of cells counted did not influence the result, because CMV-specific T-cells from donors D1 and D5 were detected irrespective of the number of CD8+ T-cells in the test. However, for the influenza-specific cells, positivity was registered in only 75% of all tests performed (36 of 48 tests). Strikingly, we observed a marked difference for the results derived from those tests involving less than 30,000 CD8+ T-cells (56% success in detection) as compared to tests performed with more than 30,000 CD8+ T-cells (84%). Fig. 2a Subgroup analysis of tetramer results from phase I/2005. Bars indicate the percentage of positives that could be detected by tetramer staining. The first group of bars shows the results for all of the six detectable positives, the second group shows results from stainings with the CMV-tetramer and the third group of columns shows results from stainings with the influenza-tetramer. The open bars in each group represent all tests performed, grey bars represent results obtained in tests that were performed on more than 3 × 104 CD8+ T-cells and black bars represent results obtained in tests that were performed on less than 3 × 104 CD8+ T-cells. The boxes within each bar indicate the fraction of tests with a positive result. The asterisk indicates a P-value < 0.05 by Chi-square analysis. b Subgroup analysis of ELISPOT results from phase I/2005. The bars indicate the percentage of positive reactivities detected by IFNγ ELISPOT assays. The open bar shows the percentage of all reactivities detected by all 11 centers that performed the ELISPOT assay as the functional test. Criteria for division of centers into two subgroups were based on the following requirements: do not use allo-APC (first subgroup analysis), use a resting time (second subgroup analysis) or use equal or more than 400,000 PBMC per well (third subgroup analysis). Grey bars always represent centers that were in conformity with the indicated minimum requirement, black bars show results from centers that did not fulfil that requirement. The boxes within each column indicate the fraction of centers in each category. The asterisks indicate a P-value < 0.05 in Chi-square analysis In conclusion, the ability to detect antigen-specific T-cell reactivities by tetramer staining was mainly affected by the number of CD8+ T-cells stained and analyzed, especially when the antigen-specific T-cells were present at low or moderate frequencies. We therefore modified our guidelines for the tetramer assay and recommended staining at least 1 × 106 PBMC and analyzing all cells in the tube. In addition, we provided an example of how optimal cell gates and dot-plot quadrants could be selected. ELISPOT assays are heterogeneous and require standardization The ELISPOT analyses were performed according to 11 more or less different protocols. The most discernible differences that were observed in these protocols concerned (1) the different types of multi-screen plates, (2) the serum origin, (3) the use of duplicates, triplicates or quadruplicates, (4) the use of allogeneic APC, (5) the inclusion of a resting phase after thawing the PBMC, (6) the number of PBMC per well, (7) the type of antibodies used, (8) the type of spot-reader, and the (9) enzyme and substrate for staining of the spots. Each center also used a different plate protocol (distribution of the wells, number of replicates, control tests). The influence of each of these parameters on the number of positive responses was studied by further analysis in which the laboratories were divided into two subgroups. As a result, several criteria were identified which could help to improve the sensitivity and comparability of detection. All data sets (duplicates, triplicates or quadruplicates) were first analyzed by Student t test for unpaired samples (“Materials and methods”). In our panel, one center used quadruplicates, nine centers used triplicates and one center performed the ELISPOT analysis in duplicates. Due to the variety in the replicates, responses measured by duplicate wells failed to pass the Student t test more often as compared to triplicates. Overall, the 11 centers were able to detect 50% of all possible reactivities in this panel phase (Table 2; Fig. 2b). In a subgroup of three laboratories (Z5, Z6 and Z8), an allogeneic APC population (T2 or K562-A*0201 cells) was added for binding and presentation of the synthetic peptides. The three centers that used allo-APC detected only 28% of all responses, while the other centers detected 58% of all responses. In five laboratories (Z3, Z4, Z7, Z8, and Z9) PBMC were thawed, and then incubated in culture medium at 37°C. After this resting phase of 2–20 h, living cells were washed, counted and seeded into ELISPOT plates. Laboratories using a resting phase detected 73% of the positive reactivities (22 out of 30 potentially positive tests). No significant difference in the ability to detect antigen-specific T cells was found using shorter or longer resting-times. In contrast, the laboratories that did not use a resting procedure detected only 30% of all positives (Fig. 2b). Finally, the number of cells seeded per well differed considerably between all participants and ranged from 1 to 6 × 105 PBMC. We divided the laboratories arbitrarily into two groups, those using either more than 4 × 105 PBMC (Z4, Z7, Z8, and Z9) or less than 4 × 105 PBMC (Z1, Z2, Z3, Z11 and Z12). The first group detected 71% of all positive samples, whereas the second group was able to detect only 43% of all positives (Fig. 2b). Centers Z5 and Z6 used a defined number of separated CD8+ T-cells in the ELISPOT and were therefore not included in this subgroup analysis. None other of the nine depicted protocol variables had any obvious impact on the detection of specific T-cells. As a conclusion from these results, four minimum requirements were formulated for the ELISPOT protocol: (1) perform triplicates for each test antigen (2) do not use allo-APC (3) add a resting time to increase the proportion of living cells seeded and (4) use a minimum number of 4 × 105 PBMC per well. Phase II/2006 of the interlaboratory testing project—general aspects To formally prove that the requirements formulated for tetramer staining and ELISPOT analysis increase the ability of the participants to detect antigen-specific CD8+ T-cells and reduce the inter-center variability, we decided to repeat the analysis in a second phase of the panel, with the same participants (phase II/2006). In this round, all groups were asked to follow our modified guidelines for the tetramer- and the ELISPOT-assays. Again, all PBMC samples were prepared and pre-tested in one central lab and peptide antigens and PE-conjugated tetramers were also provided from one source. As one investigator had meanwhile moved to another lab, we added a 13th center to the group. PBMC from seven selected healthy HLA-A*0201-positive donors and 1 HLA-A*0201-negative donor (D3) were required to be analyzed for the presence of HLA-A*0201-restricted CMV-specific T cells and for influenza-specific T-cells. The mean number of recovered cells after thawing was sufficient to perform the tests. When all the data were combined, it became clear that subjects D2, D5 and D8 possessed CMV-specific CD8+ T-cell subsets, and D1, D2, D4, D6 and D7 possessed influenza-specific CD8+ T-cells. Therefore, each laboratory could theoretically have measured eight positives (3× CMV and 5× Influenza) in this second phase. Analysis of CD8+ T-cell tetramer binding using the new guidelines In the second phase, a total of 104 tests were performed to detect the eight possible tetramer reactivities. Following the modified guidelines for tetramer staining, the mean number of CD8+ T-cells that were counted in each separate test increased markedly (+36%): a mean of about 49,000 CD8+ cells were analyzed in the phase I (n = 68 tests) and a mean of 67,000 CD8+ T-cells in phase II (n = 104 tests). The number of cells per test ranged from 12,000 to 467,000 CD8+. In 81% (84 of 104) of the tests >30,000 CD8+ were counted (compared to 66% of all relevant tests in the first phase). Table 3 shows (I) the minimum, mean and maximum frequencies of antigen-specific T-cells, (II) the results obtained from the individual centers Z1–Z13, and (III) the number and percentage of centers that detected each T-cell specificity. Donors D2, D5 and D8 showed very strong reactivities with the CMV-tetramer, with mean frequencies of 1/45 CD8+ T-cells, 1/37 CD8+ T-cells, and 1/19 CD8+ T-cells, respectively. All 13 laboratories were able to detect these populations (Table 3). All but one center detected the influenza-specific cells present at high frequencies in donors D6 (1/1116 CD8+ T-cells) and D7 (1/347 CD8+ T-cells). Donors D1, D2 and D4 possessed fewer specific cells (1/3,739, 1/3,573 and 1/5,278 CD8+ T-cells) which were found by 12, 9 and 9 centers, respectively. Three laboratories also reported influenza tetramer-binding CD8+ cells in D5 or D8. According to the results of the other centers as well as from the ELISPOT (see below), these stainings were considered as false positive (not shown). One center (Z13) was not able to detect any of the influenza-specific CD8+ T-cell reactivities. Finally, no tetramer+ cells were described in the HLA-A*0201-negative donor (D3). Table 3Overview of tetramer results from phase II/2006 of the CIMT monitoring panelD1 FluD2 CMVD2 FluD4 FluD5 CMVD6 FluD7 FluD8 CMVMin7,1437710,00010,000603,33386942(I)Medium3,739453,5735,278371,11634719Max1,538281,2502,500305712028Z13,333 453,3335,0003976929410(II)Z24,000303,3335,000301,10025021Z34,00047–5,0004558827020Z41,538712,8576,6663376926342Z56,666542,500–431,42837724Z65,00077–10,000601,66686927Z72,857473,3336,6663883329022Z82,000353,3333,333316662448Z93,3333110,0003,333323,33362520Z103,333282,222–3095220213Z111,666361,2502,5003257121515Z127,41337––357142608Z13–53––34––20Detected by12/1313/139/139/1313/1312/1312/1313/13(III)Detected %9210069691009292100(I) Minimum, mean and maximum frequencies of antigen-specific T-cells(II) Results obtained from the individual centers Z1–Z13. All frequencies are indicated as 1 per x counted CD8+ T-cells(III) Number and percentage of centers that detected each of the eight possible responses Analysis of CD8+ T-cell responses by ELISPOT following the introduction of a set of four rules In this second phase, all laboratories performed ELISPOT analysis following local protocols, all of which conformed to the newly introduced minimum requirements. Table 4 shows (I) the minimum, mean and maximum frequencies of antigen-specific cells, (II) the results obtained from the individual centers Z1–Z13, and (III) the number and percentage of centers that detected the response. High frequency T-cell responses against CMV could readily be detected by all 13 centers in donors D5 and D8 and by 12 of 13 in donor D2. Failure of center Z4 to detect the CMV reactivity in donor D2 was due to a very high background of the medium control. The number of spots representing IFNγ-producing cells after influenza-peptide stimulation was generally lower, and consequently, the influenza-specific T-cell responses in subjects D1, D2, D4 and D6 were detected by fewer laboratories (four centers for D1, three centers for D2, two centers for D4 and ten centers for D6). The high numbers of influenza-specific T-cells present in D7 were detected by all 13 laboratories (Table 4). Table 4Overview of IFNγ ELISPOT results from phase II/2006 of the CIMT monitoring panelD1 FluD2 CMVD2 FluD4 FluD5 CMVD6 FluD7 FluD8 CMVMin44,1181,79133,80358,8241,74548,38714,7201,698(I)Medium28,8231,08816,39549,96099914,2654,6691,023Max10,3453961,23141,0963914,4581,706269Z144,118596––59648,38714,720318(II)Z2–1,732––1,7454,7392,2221,698Z3–1,333––1,117–2,9821,292Z4––––774–2,273447Z5–997––1,15717,6473,8271,209Z610,3451,03114,151–1,006–1,7061,005Z7–1,317––1,06110,0006,0001,661Z832,258396–58,8243914,4583,623269Z928,571966–41,09691214,4238,9551,081Z10–8471,231–8067,4263,052696Z11–1,04433,803–1,08720,3393,6701,273Z12–1,791––1,38710,6193,0571,583Z13–1,008––9494,6154,615770Detected by4/1312/133/132/1313/1310/1313/1313/13(III)Detected %3192231510077100100(I) Minimum, mean and maximum frequencies of antigen-specific cells(II) Results obtained from the individual centers Z1–Z13. All frequencies are indicated as 1 per x seeded PBMC(III) Number and percentage of centers that detected each of the eight possible responses Comparison of the results obtained in both phases When the mean frequencies of all T-cell responses in both testing rounds were compared, it became clear that there was a difference in the distribution of reactivities (Fig. 3). In the tetramer assay, the mean T-cell frequency of the six possible positives in the first phase was 1 per 2,083 CD8+ T-cells. This value was 1 per 1,769 CD8+ T-cells for the eight possible positives in the second phase. Similarly, the mean T-cell frequency of the responses detected in IFNγ ELISPOT was 1 per 22,369 PBMC for Phase I/2005 but 1 per 14,653 PBMC for Phase II/2006. To allow a comparison of the overall performance in both phases of the panel, we therefore decided to define theoretical thresholds for high, moderate and low T-cell responses and then to compare data of the participating laboratories within these groups. Fig. 3Distribution of antigen-specific T-cell frequencies in the two testing phases as obtained by tetramer staining (a) and IFNγ ELISPOT assays (b). The figure shows the six reactivities (filled circle) and the calculated mean of all reactivities from phase I/2005 (filled line) as well as the eight reactivities (open circle) and calculated mean of all reactivities from phase II/2006 (open line). The frequency of antigen-specific T-cells is indicated on the y-axis as 1 per x counted CD8+ T-cells for the tetramer test and as 1 per x seeded PBMC for the ELISPOT assay In order to define such thresholds for low, medium and high T-cell responses, we first displayed the probability of detecting each of the 14 different reactivities as a value in a coordinate system and inserted a trendline. For both the tetramer assay and the ELISPOT assay, we observed a clear correlation between the frequencies of antigen-specific T-cells and the number of participating centers that were able to detect these populations. We then calculated the theoretical frequencies at which 90% (y = 90) and 50% (y = 50) of all participants could detect a given response (Fig. 4a, b) and used these two thresholds to divide all reactivities into three distinct classes of T-cell responses (“high”, “moderate” and “low”). Fig. 4Probability of detecting a reactivity by a tetramer staining, or b IFNγ ELISPOT assay. A trendline was inserted on the basis of results from all 14 reactivities from both phases of the panel. The figure shows the six reactivities from phase I/2005 (filled squares) and the eight reactivities from phase II/2006 (open squares). The frequency of antigen-specific T-cells is shown on the x-axis in 1 per x counted CD8+ T-cells for the tetramer assay (a) or 1 per x seeded PBMC for the ELISPOT assay (b). X-values for y = 90% and y = 50% are indicated by the broken lines For the tetramer assay, T-cell frequencies exceeding 1 per 1,200 CD8+ T-cells were therefore classified as “high”, whereas frequencies of less than 1 per 7,650 CD8+ were classified as “low” (Fig. 4a). Following the same rules for the ELISPOT assay, T-cell responses of at least one IFNγ spot per 2,850 PBMC can be considered as “high” and T-cell responses of less than one spot per 19,000 PBMC as “low” (Fig. 4b). With these calculated assay-specific thresholds for high, moderate and low T-cell responses, we compared the results obtained in the two phases. For the tetramer assay, the ability to detect high frequency T-cells (>1 per 1,200 CD8+) did not differ in the two phases, and was not influenced by the number of CD8+ analyzed, as previously seen for each of the two phases separately (Fig. 5a). However, for moderate and low T-cell frequencies, we found that they could be successfully detected in only 54% of cases in the first phase but this improved to 77% in the second phase. Moreover, here, the number of cells counted did have an impact on the ability to detect low frequency T-cells. In the first phase, only 14% were detected when less than 30,000 CD8+ were counted, as compared to 71% when more than 30,000 CD8+ T-cells were counted. The same trend was observed in phase II/2006, but in this case 40% of assays with less than 30,000 CD8+ successfully detected the moderate to low T-cell frequencies compared to 83% counting more than 30,000 CD8+ (Fig. 5a). Fig. 5a Percentage of reactivities actually detected by tetramer staining. The first two groups of bars show the detection rate for the nine high reactivities (>1 per 1,200 CD8+ T-cells) in phase I/2005 and phase II/2006. The next two groups of bars show the detection rate for five moderate to low reactivities (<1 per 1,200 CD8+) in phase I/2005 (third group) or phase II/2006 (fourth group). The open bars represent all tests performed, grey bars represent results obtained in tests that were performed on more than 3 × 104 CD8+ T-cells and filled bars represent results obtained in tests that were performed on less than 3 × 104 CD8+ T-cells. b Percentage of reactivities detected in IFNγ ELISPOT assays. The first two groups of bars show the rate of detection of the four high reactivities (>1 per 2,850 PBMC in phase I/2005 and phase II/2006. The next two groups of columns show the rate of detection for the ten moderate to low reactivities (<1 per 2,850 PBMC) in phase I/2005 and phase II/2006. The open bars represent the performances of all centers in the respective panel phase, grey bars represent results obtained from the five centers that already fulfilled at least three of the four minimum criteria in phase I/2005 and filled bars represent results obtained from centers that fulfilled less than three of the four minimum criteria in phase I/2005 We then analyzed the capacity of the laboratories to measure either high T-cell responses (>1 per 2,850 PBMC) or low to moderate T-cell responses (<1 per 2,850 PBMC) in the ELISPOT assay. This analysis was performed for two defined subgroups of participants. The first subgroup included those five centers (Z3, Z4, Z7, Z8 and Z9) that already fulfilled three or four of the requirements in the first phase of the panel. These five centers did not have to introduce any change or at least no major changes to their protocol for the repetition of the experiments in phase II. The second subgroup included the new center Z13 (led by a colleague that had been in a laboratory that only fulfilled one of four requirements in phase I) and all others that had fulfilled only one or two of the four requirements in the first phase. All laboratories in this second group had to introduce marked changes to their locally established protocols. Similar to the tetramer analysis, the new requirements were not necessary to detect antigen-specific responses among the category of high T-cell frequencies in either the first or second phases (Fig. 5b). However, applying the set of rules defined in phase I markedly improved the capacity of centers to detect the low to moderate T-cell responses. The first subgroup detected a total of 68% of the low to moderate reactivities in phase I, whereas the second subgroup detected only 20% (Fig. 5b). After harmonization of the protocols, both subgroups performed equally well. In addition, the inter-group variability in detecting positive responses was reduced in phase II (percentage of detected responses ranged from 38 to 88% with a mean of 67 ± 16%) as compared to phase I (percentage of detected responses ranged from 0 to 100% with a mean of 55 ± 33%). Experience does not equal performance Among the 13 centers that had participated in phase II, tetramer stainings had been performed for 1–8 years. Similarly, the experience in the ELISPOT technology varied between 1 and 10 years. For both techniques, we could not find any correlation between the years of experience and the ability to detect T-cell responses, not even among the subgroups of moderate or low T-cell responses (not shown). Discussion Whenever new techniques are introduced to the scientific community, they are first only available to a small group of expert laboratories. If these assays are robust and applicable for specific research or routine applications, they spread to the international community. In general, the “original” protocol then undergoes several adaptations in order to meet specific needs. On the one hand, changes can be beneficial and result in the improvement of protocols. On the other, this evolutionary process leads to employment of many different protocol variants, limiting comparison of the study results obtained by different laboratories. Thus, standardization approaches should be omitted during the initial development but are absolutely required when assays have become firmly established. In recent years, several activities aiming at the harmonization of techniques used to monitor the presence of antigen-specific T-cells have been initiated for ELISPOT [28–31], tetramer staining [32] and ICS [33–36]. While these studies showed the feasibility, general applicability and the diversity of performance among participants, they were not designed to either systematically investigate the influence of distinct protocol variables nor to test whether changes to these parameters can lead to a global improvement of the group. The CIMT monitoring panel is the first initiative that has now introduced the two-step approach proposing a strategy where technical variables that influence the performance of a defined assay are first systematically identified (“first step”) followed by a new testing phase where resultant protocol changes are validated under controlled conditions within the same group of investigators (“second step”). As soon as a number of protocol variables that might have influenced the sensitivity and the quality of the tests were identified in the first phase of our study, it was decided to validate this finding in a second phase. Because this two-step approach was not initially foreseen, the second phase was performed with PBMC samples obtained from different donors than those used in the first round. The distribution and the frequencies of detectable T-cell responses directed against the chosen model antigens were different in the first and second group of donors (Fig. 3) precluding a direct comparison of the results obtained in both phases of the panel. To circumvent this problem, two assay-specific frequency thresholds were introduced that allowed us to distinguish classes of T-cell responses (low, moderate and high) (Fig. 4a, b). Clearly, high-frequency T-cell responses were detected irrespective of the protocol used and as such did not allow the identification of factors that exert a strong influence on the sensitivity and variability of the protocols used. Relevant parameters could only be detected when the comparison was focused on the detection of T-cells that are present at low to moderate frequencies in PBMC. This finding should be taken into account when selecting model antigens for use in monitoring panels [37], in particular by laboratories that are interested in the detection of peripheral tumor-specific T-cells, which are often present at low frequencies, even after vaccination. Although our experiments do not specifically address the question of detection limits for the ELISPOT and tetramer assays, we could detect a high variability in the sensitivity of protocols used by the different participants. The majority of labs (y = 90%) is able to detect responses with a frequency above 1 per 1,200 CD8+ T cells in the tetramer assay or responses with a frequency above 1 per 2,859 PBMC in the ELISPOT. Note that some of the centers could reliably detect a response with a frequency of about 1 per 8,000 CD8+ T cells in the tetramer assay and about 1 per 40,000 PBMC in the ELISPOT assay. These low frequencies are in the range of that is commonly reported as the detection limit for internally validated protocols for both technologies [39, 40, own unpublished observations]. Another important task of standardization efforts should be to decrease the variation of results obtained in a group of several laboratories down to the stable and low values (15–30%) that can be reproducibly found within single labs. In order to quantify the variation of results among laboratories we calculated the coefficient of variation for all 14 reactivities of the two panel phases. The CVs were determined on the base of centers that were able to detect the respective T cell response and the results are shown in supplementary Tables S1a, b. As expected, the CVs we found in our inter-laboratory testing project were higher than those reported from intra-center analysis [39, 40]. In the ELISPOT assay, the background spot numbers obtained by the different participants varied greatly, but we were unable to correlate this finding to a distinct variable. Since the spontaneous cytokine secretion impacts significantly on the sensitivity of this assay, factors that especially influence the non-specific spot production, possibly the medium type or serum source, will need to be systematically analyzed in a separate study. The main conclusions from our study have been drawn on the basis of subgroup analyses. Although the CIMT panel in general (13 centers in this initial action), and consequently the subgroups formed during the analysis were rather small, we could already identify statistically significant differences in the ability to detect positive responses. We concluded that the number of counted CD8+ T-cells is the most influential crucial factor for the tetramer assay and that the combination of a resting-time and a high number of PBMC leads to increased sensitivity in the ELISPOT assay. This suggests that the impact of the identified technical variables on the quality of the assays is high. In order to identify those protocol variables that lead to more subtle differences, a larger group of participants would be needed. In addition to the systematic identification of variables that correlate with sensitivity/insensitivity of various assays, inter-laboratory testing projects also allow the rapid evaluation of individual performance among a group. Interestingly, the finding that experienced laboratories did not perform better than laboratories which recently applied these techniques strongly suggests that non-optimal protocols, once established in a lab, can commonly be maintained for several years. Periodic comparison of local protocols with those of other centers is recommended. Even if a new staff member uses an established protocol, it is recommended to have them participate in inter-laboratory testing/teaching exercise. Regular participation in multi-center comparisons could thereby help to optimize and validate participants’ performance over time and to maintain sensitive protocols or minimal standards. This is of great importance when material from expensive clinical trials has to be analyzed. All data from the CMV-serology, from the pre-testing experiments and from the results generated by the participating laboratories in ELISPOT and tetramer staining were taken together for each donor in order to qualitatively validate the presence of CMV- and influenza-specific T-cells. To estimate the quantity, i.e. the frequency of specific T-cells in each donor, we calculated the average of all qualitatively positive results, as well as the standard deviations. This procedure constitutes only an approximation of the real number of antigen-specific cells present in a given sample, and cannot be taken as a method for determining absolute T-cell frequencies. Cell samples that contain pre-defined numbers of antigen-specific T-cells (e.g. spiked T-cell clones), especially tumor-reactive T-cells, are not easily available for use in multi-center comparisons, although such standard samples are urgently needed. We see this as one major bottle-neck for the optimization and standardization of immunomonitoring techniques. Methods to generate such standard samples for broader use will therefore be elucidated with high priority in the near future for the next phases of this international collaboration. Another big challenge will be to define accepted rules for the settings of the equipment used in these analyses (flow cytometer or ELISPOT reader) in order to uniformly process and analyze the raw data. Ten from eleven laboratories that performed the ELISPOT assay in the first phase used an ELISPOT reader for spot counting. It is known that spot counts between centers can differ significantly and this may be explained by the use of different reading machines, different settings for the same type of machine or by the experience of the operator. Within this group, four different commercially available reading systems were used (supplementary Table S2). We were not able to identify differences between the types of ELISPOT readers. A new ELISPOT panel phase is currently in preparation, that will specifically focus on the performances of different ELISPOT readers and try to introduce tools to control inter center variation. In addition, none of the participant reported on the use of live/dead cell discrimination on thawed PBMC samples for the FACS-based experiments. Whether the combination of staining with Ab/HLA-tetramers and vital dyes or with a resting phase is beneficial for increasing the sensitivity of the tetramer staining assay could be addressed in future testing actions. Results from a proficiency panel of 36 laboratories from nine different countries in which the ELISPOT assay was validated are now also being reported [41]. This initiative, conducted under the aegis of the Cancer Vaccine Consortium (CVC), was mainly designed to offer an external validation to the participating laboratories but the in depth analysis of the obtained data sets lead to similar findings and recommendations as the CIMT monitoring panel. It confirmed that a resting phase of cells prior to addition to the ELISPOT plates is advantageous and should therefore be generally recommended. Furthermore, a long year experience in a technology did not guarantee for a sensitive test and failure to detect specific T cell responses concentrated on the weak responses. The fact that two independent initiatives come to similar findings is surely notable and shows the necessity to carry on running proficiency panels. Last but not least, we would like to stress that even the best guidelines and protocols alone cannot guarantee good performance. Monitoring of antigen-specific T-cell responses requires skills as well as experience. Participation in immunomonitoring panels cannot compensate for the need to constantly educate and train staff and to develop specific expertise for covering individual needs. Nevertheless, we strongly believe that by organizing further two-step inter-laboratory testing projects, the CIMT monitoring panel will be able to improve the sensitivity of the assays used for immunomonitoring as well as to actively participate in the harmonization of these assays, which is required to enable the comparison of immunotherapeutic trials performed in different centers. Electronic supplementary material Below is the link to the electronic supplementary material. ESM (PPT 82 kB)

Histochem_Cell_Biol-3-1-2137945

Physiological importance of RNA and protein mobility in the cell nucleus

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways. Introduction It is an irony of science that the discovery of fundamental principles is often delayed. A case in point is the cell biological investigation of the cell nucleus. The nucleus is arguably the functionally most prominent cellular organelle as it houses the vast majority of the genetic information and is the exclusive site of essential processes including transcription, RNA processing, replication and DNA repair. Yet, our understanding of how the nucleus is organized and how nuclear architecture contributes to cellular function is only now emerging and still lags behind that of most other cellular compartments. Fortunately, pioneering studies over the past decade have uncovered several of the fundamental principles which determine nuclear architecture and function (Lanctot et al. 2007; Misteli 2001; Spector 2003). These studies have revealed the presence of distinct structural elements, particularly lamin proteins and possibly short actin filaments, within the nucleus and they have led to the identification of a large number of distinct nuclear subcompartments in which specific nuclear functions, such as the synthesis and processing of ribosomal RNA in the nucleolus, occur (Burke and Stewart 2006; Gruenbaum et al. 2005; Handwerger and Gall 2006; Hernandez-Verdun 2006; Lamond and Sleeman 2003) (Fig. 1a). In addition, it has become clear that the genome is non-randomly organized within the spatial confines of the nucleus with specific chromosomes and genes preferentially localizing to specific sites (Lanctot et al. 2007; Meaburn and Misteli 2007) (Fig. 1b, c). These observations paint a picture of a highly complex and heterogeneous cell nucleus. They also indicate that some properties of the nucleus are counterintuitive. For example, despite the requirement to accommodate ∼2 m of DNA within the cell nucleus of a diameter of typically ∼10 μm, chromatin is estimated to only occupy about 15% of the nuclear volume. Fig. 1Nuclear architecture and function. The mammalian cell nucleus contains a large number of non-random and structural features. a Nuclear lamins (green) form a network of architectural elements at the periphery. This nuclear lamina is implicated in mechanical support of the nucleus, organization of chromatin (red/blue) in the nuclear interior, as a platform for signaling events and in the sequestration of nuclear proteins. b, c Genomes are non-randomly organized within the nucleus. b Each chromosome occupies a distinct chromosome territory (red chromosome 14; green chromosome 17 in human breast epithelial cells). c Pericentromeric heterochromatin regions (white) from multiple chromosomes often cluster into chromocenters. Bar 6.3 μm. Image courtesy: a Gianluca Pegoraro, b Karen Meaburn, c Manjari Mazumdar, all National Cancer Institute, NIH A key question in understanding nuclear architecture is how protein and RNAs move within the nucleus. This is a key issue if we are to gain a full appreciation of what the nuclear environment is like. More importantly, the mode of nuclear motion of molecules has physiological implications as it affects how proteins find their target sequences in the genome, how RNAs are exported from the nucleus upon transcription and how proteins are sequestered within the nucleus for regulatory purposes. Some initial insights into these issues are summarized in broad terms here. The motion of RNA and proteins in the cell nucleus We have long known that proteins and RNAs must be able to move within the cell nucleus. Messenger RNAs traverse the nucleus and are promptly exported from the nucleus after their synthesis and complete processing. Newly synthesized proteins on the other hand are imported into the nucleus and rapidly disperse throughout its volume, implying effective intranuclear trafficking. Furthermore, proteins rapidly relocalize within the nucleus upon experimental or physiological changes in conditions. For example, inhibition of protein synthesis leads to rapid and dramatic relocalization of a large number of nucleolar proteins (Andersen et al. 2005), and pre-mRNA splicing factors swiftly respond to transcriptional activation and inhibition (Jiménez-García and Spector 1993; Misteli et al. 1997). How proteins and RNAs move within the nucleus and how they find their targets, however, was unknown for long. It was not clear whether movements are energy dependent, whether they occur by directed transport or what the speed of intranuclear trafficking is. These fundamental questions have recently been answered (Misteli 2001). The investigation of intranuclear protein and RNA dynamics was made possible by the development of techniques to visualize and measure the motion of these molecules by time-lapse microscopy. Tracking of proteins by photobleaching methods revealed a surprising degree and speed of intranuclear trafficking. The analysis of tracers such as GFP or dextran which do not undergo specific interactions with DNA or other proteins indicated that the nuclear environment is permissive for rapid diffusional motion (Görisch et al. 2005; Misteli 2001; Seksek et al. 1997). The diffusion of molecules within the nucleus is only limited by the steric constraints imposed by chromatin and nuclear bodies (Görisch et al. 2005; Misteli 2001; Seksek et al. 1997). The typically measured diffusion coefficients of 10–100 μm2 s−1 are similar to those observed in the cytoplasm and are only ∼5 times less than that in solution. As expected for diffusion, this rapid motion is energy-independent and non-directional. RNA motion within the nucleus is similarly rapid and non-directional. Several methodological approaches involving fluorescently labeled, microinjected, engineered or endogenous RNAs demonstrate that ribosomal RNAs as well as polyA-RNAs move freely in a non-directional manner with a diffusion coefficient of 0.03–0.1 μm2 s−1 within the nucleus (Ritland-Politz et al. 2006; Shav-Tal et al. 2004). A diffusion coefficient of this magnitude is sufficient to ensure transport of an RNA particle from deep within the nucleus to the cytoplasm within a few minutes, consistent with biochemical observations on kinetics of RNA maturation and transport. Thus energy-independent, diffusion-based movement of RNA particles alone can account for the observed kinetics of RNA export, and no active mechanisms are required to ensure rapid export. An impressive demonstration of the non-directional motion of mRNA comes from studies in which a nascent RNA is visualized at its site of transcription and its export to the cytoplasm measured in living cells (Ritland-Politz et al. 2006; Shav-Tal et al. 2004). These studies show that RNAs synthesized from genes positioned in proximity to the nuclear envelope diffuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore. This observation powerfully demonstrates the non-directed, diffusion-based motion of RNAs in the nucleus. Trafficking as a means of targeting One of the conceptually most challenging problems in cell biology is the question of how molecules find their specific targets within a cell or within an organelle. This problem is particularly complex in the cell nucleus where transcriptional regulators need to find their specific target genes amongst the myriad of potential binding sites within the genome. Somewhat counter-intuitively, the non-directional, but rapid, motion of proteins within the cell nucleus provides a means to ensure targeting of proteins to specific genome locations (Misteli 2001). The power of targeting by random diffusion within the nucleus is best illustrated when considering how a transcription factor finds its target genes but it also applies to targeting of proteins to nuclear compartments or any other nuclear site. Proteins a priori do not know where their targets are and we do know of any directed transport systems, such as a molecular motor-based mechanism, that would bring a factor to a specific gene or a specific location within the nucleus. Thus, the only way for a transcription factor to find its target is to scan the genome. The intrinsic ability of proteins to rapidly move within the nucleus by diffusion-based mechanisms permits such genome scanning (Misteli 2001). This occurs by a transcription factor freely diffusing within the nucleoplasm until it interacts by chance with chromatin. The molecule will now probe whether the sequence it has encountered is a specific binding site such as in the promoter of one of its target genes. If it is, the transcription factor will be captured and stably associated with its specific target site. If the sequence is not a binding site, the molecule, after a short interaction, will dissociate from chromatin and continue its diffusional journey through the nucleus. It might appear at first glance that such a stop-and-go model for genome scanning would be insufficiently effective. However, the observed dynamic properties of transcription factors are entirely consistent with this model. We know that the residence time of most transcription factors even on specific DNA binding sites is in the order of a few seconds and that their interaction with non-specific sites is even faster, most likely in the order of tens of milliseconds (Gorski et al. 2006; Houtsmuller et al. 1999; McNally et al. 2000; Phair et al. 2004; Sprague et al. 2006). Assuming these time scales for binding, one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space. Considering that most transcription factors exist in several thousand copies and have multiple target genes, their random diffusional motion is entirely sufficient to ensure a steady supply at their target genes. This assumption is further supported by direct measurement of transcription factor flux on an endogenous rRNA promoter demonstrating the collision of several hundred molecules per second (Dundr et al. 2002). Since proteins similarly move by diffusional motion within the cytoplasm, it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins find their targets. A key feature, and a requirement, in a genome-scanning model of targeting is that the interactions of proteins with chromatin are transient. This has been confirmed by photobleaching methods on a large number of DNA binding proteins (Gorski et al. 2006; Phair et al. 2004; Sprague et al. 2006). The transient nature of protein–chromatin interactions is important for three reasons. First, it allows proteins to maintain a high rate of motion and thus allows faster scanning. Were protein–chromatin interactions static, they would get stuck at non-specific or incorrect binding sites which would slow down their overall motion. Second, the short-life of protein–chromatin interactions continuously makes available binding sites which can then be scanned by diffusing transcription factors. If proteins interacted for extended periods of time on chromatin, non-specific or improper binding would block access of the correct factors. Third, the dynamic dissociation allows for change. Were protein complexes permanently bound to their target sites, changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex, presumably by dedicated and specialized molecular machinery. In contrast, in a dynamic binding model the natural flux of proteins provides a window of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle. Protein dynamics as the key for formation of nuclear compartments A hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur (Handwerger and Gall 2006; Hernandez-Verdun 2006) (Fig. 2). The prototypical nuclear compartment is the nucleolus, a distinct intranuclear compartment in which ribosomal RNAs are synthesized and partially processed. Other prominent nuclear domains include splicing factor compartments which serve as storage and assembly sites for spliceosomal components, and the Cajal bodies which are possibly involved in maturation of small nuclear RNPs. The structure of intranuclear bodies is not determined by a membrane, and the principles underlying their biogenesis are extremely poorly understood. The recently revealed dynamic properties of proteins in nuclear compartments give a hint as to the principles involved in subnuclear compartment assembly. Fig. 2Intranuclear compartments. The mammalian cell nucleus contains a larger number of distinct intranuclear compartments. The nucleolus is the site of ribosomal RNA synthesis and is a prototypical nuclear body. The complex organization of the nucleolus is revealed by multi-color staining of distinct nucleolar components. RPA43 RNA polymerase I transcription factor, Fibrillarin snRNP component, B23 rRNA processing factor. Image courtesy of Miroslav Dundr, Rosalind Franklin University of Medicine and Science, Chicago The key property in understanding the formation of nuclear compartment is the remarkable fact that the association of proteins with their compartments is highly dynamic (Dundr et al. 2004; Kruhlak et al. 2000; Phair and Misteli 2000). Typical residence times of proteins within a compartment are in the seconds range. In practical terms the dynamic behavior of nuclear body proteins means that they undergo repeated rapid cycles of association and dissociation between the nuclear body and the nucleoplasm. As a consequence, a nuclear body is in perpetual flux and its structure is determined by the ratio of on-rate versus off-rate of its proteins. Given these observations, the simplest scenario to explain the formation of nuclear subdomains is a self-organization model in which the collective sum of all interactions amongst proteins in a body establishes and determines its structure (Carrero et al. 2006; Misteli 2007). In this model, nuclear bodies are essentially dynamic protein aggregates which form as a consequence of multiple, transient interactions amongst a large number of proteins. This is consistent with the lack of defining intranuclear membranes and with the absence of any known dedicated structural elements within intranuclear bodies. While dynamic observations on nucleoli, splicing factor compartments and Cajal bodies support this view, this model has not been rigorously tested experimentally. A key prediction, and a way to experimentally address this fundamental question, is that it should be possible to create nuclear bodies de novo and that any nuclear body component should be able to nucleate the formation of a body. Experimental systems to test these predictions are now available, and we are eagerly awaiting the results of these key experiments. The importance of protein and RNA dynamics in regulatory events The observation of dynamic properties of proteins in the cell nucleus of living cells has suggested that dynamic trafficking is an intrinsic property of proteins and RNAs. The dynamic behavior of proteins and RNAs clearly contributes to their proper function. However, is dynamic trafficking important for physiological regulation? Several observations demonstrate that dynamic trafficking of both proteins and RNA indeed can have regulatory function by several means. Of particular importance as a regulatory mechanism is intranuclear sequestration (Fig. 3). Accumulation of a protein at a particular nuclear site or within a nuclear compartment can serve to either increase the local concentration of a factor at that site or to reduce its abundance in the rest of the nucleus. Several proteins are now known to accumulate, in particular nuclear locations in response to physiological cues and their sequestration is thought to contribute to their cellular function. Fig. 3Intranuclear retention and sequestration as a regulatory means. a Retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export. b Sequestration of proteins at the nuclear periphery, particularly via interaction with the lamina (blue) controls their nucleoplasmic concentration and their availability at target genes. Sequestration can act both as a negative or a positive regulatory mechanism. Sequestration of an activator leads to repression, sequestration of a repression leads to activation. c Retention of partially or fully processed RNA within intranuclear compartments modulates RNA export rate Prototypical example of sequestration as a regulatory mechanism is the accumulation of the ubiquitin ligases MDM2 and the von Hippel-Lindau tumor suppressor protein VHL in the nucleolus (Mekhail et al. 2005, 2007). These two proteins are responsible for the ubiquitination of the p53 tumor suppressor and the hypoxia-induced factor HIF, respectively. Modification of these two targets leads to their nuclear export and degradation in the cytoplasm. Since ubiquitination is a major regulatory mechanism in their function, the concentration of the ubiquitin ligase in the nucleus significantly determines their fate. Both MDM2 and VHL diffuse freely within the nucleus. However, in response to physiological cues, both MDM2 and VHL can become sequestered in the nucleolus, thus reducing their nucleoplasmic concentration and the extent of ubiquitination of their targets (Mekhail et al. 2005, 2007) (Fig. 1a). Sequestration of these proteins appears to occur by increased retention of diffusing molecules within the nucleolus. How this retention is regulated is currently unclear (Mekhail et al. 2005, 2007). The possibility that retention is a highly controlled mechanism comes from observations on nucleostemin, a nucleolar protein implicated in stem cell maintenance and cancer proliferation (Meng et al. 2007; Tsai and McKay 2005). The retention of nucleostemin in the nucleolus is controlled by a GTP–GDP binding cycle which might be directly or indirectly linked to signaling pathways. In addition to nucleostemin the localization of several additional GTP-binding domain containing nucleolar proteins seems to be controlled in a similar fashion (Meng et al. 2007; Tsai and McKay 2005). Intranuclear sequestration can also be a means of negative regulation. The basic helix–loop–helix transcription factor Hand1 is expressed in trophoblast stem cells and is required for their differentiation along several lineages (Martindill et al. 2007). Hand1 is sequestered in the nucleolus and upon stimulation during differentiation is rapidly released and moves into the nucleoplasm where it presumably acts on target genes. Hand1 is retained in the nucleolus by its interaction with I-mfa, and this interaction is sensitive to phosphorylation of Hand1. Upon phosphorylation Hand1 dissociates from I-mfa and is released from the nucleolus. Interestingly, both the sequestration of Hand1 in the nucleolus and its release are functionally important. Premature release of Hand1 triggers differentiation, whereas the presence of Hand1 is also required to maintain the stem-cell potential of trophoblast stem cells. Thus nucleolar sequestration of this key cell fate regulator serves a dual regulatory role (Martindill et al. 2007). Most examples of intranuclear sequestration involve the nucleolus. However, the nuclear lamina has recently also emerged as a major site for transcription factor sequestration (Heessen and Fornerod 2007) (Fig. 1b). Prominent transcription factors which associate with the nuclear periphery either via interaction with the lamins or with the inner nuclear membrane proteins include c-fos, Oct-1 and Rb (Imai et al. 1997; Ivorra et al. 2006; Johnson et al. 2004). It is tempting to speculate that the association of these factors with the periphery reduces the intranuclear concentration and thus their availability at target genes (Heessen and Fornerod 2007). A physiological role for such peripheral sequestration is most strongly suggested by observations on the pro-proliferation factor c-fos which associates with the lamina in quiescent cells but is released upon entry of cells into the cell cycle correlating with activation of some of its target genes (Ivorra et al. 2006). Similarly, release of Oct-1 from the lamina correlates with activation of collagenase, one of its prime target genes (Imai et al. 1997). The sequestration of transcription factors at the nuclear periphery is a potentially powerful and simple mechanism of transcriptional regulation. It will be important to determine how widespread this mechanism is and how precisely the association of transcription factors with the lamina is controlled. Dynamic trafficking of RNA is similarly used as a regulatory mechanism (Prasanth and Spector 2007). In particular, it appears that RNA retention can act as a quality control mechanism for RNA export. For one, incompletely spliced or processed RNAs are retained at their sites of transcription and not released into the nucleoplasm for export. In addition, pools of stable polyadenylated RNAs are enriched in the nuclear splicing factor compartments, although their function remains unclear (Huang et al. 1994). But retention is also emerging as a mechanism for regulation of specific RNAs. A non-coding RNA transcribed from the mouse Cat2 gene encoding a cationic amino acid transporter is retained in the nucleus via a dedicated 3′ end containing extensive repeat sequences (Prasanth et al. 2005). These sequences are responsible for accumulation of the RNA in a nuclear compartment identified as paraspeckles (Fig. 1c). This retention serves a physiological function because upon stress the Cat2 RNA is cleaved, released from paraspeckles and rapidly exported into the cytoplasm for translation, thus ensuring a rapid physiological stress response. Although the Cat2 RNA is to-date the best characterized example of such retention, a similar mechanism is likely at work for the migration-stimulating factor (MSF) mRNA whose 3′ end resembles that of Cat2 and is retained within the nucleus (Kay et al. 2005). Under conditions of MSF secretion the RNA appears to be released from the nucleus and rapidly translated. A further candidate for control via nuclear retention is the neuron-specific gomafu RNA which is retained in intranuclear compartments. Interestingly, gomafu does not seem to encode for any protein and might thus be a non-coding regulatory RNA which is retained in the nucleus (Sone et al. 2007). Conclusions The past few years have seen a dramatic change in how we view the cell nucleus. We have come to appreciate the presence of distinct structural elements within the nucleus, the presence of a multitude of intranuclear bodies and the fact that genomes are non-randomly organized within the nuclear space. One of the most consequential findings has been the realization that just about every aspect of nuclear organization is highly dynamic. Both proteins and RNAs move rapidly within the nucleus, and they only transiently interact with chromatin and nuclear bodies. It is now clear that the dynamic nature of nuclear components is a fundamental property and has implications for how molecules are targeted to their final destinations and for how intranuclear compartments form. Most importantly, it is becoming clear that the dynamic properties of nuclear proteins are critical for various mechanisms of physiological regulation, particularly via sequestration and retention of proteins and RNA. It is likely that these recently discovered examples of regulation by modulation of dynamic interactions within the cell nucleus are only the proverbial tip of the iceberg, and it stands to reason that we should consider the contribution of dynamic interactions and trafficking in any nuclear event we investigate in the future.

Mod_Rheumatol-4-1-2279153

Retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a rheumatoid arthritis management group in Japan: one-year clinical outcomes (RECONFIRM-2)

Biologics targeting TNF have brought about a paradigm shift in the treatment of rheumatoid arthritis (RA) and infliximab, anti-TNF-α chimeric monoclonal antibody, was marketed in 2003 in Japan. We previously reported on the RECONFIRM study, a retrospective clinical study on the efficacy of infliximab therapy in a RA management group in Japan, where we evaluated the clinical response after 22 weeks of the therapy in 258 patients. The study reported here was aimed at reconfirming the clinical efficacy of the infliximab therapy and demographic factors related to the efficacy over a 54-week study period in 410 RA patients in the same study group. Infliximab was infused according to the domestically approved method, and the clinical response was evaluated following 54 weeks of infliximab therapy using the European League Against Rheumatism (EULAR) response criteria. Disease activity was assessed by DAS28-CRP (Disease Activity Score including a 28-joint count/C-reactive protein). Infliximab was discontinued in 24.4% of the 410 patients at 54 weeks and 9.3% and 8.1% discontinued the therapy due to adverse events and inefficiency, respectively. Average DAS28-CRP decreased from 5.5 at week 0 to 3.1 at week 54 after the therapy. Patients in remission and those showing low-, moderate-, and high-disease activity changed from 0.0, 1.0, 9.0 and 90.0%, respectively, at the start of the study to 27.6, 11.7, 34.4 and 26.3%, respectively, at week 54. Younger age, RF-negativity and low scores of DAS28-CRP showed significant correlations with remission at week 54. EULAR response criteria—good, moderate, and no response to infliximab—were 37.0, 41.7 and 21.2%, respectively. In conclusion, we reconfirmed the clinical efficacy of infliximab and demographic factors related to the efficacy over a 54-week study period in 410 Japanese patients with RA using DAS28-CRP and EULAR response criteria. Introduction Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that causes significant morbidity and mortality. RA patients should be started with DMARDs as early as possible, and among multiple DMARDs methotrexate (MTX) is considered the anchor drug and should be used first of all. However, even the use of MTX often fails to control disease activity and to prevent structural damage, and so more effective treatment strategies are needed. TNF-α plays a pivotal role in the pathological processes of RA through the accumulation of inflammatory cells and the self-perpetuation of inflammation, leading to cartilage and bone destruction. The combinational use of biologics targeting TNF-α and MTX has revolutionized the treatment of RA, producing significant improvements in clinical, radiographic, and functional outcomes that were not previously observed [1–5]. Infliximab, anti-TNF-α chimeric monoclonal antibody, has been marketed since July 2003 in Japan and currently provides high efficacy and potential adverse events. Although global evidence of the efficacy and safety of infliximab has accumulated, including the ATTRACT study, ASPIRE study and many others [6–10], there is no well-established firm evidence of the efficacy of this agent in Japan. Only the RECONFIRM study, a retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a RA management group in Japan, has reported clinical evidence on its efficacy and safety; this study was performed in several major rheumatology centers in Japan [11]. However, the RECONFIRM study evaluated the clinical response following only 22 weeks of infliximab therapy in 258 patients. In this RECONFIRM-2 study, we assessed the clinical efficacy and safety of infliximab and MTX over a 54-week study period when used in 410 RA patients in the same group, in order to reconfirm not only its clinical efficacy but also demographic factors related to the efficacy. Patients and methods Data and information on RA patients that fulfilled the diagnostic criteria of the American College of Rheumatology (ACR) were collected from three major rheumatology centers in Japan, including the First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu; the Division of Rheumatology and Clinical Immunology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical University, Saitama; and the Institute of Rheumatology, Tokyo Women’s Medical University. All patients that received infliximab treatment in each institution by December 2005 were registered with this retrospective study. Demographic data, including disease duration and concomitant therapy, were collected from medical charts. The following parameters were evaluated before and at 54 weeks after the initial infliximab infusion: tender joint count (TJC) 28, swollen joint count (SJC) 28, patient’s assessment of pain on a visual analog scale (patient’s pain VAS), patient’s global assessment of disease activity (patient’s global VAS), physician’s global assessment of disease activity (physician’s global VAS), and C-reactive protein (CRP). Infliximab therapy Infliximab was infused to patients at zero, two, and six weeks and thereafter every eight weeks at a dose of 3 mg/kg according to the drug labeling and the guidelines of the Infliximab Study Group in the Ministry of Health, Welfare and Labor in Japan [12]. Concomitant use of MTX was instituted in all cases, although the dose of MTX was determined by each attending physician. Therapeutic response Disease activity was assessed by Disease Activity Score, including a 28-joint count (DAS28)-CRP that was calculated according to the authorized formula (http://www.das-score.nl/). The value of DAS28-CRP is reported to be less than the original DAS28 using the erythrocyte sedimentation rate (ESR), and we used a threshold of 4.1 instead of the original 5.1 as the cut-off for high activity and 2.7 instead of 3.2 as the cut-off for low activity. Thus, we defined a value of DAS28-CRP >4.1 as high activity, 2.7–4.1 as moderate activity, <2.7 as low activity, with <2.3 being defined as remission [9]. The response to infliximab therapy at 22 weeks was evaluated by the European League Against Arthritis (EULAR) response criteria using 4.1 and 2.7 as the thresholds for the high and low disease activities, respectively [13]. Secondary insufficiency to infliximab was defined as patients with good or moderate EULAR response at week 22, and whose DAS28 increased to >0.6 or >1.2 from week 22 to 54. Discontinued subjects The continuation rate of infliximab therapy was calculated by all causes of discontinuation. Cumulative hazards and associated factors were analyzed in relation to three types of discontinuation: adverse events, inefficacy, and remission. Statistical analysis Baseline characteristics of patients are summarized in Table 1 using the mean, standard deviation, median, 25%, and 75% values for continuous variables. The continuation rate of infliximab therapy was calculated using the Kaplan–Meier estimator in pooled samples of three institutes. The discontinuation-cause specific hazards were estimated using the Nelson–Aalen estimator. Next, risk factors for the discontinuations were evaluated using the stratified proportional hazards model in order to adjust for differences among institutions. The clinical efficacies of infliximab were evaluated using a multivariate logistic regression to adjust for institutional effects. In this analysis, the last observed DAS28-CRP values were used for discontinued patients. All multivariate analyses were conducted using the variables gender, age, duration of disease, positive/negative rheumatoid factor (RF), concomitant MTX dose, concomitant prednisolone (PSL) dose, and DAS28-CRP at baseline. All reported P-values are two-sided and are not adjusted for multiple testing. The significance level was set at a P value of <0.05. Table 1Baseline characteristics of patients in three institutions of rheumatology in JapanMeanSDMin25%Median75%MaxFemale (%)87.6––––––Age53.112.71946556280Duration9.48.8036.61354Stage3.01.012344Class2.20.512224RF positive (%)87.6––––––RF (titer)213331139962412980MTX dose7.82.0068820PSL dose3.83.7004522.5CRP3.32.801.182.74.713.7TJC2810.57.30591528SJC2810.66.106101428GH63.121.9049.36680100DAS28-CRP5.51.11.94.85.66.38.0MTX methotrexate, PSL prednisolone, RF rheumatoid factor, CRP C-reactive protein, GH general health, TJC tender joint count, SJC swollen joint count, DAS disease activity score Results Baseline demographic and clinical characteristics The baseline demographic and clinical characteristics of 410 patients receiving infliximab therapy are summarized in Table 1. Patients had active disease at baseline as evidenced by high mean counts of tender and swollen joints, CRP levels, and the DAS28. Age, sex, and disease duration were similar among these three institutes, while the %user and dose of MTX or PSL were divergent and the disease activities as assessed by TJC, SJC, GH, and DAS28 was also different. Continuation of infliximab therapy Infliximab was discontinued in 100 cases (24.4%) among 410 patients during a 54-week period and the survival rate for infliximab use was comparable among three institutes by week 54 after the treatment according to Kaplan–Meier analysis (Fig. 1a). Cumulative hazards of the discontinuation during the 54-week infliximab therapy were different among the causes of discontinuation; discontinuation due to adverse events, inefficiency, remission and other causes (such as change of hospitals/clinics and economic reasons), were 0.093, 0.081, 0.007 and 0.063, respectively (Fig. 1b). Although the cause of the discontinuation was similar among three institutes, adverse events were higher in Center 1 than in the other two institutes, and remission and other causes including economic problems of the patient were greater in Center 3 than in the other two (data not shown). In 100 patients who terminated infliximab treatment, stratified Cox regression was performed to analyze factors associated with the discontinuation of the infusion. Male, older age and RF-negativity were significantly associated with the discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for the discontinuation due to maintained remission or a lack of efficacy (Table 2). Fig. 1Continuation of the infliximab therapy in RA patients for 54 weeks. a Survival rate of RA patients treated with infliximab (n = 410, total and three institutes) during the 54-week therapy. b Cumulative hazards of the discontinuation of infliximab therapy by week 54 of the treatmentTable 2Results from a Cox regression analysis performed to examine the factors related to the discontinuation of infliximab therapyVariableAE (n = 38)Inefficacy (n = 33)Remission (n = 3)Coef.HRCLCUPCoef.HRCLCUPCoef.HRCLCUPAge0.0371.0381.0061.0700.020−0.0140.9870.9571.0170.38−0.0210.9800.8881.0810.68Gender−0.9400.3910.1770.8630.0200.1601.1730.3523.9100.79–––––RA duration0.0181.0180.9831.0540.3200.0051.0050.9641.0460.83−0.1140.8930.6221.2800.54RF (±)−0.9490.3870.1710.8780.0231.5454.6860.63534.5730.13−1.1500.3170.0234.2910.39MTX dose0.0961.1010.9201.3180.290−0.1420.8670.7091.0600.160.3391.4030.7992.4650.24PSL dose−0.0660.9360.8481.0330.1900.0031.0030.9081.1090.95−0.6360.5290.1871.5000.23DAS (0 week)0.2051.2270.8901.6920.2100.1131.1200.8151.5370.49−0.4740.6230.1642.3670.49Coef. coefficient, HR hazard ratio, CL 95% confidence lower limit of HR, CU 95% confidence upper limit of HR Efficacy of infliximab therapy The average DAS28-CRP before starting infliximab was 5.5 ± 1.1, and this decreased to 3.4 ± 1.2, 3.2 ± 1.4, 3.1 ± 1.3 at weeks 2, 22 and 54, respectively, after the infliximab therapy (Fig. 2). Disease characteristics at baseline and after 2, 22 and 54 weeks of the inflilximab therapy were as follows: tender joints count (10.5, 4.2, 3.2 and 2.9), swollen joints count (10.6, 4.2, 2.7 and 2.3), GH (63, 34, 33 and 33 mm), and CRP (3.3, 1.0, 1.5 and 1.1 mg/dl) (Fig. 3). Before starting infliximab, the proportions of patients showing low, moderate, and high disease activity were 1.0, 9.0 and 90.0%, respectively. At week 22, patients in remission (defined as DAS28-CRP <2.3) and those showing low (<2.7), moderate (2.7–4.1), and high disease activity (>4.1) had changed to 27.8, 12.0, 32.4 and 27.8%, respectively, and at week 54 patients in remission and those showing low, moderate, and high disease activity were 27.6, 11.7, 34.4 and 26.3%, respectively (Fig. 4). Thus, approximately 27–28% of the patients satisfied the remission criteria at week 22 and still remained at week 54 after the infliximab therapy. Also, when the responses were evaluated by the EULAR response criteria, the proportions for good, moderate, and no response to infliximab as measured by DAS28-CRP were 37.3, 43.7 and 19.0%, respectively, at week 22 and 37.0, 41.7 and 21.2%, respectively, at week 54 (Fig. 5). Fig. 2Longitudinal analysis of DAS28 values during the 54-week study of patients using infliximab. Line in the box represents the median and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig. 3Longitudinal analysis of a SJC28, b TJC28, c CRP, and d GH values during the 54-week study of patients using infliximab. Line in the box represents the median, and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig. 4Changes in DAS28 values during the 54-week study of patients using infliximab. The ratios of patients who demonstrated high disease activity (defined as DAS28-CRP >4.1), moderate activity (2.7–4.1), low activity (<2.7) and remission (<2.3) at each observation point during the 54-week study are shownFig. 5The response to infliximab therapy during the 54-week study. The ratios of patients whose responses were evaluated by the European League Against Arthritis (EULAR) response criteria are shown Demographic factors related to the clinical efficacy of infliximab therapy In order to clarify demographic factors related to the clinical efficacy of infliximab therapy, we performed a multivariate analysis adjust for institutional differences. Younger age, RF-negativity and lower levels of DAS28-CRP were significantly associated with the clinical remission induced by infliximab therapy at week 54, whereas gender, duration of the disease, dose of MTX and dose of PSL did not show a significant association (Table 3). On the other hand, most of the demographic factors, except for older age, did not affect secondary insufficiency from week 22 to 54 after the infliximab therapy according to the logistic regression analysis (Table 4). Table 3Results from the logistic regression analysis used to examine the factors related to clinical remission at weeks 22 and 54 induced by infliximab therapyVariablesWeek 22 (n = 113)Week 54 (n = 112)Coef.ORSECLCUPCoef.ORSECLCUPIntercept1.5304.6171.0340.60835.0670.1391.7705.8681.0100.81142.4450.080Center 2 vs. Center 10.1741.1900.3590.5882.4050.6290.5641.7580.3500.8863.4880.107Center 3 vs. Center 11.0722.9220.3241.5495.5100.0010.9892.6890.3251.4235.0820.002Age−0.0140.9860.0100.9671.0060.168−0.0240.9760.0100.9570.9950.014Gender0.5631.7550.3920.8133.7870.1520.1541.1660.3700.5652.4090.677RA duration−0.0070.9930.0150.9641.0230.6400.0101.0100.0140.9821.0390.491RF (±)−0.4130.6620.3390.3401.2870.224−0.7420.4760.3290.2500.9080.024MTX dose0.0421.0430.0680.9131.1930.5350.0051.0050.0660.8841.1430.936PSL dose−0.0630.9390.0350.8761.0050.070−0.0580.9440.0340.8821.0090.092DAS28-CRP−0.4640.6290.1160.5010.7890.000−0.2760.7590.1120.6090.9450.014Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of ORTable 4Results from the logistic regression analysis used to examine the factors related to the secondary inefficiency from week 22 to 54 during infliximab therapyVariablesWeek 54–22 >0.6 (n = 85)Week 54–22 >1.2 (n = 47)Coef.ORSECLCUPCoef.ORSECLCUP(Intercept)−1.4610.2321.0790.0281.9230.176−4.3310.0131.4390.0010.2210.003Center 2 vs. Center 1−0.0080.9920.3500.4991.9700.9810.0741.0770.4400.4552.5490.866Center 3 vs. Center 10.0711.0730.3330.5592.0600.8310.0541.0560.4260.4582.4330.899Age0.0191.0190.0110.9981.0420.0790.0321.0320.0141.0041.0620.028Gender−0.2830.7530.3580.3731.5200.4290.2501.2840.5110.4713.5000.625RA duration−0.0290.9720.0170.9411.0040.083−0.0200.9800.0200.9421.0190.313RF (±)−0.2330.7920.3660.3871.6220.5240.1641.1780.5120.4323.2120.749MTX dose−0.0010.9990.0690.8731.1440.9900.0391.0400.0860.8791.2300.649PSL dose−0.0150.9850.0360.9181.0570.6820.0191.0190.0450.9341.1120.676DAS28-CRP (0 week)−0.0330.9670.1180.7671.2190.779−0.0110.9890.1480.7391.3230.941Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of OR Discussion The RECONFIRM-2 study was designed to fully evaluate the effect of infliximab used in combination with MTX on the clinical results during a 54-week study period in DMARD-resistant RA patients of a RA management group in Japan. The safety profile of infliximab therapy for a total of 5,000 cases was investigated in Japan using an all-case registered post-marketing surveillance system, and the entire profile of adverse events related to infliximab therapy was clearly identified [14]. The assessment of efficacy, however, was based only on the physician’s general evaluation and not on quantitative measures such as EULAR criteria. In this report, therefore, efficacy data based on DAS28-CRP and EULAR improvement criteria were intensively assessed. The average DAS28-CRP before starting infliximab was 5.5, it decreased to 3.2 at week 22 and it remained steady at 3.1 by week 54 after the infliximab therapy. At week 22 after the therapy, about 28% and 40% of patients satisfied the remission and low disease activity criteria, respectively, and these effects remained (28 and 39%, respectively) by week 54. Also, about 37% of the patients exhibited good response according to EULAR criteria continuously from week 22 to 54. These results reconfirmed that the clinical efficacy of infliximab at week 22 was maintained until week 54 in most of the patients treated with infliximab. Thus, appropriate treatment with infliximab and MTX in this study group could minimize secondary insufficiency of the therapy for at least one year. On the other hand, the discontinuation of infliximab was observed in approximately 24% of the patients during the 54-week period. This rate of discontinuation of the therapy was comparable to that obtained in other countries: 26.7% in the Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy (ATTRACT) international study carried out in the USA and the EU, 34.6% in a German domestic study and 26% in a French group study during the first one-year period [7, 15, 16]. Cumulative hazards differed among causes of discontinuation; discontinuations due to adverse events, inefficiency, remission and other causes (including changes of hospital/clinic and economic problems) were 0.093, 0.081, 0.007 and 0.063, respectively. Male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, including infusion reactions (N = 7), toxicoderma (4), bacterial pneumonia (3), pneumocystis jirovecii pneumonia (3), but more than two-thirds of the withdrawals due to adverse events were observed within the first 14 weeks after the therapy. In this study, to clarify how predisposing factors from the demographic characteristics of RA patients were related to the clinical efficacy of infliximab therapy, a multivariate analysis using a logistic regression was performed. In this study, multiple variables including sex, age, duration of disease, stage, class, positive/negative RF, concomitant MTX dose, concomitant PSL dose, and initial levels of CRP, TJC, SJC and GH were assessed. It is worth noting that, among multiple variables, younger age, RF-negativity and lower levels of DAS28-CRP at the baseline were significantly correlated with the clinical remission induced by infliximab therapy at week 54. These results imply that the timely use of MTX and infliximab can be strongly recommended for younger RA patients that show RF-negativity in order to efficiently achieve clinical remission. On the other hand, male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for discontinuation as a result of inefficacy, and only older age affected secondary insufficiency from week 22 to 54 after the infliximab therapy. Although it is intriguing that male gender predisposes for discontinuation as a result of adverse events, these results provide the first information that can be used to facilitate the more efficacious use of infliximab and MTX in the daily practice of rheumatologists. Taken together, this REOCNFIRM-2 study reconfirms the clinical efficacy of treatment with infliximab and MTX in Japanese RA patients using the DAS28-CRP and EULAR response criteria. Among 410 patients with active RA, approximately 28% and 39% of the patients satisfied the remission and low disease activity criteria, and good response according to the EULAR criteria was achieved in 37% of the patients treated with infliximab plus MTX during the 54-week study period. The clinical efficacy of infliximab was maintained from week 22 to 54 after the treatment, and secondary insufficiency after week 22 was marginal after the appropriate treatment in this study group. Several demographic factors, including male gender, RF-negativity and lower scores of DAS28-CRP, were significant predisposing factors for remission. The promising effectiveness of infliximab at improving measures of disease activity in RA patients has led to this therapy becoming one of the key advances in the management of RA. Thus, this study is important because it provides obvious and invaluable evidence concerning the efficacy of the combinational use of infliximab and MTX, and can guide the real clinical use of infliximab in the future.

Eur_J_Epidemiol-3-1-2039783

From evidence based bioethics to evidence based social policies

In this issue, Norwegian authors demonstrate that causes of early expulsion out the workforce are rooted in childhood. They reconstruct individual biographies in administrative databases linked by an unique national identification number, looking forward 15 years in early adulthood and looking back 20 years till birth with close to negligible loss to follow up. Evidence based bioethics suggest that it is better to live in a country that allows reconstructing biographies in administrative databases then in countries that forbid access by restrictive legislation based on privacy considerations. The benefits of gained knowledge from existing and accessible information are tangible, particularly for the weak and the poor, while the harms of theoretical privacy invasion have not yet materialised. The study shows once again that disadvantage runs in families. Low parental education, parental disability and unstable marital unions predict early disability pensions and premature expulsion out gainful employment. The effect of low parental education is mediated by low education of the index person. However, in a feast of descriptive studies of socio-economic causes of ill health we still face a famine of evaluative intervention studies. An evidence based social policy should be based on effective interventions that are able to break the vicious circles of disability handed down from generation to generation. Natural selection is for biology what the human life course is for epidemiology: an overarching framework needed to understand the occurrence of disease in the biography of the individual person [1]. The life course of human beings as a history of health and disease starts long before conception, in the genes and life course of their parents. The odds at facing a successful life are entirely different if conception started with the rape of a young teenager by an HIV positive warrior in a horrifying African civil war or as the consequence of the deep desire to raise a child among a healthy and wealthy European loving couple. To expect a life in good health, healthy parents have to provide their offspring with good genes, food, shelter, love, an upbringing and an education. However, a general problem of human life course epidemiology is that the human life course is a lot longer than the scientific career of epidemiologists. While there is a scope for ambitious programmes of prospective research spanning multiple generations, such programmes will nevertheless run into questions not addressed by the original design. In this issue, the Norwegian study of Gravseth et al. show how existing data from administrative databases can be put to good use to understand disabling processes originating in youth [2]. Evidence based bioethics As in other Scandinavian countries, several databases (here the medical birth registry, benefit and income registries in the National Insurance Administration, the education register of Statistics Norway, and the Central Population Register) can be linked by a unique national identification numbers of the child and the parents. In many European countries, such linkage is considered a breach of personal autonomy through invasion of privacy. Bioethical theory is very concerned with the potential of abuse of knowledge, but seems to neglect the potential of harm by lack of available but inaccessible knowledge [3]. Gravseth et al. shows how linked administrative databases can yield important insights in the human life course, supporting policy decisions protecting the poor and the weak. Bioethical theory mistrusts human nature, suggesting that the risk of abuse of private information from linked administrative databases is high. This has never been confirmed by empirical evidence. Bioethics should consider the possibility that untested theories supported by unproved assumptions may look good in theory, but in practice waste healthy life. Medicine knows a large array of such theories. Although critical of parenting experts, we too put our first babies to sleep on their front, likely following advice that started by the celebrated parenting expert, Dr Spock. In 1970, a statistically significantly increased risk of cot death for front sleeping compared with back sleeping (pooled odds ratio 2.93; 95% confidence interval 1.15, 7.47) could already be identified from the then available studies [4]. Preferring the cool theory of babies developing their brain and body better by looking up to spot their mother over the neglected harsh evidence has caused the death of 50,000 babies worldwide, this after 1970 only [4]. Where empirical investigation is possible, bioethical theory needs corroboration by observable evidence [5]. If evidence of harms and benefits is available, as with the long-standing practice of data linkage in the Scandinavian countries, observable evidence of benefits should have precedence over theoretical considerations based on unproved assumptions. The use of administrative databases The use of administrative databases can be very tantalising: they carry a lot of information, but what one wants is not available or not reliable [6]. However, as in all study designs, its’ use depends on one’s aims. Flagging persons in a population register and being informed by a mortality register of their death will obtain survival figures at low costs. Death is one of those few health states even epidemiologists do not argue about. Follow up is always close to 100%. An important principle of administrative databases is that they don’t follow patients but money. As soon as money is at stake, administrative registers become highly reliable to count processes amenable to financing, and loss to follow-up is close to negligible. Eligibility for benefits may change over periods, but the fact of being reimbursed is coded extremely well. One of the strong characteristics of administrative databases is their completeness, even over very long periods. In the article of Gravseth et al., early disability pension registered in 1988–2003 is a highly reliable proxy of definitive expulsion from paid work. Looking back to the medical birth registry in 1967–1976 and a limited number of parental characteristics between ages 0 and 18 of the child, the authors have explored the consequences of a limited numbers of determinants in youth for later expulsion out of work. Shaping the life course Losing the capability to do paid work between the ages 20 and 35 is a highly relevant health outcome, as defined by Sen’s capability approach [7]. Being unable to work at young adult age is a heavy burden of disability. In terms of attributable risks, education is by far the most important determinant, explaining 57% of the risk of early disability. Here, the administrative data show their inherent weakness: they contain little information of the process leading to low levels of education, be it personal factors related to the health of the child or social factors related to its’ family and living situation. However, the stratified analysis suggests that low education of the child finds it roots in low education of parents. It is worrying that even in a country as Norway, with one of the highest human development indices in the world, disadvantage as expressed by low education of parents and parental disability is still handed down from generation to generation. Last but not least, the relative importance of maternal marital status shows the importance of stable marital unions to raise healthy children. With low European fertility and postponement of first pregnancy after the age of 30, policy makers are tempted to try to push women into earlier childbirth. This policy may be ill advised, if it leads to more children of less well educated younger mothers in less stable unions. Moving to evidence based policy Gravseth et al. show, by using linked administrative databases, that causes of early disability in young adulthood are rooted in childhood and in parental disadvantage. A consequence is that interventions targeting the adults at risk will likely add more to the problems than to the solutions. The horns of the dilemma to give or to withhold a disability pension are terribly sharp. An early disability pension is a social death sentence, crippling the individual by removing incentives to seek work and declaring him or her definitively incurable. But those who are effectively crippled won’t be helped by the misery of repeated frustrations in a labour market in which they cannot compete. Studies carefully designed to identify determinants of irreversible disability are needed, inclusive randomised controlled experiments evaluating pension decisions as important health care interventions. I opened this editorial by making the strong comparison between the little African girl, pregnant after being raped and the highly educated European women in her 30s. However, even in Norway, disadvantage is still a disease that runs in families. We have now a descriptive feast of the lifelong health consequences of social disadvantage and low education. However, the evidence supporting effective interventions is remaining an evaluative famine [8]. Successful interventions need to target the vicious circles of disadvantage that are handed down from generation to generation. We need theories and models that identify potentially effective interventions and study designs able to collect the evidence that supports the effectiveness. The transgenerational aspects of life course epidemiology may be a hard nut to crack in evaluative research. But as good health starts with a good education, good education will always be a worthy proxy.

Diabetologia-3-1-1914282

The effect of moderate alcohol consumption on adiponectin oligomers and muscle oxidative capacity: a human intervention study

Aims/hypothesis The aim of this study was to investigate whether moderate alcohol consumption increases plasma high molecular weight (HMW) adiponectin and/or muscle oxidative capacity. Introduction Moderate alcohol consumption is associated with a decreased risk of type 2 diabetes compared with abstention [1], which could be explained by improved insulin sensitivity [2]. The underlying mechanism for this is not clear, but several pathways may be involved. First, moderate alcohol consumption increases plasma adiponectin concentrations, which could precede changes in insulin sensitivity [2]. Adiponectin improves insulin sensitivity by increasing muscle fat oxidation and/or decreasing intramyocellular triacylglycerols (IMTGs) [3]. Adiponectin is present in plasma as a trimer, hexamer or high molecular weight (HMW) form, the latter possibly being the most relevant in the aetiology of insulin resistance [4]. Second, moderate alcohol consumption acutely affects energy expenditure, diet-induced thermogenesis, lipolysis and lipid oxidation [5]. These changes may result from acetate production from ethanol, which is converted to acetyl-CoA mainly in peripheral tissue such as muscle [5]. Such acute changes could cumulatively affect oxidative capacity and insulin sensitivity. This is the first human study to investigate whether chronic moderate alcohol consumption affects plasma adiponectin oligomers and muscle oxidative capacity. Materials and methods In a randomised, controlled, crossover trial, conducted at TNO Quality of Life, 19 healthy, moderate-drinking, lean (BMI 18–25 kg/m2, n = 11) and overweight (BMI ≥27 kg/m2, n = 8) men (aged 18–40 years), consumed 100 ml whisky (32 g alcohol per day; Famous Grouse Scotch Whisky, 40% vol, Perth, Scotland) or mineral water (Spa Reine, Spa, Belgium) daily for 4 weeks. For the last 7 days of each treatment period the diet was fully controlled, and treatments were separated by a 2 day wash-out period. This sample size was sufficient to detect ∼15% difference of β-3-hydroxyacyl-CoA dehydrogenase (β-HAD) and citrate synthase activity in this crossover trial and correlation coefficients of 0.55 or higher with 80% power and accepting an alpha of 0.05. At the end of each treatment period, muscle biopsies from the vastus lateralis muscle were taken to assess β-HAD, cytochrome c oxidase and citrate synthase activity [6], IMTG [7] and succinate dehydrogenase activity [8]. Fasting blood samples were collected and an OGTT was performed to calculate whole-body insulin sensitivity. Subjects gave written informed consent and the University Medical Center Utrecht Medical Ethics Committee approved the protocol. The study was conducted according to the Declaration of Helsinki (2000) and the International Conference on Harmonisation Guidelines for Good Clinical Practice. Data were analysed using the SAS statistical software package (SAS/STAT version 8, SAS Institute, Cary, NC, USA). Treatment effects were assessed by ANOVA, using general linear modelling, with BMI, period, treatment and treatment order included in the model. Two-sided p values below 0.05 were considered statistically significant. Results Compliance to treatments was good as assessed by measurements of urinary ethyl glucuronide every 5 days, showing one negative sample during the whisky-drinking and no positive samples during the water-drinking period. Another indication was the 11% increase (p < 0.001) of serum HDL-cholesterol after whisky compared with water consumption. Table 1 shows results on insulin sensitivity, adiponectin and muscle enzyme activities. Insulin sensitivity was not changed after moderate alcohol consumption, but plasma adiponectin concentrations increased from 7.9 ± 0.2 mg/l after water to 9.0 ± 0.2 mg/l (means±SEM) after whisky consumption. Moderate alcohol consumption tended to increase HMW and medium molecular weight (MMW) adiponectin, but not low molecular weight (LMW) adiponectin. Differences in insulin sensitivity and adiponectin and its oligomers tended to be more pronounced among lean than overweight men (Table 2). Muscle oxidative capacity measured by β-HAD, cytochrome c oxidase and citrate synthase activity did not differ between treatments. A borderline significant interaction between treatment and BMI was observed for cytochrome c oxidase (p = 0.072) and citrate synthase (p = 0.102) activity. Moderate alcohol consumption tended to increase cytochrome c oxidase (+23.7%; 95% CI −3.9 to +51.2%, p = 0.08) and citrate synthase (+24.7%; 95% CI −7.7 to +57.2%, p = 0.11) activity among lean, but not overweight men (Table 2). Succinate dehydrogenase activity in mixed muscle fibres decreased (p = 0.03) by 15% after moderate alcohol consumption. IMTGs did not differ, despite a 15–20% difference between both treatments. These results were not different for lean and overweight men (Table 2). After whisky consumption, HMW correlated positively with activities of citrate synthase (r = 0.64, p = 0.004), cytochrome c oxidase (r = 0.59, p = 0.009) and β-HAD (r = 0.46, p = 0.056). Activities of citrate synthase (r = 0.44, p = 0.07), cytochrome c oxidase (r = 0.32, p = 0.20) and β-HAD (r = 0.44, p = 0.07) also tended to correlate with MMW adiponectin, but not LMW adiponectin. A similar pattern was observed for correlations of adiponectin oligomers with HDL-cholesterol (HMW: r = 0.55, p = 0.014; MMW: r = 0.55, p = 0.014; LMW: r = 0.21, p = 0.38). Table 1Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities after 4 weeks of consumption of whisky or mineral water in 19 lean or overweight men Mineral waterWhisky% Changep valueInsulin sensitivity indexa10.6 ± 1.39.6 ± 1.3−9.40.61Adiponectin (mg/l)7.9 ± 0.29.0 ± 0.212.50.0008 HMW0.7 ± 0.11.1 ± 0.157.1 0.074 MMW4.0 ± 0.24.5 ± 0.212.50.065 LMW3.2 ± 0.33.5 ± 0.39.40.442HbA1c (%)4.9 ± 0.024.8 ± 0.02−2.00.023Muscle β-HAD (mol/μg protein)4.22 ± 0.484.37 ± 0.493.60.827Muscle citrate synthase (mol/μg protein)0.66 ± 0.050.75 ± 0.0513.60.262Muscle cytochrome c oxidase (mol/μg protein)1.48 ± 0.121.67 ± 0.1212.80.262Intramyocellular triacylglycerols (AU) Type 10.027 ± 0.0040.022 ± 0.004−18.50.339 Type 20.010 ± 0.0020.008 ± 0.002−20.00.489 Mixed0.019 ± 0.0030.016 ± 0.002−15.8 0.429Succinate dehydrogenase activity (AU) Type 10.084 ± 0.0050.075 ± 0.004−10.70.122 Type 20.057 ± 0.0030.052 ± 0.003−8.80.221 Mixed0.073 ± 0.0030.062 ± 0.003−15.10.028Data are presented as means±SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15]Table 2Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities (means±SEM) after 4 weeks of consumption of whisky or water in lean and overweight subjects Lean groupOverweight groupWaterWhisky% Changep valueWaterWhisky% Changep valueInsulin sensitivity indexa13.3 ± 1.913.0 ± 1.9−2.30.937.8 ± 1.64.6 ± 1.6−41.0 0.21Adiponectin (mg/l)8.1 ± 0.39.4 ± 0.316.10.017.7 ± 0.28.5 ± 0.210.40.08 HMW0.8 ± 1.21.2 ± 0.250.00.120.8 ± 0.10.8 ± 0.100.62 MMW4.2 ± 0.34.8 ± 0.314.30.133.9 ± 0.24.1 ± 0.25.10.52 LMW3.2 ± 0.43.4 ± 0.46.30.693.1 ± 0.43.5 ± 0.412.90.50HbA1c (%)4.9 ± 0.024.8 ± 0.02−2.00.044.9 ± 0.024.9 ± 0.0200.53Muscle β-HAD (mol/μg protein)4.67 ± 0.505.10 ± 0.459.20.553.46 ± 0.903.69 ± 1.016.70.87Muscle citrate synthase (mol/μg protein)0.73 ± 0.080.92 ± 0.0726.00.110.60 ± 0.050.54 ± 0.06−10.00.51Muscle cytochrome c oxidase (mol/μg protein)1.72 ± 0.152.12 ± 0.1423.30.081.33 ± 0.131.13 ± 0.15−15.00.36Intramyocellular triacylglycerols (AU) Type 10.022 ± 0.0060.016 ± 0.005−27.30.500.032 ± 0.0060.028 ± 0.006−12.50.61 Type 20.006 ± 0.0020.008 ± 0.00233.30.410.013 ± 0.0030.007 ± 0.003−46.20.14 Mixed0.014 ± 0.0040.013 ± 0.003−7.10.860.024 ± 0.0040.019 ± 0.004−20.80.40Succinate dehydrogenase activity (AU) Type 10.086 ± 0.0050.080 ± 0.005−7.00.400.084 ± 0.0090.069 ± 0.008−17.90.29 Type 20.060 ± 0.0050.055 ± 0.005−8.30.500.054 ± 0.0050.048 ± 0.004−11.10.34 Mixed0.076 ± 0.0050.066 ± 0.005−13.20.160.069 ± 0.0050.057 ± 0.004−17.40.15Data are presented as means±SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15] Discussion This study showed that moderate alcohol consumption increases adiponectin concentrations, consistent with previous reports [2]. We have now observed that the alcohol-induced increase of adiponectin may be oligomer specific. Moderate alcohol consumption increased particularly HMW adiponectin, MMW adiponectin to a lesser extent, but not LMW adiponectin. These results are in line with those of Bobbert et al. [9] showing a similar pattern of changes in adiponectin oligomers after moderate weight reduction. Studies with thiazolidinedione or rigorous weight reduction show similar, but more pronounced results [10]. HDL-cholesterol concentrations correlated particularly with HMW adiponectin, as previously shown [9]. We have now also observed that HMW and MMW adiponectin are correlated with markers of muscle oxidative capacity, in line with reports of increased fat oxidation after adiponectin infusion [3] and a recent study showing that adiponectin increases muscle oxidative capacity [11]. Our results tend to confirm these findings and show that these relationships may be specific to HMW adiponectin. Altogether, this could provide an underlying mechanism for the proposed importance of HMW adiponectin in the aetiology of insulin resistance [4]. Despite this, the alcohol-induced increase of adiponectin and specifically HMW adiponectin did not affect muscle oxidative capacity, IMTG content and insulin sensitivity. As some subtle differences were present, we cannot completely rule out the hypothesis that changes in IMTG content and/or oxidative capacity could occur. Although it did not reach significance, we observed a 15–20% lower IMTG content after whisky than water consumption, which is of similar magnitude to that observed for a weight loss and physical activity intervention [12]. Furthermore, moderate alcohol consumption tended to increase muscle citrate synthase activity among lean men, but succinate dehydrogenase activity declined after moderate alcohol consumption in mixed muscle fibres. All citric acid cycle and respiratory chain enzymes are thought to change in parallel to perturbation [13]. Our findings with citrate synthase and succinate dehydrogenase, however, are contradictory to this notion. This could simply be due to the measurement of ex vivo oxidative capacity or to chance. Alternatively, moderate alcohol consumption could differentially affect various enzymes in oxidative pathways such as the citric acid or glyoxylate cycle as suggested by Kokavec and Crowe [14]. Because acetyl-CoA from ethanol oxidation is generated independently from activation of pyruvate dehydrogenase complex, it may not affect the citric acid cycle. Alcohol may instead affect the glyoxylate cycle that bypasses part of the citric acid cycle, including succinate dehydrogenase [13, 14]. Strengths of this study are its randomised, controlled crossover design. We assessed compliance to study treatments several times throughout the study and observed no significant deviations. It is therefore unlikely that our results are confounded by diet or lifestyle. Our study was, however, limited by a slightly small sample size for certain contrasts such as analyses in subgroups of lean and overweight men or of IMTG content. Therefore these results may be somewhat preliminary and need to be confirmed with larger sample sizes. Second, because insulin sensitivity was not the primary endpoint of this study, an OGTT was used to assess insulin sensitivity. However, we used an insulin sensitivity index that correlates well with the gold standard, the hyperinsulinaemic–euglycaemic clamp technique [15]. In addition, our results on insulin sensitivity do not differ from our previous studies using the clamp technique [2]. We therefore believe that using this insulin sensitivity index has not influenced our results to a large extent. In conclusion, this study shows that 4 weeks of daily moderate alcohol consumption increases adiponectin concentrations and particularly HMW adiponectin concentrations, but does not affect insulin sensitivity and oxidative capacity. Concentrations of HMW and MMW adiponectin were positively associated with muscle oxidative capacity.

Appl_Microbiol_Biotechnol-4-1-2243256

Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (KM = 8.2 μM, kcat = 26 s−1). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh. Introduction Flavoprotein oxidases catalyze the oxidation of a wide range of compounds, while at the same time they reduce oxygen to hydrogen peroxide. They are valuable biocatalysts for the oxidative activation of biomolecules, as they usually selectively oxidize their substrate at a specific position, leaving other positions unaffected. Due to their ability to use molecular oxygen as electron acceptor, no expensive coenzymes like NAD(P)H are needed. This makes oxidases inexpensive and rather straightforward in usage compared to other redox enzymes. By far, the best studied oxidase is glucose oxidase from Aspergillus niger. This enzyme has been applied for decades, mostly for diagnostic applications (Wilson and Turner 1992). Another example is pyranose oxidase (glucose-2-oxidase) from Peniophora gigantea, which oxidizes specifically the C2 position of glucose and has been applied for the chemoenzymatic synthesis of rare sugars and sugar-based synthons (Giffhorn et al. 2000). Human monoamine oxidases (MAO-A and MAO-B) are important for the oxidation of neurotransmitters (Abell and Kwan 2001). An engineered mutant of the homologous A. niger enzyme (MAO-N) has been applied in an elegant biocatalytic deracemization process to obtain enantiomerically pure chiral amines (Carr et al. 2005; Dunsmore et al. 2006). So far, the most described oxidases are from eukaryotic origin, and heterologous expression of the recombinant protein in Escherichia coli for large scale enzyme production can be problematic. In a search for novel bacterial oxidases, we looked in sequenced genomes and identified genes encoding novel oxidases. By this, we discovered oxidases primarily acting on alditols (Heuts et al. 2007) and phenolic compounds (Jin et al. 2007). These oxidases, both from actinomycetes, could be highly overexpressed in E. coli. Another way of searching for novel bacterial oxidases is to screen unsequenced bacterial genome libraries directly for genes encoding enzyme activity. As actinomycetes appear to be rich in oxidases, we have chosen to explore these organisms for relevant oxidases. Using a plate-based screening protocol for oxidase activity (Alexeeva et al. 2002), we screened a genomic DNA library of the actinomycete Rhodococcus erythropolis NCIMB 11540 to find novel oxidases acting on polyols and/or amines. In this paper, we describe the discovery and characterization of a novel flavin-containing oxidase primarily acting on putrescine. This putrescine oxidase (PuORh) shares 67% sequence identity with the enzyme from Micrococcus rubens (PuOMr; Ishizuka et al. 1993). We show that PuORh is highly overexpressed in E. coli and can be easily purified. Using a structural model, we were able to identify a glutamate residue (Glu324) that is crucial for substrate binding, and we could alter the substrate specificity by protein engineering. Materials and methods Chemicals Restriction enzymes were from Roche and New England Biolabs. One-shot electrocompetent E. coli TOP10 cells and the TOPO TA Cloning Kit were purchased from Invitrogen. Plasmid isolation was performed using the Qiagen DNA purification kit. Oligonucleotides were purchased from Sigma. Nitrocellulose filters (Protran BA85 132 mm, 0.45 μm pore size) were from Schleicher and Schuell BioScience, Dassel (Germany). Deprenyl and rasagiline were a kind gift from Prof. A. Mattevi (University of Pavia, Italy). All other chemicals were of analytical grade. The genomic DNA library from R. erythropolis NCIMB 11540 was provided by DSM (Geleen, The Netherlands). Constructs were sequenced at GATC Biotech (Kostanz, Germany). Plate-based screening method for oxidase activity A gene library of R. erythropolis NCIMB 11540 in pZErO-2 was screened for oxidases using the plate-based oxidase activity screening method adapted from the group of Turner (Alexeeva et al. 2002). This gene library was constructed by partial digestion of genomic DNA from R. erythropolis NCIMB 11540 by Sau3A1. Fragments of 4–10 kb were isolated, ligated in pZErO-2 (cut with BamHI), and followed by transformation of E. coli DH10B. Colonies were collected from plate, stored as glycerol stock, and plasmids were isolated. The total amount of plasmids contained a mean insert size of 6.0 kb and 1% of self-ligated vector molecules. Electrocompetent E. coli TOP10 cells were transformed with the gene library, and the transformed cells were diluted in Luria–Bertani (LB) medium to obtain single colonies on plate. The diluted cell suspensions were plated on nitrocellulose filters. The filters were placed on top of LB agar containing 0.05 mg/ml kanamycin and incubated for 48 h at 30°C. Subsequently, the nitrocellulose filters were transferred to empty petri dishes and stored at −20°C to partially lyse the cells. Each filter was submersed with 50 ml 50 mM sodium phosphate buffer pH 7.5 containing 1% (w/v) agarose, 2 U/ml Horseradish peroxidase (HRP) and a mixture of 1 mM 4-chloro-1-naphthol, 100 μM cholesterol, 10 mM xylitol, 10 mM sarcosine, 10 mM l-alanine, 10 mM d-glucose, 10 mM d-galactose, 10 mM lactose, and 10 mM d-glucosamine. Plates were incubated at room temperature and regularly checked by visual inspection for color formation in and around colonies. Positives were picked and cultivated overnight in 5 ml liquid LB medium and subjected to another screening cycle to assure that color formation was caused by single clones. Sequence analysis To identify unique positive clones, plasmids were isolated and subjected to restriction analysis by EcoRI. Inserts were sequenced and open reading frames (ORFs) were identified using the ORF Finder tool of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). A BLAST search (blastp) was performed with the protein sequence of PuORh using the BLAST function of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). Cloning and expression of the gene encoding PuORh To overexpress the novel putrescine oxidase, the corresponding gene (puoRh) was amplified from the fragment of genomic DNA of R. erythropolis using the following primers: puoRh_fw: 5′-GCTCCATATGCCTACTCTCCAGAGAGATG (NdeI site shown in italics) and puoRh_rv: 5′-GCTCAAGCTTTCAGGCCTTGCTGCGGGCG (HindIII site shown in italics). The amplified gene was purified from gel and ligated between the NdeI and HindIII restriction sites of the pBAD/Myc-His vector (Invitrogen). The plasmid carrying the puoRh gene (pBADpuoRh) was transformed to CaCl2-competent E. coli TOP10 cells and spread on LB agar plates containing 50 μg/ml of ampicillin. E. coli TOP10 cells containing pBADpuoRh were tested for overexpression of the protein at 17, 30, and 37°C and at arabinose concentrations of 0, 0.00002, 0.0002, 0.002, 0.02, and 0.2% (w/v). Cell extracts and cell-free extracts of E. coli TOP10 containing pBADpuoRh were analyzed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel to find the best conditions for overexpression of soluble PuORh. Enzyme purification To obtain purified protein, E. coli TOP10 containing pBADpuoRh was cultivated for 24 h at 30°C in 1 l of terrific broth medium containing 50 μg/ml ampicillin and 0.02% (w/v) arabinose. Cells were harvested by centrifugation for 15 min at 6,000 rpm at 4°C. The supernatant was discarded, and the pellet was suspended in 40 ml of 50 mM Tris–HCl pH 7.5 and sonicated for 10 min to break the cells. The cell extract was centrifuged for 30 min at 15,000 rpm at 4°C to remove the broken cells and obtain cell-free extract. From this cell-free extract PuORh was purified using a Q-Sepharose anion exchange column. Unbound protein fractions were washed from the column with 50 mM Tris–HCl buffer pH 7.5. PuORh was eluted from the column with a 50 mM Tris–HCl buffer pH 7.5 by increasing the KCl concentration. Eluted fractions containing PuORh were combined, concentrated using an Amicon filter, and desalted using a HiPrep 26/10 Desalting Column (Amersham Biosciences). Enzyme activity assay and determination of steady-state kinetic parameters PuORh activity was measured at 25°C using a peroxidase-coupled assay containing 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as the chromogenic substrate (Childs and Bardsley 1975). The H2O2 formed by PuORh can be coupled to the Horseradish-peroxidase-mediated oxidation of ABTS resulting in the formation of a green product that can be measured at 420 nm (ɛ420 = 35.7 mM−1 cm−1). The standard assay mixture with ABTS (HRP-ABTS assay) contained 50 μM putrescine, 50 mM Tris–HCl buffer, pH 8.0, 100 μM ABTS, 5 U of Horseradish peroxidase, 10 μl of enzyme dilution (appropriately diluted), and a fixed amount of substrate. The reaction was started with adding the enzyme solution. Alternatively, 0.1 mM 4-aminoantipyrine (AAP) and 1.0 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) were used as chromogenic substrates for HRP (Federico et al. 1997; HRP-AAP/DCHBS assay). The oxidation of these substrates results in the formation of a purple product, which can be measured at 515 nm (ɛ515 = 26 mM−1 cm−1). For the determination of kinetic parameters, PuORh activity was measured at different substrate concentrations at 25°C using the HRP-ABTS enzyme assay. The kinetic parameters of the mutant enzymes (PuORh Glu324Ala and PuORh Glu324Leu) for putrescine were determined by measuring directly the H2O2 production at 240 nm (ɛ420 = 43.6 M−1 cm−1). Data were fitted with Origin 7.0 using the Michaelis–Menten equation for enzyme kinetics. Influence of temperature and pH on enzyme activity and stability To determine the optimal temperature for PuORh activity, the oxidase activity was measured at temperatures between 20 and 60°C. Before adding the enzyme, the assay mixture was equilibrated for 20 min to assure the right temperature. The influence of temperature on the enzyme stability was determined by incubating 1-ml portions of the enzyme at 4, 30, 37, and 50°C. Twenty-microliter samples were taken, placed on ice after which activity was measured. In both experiments, the activity was determined with the HRP-AAP/DCHBS activity assay using 50 μM putrescine and 0.01 μM PuORh. The pH optimum for putrescine oxidase was determined by measuring the activity at different pH values at 25°C. The following buffers were used: 50 mM Pipes buffer (pH 6.4–7.2), 50 mM Tris–HCl (pH 7.4–8.8), and 50 mM Ches buffer (pH 8.7–9.6). The enzyme activity was measured using the HRP-AAP/DCHBS assay. Inhibition experiments Rasagiline, deprenyl, and cyclopropylamine were tested to probe whether they were able to inhibit PuORh by the formation of a covalent adduct with the flavin adenine dinucleotide (FAD) cofactor, as is the case for monoamine oxidase B (Binda et al. 2005). For 900 μl of a 20 μM PuORh solution, an absorbance scan was recorded from 650–300 nm. After the addition of 100 μl of 1 mM inhibitor (final inhibitor concentration, 100 μM), absorbance spectra were recorded regularly in time to see if addition of the inhibitor would cause an alteration of the typical FAD spectrum of PuORh. For cyclopropylamine, also a final inhibitor concentration of 1 mM was tested as described above. Butylamine, aminoethanol, ethylenediamine, 1,3-diaminopropane, and (2-aminoethyl)-trimethylammonium were tested as competitive inhibitors of PuORh. For this, the Michaelis constant (KM) of PuORh for putrescine was determined for three different concentrations for each of these amino compounds with the HRP-ABTS assay. Inhibition constants (KI) for each compound were calculated with the following formula: Where [I] represents the inhibitor concentration, KM,inhibitor the apparent Michaelis constant in presence of the inhibitor and the KI the inhibition constant. Analytical methods All absorbance spectra were recorded in 50 mM Tris–HCl pH 8.0 at 25°C on a Perkin Elmer Lambda Bio40 spectrophotometer. From a cuvet containing 5 μM PuORh all oxygen was removed by flushing with argon and an absorbance spectrum was recorded from 650 to 300 nm. After adding 50 μM putrescine, another spectrum was recorded for the reduced enzyme. Reoxidation was monitored by collecting spectra in time after the cuvet was exposed to air. A spectrum of the unfolded enzyme was recorded by adding 0.1% SDS and heating for 5 min at 80°C. Modeling and mutant construction Based on the structure of MAO-B in complex with rasagiline (PDB/1S2Q; Binda et al. 2004b), a model of PuORh was made using the CHPmodels 2.0 Server. Mutants were constructed by Quick Change PCR. PuORh Glu324Ala was made by a substitution of the codon GAG for GCG using the following primers: puoRhGlu324Ala_fw, 5′-CGAGGTAGTGCAGGCGGTGTACGACAACACC, and puoRhGlu324Ala_rv, 5′-GGTGTT GTCGTACACCGCCTGCACTACCTCG (mutated codon underlined). For construction of PuORh Glu324, the codon GAG was replaced by CTG using the following primers: puoRhGlu324Leu_fw, 5′-CGAGGTAGTGCAGCTGGTGTACGACAACACC, and puoRhGlu324Leu_rv, 5′- GGTGTTGTCGTACACCAGCTGCACTACCTCG (mutated codon underlined). Both mutants were expressed in E. coli TOP 10 and cultivated and purified in the same way as wild-type PuORh. Nucleotide sequence accession number The nucleotide sequence of the PuORh encoding gene (puoRh) has been submitted to GenBank under accession number EU240877. Results Identification of a novel putrescine oxidase To identify novel oxidases acting on alcohols and/or amines, a genomic library from the actinomycete R. erythropolis NCIMB 11540 was screened using a plate-based oxidase assay. Positive clones, expressing oxidase activity, could be identified by their purple color formation. From 55,000 transformants, 5 were observed to produce a purple color around the colony. Retransformation to E. coli TOP10 cells and repeated screening on plate resulted in three positive clones, which showed oxidase activity. Restriction analysis with EcoRI and PvuI showed a different insert for each clone indicating that these were independent clones. Sequencing of one of these clones revealed an inserted fragment of genomic DNA of 5,311 bp. On this fragment, an ORF was identified which shares 67% sequence identity (at the amino acid level) with putrescine oxidase (EC 1.4.3.10) from M. rubens, a FAD-containing amine oxidase that catalyzes the oxidative deamination of putrescine (Ishizuka et al. 1993). When we tested the cell extract of the clone containing the above-mentioned insert of 5,311 bp, we found that it was indeed active with putrescine. The other two positive clones probably contained the same gene, as for both clones by using the primers puoRh_fw and puoRh_rv a PCR product was seen on gel. Gene cloning, overexpression, and protein purification For overexpression, the PuORh encoding gene was cloned into the pBAD/Myc-His A vector behind the araBAD promotor (without His-tag), and the plasmid carrying the puo gene was transformed to E. coli TOP10. Several temperatures (17, 30, and 37°C) and inducing conditions (0–0.2% L-(+)-arabinose) were tested to find the optimal conditions for overexpression. The gene was well expressed at arabinose concentrations of 0.02 and 0.2% as judged by a dominant protein band of ±54 kDa on SDS-PAGE. At 17 and 30°C, the protein was mainly present in the soluble fractions while at 37°C the unsolube fractions contained most of the overexpressed protein. The overexpressed protein did not show any fluorescence under UV-light upon SDS-PAGE, which is an indication that this protein does not contain a covalently histidyl-bound flavin cofactor (Fraaije et al. 1997). PuORh was purified from a 1-l culture containing 0.02% arabinose that was grown for 24 h at 30°C. Due to the high level of overexpression (200 mg), PuORh could be easily purified in one-step by anion exchange chromotography. SDS-PAGE analysis of the purified protein revealed that, besides the major protein band present at around ∼54 kDa, there is a minor band corresponding to a protein mass which is approximately 3 kDa larger (Fig. 1, lane A). It was concluded that this must be the product of the same gene with a His-tag extension, as this minor protein band could be removed by an additional purification step using a Ni-agarose column. (Fig. 1, lane B). Although in the plasmid pBADpuoRh the stop codon (TGA) was properly introduced, it is known that in some cases, this stop codon can be translated (MacBeath and Kast 1998). With the constructed plasmid, this would indeed result in a 3 kDa larger protein. Fig. 1SDS-PAGE gel of purified recombinant PuORh. Lane A Purified PuORh. Lane B Purified PuORh after removal of His-tagged PuORh. Lane C Low molecular weight marker Spectral and molecular properties of PuORh PuORh shows a typical flavoprotein spectrum with absorbance maxima at 377 and 459 nm (Fig. 2). In the presence of 5% trichloroacetic acid, PuORh precipitated and was visible as a white pellet after centrifugation. The supernatant contained a yellow color, and its absorbance spectrum with maxima at 375 and 450 nm was typical for that of free FAD. This shows that the FAD cofactor in PuORh, like in PuOMr, is not covalently bound. At room temperature, the addition of 0.1% SDS did not unfold PuORh, indicating this flavoprotein is rather robust. When 50 μM putrescine was mixed with PuORh under anaerobic conditions, the FAD cofactor was completely reduced (Fig. 2, dotted line). Heating the same sample for 5 min at 80°C completely unfolded the enzyme as can be seen from the resulting free FAD UV/VIS spectrum (Fig. 2, dashed line). The reduced enzyme could be rapidly and fully reoxidized by addition of oxygen, indicating that the enzyme is a true oxidase. From the difference in absorbance between protein-bound FAD and free FAD, the molar extinction coefficient for PuORh (ɛ459) was calculated, 11.0 mM−1 cm−1. Based on the protein concentration as determined by Wadell’s method (Wolf 1983) and the FAD concentration (A459), a protein/FAD ratio of 0.5 was calculated. This is consistent with what has been described earlier for PuOMr (Desa 1972). Incubation of PuORh with additional FAD did not yield an increase of FAD incorporation or oxidase activity. Gel filtration experiments revealed that PuORh is mainly present as a dimer of ±100 kDa. This corresponds well with the theoretical monomeric mass of 49,375 Da. Fig. 2Spectral properties of PuORh. Absorbance spectra of oxidized native (solid line) and unfolded (dashed line) PuORh are shown. After addition of 50 μM putrescine under anaerobic conditions, the native enzyme is fully reduced (dotted line) Temperature and pH dependence of activity and stability To investigate the effect of temperature on enzyme activity and stability, both were determined at different temperatures. The optimal temperature for enzyme activity was found to be 30°C. Temperature stability experiments revealed that PuORh is a reasonable thermostable enzyme. At 50°C, half of the activity was lost after 2 h, while at 37°C, such a degree of inactivation was reached only after 1 day. PuORh has a rather sharp pH optimum for activity with putrescine with a peak around pH 8. Below pH 6.4, no significant activity can be detected. Substrate specificity Several alcohols, amino alcohols, and amines were tested as substrate, and if they turned out to be converted by PuORh, the steady-state kinetic parameters were determined at pH 8.0. Enzyme activity was found with several aliphatic diamines, amino alcohols, and polyamines. From Table 1, it is clear that putrescine is by far the best substrate. With aromatic amines and amino alcohols, no activity was observed. An increase in carbon chain length of the diamine from 4 to 6 C-atoms resulted in a more than 1,000-fold drop in catalytic efficiency (kcat/KM). For diamines containing 3 or 2 C-atoms and for n-butylamine, no activity could be detected. These amines were found to strongly inhibit the enzyme (Table 2). Apparently, they bind efficiently but are not converted. Clearly, a minimum of two amino groups is required to be a substrate for PuORh, and these amino groups should be preferably 4 C-atoms apart. Polyamines like spermine and spermidine were also converted by PuORh but are poor substrates. Compared to putrescine, 4-amino-1-butanol is also a poor substrate, while 1,4-butanediol is not accepted at all. This again indicates that two amino groups are essential for efficient catalysis, and it confirms that PuORh is a true amine oxidase. Inhibition studies showed that aminoethanol is a very effective competitive inhibitor of PuORh (KI = 1.8 μM). Apparently, this amino alcohol binds very strongly in the active site, while it cannot be oxidized. When we tested 2-hydroxyputrescine, which contains an aminoethanol moiety, we found that this compound is readily oxidized. l-Ornithine was found to be a very poor substrate. Apparently, the presence of a negatively charged and relative bulky carboxylic acid group at the C1 position prevents efficient amine oxidation. Taken together, it can be concluded that PuORh has a narrow substrate specificity and is very selective for putrescine. Table 1Steady-state kinetic parameters of PuORh at pH 8.0.ND Not determinedaValues of putrescine oxidase from Micrococcus rubens (Okada et al. 1980)Table 2Inhibition constants of competitive inhibitors of PuORhaValues of putrescine oxidase from Micrococcus rubens (Swain and Desa 1976) Model structure of PuORh reveals a key role for Glu324 in substrate binding PuORh shares 32% sequence identity with human monoamine oxidase B (MAO-B), which is a 59 kDa FAD-containing protein involved in the oxidation of neurotransmitters and other arylalkylamines like benzylamine and phenylethylamine (Shih et al. 1999). Based on the structure of MAO-B (Binda et al. 2004b), it was possible to construct a model for PuORh. The C-terminal membrane anchor of MAO-B is absent in the resulting model structure of PuORh. This could explain why PuORh is well expressed in the cytosol as a soluble protein in contrast to MAO-B, which is membrane associated (Binda et al. 2004a). In the model, the active site of the PuORh exhibits a similar architecture as that of MAO-B (Fig. 3). The catalytically important “aromatic cage,” present in flavin-containing amine oxidases (Li et al. 2006), is formed by His432 and Tyr395. The other two key amino acid residues, Lys296 and Trp385, involved in non-covalent FAD binding (Binda et al. 2002b) can also be found in the model of PuORh. However, there are also some striking differences. In MAO-B, FAD is covalently attached to Cys397 at the C8α position of the flavin. PuORh has an alanine residue at this position (Ala394), which precludes covalent binding of FAD. Furthermore, the active site of PuORh seems to be narrower than that of MAO-B due to the presence of Trp60 and Met173 in PuORh (Tyr60 and Cys172 in MAO-B). The model of PuORh also shows a glutamic acid residue (Glu324) pointing towards the N5 of the FAD, while MAO-B contains a tyrosine at this position (Tyr326). The presence of such a negatively charged amino acid in the active site has already been predicted for PuOMr several decades ago by Swain and Desa. They suggested the presence of a negatively charged carboxyl group in the active site, as PuOMr was found to be irreversibly inactivated by carbodiimides (Swain and Desa 1976). The presence and position of Glu324 can well explain the difference in substrate specificity between MAO-B and PuORh. PuORh prefers short aliphatic diamines, while MAO-B is active with a range of aromatic monoamines. Probably, Glu324 is involved in binding the protonated amino group of the diamine substrate, while the other amino group is positioned in the “aromatic cage” near the N5 of the flavin. Such a mode of binding for putrescine is consistent with our PuORh model. To confirm this hypothesis, we mutated Glu324 into an alanine (PuORh Glu324Ala) and a leucine residue (PuORh Glu324Leu) and found that the catalytic efficiency for putrescine drops, respectively, 100,000 and 200,000-fold (Table 3). Fig. 3Active site residues in the structure of MAO-B and in the model of PuORh. In MAO-B, the inhibitor rasagiline is covalently bound to the flavin. In the model of PuORh, the substrate putrescine is modeled in the active siteTable 3Steady-state kinetic parameters for putrescine of wild-type PuORh and of the mutants Glu324Ala and Glu324Leu at pH 9.0. Km (μM)kcat (s−1)kcat/Km (s−1 mM−1)PuORh WT3.5 ± 0.620.7 ± 1.15900PuORh Glu324Ala110,000 ± 20,0006.1 ± 0.40.05PuORh Glu324Leu110,000 ± 20,0003.2 ± 0.20.03 The distance between Glu324 and the N5 of the isoalloxazine ring of the flavin cofactor (9 Å) may explain the specificity for the specific chain length of the substrate. To be a substrate, a minimal carbon chain length of 4 C-atoms is required. Shorter diamines can bind to Glu324 but cannot reach the flavin and therefore are strong competitive inhibitors of PuORh (Table 2). In case of longer diamines or polyamines, the position of the amino group that is oxidized is less favorable compared to the case of putrescine resulting in a decrease in catalytic efficiency. Aliphatic monoamines like n-butylamine will bind to Glu324 via the amine moiety but cannot be oxidized and therefore are also inhibitors of PuORh. Initial substrate screening using 96-wells plates for the mutants PuORh Glu324Ala and PuORh Glu324Leu revealed that both showed some activity with aromatic amines like phenyl-1-butylamine and with aliphatic monoamines (e.g., kcat/KM for n-butylamine = 0.001 s−1 mM−1 at pH 9.0 for PuORh Glu324Leu). Unfortunately, accurate determination of the kinetic parameters of the mutant enzymes is not straightforward due to their low substrate affinity, which requires the use of high concentrations of amines, causing interference with the peroxidase-based assay. Discussion In an attempt to discover novel bacterial oxidases with biocatalytic potential, a putrescine oxidase from R. erythropolis NCIMB 11540 was identified. This putrescine oxidase was discovered by using a plate-based screening method for oxidase activity, which has been used before to screen for improved mutants during directed evolution experiments (Alexeeva et al. 2002). Our study shows that this method is also valuable for the discovery of novel bacterial oxidases of unsequenced genomes. Although the physiological substrate of the discovered putrescine oxidase, 1,4-diaminobutane, was not present during screening, clones expressing this oxidase could still be detected based on their oxidase activity. As polyamines like putrescine, spermine, and spermidine are present in most living cells, including E. coli (Tabor and Tabor 1984), this resulted in the formation of hydrogen peroxide and detection of positive clones. The physiological role of putrescine oxidase in R. erythropolis is probably related to polyamine degradation (Large 1992), which is supported by the presence of a neighboring gene encoding a putative aldehyde dehydrogenase on the sequenced DNA fragment (data not shown). PuORh displays 67% sequence identity with PuOMr, which is the only bacterial putrescine oxidase that has been characterized so far. A BLAST search with the amino acid sequence of PuORh resulted in a high number of homologous (putative) flavin-containing amine oxidases, which are widely distributed among most kingdoms of life. The seven closest homologs (sequence identity >65%) can all be found in actinomycetes. These sequences all contain the active site glutamate (Glu324), involved in substrate binding, the alanine residue (Ala394), which excludes covalent FAD binding, and the residues, which limit the size of the active site cavity (Trp60 and Met173). Most likely, they represent a clade of orthologous putrescine oxidases, which are not active with monoamines, within the family of (putative) flavin-containing amine oxidases (Supplementary information, ESM 1). PuORh shares some properties with PuOMr. Both enzymes are soluble dimeric proteins of approximately 100 kDa. Interestingly, both contain only 1 mol of non-covalently bound FAD per mole of dimer, which is rather unique among flavoproteins. The substrate specificity of PuORh is very narrow, like PuOMr, the enzyme is very specific for putrescine. Polyamines are also accepted, but aliphatic monoamines are not converted at all. In general, PuORh appears to have a higher affinity for its substrates than PuOMr (Km for putrescine = 8.2 vs 38 μM). The narrow substrate specificity of PuOMr for di- and polyamines was already rationalized by the suggestion of the presence of an ‘anionic point’ in the active site. This anionic point, likely due to a carboxylate function, binds one positively charged amino group of the substrate, while another amino group is oxidized (Swain and Desa 1976; Okada et al. 1979). Based on sequence alignment between PuORh and PuOMr and a structural model of PuORh, we can now identify Glu344 as the anionic point in PuOMr. The presence of a carboxylate function as a manner to bind a positively charged amino group is not restricted to putrescine oxidase. In polyamine oxidase (PAO), a negatively charged ‘carboxylate ring’ can be found at one side of its substrate tunnel to guide polyamine substrate molecules into the active site (Binda et al. 1999). In addition, in PAO, two glutamate residues (Glu62 and Glu170) are present in the active site opposite to the N5 of the flavin. However, these residues have been suggested to be protonated (Binda et al. 1999). Moreover, in PAO, Glu62 and Glu170 are in close proximity to a substrate secondary amine group, which will be oxidized, while in PuORh, Glu324 interacts with the (primary) amine group, which will not be oxidized. The amine group to be oxidized in PuORh is placed between His432 and Tyr395, near the N5 of the isoalloxazine ring. This structural feature resembles the “aromatic cage” found in other flavin-containing amine oxidases, like MAO-A (De Colibus et al. 2005), MAO-B (Binda et al. 2002a), and PAO (Binda et al. 1999). For MAO-B, this “aromatic cage” has been shown to play a steric role in substrate binding and in flavin accessibility and helps to increase the substrate amine nucleophilicity (Li et al. 2006). Polyamines like putrescine, cadaverine, spermine, and spermidine are related to cell growth and differentiation processes. Increased levels in body fluids and tissue occur in cancer patients, and therefore, polyamines represent important cancer markers (Casero and Marton 2007). Polyamines are also used to monitor food freshness, as an increased level can be found in spoiled food due to amino acid degradation (Bardócz 1995). Due to efficient heterologous expression and its catalytic properties, PuORh may develop as a valuable diagnostic enzyme for the detection of low amounts of putrescine and polyamines. Moreover, based on the structural model, enzyme redesign may also allow creation of mutants that can be used for synthetic purposes. Electronic supplementary material Below is the link to the electronic supplementary material. ESM 1 Unrooted phylogenetic tree representation of PuORh homologs, which exhibit over 30% sequence identity (at the amino acid level) throughout the whole sequence. In gray, the clade of putrescine oxidases. MAO-B Human monoamine oxidase B; PuORh putrescine oxidase from Rhodococcus erythropolis NCIMB 11540; PuOMr putrescine oxidase from Micrococcus rubens. (DOC 36 kb)

Breast_Cancer_Res_Treat-3-1-2137941

The spectrum of ATM missense variants and their contribution to contralateral breast cancer

Heterozygous carriers of ATM mutations are at increased risk of breast cancer. In this case-control study, we evaluated the significance of germline ATM missense variants to the risk of contralateral breast cancer (CBC). We have determined the spectrum and frequency of ATM missense variants in 443 breast cancer patients diagnosed before age 50, including 247 patients who subsequently developed CBC. Twenty-one per cent of the women with unilateral breast cancer and 17% of the women with CBC had at least one ATM germline missense variant, indicating no significant difference in variant frequency between these two groups. We have found that carriers of an ATM missense mutation, who were treated with radiotherapy for the first breast tumour, developed their second tumour on average in a 92-month interval compared to a 136-month mean interval for those CBC patients who neither received RT nor carried a germline variant, (p = 0.029). Our results indicate that the presence of ATM variants does not have a major impact on the overall risk of CBC. However, the combination of RT and (certain) ATM missense variants seems to accelerate tumour development. Introduction Homozygous or compound heterozygous germline mutations in the ATM gene cause the autosomal recessive disorder ataxia-telangiectasia (A-T). This progressive neurological childhood disease is characterized by cerebellar degeneration, immunological defects, extreme sensitivity for ionising radiation and increased risk for cancers, particularly lymphomas [1]. ATM mutations identified in A-T families can be classified in three categories; truncating mutations, mutations that lead to some expression of mutant protein that lacks kinase activity and missense mutations with reduced kinase activity (http://chromium.liacs.nk/lovd/). Heterozygous pathogenic ATM mutation carriers, ∼0.5–1% of the general population, do not display the symptoms observed in A-T patients. Several epidemiological studies have consistently shown elevated rates of breast cancer among female blood relatives of patients with A-T [2, 3]. Thompson et al. have shown that the overall relative risk in carriers was 2.23 [95% confidence interval (CI) 1.16–4.28] compared to the general population and 4.95 (95% CI 1.9–12.9) in those younger that age 50. A large review showed that ATM mutations are more frequent in breast cancer patients selected on the basis of a family history of breast cancer than in unselected patients [4]. Besides pathogenic ATM mutations, a large number of ATM variants (common polymorphisms and unclassified variants) have been described, which were found in cancer patients as well as in the general population. It has been hypothesized that the cancer risk among ATM heterozygotes might be related to mutation type, suggesting that particularly missense mutations are associated with an increased risk [5, 6]. However, two recent studies by Thompson et al. and Renwick et al. showed that pathogenic ATM mutations that cause A-T are breast cancer susceptibility alleles [2, 7]. This argues against the hypothesis that missense rather that truncating are associated with breast cancer. Women with breast cancer have in general a three to fourfold increased risk of developing a new primary cancer in the opposite breast [8]. The contralateral breast cancer (CBC) risk might be explained by the same genetic and hormonal factors that caused the first breast cancer. Treatment related factors, e.g. radiotherapy for primary breast cancer, may also contribute to the development of cancer in the contralateral breast [9] (our own data, manuscript under review). To evaluate whether germline ATM missense variants are significantly associated with CBC risk (results regarding ATM truncating mutations are reported elsewhere) and whether treatment modifies this risk, we conducted a case-control study in which we assessed the ATM missense mutation spectrum and frequency in women who developed their first breast cancer before age 50, with and without a second primary breast cancer. Methods Patients The consecutive breast cancer patients included in this study were all selected from the hospital tumour registries of The Netherlands Cancer Institute, Amsterdam (NKI-AVL) or The Dr. Daniel den Hoed Cancer Center/Erasmus Medical Center, Rotterdam (DDHK). Of all patients that were invited to participate we achieved an 80% response rate. The breast cancer patients were included if their (first) breast cancer was diagnosed before age 50 (n = 443). For CBC we required an interval of at least 1 year (n = 247). The unilateral breast cancer patients (UBC) patients all had to be disease-free (of a second breast cancer) for at least 5 years. The first 57 CBC patients were individually age-matched (1:3) to UBC controls. All patients had invasive breast carcinoma and were treated with surgery. Of the CBC patients 169 did and 78 did not receive radiotherapy treatment for their primary breast tumour. Average age at diagnosis for the first breast cancer in the RT group was 41.2/41.3 years (mean/median) and the non-exposed group 42.0/43.2 years (mean/median). Detailed treatment data, disease and patient characteristics were obtained from medical records and risk factor questionnaires (data not shown) [10, 11]. Patients were asked to donate a 20 ml blood sample or permission for use of paraffin-embedded tissue blocks and patients gave their written informed consent for mutation analysis. This study received approval of the Medical Ethical Committees of NKI-AVL and DDHK. Genomic DNA isolation Genomic DNA was either isolated from peripheral blood lymphocytes with DNAzol (Invitrogen, Breda, The Netherlands) methods according to the manufacturer’s instructions, or from three 10-μm paraffin embedded normal tissue slides according to standard protocols [12]. For histopathological examination we used a hematoxylin-eosin stained slide. Mutation analysis The complete ATM Open Reading Frame (ORF) was analysed, each exon (exon 4-65) and all intron-exon boundaries were screened for germline mutations using Denaturing Gradient Gel Electrophoresis (DGGE) identifying ∼90% of all ATM mutations and polymorphisms (details from the author upon request). All aberrations were confirmed with genomic sequence analysis, performed using the ABI PRISM BigDyeTerminator Cycle Sequencing Ready Reaction Kit Version 3.1 (Applied Biosystems, Nieuwerkerk a/d yssel, The Netherlands). Sequencing products were analysed with the ABI PRISM 3700 DNA Analyzer and corresponding software. Statistical analysis Statistical analyses were performed using standard methods for analysis of case-control studies [13]. We compared the mutation frequency between UBC and CBC and between CBC cases previously treated with RT and cases not-treated with RT. Odds ratios (ORs) and 95% CI were calculated to evaluate the association between mutation carriers status and breast cancer risk. We have used the Mann–Whitney test to determine whether the difference between the intervals between the two breast cancers of the CBC patients was significant. All analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Results and discussion ATM germline mutations In the present study, we have used the DGGE method to screen the complete ATM ORF to obtain insight in the ATM missense mutation spectrum in (contralateral) breast cancer patients. With DGGE we were able to confirm all the previously identified truncating mutations. A subset of the CBC patients described in this study had been screened in the past for ATM truncating mutations with the Protein Truncating Test, revealing seven ATM truncating mutations (including a non-sense mutation and small insertions and deletions; generating stop codons within a previously functional protein coding sequence causing premature termination of translation of the protein) [10]. Among all 443-breast cancer patients that were tested in this study with DGGE we detected a large number of ATM silent mutations (presumed neutral polymorphisms, data not shown and excluded from all analyses) and missense mutations (causing an amino acid substitution in the coded protein, most common ones; i.e. D1853N, not included in further analysis). ATM missense mutation spectrum In our study cohort we have detected 35 distinct ATM missense variants and 6 distinct truncating mutations. Several of the detected missense variants have been reported in the ATM database as being detected in A-T patients/or as polymorphisms (Table 1). None of the missense variants identified in this study are known as pathogenic A-T causing missense mutations. Seventeen of the missense variants have not been reported previously. Eleven missense variants were exclusively found in the CBC group and 10 exclusively in the UBC group. Whether this distinction in the spectrum indicates an association between particular variants and bilateral breast cancer risk cannot be concluded from the small numbers obtained in this study population. The ATM protein has several functional domains and the identified missense variants are located throughout the ORF. Potential functional implications of the newly identified unclassified variants remain to be established. Table 1ATM missense variant and truncating mutation spectrum in contralateral and unilateral breast cancer patientsMissense variantsAmino acid changeCBC n = 247UBC n = 190Databasea or literature37C>TR13C1[10]146C>GS49C55database162T>CY54H21 [4], [14]378A>TD126E1database1009C>TR337C1Novel1132A>GS377G1Novel1229T>GV410A21 [4]1810C>TP604S1database2119T>CS707P78database2276G>A S759NNovel2336T>CM779T1Novel2414G>AR805Q2Novel2572T>CF858L43database2650C>TP884S12650C>TP884S1Novel2614C>TP872S [15]3161C>GP1054R813database3925G>AA1309T11 [16]4138C>TH1380Y1database4258C>TL1420F54database4324T>CY1442H2Novel4362A>CK1454N1database4477C>GL1493V1Novel4664T>AL1555H1Novel4722G>TL1574F1Novel5044G>TD1682Y1database5071A>CS1691R22database5557G>AbD1853N3549database5558A>TD1853V31database5741A>GD1914GNovel6067G>AG2023R1database6820G>AA2274T1database6919C>TL2307F1 [14]7446G>AM2482I1Novel7874A>GD2625G1Novel8659C>GH2887D1NovelTruncating mutationsIVS10-6T>G419X12database [10], [17]1563delAG521X1database1660delA554X1NovelIVS14 + 2T>Gdel 601-6331database2572insTF858X1Novel3115A>TR1039X1Novela http://chromium.liacs.nk/lovd/b Not included in frequency analysis Despite the fact that ATM plays a role in breast cancer risk, the role of most distinct ATM missense variants remains unclear. Some studies tried to predict the relevance of each particular mutation on basis of co-segregation with breast cancer in families, the location in a functional domain or interference with the splicing machinery. Only a few studies present functional analysis that are necessary to assess the biological impact of unidentified variants found frequently in ATM [18–20]. ATM missense mutations and contralateral breast cancer Twenty-one per cent of the patients carried at least one ATM germline variant (missense and truncating; Table 2). Among the patients with CBC (n = 247) we identified in total 55 ATM variants in 45 individuals (18%); 51 missense variants and 4 truncating mutations (Table 2). Eight CBC patients had multiple ATM missense variants and 2 patients carried both a missense and a truncating ATM mutation. In the women with UBC (n = 196) we identified 52 ATM variants in 46 individuals (23%); 48 missense and 4 truncating mutations. Three UBC patients carried double missense and 3 patients both a truncating and a missense variant. Although it is known from the literature that ATM missense variants might be involved in breast cancer pathogenesis, the identified 17% missense variant carriers among the CBC patients compared to the 21% missense variants among the UBC patients indicate that there is not a significantly increased risk for bilateral breast cancer among ATM missense variant carriers, OR 0.77 (95% CI 0.48–1.24). Table 2ATM variant frequencies in all breast cancer patients diagnosed under age 50 and according to uni- or contralateral breast cancerBreast cancer patients withAll patients n = 443CBC n = 247UBC n = 196Total ATM variantsa55: 51 missense and 4 truncating52: 48 missense and 4 truncatingAt least one ATM variant91 (21%)45 (18%)46 (23%)At least one ATM missense variant85 (19%)43 (17%)42 (21%)Only one ATM truncating mutations321One truncating and one missense variant523Double missense variants1183a Not included are the most common and silent variants Association with radiation treatment Women at high risk for developing breast cancer may respond differently to radiation exposures associated with screening and treatment, than the general population. Candidate-genes like ATM are implicated in maintenance of genome integrity. Their involvements in breast cancer susceptibility as well as their role in DNA-damage repair signalling make them excellent candidates for a role in radiation-induced breast cancer [21]. Recently, we showed that women with a pathogenic germline mutation in a DNA repair pathway gene (e.g. BRCA1, BRCA2, CHEK2 and ATM) have an over 2-fold increased risk of developing radiation-associated breast cancer (manuscript under review). Therefore, we now investigated whether exposure to ionising radiation had a greater biological impact on certain ATM genotypes than on others. We did not detect a significantly increased risk of developing radiation-associated CBC among missense mutation carriers. Among those 169 CBC patients who had developed a second primary breast tumour following radiotherapy for their first breast tumour we identified 19.5% ATM missense variants carriers compared to 13% among those CBC patients who did not receive RT, the OR from this case-only analysis is 1.65 [95% CI (0.77–3.55) p = 0.2]. Furthermore, we have observed that 21% of the UBC patients, who received RT but did not develop a CBC carried an ATM missense variant, compared to 19.5% of the CBC patients that received RT for their first tumour [OR 0.86 (95% CI 0.52–1.43)]. These results suggest that RT is not a strong risk factor for the development of CBC among carriers of those ATM missense variants. It has however been shown that particular alterations in the ATM gene are associated with increased radiation sensitivity [22–24]. Gutierrez-Enriquez et al. showed that lymfoblastoid cell lines carrying the ATM variant 3161G (linked to 2572C) was associated with increased in vitro chromosomal radio-sensitivity, perhaps by interfering with ATM function in a dominant-negative manner [22]. We found this particular variant allele (3161G/2572C) exclusively in our CBC group exposed to radiotherapy (four times) and not in the non-RT-exposed CBC group. This finding supports the hypothesis that particular ATM variants might play a differential role in radiation response. Although a subset of the missense variants was only detected in the RT exposed subpopulation, individual numbers were probably too small to detect a significant effect of particular mutations associated with treatment. We observed that CBC patients with an ATM missense variant had an mean interval between the first and second breast tumour of ∼101 months, compared to 122 months for non-carriers CBC patients (p = 0.085). Interestingly, the combination of radiation treatment and a missense variant resulted in an even shorter mean interval of a 92 months in the CBC patients compared to a 136-month interval for CBC patients who neither received RT nor carried a germline variant (p = 0.029). These data suggest that carrier-ship of an ATM missense variant may accelerate the development of a second tumour and decreases the age at onset of the second breast tumour, especially in case of exposure to RT. The suggestion of a shorter induction period of RT-associated breast cancer in patients, who carry an ATM missense mutation, while the proportion of patients with missense variants was similar in CBC and UBC cases, might be attributable to a different spectrum of mutations in those patients who developed CBC. A big challenge in such a study remains to assess which particular missense mutations have an impact on ATM function. Large association studies, as performed by the Breast Cancer Association Consortium (coordinated by Doug Easton and Paul Pharoah, Cambridge), and functional studies are clearly necessary to determine the importance of particular variants and their contribution to the breast cancer risk.

Eur_J_Epidemiol-4-1-2270357

Data splitting as a countermeasure against hypothesis fishing: with a case study of predictors for low back pain

There is growing concern in the scientific community that many published scientific findings may represent spurious patterns that are not reproducible in independent data sets. A reason for this is that significance levels or confidence intervals are often applied to secondary variables or sub-samples within the trial, in addition to the primary hypotheses (multiple hypotheses). This problem is likely to be extensive for population-based surveys, in which epidemiological hypotheses are derived after seeing the data set (hypothesis fishing). We recommend a data-splitting procedure to counteract this methodological problem, in which one part of the data set is used for identifying hypotheses, and the other is used for hypothesis testing. The procedure is similar to two-stage analysis of microarray data. We illustrate the process using a real data set related to predictors of low back pain at 14-year follow-up in a population initially free of low back pain. “Widespreadness” of pain (pain reported in several other places than the low back) was a statistically significant predictor, while smoking was not, despite its strong association with low back pain in the first half of the data set. We argue that the application of data splitting, in which an independent party handles the data set, will achieve for epidemiological surveys what pre-registration has done for clinical studies. Introduction The concept of statistical significance may be the single-most important mathematical invention for applied science. Its use has become so widespread and commonplace that many non-mathematical readers may not be actively aware of its true meaning. To briefly review, the statement “X is correlated with Y at significance level alpha” signifies, “If no true correlation between X and Y exists, the probability of obtaining the observed correlation is less than alpha.” The P-value of a test is therefore a measure of surprise; the smaller the P-value, the greater the surprise. Standard practice has been to set alpha at 0.05, which literally allows for a 5% chance of erroneously reporting a significant finding (Type I error). One cannot interpret the P-value as a probability of having made a Type I error, so 5% significance does not imply that the conclusion is correct with a 95% probability. Such statements are meaningful only in a Bayesian context where one assigns a priori probabilities to hypotheses. The present article addresses non-Bayesian (frequentist) analysis, which is by far the most common in epidemiology. Ioannidis [1] purports that most scientific findings are likely to be false, despite being reported as statistically significant. One of his arguments, which we support, is that the pressure to publish creates an incentive for researchers to simultaneously address a large number of hypotheses and selectively report only “significant” results. This conduct is labelled hypothesis fishing or data dredging. (In older papers the term data mining has also been used, but the meaning of this has shifted toward discovery of valid patterns in databases.) Also, the investigator may run different statistical tests (e.g. parametric and non-parametric tests) for any given hypothesis, which is a less recognized form of hypothesis fishing. In an epidemiological and health services setting, complex data sets based on statistical surveys are commonplace, in which hundreds of variables are collected from thousands of people. Data collection for a survey requires enormous effort, with both individual and collective demands on the researchers and respondents. From a purely economic point of view, it appears logical to “turn the data set upside down,” searching for anything of interest buried in it. Therein lies the temptation to launch a large-scale fishing expedition for all potentially interesting hypotheses. Similar to real-life commercial ocean fishing, which requires adjusting the mesh size of the nets upward, researchers investigating multiple hypotheses need to adjust the level of statistical significance down in order to preserve the meaning of statistical significance. The general rule of thumb is to divide alpha by the number of hypotheses, referred to as a Bonferroni correction [2]. However, in accordance with Ioannidis [1], we contend this method is rarely applied. The limited use of Bonferroni correction may result from uncertainty surrounding the exact number of hypotheses “fished for,” but some researchers may also be motivated to keep the “catch.” In epidemiological research, upwards of 100 possible hypotheses are common, and setting the alpha at 0.0005 eliminates much of the fun in a fishing expedition. As a consequence, researchers may be tempted to discuss only a handful of “significant” results, failing to mention the 95 that proved non-significant. Hypothesis fishing renders P-values almost completely meaningless, and we consider it to be a serious problem for epidemiological survey analysis. The present paper recommends a very simple countermeasure against hypothesis fishing, based on data splitting. The procedure is similar to two-stage analysis of microarray data. We argue that if our method could be applied for survey data in general, it could do for epidemiological studies what pre-experiment registration of RCTs has done for medical experiments. The rest of the article is laid out as follows: First we review some mathematical methods that might be considered useful for counteracting hypothesis fishing. Then we explain our method in detail, and compare it to two-stage analysis of microarray data, followed by a case study where the method is applied to an analysis of low back pain. The article ends with discussion and conclusion. Mathematical remedies (that fail to solve the problem) If one considers hypotheses fishing to be a mathematical problem, it is reasonable to look for mathematical solutions. There is a large mathematical literature that relates to model building and multiple hypotheses, and we will only try to point out the main themes. The simplest way of handling the problem of multiple hypotheses is to reduce the significance level through Bonferroni correction. It is also possible to use so-called false discovery rate (FDR) [3], which is a way of controlling the expected percentage of rejected null-hypotheses (discoveries) that are falsely rejected. If all the null-hypotheses are true, FDR is equivalent to Bonferroni correction, but otherwise it is less strict. As we mentioned in the introduction, it can be hard to keep track of the number of hypotheses one is really addressing, which makes the use of Bonferroni or FDR cumbersome. Complex mathematical methods exist that adjust for the effect of model selection on inference (for example, see Madigan and Raftery [4]). In order to utilize these methods, however, one must describe the model-building strategy in a formal mathematical way. Faraway [5] has analysed various data splitting approaches similar to the one we apply, and compares them to mathematical inference adjustment methods. Based on his simulation study, he concluded that data splitting costs more in reduced accuracy than it gains in “honesty”. This conclusion is to be expected, because adjustment methods utilize information that the splitting methods disregard. In our present setting, however, accurate information on modelling procedure is not available, so adjustment methods are not an option. The task of choosing a set of hypotheses is—at least superficially—related to the problem of choosing the set of predictors to include in a statistical model for the data. An overview of this field is given by Hastie et al. [6], and we only mention a few key concepts. An obvious goal in statistical modelling of a given set of data is to develop a model that fits the data well, and a model will always fit better when the set of predictors is increased. However, if one includes too many predictors, the model is prone to imitating random properties of the data set, which are not present in the underlying sampling distribution of the data. Therefore, one needs ways to make trade-offs between model fit and model size. This can be done directly through various information criteria, such as Akaike’s Information Criterion (AIC), the Bayesian Information Criterion (BIC), or the Divergence Information Criterion (DIC). A different approach called cross validation is to split the data set in two parts, using one part for estimating model parameters, and the other for evaluating model fit. A problem with this method is that only half of the data is used for each task. A clever remedy is to leave one data point out at the time, estimate the model from the remaining data points, and measure model fit by taking the average fit on the points left out (leave-one-out cross validation). We mention these methods mainly to point out that they are not very relevant for controlling hypotheses fishing. The use of information criteria or cross validation helps in trading model fit for model size, but does not produce adjustments for the total number of variables. The cross validation procedure of splitting the data set, using one part to estimate model parameters and the other for validation is deceptively similar to the method we apply. But the purpose of validating a model’s predictions is entirely different from our purpose of conducting sound hypothesis testing. In addition to the inherent difficulties related to mathematical ways of dealing with hypothesis fishing, we do not see the problem as mainly a mathematical one. Some researchers may not be aware of the fact that P-values lose their meaning when the hypotheses are chosen from a large pool of undocumented ones. Others may be vaguely aware of the problem, but choose not to address it unless reviewers demand it. Reviewers, on the other hand, may not feel inclined to insist on purity beyond what they have done in their own scientific work. The solution: data splitting We recommend splitting the data set randomly into two sections (Parts 1 and 2). This allows the investigators to identify hypotheses in Part 1 of the data set, while remaining blind to Part 2 until the hypotheses are specified. True hypothesis testing is then performed using only Part 2 of the data. At this point there is no second-guessing. If the alpha-level is set to 0.05, and the P-value in Part 2 is 0.051, the result is by definition not significant, even if it received a P-value of 0.001 in Part 1. In such cases, there will be a temptation to “make a compromise” by computing the average of the P-values from Parts 1 and 2, but this is not allowed. Because the data in Part 1 was used to construct the hypotheses, it is tainted, and cannot take any part in the hypotheses testing. This procedure is strict with respect to identifying statistical significance. Once a hypothesis is supported, however, the entire data set is used for estimating the effect size. Thus, the purpose is to ensure the proper use of the term statistical significance. Once a significant finding is established, though, it is preferable to obtain the most accurate parameter estimates possible. Models: hypothesis variables, and confounders Epidemiological hypotheses are usually formulated within the framework of a model. Assume the hypothesis is that eating mushrooms increases the risk of cancer. To test this hypothesis, one would build a model with predictors like age, gender, smoking status, as well as mushroom habits, in order to control for these confounding factors. (If old people eat more mushrooms, excluding age from the model would give an incorrect positive association between mushroom eating and cancer.) From a purely computational point of view, no differences exist between predictors associated with hypotheses and confounders, yet the semantics are very different. The confounders are included only as a means of estimating the causal link between the cause (mushrooms) and its hypothesized effect (cancer). Researchers often use P-values as a road map (in addition to literature reviews and general medical knowledge), when deciding which variables should be included as confounders. A common piece of advice is to include confounders demonstrating an association at a P-value below 0.1 or 0.2. Despite this rationale for including a confounding factor, no similar demands are made for inclusion of the confounder in Part 2. The chosen set of confounders provides the framework within which the hypothesis is defined, and it is not the framework that is being tested. In the mushroom example, the hypothesis is that mushroom eating is associated with cancer when controlling for age, gender, and smoking, not that controlling for each of these factors is necessary. Size of Part 1 and Part 2 When deciding upon the relative size of Parts 1 and 2, a trade-off exists between the need to identify hypotheses by exploration in Part 1, and the need to achieve statistical significance in Part 2. An even split may be reasonable in cases where the need for exploration is high, particularly if the data set is large, so that half of the data set is sufficient to achieve statistical significance for stronger effects. In cases where greater domain knowledge is available based on the existing literature, a smaller Part 1 is reasonable, especially when the sample size is small. Multiple hypotheses It is possible to investigate multiple hypotheses within our splitting regime, using Bonferroni corrections. Assume, in the mushroom-cancer example, that the analysis of Part 1 also provided strong support for the hypothesis that eating bananas protects against cancer. One might then choose to include both mushroom habits and banana habits as hypothesis variables, and consequently divide alpha by 2 (the number of hypotheses). If either mushroom or banana habit fails the significance test in Part 2, it will still be in the model, as a confounder. It might be the case that both banana and mushroom habits get P-values that fall between alpha/2 and alpha. In this situation, both hypotheses would have passed the significance test individually, but the choice to include two hypotheses resulted in failure of both variables to reach significance. Many non-statisticians would argue that scientific procedures and statistical analysis should be objective, with conclusions based on ‘hard facts’, independent of arbitrary choices of hypotheses. Unfortunately, this is not the case if we wish to claim statistical significance. The mushroom-banana example is a clear case in which investigators should reduce the alpha-level to account for multiple hypotheses. At the other extreme, if independent research groups investigating different research questions based on independent data sets, their combined effort is obviously not a case of ‘multiple hypothesis testing’. A grey area exists with partially overlapping data sets, hypotheses, and research groups, often making it difficult to decide whether Bonferroni corrections are called for. A pragmatic solution may be to view a published article as a unit, and apply Bonferroni corrections within each one. Relation to two-stage analysis in genetics Readers who are familiar with microarray analysis will recognize that our data splitting method is similar to two-stage analysis, as it is routinely performed in genetics [7]. There are a few differences, however. In a microarray context the set of possible hypotheses is given by the number of genes, and the FDR method is normally used to limit the number of incorrect findings. Rather than primarily counteracting hypothesis fishing, microarray two-stage analysis is usually motivated by cost effectiveness: By screening out promising candidates first, and then evaluating them, researchers can make a higher number of valuable discoveries for each monetary unit spent. In a microarray setting the procedure is also likely to be more automatic, as interesting genes are filtered out in two more or less mechanical steps of analysis. In our epidemiological application, on the other hand, there will be a man-in-the-loop, as the researcher builds a model with hypothesis variables and confounders based on a combination of his domain knowledge and Part 1 of the data. Case study of low back pain in the Ullensaker study Study sample and setting Data material consisted of adults enrolled in an epidemiological survey for musculoskeletal pain (MSP) in the Ullensaker municipality, 40 km northeast of Oslo in Norway. In 1990, 4050 inhabitants born in 1918–1920, 1928–1930, 1938–1940, 1948–1950, 1958–1960 and 1968–1970 (age 20–70 years) were sent a postal questionnaire about MSP. Of these people, 67% responded. Individuals who reported low back pain (LBP) during the past year (1990) were excluded from this material (N = 1439), such that the original sample consisted of 1283 participants who were free of LBP in 1990. In 2004, a 14-year follow-up was conducted. A total of 763 participants (59%) responded and formed the present study sample. These 763 participants were randomly divided in two samples with n = 369 (Part 1) and n = 394 (Part 2), respectively. Outcome measures To identify respondents with LBP, we used the answer to the question, “During the past year, have you experienced pain or discomfort in your lower back?”. This item was based on the Standardised Nordic Pain Questionnaire [8], which is a self-report questionnaire frequently used in Scandinavian epidemiological studies. Independent variables (potential risk factors) In 1990, the survey questionnaire contained a number of socio-demographic and health-related factors, which could be included as risk factors in the present study. Socio-demographic variables were gender, age, marital status, and work status. Health-related variables were body mass index (BMI), smoking status, number of MSP sites other than the low back, duration of previous MSP, use of medication due to MSP, having been examined by a health care provider due to MSP during the last year, comorbidity, family history of musculoskeletal problems, emotional distress, leisure physical activity, participation in competitive sports, sleeping problems, and self-perceived health. Model and hypotheses A logistic regression model was developed based on Part 1 of the data set and medical expertise. The number of pain sites and smoking status were included as independent variables. Smoking status was dichotomised as smoking and non-smoking. Number of pain sites was operationalized using participant responses on the Nordic Pain Questionnaire [8]. Specifically, respondents reported whether they had experienced any pain or discomfort from the following 10 areas during the previous year: head, neck, shoulder, elbow, hand/wrist, upper back, low back, hip, knee and ankle/foot (responses were “yes/no”). The total number of pain sites was computed and categorized into the following four categories: no pain sites, 1 or 2 sites, 3 or 4 sites, and 5 or more pain sites. We also included age, which was categorized into values corresponding to the six birth cohorts: 1918–1920, 1928–1930, 1938–1940, 1948–1950, 1958–1960, and 1968–1970. In addition, gender and marital status (dichotomised into married/partnership versus living alone) were included. Results of the logistic regression model are presented in Table 1. We hypothesized that smoking would be positively associated with LBP. Therefore, a 1-tailed hypothesis test was conducted. It was also hypothesized that individual pain sites would be positively associated with LBP. To limit the number of hypotheses, though, we hypothesized that the total number of pain sites would affect LBP probability, rather than run analyses for each level of the variable. Hypotheses testing The significance level alpha was set to the usual value of 0.05. With two hypotheses, the critical P-value becomes 0.025. Results for Part 2 of the data set are illustrated in Table 2. The P-value of the pain sites variable (0.015) was below the critical value of 0.025, and therefore it is concluded that the number of pain sites was significantly associated with LBP at the 14-year follow-up. Table 1Parameter estimates from Part 1, controlling for age, gender, and marital statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0121 or 2 pain sites2.292(1.248–4.208)0.0073 or 4 pain sites2.690(1.406–5.147)0.0035 or more pain sites2.944(1.193–7.262)0.019Smoking2.079(1.285–3.363)0.003aThe reference category for number of pain sites was no pain sitesTable 2Parameter estimates from Part 2, controlling for age, gender, and marital statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0151 or 2 pain sites1.328(0.793–2.224)0.2813 or 4 pain sites1.598(0.857–2.979)0.1415 or more pain sites3.941(1.700–9.136)0.001Smoking0.993(0.627–1.571)0.487*aThe reference category for number of pain sites was no pain sites*1-sided P-value Smoking status received a 1-sided P-value of 0.487, which exceeds the limit of 0.025 by a large margin, and this variable is thus deemed non-significant. This result may seem surprising, given the variable’s strong association with the dependent variable in Part 1 of the data set (2-sided P-value = 0.003). This illustrates the dangers of hypothesis fishing: Our analysis suggests that the smoking status variable may only have been a “lucky winner” in the “P-value lottery” of Part 1. Parameter estimates Having concluded that number of pain sites has a significant effect on low back pain at follow up, we estimated the magnitude of the effect from the full data set (Table 3). Because the hypothesis test failed to give significance for smoking status, it is included only as a confounder together with age, gender, and marital status. The 2-sided P-value for smoking status was 0.040, so researchers following a hypothesis fishing procedure would probably have reported it as a statistically significant predictor. Table 3Parameter estimates of the hypotheses variable number of pain sites, from the complete data set controlling for age, gender, marital status and smoking statusPredictorOR estimate 95% CI for ORP-valueNumber of pain sitesa0.0001 or 2 pain sites1.637(1.116–2.400)0.0123 or 4 pain sites1.983(1.285–3.061)0.0025 or more pain sites3.346(1.846–6.067)0.000aThe reference category for number of pain sites was no pain sites Discussion Our study was designed to illustrate a simple and straightforward data splitting method to counteract hypothesis fishing in large-scale epidemiological surveys. This method involves splitting the data set, where the first half is used to identify hypotheses, while the remaining data is used to test the hypotheses. The data splitting procedure was illustrated using data material collected for a population-based health survey administered in Norway in 1990 and 2004. Results demonstrated that the number of pain sites (“widespreadness” of pain) was significantly associated with LBP following a 14-year follow-up. Smoking status was a strong predictor of LBP in Part 1 of the data set (hypothesis identification), but did not achieve significance in Part 2 (hypothesis testing). Therefore, this finding was dismissed as non-significant in our study. For the full data set, the P-value for smoking status was 0.040, so the traditional way of analysing epidemiological data would have given a different conclusion. In this study, the investigators had free access to the entire data set prior to data splitting and during model development. However, any temptation to “peak” at the material was successfully avoided, as indicated by the discrepant results for smoking status. This indicates that the data splitting procedure can indeed function properly in the absence of strict external control of the data. Nevertheless, we recommend that the data set be handled by an independent party, so that researchers can document claims that only Part 1 of the data set was used for model and hypothesis development. Ideally, the establishment of an independent international body is recommended to manage splitting of survey data. A fixed date for releasing data for Part 2 would be agreed upon, so that only those hypotheses specified prior to the release date would undergo a true significance test. Although several challenges and practical issues would inevitably need resolution (i.e., data collection, confidentiality, release of data), such an organization should be feasible and acceptable to the scientific community. Conclusions Results demonstrated that the number of musculoskeletal pain sites significantly predicts low back pain at a 14-year follow-up, when controlling for age, gender, marital status, and smoking. The application of the data splitting method in our study indicates its potential as an effective and useful method to counteract hypothesis fishing in population surveys. In our opinion, systematic data splitting administered by an independent party would accomplish for statistical surveys what pre-registration has already done for clinical trials.

Neurochem_Res-3-1-1832151

The Role of Presenilin and its Interacting Proteins in the Biogenesis of Alzheimer’s Beta Amyloid

The biogenesis and accumulation of the beta amyloid protein (Aβ) is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer’s disease. The presenilins and its interacting proteins play a pivotal role in the generation of Aβ from the amyloid precursor protein (APP). In particular, three proteins (nicastrin, aph-1 and pen-2) interact with presenilins to form a large multi-subunit enzymatic complex (γ-secretase) that cleaves APP to generate Aβ. Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the γ-secretase enzyme. Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme. In addition, a number of presenilin interacting proteins that are not components of γ-secretase play important roles in modulating Aβ production. This review will discuss the components of the γ-secretase complex and the role of presenilin interacting proteins on γ-secretase activity. Introduction: the generation of beta amyloid by presenilin and its binding proteins Alzheimer’s disease (AD) is a complex, progressive neurodegenerative disorder that is neuropathologically characterised by extensive neuronal loss and the presence of neurofibrillary tangles and senile plaques. While the majority of AD cases are sporadic, ∼5% of AD cases are familial (FAD) with mutations in three genes, amyloid precursor protein, presenilin 1 (PS1) and presenilin 2 (PS2) accounting for the majority of cases. A major feature of both sporadic and familial forms of AD, is the accumulation and deposition of a small peptide referred to as beta amyloid (Aβ) within brain tissue of AD sufferers. The mechanisms that underlie the disease processes are poorly understood. However, the accumulation of Aβ is thought to play a pivotal role in neuronal loss or dysfunction through a cascade of events that include the generation of free radicals, mitochondrial oxidative damage and inflammatory processes (reviewed in Refs. [1, 2]). The primary event that results in the abnormal accumulation of Aβ is thought to be the dysregulated proteolytic processing of its parent molecule, the amyloid precursor protein (APP). The APP molecule is a transmembrane glycoprotein that is proteolytically processed by two competing pathways, the non-amyloidogenic and amyloidogenic (Aβ forming) pathways. How these pathways are regulated remains unclear. However, there are many factors including diet, hormonal status, and genetic mutations that influence the processing of APP to generate Aβ (reviewed in Refs. [1, 3]). Three major secretases are postulated to be involved in the proteolytic cleavage of APP. These include α-secretase (of which the metalloproteases ADAM17/TACE and ADAM 10 are likely candidates), beta APP cleaving enzyme (BACE, formally known as β-secretase) and the γ-secretase. The α-secretase cleaves within the Aβ domain of APP thus precluding the formation Aβ and generating non-amyloidogenic fragments and a secreted form of APP (α-APPs). In the amyloidogenic pathway (Fig. 1), BACE cleaves near the N-terminus of the Aβ domain on the APP molecule, liberating another soluble form of APP, β-APPs and a C-terminal fragment (C99) containing the whole Aβ domain. The final step in the amyloidogenic pathway is the intramembranous cleavage of the C99 fragment by γ-secretase, to liberate the Aβ peptide (reviewed in Ref. [1]). Identification of multiple cleavage sites within the APP molecule has suggested the presence of other secretases, namely ε- and ζ-secretase; that generate the APP intracellular domain (AICD) and a longer fragment of Aβ (Aβ46), respectively [4–7]. Although different enzymes may exists, the same γ-secretase complex that generates Aβ may also generate AICD and Aβ46 as these products are inhibited by γ-secretase inhibitors [4–7] indicating that this enzyme has multiple cleavage sites. However, over-expression and mutagenesis studies have shown that the production of Aβ and AICD may be independent catalytic events [8, 9]. Fig 1Formation of the γ-secretase complex and Aβ generation. A schematic representation of the proposed formation of the γ-secretase complex and the enzymatic activity resulting in Aβ generation. Evidence has been provided to show that PS, nicastrin (NCT), aph-1, and pen-2 are the only components of the γ-secretase complex. Nicastrin and aph-1 are involved in the early formation of the complex, interacting with each other and forming a dimeric sub-complex. The presenilin holoprotein is incorporated into the aph-1: NCT sub-complex, nicastrin undergoes post-translational modifications and the complex is transported to the cell surface (or other Aβ generating compartments, i.e. TGN) as a trimeric complex. Pen-2 is incorporated into the complex and presenilin is cleaved forming the active γ-secretase complex. Following cleavage of APP by BACE to liberate secreted APP, the APP-C99 fragment left embedded within the membrane undergoes regulated intramembranous processing (RIP) by the γ-secretase complex, liberating Aβ1–40 or Aβ1–42 (Modified from Ref. [165]) The γ-secretase enzyme is thought to be an aspartyl protease that has the unusual ability to regulate intramembrane proteolysis (RIP) for a growing list of type 1 integral membrane proteins which include, APP, APP like proteins (APLPs), E-Cadherin, CD44, lipoprotein receptor related protein (LRP), Notch, sterol regulatory element-binding protein (SREBP), interferon response element (IRE1) and activated transcription factor 6 (ATF-6) (for recent review see [10]). The mechanism of RIP and γ-secretase activity is unknown. However a transient hydrophilic environment for catalysis within the lipid membrane must be created. Furthermore, the enzyme (or an enzyme domain) must have the ability to bend and unwind the α-helical substrates, exposing their amide bonds to hydrolysis. It is therefore conceivable that the γ-secretase enzyme is an integral protein of the lipid bi-layer and contains a number of proteins that may have different functions within an enzyme complex. The exact conformation or molecular architecture of the γ-secretase enzyme remains unknown. However, large molecular mass complexes of ∼250 kDa [11, 12], ∼500 kDa [13–17] and ∼2,000 kDa [13, 18] have been identified. Over the last few years biochemical and genetic approaches have identified four components of the γ-secretase complex, presenilins, nicastrin, anterior pharynx defective (aph-1) and presenilin enhancer 2 (pen-2). Over-expression and expression knockdown studies have provided strong evidence that these proteins are essential for γ-secretase activity [15, 19–24]. Subsequent reconstitution studies in non-mammalian cells have provided evidence that these are the only components responsible for γ-secretase catalytic activity [25–27]. However the active component and the active catalytic site of this enzymatic complex are yet to be identified. The roles of presenilin and its binding partners within the γ-secretase complex in Aβ generation Presenilin 1: the catalytic component of the γ-secretase enzyme Presenilin 1 (PS1) is thought to be the most critical component of the γ-secretase complex as evidence indicates that it is the catalytic component of this proteolytic enzyme. The finding that mutations in PS1 account for the majority of inherited early onset forms of AD and result in the overproduction of the highly amyloidogenic Aβ42 [28–30] provided the initial evidence that PS1 facilitates γ-secretase activity. Subsequent studies showed that PS1 ablation or mutagenesis of two highly conserved aspartate residues within transmembrane domains 6 and 7 of PS1 results in a reduction in Aβ levels [31–34]. Furthermore, aspartyl protease inhibitors and transition state analogue inhibitors which are designed to target the active site of the protease, all reduce Aβ40 and Aβ42 levels and have been shown to affinity label and bind to PS1 [35–37]. In addition, physical interactions between PS1 and the APP-C99 fragment have been identified [38–40]. The studies outlined above provide substantial evidence to indicate that PS1 is the catalytic subunit of γ-secretase. However, this transmembrane protein is not typical of a conventional aspartyl protease, and lacks the typical D(T/S)G motif for an aspartyl protease active site (recently reviewed in [41]. Aspartyl proteases require the presence of two aspartate residues for the hydrolysis of the peptide bond. Although PS1 lacks the typical D(T/S)G aspartyl protease active site it does contain the two aspartyl residues (D257 and D385) which are either critical for the active site on the γ-secretase complex or constitute the active site [42]. Other proteases that contain atypical aspartyl protease motifs and have weak homology with PS1 include the bacterial membrane embedded aspartyl protease, type 4 prepilin protease and the signal peptidases, which are all transmembrane proteins. These proteins share the unusual aspartyl motif GxGD and perform intramembranous cleavage of type I membrane proteins (reviewed in [43]). The topology and atypical characteristics of these proteins make them an unprecedented class of protease and as is the case for PS1, would require interaction with of other proteins within the complex. The interaction between the components of the γ-secretase complex is not sufficient to create a macromolecular enzyme complex such as γ-secretase (i.e. total mass of all the components is ∼200 kDa). Thus, dimer or trimer formation must occur within the complex. To date the only member of the complex that is known to undergo this process is PS1. The full length PS1 protein is rapidly endoproteolytically cleaved within its characteristic large hydrophilic loop into amino- and carboxy-terminal fragments (NTF/CTF) of ∼27 and ∼17 kDa, respectively [44, 45]. These fragments are thought to interact with each other to form the catalytic component of the γ-secretase enzyme since in the absence of full length PS1, co-expressed NTF and CTF were able to generate γ-secretase activity in presenilin deficient mammalian cells [46, 47]. The stoichiometry and the nature of the interaction between these fragments remain unclear. It has been shown by many studies that the NTF:CTF form a heterodimer in mammalian cells [12, 48–51] leading to suggestions that this heterodimer is the active γ-secretase [52]. However, Cervantes and colleagues [53] provided evidence that the presenilin fragments can form a tetramer by identifying heterodimers as well as NTF and CTF homodimers in yeast. Evidence for heterodimer and NTF homodimer (but not CTF homodimer) formation has been provided by photo-affinity labelled cross-linking studies [54]. This formation provides a core of aspartyl residues required for aspartyl protease activity. However, it has yet to be established whether the hypothetical “core” is formed between fragments from one PS1 molecule or multiple molecules within the complex. Further, if PS1 is the “catalytic core” of the γ-secretase complex the roles of the other interacting proteins need to be determined. Nicastrin: the substrate docking site and scaffolding for building the γ-secretase complex Nicastrin was discovered to be part of the γ-secretase complex when it was found to co-immunoprecipitate with PS1 [55]. Nicastrin is a type I transmembrane protein with a 670 amino acid long hydrophilic N-terminal domain, a transmembrane domain, and a relatively short cytoplasmic C-terminus of twenty amino acid residues [55]. The protein undergoes glycosylation and sialylation within the secretory pathway to yield a mature ∼150 kDa protein, the largest component of the γ-secretase complex [14], [56–58]. Nicastrin has been shown to be a critical component of the γ-secretase complex as inhibition of nicastrin function in vitro and in vivo results in a complete loss of APP and Notch cleavage [14, 55, 59–61]. Within this complex, nicastrin appears to be involved in the early formation of the complex, particularly in the formation of the first sub-complex between itself and aph-1 (Fig. 1). Recent evidence suggests that nicastrin is critical for the correct assembly of the γ-secretase complex within the endoplasmic reticulum and the intracellular trafficking of the complex to the cell surface [62, 63]. Furthermore, nicastrin is essential for the interaction between the complex and APP-C99 and thus may act as a receptor for γ-secretase substrates [63, 64]. Therefore, although nicastrin is a critical component, evidence to date suggests that it is unlikely to be a catalytic component of the γ-secretase complex. Instead, nicastrin acts as a scaffold for the building of the active complex, and a possible substrate docking site. Anterior pharynx defective 1 (aph-1): scaffolding partner for nicastrin and may have possible catalytic activity The aph-1 protein is a ∼30 kDa, 308 amino acid long 7-transmembrane protein [65] existing in two homologous forms, located on chromosome 1 and chromosome 15 (aph-1a & aph-1b respectively; [19]). In addition, as the result of alternate splicing, a long and short isoform of aph-1a have also been reported, with the short isoform more abundantly expressed in most tissues [21]. Although the exact function of these isoforms remains unknown, multiple γ-secretase complexes containing aph-1 isoforms are thought to exist [66, 67]. The aph-1a isoform appears to be incorporated into active γ-secretase complexes. Deletion of aph-1a causes a reduction in Aβ generation [66] and is the major isoform present in γ-secretase complexes during embryonic development [67]. The γ-secretase complexes incorporating aph-1b are thought to have a redundant function as aph-1b has been found not to be involved in the regulation of other γ-secretase components or γ-secretase activity [66]. The aph-1 protein forms a stable and intermediate ∼140 kDa complex with Nicastrin, before binding PS1. Aph-1 has been shown to interact with immature and mature forms of presenilins and nicastrin [21]. The GXXXG motif located within transmembrane 4 of aph-1 has been shown to be important in this interaction [68], [69]. The aph-1: nicastrin sub-complex appears to play a role in the stabilisation and assembly of PS1 in the γ-secretase complex (Fig 1; [24, 70, 68]). However, the exact role of aph-1 in the γ-secretase complex remains unknown. Expression studies have shown that removal of aph-1 destabilises the presenilin holoprotein and the addition of aph-1 leads to the accumulation of the holoprotein [11, 19–24, 71]. Thus together with nicastrin, aph-1 is thought to contribute the initial formation and trafficking of the γ-secretase complex and thereby provide a scaffold for the proteolytic processing of PS1 and the formation of the active site. Aph-1 is critical for the activity of the fully constructed γ-secretase complex. Evidence has been provided that as well as binding to immature components of γ-secretase in early stages of complex formation, aph-1 also interacts with the mature forms of PS1, nicastrin and pen-2 [68, 72]. Furthermore, recent evidence has been provided to suggest that this interaction occurs on the cell surface where it also binds the γ-secretase substrate, Notch and facilitates its cleavage [72]. These studies suggest that apart from the scaffolding function it shares with nicastrin, aph-1 has another function within the fully constructed, active γ-secretase complex. Structural and functional similarities between aph-1 and other proteases that possess the ability for intramembranous cleavage (such as rhomboid, [68, 73] suggests that this transmembrane protein may have an enzymatic function within the complex. This postulated novel function for aph-1 needs to be validated by future studies. Presenilin Enhancer 2 (pen-2): the elusive presenilinase or “molecular clamp” that holds the complex together? The pen-2 protein is a ∼12 kDa, 101 amino acid long protein containing two transmembrane domains and its N- and C-terminals facing the lumen of the endoplasmic reticulum [74, 75]. Pen-2 has been shown to interact with Nicastrin, aph-1, and also PS1. Without this interaction pen-2 has been shown to be degraded, possibly by the proteasome [76, 77]. Ablation of pen-2 results in significantly reduced PS1 endoproteolysis and Aβ production, suggesting that pen-2 is essential for both presenilinase and γ-secretase activity [15, 19, 22–24]. Likewise, combinational expression studies have shown that when pen-2 is co-expressed with the other components of the γ-secretase complex in a step-wise manner, endoproteolysis of the PS1 holoprotein was enhanced [11, 22, 24–27, 71]. A trimeric sub-complex between presenilin, nicastrin and aph-1 has been identified in cells lacking pen-2 [24, 70] suggesting that pen-2 may be the final protein incorporated into the γ-secretase complex (Fig. 1). Recent evidence has suggested that pen-2 is incorporated into the γ-secretase complex through an interaction with presenilins. The “DYLSF” domain of pen-2 and a “NF” motif on transmembrane 4 of PS1 have been shown to be critical for the interaction between these proteins [78–80]. The cleavage of PS1 occurs within the hydrophilic loop and is distal from the interaction site on transmembrane 4, suggesting that another domain of pen-2 is responsible for the cleavage of PS1 or pen-2 may present PS1 to the elusive “presenilinase” which may be another member of the γ-secretase complex. The C-terminal end of pen-2 has been shown to be important for γ-secretase activity since altering the length of this region by addition or deletion of residues has been shown to reduce Aβ40 and 42 generation without altering the binding of pen-2 to the complex [78] suggesting that pen-2 may have an alternative function within the complex. One suggestion put forward by Hasegawa and colleagues [78] is that the C-terminus of pen-2 may be a linker/space molecule that maintains spatial interactions between proteins within the complex. However, recent evidence using pen-2 C-terminal loss of function mutations suggest that the C-terminus acts as a “molecular clamp” holding together the presenilin fragments and the whole γ-secretase complex [81]. If this is the case then PEN-2 is an integral part of the catalytic process holding the complex together whilst the γ-secretase products are generated. The newest member of the complex: TMP21, a modulator of γ-secretase activity Recently another member of the γ-secretase complex was immuno-purified from wild-type PS1 and PS2 blastocyte derived cells [82]. The type 1 transmembrane protein termed TMP21 is a member of the p24 cargo-family which may have a signalling role in the sorting and transport of proteins from the endoplasmic reticulum to the Golgi [83, 84]. It was shown by Chen and colleagues [82] that TMP21 is a member of the complex as it was isolated in a high molecular weight presenilin complex, interact with all of the known components of the γ-secretase complex, co-localised with the complex components in the ER, Golgi and cell surface and destabilised from the complex in the absence of the presenilins and pen-2. Although the over-expression of TMP21 did not alter γ-secretase activity, its suppression resulted in and increase in Aβ40 and Aβ42. However, suppression of TMP21 did not alter the production of AICD, notch cleavage to generate NICD or cleavage of E-cadherin to generate CICD. These results are consistent with the notion that γ- and ε-secretase cleavage activities are independently regulated and indicate a role for TMP21 in modulating γ-secretase activity to generate Aβ. This role for TMP21 appears to be independent of its role in protein transport since the suppression of both TMP21 and p24a (a member of the p24 cargo family that interacts with TMP21) does not result in additional increases in Aβ production to that observed following the suppression of TMP21 only [82]. This finding led the authors to postulate that there are two pools of TMP21, a major pool that is stabilised by, p24a and has no role in Aβ production and a minor pool that modulates Aβ production, independent of p24a. Although, further investigation is required to determine the precise mechanism of action, it appears that TMP21 may function to regulate intramembrane proteolysis controlling γ-secretase activity and thus preventing the over-production of Aβ. This modulator role for TMP21 is important to consider when elucidating mechanisms on how γ-secretase activity is altered (for example by presenilin mutations) resulting in enhanced Aβ40 and Aβ42 production. Presenilin 2 (PS2): capable of forming a fully functional γ-secretase complex The presenilin proteins share an overall homology of 67% with the highest similarities in the predicted transmembrane domains. Fewer similarities are found at the N-terminus and in the central region of the hydrophilic loop [85, 86]. Although there are some structural similarities between PS1 and PS2 several lines of evidence suggest that these proteins may have distinct functions. Mice lacking PS1 die before birth and have severe skeletal and brain deformities, whilst those lacking PS2 only develop a mild pulmonary fibrosis and haemorrhage with age [33, 87–90]. Compared to neuronal cultures isolated from PS1 ablated mice, those isolated from PS2 knockout mice exhibit higher Aβ production and are less sensitive to γ-secretase inhibitors [89, 91]. Lai and colleagues [91] also provided evidence to suggest that distinct PS1 containing and PS2 containing complexes exist [91]. If this is the case, evidence to date suggests that PS2 containing complexes have different functions and have less γ-secretase processing power than PS1 containing complexes. However, recent in vitro evidence has suggested that chimeras of PS1 and PS2 fragments retain γ-secretase activity [92] suggesting that active complexes containing both PS1 and PS2 could occur. However, this is unlikely to occur in vivo as PS2 and PS1 transgenic mice have differential effects on γ-secretase activity [93]. In this study mice lacking PS1 were crossbred with transgenic mice containing wild type or mutant PS2/PS1. Compared to mice harbouring wild-type PS1, those harbouring wild-type PS2 had a ∼4-fold reduction in Aβ40 and ∼2-fold reduction Aβ42 brain levels. In addition, the authors reported that although mutations in PS1 lead to higher brain levels of Aβ40 and Aβ42 than PS2 mutations, those mice harbouring PS2 mutations had higher brain levels of Aβ42, whilst Aβ40 brain levels remain unchanged [93]. Taken together these studies suggest that PS1 containing complexes predominate in generating Aβ40 and Aβ42. However, the finding that PS2 is involved in Aβ42 (but not Aβ40) generation suggests that two Aβ generating γ-secretase complexes (γ-40 secretase and γ-42 secretase) exist, with γ-42 secretase complexes containing both PS1 and PS2. The roles of presenilin binding proteins not incorporated into the active γ-secretase complex In addition to Aβ generation, the presenilins have been implicated in a variety of intracellular processes including membrane trafficking, Notch signalling, neuronal plasticity, cell adhesion, regulation of calcium homeostasis, the unfolded protein response, and apoptosis. Therefore it is not surprising that the presenilins have a growing list of binding partners (see Table 1). Though the presenilin binding proteins that are incorporated into the γ-secretase complex are critical for the activity of this enzyme, those that are not integral components have equally important roles in modulating Aβ production. A number of non-complex presenilin binding proteins have been shown to alter γ-secretase activity (see Table 1). However, only a few have had supporting evidence that their direct interaction with the presenilins modulate γ-secretase activity. These are described below and shown in Fig. 2. Table 1Presenilin interacting proteinsInteracting proteinProposed functionInteraction with presenilins shown to modulate Aβ production ReferenceComplex componentsAPH-1Component of the γ-secretase complex; role for initial formation of the complex, or a proteolytic roleYes[19, 65, 67, 123].NicastrinComponent of γ-secretase complex; possible role as a receptor for APP-C99Yes[14, 55–58].PEN-2Component of the γ-secretase complex, role in proteolytic cleavage of the presenilins Yes [19, 24, 70, 76, 77]. TMP21Recently identified component of the γ-secretase complex, role in modulating Aβ productionYes[82]γ-secretase substratesAPP-C99Substrate for the γ-secretase complex: precursor to Aβ generationYes [38–40]E/N cadherinγ-secretase substrate; role in Cell-Cell AdhesionNo[124]IRE1γ-secretase substrate; role in unfolded protein responseNo[125, 126]LRPγ-secretase substrate; Lipid metabolismYes [119]Notch1 γ-secretase substrate; precursor to NICD generationYes [112]Interacting binding proteinsABP-280, Filamin homolog1Actin binding proteinNo[127]Bcl-2 Anti-apoptotic moleculeNo[128]Bcl-XLAnti-apoptotic moleculeNo[129]CalmyrinPossible calcium-myrstol switchNo[130]CALP- calsenilin like proteinNovel member of the calsenilin/KChIp protein familyNo[131]CalsenilinCalcium binding proteinYes[94, 96–99, 101, 102]CLIP-170Linking membrane organelles to microtubules No[132]DRALLIM-domain containing proteinNo[133]FHL2Role in PI3K/Akt activationNo[134]FKBP38Role in mitochondria mediated apoptosis No[135]GoSignalling moleculeNo[136]GSK3βWnt signalling, serine threonine protein kinase No[137]HC5/ZETASubunits of the catalytic 20S proteasomeYes[138]Met1Putative methyltransferaseNo[139]Modifier of cell adhesion protein (MOCA)Regulates proteasomal activity on APPYes[140]Omi/HtrA2Serine protease, proapoptoticNo[141]PAGNeuronal proliferation proteinNo[142]PAMP and PARLMetalloproteases No[143]PKASerine/Theronine protein kinase; β-catenin phosphorylationNo[144]PlakoglobinRole in b-catenin/Tcf-4 activityNo[145]PLD1Phospholipid-modifying enzymeYes [146]PSAPPDZ like proteinNo[147]QM/Jif1Negative regulator of c-JunNo[148]Rab proteinsVesicle mediated protein traffickingYes[149, 150]RyR2Cardiac ryanodine receptor No[151]SEL-10Ubiquitination of proteinsYes[152]SorcinCalcium binding regulator of ryanodine receptorNo[153]Syntaxin 1ASynaptic plasma-membrane proteinNo[154]Syntaxin 5ER-Golgi vesicular transportYes[155, 156]TauMicrotubule binding proteinNo[137]TelencephalinNeuron specific adhesion moleculeNo[40]TPIPTetratricopeptide repeat-containing proteinNo[157]UbiquilinUbiquitin domain-containing proteinNo[158–160]X11 family of proteinsCytoplasmic adaptor proteinsYes[110, 111]β-catenin/δ-catenin (NPARP)Wnt signalling. Cell adhesionNo[161–163] μ-CalpainCalcium-dependent thiol proteaseNo[164]Fig. 2The postulated roles for presenilin interacting proteins not incorporated into the γ-secretase complex in Aβ generation. A schematic representation of the postulated roles of presenilin interacting proteins in (A) promoting or (B) attenuating γ-secretase activity and Aβ generation. (A) Together with the formation of the γ-secretase complex, calsenilin have been shown to interact with the presenilins and promote Aβ generation when over-expressed in mammalian cells. The role of calsenilin in Aβ generation remains unclear but maybe involved in the presentation of the substrate to the γ-secretase complex. (B) The presenilin interacting protein, X11 and other γ-secretase substrates that compete for enzyme activity such as Notch fragments (i.e. NEXT) have all been shown to reduce Aβ generation when over-expressed in mammalian cells. X11 may reduce Aβ production by binding to PS and APP preventing PS mediated cleavage of APP or its presentation to the γ-secretase complex Calsenilin Calsenilin (also called DREAM or kChIP3) is a calcium binding protein, that was first identified through yeast two hybrid screening using the PS2 C-terminus as the bait protein [94]. The interaction between calsenilin and the presenilins appear to have a role in APP processing. It was initially demonstrated that calsenilin can form a complex with PS1 and PS2 and regulate presenilin fragment formation in mammalian cells [95]. This study also showed that calsenilin may facilitate presenilin mediated apoptosis, since calsenilin was shown to preferentially interact with the C-terminal fragment of PS2 that results from caspase cleavage of the presenilin holoprotein [96]. Expression studies in vivo (Xenopus) and in vitro (mammalian cells) have shown that the interaction between presenilin and calsenilin may be involved in ER calcium release and apoptosis [97, 98]. Over-expressing calsenilin in neuronal cells resulted in cell death which was enhanced by the co-expression of wild-type or PS1 mutations [99]. These studies have suggested that the interaction between calsenilin and presenilins may function in apoptotic pathways. However, several lines of evidence suggest that calsenilin may also be implicated in APP processing and Aβ generation. Initial in vitro studies showed that over-expression of calsenilin in mouse neuroblastoma cells resulted in a gene dose dependent accumulation of Aβ42 [100]. Using a γ-secretase luciferase based assay and a cell free system established by Li and colleagues [18] to characterise the γ-secretase complex in purified cell membranes, Jo and colleagues [101] recently reported that the over-expression of calsenilin resulted in an increase in γ-secretase activity. This calsenilin/presenilin, mediated activity was markedly reduced using a well-characterised γ-secretase inhibitor (L-685, 458). Furthermore, compared to wild-type mice, brain Aβ40 and Aβ42 levels were reduced in calsenilin ablated mice [102]. Together these studies would suggest that calsenilin has a second function, to facilitate γ-secretase activity. X11/Mint proteins A family of cytoplasmic adaptor proteins called X11 (also known as munc-18-interacting proteins—mints) have also shown to have a direct role in APP processing. In particular, several studies have reported that X11α and X11β stabilise APP and inhibit the secretion of Aβ and APP from neuronal cells [103–106]. The molecular mechanisms by which the X11s influence APP processing and Aβ production are not clear. However, this effect appears to be modulated by a direct interaction between X11 and APP. The interaction between the phosphotyrosine binding (PTB) domain within X-11 and the YENPTY motif within APP appears to modulate Aβ generation, since introduction of point mutations within these domains enhanced Aβ generation [103, 106–109]. The binding of X11 appears to stabilise APP, leading to its intracellular accumulation [103, 106–109]. It is conceivable that the X11–APP interaction may inhibit secretase accessibility to APP resulting in impaired Aβ secretion. The X11 proteins have multiple binding domains and have been shown to interact with the PS1 C-terminal fragment through their PDZ domain [110]. The authors also showed an interaction between PS1 and APP in the presence of X11 that was otherwise ablated in the absence of X11. This suggests that an interaction between APP and PS1 is mediated through X11. A subsequent study has reported that X11β and alcadein (and X11β-binding protein) expression prevents the binding of PS1 to APP [111]. The significance of the results from these interaction studies remains unclear. However, they do suggest that X11 proteins may act as inhibitory molecules preventing PS1 mediated proteolytic processing of APP and thus attenuating Aβ production. Competing substrates Some presenilin binding proteins reduce the likelihood of Aβ generation by competing for the catalytic activity offered by γ-secretase. One example is Notch1 which undergoes cleavage by γ-secretase to liberate an intracellular domain (NICD) that translocates to the nucleus where it co-activates transcription of genes involved in several developmental pathways [112–115]. The first evidence of substrate competition came from one study that treated neurons with the Noch1 ligand, delta, and found a dose dependent reduction in Aβ levels [116]. In contrast, the over-expression of APP in these neurons resulted in a reduction in Notch signalling [116]. Further evidence for substrate competition was provided by Kimberley and colleagues [117] where APP and Notch based substrates directly competed for γ-secretase activity in a cell free assay. Similar results were achieved in a subsequent study where cells expressing Notch showed a reduction in Aβ production [118]. Additional evidence for substrate competition has been provided recently for another substrate of γ-secretase, the cytoplasmic tail of the low-density lipoprotein receptor—related protein (LRP), which has also been shown to interact with PS1 [119]. The co- expression of the LRP C-terminal fragment and APP in CHO cells has been shown to result in a reduction in secreted levels of Aβ40 and Aβ42 [119]. Taken together, these studies not only suggest substrate competition but also the presence of a single γ-secretase. However, the presence of multiple γ-secretase complexes cannot be ruled out. As discussed above there is evidence in the literature for the formation of γ-secretase complexes with distinct functions. All of the studies described above are undertaken in conditions where there is a large amount of substrate available which could down-regulate the activity of one γ-secretase for another. Whether substrate competition occurs under physiological conditions and if there are certain conditions that promote a higher expression of one substrate over another remains to be determined. Concluding comments It is well established that together with presenilin, its binding proteins, nicastrin, aph-1 and pen-2 are the only proteins that constitute the γ-secretase complex responsible for generating Aβ from APP. The function(s) of each γ-secretase component within the complex is gradually becoming apparent. However, there are many facets of γ-secretase activity that are yet to be identified such as the domain responsible for actual catalytic activity; the mechanisms of enzymatic activity within the lipid bi-layer; and the existence of single or multiple γ-secretase complexes. Further investigation using appropriate protein expression models and reconstitution studies such as those used to validate the protein components of γ-secretase complex is required to provide some insight into the processes that underlie the catalytic activity of this enzyme. Indeed, reconstitution of the γ-secretase complex in insect cells and purification of this complex from the cells have provided researchers with the first 3D images of the structure of this intricate enzyme [120, 121]. Identification of the specific catalytic domain(s) within the complex would provide a site that could be a potential target to develop agents that specifically modulate Aβ levels without altering the other known (and unknown) activities of the γ-secretase enzyme. Recent insight into this notion was provided by the finding that certain compounds selectively block the cleavage of APP but not Notch [122]. However, it is important to note that other presenilin interacting proteins not incorporated into the complex and the pathways they are involved in play important roles in APP processing and Aβ generation. These interactions assist in many facets of γ-secretase activity, including modulating substrate/enzyme availability, and thus are important to consider when developing appropriate therapeutic strategies.

Biochim_Biophys_Acta-2-1-2396231

Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased (∼ 9-fold; 15–30 min) with later increases in expression of Klf4 and Klf6 (∼ 5-fold; 30–60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1–2 h (∼ 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1β or tumor necrosis factor α downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli. 1 Introduction C2H2 zinc finger transcription factors possess two cysteine and two histidine residues which co-ordinate Zn2+ within each finger to form a conserved DNA-binding structure. Sp1-like proteins and Krüppel-like factors (Klfs) each have three C2H2 zinc fingers at their C-termini, and Klfs are distinguished by a highly conserved interfinger space sequence [1]. The seventeen mammalian Klfs which have been identified are now designated Klf1-17 [2,3], though many have alternative names. Several were named according to the tissues in which they were originally shown to be enriched (e.g. Klf1 is erythroid Eklf, Klf2 is lung Lklf, Klf4 is gut Gklf, Klf5 is intestinal Iklf and Klf15 is kidney Kklf [2]). Other Klfs are widely expressed including Klf3 (basic Bklf), Klf6 and Klf7 (ubiquitous Uklf). Klf9 was identified as a basal transcription element binding (BTEB) protein, with Klf5 and Klf13 as homologues (BTEB2 and BTEB3, respectively). Klf10 and Klf11 were identified as early genes induced by transforming growth factor β and were named TIEG and TIEG2, respectively. Klfs bind to consensus GC-rich or CACCC sequences through the three C-terminal zinc fingers [1]. The frequency of such sequences in gene promoters, coupled with the high sequence identity between Klf proteins in their DNA-binding domains, raises questions relating to the specificity of any Klf for a particular promoter and potential functional redundancy. Nevertheless, Klfs can be clustered according to whether they act primarily as transcriptional repressors and/or activators, and the domain structure of the N-terminal regulatory regions. Klf3, Klf8 and Klf12 recruit C-terminal binding proteins (CtBPs) to repress transcription, whereas Klf9, Klf10, Klf11, Klf13 and Klf16 repress transcription through interaction with mSIN3a [1,4]. Klf15 also represses transcription, though the mechanism is not clear. Klf2, Klf4, Klf5, Klf6, and Klf7 are largely transcriptional activators [1], but may suppress gene expression in specific circumstances (e.g. Klf5 negatively regulates expression of Klf4 [5]). In addition to interactions with other proteins, post-translational modifications (e.g. phosphorylation or acetylation) regulate the transactivating activities of different Klfs [1,6]. Globally, Klfs regulate fundamental cellular responses such as growth, apoptosis, angiogenesis and proliferation. For example, Klf5 overexpression is associated with cell proliferation, whereas Klf2, Klf4 and Klf6 are more consistently implicated in cell cycle arrest [7–9]. Klf4 and Klf5 may both promote apoptosis induced by oxidative stress though the mechanism is unclear [9]. Mammalian cardiac myocytes (the contractile cells of the heart) become terminally-differentiated shortly after birth. Subsequent growth of the heart results from an increase in size of individual cardiac myocytes and, in the adult, myocytes may hypertrophy in order to maintain or increase cardiac output. This is associated with physiological and morphological changes (increases in cell size and myofibrillogenesis) and changes in gene expression including increased expression of immediate early genes (IEGs), and re-expression of genes normally expressed in early development [10]. Stimuli such as endothelin-1 (ET-1), which activate Gq protein-coupled receptors are particularly implicated in cardiac myocyte hypertrophy, and promote both the physiological/morphological changes and the changes in gene expression associated with the response [11]. In contrast, oxidative stresses or pro-inflammatory cytokines may induce cardiac myocyte apoptosis [12]. Many studies have examined the intracellular signalling pathways which are activated by various stimuli in cardiac myocytes, and these are presumed to lead to changes in gene and protein expression to promote hypertrophy or to facilitate myocyte death [13]. Many Klfs are expressed in adult hearts to a degree although the cell types in which they are expressed cannot be ascertained. For example, Klf2 is associated with endothelial cells [14,15], and cardiac endothelial cells could account for expression of Klf2 in whole heart extracts [16]. As reviewed by Haldar et al. [17], Klf13 and Klf15 are the only Klfs which have so far been shown to play a role in cardiac myocytes. Klf13 is highly expressed in adult hearts [18] and is required for normal cardiac development in Xenopus [19]. Klf15 is highly expressed in adult hearts and in cardiac myocytes [20,21]. It appears to be anti-hypertrophic since it is downregulated during hypertrophy, and overexpression of Klf15 in cardiac myocytes suppresses the morphological changes and changes in gene expression induced by hypertrophic stimuli [21]. Our microarray studies highlight the dynamic changes in expression of many genes (including Klfs) induced by H2O2 (an example of oxidative stress) or ET-1 in cardiac myocytes over 4 h [22–25]. Here, we explore further the regulation of Klf family members in cardiac myocytes in response to ET-1, H2O2 and pro-inflammatory cytokines [interleukin 1β (IL-1β) and tumour necrosis factor α (TNFα)]. The dynamic regulation of expression of multiple Klf family members suggests that, as a family, they are actively involved in regulating phenotypic responses of cells to extracellular stimuli. 2 Materials and methods 2.1 Primary culture of neonatal rat cardiac myocytes and preparation of polysomes Myocytes were dissociated from the ventricles of 2- to 4-day-old Sprague‑Dawley rat hearts by an adaptation of the method of Iwaki et al. [26] as previously described [27]. Cells were plated at 4 × 106 cells/60 mm Primaria culture dish (BD Biosciences) for 18 h in 15% (v/v) foetal calf serum, then serum was withdrawn for 24 h before experimentation. Myocytes were exposed to ET-1 (100 nM), H2O2 (0.2 mM), IL-1β (100 ng/ml) or TNFα (10 ng/ml) with or without pre-treatment (10 min) with inhibitors (10 μM U0126; 50 μM LY294002; 5 μM SB203580). Cardiac myocyte polysomes were prepared by sucrose density centrifugation (0.8–1.6 M sucrose gradients; 5 ml) as previously described [25]. Fractions were collected by upward displacement (fraction 1 from the top of the gradient, 0.8 M sucrose; fraction 12 from the bottom of the gradient, 1.6 M sucrose) whilst monitoring absorbance at 254 nm. 2.2 RNA preparation and quantitative PCR (qPCR) Total RNA and polysome RNA (fractions 6–11 of the sucrose gradients) were extracted and cDNA synthesized using reverse transcription as previously described [22,25]. qPCR was performed using a Real‑Time PCR System (Model 7500, Applied Biosystems) as described [25] using primers designed to amplify mRNA sequences across an intron (Table 1). qPCR analysis of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was performed as a control and the relative quantification protocol was used. PCR conditions were 50 °C for 2 min, 95 °C for 10 min (Jump-Start Taq polymerase activation step), followed by 40 cycles of 95 °C for 15 s and 59 °C for 60 s. Following qPCR, dissociation curve analysis was routinely performed to check for aberrant amplification products (e.g. primer‑dimers). 2.3 Western blotting Cardiac myocyte nuclear extracts were prepared and Western blotting performed essentially as described [28]. Extracts from 1.8 × 106 cells were analysed with separation of proteins on 10% (v/v) polyacrylamide gels. Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies (Santa Cruz Biotechnology Inc.; Klf6(R-173), sc-7158, 1/500 dilution). Bands were detected and analysed by scanning densitometry as described [29]. 3 Results 3.1 ET-1 regulates expression of multiple klf family members in cardiac myocytes Our microarray studies (with Affymetrix rat genome 230 2.0 arrays) of the effects of ET-1 on cardiac myocyte gene expression over 4 h [25] identified Klfs as a family of transcription factors which appeared to be dynamically regulated at the mRNA level. Mining the data specifically for Klfs, we identified rapid and transient increases in expression of Klf2, Klf4, Klf5, Klf6, Klf9 and Klf10, with concomitant downregulation of Klf3, Klf11 and Klf15 (Table 2). There was no statistically significant change in expression of Klf13 or Klf16, although Klf13 exhibited a small (non-significant; one-way ANOVA with Tukey post-test) increase in expression over 2– 4 h. Klf1 (probeset 1382033_at), Klf7 (probesets 1380363_at, 1377618_at, 1384497_at) and Klf12 (probeset 1385545_at) were not consistently called “present” with low fluorescence values and are thus expressed at minimal levels. We could not identify Klf8, Klf14 (Sp6) or Klf17 on the arrays. Using qPCR, we examined more carefully the effects of 100 nM ET-1 on Klf mRNA expression. Klf2 mRNA expression was significantly increased within 15 min, with maximal expression (∼ 9-fold relative to controls) at 30 min (Fig. 1A). Klf4 and Klf6 mRNAs were also upregulated (Fig. 1, B and C), though the relative stimulation was less (∼ 5-fold relative to controls) and the response was delayed relative to Klf2 (maximal expression at 0.5–1 h). Klf5 mRNA was upregulated from 1 h, with maximal expression at 1.5 h (∼ 13-fold relative to controls) (Fig. 1D). For these Klfs, the increase in expression was transient, with expression of Klf2 returning to baseline within 90 min and expression of Klf4, Klf5 and Klf6 declining to basal levels within 4 h. The increases in expression of Klf9 and Klf10 were relatively small (maximal expression < 2-fold at 1.5–2 h) although levels remained elevated over 4–6 h (Fig. 1, E and F). Consistent with our microarray data, Klf3, Klf11 and Klf15 were downregulated in response to ET-1. Whereas the decrease in expression of Klf3 was transient with minimal expression at ∼ 30 min (Fig. 1G), the decrease in expression of Klf11 and Klf15 was sustained over at least 6 h (Fig. 1, H and I). It is notable that the Klfs which were downregulated and those with only a small increase in expression are largely associated with transcriptional repression, whereas the Klfs with the greatest increases in expression (Klf2, Klf4, Klf5 and Klf6) are mostly associated with transcriptional activation [1,4,6]. Further studies focused on this latter group. 3.2 Regulation of Klf2, Klf4, Klf5 and Klf6 by ET-1 To determine whether Klf2, Klf4, Klf5 and Klf6 mRNAs were upregulated as IEGs (i.e. the increase in expression is regulated by pre-existing transcription factors and does not require synthesis of new proteins), we examined the effects of 20 μM cycloheximide (to inhibit protein synthesis). Cycloheximide alone increased the expression of Klf2, Klf4 and Klf6 mRNAs (Fig. 2A; probably due to activation of stress-activated signalling pathways [30,31]), but did not inhibit the increase in expression induced by ET-1. However, the increase in expression of Klf5 by ET-1 was inhibited by cycloheximide (Fig. 2B), indicating that de novo protein synthesis is required for upregulation of Klf5 mRNA. Thus, Klf2, Klf4 and Klf6 are regulated as IEGs in response to ET-1, whereas Klf5 is a second phase gene. ET-1 potently activates the extracellular signal-regulated kinases 1/2 (ERK1/2) in cardiac myocytes [32]. We used 10 μM U0126 (a selective inhibitor of the ERK1/2/5 cascades [33–35], though ERK5 is not significantly activated by ET-1 [24]) to determine whether the increase in expression of Klf2, Klf4, Klf5 and Klf6 requires ERK1/2 signalling. U0126 minimally affected basal expression of each Klf, but partially inhibited the ET-1-induced increase in expression of Klf2 (56 ± 4% inhibition at 30 min; mean ± SEM, n = 5), Klf4 (43 ± 9% inhibition at 1 h; n = 4), Klf5 (72 ± 8% inhibition at 1 h; n = 4) and Klf6 (45 ± 8% inhibition at 1 h; n = 4) (Fig. 3A). These data suggest that ERK1/2 signalling is required, at least in part, for the upregulation of Klf2, Klf4, Klf5 and Klf6 mRNAs by ET-1. Signalling through phosphoinositide 3′ kinase (PI3K) may increase expression of Klf2 [36], and p38-MAPK is activated by ET-1 in cardiac myocytes [37], so we examined the effects of 50 μM LY294002 or 5 μM SB203580 (selective inhibitors of PI3K and p38-MAPK, respectively [34]). Surprisingly, LY294002 increased basal expression of Klf2 and Klf4 in cardiac myocytes and promoted further the increase in expression induced by ET-1 (Fig. 3B), indicating that PI3K signalling is not required for the upregulation induced by ET-1 and suggesting that basal PI3K signalling negatively regulates Klf2/Klf4 expression. LY294002 did not affect Klf6 expression, but attenuated the increase in expression of Klf5 by ET-1. Since basal PI3K signalling is required for protein synthesis in cardiac myocytes [38], the effect of LY294002 on the increase in Klf5 induced by ET-1 (which requires de novo protein synthesis; Fig. 2B) may be a reflection of this. SB203580 had a minimal effect on the basal expression of Klf2, Klf4, Klf5 or Klf6, or on the increase in expression induced by ET-1 (Fig. 3B). It is unlikely that p38-MAPK signalling plays a significant role in the response. 3.3 Translation-state analysis of Klf2, Klf4, Klf5 and Klf6 mRNA expression Given the increasing evidence for translational regulation of specific mRNAs [39,40], we performed translation-state analysis of Klf2, Klf4, Klf5 or Klf6 mRNAs. We used qPCR to compare the relative levels of expression in the total RNA pool and in RNAs associated with cardiac myocyte polysomes (prepared by sucrose density centrifugation; Fig. 4, A and B). Klf2, Klf4 and Klf6 mRNAs were marginally enriched in cardiac myocyte polysomes in unstimulated cells and, following stimulation with ET-1 (1 h), the relative increase in the polysomes was at least as great as in the total RNA pool (Fig. 4, C–E). Klf2 was increased 5.6-fold in the polysomes (4.8-fold in total RNA), Klf4 was increased 6.4-fold (5.0-fold in total RNA) and Klf6 was increased 4.6-fold (4.8-fold in total RNA). Klf5 mRNA was marginally decreased in the polysomes relative to the total pool, but the increase in expression induced by ET-1 in the polysomes (4.3-fold) was slightly higher than in the total pool (3.9-fold) (Fig. 4F). We therefore expect all these Klfs to be efficiently translated into protein. Consistent with this, Klf6 protein (detected as bands of ∼ 38–40 kDa on Western blots) was increased in cardiac myocytes exposed to ET-1, with maximal expression at ∼ 1 h (Fig. 4G). It could be argued that early genes should be efficiently translated and this may be a property of all such genes. However, Irs2 was upregulated as an IEG by ET-1 (no inhibition by cycloheximide, Fig. 2A), but the mRNA was not enriched in the polysomes and the increase in the polysomes was significantly less than that in the total RNA pool (Fig. 4H). Similarly, for interleukin 1 receptor-like 1 (Il1rl1), a second phase gene (upregulation was inhibited by cycloheximide; Fig. 2B), the increase in expression in the polysomes was significantly less than in the total RNA pool (Fig. 4I). It is therefore notable that Klf2, Klf4, Klf5 and Klf6 mRNAs are efficiently recruited to the polysomes for translation into protein. 3.4 Regulation of Klf2, Klf4 and Klf6 by H2O2 and pro-inflammatory cytokines We considered whether other stimuli also regulate expression of Klf2, Klf4 or Klf6 mRNAs. H2O2 (0.2 mM) promotes cardiac myocyte apoptosis [41], but also stimulates ERK1/2 (and other pathways) [42], and stimulated a transient increase in expression of Klf2 (Fig. 5A). However, the response was less than that of ET-1 (∼ 3.6-fold) and was delayed (maximal increase at 1 h) (Fig. 1A). Consistent with our previous report [23], 0.2 mM H2O2 upregulated Klf4 and Klf6 mRNAs with maximal expression (5.0-fold) from 1 h and sustained expression over at least 6 h (Fig. 5, B and C). We have observed a similar delay in induction of other IEGs by H2O2 compared with ET-1 (e.g. Fos family members, unpublished data), which may reflect the delayed time course for activation of ERK1/2 signalling (maximal activation of ERK1/2 by ET-1 is at ∼ 5 min [43]; maximal activation by H2O2 is at 15–30 min [42]). Consistent with a role for ERK1/2 signalling, U0126 inhibited the increase in expression of Klf2, Klf4 and Klf6 by H2O2 (Fig. 5D). As with ET-1, SB203580 did not affect the response to H2O2 (Fig. 5D), whereas LY294002 enhanced the response (Fig. 5E). In endothelial cells, Klf2 is downregulated by TNFα [15]. IL-1β or TNFα profoundly and rapidly (from ∼ 1 h) downregulated Klf2 mRNA expression, a response which was sustained over at least 6 h (Fig. 6, A and B). Neither cytokine significantly affected the expression of Klf4 or Klf6 (data not shown). The decrease in expression of Klf2 in response to IL-1β was not affected by either SB203580 or U0126, although LY294002 (which itself increased expression of Klf2 mRNA, Fig. 3B) restored the baseline levels of expression (Fig. 6C). 4 Discussion 4.1 Regulation of gene expression by Klf family members Many studies of individual Klfs relate to specific cells/tissues in which they are highly expressed or for which the phenotype of a transgenic mouse suggests they may play some role. Thus, most studies of Klf2 focus on its regulation in lung tissue, endothelial cells or T cells, whereas many studies of Klf4 are in gut, endothelial cells or smooth muscle cells, and Klf5 is studied largely in gut or smooth muscle cells. Although there are indications that Klfs are transcriptionally regulated in these various cells (e.g. Klf2 expression is increased by shear stress in endothelial cells [14]), and there is an indication of interplay between Klf family members (e.g. Klf4 and Klf5 appear to operate in an antagonistic manner to regulate cell cycle progression [8]), few studies consider the regulation and function of the Klfs as a family. Our data demonstrate substantial and dynamic regulation of nine Klfs by ET-1 in a single cell type (Fig. 1) with at least three regulated as IEGs (Fig. 2A), suggesting that Klfs potentially play a much more significant role in the phenotypic responses of more cell types than has so far been considered. Klfs are defined by the high degree of homology in their C-terminal DNA-binding domains [1,6]. It is probably not surprising, therefore, that all bind to similar consensus DNA sequences or, with 17 family members, that such elements are relatively common in gene promoters. The Klfs form two principal groups of transcriptional repressors (acting through CtBPs or mSin3a) plus a group which are primarily transcriptional activators, and it could be argued that the different Klf groups all do bind to similar sequences but the effect depends on whether the Klf is an activator or repressor and, if the latter, the mode of action. An additional factor could be that different cells express different Klfs and interactions with other cell-specific transcription factors may result in differential gene expression. In cardiac myocytes, we detected expression of at least 11 Klfs (Table 2), with regulation of several family members from each of the activator/repressor groups by ET-1 (Fig. 1), suggesting that (at least in this system) neither tissue specificity nor classification according to global function is responsible for functional differences between individual Klfs. Since there is evidence for functional specificity in other cells [44,45], minor variations in primary and secondary structure may be sufficient for individual Klfs to have differing affinities for precise sequences in various gene promoters. Expression of any individual gene may therefore reflect the balance of Klfs expressed at any particular time in relation to the precise DNA-binding sites which can be accessed by them. 4.2 Regulation of expression of Klf2, Klf4, Klf5 and Klf6 Of the Klfs which were upregulated by ET-1, Klf2 mRNA expression increased very rapidly and transiently (Fig. 1A), and it was regulated as an IEG (Fig. 2A). Partial inhibition of the response by U0126 (Fig. 3A) suggests that ERK1/2 signalling is required to some degree. ERK1/2 signalling was also required for upregulation of Klf2 by H2O2 (Fig. 5D). In contrast to endothelial cells subjected to shear stress [36], the increase in Klf2 expression by LY294002 and enhanced response to ET-1 in the presence of LY294002 (Fig. 3B) indicated that PI3K signalling negatively regulates Klf2 expression in cardiac myocytes. ERK5 phosphorylation of the transcription factor MEF2 promotes Klf2 expression in other cells [46], but ERK5 is not significantly activated in cardiac myocytes by ET-1 [24], ERK1/2 do not efficiently activate MEF2 [47] and inhibition of p38-MAPKs (which also phosphorylate MEF2 [47]) by SB203580 had no effect on Klf2 expression induced by ET-1 (Fig. 3B), suggesting that MEF2 is not the principal factor involved in this context. Of the transcription factors associated with the Klf2 promoter identified by Ahmed and Lingrel [48], the most probable candidate for regulating Klf2 expression by ET-1 in our study is CREB. CREB is rapidly (within 5–10 min) phosphorylated in cardiac myocytes exposed to ET-1, a response which is inhibited by U0126 [49]. Like Klf2, the increases in expression of Klf4 and Klf6 (Fig. 1, B and C) were inhibited by U0126, (Figs. 3A and 5D), and both were regulated as IEGs (Fig. 2A). Our data are consistent with other systems in which Klf4 is regulated as an IEG by 15-deoxy-Δ12,14 prostaglandin J2 [50] or platelet-derived growth factor [51], and Klf6 is regulated as an IEG by phorbol esters or serum [52,53]. However, for either Klf4 or Klf6, the potential transcription factors or mRNA stabilisation factors which promote the increase in expression are not known. The increase in expression of Klf5 induced by ET-1 was inhibited by cycloheximide (Fig. 2B), so the principal factors promoting its expression must be newly-synthesized proteins, and the response was inhibited by U0126 (Fig. 3A), implicating ERK1/2 signalling in the response. These data are consistent with other studies in which phorbol esters or fibroblast growth factor increase Klf5 expression in smooth muscle cells in an ERK1/2-dependent manner [54] and, in fibroblasts, the increase in expression induced by phorbol esters is inhibited by cycloheximide [55]. Studies of mRNA expression raise the question of whether or not changes in mRNA expression equate to changes in protein expression. In cardiac myocytes treated with ET-1, translation-state analysis indicated that mRNAs for Klf2, Klf4, Klf5 and Klf6 were increased to a similar or greater extent in the polysomes than in the total RNA pool (Fig. 4). Since ET-1 also increases the global rate of protein synthesis in cardiac myocytes [56] (illustrated by the increase in A254 profile of polysome fractions 6–11 with concomitant decrease of monosome fractions 2–4; Fig. 4A), overall, we would predict that the rate of synthesis of each of the Klf proteins should be increased at least in proportion to the mRNA. Consistent with this, we detected a significant, transient increase in expression of Klf6 protein following stimulation with ET-1 (Fig. 4G). Although cellular stresses such as oxidative stress can generally inhibit translation, 0.2 mM H2O2 does not have a significant effect on global protein synthesis over 4 h, and our microarray data indicate that Klf2, Klf4, and Klf6 are increased to a similar extent in the polysomes as in total RNA (data not shown). 4.3 Regulating Klf-dependent gene expression in cardiac myocytes Downregulation of Klf15 is associated with the hypertrophic response of cardiac myocytes and may be required for hypertrophy to develop [21]. However, our data suggest that the situation regarding Klf-mediated regulation of gene expression in cardiac myocytes is more complex. In cardiac myocytes exposed to ET-1, we detected a rapid decrease in expression of mRNAs for three established repressors of gene expression (Klf3, Klf11 and Klf15; Fig. 1, G, H and I), with simultaneous rapid increases in expression of four Klfs more commonly associated with transcriptional activation (Klf2, Klf4, Klf5 and Klf6; Fig. 1, A–D). In response to ET-1, therefore, there appears to be an immediate overall switch towards increasing Klf-directed gene expression. However, the increases in expression of Klf2, Klf4, Klf5 and Klf6, and decrease in expression of Klf3 were transient, with a delayed increase in expression of Klf9 and Klf10, suggesting that the balance of Klf-regulated gene expression continues to change. A similar effect was detected with H2O2, with an early increase in expression of Klf2, Klf4, Klf5 and Klf6 and simultaneous decrease in expression of Klf3, Klf11 and Klf15 (Fig. 5 and [23]). In contrast, Il-1β or TNFα promoted downregulation of Klf2 expression (Fig. 6) with little effect on Klf4 or Klf6. Clearly, the genes which each of the Klfs regulates in cardiac myocytes remain to be established.

Histochem_Cell_Biol-4-1-2248605

Imaging and imagination: understanding the endo-lysosomal system

Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today. Introduction Lysosomes are the terminal degradative compartments of the endocytic pathway. They receive extracellular components via endocytosis and intracellular material via autophagy, as well as via the biosynthetic pathway (Eskelinen 2005; Klionsky 2007; Luzio et al. 2007; Sachse et al. 2002a). By modulating the degradation of a multitude of proteins, lysosomes are key organelles in the regulation of cellular homeostasis. Moreover, these catabolic functions are complemented by a group of cell type-specific lysosome-related organelles, which store and/or secrete key factors involved in a diversity of functions, such as blood clotting, antigen presentation, pigmentation and alveolar surface tension regulation (dell’Angelica et al. 2000a; Raposo et al. 2007; Weaver et al. 2002). Because of their many functions, defects in lysosome functioning can have devastating consequences, as is illustrated by the rapidly growing number of human disorders displaying a primary or secondary defect in the lysosomal system. To date, lysosomal aberrations have been found in the ‘classical’ lysosomal storage disorders, as well as in muscular and neurological disorders, numerous types of cancer, immune-deficiency diseases and pigmentation-bleeding disorders (Futerman and van Meer 2004; Kroemer and Jäättelä 2005; Mohamed and Sloane 2006; Nixon 2005; Saftig et al. 2001). Unraveling the intrinsic complexity of the endo-lysosomal system is a major challenge for cell biologists. Here, we will review some of the current insights in the mechanisms involved in lysosome biogenesis and functioning. Imaging of the endo-lysosomal system combined with imaginative visions has been instrumental for furthering the conceptual understanding of the endo-lysosomal system. Therefore, on the special occasion of the 50th anniversary of the Journal of Histochemistry and Cell Biology, we will also highlight some examples in the literature showing how imaging, especially electron microscopy (EM), has contributed to our understanding of the endo-lysosomal system today. The discovery of the lysosome The famous story of the discovery of the lysosome reads as a compelling detective novel (Bainton 1981; de Duve 2005). A major question that was faced by the cell biological society of the fifties was how cells could exert proteolytic activity without being digested by autolysis. The first indications on the existence of lysosomes came from studies designed to answer an entirely different question-i.e. to clarify the action of insulin on the liver-in which acid phosphatase activity (nowadays known as a lysosomal activity) measurements were used as a control. A combination of experiments from the labs of Christian de Duve and Albert Claude led to the intriguing observation that acid phosphatase in fresh fractions, prepared by a relatively gentle protocol, showed astonishingly low activity levels. By contrast, samples that were obtained by a crude fractionation protocol or stored for some days before assaying showed elevated levels of phosphatase activity. When this same hide-and-seek strategy was found for four additional hydrolytic enzymes, all with an acidic pH optimum and the same sedimentation characteristics as acid phosphatase, de Duve concluded that these enzymes must be contained together in particles surrounded by a membrane ‘to prevent the enzymes getting out and the substrate getting in’. The term lysosome, for lytic particle or digestive body, was introduced (de Duve et al. 1955). Novikoff, together with de Duve, provided the first EM pictures of these liver fractions that in addition to numerous mitochondria also showed an enrichment of electron dense bodies (Fig. 1). These were tentatively recognized as the ‘pericanalicular dense bodies’ previously found in intact liver cells (Novikoff et al. 1956). In these same years, Gomori had developed a protocol for acid phosphatase cytochemistry (Gomori 1952), which was subsequently optimized for EM by Novikoff, Holt, Barka and Anderson (Barka and Anderson 1962; Holt 1959; Novikoff 1961). The circle was closed when the acid phosphatase staining was applied to the ultrastructural level, thereby providing the direct evidence that acid phosphatase activity localized to the dense bodies of the liver parenchyma cells (Essner and Novikoff 1961) (Fig. 2), as well as similar bodies in various other cell types. Fig. 1Reproduction of the first electron micrograph ever that indicated the existence of lysosomes (arrows), as originally published in 1956 by Novikoff and colleagues. The sample represents a liver fraction that also contains mitochondria (MT) and endoplasmic reticulum (ER). Reproduced from Novikoff et al. (1956)Fig. 2Reproduction from Essner and Novikoff (1961). This picture shows for the first time at the ultrastructural level that acid phosphatase activity is localized in lysosomes (LY) of liver cells. BC bile canaliculus, ER endoplasmic reticulum, EX extraneous precipitate, MI microvilli, V vacuole Another major clue was provided by Werner Straus. He found that ‘droplets’ of unknown function within the proximal tubules of the kidney contained reabsorbed (i.e. endocytosed) material as well as acid phosphatase activity and other lysosomal hydrolases (Straus 1954, 1956). This observation provided the first link between lysosomal digestion and endocytic uptake of extracellular material. Hence, the concept was born of the lysosome as a membrane-bound organelle that contains acid hydrolases involved in the digestion of substances that enter the cell via endocytosis (de Duve 1959). It explained that cells digest without autolysis by setting apart the degradative enzymes together with their endocytosed substrates in a membrane-bound compartment. The answer to the riddle was found. The endocytic pathway in a nutshell Lysosomes receive extracellular substances for degradation via endocytosis: the invagination and pinching-off of membrane-bound vesicles from the plasma membrane (Fig. 3a). There are at least five highly controlled different entries into the cell, of which the ‘classical’ clathrin-mediated pathway is best-documented. However, the importance of non-clathrin-mediated pathways is becoming increasingly evident (Conner and Schmid 2003; Kirkham and Parton 2005; Mayor and Pagano 2007; Nichols 2003). Clathrin-mediated endocytosis begins with the sorting of receptors into a clathrin-coated pit at the plasma membrane, which then buds off to form a transport vesicle. These vesicles fuse with the first intracellular sorting station, the early endosome (Fig. 3a). Early endosomes have a mildly acidic pH that triggers the dissociation of some ligands from their receptors. Emptied receptors enter membrane tubules that emerge from the endosomal vacuole, resulting in transport via the recycling endosome back to the plasma membrane or, alternatively, the trans-Golgi network (TGN) (Fig. 3a). Receptors that remain ligand-bound are sequestered into small 50–80 nm diameter intraluminal vesicles (ILVs) that bud from the endosomal-limiting membrane into the endosomal lumen. Through a series of such vesicle fusion and fission events involving protein sorting, early endosomes gradually transform into late endosomes, a process called endosomal maturation. The formation of ILVs can continue up to the late endosome, which is why during endosomal maturation the number of these internal vesicles increases and late endosomes are often referred to as multivesicular bodies (MVBs, Fig. 3a). Degradation of cargo starts in late endosomes/MVBs and continues in the lysosome. Late endosomes/MVBs develop into lysosomes by multiple fusion and fission cycles (Futter et al. 1996; Luzio et al. 2007). After degradation of the lysosomal content, transporter molecules translocate sugars, amino acids and lipids across the limiting lysosomal membranes into the cytosol where they function as building blocks in the synthesis of new products. Fig. 3Sorting and transport within the biosynthetic and endo-lysosomal pathways. a Schematic representation of the interplay between the biosynthetic pathway and endo-lysosomal system depicting the main endosomal compartments that are discussed in the text. b Detailed overview of the various sorting steps at the early endosome. At the endosomal vacuole or sorting endosome, receptor–ligand complexes destined for degradation accumulate in the bilayered clathrin coat (1), upon which they are sorted into intraluminal vesicles (ILV) that form by inward budding of the vacuolar limiting membrane. Recycling cargo by default enters the membrane tubules of the tubular sorting endosome (2) from which it can recycle back to the plasma membrane via the recycling endosome, or is actively sorted towards late endosomes/lysosomes (AP-3 pathway for lysosomal membrane proteins) or TGN (AP-1 pathway). A third exit from the vacuolar endosome is provided by the endosome-to-TGN carriers (3) that mediate SNX1-dependent recycling of MPRs and sortilin to the TGN. c From the TGN, multiple pathways arise that mediate transport to the endo-lysosomal system and plasma membrane. It is still an open question to what extent these pathways use different or overlapping molecular machinery and transport carriers. AP adaptor protein complex, ER endoplasmic reticulum, M6P mannose 6-phosphate, MPR mannose 6-phosphate receptor, SAP sphingolipid activator protein, SNX1 sorting nexin 1, TGNtrans-Golgi network Defining early and late endosomes When studying the endo-lysosomal system, one is forced to continuously pose the question how to discriminate between the distinct endosomal intermediates. The distinction ‘early’ and ‘late’ endosomes often has too limited discriminating power to describe the dynamic continuum of the endo-lysosomal pathway. In a recent EM study (Mari et al. 2007), a novel method to distinguish early and late endosomes was introduced by combining morphological, kinetic and molecular criteria. Early endosomes are by definition accessible to internalized transferrin (Tf) that recycles to the plasma membrane. As a result, late endosomes contain only marginal levels of Tf. With this in mind, the number of ILVs within the endosomal vacuoles was correlated with the occurrence of internalized Tf. Tf-positive early endosomal vacuoles on average contained one to eight ILVs, whereas endosomal vacuoles with nine or more ILVs were mostly devoid of Tf and therefore designated as late endosomes. A similar analysis with various other established early endosomal marker proteins corroborated and extended this definition. For example, the early endosomal antigen 1 (EEA1) appeared to be connected with only the very early maturation stages of early endosomes (one to five ILVs), whereas Rab11 appeared only on the later maturation stages of early endosomes (four ILVs or more). The early endosomal markers Rab4 and Hrs were more evenly distributed. Although the interpretation of this study is currently limited to the cell line in which the analysis was performed, i.e. the human hepatoma cell line HepG2, these observations illustrate that distinct stages of endosomes can be better monitored with increasing means of analysis. TGN-to-endosome transport pathways To fulfill their degradative function, lysosomes must also receive lysosomal proteins. These are synthesized in the endoplasmic reticulum and Golgi complex, after which they enter the TGN (Fig. 3a). From the TGN, multiple pathways to the endosomal system arise, mediating delivery of essential components to various endosomal intermediates. Thus, lysosomes receive their components via both the endocytic pathway and biosynthetic pathway. Two classes of proteins together are essential for lysosomal functioning: the soluble lysosomal hydrolases and lysosomal membrane proteins. Each lysosomal hydrolase targets specific substrates for degradation and presently more than 50 different types are known. The heavily glycosylated lysosomal membrane proteins encompass a group of proteins with various functions. The major lysosomal membrane proteins (Marsh et al. 1987) are the lysosome-associated membrane proteins (LAMP)-1 and -2, the lysosomal integral membrane protein (LIMP)-2 and the tetraspanin CD63 [originally identified as platelet-activating glycoprotein (Pltgp40) and also often referred to as melanoma-associated antigen ME491, LIMP-1 or LAMP-3]. Initially, lysosomal membrane proteins were thought to mainly play a role in lysosomal stability and integrity, but currently new functions emerge involving chaperone-mediated autophagy and macroautophagy (reviewed by Eskelinen et al. 2003). Thus, the proper targeting of lysosomal proteins from the TGN to the endo-lysosomal system is an essential process in the biogenesis and maintenance of lysosomes. The best-known pathway for TGN-to-endosome transport is the delivery of soluble lysosomal hydrolases by the 300 kD cation-independent (CI) and 46 kD cation-dependent (CD) mannose 6-phosphate receptors (MPR) (Kornfeld and Mellman 1989; von Figura 1991). However, as will be outlined in the next paragraphs, increasing evidence indicates the existence of multiple additional or alternative pathways from the TGN to endosomes. MPR-dependent transport of soluble lysosomal hydrolases Both the CI and CD-MPR are present in almost all mammalian cell types. The lumenal domain of the CI-MPR is composed of 15 repetitive units, with two mannose 6-phosphate (M6P)-binding sites, whereas the CD-MPR has a lumenal domain of 159 amino acids, resembling one such unit, and has a single M6P-binding site. The M6P-binding sites recognize M6P-moieties, which are specifically present on the precursor forms of newly synthesized lysosomal hydrolases. Both receptors predominantly function as non-covalently bound homodimers. A specific role for each MPR in the targeting of lysosomal hydrolases has not been established, but distinct lysosomal hydrolases can exhibit different affinities for CI or CD-MPR (Qian et al. 2008; Sleat and Lobel 1997). CI-MPR can endocytose extracellular lysosomal hydrolases, whereas under physiological conditions CD-MPR is not involved in endocytosis. CD-MPR shows enhanced ligand binding in the presence of divalent cations (Hoflack and Kornfeld 1985a, b). Interestingly, the CI-MPR also has several other functions; it regulates the levels of circulating insulin-like growth factor II by binding it at the plasma membrane for delivery to lysosomes. Furthermore, it facilitates the activation of the precursor of transforming growth factor-β1, which is a growth inhibitor, and also binds retinoic acid, thereby mediating retinoic acid-induced apoptosis and growth inhibition, further supporting the idea that CI-MPR could be a tumor suppressor (reviewed by Ghosh et al. 2003a). Together, the two MPRs mediate the targeting of the huge majority of newly synthesized lysosomal hydrolases from the TGN to the endo-lysosomal system (Fig. 3c) (Hoflack and Kornfeld 1985a, b; Sahagian et al. 1981). In steady state, both CD and CI-MPR have been localized by immuno-EM to the TGN, plasma membrane, endosomes and associated vesicles (Bleekemolen et al. 1988; Geuze et al. 1984, 1985; Griffiths et al. 1988; Klumperman et al. 1993, 1998). Sorting of MPRs at the TGN was long thought to predominantly depend on binding to the heterotetrameric adaptor-protein complex (AP)-1, which also mediates the recruitment of clathrin (Fig. 4). The discovery of the Golgi-localized, γ-ear-containing, Arf-binding family of proteins (GGA) as clathrin adaptors changed this view (Boman et al. 2000; dell’Angelica et al. 2000b; Hirst et al. 2000; Poussu et al. 2000). GGAs are monomeric proteins and three different forms are known in mammals, GGA1, GGA2 and GGA3. GGAs and AP-1 might function in parallel to generate distinct MPR-containing vesicles at the TGN, thereby allowing delivery to different endosomal compartments. Alternatively or additionally, GGAs may facilitate entry of MPR into clathrin-coated vesicles by interacting with AP-1. A major clue in favor of the latter hypothesis was provided by immuno-EM showing that GGAs and AP-1 colocalize in the same clathrin-coated TGN buds (Doray et al. 2002; Puertollano et al. 2003). Furthermore, the three GGAs were found to interact with each other and also to bind directly with their hinge domain to the γ-ear domain of AP-1. Depletion of each GGA resulted in decreased levels of the other GGAs, their redistribution to the cytosol and impaired sorting of CI-MPR and the lysosomal hydrolase cathepsin D (Doray et al. 2002; Ghosh et al. 2003b). However, these findings do not rule out the possibility that different adaptor proteins can also act separately to a certain extent. Interestingly, it is not precisely known where the MPRs deliver their bound ligands. A small percentage of the MPRs travels to the cell surface, from where they are internalized by clathrin-mediated endocytosis and routed to the early endosomes. However, the majority takes a direct intracellular pathway to the endo-lysosomal system, and it is generally believed that both early and late endosomes can be recipients for TGN-derived MPR. The involvement of multiple adaptors could provide a means to establish these distinct pathways. Fig. 4Immuno-EM of a human hepatoma cell (HepG2) showing the occurrence of CIMPR (represented by 10 nm gold particles) in AP-1 (represented by 15 nm gold particles) coated TGN membranes (arrowheads). CI-MPR and AP-1 positive membranes also show the characteristic cytoplasmic dense coat indicative for the presence of clathrin. The TGN area is enclosed by distinct golgi stacks (G). Bar, 200 nm MPR-independent transport of soluble lysosomal hydrolases In addition to the well-characterized MPR pathway, several additional transport pathways for lysosomal hydrolases exist (Fig. 3c). For example, the lysosomal hydrolases lysosomal acid phosphatase and β-glucocerebrosidase do not acquire M6P residues and their transport is not depending on MPRs (Aerts et al. 1988; Waheed et al. 1988). Lysosomal acid phosphatase is a type I transmembrane protein that follows the constitutive secretory pathway to the plasma membrane and reaches the lysosome via endocytosis. In lysosomes, the transmembrane precursor is processed into a soluble form, but both proteins are enzymatically active (Waheed et al. 1988). Lysosomal acid phosphatase contains the tyrosine-based motif YRHV in its cytosolic domain, which is required for endocytosis, but does not mediate direct TGN-to-endosome targeting (Obermüller et al. 2002; Peters et al. 1990; Pohlmann et al. 1988). β-Glucocerebrosidase has no membrane spanning domain (Erickson et al. 1985). After synthesis, it is glycosylated upon which it becomes membrane-associated (Rijnboutt et al. 1991). The machinery required for proper transport of β-glucocerebrosidase was long unknown. Recently, Paul Saftig and colleagues unexpectedly identified the lysosomal membrane protein LIMP-2 as a specific receptor for β-glucocerebrosidase. LIMP-2 binds β-glucocerebrosidase in a pH-dependent manner, enabling association in the ER and guiding it all the way to the lysosome, where the complex likely dissociates because of the acidic pH (Reczek et al. 2007). Whether LIMP-2 recycles between endosomes and TGN is not known. This study for the first time implies a lysosomal membrane protein in the transport of a lysosomal hydrolase. It will be very interesting to establish whether LIMP-2 acts as a more general binding partner for lysosomal hydrolases. In addition, characterization of the molecular machinery required for transport will provide further insight into this TGN-to-endosome transport pathway. Lysosomal hydrolases that are normally modified with a M6P residue can also traffic in an MPR-independent way to the lysosomes (Fig. 3c). This became apparent by comparing different cell types from patients with I-cell disease, also known as Mucolipidosis type II. In this disease, soluble lysosomal hydrolases do not acquire M6P groups due to a deficient activity of the enzyme GlcNAc-phosphotransferase (Hasilik et al. 1981; Reitman et al. 1981). As a consequence, the lysosomal hydrolases follow the default constitutive pathway to the plasma membrane (Fig. 3c) and are thus secreted. However, in cells of non-mesenchymal origin, most lysosomal hydrolases are still targeted correctly to the lysosome (Fig. 5) (Little et al. 1987; Owada and Neufeld 1982; Waheed et al. 1982), suggesting the existence of alternative pathways. The same phenomenon was observed in a very recently established mouse model system for I-cell disease, achieved by GlcNAc-phosphotransferase knockout (Gelfman et al. 2007). Furthermore, in mice that are deficient of both MPRs, the intracellular activities of several lysosomal hydrolases are comparable to the control situation (Dittmer et al. 1999). These findings indicate that lysosomal hydrolases can follow a pathway to the lysosome that is not mediated by MPRs. Fig. 5 Immuno-EM of a B lymphoblast derived from a patient with I-cell disease showing a lysosome (L) positively labeled for the lysosomal hydrolase cathepsin D (represented by 10 nm gold particles). This picture indicates that although the MPR pathway is impaired in these cells, lysosomal enzymes can still reach lysosomes. Bar 200 nm The mechanisms and pathways of MPR-independent transport are still poorly defined. Recent studies, however, have implicated a role for the multiligand receptor sortilin as an alternative receptor for a subset of lysosomal proteins, called sphingolipid activator proteins (SAPs), which are non-enzymatic cofactors required for the degradation of glycosphingolipids. Two SAPs are known; the precursor protein prosaposin, which renders saposin A, B, C and D after proteolysis, and GM2 activator protein (AP), an essential cofactor for β-hexosaminidase A. Studies from Lefrancois and colleagues have shown that sortilin interacts with both prosaposin and GM2AP, which is independent of M6P tags, whereas depletion of sortilin induced their secretion (Lefrancois et al. 2003). It remains to be established, however, whether sortilin is the exclusive receptor for SAPs, or whether MPR has an additional role in their transport. Interestingly, sortilin, together with SorLA and SorCS1–3, forms a protein family sharing homology to the lumenal domain of the yeast vacuolar sorting protein Vps10p, which directs carboxypeptidase Y to the vacuole (Marcusson et al. 1994). Immuno-localization studies positioned sortilin predominantly in endosomes and the TGN (Mari et al. 2007), where it colocalized with CI-MPR in AP-1 and GGA3 positive clathrin-coated transport carriers, indicating that sortilin and MPRs travel via the same carriers to the endo-lysosomal system. Currently, the functions of the SorCS-subgroup are unknown, but since SorCS3 is predominantly localized at the plasma membrane it is unlikely to be involved in TGN-to-endosome trafficking (Westergaard et al. 2005). Whether sortilin or possibly SorLA could act as alternative receptors for lysosomal hydrolases is still unclear. TGN-to-lysosome transport of lysosomal membrane proteins Lysosomal membrane proteins that exit the TGN can be transported to lysosomes following either a direct or indirect pathway, involving passage over the plasma membrane. Virtually nothing is known about the carriers and molecular machinery that mediate the direct route of lysosomal membrane proteins to the lysosomes. Moreover, the contribution of this pathway remains debated (Carlsson and Fukuda 1992; Janvier and Bonifacino 2005). Important for the sorting of lysosomal membrane proteins to lysosomes are the tyrosine and dileucine-based consensus motifs within their cytosolic tails. LAMP-1, LAMP-2 and CD63 (LIMP-1) all bear a YXXØ motif, with an essential tyrosine residue, hydrophobic XX residues and the Ø residue being a bulky hydrophobic amino acid (reviewed by Bonifacino and Traub 2003). The YXXØ motif is required for endocytosis at the plasma membrane, but has also been implicated in the direct targeting from TGN to lysosomes. For TGN sorting, however, additional characteristics seem to be required; a glycine residue preceding the critical tyrosine, the XX residues being acidic and the entire motif positioned six to nine residues from the transmembrane domain. LIMP-2, the recently identified receptor for β-glucocerebrosidase, has a dileucine-based sorting motif DXXLL, also referred to as acidic cluster-dileucine motif, which has also been implicated in both internalization and TGN-to-lysosome targeting. The YXXØ motifs are recognized by the μ subunits of AP-1, AP-2, AP-3 and AP-4 and the DXXLL motif by the VHS domain of the GGAs (reviewed by Bonifacino and Traub 2003). The role of adaptor proteins in TGN sorting of lysosomal membrane proteins remains to be established. Segregation of LAMP-1 into AP-1 positive vesicles was observed in a biochemical study and also confirmed by immuno-EM, indicating exit from the TGN in clathrin-coated vesicles (Höning et al. 1996; Hunziker and Geuze 1996). However, sorting of LAMP-1 is not strictly dependent on AP-1. In mice lacking the μ1A subunit, which impairs AP-1 binding and clathrin assembly at the TGN, there was no significant increase in plasma membrane levels of LAMP-1 (Meyer et al. 2000), whereas the protein was still found in lysosomes. Depletion of clathrin in HeLa cells did result in increased levels of LAMPs at the cell surface, but not a complete redistribution of LAMPs to the plasma membrane (Janvier and Bonifacino 2005), indicating the existence of a direct TGN-to-lysosome pathway independent of clathrin (Karlsson and Carlsson 1998). Interestingly, depletion of AP-1 in HeLa cells, like in mice, again had only modest effects on LAMP transport over the surface (Janvier and Bonifacino 2005). Together, these data indicate that in addition to AP-1 and clathrin, other factors might be involved in direct TGN-to-lysosome transport of LAMPs. The presence of a clathrin-independent TGN-to-endosome pathway was also indicated by studies on cells that present antigens, like B lymphocytes, macrophages and dendritic cells, in the context of major histocompatibility complex (MHC) class II molecules (Fig. 3c). The newly synthesized MHC II molecules are transported from the TGN either via the plasma membrane or directly to so-called MHC II loading compartments (MIICs) that are lysosome-related organelles representing early and late endosomes in these cells (Kleijmeer et al. 1997). For proper transport of the integral membrane protein MHC II from TGN to MIICs, association with the type 2 transmembrane protein, invariant chain (Cresswell 1996), is important. Invariant chain contains [DE]XXX[LI]-type signals in its C-terminus, but also the cytoplasmic tail of class II was found to contain endosomal sorting information (Bakke and Nordeng 1999; Bonifacino and Traub 2003). The TGN-to-endosome pathway of MHC II/invariant chain was studied in detail in B lymphoblasts by a combined biochemical and immuno-EM approach (Glickman et al. 1996). This revealed that MHC II, together with cathepsin D, in both normal and I-cell disease B lymphoblasts entered TGN exit domains that did not contain AP-1 or clathrin and were devoid of CD-MPR. Further studies are required to establish the molecular machinery of this pathway. Interestingly, a recent study linked the TGN exit of lysosomal membrane proteins to GGA3, the same component involved in the exit of MPRs. GGAs are monoubiquitinated at their GAT (GGA and TOM) domain, which supports the idea that ubiquitin is a universal sorting signal, acting at various intracellular sites. The lysosomal-associated protein transmembrane-5 (LAPTM5), which is specifically expressed in hematopoietic cells, associates in the TGN with both the ubiquitin ligase Nedd4 and GGA3 (Pak et al. 2006). This association is required for efficient targeting of LAPTM5 to lysosomes and indicates that ubiquitination of GGA3 is possibly accomplished by Nedd4. The finding that GGAs can be involved in TGN-to-lysosome transport of a lysosomal membrane protein might explain the mild effect of AP-1 knockdown. Clearly, further studies are required to clarify this direct TGN-to-endosome transport pathway of lysosomal membrane proteins. The early endosome: from CURL to tubular sorting endosome The combined data on TGN-to-endosome transport of lysosomal hydrolases and lysosomal membrane proteins suggest the existence of multiple TGN exits for direct delivery to the endosomal system (Fig. 3c). There may be several reasons for such different pathways. For example, they allow custom tailored regulation of the delivery of distinct types of cargoes and provide a back-up system in case a given pathway is no longer available (such as in I-cell disease). The existence of alternative TGN-to-endosome pathways would also allow different cargoes to enter the endo-lysosomal system at distinct stages, which may coincide with their function and mode of sorting. In addition to understanding TGN exits, it is therefore equally important to define the recipient endo-lysosomal intermediates in lysosome biogenesis. With the refining of EM techniques, our understanding of the endo-lysosomal system has equally evolved. The technique that we now know as ‘classical’ EM, i.e. thin sections of plastic-embedded samples of chemically fixed cells, allowed our first acquaintance with the fascinating complexity of the cell’s inner world. Immunocytochemistry added another dimension by allowing the localization of enzymatic activities to specific compartments (as illustrated for acid phosphatase). The next step was the development of immunocytochemical procedures, which greatly increased the repertoire of proteins that can be visualized in the cell. One of the first published examples of immuno-EM also marked a major leap in our understanding of the endo-lysosomal system; i.e. the identification of the early endosome as sorting device. In 1983, a novel immuno-EM technique was introduced by Hans Geuze and Jan Slot, in which differentially sized gold particles were used to simultaneously localize two distinct proteins. By applying this technique to visualize a ligand-asialoglycoprotein (ASGP), which is endocytosed by liver cells for transport to lysosomes-as well as its receptor (ASGPR, which recycles after endocytosis), they for the first time visualized a protein-sorting event. The ASGPR was found in tubular membranes that lacked ASGP and were connected to vacuoles that were enriched for ASGP but depleted of ASGPR (Geuze et al. 1983) (Fig. 6, left panel). The ASGPR-containing tubules were implicated in the recycling of the ASGPR to the plasma membrane, and the ASGP-containing vacuoles were defined as ‘precursor compartments en route to the lysosome’. The entire compartment was called ‘CURL’: ‘compartment of uncoupling receptor and ligand’. Fig. 6From CURL to tubular sorting endosome. Left panel reproduction of the first electron micrograph using double-immunogold labeling, showing the uncoupling of a ligand (asialoglycoprotein (ASGP), indicated by 5 nm gold particles) from its receptor (indicated by 15 nm gold particles) in early endosomes, at that time called CURL (compartment for uncoupling receptor and ligand). The ASGP receptor is found in tubules implicated in its recycling, whereas ASGP en route to lysosomes remains in the vacuole. Originally published by Geuze et al. (1983). Right panel, reproduction from Peden et al. (2004), showing by triple labeling that the adaptor protein AP-3 (15 nm gold particles), the lysosomal membrane protein LAMP-1 (10 nm gold particles) and the recycling ASGP receptor (5 nm gold particles) co-localize in the tubules emanating from endosomal vacuoles. This work gave rise to the concept of the tubular sorting endosome Over the years, it became clear that CURL was involved in the sorting of multiple types of proteins and the modern structural equivalent of CURL is the early endosome. Indeed, the early endosome is the major cellular site, where cargo destined for recycling is sorted from cargo en route to late endosomes and lysosomes. To indicate this, early endosomes are often subdivided into the globular ‘vacuolar or sorting’ endosome (Fig. 3b), whereas the associated tubules were indicated as ‘recycling tubules’ or ‘recycling endosomes’. However, recent studies provided accumulating evidence that early endosome-associated tubules are more than just transport intermediates for recycling cargo; they are also actively involved in sorting (Fig. 3b). With the increasing number of antibodies against distinct types of cargoes and by optimizing immuno-EM techniques (Slot and Geuze 2007), it became possible to localize less abundant cargoes. This revealed that at steady state conditions minor amounts of LAMP-1 and LAMP-2 are found in the early endosome-associated tubules (Fig. 6, right panel). The importance of this localization became apparent from studies on the pigmentation-bleeding disorder Hermansky-Pudlak-syndrome-2 (HPS-2). Cells from HPS-2 patients lack a functional AP-3 adaptor complex and show an increased transport of lysosomal membrane proteins over the plasma membrane (dell’Angelica et al. 1999). A combination of biochemical and immuno-EM approaches revealed that AP-3 mediates the exit of lysosomal membrane proteins from the early endosome-associated tubules, most likely for transport to late endosomes or lysosomes (dell’Angelica et al. 1998; Peden et al. 2004) (Fig. 3b). Impairment of AP-3 function results in an increased cycling of lysosomal membrane proteins between early endosomes and plasma membrane (Peden et al. 2004), which explains the increased levels of lysosomal membrane proteins at the plasma membrane of HPS-2 cells. Importantly, early endosomes can be reached by lysosomal membrane proteins via the plasma membrane, but possibly also via a direct pathway from the TGN. This may explain why in the literature AP-3 has been implicated in the direct as well as indirect pathway to lysosomes (Ihrke et al. 2004; Rous et al. 2002). In melanocytes, AP-3 also mediates a pathway from early endosomes to melanosomes, which are lysosome-related organelles (Theos et al. 2005). Importantly, by multiple double- and triple immunogold labellings, it was shown that a particular early endosome-associated tubule could contain the recycling proteins Tf and ASGPR, the CD and CI-MPR, and LAMP-1 and LAMP-2. In addition, one single tubule could display multiple budding profiles that were positive for either the adaptor complex AP-1 or AP-3 (Peden et al. 2004). AP-1 on endosomal tubules has been implicated in the retrograde transport of the CD-MPR and of the Shiga toxin B subunit to the TGN (Meyer et al. 2000), as well as recycling of internalized Tf (Mallard et al. 1998; van Dam and Stoorvogel 2002). The presence of distinct types of recycling proteins and the association of multiple adaptor complexes indicated that the early endosome-associated tubules mediate sorting of cargo proteins to distinct destinations in the cell, i.e. plasma membrane, TGN and late endosomes or lysosomes. Therefore, these AP-3 positive compartments were introduced as tubular sorting endosomes (Figs. 3b and 6, right panel) (Peden et al. 2004). A recent review of Bonifacino and Rojas extends this concept (indicated here as ‘tubular endosomal network’), by ascribing all protein machinery currently known to be involved in endosome-to-TGN transport to distinct exits of the tubular sorting endosome (Bonifacino and Rojas 2006). Together, the available data launch the concept of a two-step rocket. In the first step, at the vacuolar domain of early endosomes, proteins destined for late endosomes-lysosomes are sorted from recycling proteins that accumulate in the attached tubules. Then, in the second step, taking place in the tubular sorting endosome, recycling proteins are sorted out to distinct destinations in the cell. Within this model, the recycling endosomes are one type of transport intermediate that evolve from the tubular sorting endosomes, mediating the recycling of cargo to the plasma membrane (Fig. 3b). Multiple sorting events at the early endosomal limiting membrane Despite the relatively simple and attractive concept of the two-step rocket, sorting at the early endosomes displays another level of complexity; recycling carriers not only evolve from tubular sorting endosomes, but also from endosomal vacuoles. A recent study showed the formation of so-called endosome-to-TGN carriers (ETCs) from the limiting membrane of early endosomal vacuoles (Fig. 3b) (Mari et al. 2007). These ETCs are so far specifically involved in the endosome-to-TGN recycling of lysosomal protein receptors, i.e. the CD- and CI-MPRs and sortilin. Importantly, ETCs constitute an entirely different population of recycling carriers than those emanating from the tubular sorting endosome. With the exception of the MPRs, ETCs are devoid of any of the cargo or machinery proteins found in the tubular sorting endosome. The associated machinery proteins of ETCs are SNX1, SNX2 (Carlton et al. 2004, 2005; Mari et al. 2007) and hVps26p (Arighi et al. 2004; Mari, personal communication). ETCs also differ from tubular sorting endosomes by morphology. By 3D-tomographic analyses, ETCs were identified as non-branched, short tubules and vesicles, not organized in a reticulum. Apart from segregating proteins into tubular sorting endosomes and ETCs, the limiting membrane of early endosomal vacuoles is involved in a third protein sorting event; i.e. segregation of proteins into a characteristic bilayered coated area that is involved in protein sorting into ILVs (Fig. 3b, see also below) (Bonifacino and Rojas 2006; Peden et al. 2004; Raiborg et al. 2002; Raposo et al. 2001; Sachse et al. 2002b). Thus, proteins present in early endosomal vacuoles may be sorted to at least three types of specialized sorting domains: tubular sorting endosomes, ETC or the bilayered coat. The formation of two types of exits from the non-coated regions of the early endosomal vacuoles predicts the existence of complementary molecular machineries that specifically recruit cargo to either of these recycling compartments (i.e. ETC or tubular sorting endosome). Future investigations are needed to further unveil the components of these machineries. Sorting to late endosomes and lysosomes The default pathway for molecules entering early endosomes is recycling back to the plasma membrane, a pathway that starts with the segregation of cargo into the tubular extensions of the early endosomes (Draye et al. 1988; Yamashiro et al. 1984). Endocytosed cargo from the plasma membrane that is not destined for recycling requires active sorting to late endosomes/lysosomes, a process mostly mediated by inclusion into ILVs (Gruenberg and Stenmark 2004; Katzmann et al. 2002; Sachse et al. 2002b). Protein sorting into ILVs is critical for growth factors and their receptors, such as growth hormone (GH) (Sachse et al. 2002b) and epidermal growth factor (EGF) (Raiborg et al. 2003; Urbe et al. 2003). Sorting into ILVs segregates these ligand–receptor complexes from the cytoplasm, thereby terminating signaling. ILV sorting requires ubiquitination and involves among others the cytoplasmic proteins Hrs and STAM (Bache et al. 2003b; Raiborg et al. 2001, 2002; Urbe et al. 2003). By immuno-EM, it was shown that at the limiting membrane of the early endosomal vacuole the GHR and EGFR are concentrated in flat, bilayered coated areas with a fuzzy outer layer of clathrin and a dense inner layer of unknown protein composition. These bilayered coated domains were enriched in Hrs (Sachse et al. 2002b). These findings led to the proposition of the ‘sorting-by-retention’ model for endosomal sorting (Sachse et al. 2002b). This model describes that receptor–ligand complexes prior to their incorporation into ILVs are retained and concentrated in the bilayered coated areas of endosomal vacuoles, whereas recycling proteins pass through the vacuolar-limiting membrane and follow the bulk flow route towards recycling endosomes. A recent study showed that the recruitment of clathrin is responsible for concentrating Hrs in these endosomal microdomains, reinforcing an active role for clathrin in protein retention (Raiborg et al. 2006). Downstream of Hrs, the targeting of ubiquitinated receptors into ILVs requires the ‘endosomal sorting complexes required for transport’ (ESCRT)-1, -2 and -3 in concert with Alix/AIP-4 and the AAA-ATPase (hVps4). hVps4 is required for disassembly and recycling of ESCRT complexes (Babst 2005; Bache et al. 2003a; Katzmann et al. 2003) and interestingly, also for the disassembly of the bilayered coat (Sachse et al. 2004). Impairment of hVps4p function gives rise to an elongated coat and decreased formation of ILVs, indicating that coat disassembly is required for the inward budding process to proceed (Sachse et al. 2004). Indeed, 3D-electron tomography of early endosomes showed that inward budding of ILVs is mostly seen at the edges of the coats (Murk et al. 2003). Notably, the sorting-by-retention model proposes a novel role for clathrin in addition to its role in the formation of transport vesicles, i.e. defining endosomal subdomains involved in protein retention. Future developments With the increasing number of molecular markers of endosomes and lysosomes, there is a growing awareness that distinct populations of endosomes and perhaps even lysosomes, with different functions, can coexist within one cell (Bökel et al. 2006; Lakadamyali et al. 2006; Möbius et al. 2003; White et al. 2006). For example, ILVs enriched in the lipid lysobisphosphatidic acid (LBPA) are found in distinct MVBs from those enriched in cholesterol (Möbius et al. 2003). Another example is illustrated in Fig. 7 showing three seemingly identical late endosomes, of which only two are reached by endocytosed BSA-gold after 3 h of uptake. One explanation is that cargo drives the formation of specific endocytic compartments. For example, addition of EGF to cells results in the specific increase of the type of MVBs that do not contain LBPA (White et al. 2006). Formation of cargo-dependent carriers can start at the plasma membrane. The recent identification of clathrin-independent endocytosis, as well as the discovery of cargo specific adaptors that lead to clathrin-coated pit formation-either in conjunction with or independent of AP-2-illustrates that alternative endocytic pathways can arise from the plasma membrane. If and how these pathways converge at the endosomal level is presently unclear (Benmerah and Lamaze 2007). EM is an indispensable tool to search for cargo-specific endosomal compartments, since by fluorescence microscopy only the labelled compartment is visible and a nearby compartment negative for the cargo under study will remain unseen. Fig. 7Electron micrograph illustrating the heterogeneity of late endosomes (LE). HepG2 cells were allowed to internalize bovine serum albumin (BSA) conjugated to 5 nm gold particles (abundant labeling) for 3 h and processed for immuno-EM of cathepsin D (10 nm gold particles, scarce labeling). Three seemingly identical late endosomes containing multiple ILVs and partially degraded material are shown, two of which are heavily loaded with BSA-gold, whereas the upper one is devoid of the endocytic tracer. G Golgi stacks; Bar 200 nm Another point of interest is that not all cargo might be recruited to ILVs by the same mechanism. Both in yeast (Bilodeau et al. 2002; Epple et al. 2003) and mammalian cells (Hislop et al. 2004), examples of cargoes are described that do not require direct ubiquitination. In case of the melanosomal protein Pmel17, which is also targeted to ILVs independently of ubiquitin and Hrs, two N-terminal lumenal subdomains seem necessary and sufficient for efficient transport (Theos et al. 2006). Possibly, these non-ubiquitinated cargoes associate with others for their association with the ESCRT machinery, but perhaps they use an alternative mechanism. Interesting in this respect is that LBPA can induce ILV formation in liposomes, in the absence of protein, suggesting that lipid-driven and ESCRT-dependent pathways may coexist (Matsuo et al. 2004). Also the lysosomal targeting of LAMP-1 and LAMP-2 remains an interesting open question, since these proteins are predominantly located on the limiting membranes of late endosomes and lysosomes, and incorporation into ILVs seems therefore not involved. By a more detailed characterization of the distinct endosomal maturation stages (Mari et al. 2007), it will be feasible to obtain a more accurate definition of the stage in which a specific cargo enters or exits the endo-lysosomal system. One of the questions that is currently addressed in our lab is whether lysosomal membrane proteins can travel directly to late endosomes or lysosomes, thereby bypassing the need of ESCRT sorting machinery. Finally, with the development of live cell imaging methods and correlative live cell imaging EM, a unique tool is provided to integrate dynamic parameters with ultrastructural protein localization studies, which opens a whole new field of analysis.

Pediatr_Radiol-4-1-2367394

Imaging findings in noncraniofacial childhood rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. This paper is focuses on imaging for diagnosis, staging, and follow-up of noncraniofacial RMS. Introduction Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma and constitutes 3–5% of all malignancies in childhood [1]. In children, the age-standardized annual incidence rate for RMS is between 4 and 7 per million [2]. This review focuses on the imaging of all RMS occurring outside the head and neck region, which comprise 40% of RMS; around 15% are genitourinary (GU) nonbladder prostate tumours (GU-NBP, i.e. paratesticular, vaginal and uterine tumours), 10% are bladder prostate tumours (BP), 15% occur in the limbs, and 20% occur in other sites (i.e. thoracic or abdominal tumours). Clinical symptoms vary widely, based on the location of the primary tumour, but in general presenting symptoms often are indolent, with nonspecific or minimal symptoms, at the start mimicking innocent general paediatric diseases. Often the duration or progression of symptoms alerts the physician to the presence of a malignant tumour. Site-specific symptoms in GU-NBP tumours are a paratesticular mass in boys that may be painful or not, while girls may present with a grape-like (botryoid) vaginal extrusion of mucosanguineous tissue or micturition problems. BP tumours may present with urinary retention, haematuria, constipation or an abdominal mass. Limb tumours often present with a painless swelling and/or with enlarged regional lymph nodes, while presentation of tumours in other locations may vary from biliary obstruction in tumours of the biliary tract, to painless masses, all depending on their primary site. Patients with metastatic disease present with more general symptoms of fatigue, weight loss, and low blood counts. As soon as a RMS is suspected an extensive work-up must be performed to get a histological diagnosis and correct staging of the tumour. Imaging of the primary site with US, MRI or CT is required in all patients at the start of the diagnostic work-up [3]. Once the diagnosis has been confirmed histologically, the most frequently involved metastatic sites will be investigated; CT of the lungs, imaging of regional lymph nodes, and a technetium bone scan are recommended for every patient. Furthermore, bone marrow aspirates and trephines should be performed in all patients, while examination of the cerebral spinal fluid is only required in patients with paraspinal and parameningeal locations. As soon as the work-up has been completed, the patient can be stratified to receive treatment according to the child’s risk group, based on the six significant prognostic factors for localized RMS that came out of a retrospective European analysis (European Paediatric Soft-tissue Sarcoma Study Group, EpSSG); histology, postsurgical status according to the Intergroup Rhabdomyosarcoma Study Group (IRSG), tumour site, node involvement, tumour size, and patient age (Table 1). Table 1EpSSG prognostic factors [28] FavourableUnfavourableHistologyEmbryonalAlveolarIRSG statusHigher grades more unfavourableTumour siteHead and neck non-parameningealAll other sitesOrbitalGenitourinary – nonbladder/prostateNode involvementN0N1Tumour size (cm)≤5>5Age (years)<10≥10 Pathology RMS is a fast-growing, primitive, high-grade, malignant mesenchymal tumour. Depending on their degree of differentiation, the tumour cells manifest features that more or less can be found in the cells of skeletal muscle. These features, essential for the diagnosis, are the presence of myofibrils and cross striations (on light and electron microscopy) and/or positive immunohistochemical staining for markers of muscle differentiation such as desmin and myoD1. For detailed descriptions of the histopathological aspects of RMS, reference should be made to Weiss and Goldblum [4] and Fletcher et al. [5]. Based on morphology, RMS is traditionally subdivided into embryonal, alveolar and pleomorphic. Pleomorphic RMS, in contrast to embryonal and alveolar RMS, almost exclusively occurs in adults (median age sixth decade), and is therefore not discussed further. Embryonal RMS is the most common type (60–70% of all RMS). The cells show a close resemblance to various stages in the embryogenesis of normal skeletal muscle (Fig. 1). Subtypes are botryoid RMS and spindle-cell RMS (Fig. 2). When arising in the submucosa, embryonal RMS may present as a fast-growing exophytic, polypoid mass. This macroscopic variant is called botryoid RMS (grape-like) and, due to its growth pattern (primary exophytic and not invasive), has a better prognosis. Fig. 1Embryonal RMS: small cells and primitive spindle-shaped cells resembling the first stages of developing normal skeletal muscle (H&E, original magnification 10×20)Fig. 2Embryonal RMS, spindle-cell type: closely packed spindle cells arranged in perpendicular crossing fascicles giving a leiomyosarcoma-like appearance (H&E, original magnification 10×20) Spindle-cell RMS comprising about 4% of all RMS, seems to behave less aggressively and is most often encountered in a paratesticular location (about 30% of all paratesticular RMS) and the head and neck [6, 7]. Histologically spindle cell RMS is characterized by parallel orientation of spindle cells with an eosinophilic, fibrillary cytoplasm and an elongated, hyperchromatic nucleus. The cells are arranged in broad fascicles (fibro-/leiomyosarcoma-like) or in short, interdigitating fascicles (storiform) and whirls with, in contrast to the broad fasciculated variant (leiomyomatous type), an abundant, partly hyalinized collagenous matrix. Alveolar RMS is composed of ill-defined, dense aggregates of poorly differentiated round or oval tumour cells that frequently show loss of cohesion (Fig. 3). This loss of cohesion and the presence of thin fibrous septa result in an alveolar pattern. In cases where the tumour cells do not show loss of cohesion, the term ‘solid alveolar RMS’ is used. Alveolar RMS represents about 20% of all RMS and has two specific translocations with specific fusion transcripts that can be detected by RT-PCR. The majority (about 55%) show a t(2;13)(q35;q14) translocation with the corresponding fusion transcript PAX3-FKHR [8]. In about 22% a t(1;13)(p36;q14) translocation is found with fusion transcript PAX7-FKHR. In rare cases, RMS with a more or less alveolar pattern lacks these translocations. Fig. 3Alveolar RMS: cluster of primitive cells with loss of cellular cohesion and bordered by dense fibrous septa, resulting in an alveolar pattern (H&E, original magnification 10×20) Both embryonal and alveolar RMS may show rhabdoid tumour-like features and anaplasia (focal or diffuse) [9, 10]. More recently, under the heading sclerosing RMS, a variant with hyalin sclerosis has been described (Fig. 4) [11]. It is unclear whether this is a distinct subtype. Fig. 4Sclerosing RMS: small cells, primitive spindle-shaped cells and eosinophilic rhabdomyoblasts in a background of hyaline sclerosis (H&E, original magnification 10×20) In 1995, the IRSG and the International Society of Paediatric Oncology (SIOP) suggested a prognostically more significant classification (Table 2) [12]. Table 2RMS: IRSG and SIOP classification, extended with new variants [12]GroupPrognosisSubtypeIBetter prognosisEmbryonal RMS, botryoid typeEmbryonal RMS, spindle-cell typeIIIntermediate prognosisEmbryonal RMS (remaining)IIIWorse prognosisAlveolar RMSIVUnclear prognosisRMS with rhabdoid featuresEmbryonal RMS with diffuse anaplasiaSclerosing RMS Imaging findings Radiography As RMS is a soft-tissue tumour, conventional radiology plays an insignificant role in its diagnosis. Localized bony erosion adjacent to the primary site is a recognized complication; this area may be hot on a 99mTc-MDP bone scan in the absence of metastatic disease in the skeleton. In contrast to the initial diagnostic work-up, where for the assessment of pulmonary metastases CT is mandatory, AP and lateral chest radiographs are used in the follow-up period. Ultrasonography US is often the first imaging modality used in children with soft-tissue masses because it is readily available, has high resolution, and can easily assess extent and vascularity of a mass. One should not forget that most soft-tissue lesions are benign, can readily be diagnosed with US, and do not need further diagnostic work-up or even treatment. On US, RMS in general shows as a well-defined, slightly hypoechoic inhomogeneous mass that can show significantly increased flow (Fig. 5). In the particular case of paratesticular RMS, US is the imaging modality of choice, although CT of the abdomen is also necessary to evaluate for retroperitoneal lymphadenopathy. In all other RMS locations additional imaging using CT or MRI is mandatory. Fig. 5A 6-year-old boy with a mass in the left scrotum. US image shows an ill-defined heterogeneous mass surrounding the testis (open arrow). The mass shows increased flow (solid arrow). Histopathology: embryonal RMS US is also of use in image-guided biopsies. Recently Sebire and Roebuck [13] systematically reviewed the pathological diagnosis of paediatric tumours from image-guided needle-core biopsies. They concluded that image-guided biopsy material was sufficient to come to a diagnosis in 94% (95%; CI 92–96%) of patients. Complications needing treatment, mostly haemorrhage requiring transfusion, were reported in only 1% of patients. For image-guided biopsies the material obtained should be transported fresh to the pathology department. Fixation should not be performed as this precludes further cytogenetic studies. CT In order to assess pulmonary metastases from RMS, CT of the chest is a mandatory examination. However, assessment of pulmonary involvement can sometimes be difficult. Although criteria such as number and size of lesions, morphology (noncalcified, round and well-defined) and location (inferior lobes, subpleural spaces, branching vessels) have been applied in adult patients, none has shown 100% specificity. According to the EpSSG guidelines for evaluating chest CT the following criteria with respect to the diagnosis of pulmonary metastases should be applied: one pulmonary or one pleural nodule over 10 mm in diameter, two or more well-defined nodules of 5–10 mm or five or more lesions >5 mm [3]. This comes with the assumption that there is no other medical explanation for these lesions. When there is a high suspicion that a small lesion is metastatic, and appears to be the only site of metastatic disease, biopsy may be performed. In the EpSSG protocol, lung biopsy is not recommended. With the introduction of multidetector CT (MDCT) the advantage of MRI of being a multiplanar imaging modality has been overtaken, and by virtue of its underlying physics, CT is superior to MRI in detecting osseous changes. The drawback of CT in children is the use of ionizing radiation. Since the seminal papers of Paterson et al. [14] and Brenner et al. [15], we should be aware of the theoretical risk of CT-induced cancer fatalities and take this into consideration especially in the work-up of children with cancer, as they have already proven their tendency to present with a malignancy early in life [14, 15]. MRI With its superior ability to depict soft-tissue changes, MRI is the primary imaging modality in RMS [16]. Although imaging protocols should be tailored for each individual patient, they should at least consist of axial T1-W and T2-W images (for anatomic detail and assessment of neurovascular structures), T1-W images perpendicular to the axial plane, and imaging after gadolinium administration. It is important that at least two series should be identical, one before and one after contrast agent administration, to be able to discern enhancement. Contrast-enhanced series are mandatory and ideally be performed with fat saturation. The imaging characteristics of RMS are relatively nonspecific. Like most soft-tissue tumours they have intermediate signal intensity on T1-W images (Fig. 6). On T2-W images they tend to be of intermediate-to-high signal intensity. If the tumour contains a high number of septa it may have a lobular shape. RMS in general show strong enhancement on postcontrast imaging (Fig. 6). In very rare instances the tumour may show a predominantly cystic appearance (Fig. 7). Dynamic series are useful in order to assess tumour vascularity, and to differentiate between postchemotherapy/surgery residual disease and fibrosis. Fig. 6A 13-year-old girl who noticed a small lump near the anus. a T1-W MR image shows a well-defined pararectal lesion (arrow). b After intravenous gadolinium administration the lesion shows homogeneous enhancement (arrow) Histopathology: alveolar RMSFig. 7An 8-year-old girl with haemolysis, fever and a mass underneath the scapula. a Coronal STIR image shows a lesion with mixed signal intensity (open arrow) and multiple enlarged lymph nodes in the neck (solid arrow). b Axial T2-W image shows multiple cystic lesions with fluid-fluid levels (open arrow). Histopathology: stage IV embryonal RMS For the surgeon, in order to plan surgery, it is of importance to describe the compartment in which the RMS is located [17]. Vascular involvement is considered to be absent if there is a normal tissue plane visible between the tumour and the vessel, or if the tumour has a less then a 180° circumferential relationship. If the tumour surrounds the vessel for more than 180°, it is considered to be encased. Two studies have addressed the use of whole-body MRI (WB-MRI) in paediatric oncology [18, 19]. In the first study with various malignant tumours WB-MRI had a superior positive predictive value for skeletal metastases compared to bone scintigraphy (94 vs. 76%, respectively), and also a significantly higher sensitivity (99 vs. 26%, respectively) [18]. In the second study, WB-MRI had a higher sensitivity (82%) than skeletal scintigraphy (71%) for the detection of bone marrow metastases, but a lower sensitivity than FDG-PET (90%) [19]. The authors of both studies concluded that WB-MRI can replace bone scintigraphy. One advantage of this approach would be the implementation of a one-stop-shop approach to childhood RMS. A drawback of MRI in young children is, however, the need for general anaesthesia. Bone scintigraphy In the current EpSSG protocols, bone scintigraphy is mandatory as part of the work-up in patients with RMS. The finding of an isolated hot spot on the bone scan should be evaluated with conventional radiography or MRI. Cogswell et al. [20] reported a retrospective series of 40, primarily adult, patients with RMS and found bone metastases in 18%. Bone scintigraphy in their study had a sensitivity of 70% and specificity 95% in the detection of metastatic disease. In contrast to this, in a Dutch study of 109 patients with soft-tissue sarcoma, bone metastases were found in only 8 patients (7%) [21]. However, of these eight patients, six reported bone pain and all had other sites of metastatic disease. The authors conclude that the yield of routine bone scintigraphy is low and that it should be reserved for symptomatic or high-risk patients only. Positron emission tomography-CT In PET-CT studies fluorine-18 fluorodeoxyglucose (18F-FDG), a radiolabelled glucose analogue, is used [22]. As 18F-FDG is a glucose analogue, it shows uptake in metabolically active cells, which most malignant tumour cells are. The combination of PET with CT, without moving the relative position of the patient, yields a higher diagnostic accuracy than PET alone (Fig. 8). In general, the CT scan will be low-dose CT scan only meant to identify anatomical structures. However, as the CT scanners in modern PET-CT systems are of high diagnostic quality, it is also possible to combine a diagnostic CT scan, e.g. for the depiction of pulmonary metastases, with a PET scan. Fig. 8A 19-year-old boy with a history of treated metastatic RMS presented with low back pain. The PET-CT image shows intense 18F-FDG uptake in the spinal canal (open arrow). Physiological excretion of the radiopharmaceutical via the kidneys is visible (solid arrows). Histopathology: embryonal RMS The literature on the use of PET-CT in children with RMS is limited to several case reports or small studies [23, 24]. Although in some cases PET-CT has been shown to be of benefit in individual patients, larger prospective studies are needed. Staging and follow-up Staging of RMS is of importance for the individual patient as it gives an indication of prognosis, and thus treatment stratification. From a broader perspective staging makes compiling data on larger patient groups for research purposes possible, enabling evaluation of the outcome of different treatment regimens. The main staging system is the postsurgical staging system developed by the IRSG. This is currently used by study groups both in the USA and now in Europe also (Table 3). The IRSG was formed in 1972 and consisted of surgeons, pathologists, oncologists, and radiation oncologists. The absence of radiologists is striking, and paediatric radiologists are still infrequently involved in development of paediatric oncology study protocols, although in the EpSSG RMS 2005, paediatric radiologists were involved in the development of the protocol. For staging regional nodes it is important to be familiar with the regional node stations. Lymph node involvement has a negative impact on prognosis, as has been shown in the SIOP Malignant Mesenchymal Tumor 89 trial [25]. Overall 5-year survival was 60% for N1 patients versus 73% in N0 patients (P = 0.03). Distant lymph node involvement upgrades a patient to stage IV disease (Fig. 7), having an adverse impact on prognosis: overall 5-year survival becomes 24% [26]. Table 3IRSG classificationStageCharacteristicsILocalized disease completely resected (regional nodes not involved)A: Tumour confined to muscle or organ of originB: Tumour infiltrating outside organ of (muscle of) originIILocalized or regional disease with total resection of gross tumourA: Primary tumour grossly resected, with microscopic residual disease (negative findings in local nodes)B: Primary tumour and positive nodes completely resectedC: Primary tumour and positive nodes resected, with evidence of microscopic residual diseaseIIIIncomplete resection of tumour or biopsy, with gross residual diseaseIVDistant metastatic disease present at diagnosis Table 4 lists the regional node stations by primary tumour site. Oval-shape lymph nodes and a short axis <1 cm are considered to be normal [3]. If the node shows peripheral enhancement or is round with a short axis of 1.5–2 cm then the node should be considered probably invaded by tumour. Besides surgical resection or needle-core biopsies, lymph node involvement can also be assessed using fine-needle aspiration (FNA). Klijanienko et al. [27] reported a review of the use of FNA in 180 tumours; 176 (97.8%) were either diagnosed accurately or as round-cell sarcoma. Table 4Regional node stations by primary tumour site. Disease with involvement of other lymph nodes than those specified in the table should be classified as stage IVAnatomical siteNode stationExtremityLower extremityInguinal, femoral, popliteal nodes (rare)Upper extremityAxillary, brachial, epitrochlear, and infraclavicular nodesGenitourinaryBladder, prostate, cervix, uterus, paratesticularPelvic, retroperitoneal nodes at renal artery level or belowVaginaRetroperitoneal, pelvic nodes at or below common iliac inguinal nodes VulvaInguinal nodesThoracicIntrathoracicInternal mammary, mediastinal nodesRetroperitoneum/pelvisPelvic, retroperitoneal nodesTrunkAbdominal wallInguinal, femoral nodesChest wallAxillary, internal mammary, and infraclavicular nodesOtherBiliaryLiver hilar nodes Perianal/perinealInguinal, pelvic nodes (may be bilateral) Only in patients with intraspinal or suspected meningeal extension (on imaging or clinical assessment) does the EpSSG RMS protocol state that craniospinal MR should also be performed. In the current EpSSG RMS 2005 protocol, risk stratification is based on six criteria that have emerged from the analyses of previous European studies: histology (embryonal vs. alveolar), postsurgical stage (IRSG), tumour site, node stage, tumour size and patient age (Table 1) [28]. During follow-up, tumour size is an important parameter in assessing tumour response. In the current EpSSG protocol complete remission is defined as disappearance of tumour both clinically and on imaging. Minor response is defined as >33% reduction in volume after three courses of chemotherapy; if not reached the patient is eligible for second-line chemotherapy. In recording tumour response the EpSSG uses volumetric evaluation; additionally the presence or absence of a posttherapeutic residue should be mentioned in the radiology report [28]. In studies in adults the use of Response Evaluation Criteria In Solid Tumours (RECIST) has been advocated by the European Organization for Research and Treatment of Cancer (EORTC), National Cancer Institute of Canada Clinical Trials Group and the National Cancer Institute (NCI) of the United States [29]. With respect to implementation in children, RECIST have been a matter of debate [30]. Recently RECIST were retrospectively applied to ten consecutive children with cancer [31]. The authors concluded that tumour size was underestimated and that in disseminated disease many lesions were either calcified or too small to measure and, therefore, that RECIST are not readily applicable in paediatric oncology. Currently the EpSSG has incorporated RECIST to be used alongside the volumetric measurements in their latest protocol in order to prospectively assess the validity of RECIST in a large patient population with a single tumour type. Tumour relapse in patients most commonly presents with locoregional disease (51%) compared to distant relapse (41%) [32] (Fig. 9). In a retrospective case-based study the use of PET-CT was advocated; this, however, needs to be evaluated in larger prospective studies [33]. Fig. 9Two years after initial diagnosis the patient shown in Fig. 6 presented at the outpatient clinic complaining of back pain. a Coronal STIR image of the pelvis shows discrete increased signal intensity in the left ischium (open arrow). b Subsequently acquired PET-CT image confirms the presence of recurrent disease in the same location (open arrow). Note excretion of tracer into the urinary bladder (solid arrow). c PET-CT image also shows a second lesion in the thoracic spine (open arrow). Additional rib and pleural metastases were also visible (not visible on this image) Tumour locations Genitourinary Approximately 25% of all RMS are GU RMS [34, 35]. As mentioned above, GU RMS can simply be subdivided into two subgroups based on different prognosis and subsequent treatment strategy, GU bladder/prostate (GU-BP) being an unfavourable location (Fig. 10). Tumours at other GU non-bladder/prostate (GU-NBP) sites, such as a paratesticular location (testes, epididymis and spermatic cord; Fig. 5), vagina or uterus (Fig. 11), have a favourable prognosis, and thus require less-intensive treatment [36–48]. There is a caveat to MRI of the bladder in RMS: after intravenous contrast medium administration, layering of contrast medium can occur making it difficult to appreciate bladder wall enhancement [49]. T2-W sequences can be particularly useful in this setting to assess bladder wall thickening. Additional cystoscopy is often warranted [50]. At the end of treatment, some residual soft-tissue thickening may persist, and on MRI it is impossible to decide whether this is residual scarring or tumour; in these cases endoscopic biopsy is mandatory. Fig. 10A 3-year-old boy with RMS of the prostate. The sagittal T1-W contrast-enhanced MR image shows the lesion invading the bladder wall. A transurethral catheter has been inserted (open arrow). Histopathology: botryoid RMSFig. 11A 2-year-old girl presented with a mass in the vagina. a Axial T1-W contrast-enhanced image shows the mass with heterogeneous enhancement. The tumour has both solid (asterisk) and fluid (open arrow) compartments. b Sagittal T2-W MR image shows the mass with mixed signal intensity. The bladder is displaced anteriorly and the uterus cannot be visualized. Histopathology: embryonal RMS Extremities RMS of the extremities (Figs. 12 and 13) is almost always of alveolar histology, tends to occur in older children and young adults, is often present with positive regional lymph nodes, and has a propensity to metastasize to unusual sites; these negative prognostic factors contribute to the relatively poor prognosis of RMS in this location [51, 52]. In approximately 12% of patients, nodal involvement is seen on imaging; however, when nodal dissection is performed the rate of nodal involvement increases to almost 50% [53]. This discrepancy between imaging findings and nodal dissection might be reduced by using high-quality state-of-the-art US. In current treatment protocols systematic biopsy of regional nodes is advocated, even if the nodes are not palpable or enlarged on imaging; sentinel node procedures are recommended whenever feasible, although the value of upgrading a patient to a higher risk group based merely on a positive sentinel node has not been studied yet. As in all tumours of the extremities, preoperative imaging plays a vital role in the depiction of the relationship between neurovascular bundles and the tumour. Fig. 12A 4-year-old girl presenting with a mass in the left lower leg. a Axial T1-W contrast-enhanced MR image shows an ill-defined mass circumferential to the fibula. Note the cortical thinning (open arrow) of the fibula. b Sagittal PD-weighted image shows diffuse bone marrow metastases. Histopathology: embryonal RMSFig. 13A 2-month-old boy with a mass in the third ray of the left foot. T1-W MR image shows a discrete lesion (open arrow) of intermediate signal intensity. Histopathology: embryonal RMS Other Chest wall Chest wall RMS (Fig. 14) is a relatively rare finding with a reported incidence of 3.7% in the IRSG II and IRSG III studies [54]. Most of the reports of chest wall involvement are either case reports or small series [55–57]. In a retrospective analysis of 15 patients, Saenz et al. [57] found a 5-year survival of 67%. Fig. 14A 4-year-old boy presenting with a mass on the right chest wall. a US image shows a heterogeneous mass in the pectoralis major muscle (the pectoralis minor is not involved; asterisk). b T1-W MR image of the chest shows a mass of intermediate signal intensity. c After intravenous contrast medium administration the lesion shows homogeneous enhancement. Histopathology: alveolar RMS Pulmonary There is an ongoing debate and controversy whether congenital cystic anomalies predispose children to intralesional development of RMS [58–62]. It has been reported that pleuropulmonary blastoma (PPB) has been mistaken for or classified as RMS arising in congenital cystic adenomatoid malformation (CCAM) on a number of occasions [63]. Despite the fact that the exact incidence in CCAM is unknown, it has prompted paediatric surgeons to resect even small pulmonary cystic lesions (Fig. 15) [64, 65]. Fig. 15A 3-year-old boy with dyspnoea. Chest CT image shows displacement of the trachea (open arrow) and oesophagus (solid arrow) to the right due to a large mass (asterisk) with accompanying pleural effusion. Histopathology: embryonal RMS Biliary tree RMS is the most common tumour of the biliary tree in childhood, although it only accounts for approximately 0.04% of all childhood tumours [66]. The initial diagnosis will, in most patients, be made on US, which may show a solid or cystic mass situated in the liver hilum, and intrahepatic bile duct dilatation (Fig. 16). MRI is mandatory for presurgical evaluation, where magnetic resonance cholangiopancreatography (MRCP) can depict the biliary tree (Fig. 16). In many patients, however, endoscopic retrograde cholangiopancreatography (ERCP) will have to be performed in order to depict intraductal irregularities (Fig. 16). Biliary tree RMS is a tumour that does not necessarily need to be fully resected in order to achieve long-term survival, as long as adequate radiotherapy is added [67]. Intraperitoneal metastases, which can also be found on follow-up, should also be born in mind. Fig. 16An 8-year-old boy presented with abdominal pain and jaundice. a US image shows a central process in the liver hilum (open arrow) and dilatation of the intrahepatic bile ducts (solid arrow). b T2-W MR image shows a circumscribed lesion with increased signal intensity (open arrow). c MRCP image shows intrahepatic bile duct dilatation. Note that the right and left duct systems do not communicate (open arrow). d ERCP image (ERCP performed in order to insert a stent in the common bile duct). Histopathology: embryonal RMS Other locations In extremely rare instances RMS can be found in other organs such as the heart, the diaphragm (Fig. 17), the omentum, the urachus and the digestive tract [68–76]. Fig. 17A 4-year-old boy was shown to have a right-sided pleural effusion on a chest radiograph. Balanced FFE sagittal MR image shows a mass (open arrow) arising from the diaphragm (courtesy of S.G.F. Robben, Academic Hospital Maastricht, The Netherlands) Congenital RMS has been reported to occur as a congenital tumour (Fig. 18) [77–81]. In congenital alveolar RMS the prognosis is reported to be extremely poor, despite otherwise adequate treatment [79]. Orbach et al. [82] reported the SIOP data on soft-tissue sarcoma in the first year of life. In their study population of 16 newborns, with a follow-up of 1.8–10.0 years, 3 out of 5 newborns with RMS survived. It has been noted that in congenital RMS the disease may be metastatic at the time of birth, with metastases described in a number of organs and in the placenta [81]. Fig. 18A 4-day-old girl born with a lump on the left foot. Antenatal ultrasonography at 20 weeks showed no abnormalities. a T1-W MR image shows a large inhomogeneous mass arising from the left foot. b Abdominal US image shows popliteal and inguinal nodal invasion, and hepatic and pancreatic metastases (open arrow). Due to the poor prognosis, no therapy was given, and the child died several weeks later. Histopathology: poorly differentiated soft-tissue sarcoma without distinct translocations Adult patients Every once in a while paediatric radiologists and paediatric oncologists receive a request for help in the management of an adult patient (Fig. 19). RMS, although seen as a soft-tissue tumour of childhood, can also occur later in life [83–86]. Compared to childhood RMS, adult RMS has a poor outcome. In a large retrospective study of 171 patients 5-year overall survival was only 40% [85]. However, the patients in this series treated according to the guidelines for treatment of childhood RMS showed survival figures comparable to those seen in children. This suggests that treatment of adult RMS should be based on paediatric protocols tailored to adults, to increase survival in this age group. In the Academic Medical Centre Amsterdam we have a working group on childhood tumours in (often young) adults that specifically deals with this challenging population. This working group consists of medical oncologists, paediatric oncologists, radiation oncologists, (orthopaedic) surgeons and a paediatric radiologist. Imaging features will in general not be of help, as the pretest likelihood of RMS in an adolescent or adult is extremely low. Fig. 19A 45-year-old man with a mass in the thigh. T1-W contrast-enhanced MR image shows a heterogeneous circumscribed mass in the vastus lateralis muscle of the right leg. Histopathology: alveolar RMS Differential diagnosis Given the wide variety of locations in which RMS can be found it is difficult to give a concise list of differential diagnoses. The site of the primary lesion determines the differential diagnosis. Keeping location out of the equation there are, however, certain tumours, such as haemangiomas/vascular malformations (Fig. 20), adult-type soft-tissue sarcomas, peripheral neuroectodermal tumours (PNET), infantile fibrosarcoma, aggressive fibromatosis, desmoplastic small round-cell tumours and rhabdoid tumours, and other more even rarer soft-tissue tumours such as nonosseous Ewing sarcoma (Fig. 21), that should be kept in mind when performing US or reading CT or MRI studies of soft-tissue tumours in childhood. Fig. 20A 1-year-old girl with a mass on the left buttock. a Duplex US image shows a highly vascularized, well-delineated heterogeneous mass that was initially thought be a haemangioma. b Coronal STIR image shows a circumscribed solid lesion that invades the pelvis via the greater sciatic foramen (open arrow). c After initial resection, with incomplete margins, tumour recurrence was seen. MR image 2.6 years after initial diagnosis shows progression of disease extending to the abdominal wall (open arrow). Histopathology: alveolar RMSFig. 21A 22-month-old boy with a mass in the right groin. MRI shows a heterogeneous lesion adjacent to the gracilis muscle (open arrow). Histopathology: extraosseous Ewing sarcoma Treatment and prognosis Treatment of RMS requires a multidisciplinary approach, where chemotherapy, surgery and radiotherapy (RT) each has its own specific role. Chemotherapy Before the introduction of chemotherapy only 25% of patients with RMS survived, despite adequate local therapy. This indirectly indicates that the vast majority of patients had at least minimal disseminated disease at diagnosis, whereas, with state-of-the-art imaging techniques and bone marrow investigations, only 15% of patients with RMS present with overt metastatic disease. Besides treating minimal disseminated disease, chemotherapy enables local therapy to be more conservative. In most patients, surgery at diagnosis consists of biopsy only. Chemotherapy is given to all patients; it reduces tumour size and extension and often allows a delay in surgery. The tumour is more likely to be completely resected and mutilating surgery avoided. Most international protocols use vincristine and dactinomycin as standard drugs. The choice of alkylating agent differs between Europe (ifosfamide) and North America (cyclophosphamide). These drugs have proved to be equally effective, but differences in toxicity exist: ifosfamide is more nephrotoxic, whereas cyclophosphamide more gonadotoxic [87]. The addition of other antineoplastic agents to vincristine, dactinomycin and alkylators in the treatment of RMS has been investigated, but no significant improvement in outcome has been found [88]. Surgery In children surgery often starts during the diagnostic phase with biopsy for histological studies, although here interventional radiology plays an increasing role. Excisional biopsy is not advocated except for paratesticular tumours. Most patients end up with postsurgical stage IRS group III. Surgery is generally delayed until after tumour reduction by chemotherapy. The surgical treatment of RMS is site-specific, but the current paradigm is complete wide excision of the primary tumour with a margin of uninvolved tissue whenever possible. Debulking and mutilating procedures should be avoided. Radiotherapy The philosophies underlying the treatment strategies in North America and Western Europe have differed in the past. North American protocols involved aggressive surgery and routine RT, except for those tumours that were microscopically radically resected at diagnosis, followed by prolonged chemotherapy regimens for up to 2 years. The SIOP-MMT (International Society for Paediatric Oncology–Malignant Mesenchymal Tumour) group advocated the use of chemotherapy and surgery in order to achieve complete remission in as many patients as possible in order to avoid RT in these often very young patients. RT often has devastating effects in growing children, potentially leading to significant cosmetic and functional problems. Radiation tolerance of growing bone is ≤20 Gy, but the radiation dose from the treatment of RMS ranges from 36 to 50.4 Gy. For certain sites this policy worked out well (e.g. orbital RMS), and in many patients RT could be avoided [89]. At most other sites, relapse rates were high, and salvage rates after relapse were low. The treatment protocols in Germany (Co-operative Weichteilsarcom Studie, CWS) and Italy (Italian Cooperative Soft Tissue sarcoma Group, ICG) were between that in North America and that of the SIOP-MMT group. In 2005, the former SIOP-MMT, CWS and ICG joined to form the EpSSG. As a result of extensive cooperation between the European and the North American groups, the similarities in treatment strategies now outnumber the differences [90]. Although RT is still deemed essential in many patients to achieve cure, the possibility of radiation-induced second malignant neoplasms must be kept in mind [91, 92]. European approach Today more than 70% of nonmetastatic RMS are cured, but survivors may suffer from sequelae [93–98]. Therefore, the SIOP-MMT group has tried in their RMS75, MMT84, MMT89 and MMT95 studies to avoid mutilating surgery and RT [93, 99, 100]. Only children ≥3 years of age with parameningeal RMS at high risk of meningeal extension and children not achieving complete remission after chemotherapy and surgery were irradiated after intensive chemotherapy. Of all survivors, 49% were treated without significant local therapy [93]. Locoregional relapse occurred in 34%. This has lead to an event-free survival (EFS) of 57% and an overall survival (OS) of 71% [93]. Alveolar histology was associated with a significantly higher risk of relapse and a much higher risk of metastases. North American approach In the studies performed by the IRSG the need for RT is based on surgical radicality, localization, and size of the primary tumour. Only completely resected tumours (IRS group I) with a favourable location (stage I; Table 5), and small tumours (≤5 cm) at unfavourable locations (stage II) did not receive RT in the IRS-IV study [87]. Grossly resected tumours with microscopic residual disease (IRS group II), and incompletely resected tumours or tumours with gross residual disease after biopsy (IRS group III) were irradiated [87, 88, 101, 102]. With this approach in the IRS-IV study an EFS of 78% and an OS of 84% were obtained. Between the IRSG and SIOP-MMT group a significant difference in OS in alveolar RMS was seen (71% vs. 38%) [90]. The salvage rate after relapse of an alveolar RMS was low, and therefore in the current European trial, RT is mandatory for all patients with alveolar histology. Table 5Pediatric Oncology Group and Children’s Cancer Group for the Study of Rhabdomyosarcoma classification [28]StageSitePrimary tumouraSizebRegional nodescDistant metastasisdIOrbitT1 or T2a or bN0, N1 or NXM0Head and neck (excluding parameningeal)Genitourinary, nonbladder nonprostateIIBladder and prostateT1 or T2aN0 or NXM0ExtremityCranial parameningealOther (including trunk, retroperitoneum, etc)IIIBladder and prostateT1 or T2aN1M0ExtremityCranial parameningealT1 or T2bN0, N1 or NXM0Other (including trunk, retroperitoneum, etc)IVAll sitesT1 or T2a or bN0 or N1M1aT1 tumour limited to original muscle or organ, T2 tumour has extension or fixation to the surrounding tissue.ba tumour equal to or less then 5 cm in greatest dimension, b tumour larger than 5 cm in greatest dimension.cN0 no clinical involvement of regional nodes, N1 clinical involvement of regional nodes, NX status of regional nodes unknown.dM0 no distant metastasis, M1 distant metastasis. As described above, European and American approaches, although historically different, have converged based on the results of successive international trials and extensive cooperation between the European and North American groups [90]. As approaches now are very similar, in this review the current EpSSG approach for the different clinical groups is discussed. Current EpSSG approach IRS group I In the IRS-I study, a randomized trial in clinical IRS group I patients, no difference was seen in survival between patients treated with chemotherapy and those treated with chemotherapy and RT [101]. A subsequent retrospective study based on the subsequent IRS-I to IRS-III trials confirmed that for embryonal RMS although there was a small difference in failure-free survival (FFS) with and without RT, OS was not significantly different [103]. On the other hand, for alveolar RMS and undifferentiated RMS, 10-year FFS was 73% vs. 44%, and OS 82% vs. 52% for patients treated with and without RT, respectively [103]. Therefore the current EpSSG study recommends RT in non-embryonal RMS only. IRS group II Evaluation of the role of RT in IRS group II patients in subsequent Cooperative Soft Tissue Sarcoma studies (CWS 81, 86, 91 and 96) showed that for embryonal RMS, EFS was significantly different for patients treated with and without RT [104]. However, OS at 5 years was not significantly different (84% vs. 77%). For patients with tumours of unfavourable histology (independent of site and size), EFS and OS were significantly better when RT was part of the treatment [104]. Therefore, in IRS group II patients, RT is recommended. It is compulsory in patients with high-risk features, but may be omitted in patients with favourable histology in whom RT may be considered too toxic when considering patient age and site of the tumour. IRS group III In patients with IRS group III tumours, RT is the only available therapy in patients who cannot receive a secondary complete resection. Patients who do receive a delayed complete resection benefit from additional RT. In the CWS trials 81–96 5-year EFS was 77% and 58% for those treated with and without additional RT, respectively [28]. OS, however, was not significantly different between the two groups: 84% and 79%, respectively. RT is, therefore, usually indicated except in patients with a favourable site and histology. Prognosis As malignant tumours in childhood are rare diseases, most children with cancer have been included in international treatment protocols. This way survival for localized disease (85% of patients) has improved from 25% in the early 1970s to 75% in the most recent international trials [101, 105–107]. Although results for localized RMS have improved dramatically in the past decades, patients with disseminated disease still have a dismal prognosis, with a 5-year OS of 24% [26]. A way to reduce long-term sequelae of RT may be by using brachytherapy, especially in girls with genital tract RMS and patients with bladder-prostate, extremity, and head and neck RMS [100, 108–111]. Other options are the use of modulated RT (IMRT) and proton therapy [112, 113]. The postoperative patient Image interpretation and management of the patient after surgery and often RT are challenges (Fig. 22). Most important is proper knowledge of the surgical procedure and/or the radiation field. The following postoperative changes can be encountered in children treated for RMS: HaematomaOedemaSoft-tissue infection/abscessCalcificationForeign bodiesMuscle flaps/fat padsDistorted anatomyRadiation effect Figure 23 shows a possible decision tree to manage postoperative findings on follow-up imaging. Fig. 22A 6-year-old boy with a history of treated bladder RMS. At surgical resection the urethra was damaged leading to a persistent urinoma in, after RT, nonvital tissue. MRI image after treatment shows a mass (asterisk) between the urethra (open arrow) and the rectum (solid arrow). This mass is a vascularized gracilis muscle flap used to repair the defect. Without proper knowledge of the surgical history of the patient, this might have been interpreted as tumour recurrence. Histopathology: embryonal RMSFig. 23Flow chart for posttreatment lesions found on MRI. SI signal intensity, CE contrast-enhanced [114] Conclusion In this review we have discussed the findings of RMS outside the craniofacial region. The treatment of RMS requires a multidisciplinary approach, in which paediatric oncologists, radiologists, paediatric surgeons, pathologists and radiation oncologists all play a vital role. Although they are the most common soft-tissue tumour of childhood, these still rare tumours should be evaluated and treated in specialized centres.

Obes_Surg-4-1-2367390

Delayed Intrathoracic Gastric Perforation After Obesity Surgery: A Severe Complication

We describe a case of a patient with an intrathoracic gastric perforation, 6 months after she underwent a gastric banding procedure for the treatment of morbid obesity. After an urgent laparotomy during which the stomach was replaced and oversewn, she recovered uneventfully. The possible mechanism of this severe complication is discussed. Introduction Gastric banding is a frequently performed operation for patients with morbid obesity. Complications have been described extensively. We describe a rare and severe complication of an intrathoracic stomach in a patient with gastric banding, 6 months after the initial procedure, and its treatment. Case Report A 37-year-old woman was admitted to our emergency department with acute onset of abdominal pain, vomiting, and dyspnea. Six months earlier, she had undergone laparoscopic adjustable gastric banding (Lap-Band®)for severe obesity. Physical examination now showed an anxious, dyspneic woman with a respiratory rate of 45 per minute. There was a tachycardia (140/minute) and blood pressure difference between the left (60/40 mmHg) and the right arm (110/70 mmHg). Auscultation of the chest revealed diminished breathing sounds on the left side. Chest x-ray showed an intrathoracic position of the stomach through a paraesophageal hernia with a shift of the mediastinum to the right (Fig. 1). A small pneumothorax was seen. Fig. 1Intrathoracic position of the stomach with a shift of the mediastinum to the right After a nasogastric tube was inserted, the patient was intubated because of respiratory insufficiency. A computed tomography (CT) scan of the chest showed a complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax (Fig. 2). The condition of the patient worsened and she was taken to the operating room for urgent laparotomy. Fig. 2Complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax During laparotomy an ischemic gastric perforation was identified at the orifice of the paraesophageal hernia, with spill of gastric content into the abdomen and thoracic cavity. The intrathoracic stomach was reduced into the abdomen. The gastric perforation was oversewn. The Lap-band was removed, and a Nissen fundoplication was performed. At last, a thoracic drain was placed to treat the pneumothorax. The patient was treated with intravenous antibiotics (metronidazole and cefuroxime) for 5 days. After 2 days, she was able to leave the Intensive Care Unit. Her postoperative course was uneventful. Thirteen days after surgery, she left the hospital. Discussion Gastric perforation after gastric banding has been described earlier [1]. In all reported cases, the perforation was caused by erosion of the band through the stomach with an incidence of 0.3–3% of the patients. Furthermore, herniation of the stomach into the thoracic cavity is a well-known entity. It has been described as a late complication after Nissen fundoplication, even in combination with intrathoracic perforation [2–4]. We describe the first case in which both perforation and herniation have occurred simultaneously after obesity surgery and even not as an immediate consequence of the operation. In our case, the gastric band was not the primary cause of the perforation. Most probably, the stomach had moved into the thoracic cavity through a preexistent paraesophageal hernia. Because of air-trapping distal to the band accompanied by having a copious meal, the intrathoracic stomach distended, rotated, and perforated at the orifice of the hernia. The acute onset of pain can be explained by the perforation and the dyspnea appears to be caused by mediastinal shift as well as the pressure atelectasis of the left lung. Retrospectively, our patient was known to have a paraesophageal hernia before she underwent gastric banding. The possible severe complications of this combination should be considered. A Nissen fundoplication before or in the same operation could be an option for this subgroup of obese patients.

J_Biol_Inorg_Chem-3-1-2099461

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM−1 cm−1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s−1, kobs2 = 1.9 s−1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 × 10−2 s−1, kobs4 = 2.18 × 10−2 s−1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed. Introduction Tungstoenzymes and molybdoenzymes typically catalyze the generic n = 2 reaction: R + H2O ↔ RO + 2[H], where R is either an oxoanion (e.g., sulfite) or a relatively small organic metabolite (e.g., an aldehyde). On the basis of structural (cofactor and protein) and functional properties molybdoenzymes are classified in three families: the sulfite oxidase family, the xanthine oxidase family, and the dimethyl sulfoxide reductase (DMSOR) family [1]. The latter also encompasses a limited number of tungsten-containing formate dehydrogenases. The majority of the tungstoenzymes, however, cannot be classified in any of these three families; they form a separate family of aldehyde oxidoreductases (AORs) together with a few apparently less common AOR molybdoenzyme members [2]. The mechanism of action of molybdoenzymes has been extensively studied for several decades, especially for xanthine oxidase [3]. In contrast, attempts to understand the reaction mechanism of tungstoenzymes, in particular for the members of the AOR family, are more recent and of limited extent, although the available structural information on tungstoenzymes and that on molybdoenzymes are comparable. The present study leads to the first proposal of a complete catalytic cycle of the AOR family member formaldehyde oxidoreductase (FOR). The cofactor common to the enzymes of all four families consists of a single Mo(VI/IV) or W(VI/IV) ion coordinated by the dithioleno sulfurs of one or two three-ring pterin ligands sometimes with an additional nucleotide attached through a phosphoester bond [4]. All tungsten systems appear to be nonnucleotide dipterin systems with an additional magnesium phosphate coordination between the pterins [5, 6]. The coordination number of the Mo or W is 4–7; extra ligands are provided by an amino acid side chain and/or by O,S of a small, nonprotein ligand. The vast majority of the Mo/W enzymes have additional prosthetic groups for interaction with a second substrate (e.g., NADH) and/or for electron transfer with a protein natural redox partner (e.g., ferredoxin). These heme, flavin, and/or iron–sulfur prosthetic groups are strongly colored to the extent that optical monitoring of the relatively weakly colored (see below) metallopterin has thus far not been tried for mechanistic studies of these complex enzymes. Electron paramagnetic resonance (EPR) spectroscopy is not a competitive alternative, because the only EPR-detectable oxidation state, Mo(V) or W(V), does not appear to be a competent intermediate in the primary, n = 2 reaction. A small group of molybdoenzymes, exemplified by DMSOR, are exceptional because these proteins contain no other prosthetic groups in addition to the active-site molybdopterin. Furthermore, substitution of W for Mo in Rhodobacter capsulatus DMSOR affords an active enzyme with an unmodified 3D structure. The Mo(VI) and W(VI) metallopterin UV–vis spectra of this enzyme have been reported to exhibit several broad peaks in the visible and near-UV region with extinction coefficients of the order of ε ≈ 2 mM−1 cm−1 [7], and the molybdopterin absorption has been used as monitor in a pre-steady-state kinetics study [8]. Following the DMSOR, the next step up in optical complexity is found in the AOR family of tungstoenzymes, which all contain—in addition to the tungstopterin active site—a single [4Fe–4S](2+;1+) cluster for electron transfer. The visible spectrum of the iron–sulfur cubane consists of a single broad line at approximately 400–430 nm, which is not only a simpler pattern to that found for [2Fe–2S] clusters (e.g., in the xanthine oxidase family of molybdoenzymes), but it also has a significantly smaller extinction coefficient than flavin or heme prosthetic groups, and so chances are more favorable for monitoring the weaker absorptions from the metallopterin cofactor in these complex enzymes. The homotetrameric (4 × 69 kDa) Pyrococcus furiosus FOR is a member of this family, and we describe here a study of this enzyme as the first example of pre-steady-state kinetics research of a complex, group-6 metalloenzyme using the color of the active center as the monitor. Our choice of FOR was also indicated by the availability of a 3D structure, which was the basis of an initial proposal of part of the enzyme’s working mechanism [6], and was further stimulated by the unusual behavior of the enzyme’s substrate in aqueous solution [9, 10]. Materials and methods P. furiosus (DSM 3638) was grown at 90 °C under anaerobic conditions with starch as a carbon source, as previously described [11]. Cells were broken by osmotic shock, diluting with 5 vol 30 mM tris(hydroxymethyl)aminomethane (Tris)/HCl, pH 8.0, containing, 0.1 mg ml−1 DNase I, 0.1 mg ml−1 RNase, and 1 mM cysteine. A cell-free extract was obtained as the supernatant after 30 min centrifugation at 15,000g. “High-activity” FOR was purified as reported previously [9, 10]. Samples were anaerobically purified in 20 mM Tris/HCl buffer, pH 8.0. An extra column of (diethylamino)ethyl (2.0 cm × 10 cm) was equilibrated with 20 mM piperazine/diethanesulfonic acid buffer, pH 6.4. A 100-ml gradient was used from 0 to 0.5 M NaCl. Ferredoxin was purified as reported previously [12]. Samples were anaerobically purified in 20 mM Tris/HCl buffer, pH 8.0. Protein concentration was determined using the bicinchoninic acid method using bovine serum albumin as the standard. Subunit molecular weight and degree of purity were determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis using a Phast system (GE Healthcare). FOR activity was routinely assayed at 80 °C, under anaerobic conditions, with formaldehyde, or glutardialdehyde, as the substrate and 3 mM benzyl viologen as the electron acceptor in 50 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Epps) buffer, pH 8.4. Steady-state kinetics of FOR were determined by activity measurements in triplicate in an optical assay with different concentrations of formaldehyde (0.5, 1, 2, 5, 10, 25, 50 mM) as a substrate. Benzyl viologen (ε600 = 10.6 mM−1 cm−1 [13]) or ferredoxin (ε400 = 17.0 mM−1 cm−1 [14]) were used as an electron acceptor at various concentrations in 50 mM Epps buffer, pH 8.4, and  80 °C, where one unit is defined as 2 μmol electron acceptor reduced per minute. The concentrations of benzyl viologen and ferredoxin were, respectively, 50–3,000 and 10–200 μM, and the ferredoxin concentration is per dimer [15]. The UV–vis spectrum was recorded with a Hewlett-Packard 8452A diode-array spectrophotometer. The optimal excitation wavelength for fluorescence measurements was obtained using a Shimadzu RF-5001PC spectrofluorophotometer. Pre-steady-state studies were carried out under anaerobiosis in 50 mM Epps buffer, pH 8.4, at 50 °C with formaldehyde (25 mM) and formaldehyde-d2 (25 mM) as a substrate using an SX.18 MV stopped-flow apparatus equipped with a sequential mixing capability and a diode-array rapid-scan detection system (Applied Photophysics). The UV–vis data were obtained by recording 400 spectra at different time scales (16, 65 and 250 s). A spectrum obtained 2 ms after mixing was taken as the “oxidized” reference spectrum. Fluorescence data were obtained by exciting the samples at 280 nm, and the change in maximum fluorescence emission was measured at 340 nm. Results Steady-state kinetics Steady-state kinetics studies at 80 °C were performed on the combinations FOR/formaldehyde/benzyl viologen and FOR/formaldehyde/ferredoxin. FOR catalyzes a two-electron transfer from formaldehyde to the one-electron acceptor benzyl viologen or ferredoxin. This results in a reaction with three substrates; one formaldehyde molecule and two benzyl viologen or two ferredoxin molecules. The possible mechanisms for this reaction are so-called enzyme-substitution mechanisms: the triple-transfer mechanism or the concerted-substitution mechanism [16]. Triple-transfer mechanism: Concerted-substitution mechanism: The equation for the reaction rate can be simplified, when substrates B and C (ferredoxin or viologen) are the same. The results have the same form as equations for the reaction rate of an enzyme with two substrates. The triple-transfer mechanism will transform into the substituted-enzyme mechanism for two substrates and the concerted-substitution mechanism will transform into the ternary-complex mechanism for two substrates [16]. The apparent KM values for the second and third substrates are identical, and the measured KM is twice the real KM of the second or third substrate [16, 17]. To predict whether the systems function according to a ternary-complex mechanism or a substituted-enzyme mechanism, one substrate was varied, while the other substrate concentration was held constant. Substrate inhibition of one of the substrates is described by Eq. 3, where KsiB is the constant that defines the strength of the inhibition of substrate B with concentration b: Primary plots of the concentration of the inhibitor divided by the reaction rate (b/v) versus the concentration of the inhibitor (b) are parabolic and intersect at a common point on the b/v axis. The primary plots of the concentration of the noninhibiting substrate divided by the reaction rate (a/v) versus the concentration of the noninhibiting substrate (a) are linear and intersect at a positive value of the concentration of noninhibiting substrate [16]. A more complex situation occurs when both substrates show substrate inhibition. At low concentrations of both substrates the shapes of both curves are parabolic. Substrate inhibition by one substrate at low concentrations of the other provides strong positive evidence that the substituted-enzyme mechanism applies [16]. The plots of a/v versus a and b/v versus b, with substrate A is formaldehyde and B is benzyl viologen or ferredoxin, are presented in Figs. 1 and 2. All intersection points are situated within experimental error at positive values of the substrates. The parabolic shape of all the figures indicates, as stated above, substrate inhibition by both substrates. All four graphs in Figs. 1 and 2 are consistent with a substrate substituted-enzyme mechanism. Fig. 1Primary plots for Pyrococcus furiosus formaldehyde oxidoreductase with formaldehyde as a substrate and benzyl viologen as an electron acceptor. Formaldehyde was used in concentrations of 1, 2, 5, 10, 25, and 50 mM. Benzyl viologen was used at concentrations of 50, 100, 500, 1,000, 2,000, and 3,000 μM. Each data point is the average value from three measurementsFig. 2Primary plots for P. furiosus formaldehyde oxidoreductase with formaldehyde as a an substrate and ferredoxin as electron acceptor. Formaldehyde was used at concentrations of 0.5, 2, 5, 10, 25, and 50 mM. Ferredoxin was used at concentrations of 10, 20, 40, 80, 100, and 200 μM. Each data point is the average value from three measurements One-substrate Michaelis–Menten kinetics was used to fit the data and to calculate KMapp and Vmaxapp at the different concentrations of substrate and acceptor. To calculate the KM and the Vmax of the different substrates the KMapp and vapp data were fitted using the equations for the apparent values of the Michaelis–Menten parameters for the substituted-enzyme mechanism [16]: with equivalent expressions for substrate A. The KM and Vmax values of formaldehyde measured with excess benzyl viologen as an electron acceptor (Table 1) are in agreement with previous measurements [10, 18]. The KM value for formaldehyde (3.8 mM) with ferredoxin as an electron acceptor is approximately 3 times less than when benzyl viologen is used as the acceptor. The conversion rate of formaldehyde with ferredoxin as the electron acceptor is 6 times less than when benzyl viologen is used as the acceptor. The values for KM are unusually high for a physiological substrate. We have previously provided experimental evidence indicating that only “free” formaldehyde (i.e., the nonhydrated form which is 0.2% at 80 °C) is a substrate for FOR [10]. The corrected KM values for free formaldehyde with acceptor benzyl viologen or ferredoxin are, respectively, 71 and 21 μM. Note that Vmax decreases by a factor of 4 when the temperature is lowered from 80 to 20 °C, and that KM for free formaldehyde is temperature-independent [10]. Table 1 Catalytic properties of formaldehyde oxidoreductase determined at 80 °C with formaldehyde as the substrate and ferredoxin or benzyl viologen as the electron acceptor Electron acceptor Benzyl viologen Ferredoxin KM total formaldehyde (mM) 13 ± 2 3.8 ± 0.5 KM free formaldehyde (μM) 71 ± 10 21 ± 3 Vmax formaldehyde (s−1) 28 ± 1a 4.5 ± 0.5 KM electron acceptor (μM) 73 ± 19 14 ± 7 Vmax electron acceptor (s−1) 32 ± 2 6.2 ± 3.2 KM glutardialdehyde (mM) 12 ± 2 Vmax glutardialdehyde (s−1) 13 ± 1 The ratio of activities at 80 °C compared with 50 °C is 2.8 under standard conditions (see “Materials and methods”) with 50 mM total formaldehyde aIn our previous work [10] Vmax = 54 s−1 was reported on a per tungsten atom basis; this is equal to 28 s−1 on a per protein molecule basis Varying the formaldehyde concentration gives the KM and Vmax values for the electron acceptors. The KM value of benzyl viologen of 73 μM is comparable to values found in literature for other enzymes that react with benzyl viologen as an electron acceptor. The KM value of ferredoxin (14 μM) is, however, uncharacteristically low: previously reported literature values for FOR and AOR are 100 and 200 μM, respectively [9, 19]. An explanation for this discrepancy may be found in the fact that high concentrations of substrate, up to 50 mM of total formaldehyde for FOR, have been used in previous studies to determine KM of ferredoxin. In the present work concentrations from 0 to 10 mM were used to determine KMapp and vapp. When activity was measured with high concentrations of formaldehyde (i.e., resulting in significant substrate inhibition) the apparent KM of ferredoxin increased to approach values found in the literature (data not shown). In a previous study alternative substrates for FOR were reported with KM values of the order of a few millimoles per liter. The lowest KM value was found for glutardialdehyde (KM = 0.8 mM) with benzyl viologen as an electron acceptor [9]. We have been unable to reproduce these results. In our hands the KM for glutardialdehyde was determined to be 12 mM, i.e., approximately the same as the apparent KM for total formaldehyde. Pre-steady-state kinetics Pre-steady-state kinetics studies were performed on FOR (20 μM) and formaldehyde at 50 °C. Formaldehyde was used at a concentration of 25 mM (50 μM free formaldehyde). UV–vis spectra were recorded at 40- and at 160-ms intervals. At least two spectra per time point were averaged for noise reduction. In the spectrum of oxidized FOR the broad feature of the [4Fe–4S]2+ cluster dominates. However, close inspection reveals weak (εmax ≈ 1–2 mM−1 cm−1) absorptions at higher wavelengths similar to those reported for the single-cofactor DMSOR [20]. After subtraction of the spectrum at t = 2 ms, difference spectra were obtained (Fig. 3), at 0.1, 0.5, 1, 2, 5, 10, and 16 s, which reveal peak shifts and a lack of isosbestic points. Fig. 3Difference in absorbance versus wavelength measured in pre-steady-state experiments with formaldehyde as a substrate at times 0.1, 0.5, 1, and 2 s (a) and 2, 5, 10, and 16 s (b). The reference spectrum was taken at t = 2 ms. c UV–vis spectra of the oxidized and the substrate-reduced form (after 65-s incubation time) of P. furiosus formaldehyde oxidoreductase From the difference spectra five different wavelengths were selected: 335, 390, 435, 595, and 695 nm. At 595 and 695 nm, approximately corresponding absorption bands can be found in the spectrum of oxidized DMSOR from R. capsulatus [8]. Amplitude versus time traces of the 40 and 160 ms spectra at the different wavelengths were combined. After 65 s the time traces for all the wavelengths essentially leveled off (Fig. 4). Data points were taken up to 250 s but no further significant changes were found (not shown). Fig. 4Stopped-flow UV–vis traces obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at 335, 390, 435, 595, and 695 nm The major changes at the different wavelengths occur in the first seconds. Three phases starting at different times can be identified in Fig. 5a. The first phase starts at or before t ≈ 20 ms; the second and the third phases start at approximately 100 and 300 ms, respectively. Changes in the amplitude versus time traces at 390 and 435 nm start at or before 20 ms; the trace of the wavelength of 595 nm follows the trend of the 335 nm trace, with the difference that all phases start approximately 200 ms later. All data at the different wavelengths were globally simulated with a four-phase exponential least-squares fit (Table 2). Fig. 5Stopped-flow UV–vis traces plus fits on a logarithmic time scale obtained for the reduction of P. furiosus formaldehyde oxidoreductase by a formaldehyde or b deuterated formaldehyde at 335, 390, 435, 595, and 695 nm. c Stopped-flow fluorescence trace plus fit obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at an excitation wavelength of 295 nm and an emission wavelength of 340 nm. d Stopped-flow UV–vis traces plus fits obtained for the binding of formate by P. furiosus formaldehyde oxidoreductase at 335, 390, 435, 595, and 695 nmTable 2Rate constants from pre-steady-state kinetics of formaldehyde oxidoreductase plus formaldehyde or deuterated formaldehyde or formate at 50 °CUV–visFluorescenceCH2OCD2OFormateCH2Ok1 (s−1)4.71.120.514.7k2 (s−1)1.90.942.00 × 10−21.9k3 (s−1)6.10 × 10−26.07 × 10−26.10 × 10−2k4 (s−1)2.18 × 10−22.26 × 10−2 A previous steady-state kinetics study on the substrate kinetic isotope effect of FOR showed a significant primary isotope effect for C–H/D bond breaking. The specific activity of FOR dropped by a factor of 3 when deuterated formaldehyde was used as a substrate [21]. In the present work the pre-steady-state kinetics of FOR with formaldehyde-d2 was determined in the same fashion as for the experiments with formaldehyde. The amplitude versus time traces at selected wavelengths of FOR incubated with formaldehyde-d2 resemble the traces of the different wavelengths when formaldehyde was used as the substrate; the phases are similar, only the duration and the onset of the changes of states are different. Again the data were fitted with a four-phase exponential fit (Fig. 5b; Table 2). Tryptophan residues are excellent probes for protein conformational changes. In FOR a single tryptophan (Trp441) can be found in the active site [6]. A fluorescence stopped-flow experiment was performed using 280 nm as the excitation wavelength and 340 nm as the emission wavelength. The concentrations of FOR and formaldehyde used were the same as in the previous UV–vis stopped-flow experiments. Traces were measured on different time scales: 1, 10, and 100 s, and were combined (Fig. 5c). The data can be fitted to a three-phase exponential model. The noise levels however are high. The fluorescence data were fitted simultaneously with the UV–vis data of Fig. 5a, affording a consistent set of parameters (Table 2). Formaldehyde is oxidized to formate by FOR with reduction of the W(VI) center. We checked whether FOR can oxidize formate to carbon dioxide. Activity measurements in an optical assay were done at 80 °C using formate as the substrate and methyl viologen as the electron acceptor. FOR was unable to oxidize formate. Pre-steady-state binding experiments with oxidized FOR using formate as the nonreducible substrate were done at the same time intervals as for the experiments with formaldehyde (Fig. 5d). Although formate is not converted to carbon dioxide by FOR, absorption changes are found in the time traces at the different wavelengths. The first phase starts at t ≈ 20 ms; the second and the third phases start at approximately 100 and 300 ms, respectively. The time trace at 335 nm resembles the trace obtained with formaldehyde. The time traces at 390 and 435 nm are not similar to each other in contrast to when formaldehyde was used as the substrate. The time traces at 595 and 695 nm are essentially unaffected by the formate. All data at the different wavelengths were globally fitted with a two-phase exponential least-squares model (Table 2). Discussion Steady-state kinetics An initial proposal for part of the working mechanism of P. furiosus FOR was deduced by Hu et al. [6] from the enzyme’s 3D structure: two electrons are transferred from the substrate to the tungsten center. The pterin acts as a noninnocent ligand and transfers the electrons via the N8 atom one by one through Cys491 to the iron–sulfur cluster of FOR. The iron–sulfur cluster of ferredoxin accepts an electron through its Asp14 [6]. The electron is then transferred from the iron–sulfur cluster of ferredoxin to a membrane-bound hydrogenase [22]. After reduction of the iron–sulfur cluster of ferredoxin, the electron transfer comes to a halt. Only one electron at a time can pass through this route. Two ferredoxin molecules are necessary to transport both electrons; alternatively, a ferredoxin dimer [15] can twice in a row bind to FOR and accept an electron. This means that a two-substrate mechanism is insufficient to describe this three-substrate system. The second and the third substrates are both ferredoxin. This results in steady-state Michaelis–Menten equations that have the same form as equations for the reaction rate of an enzyme with two substrates; however, the measured KM for ferredoxin will be twice the “real” KM. The triple-transfer mechanism for three substrates with substrates two and three (ferredoxin) the same is consistent with the experiments. Both substrates, either formaldehyde and ferredoxin or formaldehyde and benzyl viologen, inhibit FOR. The KM values of 20–70 μM for free formaldehyde are in agreement with our previous studies [10]. When a nonphysiological electron acceptor such as benzyl viologen is used then the KM for formaldehyde is 3–4 times higher. It is possible that when ferredoxin binds to FOR the enzyme experiences a slight conformational change, resulting in a lower KM for formaldehyde. In these studies a KM of 14 μM was determined for ferredoxin, which is 5 times lower than the KM of benzyl viologen. The high KM reported in the literature for ferredoxin was determined with high concentrations of formaldehyde. An explanation for this high value is that at high concentrations (50 mM) formaldehyde inhibits and partially denatures FOR. Pre-steady-state kinetics Pre-steady-state kinetics studies were performed at 50 °C. This relatively low temperature was dictated by technical limitations of the stopped-flow apparatus. FOR activity at 50 °C is 2.8 times less than at 80 °C, which affords an increased time resolution of enzyme intermediates. Difference spectra from the pre-steady-state data revealed peak shifts and a lack of isosbestic points. These features are an indication that several processes are happening in the first seconds of the reaction. The visible spectrum of the iron–sulfur cubane consists of a single broad line at approximately 400–430 nm; the relatively weakly colored metallopterin is found at different wavelengths. A minimal first-order exponential fit model was used to fit the pre-steady-state data. The UV–vis data could be fitted globally with a three-phase exponential fit. Two fast processes and one slow process were found. The fluorescence data were initially modeled with a two-phase exponential fit, resulting in one fast and one slow process. The fast and slow processes deduced from the fluorescence and from the UV–vis data were clearly different. A four-phase exponential model afforded a significantly improved fit to the UV-data (two fast and two slow processes), and the fluorescence data were fitted more accurately with a three-phase exponential model (two fast processes and one slow process). The complete set of fluorescence and UV–vis data obtained with the same enzyme and substrate were then fitted in a global analysis with a three-phase and a four-phase exponential model, respectively. The same four-phase model was used to fit the pre-steady-state data obtained using formaldehyde-d2 as the substrate. The fit is comparable with the fit of formaldehyde but the rate constants of the first and second phases are lower, as expected from the steady-state studies. The first phase is 4 times slower (k1 = 1.12 s−1) than with formaldehyde as the substrate. The rate constant of the second phase (k2 = 0.94 s−1) is lower than when formaldehyde is used as the substrate, consistent with a previously reported steady-state kinetics experiment [21]. The rates for the first two phases with CD2O as the substrate are not significantly different within experimental error. The rate constant for intramolecular electron shuffling appears to be unaffected by the use of formaldehyde-d2. Residuals of the individual fits plotted versus time were essentially randomly distributed around the x-axis, thus attesting to the quality of the global fit. Several intermediate species are formed during the first seconds of the reaction. These results are in contrast with previous studies on molybdenum–DMSOR, where in stopped-flow experiments clear isosbestic points were found, and analysis of the time course produced a simple exponential, indicating a simple concerted two-electron reduction of the enzyme with no intermediate species [23]. Two alternative interpretations can be envisioned for the three phases in pre-steady-state kinetics (Fig. 6). Fig. 6Proposed alternative models for the pre-steady-state phases of fully oxidized P. furiosus formaldehyde oxidoreductase reacting with formaldehyde. Rate constants with capital letters as a subscript involve binding, oxidizing the substrate, releasing the product, and rearranging electrons between the tungsten center and the iron–sulfur cluster. Alternatively, rate constants with numbered subscripts involve oxidation of substrate, a conformational change leading to weakened product binding, release of product, and electron rearrangement FOR is represented in Fig. 6 as a rectangle with two redox groups, a tungstopterin (W) with oxidation states 6+, 5+, or 4+, and a [4Fe–4S] cubane cluster with oxidation states 2+ or 1+. On the left of W is an empty binding pocket which can be occupied by substrate (A), by product (P), or by ready-to-leave deactivated product (P*). Occupation by A means that the enzyme has acquired a molecule of substrate in the previous reaction. Similarly, a W capped by a double-bonded O means that the enzyme has previously acquired an oxygen (protonation undefined) from the solvent. The protein is initially in the fully oxidized form; the tungsten center is 6+ and has an oxygen atom bound that was previously acquired from a water molecule, and the iron–sulfur cluster is 2+. In the first model the initial step (kA = 4.7 s−1) observed in the optical monitoring is taken to be the overall binding process of formaldehyde to the enzyme. No changes to the formal oxidation state of the tungsten center and iron–sulfur cluster are assumed to occur in this stage. Subsequently, in what appears to be a single event, the oxygen atom is transferred, the substrate is oxidized to the product (kB = 1.9 s−1), and the tungsten center receives two electrons from the substrate and is reduced to 4+. The product then leaves the protein (kC = 6.10 × 10−2 s−1). In this step there are no changes in the oxidation states of the tungsten center and the iron–sulfur cluster. The active site of FOR has been proposed by Hu et al. [6] to be closed off from the solvent by a lid provided by the protein itself. It is possible that the substrate, free formaldehyde, is so small that it will not experience significant resistance in reaching the active site. The bigger and charged product may have more difficulty getting free of the enzyme, resulting in a low rate. The protein will then, in the absence of an external electron acceptor, redistribute (to a minor extent; see below) its electrons over the tungsten center and the iron–sulfur cluster (kD = 2.18 × 10−2 s−1). The tungsten center will become 5+ and the iron–sulfur cluster 1+, consistent with previously determined redox properties for “high-activity” enzyme [10]. An isotope effect of 4.2 is found for the rate constant of the first step (Table 1), which seems to be unrealistically large for a noncovalent binding event, and would rather seem to point toward an event involving the breaking and making of covalent chemical bonds. Therefore, the following would appear to be a rather more likely interpretation of the stopped-flow data. Alternatively, if the entry and binding of the substrate does not lead to a detectable change in the optical absorption, then the first step (k1 = 4.7 s−1) can be the two-electron reduction of the tungsten. In this model the second step (k2 = 1.9 s−1) is a conformational change leading to an “open” state with decreased affinity for the product formed. The third step (k3 = 6.10 × 10−2 s−1) then is the ready release of this now weakly bound formate. The pre-steady-state phase ends with the two-electron-reduced intermediate which, in the absence of an external electron acceptor, slowly, and only partially converts, by electron shuffling, to the EPR-detectable, paramagnetic side product. Previous EPR studies on FOR revealed this slow electron shuffling at 20–60 °C between the tungsten center and the iron–sulfur cluster upon reduction with formaldehyde or with dithionite [10]. Only after minutes the W(V) signal appeared in the EPR measurements. This observation is in qualitative agreement with the low rate constants found here for the decay of the fourth state. The pre-steady-state data from the experiments with formate and FOR were fitted with a two-phase exponential model: there is one fast process and one slow process. Formate is not oxidized or reduced by FOR; therefore, the fast process (kobs1 = 0.51 s−1) could be the entry and the binding of formate to the active site of FOR. As in the FOR/formaldehyde system the slow process (kobs2 = 2.00 × 10−2 s−1) is probably a nonphysiological process: the protein reaches a dead end. Excluding the very slow electron redistribution step in the absence of external electron acceptor (the last step in Fig. 6) a complete cycle could now be written in which the enzyme shuttles between fully oxidized, one-electron-reduced, and two-electron-reduced, by inclusion of the reoxidation steps of the two-electron-reduced intermediate through serial reactions with two molecules of oxidized ferredoxin. However, such a scheme would be inconsistent with steady-state turnover rates, because the rate constant (k3 or kC) of the product-release step (the penultimate step in Fig. 6) is more than an order of magnitude less than kcat. On the other hand, it is conceivable, that the one-electron-reduced intermediate reacts with a second substrate molecule to become three-electron-reduced (with respect to the resting enzyme). This would lead to the cycle presented in Fig. 7 in which the enzyme shuttles between one-electron-reduced, two-electron-reduced, and three-electron-reduced. Fig. 7Proposed model for the catalytic redox cycle of P. furiosus formaldehyde oxidoreductase. A is the substrate formaldehyde; P is the product formate. The cube-enclosing circle represents the natural redox partner, ferredoxin. Pre-steady-state kinetics measures either the steps with rate constants kA through kD or, alternatively, those with constants k1 through k4. The fully oxidized enzyme is reductively activated by the reaction with formaldehyde to enter a redox cycle in which the enzyme shuttles between one--electron-reduced, two--electron-reduced, and three-electron-reduced states. See the text for additional explanation Under this model the steps observed in the pre-steady-state experiments would actually constitute an activation process. Note that once the steady state of this cycle has been reached, the enzyme reacts sequentially with one formaldehyde and two ferredoxin molecules consistent with the “A+2B” Michaelis–Menten steady-state analysis. The proposed reaction cycle of Fig. 7 is based on a combination of steady state-kinetics, pre-steady-state kinetics, and EPR studies in the absence of an electron acceptor and available structural data. Future pre-steady-state kinetics studies in the presence of excess natural electron acceptor ferredoxin may put the model to a rigorous, be it experimentally challenging, test.

Ann_Hematol-4-1-2275303

Practical recommendations on the use of lenalidomide in the management of myelodysplastic syndromes

Lenalidomide, an oral immunomodulatory agent, has received approval in the USA from the Food and Drug Administration (FDA) for the management of myelodysplastic syndromes (MDS) classified by the International Prognostic Scoring System (IPSS) as low risk or intermediate-1 risk and with a deletion 5q (del(5q)) cytogenetic abnormality. Although some patients with del(5q) have a relatively good prognosis, all del(5q) patients will become transfusion-dependent at some point during the course of their disease. The results of two clinical trials in more than 160 patients with MDS have demonstrated clear therapeutic benefits of lenalidomide, with >60% of patients achieving independence from transfusion during therapy, irrespective of age, prior therapy, sex, or disease-risk assessment. The recommendations presented in this review will aid the safe administration of lenalidomide for the treatment of patients with low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality, and they will help physicians avoid unnecessary dose reduction or interruption, thus assuring the best efficacy for patients. Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and leading to peripheral cytopenias and a genetic instability with enhanced risk of disease transformation to acute myeloid leukemia (AML). In the general population, MDS affects approximately five in every 100,000 individuals and is regarded as a relatively rare disease [1]. However, its incidence is highest in people aged >70 years, and in this age group, MDS is the most frequently occurring malignant hematological disorder: twice as frequent as AML, more common than chronic lymphocytic leukemia, and more common than all other malignant lymphomas combined [2]. All patients diagnosed with MDS will eventually die from their disease, and in about 30%, the disease will progress to AML [3]. Between 40% and 60% of patients with primary MDS and about 90% of patients with secondary MDS have chromosomal abnormalities at the time of diagnosis [4]. Chromosomal abnormalities may occur as a single abnormality or as part of a complex karyotype; disease severity increases with the number of abnormalities. The most common chromosomal abnormalities are deletions of chromosome 5 and chromosome 7 and trisomy of chromosome 8 [5]. In a subgroup of patients who have an isolated deletion 5q (del(5q)), termed 5q-syndrome, the clinical profile is distinct [6]. Typically, this clinical profile includes macrocytic anemia, a normal to increased platelet count, mild leukopenia, hypolobulated megakaryocytes in the bone marrow, a medullary blast count <5%, and an isolated del(5q) abnormality including a common deleted region between 5q31 and 5q33 [4, 7]. Consistent with the observation that disease severity is related to the number of chromosomal abnormalities, patients with del(5q) plus additional cytogenetic abnormalities have a worse prognosis than those with an isolated del(5q) abnormality [3, 5]. On the basis of the results of an international phase II trial in 148 patients [8], the oral immunomodulatory, antiangiogenic, and antineoplastic agent lenalidomide (Revlimid®; Celgene Corporation, NJ, USA) was approved in December 2005 by the US Food and Drug Administration (FDA) for the treatment of patients with transfusion-dependent International Prognostic Scoring System (IPSS) classified low-risk to intermediate-1-risk MDS with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. The first 46 patients recruited received lenalidomide 10mg/day for 21days every 28-day cycle, for up to 24weeks. After a protocol amendment, the remaining 102 patients received lenalidomide 10mg/day on a continuous dosing schedule [8]. Overall, 67% of patients who were initially transfusion-dependent achieved transfusion independence, and another 9% achieved a decrease (≥50%) in the total number of transfusions required during lenalidomide therapy [8]. Forty-five percent of the 85 evaluable patients in this study achieved a complete cytogenetic response, irrespective of chromosomal complexity [8]. In January 2007, an international group of MDS specialists met to discuss the practical management of lenalidomide in patients with transfusion-dependent, IPSS-classified low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality. The recommendations presented here are based on the clinical data derived from the current literature [8, 9], from the known clinical profile of lenalidomide in other disorders, and the clinical experience of the panel members in the practical use of this agent. Although lenalidomide has been used in IPSS-classified intermediate-2-risk and high-risk patients and in patients without an associated del(5q) abnormality, the recommendations presented here focus on the current approved indication of the drug in the USA. Selection of patients for lenalidomide therapy The FDA approved lenalidomide for patients with transfusion-dependent anemia due to low- or intermediate-1-risk MDS associated with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. This is an important limitation, as patients with a complex karyotype and del(5q) are eligible for treatment only if they have no more than one cytopenia (i.e., anemia, neutropenia, or thrombocytopenia) and their medullary bone marrow blast count is <5%. Furthermore, patients with a bone marrow blast count up to 10% receive a score of 0.5 points in the IPSS classification (Table 1) [3]. Therefore, in order to remain in the IPSS intermediate-1-risk category, one additional chromosomal abnormality (not involving chromosome 7) or two or more peripheral cytopenias are allowed, but not both. Although teratogenicity has not been reported with lenalidomide, as it is an analogue of thalidomide, women of childbearing potential should have two negative pregnancy tests performed within 14days prior to lenalidomide intake. Table 1International prognostic scoring system for myelodysplastic syndromes: survival and evolution of acute myeloid leukemia Score valuePrognostic variable00.51.01.52.0Bone marrow blasts, %<55–10–11–2021–30KaryotypeaGoodIntermediatePoor  Cytopeniasb0/12/3   Scores for risk groups are as follows: Low, 0; Int-1, 0.5–1.0; Int-2, 1.5–2.0; and High, ≥2.5aGood: normal, −Y, isolated del(5q), isolated del(20q); poor: ≥3 abnormalities or chromosome 7 anomalies; intermediate: other abnormalitiesbNeutrophils <1800 × 106/l, hemoglobin <10 g/dl, platelets <100 × 109/lTable reproduced with permission from [3] © The American Society of Hematology Lenalidomide therapy is effective regardless of prior erythropoietin [8, 9] or prior thalidomide therapy [9], and those previous therapies should not be regarded as contraindications for its use, although concurrent therapy with erythropoietin is not recommended, owing to concerns regarding venous thromboembolism. Clinical data have shown that the frequency of response to lenalidomide is similar across all age groups evaluated [8]. Importantly, the overall adverse-event burden did not increase in older patients, but serious adverse events were more frequent in patients aged over 65years compared with younger patients (54% vs. 33%, respectively) [10]. Although the impact of renal impairment on lenalidomide efficacy or toxicity in patients with MDS has not been evaluated, the risk of toxicity is expected to be greater in patients with impaired renal function because lenalidomide is renally excreted [11]. In patients with known renal impairment, the expert panel support the dosing schedule proposed by Chen et al., which is based on creatinine clearance (Table 2) [11]. As advancing age is associated with declining renal function, it is recommended that patients older than 65years who are prescribed lenalidomide are monitored carefully throughout their treatment (Table 3). Table 2Recommended dose adjustments for patients with impaired renal functionaRenal impairmentDoseMild10 mg (full dose) every 24 h (80 > CLcr ≥ 50 ml/min)Moderate5 mg every 24 h (30 ≤ CLcr < 50 ml/min)Severe5 mg every 48 h (CLcr < 30 ml/min, not requiring dialysis)End-stage renal disease5 mg three times a week after each dialysis (CLcr < 30 ml/min, requiring dialysis)aRecommendations based on a pharmacokinetic study by Chen et al. [11]CLcr creatinine clearanceTable adapted from [11] © 2007 SAGE Publications. Adapted by permission of SAGE Publications, Inc.Table 3Recommendations for laboratory monitoring during treatmentFunctionTestRecommendation(s)Kidney functionCreatinineEvery 4 weeks in patients aged 65 years and olderBloodFBCWeekly monitoring of full blood count mandatory for the first 2 months (it may be continued for 5 months).Biweekly or monthly monitoring should be considered thereafter, depending on hematological status.If treatment is interrupted in patients who had a previous episode of neutropenia or thrombocytopenia while on lenalidomide treatment, the same monitoring guidelines apply at re-initiationThyroid functionTSH, T4Monitor every month during the course of treatmentIn case of loss of response during lenalidomide treatmentGonadal functionTestosteroneIn case of loss of response during lenalidomide treatmentDigoxinDigoxinIn patients concomitantly taking Digoxin, the plasma level should be monitored periodicallyPregnancy testUrine testDay −14 and day 0 at initiation of therapy, monthly thereafter (in women of childbearing potential)Bone marrowBM aspiration and cytogenetic testing; Trephine biopsy optionalAt commencement of therapy.In case of loss of response to rule out progressive disease or cytogenetic evolutionBM Bone marrow, FBC full blood count, T4 thyroxine, TSH thyroid-stimulating hormone Likewise, the safety, efficacy, and pharmacokinetics of lenalidomide therapy have not been fully investigated in patients with evidence of hepatic impairment, or those with frank hepatic dysfunction. Clinical trials conducted to date have excluded patients with inadequate hepatic function, with individual trials using different serum transaminase measures as exclusion criteria; most trials excluded patients with serum transaminase levels greater than three times the upper limit of normal. Pharmacokinetic studies have revealed that the co-administration of lenalidomide with digoxin may increase the maximal digoxin concentration, although the area under the concentration–time curve remained unchanged [10]. Importantly, lenalidomide does not interact with the cytochrome P450 system, and so, can be confidently co-administered with other medications metabolized by this route (Celgene data on file) [10]. This latter finding is particularly relevant for older patients, who may be more likely to be receiving concomitant medications for co-morbidities. The expert panel concurred with the current prescribing information for lenalidomide and recommended that patients taking digoxin should have their digoxin plasma levels monitored periodically (Table 3). Treatment with lenalidomide in del(5q) MDS The time-to-response during lenalidomide therapy was evaluated in the larger of the two pivotal clinical trials [8]. For most patients, the median time to initiation of the transfusion-independent period was 4.6weeks, but responses have been noted to take up to 12months [8]. Therefore, the expert panel recommends a treatment duration of at least 4months in order to obtain an initial response (Table 4). Patients were followed for a median of 104weeks, but as more than half of them remained transfusion-free at this assessment point, the median duration of transfusion independence could not be calculated. Encouragingly, among the pooled patients with MDS with a del(5q) abnormality from the two studies [8, 9], transfusion independence and major erythroid response were maintained for a median duration of at least 2years [12]. Even though there are anecdotal reports of long-lasting erythroid remissions in some patients with hematological complete response who discontinued lenalidomide, the panel recommends continuation of lenalidomide treatment in responders for as long as it is tolerated. In patients where the dose has initially been reduced for mild adverse events, dose escalations may be considered (Table 4). In responders with high ferritin levels due to long transfusion histories who achieve normal hemoglobin levels, lenalidomide treatment should be continued and therapeutic phlebotomy be considered to reduce the secondary iron overload. In responders who discontinue due to adverse events, the panel recommends checking for response continuation before initiating another treatment (Table 4). Preliminary data show that prolonged treatment with lenalidomide does not increase the risk of transition to acute myeloid leukemia [13]. Of note, there are reports of patients acquiring additional cytogenetic abnormalities while on lenalidomide [8], but at this time the relevance of this phenomenon is not entirely clear. Until there is data to the contrary, patients with continuous hematological response should remain on the drug. Patients losing their response and developing a complex karyotype should be approached as they would be in the absence of lenalidomide, and alternative therapies, including transplantation, should be considered. Table 4Recommendations for treatment duration Recommendation(s)Initial treatmentTreatment should be continued for at least 4 months in order to obtain an initial responsePatients who Have a complete hematological responseContinue lenalidomide therapy for as long as it continues to be well tolerated to avoid relapse (both erythroid and cytogenetic) Have a partial responseContinue lenalidomide therapy, and consider an escalation of the lenalidomide dose to a maximum of 10 mg per day, if tolerable Discontinue treatment because of adverse eventsPatients should not begin another therapy immediately; it is recommended to wait 8–12 weeks to determine whether the response continues Tolerability of lenalidomide in del(5q) MDS Lenalidomide has been shown to be well tolerated in the two clinical trials reported to date [8, 9]. The most commonly reported adverse events of grade 3 or 4 severity were neutropenia (55%), thrombocytopenia (44%), anemia (7%), leukopenia (6%), rash (6%), diarrhea (3%), pruritus (3%), pneumonia (3%), and fatigue (3%) [8]. Management of hematological adverse events Hematological adverse events, including neutropenia and thrombocytopenia, were the most common adverse events with lenalidomide therapy and the most frequent reasons for dose adjustment [9]. As such, thrombocytopenia and neutropenia should be expected to occur in most patients—in fact, they appear to be associated with a higher likelihood of a response to therapy [14]. In general, regular monitoring of blood cell counts is recommended (Table 3). The majority of hematological adverse events (62%) occurred early in the course of treatment (within the first 8weeks) [8]. Grade 3 or 4 neutropenia was reported in 55% of patients [8]. Monitoring and, in some cases, additional treatment with granulocyte colony-stimulating factor (G-CSF) is recommended (Tables 3 and 5, and Fig. 1). Neutropenic sepsis was the only reported cause of death (occurring in three patients) that was regarded as possibly related to the study drug [8], therefore, patients should receive clear guidance on how to react in the event of febrile neutropenia (Table 5). Grade 3 or 4 thrombocytopenia was observed in 44% of patients [8]. In some thrombocytopenic patients, interruption of lenalidomide treatment may be necessary (Table 5 and Fig. 1). Lenalidomide has not been tested in patients with a baseline neutrophil count <500 × 106/l or baseline platelet count <50 × 109/l. Fig. 1Recommendations for the management of a neutropenia and b thrombocytopenia. aNo consensus was reached, recommendation is based on the opinion of some of the panel members G-CSF granulocyte colony-stimulating factorTable 5Recommendations for the management of hematological adverse events Recommendation(s)NeutropeniaFor treatment recommendations for neutropenia see Fig. 1Febrile neutropeniaProvide patients with clear guidance on how to react in the event of febrile neutropenia (patient education, specialized hematological care at all times, and broad-spectrum antibiotics within 3 h of fever onset)ThrombocytopeniaFor treatment recommendations for thrombocytopenia see Fig. 1VTEVTE prophylaxis is not generally recommended in patients with MDS. Combining lenalidomide with erythropoietin is also not recommended.If erythropoietin is used, be aware of a potentially increased risk of VTE. Patients should be informed about the risk of VTE and monitored for symptomsIf VTE does occur, interrupt lenalidomide treatment, treat the VTE, and carefully re-introduce lenalidomide once stable anticoagulation has been establishedPolycythemiaLenalidomide should be continued and phlebotomy considered, depending on ferritin levels. Although polycythemia is usually transient, treatment interruption may be necessary if additional risk factors for VTE are presentMDS myelodysplastic syndromes, VTE venous thromboembolism In the opinion of the panel, patients presenting with a neutrophil count <1,000 × 106/l prior to the start of lenalidomide therapy can still be treated with lenalidomide if G-CSF is co-administered. When neutropenia occurs during lenalidomide therapy, interruption of the lenalidomide treatment schedule might be necessary (Table 5 and Fig. 1). When thrombocytopenia (platelet count <25 × 109/l) is diagnosed at presentation, based on the rationale that once a response is achieved thrombocytopenia is likely to resolve, some experts would consider administering lenalidomide with platelet support (Table 5 and Fig. 1). However, other experts advise against the use of lenalidomide in patients with a platelet count <50 × 109/l because there is a risk of sustained deterioration of thrombocytopenia, despite an erythroid response. In some patients responding to lenalidomide, polycythemia may occur [8]; if appropriate, therapeutic phlebotomy or interruption of lenalidomide therapy may be considered (Table 5). Grade 3 or 4 venous thromboembolism (VTE) was observed in 3% of patients [8]. Generally, the risk of VTE is increased if there is a history of superficial vein thrombosis, or previous VTE [15, 16]. It is unknown if this applies to patients treated with lenalidomide as well. Nevertheless, these patients should be carefully monitored. In patients with a history of previous VTE, low-molecular-weight heparin should be used to prevent recurrent thrombosis. Although aspirin is effective in the prevention of thrombosis in multiple myeloma, aspirin cannot be recommended alongside lenalidomide as lenalidomide frequently leads to grade 3 or 4 thrombocytopenia in patients with MDS with del(5q). There are insufficient data on low-dose coumadin treatment for the prophylaxis of VTE, and therefore the panel did not recommend its use. Of particular relevance to patients with MDS, VTE risk is increased by concomitant erythropoietin use [17]. Since the incidence of VTE in patients with lenalidomide-treated MDS is generally low [8], prophylaxis in patients without antecedents of VTE is not recommended. If VTE does occur, it should be treated according to standard protocols. Treatment with lenalidomide should be interrupted until stable anticoagulation is achieved and then carefully reintroduced (Table 5). Once VTE has occurred during lenalidomide therapy, patients must remain on anticoagulation therapy as lenalidomide treatment continues. Management of non-hematological adverse events Unlike thalidomide, lenalidomide does not lead to dose-dependent peripheral neuropathy or somnolence [18], but other non-hematological adverse events (NHAEs) may occur. The most common NHAEs (all grades) associated with lenalidomide therapy are diarrhea (49%), pruritus (42%), rash (36%), and fatigue (31%) [10]. The most commonly reported grade ≥3 NHAEs were rash (6%), fatigue (3%), diarrhea (3%), and pruritus (3%) [8]. In general, should NHAEs arise during lenalidomide therapy, alternative causes should be ruled out and symptomatic treatment initiated (Table 6). Dry skin and pruritus occur regularly, and itching of the scalp is a characteristic adverse event during the first few weeks of therapy. These NHAEs are usually self-limiting and seldom need treatment. Unselective antihistamines (e.g., clemastine) are usually helpful. In severe cases, a short course of systemic corticosteroids (10mg of prednisone or equivalent), or local steroid application may be helpful. Rash often resolves spontaneously with time [19], and the panel do not recommend discontinuation of lenalidomide treatment should rash arise; however, in patients with severe or persistent rash, the temporary interruption of lenalidomide may be necessary (Table 6). Table 6Recommendations for management of non-hematological adverse events (NHAEs)NHAERecommendation(s)RashUsually resolves within 2–3 weeks, no interruption of lenalidomide treatment neededIf required treat with unselective antihistamines (e.g. clemastine), topical steroids, or a short course (14 days) of oral 10 mg prednisoneIf rash is severe or persists after treatment, lenalidomide should be interrupted until rash resolves. In the experience of the panel, lenalidomide can be restarted thereafter without recurrence of rashDiarrheaTreat symptomatically after ruling out other underlying causesHypothyroidismIn case of hypothyroidism, thyroid replacement therapy must be initiatedOther NHAEsTreat symptomatically after ruling out other underlying causes such as anemia or autoimmune disorders Diarrhea is a frequent problem and may impact on a patient’s quality of life. Patients with known lactose intolerance should add lactase to their diet, as lenalidomide capsules contain small amounts of lactose. Diverse symptomatic therapies have been reported to be effective in those patients, including, but not limited to, loperamide, pipaverium bromide, uzara root extract, and tinctura opii. Muscle cramps have been reported by a number of patients after a variable time of lenalidomide intake. Magnesium dietary supplements may be tried, but are usually of limited value. Quinine sulphate (up to 200mg thrice-daily) works well, however, physicians should check for other cytochrome-P450-interacting drugs (e.g., sotalol, terfenadine, astemizole, voriconazole, erythromycin, rifampicin, and cisapride) that might prolong QT time in these patients. Patients who experience fatigue will be likely to benefit from counselling [20], and in cases of severe fatigue, dose reduction may be needed. Hypothyroidism has been reported in approximately 7% of patients [10]. It is almost exclusively of autoimmune cause. Patients should be screened for hypothyroidism every other month during the course of treatment (Table 3). If hypothyroidism is confirmed, hormone replacement therapy is indicated (Table 6). Patients losing their response to lenalidomide during treatment may have developed either hypothyroidism or hypogonadism, and should be screened for both conditions. Conclusions The approval of lenalidomide for the treatment of MDS patients with low-risk or intermediate-1-risk disease with a deletion of chromosome 5q represents a significant step forward for this underserved group of patients. The key goal of the strategies presented here is to avoid unnecessary dose delays and reductions, and to maximize probability of response. Patient selection is straightforward, as efficacy does not appear to be influenced appreciably by relevant prognostic factors including age, prior erythropoietin therapy, sex, French–American–British type, or IPSS score. Treatment should be continued for a minimum of 4months to ensure treatment response. Neutropenia and thrombocytopenia are the most common adverse events during lenalidomide therapy but can be managed by the introduction of G-CSF and dose interruption. Other side-effects are generally manageable, and a range of simple (prophylactic) interventions are recommended to address the more common and more serious associated adverse events.

Environ_Manage-2-2-1705511

Perceived Conflicts Between Pastoralism and Conservation of the Kiang Equus kiang in the Ladakh Trans-Himalaya, India

An emerging conflict with Trans-Himalayan pastoral communities in Ladakh’s Changthang Plateau threatens the conservation prospects of the kiang (Equus kiang) in India. It is locally believed that Changthang’s rangelands are overstocked with kiang, resulting in forage competition with livestock. Here, we provide a review and preliminary data on the causes of this conflict. Erosion of people’s tolerance of the kiang can be attributed to factors such as the loss of traditional pastures during an Indo-Chinese war fought in 1962, immigration of refugees from Tibet, doubling of the livestock population in about 20 years, and increasing commercialization of cashmere (pashmina) production. The perception of kiang overstocking appears misplaced, because our range-wide density estimate of 0.24 kiang km−2 (± 0.44, 95% CL) is comparable to kiang densities reported from Tibet. A catastrophic decline during the war and subsequent recovery of the kiang population apparently led to the overstocking perception in Ladakh. In the Hanle Valley, an important area for the kiang, its density was higher (0.56 km−2) although even here, we estimated the total forage consumed by kiang to be only 3–4% compared to 96–97% consumed by the large livestock population (78 km−2). Our analysis nevertheless suggests that at a localized scale, some herders do face serious forage competition from kiang in key areas such as moist sedge meadows, and thus management strategies also need to be devised at this scale. In-depth socioeconomic surveys are needed to understand the full extent of the conflicts, and herder-centered participatory resolution needs to be facilitated to ensure that a sustainable solution for livelihoods and kiang conservation is achieved. One of the seven equid species in the world, the kiang Equus kiang, occurs in parts of China and India, with small populations also reported from Pakistan and Nepal. Unlike the Asian wild ass E. hemionus, of which the population has declined drastically over the last century, kiang continues to have a wide distribution with fairly large populations (Schaller 1998). Within India, Ladakh (approximately 75° 50′ to 75° 80′ E; 32° 30′ N to 32° 37′ N) remains a stronghold for the kiang (Fox and others 1991; Shah 1996), where local Buddhist communities have been fairly tolerant of the species, and large herds of kiang can be relatively easily seen in eastern Ladakh. Kiang is classed as a Least Concern species under the IUCN Red List category; however the Western Kiang, E. k. kiang, which occurs in Ladakh, is classed as “data deficient” (Shah 2002). Yet, with growing integration of the local economy of Ladakh with better developed cash markets, pastoral communities are fast losing their tolerance towards the kiang and it is increasingly seen as a competitor of livestock (Fox and others 1991). Ironically, its relatively large population and conspicuousness are becoming concerns for the continued conservation of the species. Although the species is not persecuted (except for driving them away from pastures by herders on horseback) in Ladakh largely due to the Buddhist beliefs of the local pastoral communities, this perceived conflict is currently one of the most serious issues being faced by the local district administration and the wildlife department, with vehement demands for compensation. Indeed, a decreasing tolerance for the species may lead in future to a worsening willingness to preserve it. There is a belief among the administration that Ladakh’s rangelands are presently overstocked by kiang, and that they are degrading the pastures (Anon 2003). Over the last decade, as a measure to protect forage from kiang and make it available to livestock, the local government and some nongovernmental organizations have started fencing the most productive pastures, the sedge meadows near rivers and streams, thereby increasing forage availability for livestock and reducing the available habitat for the kiang (Richard 1999; Bhatnagar and Wangchuk 2001). The purpose of this article is to document this intensifying conflict between pastoralism and conservation of the kiang in Ladakh. The kiang subspecies in question is categorized as “data-deficient” by the IUCN (Moehlman 2002). We examine the primary ecological, socioeconomic, and political aspects of kiang–human conflict, and summarize the results of our rangewide surveys of the kiang population in Ladakh. We also discuss the growing linkages between a traditional way of life and the global cashmere market, and their consequences for conservation of the kiang. Methods Study Area Administratively, the Ladakh region in the state of Jammu and Kashmir (India) is divided into two districts: the western Kargil district and the eastern Leh district. Leh district is a high-altitude cold desert spread over approximately 45,000 km2. This rain-shadow region is semiarid to arid, with winter temperatures dropping to below −25°C. Western and central Leh are mostly rugged, relatively lower in elevation, and inhabited by agropastoralists. The eastern part of the district, called Changthang, on the other hand, comprises high plateaus and rolling hills (usually >4000 m) interspersed with lake and river basins that have moist patches of relatively dense graminoid vegetation. Most of the remaining arid region is dominated by medium to sparse steppe vegetation. Changthang covers approximately 20,000 km2 and is inhabited by the nomadic Changpa community and Tibetan refugees, both of whom primarily rear goat and sheep. According to the human census in 2001 conducted by the government, the population of the Leh district was 117,637, with the majority (85%) being in the western region. The population is sparse in the Changthang (13,444) with a density of approximately 0.7 km−2. Ladakh is home to a diverse assemblage of wild carnivores, ungulates, and birds (Pfister 2004). Within Ladakh, the kiang occurs in the eastern Changthang part of the Leh district. Our survey covered most of this region, from the Pangong Tso in the North, along the eastern border with Tibet, to Hanle in the south (Figure 1). We divided the entire region into five blocks based on their location and overall similarity in terms of topography and vegetation, and surveyed each block (Table 1). Our repeated counts of kiang were conducted in the Hanle Valley. This valley (79° 0′ 45″ E, 32° 37′ 29″ N; approximately 2800 km2) extends from the Zanskar mountains in the south to the Indus Valley in the north into which the Hanle River drains at Loma. The higher reaches are primarily rolling mountains and plateaus, while the mid and lower portion is a wide valley fringed by a high range exceeding 5000 m on the east, and a lower, more gradual ridge on the west. Fig. 1The five blocks surveyed for kiang in eastern Ladakh during 2000 to 2003. The estimated “viewscape” is based on modeling for areas visible within 3 km on either side of the road (see text for details). Distribution of kiang in Ladakh is also shown (based on Chundawat and Qureshi 1999). We also surveyed some additional adjacent areas with similar habitat.Table 1Density of kiang in the surveyed blocks of eastern Ladakh, IndiaSurvey blockTransect length (km)Area (km2)No. KiangKiang density (km−2)Loma-Demchok (Indus)822882490.86Hanle Valley66347780.23Chushul-Loma63299190.06Parma Valley7436090.03Pongong Tso80359100.03Total36516533650.24 (95% CL ± 0.44) Data Collection and Analysis Through archival research and field observations, we documented the intensifying human–kiang conflict in Ladakh. We describe the perceptions of the Ladakhi administration regarding this conflict, and the efforts they have undertaken to address it. Through archival research, we also examine recent land-use changes in eastern Ladakh, describe the growing pashmina or cashmere industry, and its fallouts for the kiang–human conflict. Literature was also surveyed for obtaining information on the past status and distribution of the kiang. We also obtained data on livestock populations from the Sheep Husbandry Departments in Nyoma and Leh, to estimate the livestock density in eastern Ladakh. We had informal conversations with over 50 herders to learn about their perceptions regarding conflicts. Our data on the kiang population in Ladakh come from rangewide surveys conducted in the year 2000. The study area was surveyed from a slow-moving vehicle (traveling at 10–12 km/hour) and areas on either side were periodically scanned using an 8× binocular and/or 20× spotting scope. On every sighting, animals were counted and classified, and habitat variables such as topographic feature, elevation, slope, aspect, dominant plant species, and approximate plant cover were recorded. Classification of kiang by sex was not possible in most instances due to the similarity between the sexes, especially when seen from a distance. A total distance of 365 km was covered in the Changthang, traveling on an average 60–70 km per day. We also covered approximately 50 km on foot in the upper catchment of the Hanle Valley. Subsequently, in the spring of 2001, 2003, and 2004, we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the Hanle Valley, which had emerged as an area with moderate to high kiang density during the 2000 survey. Density for kiang and livestock was calculated by estimating the area covered in each segment using Geographical Information System (GIS) tools. The area surveyed along the roads was estimated by creating a “viewscape” in ArcInfo, which effectively created polygons of all area visible from the road. The base map used for this estimation was the National Imagery and Mapping Agency’s Vector Map Level 0 (Digital Chart of the World) Edition: 5. This was integrated with the Digital Elevation Model (resolution 90 m or 3 arc seconds) Shuttle Radar Topography Mission (SRTM) data from the Space Shuttle mission (for the year 2000) obtained from ftp://e0mss21u.ecs.nasa.gov/srtm/ to generate maps of the study area with numerous layers of information that included elevation, aspect, and drainage. After creating the viewscape, we used a cutoff distance of 3 km on either side of the road, within which all visible areas were calculated. This was generally our maximum sighting distance for the kiang (most Rangefinders do not work at this distance). We estimated the extent of the surveyed areas (through viewscapes) for both the rangewide surveys as well as the region of our repeated kiang counts in Hanle Valley. As a preliminary exercise to understand the extent of forage consumption by kiang vis-à-vis livestock, we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake (as percent of body mass) among herbivores (Foose 1982). In the absence of population structure data for both kiang and livestock, we used adult body masses (averaged between sexes) from Mishra and others (2002). We used the established relationships for foregut fermenters (goat, sheep, cattle, yak, cattle-yak hybrids) and hindgut fermenters (kiang, horses, donkeys), as appropriate. Forage quality in the highly seasonal cold desert steppe of Ladakh is expected to vary strongly between seasons, with high-protein, low-fiber forage being available in summer, and low-protein, high-fiber forage in winter. We therefore estimated forage consumption by each species separately for winter and summer, using the relationships for low-protein, high-fiber grass hay diet for winter (4.5–7.4% crude protein and 65–70% cell wall) and high-protein, low-fiber legume hay diet for summer (17.4–22.2% crude protein and 31–56% cell wall) (Foose 1982). For hindgut fermenters, daily forage intake as a percent of body mass (W) in summer was estimated as 13.8W−0.315, and 6.95W−0.236 in winter. Similarly, for the ruminants (goat, sheep, cow, dzo/dzomo, yak), it was estimated as 7.31W−0.231 in summer and 4.04 W−0.184 in winter (Foose 1982). Results and Discussion Genesis of the Conflict: Kiang Population Changes in Recent History Kiang inhabits the open plains and rolling mountains of the Changthang plateau in eastern Ladakh at altitudes between 4000 m and 5500 m. It has a total estimated range of approximately 7400 km2 in Ladakh (Chundawat and Qureshi 1999; Figure 1). The species was common in Ladakh during the earlier part of the 20th century (Stockley 1936), but is believed to have declined substantially with the buildup of armed forces in Ladakh during the war between India and China in 1962 (Fox and others 1991). There are no population estimates available for the period before or immediately following this war, but it is believed that the population had recovered by the 1980s. The only published estimate of their population comes from the late 1980s when approximately 1500 animals were estimated to occur in Ladakh (Fox and others 1991; Shah 2002). It thus appears that for at least a decade after the war, the kiang population remained relatively small in Ladakh. The genesis of the present conflict can be traced back to the early 1980s, by which time their population had apparently recovered (Fox and others 1994). The prevalent belief in Ladakh during that time, and one that continues until today, is that the kiang population has drastically increased in eastern Ladakh because many animals have moved from Tibet to the Indian side of the border to escape persecution (Fox and others 1991; Anon. 2003). Even in the 1980s, there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock, and demands that the government should “drive the kiang back into Tibet” (Fox and others 1991). Intensification of the Conflict: Changing Face of Pastoralism in Ladakh The Tibetan plateau was inhabited by nomadic hunting people 30,000 years ago, and nomadic pastoralists have been herding livestock on the plateau for at least two millennia (Schaller 1998). The nomads, who became Buddhists presumably in the last millennium, live in tents (ribos) in seasonal camps. In the past, the few nomadic groups inhabiting eastern Ladakh belonging to the Changpa community traveled distances exceeding 100 km during their seasonal migrations (Phuntsok 2000; Hagalia 2004). This has recently changed, and most seasonal movements are now restricted to within 40–50 km (Hagalia 2004). After the 1960s, there has been a steady increase in the population of the Changpa and their livestock, presumably facilitated by access to better health care, provisions, and subsidies, both from the government as well as the army (Chaudhuri 2000; Phuntsok 2001; Bhatnagar and Wangchuk 2001). Supplemental forage provided by the government in the event of severe winters now offsets most of the starvation-related livestock mortality, which in the past was perhaps an important factor limiting the livestock population. The 1962 war between India and China also led to the curtailment of movement of the Changpa herders from eastern Ladakh into parts of Tibet, considerably reducing the availability of grazing land. Many of their pastures such as Skagzung, the primary winter pasture of the Rupshu Changpas (Figure 1), became unavailable, effectively escalating the stocking density in eastern Ladakh. Furthermore, a large number of Tibetan refugees have moved into eastern Ladakh with their livestock herds since the late 1950s, thus contributing to a substantial increase in the number of herding families and the livestock population (Bhatnagar and Wangchuk, 2001; Hagalia 2004). As per the government records, the livestock population in Ladakh (including Changthang) has doubled in about two decades, from approximately 212,500 in 1977 to 487,000 in 1999 (Richard 1999; Bhatnagar and Wangchuk 2001). In 1999, Changthang constituted 44% of Ladakh’s livestock population, most of which (94%) were sheep and goats. One of the most important livestock products from the region is the pashmina or cashmere, a fine luxury fiber derived from the underwool of the local Changra goat. Traditionally, more than 95% of cashmere from Ladakh has been used for barter with or sale to traders from the neighboring Kashmir region under a treaty with the erstwhile kingdom of Kashmir dating from 1684 (Rizvi 1999). Over the last two decades, however, cashmere production in Ladakh has been promoted by the government in a substantial way. This has included setting up of goat breeding farms, efforts at improving yield, and provisioning of veterinary services to nomads that has brought down the kid mortality rates to 2–3% from an estimated 30–40% (Jina 1995). However, the efforts at increasing the per animal cashmere yield have apparently had very limited success (Jina 1995), suggesting that increasing cashmere production necessitates an increase in the goat population. More recently, with part support from the central government of India, the International Fund for Agricultural Development and the UNDP, the Ladakh administration has set up a cashmere de-hairing plant, expected to result in considerable added value to the cashmere from Ladakh (Joshi and Morup 2003; Mital 2004). Presently, Ladakh contributes merely 30,000 kg (0.37%) to the global cashmere production and the administration is eager to increase Ladakh’s contribution in global trade. The recent intensification of human–kiang conflict needs to be viewed against these socioeconomic and political developments. The human and livestock populations in kiang habitat have significantly increased in the last four decades due to factors ranging from better healthcare to the influx of refugees. Furthermore, promotion of production and value of cashmere has meant that livestock rearing is fast becoming a cash-based enterprise that increasingly caters directly to the lucrative global cashmere market. Herders are realizing the importance of every bit of pastureland in maximizing cashmere production and see kiang as a direct threat to pasture production. Against this background, it is perhaps not surprising that people are fast losing their tolerance of the kiang. The Ecological Context: Current Kiang Population and Relationships with Livestock In our rangewide survey of the kiang, we surveyed an estimated area of 1653 km2, which is 22% of the total range of the kiang in Ladakh (7400 km2; Chundawat and Qureshi 1999). We recorded a total of 181 distinct kiang groups totaling 365 animals. The average density in the surveyed blocks was 0.24 (95% CL ± 0.44) kiang km−2, with the highest abundance along the upper Indus from Loma to Demchok followed by the Hanle Valley (Table 1, Figure 1). With an overall livestock population of 210,000 (as per unpublished records from the Nyoma office of the Sheep Husbandry Department (2004)) in the Nyoma and Durbuk blocks that comprise the Changthang region of Ladakh, the livestock density in the region was 11 km−2. Thus, in terms of density, kiang represents only 2% of the total herbivore population (kiang and livestock together) in the Changthang, with the bulk being contributed by goats (57%). The group sizes of kiang observed during the rangewide survey varied from 1 to 74. The mean group size was 2.8 (95% CL ± 0.8), with most of the sightings (55%) being of solitary animals. Most kiang were seen in open wide valleys (79%, n = 202), followed by stony slopes (19%) and very rarely in broken areas (2%). There were no sightings on slopes steeper than 30°. We observed kiang between elevations of 4100 and 5500 m, with most sightings between 4200 and 4300 m. Most kiang groups (73%) were seen on sedge (Carex spp. and Kobresia spp.) and grass (Stipa spp.) meadows in the valley bottom, which were occasionally interspersed with bushes of Caragana sp. These meadows appear to be critical habitat for kiang during summer and winter as also reported by Schaller (1998), but are limited in extent to narrow discontinuous strips in moist areas along valleys. The remaining sightings were in areas that had sparse to dense Artemisia steppe (25%; with Tanacitum sp. and Sedum sp. being the other dominant plants) and high altitude forb meadows (2%; Saussurea spp., Salsola sp., and Polygonum sp. as the dominant plants). At a landscape level covering entire eastern Ladakh, of the total daily forage consumption by large herbivores, we estimated that kiang consume 10–11% and livestock 89–90% (Table 2). Among livestock, goats were estimated to consume the maximum forage (41% to 43% of the total forage consumed), followed by sheep (26% to 27%). Table 2Herbivore density and estimated forage consumption by kiang vis-à-vis livestock in ChangthangSpeciesBody weight(kg)Density(animals km−2)Total forage consumption in summer (kg km−2)Percent forage consumption in summerTotal forage consumption in winter (kg km−2)Percent forage consumption in winterKiang2750.241.6101.211Livestock  Donkey900.040.110.11  Horse2480.150.950.76  Cattle1910.160.740.54  Yak2980.261.591.110  Yak cattle hybrids2270.010.03<10.02<1  Sheep343.984.4272.926  Goat336.446.9434.541Livestock total112111.0314.5909.789Total11.2716.0810.96Body weights are taken from Mishra (2001) and have been averaged between sexes. Note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult. Livestock density was calculated only for adult animals. Our subsequent estimate of the kiang population in the Hanle Valley (the region between Rongo and Kalangtartar) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang/km2 (95% CL ± 0.08). We estimated that on an average, kiang remove only 3% to 4% of the total forage consumed in the Hanle Valley, with the greater part being consumed by goats (45% to 47%) followed by yaks (20% to 21%) and sheep (20%) (Table 3). Table 3Herbivore density and estimated forage consumption by kiang vis-à-vis livestock in the Hanle ValleySpeciesBody weight (kg)Density(animals km−2)Total forage consumption in summer (kg km−2)Percent forage consumption in summerTotal forage consumption in winter (kg km−2)Percent forage consumption in winterKiang2750.563.632.84Livestock  Donkey9000000  Horse2481.579.587.49  Cattle1910.662.721.92  Yak2983.9122.82016.521  Yak cattle hybrids22700000  Sheep3421.5223.72015.519  Goat3350.6954.54735.545Livestock total112178.34113.29776.796Total78.9032.479.6 Synthesis of the Conflict: Is It Real or Perceived? Most wars in the last five decades have resulted in detrimental effects on wildlife (Blom 2000; Dudley and others 2002; Mishra and Fitzherbert 2004), and the war between India and China in 1962 was no exception, particularly from the viewpoint of kiang conservation. Our review suggests that the kiang population in Ladakh declined drastically in the years during and after the war between India and China in the 1960s, possibly due to hunting, land mines, and disturbances associated with the war. Thus, for two decades during and after the war, a generation of herders presumably saw and became used to a very low density of kiang in Ladakh’s rangelands. Not surprisingly, the genesis of kiang–human conflict in Ladakh can be traced back to the 1980s, the period by which the kiang population had recovered. It seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km2 has led to the perception to a generation of nomads that Ladakh’s rangelands are now overstocked with kiang. Available estimates from three regions in Tibet place the density of kiang between 0.15 and 0.39 kiang km−2 (Schaller 1998), suggesting that the kiang density in Ladakh is comparable with those in Tibet. Furthermore, our results show that the share of forage consumed by the kiang vis-à-vis livestock is relatively small (10–11% in the whole of eastern Ladakh, and 3–4% in Hanle Valley). Thus, at a landscape level, the perception of the overabundance of kiang in Ladakh, and of kiang compromising cashmere production, is clearly unfounded. Nevertheless, there is variation in kiang density locally (0.03 to 0.86 kiang km−2 recorded in our surveys). Kiang occasionally congregate in large herds and can together remove considerable forage from the low-productivity Trans-Himalayan pastures. Using the data in Table 2, a group of 70 kiang (the largest group seen during the survey was 74) can be estimated to potentially consume 199–254 kg dry forage in a single day. The peak standing biomass in sedge meadows, which seem to be important kiang habitats, has been estimated in the adjoining region of Spiti at 857 kg ha−1 (Mishra 2001). These habitats seem critical for both kiang and livestock, but cover a small fraction of the landscape, with an estimated 150 km2 in the entire approximately 20,000 km2 of Changthang. Illustratively, this means that in a single day a large group of 70 kiang can potentially consume more than a quarter of the standing biomass from 1 ha of a key resource area. This consumption is also very similar to the estimated daily forage consumption by a herd of 200 goats (approximately 215 kg). Because of the long winters (November to April) and short summers (July–August), the potential for regrowth during summer is low, aggravating the impact. Considering the potential of large kiang herds to remove significant amounts of forage, together with the fact that grazing areas have been reduced and the livestock population in Ladakh more than doubled in the last 30 years, it is not difficult to see that at a localized scale of the valued sedge meadows, some herders must be facing serious forage competition from the kiang. Given the intensifying commercialization of cashmere production in Ladakh and its integration with global markets, this localized forage competition is, perhaps understandably, no longer tolerated, and the conflict needs to be addressed immediately. However, it must be borne in mind that the problem seems to be evident at local scales, and will need to be addressed at that scale. In the absence of any scientific understanding of the conflict so far, or efforts to document it, this local conflict has already begun to take on a serious political dimension. The politicians and the district administration in Ladakh are blaming the kiang for compromising cashmere production, and the wildlife department is being looked upon to provide a solution. The only on-ground reaction of the district administration has been to fence large productive sedge and grass meadows from the kiang. In the Hanle Valley alone, an estimated 120 ha of meadows have been fenced already. Three fourths of our total kiang sightings during the rangewide surveys were in such meadows, suggesting that this is an important habitat for the species, and continued fencing of this habitat may cause a decline in the kiang population of Ladakh. How can this conflict be managed? Although our review of the recent history of political and socioeconomic developments in kiang habitat and our estimates of density and forage consumption of kiang and livestock bring to light interesting dimensions of the kiang–human conflict, the current level of understanding of the kiang’s ecology and its relationships with livestock is still very preliminary. Intensive research needs to be promoted to assist in better management of the species, and its conflicts with humans. Monitoring programs need to assess kiang and livestock density, as well as the land use changes in kiang habitat. Our article shows that the problem of kiang–human conflict is locally concentrated in the key resource areas that contain the sedge meadows, and management strategies need to be devised at that local scale. Surveys are needed to identify all high-conflict areas, and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang. It needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in Ladakh. The kiang is a striking and conspicuous wild animal that may provide a potential for ecotourism. Finally, after taking up the above interventions, it is important that fences on the sedge meadows to exclude kiang be removed and the practice of fencing discontinued because they lock out key resource areas from the kiangs’ range. Without access to key resource areas, kiang and other wild herbivores, such as the Tibetan gazelle, Procapra picticaudata, may face severe population declines in Ladakh.

Semin_Immunopathol-3-1-2071951

Complement in glomerular injury

In recent years, research into the role of complement in the immunopathogenesis of renal disease has broadened our understanding of the fragile balance between the protective and harmful functions of the complement system. Interventions into the complement system in various models of immune-mediated renal disease have resulted in both favourable and unfavourable effects and will allow us to precisely define the level of the complement cascade at which a therapeutic intervention will result in an optimal effect. The discovery of mutations of complement regulatory molecules has established a role of complement in the haemolytic uremic syndrome and membranoproliferative glomerulonephritis, and genotyping for mutations of the complement system are already leaving the research laboratory and have entered clinical practice. These clinical discoveries have resulted in the creation of relevant animal models which may provide crucial information for the development of highly specific therapeutic agents. Research into the role of complement in proteinuria has helped to understand pathways of inflammation which ultimately lead to renal failure irrespective of the underlying renal disease and is of major importance for the majority of renal patients. Complement science is a highly exciting area of translational research and hopefully will result in meaningful therapeutic advances in the near future. Introduction The complement system involves approximately 30 plasma- and membrane-bound proteins (reviewed in [1, 2]). These proteins play an important role in anti-microbial defence and the clearance of immune complexes and apoptotic and necrotic cells. The role of complement is not restricted to the innate immune system but includes important functions in the regulation of the adaptive immune response. Although complement undoubtedly contributes to tissue damage in numerous forms of glomerulonephritis, we will also see that complement protects against immune-mediated tissue damage in a number of settings. In the following, we will first give an introduction to the pathways of complement activation and their regulation. This will be followed by a review of glomerular renal diseases in which complement plays a prominent role. The role of complement in non-glomerular renal disease entities such as transplantation and ischaemia/reperfusion damage falls outside the scope of this study and is reviewed in references [3] and [4]. Overview of the complement pathways The complement system consists of three different pathways that all converge in the activation of the central complement molecule C3 (Fig. 1). Sufficient activation of C3 will then lead to the formation of the membrane attack complex. Fig. 1Schematic overview of the three pathways of complement activation The first component in the activation of the classical pathway is C1. Binding of at least two bindings sites of C1q to antigen-bound IgG or IgM, acute phase proteins such as CRP or dead cells leads to conformational changes that result in the activation of the associated serine proteases C1r and C1s. Activated C1s cleaves C4 into C4a and C4b. C4b then covalently binds to nearby structures. The bound C4b then binds C2 whereupon the smaller C2b fragment is cleaved off, resulting in the formation of the C4bC2a complex, which is the classical pathway C3 convertase. The lectin pathway is related to the classical pathway and uses the same C3 convertase, C4bC2a. The initiation molecules of the lectin pathway, mannose-binding lectin (MBL) and the ficolins, recognise carbohydrate ligands present on a wide range of microorganisms in a pattern-like fashion. The interaction of MBL with its ligand leads to the activation of the MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3). MASP-2 then cleaves C4 and subsequently C2 leading to the formation of the C3 convertase which is identical to the classical route C3 convertase, C4bC2a. MBL consists of up to six trimeric subunits which are arranged in a structure similar to C1q. Frequently occurring single nucleotide polymorphisms within exon 1 of the MBL-2 gene and polymorphisms of the promoter region explain the up to 1,000-fold inter-individual variation in MBL plasma levels. The activation of the alternative route of complement depends on spontaneous activation of the C3 molecule by hydrolysis of the internal thioester bond of C3. This results in the formation of the C3b-like molecule C3(H2O). Hydrolysed C3 then binds to factor B. This interaction renders factor B susceptible to cleavage by factor D resulting in the release of the Ba fragment and the formation of the C3 convertase C3(H2O)Bb. This initial convertase constantly cleaves C3 at a low rate generating C3b. This constant low rate generation of C3b is referred to as the “tick over” of the alternative pathway. The generated C3b can interact with factor B to form the more active alternative pathway C3 convertase C3bBb. The majority of the generated C3b is rapidly inactivated by circulating factor I together with its co-factors, factor H (fH) and membrane co-factor protein (MCP). However, if C3b binds to an activator surface (e.g. a bacterial wall or damaged tissue), the molecule is protected against inactivation and further amplification of the alternative route will occur. Properdin is an important positive regulator of the alternative route. Binding of properdin leads to stabilisation of the labile C3 convertase and promotes the assembly of a complement-activating lattice by further binding factor B and C3b molecules [5–7]. The early activation steps of the classical, lectin and alternative route of complement activation converge in a common terminal pathway. The addition of a further C3b molecule to the C3 convertase complex leads to the formation of C3bBbC3b in the case of the alternative pathway and to the formation of C4bC2aC3b in the case of both the classical and lectin pathways. These C5 convertases then initiate the assembly of the membrane attack complex by cleavage of C5 to C5a and C5b. C5a can then function as a potent anaphylotoxin. The newly formed C5b forms a tri-molecular complex by binding C6 and C7. After inserting into a cell membrane, this complex binds C8 and multiple C9 molecules. This results in the completion of the pore-forming membrane attack complex (C5b-9). This complex can lead to cell lysis and, in the absence of complete lysis, to cell activation. Regulation of complement activation The complement system consists of numerous regulatory molecules that protect the host from uncontrolled tissue destruction and activation by the complement system. Recently, defective complement regulation has been shown to play an important role in the pathogenesis of some forms of the haemolytic uremic syndrome (HUS) and membranoproliferative glomerulonephritis (MPGN). The role of complement in these diseases will be discussed in more detail below. C1-inhibitor is a powerful inhibitor of the classical pathway of complement activation. It binds to activated C1r and C1s and causes dissociation of these inactivated enzymes from C1q. Recent data shows that C1 inhibitor also inhibits the activation of the lectin pathway by inactivation of MASP-2 and, at higher concentrations, fluid phase activation of the alternative pathway [8]. Factor I is a circulating serine protease that proteolytically degrades C3b and C4b in the presence of the co-factors fH and C4-binding protein (C4bp). Next to its function as a co-factor, fH also inhibits activation of the alternative pathway by binding to C3b and displacing Bb from the C3 convertase complex. Similarly, C4bp regulates activation of the classical and lectin pathway by displacing C2a from C4b. Both fH and C4bp promote the degradation of the C3 and C5 convertases of the respective pathways. Cell-membrane-bound inhibitors of complement activation also contribute to the defence against inappropriate tissue damage by homologous complement. Decay-accelerating factor (CD55) exerts its effect early in the complement cascade by inhibiting the activation of C3 by preventing the formation and accelerating the decay of both the alternative and classical pathway C3 and C5 convertases. Membrane co-factor protein (MCP, CD46) serves as a co-factor for the cleavage of C3b and C4b by factor I. CD59 interacts with the final section of the complement activation pathway by inhibiting the formation of C5b-9. Complement receptor 1 (CD35, CR1) also functions as a complement regulator by accelerating the decay of the C3 convertases. A functionally intact soluble form of CR1 can be detected in plasma [9]. Recently, a new complement receptor, the human complement receptor of the immunoglobulin superfamily (CRIg), has been described [10]. CRIg is present on macrophages in both humans and mice and plays a role in pathogen clearance. It binds to C3b and selectively inhibits the C3 and C5 convertases of the alternative pathway. A recombinant soluble form of CRIg suppressed inflammation in two murine models of arthritis [11]. Immune-complex-mediated glomerulonephritis Immune complex glomerulonephritis is a good example for the dual role of the complement system. Immune complexes can either be deposited in the glomerulus by passive deposition from the circulation or by in situ formation via binding of antibody to local antigens. Alternatively, local formation of immune complexes may occur when a circulation antigen is recognised by antibodies after deposition in the glomerulus (planted antigen). Subepithelial complement deposition as found in membranous nephropathy leads to a non-inflammatory complement-mediated damage because the anaphylotoxins produced during the local activation do not reach circulating leucocytes. Subendothelial deposition of complement factors is associated with a brisk inflammatory response because the produced anaphylotoxins easily come into contact with circulating cells. Subendothelial immune complex deposition is typical of proliferative lupus nephritis. Various studies have underscored the role of complement in immune-complex-mediated glomerulonephritis. Complement depletion by treating rats with aggregated human IgG resulted in a marked decrease of neutrophil influx and renal damage in a nephrotoxic serum model of acute glomerulonephritis [12]. Later studies demonstrating a beneficial effect of complement depletion in the non-inflammatory Heymann nephritis model of membranous nephropathy showed that complement-mediated damage is not dependent on the influx of inflammatory cells [13]. The contribution of terminal pathway of complement-mediated injury was established in various glomerulonephritis models in C6-depleted or C6-deficient rats. Renal damage is ameliorated in both the anti-Thy-1 and the passive Heymann nephritis models in the absence of C6 [14, 15]. As a follow-up to these findings, soluble CR1 was successfully used to treat the disease in both models [16]. A recent paper pointed towards an important role of the alternative pathway of complement activation in mouse models of type I and type II cryoglobulinemia [17]. The glomerular influx of neutrophils was significantly less in mice deficient for C3, factor B and C5, whereas C1q deficiency had no protective effect, suggesting involvement of the alternative pathway or lectin pathway. It is interesting to note that many of the complement-deficient models of renal disease show spontaneous or worsened renal disease. This is compatible with the observation that the complement system plays an important role in the clearance of immune complexes from the circulation and in the solubilisation of deposited immune complexes. Immune complexes are rapidly opsonised with C4b and C3b. These complement components mediate the binding of the immune complexes to CR1 on erythrocytes. The complexes are then stripped off the erythrocytes when they pass through the liver or spleen. Thus, CR1-mediated clearance plays an important role in the handling of immune complexes and in keeping soluble immune complexes away from the endothelial surface thereby preventing vascular injury. Numerous animal models of immune-complex-mediated renal disease demonstrate a protective role of complement. C3 deficiency did not protect mice from the formation of immune complexes and proteinuria in a planted antigen model of immune complex nephritis [18]. This can be explained by the role of Fc-receptor-mediated activation of either resident or infiltrating cells at the site of injury. In the complete absence of complement activation, deposition of immune complexes can still lead to renal damage via Fc-receptor-mediated cell activation [19, 20]. An interesting recent study demonstrated that factor-D-deficient mice spontaneously develop immune complex glomerulonephritis with mesangial deposition of IgM and C3 [21]. Apparently, amplification of C3 activation is necessary for the processing or dissociation of IgM-containing immune complexes in the kidney. The important role of the complement system in immune complex clearance is underscored by the finding that humans with complement deficiency are prone to immune-complex-mediated disease. Systemic lupus erythematosus (SLE) is a highly relevant example for this dual role of the complement system as will be discussed in the next section. Role of complement in lupus nephritis The degree of peripheral complement consumption and the heavy glomerular deposition of complement in SLE nephritis point towards an important role of complement in lupus nephritis. Various approaches at inhibiting complement activation have been successful in treating experimental lupus nephritis. Wang et al. prevented the development of glomerulonephritis with an anti-C5 antibody in lupus-prone NZB/W mice. Both treatments with the soluble rodent complement inhibitor rCrry and transgenic expression of this CR1-like molecule limited renal damage in the MLR/lpr mouse model of SLE [22, 23]. However, in humans, deficiencies of the early complement proteins C1q, C2 and C4 are associated with an increased risk of developing SLE [24]. Similarly, mice with C1q or C4 deficiency develop an autoimmune disease which resembles SLE [25–27]. Next to the loss of clearance of immune complexes, complement deficiency may also lead to auto-immune phenomena due to defective clearance of apoptotic cells. C1q binds to apoptotic cells [28] and C1q-deficient mice are impaired in their capacity to clear these cells [29]. Defective clearance of this rich source of auto-antigens may contribute to the emergence of auto-immunity in patients with SLE. Taken together, it seems that, in lupus, the early components of the classical pathway of complement activation are beneficial due to their role in the clearance of immune complexes and apoptotic cells. Probably, the damage caused by Fc-receptor-mediated mechanisms in the presence of an increased deposition of immune complexes overrides the benefit of complement inhibition in these models. However, the inhibition of complement activation downstream of C3 may be a promising therapeutic approach. Lupus nephritis is strongly associated with the presence of anti-C1q antibodies. These antibodies are present in 30–40% of SLE patients [30] and correlate with active lupus nephritis with a sensitivity of 87 to 97% and a specificity of 92% [31, 32]. Antibodies against MBL are also present in SLE, but no association with disease activity was detected [33]. The strong association of anti-C1q antibodies with active lupus nephritis suggests a pathogenic role of these antibodies. To study this question, our group has generated homologous mouse anti-mouse C1q antibodies. The administration of these antibodies to healthy mice resulted in deposition of C1q in the glomeruli together with an influx of granulocytes. However, this was not accompanied by a reduction of renal function or significant proteinuria [34]. When mice were pre-treated with a sub-nephritogenic dose of rabbit anti-C1q antibodies, the subsequent administration of mouse anti-C1q antibodies resulted in an increased deposition of immunoglobulin. The use of mice deficient for C3, C4 or for all three Fc gamma receptors showed that both complement and Fc-gamma-receptor-mediated damage is involved in this model. IgA nephropathy Mesangial IgA deposition is the hallmark of IgA nephropathy. Early studies on complement deposition in IgA nephropathy reported co-deposition of C3 and properdin [35]. Because C1q and C4 are usually not detected in kidneys with IgA nephropathy, this complement deposition was thought to be caused by alternative pathway activation. Mesangial deposition of C5b-9 is also present and underscores the possible pathogenic importance of complement activation in this setting [36]. More recent studies suggest a role of the lectin pathway of complement activation in IgA nephropathy. Co-deposition of IgA and MBL has been described by several authors in both IgA nephropathy [37–39] and the related entity Henoch Schönlein purpura [40]. Hisano et al. found a relation between the presence of MBL deposition and the severity of the disease [41]. The discovery of MBL-binding properties of IgA has provided a mechanistic explanation for the link between IgA and MBL deposition [42]. MBL binds to polymeric IgA in a calcium-dependent fashion via its lectin domain, suggesting an interaction with carbohydrates exposed on the IgA molecule. The binding of MBL to IgA results in complement deposition and offers an explanation for the complement deposition found in IgA nephropathy. A recent publication underscored the potential clinical importance of MBL deposition in IgA nephropathy [43]. About 75% of the 60 biopsies in this study were negative for MBL and C4d, indicating that the C3 and C5b-9 deposition in these patients is most probably caused by the alternative pathway. The 25% of biopsies in which glomerular MBL deposition was found were positive for C4d in the absence of Clq, indicating complement activation via the lectin pathway. MBL deposition in the glomerulus was associated with both clinical and histological markers of more severe renal damage including more severe proteinuria, renal failure, extracapillary proliferation, glomerular sclerosis and interstitial fibrosis. The MASP-associated lectin, L-ficolin, was also present in these biopsies and may contribute to the activation of the lectin pathway next to MBL. Membranoproliferative glomerulonephritis Complement research has greatly contributed to the understanding of the pathophysiology of type II membranoproliferative glomerulonephritis (dense deposit disease) [44]. MPGN type II is characterised by deposits within the glomerular basement membrane together with staining for C3 along the glomerular basement membrane. In contrast to MPGN type I, deposition of immunoglobulins is usually not detected. The glomerular complement deposition is usually accompanied by decreased circulating C3 levels and alternative route activity. More than 80% of patients with MPGN type II are positive for serum C3-nephritic factor (C3NeF) [45]. C3NeF is an antibody directed against the alternative pathway C3 convertase. The binding of C3NeF to C3bBb prolongs the half-life of the C3 convertase by slowing down the dissociation of Bb form C3b [46]. One of the mechanisms by which C3NeF increases the half-life of C3bBb is by inhibition of fH-mediated inactivation of the convertase [47]. Consistent with complement activation by stabilisation of alternative pathway convertase activity, serum complement profiles of patients with MPGN II show predominant depletion of C3 with no consumption of C1q and C4. The glomeruli of affected kidneys show marked deposition of C3 along the glomerular capillary walls without deposition of C1q, C4 or immunoglobulins. MPGN II is also associated with acquired partial lipodystrophy. This entity is also associated with the presence of C3NeF and marked C3 depletion. In vitro data shows that C3NeF is capable of inducing alternative-pathway-mediated damage on adipocytes [48] The role of a deregulated alternative pathway in MPGN type II was highlighted by the discovery of FH mutations in both humans [49] and pigs with MPGN type II. Mice with a targeted deletion of FH have significantly reduced levels of C3 and consistently develop MPGN with deposition of C3 in the capillary walls [50]. If fH-deficient mice are also deficient for factor B, they cannot activate the alternative pathway and no renal disease develops. A chronic serum sickness model of immune complex disease demonstrated increased deposition if IgG immune complexes with increased C3 deposition in fH-deficient mice compared to wild-type mice. The fH-deficient mice developed diffuse proliferative glomerulonephritis, while the wild-type mice were protected against glomerular pathology. These findings indicate a role of fH in processing immune complexes and protecting the glomerulus against immune-complex-mediated disease. Patients with MPGN type II develop ocular lesions which are similar to the drusen that are found in patients with age-related macular degeneration (AMD). The finding of a close association of AMD with factor H mutations suggests that complement is also involved in the pathogenesis of this visually disabling disease [51–54]. A recent study into the role of the terminal complement pathway in MPGN has pointed towards the potential of therapeutic inhibition of the complement system in MPGN [55]. Mice deficient for both fH and C5 developed less severe glomerulonephritis with better renal function, lower mortality and reduced glomerular cellularity in comparison to fH-deficient mice with normal C5. It is interesting to note that C5-deficient mice were not protected against proteinuria, suggesting that the glomerular C3 activation is sufficient to disrupt the glomerular permselectivity. The induction of heterologous nephrotoxic serum nephritis in fH-deficient mice resulted in markedly increased renal damage when compared to wild-type mice. The renal damage was clearly reduced in fH-deficient mice lacking C5 whereas C6-deficient mice were not protected. These findings demonstrated that the complement-mediated renal damage depended on the formation of the anaphylotoxin C5a but not on the formation of C5b-9. The role of C5 was supported by the reduction of proteinuria and glomerular neutrophil accumulation after treatment with an antibody against C5. These observations suggest that anti-C5 treatment could serve as a treatment option in MPGN type II. Complement and the atypical haemolytic uremic syndrome The haemolytic uremic syndrome is characterised by microangiopathic haemolytic anemia, consumptive thrombocytopaenia and the formation of microvascular thrombi. The vascular damage is particularly severe in the kidney and can lead to acute renal failure. Most cases of HUS are associated with diarrhoea and are caused by the verotoxin-producing Escherichia coli strain O157:H7. The less common form that is not associated with diarrhoea is referred to as atypical HUS (aHUS). Especially in children, the outcome and prognosis of diarrhoea-associated HUS is good, whereas atypical HUS is associated with substantial chronic renal failure and mortality. The familial occurrence of aHUS [56] and the occasional finding of complement consumption [57] and deposition [58] in familial aHUS suggested a hereditary defect in alternative pathway activation or control. Importantly low levels of C3 persisted in patients with familial HUS after remission of the disease and low C3 levels were also detected in unaffected relatives. The discovery of fH mutations in families with aHUS confirmed this hypothesis [59, 60]. Until now, more than more than 100 fH mutations have been described [61]. These can be searched in an interactive HUS database (http://www.FH-HUS.org). The fH mutation frequency is 38% in familial forms of aHUS and 20% in sporadic forms [62]. In contrast to the fH mutations in patients with MPGN type II, patients with aHUS are usually heterozygous for the fH mutation. The penetrance is around 60%. Patients usually have normal levels of circulating fH protein, but reduced C3 levels are found in about 50% of the aHUS cases with a fH mutation [62]. Also in contrast with the mutations associated with MPGN, the fH mutations in patients with aHUS are located in the C-terminal region which is important for binding to cellular surfaces [63–65] via an interaction with C3b deposited in the surface of these cells [66]. Because mutated fH cannot bind to surface-bound C3b, circulating factor B can associate with the C3b and C3 convertase is formed, leading to unopposed complement activation on the endothelium. The creation of a transgenic mouse that lacks the exons encoding for the C-terminal region of fH that is responsible for the binding to cellular surfaces has resulted in an aHUS model that is highly similar to the human disease [67]. These mice have a preserved capacity to regulate fluid phase complement activation and did not develop glomerulonephritis. The absence of systemic complement depletion in the presence of defective endothelial protection against complement attack led to a typical picture of HUS including the formation of glomerular microthrombi, fragmentocytes in peripheral blood and thrombocytopenia. Next to fH mutations, other mutations in complement regulatory proteins have been discovered in patients with aHUS. MCP mutations are found in approximately 14% of the patients with aHUS [62] and until now 43 mutations have been reported [61]. The course of the disease is milder in patients with MCP mutations, and plasma therapy does not seem to contribute to the outcome. Factor I mutations are quite rare and are found in about 4.5% of the patients with aHUS. More recently, two factor B mutations have been discovered [68]. One of these mutations increases the affinity of factor B for C3b, while the other increases the half-life of C3bBb. Both mutations result in an increased activity of the alternative pathway. Taken together, the clinical and experimental findings clearly point towards an important role of complement regulation in the pathogenesis of aHUS. However, until now, mutations of complement-regulatory proteins are only found in about 50% of the affected patients and family members of affected patients can share the mutations without manifesting aHUS. It seems that both additional predisposing factors and triggering circumstances, e.g. infections, are necessary to initiate the full-blown microangiopathy of aHUS. Nevertheless, it is clinically useful to screen patients with aHUS for the known mutations because the findings may influence the prognosis and therapeutic decisions. Although evidence is lacking, patients with fH deficiency are usually treated with plasma therapy [69]. On the other hand, MCP is a membrane-bound protein and there is no rationale for plasma substitution in patients with MCP mutations and aHUS. As mentioned above, retrospective data do not indicate that plasma therapy results in improved outcomes in these patients [62]. However, patients with MCP mutations do well after kidney transplantation [70] as functional MCP is present on the endothelium of the transplanted kidney whereas patients with fH mutations have a very high rate of disease recurrence and graft loss after kidney transplantation [71]. Preemptive plasma therapy may be an option in these patients, but data are lacking to support this approach. Others have attempted combined liver and kidney transplantation in children with fH-associated HUS, but liver transplantation is associated with greatly increased risks when compared with kidney transplantation alone, and this procedure has not been uniformly successful until now [72–74]. Complement and progressive renal damage Independent of the underlying renal disease, proteinuria is associated with tubulointerstitial fibrosis and a progressive loss of renal function [75–77]. A number of mechanisms by which proteinuria may cause renal function loss have been suggested. These include oxidative damage induced by transferrin, lysosomal rupture caused by overload of the resorptive capacity for urinary proteins and pro-inflammatory effects of albumin-bound free fatty acids [78–81]. For many years, complement deposition along the brush border has been noted in proteinuric renal disease [82]. C5b-9 is found in urine from patients with various proteinuric renal diseases including diabetic nephropathy [83]. The C5b-9 in the urine is very probably generated within the tubulular lumen due to an intrinsic complement-activating property of the tubular cells [84, 85]. The exact mechanism of tubular complement activation is not understood, but tubular ammonia production [86] and a low expression of complement regulatory proteins on the apical cell surface [87] are thought to contribute. The insertion of sublytic amounts of C5b-9 in the cell membrane of tubular cells leads to the production of pro-inflammatory cytokines [88, 89] and collagen. In vivo evidence for the role of complement in proteinuria-mediated renal damage has been derived from studies in C6-deficient rats. Rats with an inherited C6 deficiency were protected against tubulointerstitial damage in both the remnant kidney model [90] and the puromycin model of proteinuric renal disease [91]. A therapeutic intervention with either the murine complement inhibitor Crry or CD59 targeted to the renal tubulus resulted in improved renal function and less interstitial damage when compared with untreated animals [92]. Conclusions Increasing knowledge about the complement system has taught us about both the protective and harmful roles of complement in renal disease. In the course of this review, it has repeatedly become clear that complement inhibition early on in both the classical and alternative pathways is associated with the risk of increased deposition of immune complexes and the resulting damage may outweigh the benefit. On the other hand, it seems that complement inhibition distal of the formation of the C3 convertases is safe and offers more promising therapeutic options for renal diseases for which no satisfying treatment has been established until now. Independently of these promising therapeutic prospects, complement has become an invaluable tool in the diagnosis and monitoring of renal disease and results of complement studies have a strong impact on day-to-day decision making in the care of our patients with renal disease.

J_Chem_Ecol-3-1-2039843

Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae. Introduction Most plants are not simply homogeneous resources to herbivorous insects but produce discrete organs with different functions. In addition to the vegetative leaf tissues, plants produce reproductive organs (flowers, seeds) that may exhibit significant differences in levels of both nutrients and defensive secondary metabolites. Specialized guilds of herbivores are known to exploit specific plant structures (Schoonhoven et al. 2005). Flowers and seeds may be attacked by insects that rarely feed on leaf tissues, and vice versa. Other herbivores exhibit more plasticity in food selection and will readily feed on all available parts of the plant. Even the latter herbivore types display some preference for a specific plant structure, presumably as a means of obtaining optimal nutrient intake, limiting competition, or obtaining enemy-free space. The cabbage white butterflies Pieris brassicae L., Pieris rapae L., and Pieris napi L. are specialized on the plant family Brassicaceae. Interactions between these butterflies and their host plants have been explored in detail, and they have acquired the status of a model system in the field of insect–plant biology (Feltwell 1982; Chew and Renwick 1995; Renwick 2002). Glucosinolates, characteristic of the Brassicaceae, play a crucial role as chemical mediators of these interactions. Adult females of P. brassicae and P. rapae, as well as their larvae, exploit glucosinolates as token stimuli during selection of host plants for oviposition and feeding (Renwick et al. 1992; van Loon et al. 1992; Moyes et al. 2000; Schoonhoven and van Loon 2002). The majority of Pieris–Brassica interaction studies has focused on cultivated forms of Brassica oleracea L., which are biennials. Other wild crucifers, such as the annual black mustard, Brassica nigra L. (Koch), are reported to be host plants for P. brassicae in Europe as well (Harvey et al. 2003). We studied within-plant feeding site location in detail on three genotypes of B. nigra. We included the analysis of leaf and flower glucosinolates to assess whether a chemical basis for selection of either plant organ in terms of quality or quantity of these compounds could be determined. In addition, we investigated whether growth rates differed between caterpillars feeding on either leaves or flowers to assess whether the observed preference behavior would confer a nutritional benefit. Methods and Materials Plants Seeds of B. nigra var. abyssinica A. Braun from three early flowering accessions were obtained from the Centre for Genetic Resources (CGN, Wageningen, The Netherlands). The accessions were: A, CGN06619 (feral population, collected in 1975 from the Peloponesus, Greece); B, Junius CGN06618 (advanced cultivar, collected in 1977 in Germany); and C, Giebra CGN06620 (advanced cultivar, donated in 1965 by the Botanical Garden of the Justus Liebig University, Giessen, Germany). Sowing was done on April 7, 2004, in a greenhouse at 22 ± 2°C and 60 ± 5% RH. Two weeks later, plants were individually transplanted into 1.5-l pots and transferred to another greenhouse compartment on April 30, 2004. This compartment was kept at 22 ± 2°C, 60% RH, natural light supplemented by additional illumination by 500-W SON-T lamps (Philips, Eindhoven, The Netherlands) for 16 hr per day. Flower bud formation and flower opening were recorded daily to determine developmental stage (Harper and Berkenkamp 1975). Insects Pieris brassicae caterpillars were obtained from a laboratory strain established in 2004 and reared on Brussels sprouts (B. oleracea var. gemmifera cv Cyrus) plants grown in a climatized greenhouse, under the same conditions as given above for B. nigra plants. The colony was maintained in a climatized room at 22 ± 1°C, RH 40 ± 5%, and a photoperiod of L16:D8. Caterpillar Position on the Plant On the first day of an experiment, newly hatched P. brassicae larvae were collected from the laboratory culture and taken to the greenhouse. Three caterpillars were placed on the first true leaf below the lowest flowering branch of a plant in growth stage 4.1 (Harper and Berkenkamp 1975). Total numbers of plants used from each accession were A, 8; B, 7; and C, 9. Caterpillar position on the plants was scored each morning in four categories: on a true leaf, on a small leaf in the inflorescence, on a flower, and on the stem. Caterpillar instar stage was recorded daily until the fifth instar was reached. In the late fifth instar, caterpillars that had entered the wandering phase left the plants. Caterpillar Growth on Leaves and Flowers Three neonate caterpillars were inoculated on each of 20 plants of accession A, on a mature leaf just below the inflorescence. The inoculation took place when plants had just started to flower (growth stage 4.1). Just after caterpillars had molted to the third instar, they were transferred to the lowest branches of the inflorescence on half of the plants. We applied a specially constructed water barrier to prevent caterpillars from migrating from leaves to the inflorescence or vice versa, while not influencing the microclimate around the plant (Fig. 1). Fresh body weights of larvae were determined to the nearest milligram on a Mettler electronic balance at three time points separated by 3-d intervals. Fig. 1Schematic drawing (top and lateral views) and dimensions of water barrier applied around the stem of B. nigra plants to prevent movement of caterpillars from leaves to flowers and vice versa. A circular water container was manufactured of plastic. The middle circle consisted of a central platform, preventing drowning of caterpillars feeding on the inflorescence in the rare events of falling down or downward migration. In the center of the platform, a hole allowed the main stem to grow through. The platform was put in place around the main stem just below the branch carrying the first flower buds. The circular opening between the central hole and the stem was blocked with soft foam (not drawn) Preparation of Plant Samples for Glucosinolate Analysis Ten seedlings of each of the three accessions were individually transplanted to 1 1/2-l pots. Plants were maintained at 22 ± 2°C, RH 60 ± 5%, during the 16-hr photophase (8 Philips SON-T Agro 400-W sodium lamps) and 8-hr scotophase.At day 47 after sowing, leaf and flower samples were taken from all 10 plants of each accession. After sampling, five plants of each accession were designated untreated (controls). Three groups of 10 newly hatched P. brassicae caterpillars were placed on three leaves of the five treated plants. As soon as the caterpillars had migrated to the flowers, which took place late in the second or early in the third instar (days 5–6), leaves damaged by the caterpillars were sampled. Leaves of a similar age were sampled from the control plants and intact flowers were sampled from both control and treated plants.The flower and leaf samples were kept in paper envelopes and stored in a −20°C freezer before and after freeze-drying. Samples were freeze-dried in the envelopes for 2 d (Labconco Freedry system). After freeze-drying, the envelopes were sealed in plastic and sent to the laboratory of NA in Denmark for chemical analysis. Determination of Glucosinolate Content Freeze-dried samples were weighed accurately into 10-ml centrifuge tubes (excluding the petiole part of leaves). For samples below 300 mg, the entire sample [27–257 mg dry weight (DW)] was used to avoid errors associated with subsampling, but from a few samples above 300 mg, a representative subsample was taken. Each sample was extracted 3× in 70% boiling aq. methanol with benzylglucosinolate as internal standard. The combined extracts were adjusted to 10.00 ml, of which an amount was applied to an anion exchange column (Agerbirk et al. 2001a). To keep within the linear range of the additional sample preparation steps, the amount of crude extract applied depended on the weight of the sample extracted as follows (DW extracted/volume applied): 25–75 mg/8 ml, 75–150 mg/4 ml, and 150–300 mg/2 ml. Glucosinolates were converted to desulfoglucosinolates (Agerbirk et al. 2007), which were eluted and quantified by high-performance liquid chromatography (HPLC) (Agerbirk et al. 2001a) and identified by comparison of retention times and diode array UV spectra with those of authentic standards (Agerbirk et al. 2001b). A number of unidentified trace peaks with areas typically less than 0.2% of the desulfosinigrin area were ignored. Generally accepted relative response factors were used for calculation of glucosinolate levels based on the HPLC peak areas (at 229 nm, band width 8 nm) of the desulfoderivatives (Wathelet et al. 2004). The recoveries of internal standard in the three extraction series were [mean (SD)] 99% (6%), 97% (13%), and 98% (4%), as compared to HPLC peak areas obtained after application of the internal standard only to DEAE columns in parallel control experiments. Preliminary experiments without the addition of internal standard (comprising both leaf and flower samples from plants both with and without caterpillars feeding) confirmed the absence of benzylglucosinolate as an endogenous glucosinolate in the material, in accordance with previous reports (Feeny and Rosenberry 1982; Sang et al. 1984), allowing the use of this glucosinolate as internal standard. The identities of all desulfoglucosinolate peaks were confirmed by liquid chromatograph mass spectrometry (LC–MS) of representative samples with an Agilent 1100 Series LC (Agilent Technologies, Waldbronn, Germany) coupled to a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). An XTerra MS C18 column (Waters, Milford, MA, USA; 3.5 μM, 2.1 × 100 mm) was used at a flow rate of 0.2 ml min−1. The mobile phases were as follows: A, 0.1% (v/v) HCOOH and 50 μM NaCl; B, 0.1% (v/v) HCOOH and 80% (v/v) MeCN. The gradient program was 0 to 4 min, isocratic 2% (v/v) B; 4 to 10 min, linear gradient 2 to 8% B; 10 to 30 min, linear gradient 8% to 50% (v/v) B; 30 to 35 min, linear gradient 50 to 100% (v/v) B; 35 to 40 min, isocratic 100% B. The mass spectrometer was run in electrospray mode, observing positive ions. Mass spectral data were treated with the native DataAnalysis software.The extraction procedure was somewhat simplified compared to the standard protocol (Agerbirk et al. 2001a, originally optimized for seed extraction), as variable sample weights were accepted, and mechanical homogenization during extraction was avoided. These simplifications were justified by results of initial control experiments: A number of both leaf and flower samples of varying weights (up to 400 mg) were first subjected to the extraction described above. Then, the residue was further extracted for another three times, but this time with mechanical homogenization. In all cases, low relative amounts of glucosinolates (1–16% compared to the first extract) were detected in the extract obtained with mechanical homogenization (with a tendency for increasing percentage with increasing sample weight). Proportional amounts of internal standard were similarly recovered in all the extracts obtained with mechanical homogenization, showing that the internal standard had already been completely mixed with endogenous analytes during the extraction without homogenization. Hence, it was concluded that the ratio of internal standard to endogenous glucosinolates extracted without mechanical homogenization was representative of their initial concentrations. Statistical Analyses Proportion of days caterpillars spent on different positions on the plant was treated as binomial data. Distributions over the four positions were compared by using the Kolmogorov–Smirnoff test (GenStat release 8.11; Anonymous 2005). Caterpillar weights were analyzed by analysis of variance. The amount (μmol/g DW) of each glucosinolate detected in the leaf and flower samples were analyzed with a generalized linear model after a logarithmic transformation. The effect of time (before and during caterpillar feeding), treatment (undamaged and damaged by caterpillars), plant tissue (leaf and flower), and genotype (accessions) and all possible two- and three-way interactions were investigated (GenStat release 8.11). Results Position on the Plant First and second instars fed primarily on leaves, whereas third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed (Fig. 2). The distribution of caterpillars over the four positions was statistically similar for the three genotypes (Kolmogorov–Smirnoff test, P > 0.05). Fig. 2Frequency distribution of caterpillar position on B. nigra plants observed daily over a period of 12–13 d. On day 1, neonates were introduced on a true leaf just below the inflorescence. Four organ positions were distinguished: on a true leaf, on a small leaf in the inflorescence, on a flower, or on a stem in the inflorescence. Percentages are averaged based on three caterpillars per plant for eight, seven, and nine plants of accessions A, B, and C, respectivelyCaterpillars fed in discrete meals two to three times each hour, interspersed with phases without feeding. On a leaf, after termination of a meal, they moved away from the freshly damaged wound, after which they rested most of the time elsewhere on the leaf until they moved back to the feeding site. By contrast, in the inflorescence, during a single meal, caterpillars moved from flower to flower along flowering branches. A fifth-instar caterpillar consumed on average 135 ± 21 buds and flowers. When they were not feeding, P. brassicae larvae rested on stems immediately adjacent to the flowers. Caterpillar Growth Caterpillars feeding on flowers of accession A since molting to the third instar had significantly higher fresh body weights on each of the three measuring points (mid-L4 and early and mid-L5 and late L5; Fig. 3) than those that were confined to leaves (P < 0.01, generalized linear model). Fig. 3Fresh body mass of P. brassicae on either flowers (circles) or leaves (squares) of B. nigra in mid-fourth, mid-fifth, and late-fifth instar developing on B. nigra, accession A. Mean and SEM are plotted for 30 caterpillars Glucosinolate Levels Five glucosinolates were identified: the aliphatic allylglucosinolate (sinigrin), which occurred in large amounts, and low levels of four aromatic glucosinolates including 2-phenylethylglucosinolate and the three indole glucosinolates indol-3-ylmethylglucosinolate (glucobrassicin), 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Despite the minor levels of the identified aromatic glucosinolates, they were quantified and included in the statistical analyses because of their well established chemosensory influences on Pieris species (Schoonhoven and van Loon 2002). No other significant peaks were detected. 2-Phenylethylglucosinolate was either below the detection limit in leaves (accession C) or present at very low levels (range 0.001–0.013 μmol/ g DW). We carefully distinguished the peak with the exact retention time of (desulfo) 2-phenylethylglucosinolate from an unidentified trace peak that was more intense in leaf chromatograms (estimated levels 0.03–0.35 μmol/g DW in leaves, 0.01–0.08 μmol/g DW in flowers), that had a retention time 0.7 min longer than that of (desulfo) 2-phenylethylglucosinolate, and that did not coelute with the authentic standard of 2-phenylethylglucosinolate after spiking.Genotype and tissue type were the most important factors affecting the levels of identified glucosinolates, and these factors showed a significant interaction (Table 1). No significant interactions were found between treatment and tissue (P > 0.05 for all glucosinolates). Glucosinolate concentrations have been graphically depicted for accession A in Fig. 4. Sinigrin was by far the most abundant compound in all three genotypes, with levels at least 44 times higher (range 44–305) in leaves and at least 87 times higher (range 87–447) in flowers of any of the three genotypes and treatments than the second most abundant compound, 4-hydroxyglucobrassicin. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. Sinigrin levels were ca. fivefold higher in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in leaves were higher than in undamaged plants (P < 0.05; Table 1). In flowers of accession A (Fig. 4), there was a similar tendency for induction of sinigrin by caterpillar feeding, but such induction was not seen with flowers from the other two genotypes (results not shown). Levels of other glucosinolates showed moderate variations that were not affected by the experimental variables (Table 1, Fig. 4 and results not shown). There was no systematic induction of any indole glucosinolate or of total indole glucosinolates in response to caterpillar feeding (results not shown). Table 1Results from generalized linear model analysis on log-transformed data of glucosinolate concentrations in two tissue types (leaves and flowers) for three genotypes of B. nigraFactorSinigrinPEGB4OHGB4MeOGBTimensnsns***nsTissue******ns***nsTreatment*nsns*nsGenotype************nsTissue × genotype*****ns*nsTreatment means presence or absence of third-instar caterpillars feeding on flowers during 4 d. Effect of the factor “time” refers to changes over 4 d in intact plantsPE = phenyethylglucosinolate; GB = glucobrassicin; 4OHGB = 4-hydroxyglucobrassicin; 4MeOGB = 4-methoxyglucobrassicin*P < 0.05; **P < 0.01; ***P < 0.001Fig. 4Concentrations of five glucosinolates (mean + SEM) in leaves and flowers of 10 plants of accession A. For both leaves and flowers, the initial concentration just before treatment, the concentration in leaves or flowers of plants damaged by feeding caterpillars during 4 d, and the concentration determined in leaves or flowers of intact plants 4 d after caterpillars were introduced on the treated plants Discussion Our data show that P. brassicae third, fourth, and fifth instars preferentially feed on B. nigra flowers. Preference was absolute: although leaf material was available in surplus, caterpillars fed exclusively on flowers. Historically, P. brassicae has been commonly considered a folivore (Feltwell 1982), with its impact on plants examined primarily from this perspective. To the best of our knowledge, this is the first detailed study showing that P. brassicae larvae preferentially feed on the buds and flowers of its host plant. The status of P. brassicae as an agricultural pest has invariably resulted in a focus on cultivated, biennial forms of B. oleracea in studies of its host plant relationships. We also observed flower and silique feeding under field conditions (Smallegange et al., unpublished results). The impact of folivores on plants has both direct and indirect effects. Directly, these herbivores reduce the leaf area available for photosynthesis, thereby decreasing the plants’ ability to garner resources for defense and reproduction (Marquis 1984; Mothershead and Marquis 2000; Schoonhoven et al. 2005). Indirectly, leaf herbivory can delay flowering (Meyer and Root 1993; Strauss et al. 1996) or can alter developing flowers both physically (Strauss et al. 1996; Lehtilä and Strauss 1999; Mothershead and Marquis 2000) and chemically (Lohman et al. 1996; Karban and Baldwin 1997). This may cause a reduction in plant attractiveness to pollinators or in the efficiency of pollen delivery, resulting in decreased pollination and, subsequently, reduction of seed set and overall fitness of the plant. Florivory, feeding on flowers, has received less attention than folivory or feeding on other vegetative plant parts despite its common occurrence and potentially high impact on plant fitness (recently reviewed by McCall and Irwin 2006). Feeding on flowers causes damage to structures essential for plant reproductive output (Juenger and Bergelson 1997; Adler et al. 2001). Indirectly, petal damage leads to a reduction in pollinator visitation that not only reduces reproductive success but also affects male fitness (via pollen removal) of the plant as well (Karban and Strauss 1993; Krupnick and Weis 1998; Adler et al. 2001). However, the compensatory abilities of plants can partially overcome the impact of floral herbivory (Hendrix 1988; Krupnick and Weis 1998). Removal of reproductive organs as a preferred mode of feeding has evolutionary consequences, as this type of attack is likely to exert a stronger selection pressure than leaf feeding, especially in annual species like B. nigra. Although many plant species accumulate higher concentrations of defensive secondary metabolites in reproductive organs than in vegetative organs (Schoonhoven et al. 2005), in B. nigra, these higher levels are not sufficient to deter the specialist feeder P. brassicae. A putative barrier to florivory by generalist herbivores lies in the higher quantities of secondary plant compounds that are typically found in these organs relative to the levels reported for leaves (Rask et al. 2000; Fahey et al. 2001; Brown et al. 2003; Strauss et al. 2004; this study). The location of high concentrations of defensive secondary plant compounds, including glucosinolates, in reproductive structures is consistent with the optimal distribution of chemical defenses predicted by plant defense theory (Zangerl and Bazzaz 1992; van Dam et al. 1996; Wallace and Eigenbrode 2002; Strauss et al. 2004). Myrosinases, β-thioglucosidase enzymes that convert glucosinolates into products such as isothiocyanates and nitriles, which are volatile in the case of, e.g., sinigrin and 2-phenylethylglucosinolate, occur in different forms in flowers and leaves (Rask et al. 2000). By using headspace techniques, isothiocyanates and nitriles have been reported from mechanically macerated bud samples of several Brassica species (Tollsten and Bergström 1988). Phenylacetonitrile was detected in headspace samples of intact B. rapa L. flowers (Omura et al. 1999). Benzylcyanide was released in significantly higher amounts from Brassica napus L. in midflowering than in bud stage (Jönsson et al. 2005). High levels of isothiocyanates are potentially toxic to both specialist and generalist herbivorous insects (e.g., Li et al. 2000; Agrawal and Kurashige 2003), while even low levels may be detected by specialized sensory receptors and assist specialized herbivores in host plant finding (Renwick et al. 2006). The growth rate we observed for caterpillars feeding on flowers was remarkable. The feeding site preference of L3–L5 caterpillars presents a case of within-plant selective foraging, the adaptive value of which might be the ingestion of higher nutritional content in flowers, known to act as nutrient sinks, compared to leaves. Nutrient acquisition during the larval stage can affect pupal survival, longevity, and fecundity of Lepidoptera (Feltwell 1982; Van der Meijden et al. 1984; Metspalu et al. 2003). For example, female pupae of Tyria jacobaeae were significantly larger when caterpillars were reared on leaves and inflorescences of Senecio jacobaea plants than when reared on leaves only. This is advantageous because large pupae have a higher eclosion success, and it may explain why T. jacobaeae females prefer flowering plants for oviposition (Vrieling and de Boer 1999). Faster growth has often been interpreted as a mortality-reducing factor. It is assumed that by reducing the amount of time that larvae are early instars, when they are most vulnerable to attack by natural enemies, they will experience higher survival (the ‘slow-growth–high-mortality hypothesis’ sensu Clancy and Price 1987; Benrey and Denno 1997). This has been experimentally confirmed under field conditions for the interaction between P. rapae feeding on B. oleracea and its predators and parasitoids such as Cladophora glomerata (Loader and Damman 1991; Benrey and Denno 1997). However, for other tritrophic interactions, faster growth was associated with higher mortality due to natural enemies (e.g., Clancy and Price 1987). Biere et al. (2002) found that larvae of the moth Hadena bicruris exhibit a strong preference for seeds of their food plant, Silena latifolia, over leaf tissues. Herbivore development proceeded more rapidly on seeds, and the larvae were less susceptible to parasitism from their main endoparasitoid, Microplitis tristis. Herbivorous insects specialized on glucosinolate-containing plants typically avoid the formation of toxic isothiocyanates by employing specialized detoxifying mechanisms. In the case of P. brassicae, this is accomplished by a nitrile specifier protein (NSP) in the gut that changes the products of the myrosinase-catalysed hydrolysis of glucosinolates from isothiocyanates to relatively harmless nitriles (Wittstock et al. 2004), which may be further metabolized before excretion depending on side chain structure (Agerbirk et al. 2006; 2007). Shortly before this mechanism was reported, it was discovered that allyl isothiocyanate, the volatile hydrolysis product of sinigrin, is toxic to the specialist feeder P. rapae when feeding on an artificial diet (Agrawal and Kurashige 2003). Assuming a 20% DW in the artificial diet as used by Agrawal and Kurashige (2003), the maximum concentration to which P. rapae caterpillars were exposed in their study was 8.5 μmol/g DW. This dose caused significant negative effects on growth rate and inhibited development. In contrast, sinigrin levels in B. nigra flowers were more than 10 times higher (Fig. 4) and flowers sustained higher growth rates of P. brassicae than leaves, which had average levels of sinigrin two times higher than the corresponding amount of allylisothiocyanate as applied in the artificial diet. Assuming that isothiocyanates were produced in significant amounts during ingestion of glucosinolate-containing foliage by P. rapae, an involvement of the glucosinolate–myrosinase system in defense against this specialist species was suggested. Brassica nigra plants that had been induced by previous P. rapae feeding damage had a 27% increase in sinigrin concentration, but also 43% higher trichome density (Traw and Dawson 2002). Our data confirm the reported induction of sinigrin in leaves upon feeding, but also demonstrate that the magnitude of the induction is minor relative to the difference between leaves and flowers. With the elucidation of the NSP-based mechanism for detoxification of the glucosinolate–myrosinase system, the suggested involvement of the glucosinolate–myrosinase system and defense to specialists now needs to be re-evaluated. Formation of isothiocyanates is to be expected only if the myrosinase activity in the plant is so high that available NSP cannot completely direct the reaction towards nitriles, combined with glucosinolate levels sufficiently high to produce toxic levels of isothiocyanates. The selective foraging on flowers by P. brassicae documented here demonstrates that the caterpillars are able to detoxify even high levels of sinigrin, such as those present in B. nigra flower tissue. Not only high glucosinolate levels but also high myrosinase activity would be needed to exert toxic effects on Pieris caterpillars, as has been experimentally demonstrated for another glucosinolate-adapted herbivorous insect, Plutella xylostella (Li et al. 2000). Glucosinolates occur in cells of all organs of Brassicaceous plants, whereas myrosinases can be found in scattered glucosinolate-free cells. It remains to be examined whether flower buds of different age, flowers, and siliques of B. nigra have cells that contain myrosinases. In the related B. napus L., expression of the MYR1-gene coding for particular myrosinase isoforms has been demonstrated to occur in all organs investigated, including petals (Falk et al. 1992), although myrosinase was absent in very young flower buds in this species (Andréasson et al. 2001). Myrosinase gene promoters fused to beta-glucuronidase as a reporter showed expression of myrosinase in idioblast myrosin cells in immature and mature seeds and phloem myrosin cells of B. napus (Thangstad et al. 2004). Data on headspace composition of inflorescences actually fed upon by Pieris spp. are not available. Follow-up studies on the types and quantities of volatile breakdown products emitted by herbivore-damaged flowers are required to quantify myrosinase activity levels in flowers. If these levels are low, the better performance associated with flower feeding might be reconciled with a role of the glucosinolate–myrosinase system in defense against the specialist P. brassicae. However, a likely and simple explanation of the better performance associated with flower-feeding may be that the caterpillars are able to efficiently detoxify the glucosinolate–myrosinase defense system in both organs, and that flowers, major nutrient sinks on a plant, are more nutritious than leaves. The fact that early instars did not migrate to flowers may be an adaptation to factors other than nutrient content: leaves are the site of oviposition, are present earlier in the season than flowers, and may provide better refuge from natural enemies for small instars. We have never observed P. brassicae or P. rapae ovipositing on flowers or flowering stems. Neither P. rapae nor P. brassicae sequester glucosinolates for their own defense against natural enemies (Müller et al. 2003), in contrast to what had been published earlier by Aplin et al. (1975). The intensely green, clear regurgitant these caterpillars instantly, and typically, produce when attacked may contain highly concentrated glucosinolate hydrolysis products (nitriles) stored in the foregut. Presence of concentrated glucosinolate hydrolysis products may explain the obvious repellent effect of regurgitant to natural enemies such as Cotesia wasps and predators (Brodeur et al. 1996). Consequently, caterpillars may prefer flower tissues that contain high levels of these compounds, thus boosting their defense against both specialist and generalist natural enemies (Soler et al. 2005). Behavioral experiments with parasitoids and predators known to act as common natural enemies of P. brassicae (Feltwell 1982) are required to test this hypothesis.

Hum_Reprod-1-1-2387218

Cell identity in the preimplantation mammalian embryo: an epigenetic perspective from the mouse

The early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation. Following fertilization, a single cell, the zygote, will give rise to all tissues of the organism. The first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm, a differentiated tissue that envelopes the inner cell mass. The question of when and how the cells start to be different from each other in the embryo is central to developmental biology: as cell fate decisions are undertaken, loss of totipotency comes about. Although the blastomeres of the preimplantation embryo are totipotent, as the embryo develops some differences appear to develop between them which are, at least partially, related to the epigenetic information of each of these cells. The hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented. Although there are now some indications that epigenetic mechanisms are involved in cell fate determination, much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division. These considerations are further discussed in the context of preimplantation genetic diagnosis: does it matter to the embryo which cell is used for genetic diagnosis? The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future. Embryonic development starts from a single cell, the zygote. In this cell, the two gametes convey and contribute information to start a new developmental programme. The formation of the newly fertilized zygote constitutes therefore the climax of totipotency because of the resulting zygote’s inherent ability to produce all cell types in a new organism. In the mammalian embryo, the first differentiative event occurs as inner cells form upon cell division at the 8-cell stage. As a result of this division, an ‘inner’ and an ‘outer’ population of cells can be distinguished in the 16-cell stage embryo, which will respectively occupy different positions in the morula (Johnson and Ziomek, 1981). Much work has been done in trying to understand how cell polarity develops in the inner and outer cells, but I will not deal with this topic here and instead will refer the reader to an excellent review published elsewhere (Johnson and McConnell, 2004). The inner cells will develop into the inner cell mass (ICM) and the outer layer of cells will differentiate into the trophectoderm (Tarkowski and Wroblewska, 1967; Ziomek and Johnson, 1982). Morphologically, however, the first overt signs of differentiation are evident only at the blastocyst stage, with the formation of the trophectoderm, which is the first differentiated tissue to form as an epithelial layer that envelops the ICM (Fig. 1A). The latter, in contrast to the trophectoderm, retains its pluripotent character and the ability to self renew. While the ICM will give rise to the embryo proper, the trophectoderm will give rise to the extraembryonic tissues that will support development of the embryo during gestation. The ICM will also give rise to yet another extraembryonic tissue, the primitive endoderm, which is first visible as a cuboidal layer of cells lining the blastocoelic cavity on the fourth day of development. The trophectoderm and the ICM each display molecular identity, which is reflected in part by the expression of specific genes that are, for the ICM, involved in its specification and the maintenance of pluripotency (such as Nanog and Oct4) or, for the trophectoderm, that are required for its differentiation (such as Cdx2) (Palmieri et al., 1994; Nichols et al., 1998; Chambers et al., 2003; Mitsui et al., 2003; Strumpf et al., 2005). The bifurcation of these two lineages is complete at the late blastocyst stage. At this stage, cells from the ICM do no longer have the potential to form trophectoderm derivates in vivo upon transplantation (Rossant and Lis, 1979), indicating that these cells have lost their totipotency and that lineage allocation has definitely occurred. Figure 1: Cell lineages of the mammalian blastocyst and epigenetic marking (A) Representation of the lineages in the mammalian blastocyst on the third day of gestation (E3.5). The blastocyst is composed of two distinct populations of cells: the trophectoderm (red) and the inner cell mass (ICM, green), which display molecular identity and epigenetic asymmetries. The embryonic-abembryonic regions (dotted line) of the blastocyst are determined by the position of the ICM, which lies within the embryonic region of the blastocyst. (B) Diagram illustrating some of the epigenetic marks. The DNA wrapped around the nucleosome (beige cylinders) is shown as light blue. The DNA is subject to DNA methylation, which constitutes one of the main epigenetic players. The core histones (beige) that form the nucleosome can be covalently modified (by acetylation, methylation, phosphorylation), particularly on their N-terminal tails. Each of these marks can have an effect on how the information contained in the DNA is read by modulating downstream events such as transcriptional activation or repression. For example, histone methylation (Me) can have a positive effect on transcription (green) or a repressive one (red). The marks can be present in different combinations and may change during the cell cycle. (C) Model for epigenetic marking and lineage allocation. In this model, an epigenetic mark would be laid down in a given cell during development. There could be other epigenetic event(s) that reinforce and/or are influenced by the first marking event. Cumulatively, this could result in determination of the fate of that cell towards a lineage in the blastocyst. The lineage specific marks could stabilize such cell identity and might be necessary for further differentiation. Alternatively, the acquisition of these epigenetic marks could be the result of cell fate determination. One should also consider that not only the nature of the mark would be important, but also the different regions of the chromatin that would be affected by such marks. Because the cells in the preimplantation embryo are totipotent and because the chromatin will still need to be dynamically remodelled during subsequent development, flexibility should be an important component of epigenetic mechanisms taking place during early development. As cell fate decisions are taken, a concomitant loss of totipotency takes place Investigations during recent years have recreated an interest in whether the blastomeres of the mammalian embryo are truly alike throughout preimplantation development before the first differentiative division mentioned above. In other words, whether they acquire a ‘fate’ or whether they start to differ from each other prior to their spatial ‘inner/outer’ allocation within the embryo upon the formation of the morula. Of course, if this were to be the case, the big challenge would be to ascribe molecular mechanisms to these processes. The question of when and how the cells start to be different from each other is not a trivial one. In particular, because as the first cell fate decisions are undertaken, a concomitant loss of totipotency occurs. The developmental time window when this first cell fate decision occurs comprises a number of epigenetic events (Morgan et al., 2005; Surani et al., 2007). These events include the reprogramming of the parental chromatin. Whether such epigenetic events are the cause or the consequence of reprogramming remains an exciting open question, but it is probably a combination of the two. Moreover, the two lineages of the blastocyst exhibit some epigenetic asymmetries. This mini-review is divided in two parts, the first one will deal with the main epigenetic mechanisms that are known to occur during mammalian preimplantation development. The second one will give an overview on data obtained through experimental embryology manipulations and lineage tracing observations to study cell fate in the early mouse embryo. Epigenetic mechanisms in the preimplantation embryo In general terms, epigenetic mechanisms include DNA methylation, covalent modification of histones, chromatin remodelling and histone replacement through incorporation of the so-called histone variants (Fig. 1B). Histone marks have emerged as one of the main players involved in epigenetic mechanisms (Kouzarides, 2007). Histone modifications can be highly dynamic, or have a function in epigenetic memory. Although it is still unclear whether they are the actors of the epigenetic information or the epigenetic information itself, it is evident that covalent modifications of histones are essential components of the epigenome. Histones can be modified by a number of enzymes that mediate methylation, acetylation, phosphorylation, ubiquitynation and ADP-ribosylation of specific amino acid residues (reviewed in Kouzarides, 2007). By and large, the highest density of modifications so far described occurs in histone H3, particularly on its tail. The effects of these modifications on the chromatin and on cellular processes are very diverse, and a modification of the same residue can even have opposite effects depending on the type of modification. For example, trimethylation of H3K9 is considered as a repressive mark, whereas acetylation of the same lysine has a positive effect on transcription (Bannister and Kouzarides, 2005). Likewise, methylation of arginine residues can have a positive effect on transcription (Chen et al., 1999) or a repressive effect (Pal et al., 2004), depending both on the targeted residue and on whether the methylation is symmetric or asymmetric. For some of the modified residues, there is a very clear view of the outcome of an eventual modification: H3K9me3 creates a specific docking site for the heterochromatin protein 1 (HP1), which subsequently recruits the H3K9 methyltransferase Su(var)3-9 and reinforces an autoregulatory loop for heterochromatin formation and maintenance (Bannister et al., 2001; Lachner et al., 2001; Nakayama et al., 2001). The levels of regulation of epigenetic events in the preimplantation mouse embryo are multiple. They include the regulation of the subcellular localization of DNA methyltransferase activity, highlighted by the cytoplasmic retention of Dnmt1o (Carlson et al., 1992); the exclusion of a particular histone modification from the chromatin, which is exemplified by the lack of detection of H3K9me3 in the paternal pronucleus after fertilization resulting in an asymmetry of histone marks between the two pronuclei (Arney et al., 2002; Santos et al., 2005), the differential incorporation of chaperons and histone variants in the parental chromatin (van der Heijden et al., 2005; Torres-Padilla et al., 2006) and the acquisition of highly specific histone variants in the gametes (Clarke et al., 1992; Tanaka et al., 2001; Govin et al., 2007). Further, the maternal and paternal pronuclei exhibit different patterns of global DNA methylation: while the paternal pronucleus is rapidly demethylated—presumably through an active mechanism—right after fertilization, the maternal pronucleus is only passively demethylated through the subsequent rounds of replication and cell division that follow the first mitosis of the embryo (Mayer et al., 2000). Moreover, while the centromeric and pericentric paternal chromatin remain DNA methylated, the maternal DNA loses methylation in such regions (Rougier et al., 1998). The changes in the levels of DNA methylation as development proceeds in the preimplantation embryo are dynamic. Global levels of DNA methylation have been analysed by immunofluorescence, bisulphate sequencing and restriction digestion (Rougier et al., 1998; Mayer et al., 2000; Santos et al., 2002; Aranyi and Paldi, 2006). Bisulphate sequencing and restriction digestion have also been used to analyse the methylation status of repeat sequences (such as L1 and IAP repeats) and some single-copy sequences (such as actin) (Howlett and Reik, 1991; Oswald et al., 2000). These studies have revealed that although global levels of DNA methylation decrease until the blastocyst stage, changes in DNA methylation do not occur to the same extent on all genes. Remarkably, imprinted genes (such as H19) and some repeat sequences (such as IAPs) do not undergo demethylation (Tremblay et al., 1997). During early stages of development, decisions involved in cell fate determination and pluripotency have to be assumed. These processes require the chromatin to be dynamically remodelled to ensure its plasticity. This implies that the mechanisms involved in regulation of chromatin structure need to ensure stability across generations and cell division, but they also need to be flexible (Reik, 2007). The double nature of a covalent modification either on histones and/or on the DNA as dynamic (because in principle it can be added and removed) and at the same time its potential ability to propagate a memory, fits well with these aforementioned needs. Moreover, in keeping with the importance of epigenetic mechanisms during early development, the possibility for an epigenetic mark(s) underlying these phenomena appears very attractive. Concerning the epigenetic asymmetries of the ICM and the trophectoderm, the ICM displays, in global terms, higher levels of DNA methylation compared with the trophectoderm (Dean et al., 2001; Santos et al., 2002). Specific histone marks such as trimethylation of lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3, respectively) are enriched in the ICM compared with the trophectoderm (Erhardt et al., 2003). Likewise, the trophectoderm retains an imprinted form of X inactivation, where the paternal X chromosome is silenced (Heard and Disteche, 2006). This is in contrast to the ICM, where there is reactivation of the inactive X chromosome and a subsequent round of inactivation occurs at random in which either the maternal or the paternal chromosome is inactivated (Mak et al., 2004; Okamoto et al., 2004). The aforementioned epigenetic asymmetries of the two lineages of the blastocyst are evident once lineage allocation has taken place and might reinforce their molecular identity. However, epigenetic asymmetry could also act as a driver for lineage allocation, in which case, the former would precede the latter (Fig. 1C). This constitutes a fascinating current working hypothesis. Development of cell identity in the mouse embryo From experimental embryology, we have learnt from pioneer experiments performed in the 50’s that after mechanical separation of the blastomeres of a 2-cell stage embryo and transfer into foster mothers, each of these two cells gives rise to an adult mouse (Tarkowski, 1959). This indicates that mouse embryos are very flexible in what people have referred to as developmental potential. Derivation of twins from mouse blastomeres at later stages of development (e.g. 4-cell stage or later) has not been possible. Although this has been linked to the low number of cells present in the resulting embryos and hence their inability to form an ICM with a normal cell number, rather than to their developmental potential or identity (Tarkowski and Wroblewska, 1967; Rossant, 1976). Indeed, when random single 4- and 8-cell stage blastomeres are aggregated with ‘carrier’ blastomeres, their progeny is able to contribute to all the tissues of the embryo (Kelly, 1977; Tarkowski et al., 2005), and in this sense, the blastomeres were considered to be totipotent. So, what are these carrier cells providing? Is it a simple matter of cell number? and/or of an appropriate environment? It is also possible however that some subtle intrinsic differences of these blastomeres might be masked by the limitations of the outcome of transplantation procedures, given that the results of these studies are very often far from 100% and the transplantation efficiency is never absolute. Whether this is solely related to technical difficulties linked to these challenging manipulations or to an intrinsic property of specific blastomeres of the embryo is impossible to ascertain. Despite a controversial viewpoint on whether there is any polarity in the early mouse embryo or not, most reports coincide with the interpretation that a blastomere at the 2-cell stage contributes to both the ICM and the trophectoderm (reviewed in Edwards and Beard, 1997; Zernicka-Goetz, 2006). The suggestion of a given blastomere at the 2-cell stage having a ‘preferential’ fate towards either of the blastocyst lineage is not resolved and some researchers have suggested that a slight, but distinct difference in the fate of 2-cell stage blastomeres might be disturbed by experimental manipulations (Alarcon and Marikawa, 2005; Hiiragi et al., 2006). The role of extrinsic factors to the embryo, such as the shape of the zona pellucida, in axis specification of the blastocyst is also a matter of controversy (Gardner, 2007; Kurotaki et al., 2007). However, there are some indications that a bias for a blastomere to contribute to a given region of the embryo in the blastocyst could already exist at the 4-cell stage (Fujimori et al., 2003; Piotrowska-Nitsche and Zernicka-Goetz, 2005). These conclusions are mostly based on lineage tracing experiments of labelled blastomeres, and their degree of invasiveness is debatable. Some groups have used the plane of division in relation to the animal–vegetal axis of the embryo as a sort of guideline to distinguish and characterize blastomeres according to their cleavage plane. By convention, the animal pole is demarcated by the position of the second polar body (which is extruded after resumption of meiosis II upon fertilization) and hence the vegetal pole lies on the opposite side (Fig. 2A). The division from the 2- to the 4-cell stage would segregate for the first time the ‘animal’ and the ‘vegetal’ components of the zygote if it occurs equatorially, that is, perpendicular to the animal–vegetal axis of the conceptus (Gardner, 2002). Thus, whereas a cell that derives from a meridional division (parallel to the animal–vegetal axis) inherits both components, an equatorial division gives rise to an ‘animal’ and a ‘vegetal’ blastomere (Fig. 2B). By looking into the plane of division and the order at which this division occurs from the 2- to the 4-cell stage and subsequent lineage tracing, a subgroup of embryos was identified where it is possible, with a relatively high probability, to predict the future position of the blastomeres in the blastocyst (Piotrowska-Nitsche et al., 2005). This group of embryos are referred to as ME embryos (for Meridional–Equatorial, reflecting the type of cleavage plane and order of division that generated them (Fig. 2B) (Piotrowska-Nitsche et al., 2005; Piotrowska-Nitsche and Zernicka-Goetz, 2005). Although the ME type embryos constitute only a small part (20%) of a complete litter, they provide a very nice system where it is possible to explore the foundations of differentiation in the embryo. Indeed, the ‘vegetal’ blastomere would most often populate the abembryonic region of the blastocyst, which contains mainly mural trophectoderm. Figure 2: Blastomere division planes according to the animal-vegetal axis in the embryo (A) The animal–vegetal (A–V) axis of the preimplantation embryo is demarcated, by convention, by the position of the second polar body, which marks the animal pole. The opposite side to the site of extrusion of the polar body is, by default, the vegetal pole of the embryo. (B) The division pattern from 2-to- 4-cell stage of a typical embryo that undergoes one meridional (M) and one equatorial (E) division (ME embryo) is represented. The cleavage plane is depicted by a red dashed line (embryo on the left). The blastomere that divides earlier is represented on the left. A Meridional division has a cleavage plane that is parallel to the A–V axis of the embryo and hence gives rise to two cells containing both ‘animal’ and ‘vegetal’ components (two cells with pink and yellow motifs on the embryo depicted on the right). In contrast, when a 2-cell stage blastomere divides equatorially, a segregation of the ‘animal’ and ‘vegetal’ cytoplasm occurs and follows derivation of an ‘animal’ (pink) and a ‘vegetal’ (yellow) blastomere In looking for epigenetic marks that could be involved in an eventual cell fate decision of the blastomeres of these ME embryos, it was found that the ‘vegetal’ blastomere displays the lowest levels of dimethylated arginine 26 of H3 (H3R26me2). If H3R26me2 participates in lineage allocation, one might predict that modulating the levels of histone arginine methylation, would have an effect over cell fate. Overexpression of the histone methyltransferase that methylates this residue on H3, PRMT4/CARM1, into individual blastomeres not only induced upregulation of Nanog and Sox2, but also resulted in an almost complete allocation of these blastomeres into the ICM compartment (Torres-Padilla et al., 2007). It is interesting to note that in the mouse, the differences described in histone H3 arginine methylation appear at the 4-cell stage (Torres-Padilla et al., 2007), that is. as early as one cell cycle after the major wave of embryonic genome activation occurs (Schultz, 2002; Hamatani et al., 2004), which suggests that these events might be, at least in part, linked to the transcriptional programme of the embryo. The developmental stage at which genome activation occurs in other mammalian species varies considerably: it takes place at the 1-to- 2-cell stage in mice, the 4-to- 8-cell stage in cows and humans, and the 8-to- 16-cell stage in sheep and rabbits (Schultz and Heyner, 1992). Would this anticipate a different timing for an eventual ‘cell fate path’ for other species? Normal fertile adults can be derived from single blastomeres from 2-, 4- and 8-cell stage embryos in the rabbit, sheep and cattle (Moore et al., 1968; Willadsen, 1981; Willadsen and Polge, 1981). Thus, these species indeed support blastomere isolation and further development at later stages than the mouse does. Transplantation of isolated 4-cell blastomeres into morula stage embryos has demonstrated that the blastomeres at the 4-cell stage are totipotent (Kelly, 1977). In this context, it is important to note that aggregating the ‘vegetal’ cell from ME embryos to form chimeric embryos, showed that this cell is able to contribute to all tissues in the embryo. However, aggregating the same blastomere with other ‘vegetal’ blastomeres from ME embryos exclusively, results in a failure to proceed through development (Piotrowska-Nitsche et al., 2005). Thus, the environment where the blastomeres develop is crucial for the success of the embryo throughout development, and in a ‘normal’ situation, where the embryo has not been perturbed and a given cell develops in its niche, some differences appear to develop, which are, at least partially, related to the epigenetic information of each of them (Torres-Padilla et al., 2007). If epigenetic asymmetries of the early embryo are related to lineage allocation, it is still uncertain whether they are a cause or a consequence for lineage choice. Also, it remains unknown whether such epigenetic asymmetries would affect only particular regions of the genome. For example, whether genes involved in specification of the ICM such as Sox2 and Nanog would all be targeted by the same epigenetic marks in the same blastomere or whether such marks would vary among genes and/or among blastomeres. Further, are ‘inner’ and ‘outer’ cells at the 16-cell stage distinguishable in terms of their chromatin landscapes? The experiments showing that blastomeres are able to respond to the overexpression of a histone modifier and change their fate, indicate that these cells have not yet acquired a ‘fix’ destiny, but that they can still be responsive to some kind of signals. These experiments have an important impact on showing that manipulating the epigenetic information can affect cell fate in the preimplantation embryo, in line with the importance of epigenetic mechanisms being crucial for early development. Moreover, these results do illustrate that such cells can still be flexible and accommodate themselves after a perturbing event (in this case, overexpression of a histone methyltransferase and the downstream effects on the information that is. imparted through specific histone modifications). These studies have originated some interest from the part of the medical community, particularly, in the context of preimplantation genetic diagnosis (PGD) (Goldman, 2007). Does it matter to the embryo which cell is used for genetic diagnosis? Might the death of one of these cells have an effect on subsequent development? This question is equally valid on the impact of cell loss upon cryopreservation (Cohen et al., 2007). Although these are very delicate questions with very likely no easy answer, from the perspective of the mouse embryo, at least four things are to be considered. A tendency for a blastomere of some 4-cell stage embryos to contribute to a given region of the embryo has been documented. Second, the blastomeres in the 4-cell stage show clear differences not only in the levels of histone methylation, but also in their transcriptional activiy when they develop without being perturbed. However (third), the cells undergo a redirection of cell fate when a histone methyltransferase is overexpressed, indicating that they can readapt. Finally, the environment in which cells develop seems to be crucial for completing development and somehow the remaining cells in the embryo could compensate provided they are somehow different from each other. It is also important to note that a 4-cell stage mouse embryo might correspond to a very different developmental stage than a 4-cell embryo in other mammalian species, as illustrated by the differences in the onset of genome activation between them. Indeed, PGD is most often performed at the 8-cell stage and some reports document a better rate of development when 6-to- 9-cell stage embryos are diagnosed, as opposed to 3-to- 4-cell stage embryos (Wang et al., 2007). Moreover, the effects of in vitro fertilization procedures and culture on embryonic development are also extremely important, as they have been shown to alter epigenetic information in the mouse (Li et al., 2005). As a final consideration, I would like to leave the reader with an open perspective of some ongoing questions in the field. Although there are strong indications that epigenetic mechanisms are involved in cell fate determination, we are still far from establishing a direct link between an epigenetic mark(s) and the derivation of a particular cell lineage in the embryo. Much work is still to be done to determine how these mechanisms are set in play upon fertilization and how they are transmitted during subsequent cleavage stages. Also, what other epigenetic marks contribute to the inheritability of cell fate decisions? How do these marks relate to and influence each other? Are different lineage-specific genes marked by a different combination(s) of epigenetic marks? Do the marking of these genes occur at different stages of development? It is also tempting to expand these notions into the stem cell field and question whether these mechanisms would also underlie the intrinsic self renewal ability of adult stem cells and their potential to differentiate into other cell types. The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future. Funding The author acknowledges support from the PNRRE/INSERM.