Unexpected Role of Surface Transglutaminase Type II in Celiac Disease
2005; Elsevier BV; Volume: 129; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.07.054
ISSN1528-0012
AutoresLuigi Maiuri, Carolina Ciacci, Ida Ricciardelli, Loredana Vacca, Valeria Raia, Antonio Rispo, Martin Griffin, Thomas B. Issekutz, Sonia Quaratino, Marco Londei,
Tópico(s)Autoimmune Bullous Skin Diseases
ResumoBackground & Aims: In celiac disease (CD), transglutaminase type II (TG2) has 2 fundamental roles: (1) as the autoantigen recognized by highly specific autoantibodies and (2) the modifier of pathogenic gliadin T-cell epitopes. It follows that inhibition of TG2 might represent an attractive strategy to curb the toxic action of gliadin. Here we studied the validity of this strategy using the organ culture approach. Methods: Duodenal biopsy specimens from 30 treated patients with CD, 33 untreated patients with CD, and 24 controls were cultured with or without gliadin peptides p31-43, pα-9, and deamidated pα-9 for 20 minutes, 3 hours, and 24 hours. In 31 patients with CD and 16 controls, TG2 inhibitor R283 or anti-TG CUB 7402 or anti-surface TG2 (6B9) mAbs were used in cultures. T84 cells were also cultured with or without peptides with or without TG inhibitors. Mucosal modifications after culture were assessed by immunofluorescence, in situ detection of TG activity, confocal microscopy, and fluorescence-activated cell sorter analysis. Results: The enzymatic inhibition of TG2 only controlled gliadin-specific T-cell activation. The binding of surface TG2 contained gliadin-specific T-cell activation and p31-43–induced actin rearrangement, epithelial phosphorylation, and apoptosis, both in organ cultures and T84 cells. Conclusions: These data indicate a novel and unexpected biological role for surface TG2 in the pathogenesis of CD suggesting a third role for TG2 in CD. These results have a specific impact for celiac disease, with wider implications indicating a novel biologic function of TG2 with possible repercussions in other diseases. Background & Aims: In celiac disease (CD), transglutaminase type II (TG2) has 2 fundamental roles: (1) as the autoantigen recognized by highly specific autoantibodies and (2) the modifier of pathogenic gliadin T-cell epitopes. It follows that inhibition of TG2 might represent an attractive strategy to curb the toxic action of gliadin. Here we studied the validity of this strategy using the organ culture approach. Methods: Duodenal biopsy specimens from 30 treated patients with CD, 33 untreated patients with CD, and 24 controls were cultured with or without gliadin peptides p31-43, pα-9, and deamidated pα-9 for 20 minutes, 3 hours, and 24 hours. In 31 patients with CD and 16 controls, TG2 inhibitor R283 or anti-TG CUB 7402 or anti-surface TG2 (6B9) mAbs were used in cultures. T84 cells were also cultured with or without peptides with or without TG inhibitors. Mucosal modifications after culture were assessed by immunofluorescence, in situ detection of TG activity, confocal microscopy, and fluorescence-activated cell sorter analysis. Results: The enzymatic inhibition of TG2 only controlled gliadin-specific T-cell activation. The binding of surface TG2 contained gliadin-specific T-cell activation and p31-43–induced actin rearrangement, epithelial phosphorylation, and apoptosis, both in organ cultures and T84 cells. Conclusions: These data indicate a novel and unexpected biological role for surface TG2 in the pathogenesis of CD suggesting a third role for TG2 in CD. These results have a specific impact for celiac disease, with wider implications indicating a novel biologic function of TG2 with possible repercussions in other diseases. Transglutaminase type II (TG2) has emerged as an essential player in celiac disease. It is the main1Dieterich W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Schuppan D. 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Maxwell M.D. Localization of cellular transglutaminase on the extracellular matrix after wounding characteristics of the matrix bound enzyme.J Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (147) Google Scholar intracellularly, primarily in the cytoplasm but also within the nucleus,14Lesort M. Attanavanich K. Zhang J. Johnson G.V. Distinct nuclear localization and activity of tissue transglutaminase.J Biol Chem. 1998; 273: 11991-11994Crossref PubMed Scopus (157) Google Scholar and on the cellular surface.15Martinez J. Chalupowicz D.G. Roush R.K. Sheth A. Barsigian C. Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers.Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (113) Google Scholar All these different localizations are obviously characterized by diverse functions of TG2. Remarkably, as in the case of surface expression, enzymatic activity is not always necessary for the biologic activity of TG2.16Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.J Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (432) Google Scholar, 17Akimov S.S. Belkin A.M. Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin a role in TGFbeta-dependent matrix deposition.J Cell Sci. 2001; 114: 2989-3000Crossref PubMed Google Scholar Studies performed among the others by our group18Maiuri L. Picarelli A. Boirivant M. Coletta S. Mazzilli M.C. De Vincenzi M. Londei M. Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.Gastroenterology. 1996; 110: 1368-1378Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar have also pointed to an alternative but complementary activation by gliadin peptides of the innate immune system. In the present study, we explored whether TG2 is induced by gliadin challenge and whether the same peptide that we reported to provoke activation of the innate immune system modulated TG2 induction/up-regulation. We further tested whether inhibition of TG activity, by means of a highly specific pharmacologic site-directed inhibitor20Freund K.F. Doshi K.P. Gaul S.L. Claremon D.A. Remy D.C. Baldwin J.J. Pitzenberger S.M. Stern A.M. 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Unexpectedly, surface transglutaminase type II (sTG2) binding further controlled all the "nonadaptive" but disease-specific modifications induced by gliadin "innate" peptide challenge. These results open a new scenario in the pathogenic picture of celiac disease and also indicate a novel and unforeseen biologic role for sTG2. Duodenal multiple endoscopic biopsies were performed for diagnostic purposes in 30 patients with celiac disease on a gluten-free diet (TCD) (mean age, 37.0 years; range, 21–55 years), 33 untreated patients with celiac disease (mean age, 33.4 years; range, 21–55 years), and 24 control patients without celiac disease (mean age, 34.7 years; range, 20–52 years) affected by esophagitis (13 patients) or irritable bowel syndrome (11 patients). Informed consent was obtained from all individuals, and the study was performed according to the Ethical Committee of the University of Naples (no. 71/01 protocol). One specimen from each patient was used for diagnosis; the other samples were cultured in vitro as described in the following text. Only HLA-DQ2–positive patients were studied because HLA-DQ2–restricted epitopes were studied.6Arentz-Hansen H. Korner R. Molberg O. Quarsten H. Vader W. Kooy Y.M. Lundin K.E. Koning F. Roepstorff P. Sollid L.M. McAdam S.N. The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.J Exp Med. 2000; 191: 603-612Crossref PubMed Scopus (571) Google Scholar Peptides pα-9(57–68) and pα-2(62–75) and their deamidated forms, p31-43, and human thyroid peroxidase 535–551 as previously described19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar were synthesized using Fmoc chemistry22Quaratino S. Duddy L.P. Londei M. Fully competent dendritic cells as inducers of T cell anergy in autoimmunity.Proc Natl Acad Sci U S A. 2000; 97: 10911-10916Crossref PubMed Scopus (40) Google Scholar and used at 20 μg/mL. Purity was determined by high-performance liquid chromatography purification and mass spectrometry. Duodenal biopsy specimens were cultured in vitro for 20 minutes, 3 hours, or 24 hours as previously reported.18Maiuri L. Picarelli A. Boirivant M. Coletta S. Mazzilli M.C. De Vincenzi M. Londei M. Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.Gastroenterology. 1996; 110: 1368-1378Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Maiuri L. Ciacci C. Auricchio S. Brown V. Quaratino S. Londei M. Interleukin 15 mediates epithelial changes in celiac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Biopsy specimens from 9 patients with celiac disease and 6 controls were incubated for 3 hours with p31-43 and rechallenged for 21 hours with medium or pα-2, pα-9, the deamidated form of pα-9 (in 3 cases), and irrelevant thyroid peroxidase 535–551 as previously reported.19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar R283 (250 μmol/L),24Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts the active-state conformation of the enzyme does not affect cell motility but is important for its secretion.J Biol Chem. 2002; 277: 16567-16575Crossref PubMed Scopus (122) Google Scholar CUB 7402 mAb (NeoMarkers, Fremont, CA), 6B9 mAb,25Mohan K. Pinto D. Issekutz T.B. Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration.J Immunol. 2003; 171: 3179-3186Crossref PubMed Scopus (53) Google Scholar or isotype control immunoglobulin (Ig) G1 mAbs (Dako, Copenhagen, Denmark) were also added in cultures from 22 patients with celiac disease and 10 controls to the medium containing p31-43 and in cultures from 9 patients with celiac disease and 6 controls to the previously described sequential combination of peptides during the whole experiment. mAbs were used between 5 and 20 μg/mL with comparable results. Transwell cultures of T84 cells were performed as described elsewhere.26Abreu M.T. Palladino A.A. Arnold E.T. Kwon R.S. McRoberts J.A. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.Gastroenterology. 2000; 119: 1524-1536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Transepithelial resistance was evaluated using a Millipore Millicell-ERS voltmeter (Millipore, Watford, UK), and experiments were performed when transepithelial resistance was 2000 Ω × cm2.26Abreu M.T. Palladino A.A. Arnold E.T. Kwon R.S. McRoberts J.A. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.Gastroenterology. 2000; 119: 1524-1536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Peptides were used at 20 μg/mL mAbs (6B9, CUB 7402, or isotype control IgG1) at 5–20 μg/mL. For apoptosis studies, T84 cells were cultured with peptides p31-43, pα-9, or pα-2 for 24 hours. Neutralizing anti-Fas (M38; Immunex, Seattle, WA) (10 μg/mL), 6B9,27Maiuri L. Ciacci C. Raia V. Vacca L. Ricciardelli I. Raimondi F. Auricchio S. Quaratino S. Londei M. FAS engagement drives apoptosis of enterocytes of coeliac patients.Gut. 2001; 48: 418-424Crossref PubMed Scopus (76) Google Scholar CUB 7402, or isotype control were added 2 hours before p31-43. Four-micrometer frozen tissue sections of biopsy samples from each patient before and after in vitro cultures were fixed in acetone for 10 minutes. The sections were individually incubated for 2 hours at room temperature with the following antibodies: anti-phospho-tyrosine PY99 mAb (1:80, mouse IgG2b; Santa Cruz Biotechnology, Santa Cruz, CA), CUB 7402 (1:50, mouse IgG; NeoMarkers, Copenhagen, Denmark), 6B9 mAb (10 μg/mL), CD8 (1:100, mouse Ig; Dako), CD3 (1:100, mouse Ig; Dako), and CD25 (1:30, mouse Ig; Dako). The antigen expression and distribution were visualized by indirect immunofluorescence as previously described.18Maiuri L. Picarelli A. Boirivant M. Coletta S. Mazzilli M.C. De Vincenzi M. Londei M. Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.Gastroenterology. 1996; 110: 1368-1378Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Maiuri L. Ciacci C. Auricchio S. Brown V. Quaratino S. Londei M. Interleukin 15 mediates epithelial changes in celiac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Actin reorganization was visualized by 2 hours of incubation with 0.3 μmol/L fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma, Poole, England). Two-color immunofluorescence with anti-CD25 and anti-CD3 (1:50, rabbit polyclonal; Dako) antibodies were performed as previously described.19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar Isotype control mAb (IgG1 or IgG2), isotype-matched nonimmune Igs, or isotype-matched antibodies against inappropriate blood group antigens were used as control of specificity. At least 5 sections of each sample were tested with each primary antibody and the appropriate control, as previously reported.18Maiuri L. Picarelli A. Boirivant M. Coletta S. Mazzilli M.C. De Vincenzi M. Londei M. Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.Gastroenterology. 1996; 110: 1368-1378Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Maiuri L. Ciacci C. Auricchio S. Brown V. Quaratino S. Londei M. Interleukin 15 mediates epithelial changes in celiac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Morphometric analysis of data was performed as detailed in our previous reports18Maiuri L. Picarelli A. Boirivant M. Coletta S. Mazzilli M.C. De Vincenzi M. Londei M. Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.Gastroenterology. 1996; 110: 1368-1378Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 23Maiuri L. Ciacci C. Auricchio S. Brown V. Quaratino S. Londei M. Interleukin 15 mediates epithelial changes in celiac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 27Maiuri L. Ciacci C. Raia V. Vacca L. Ricciardelli I. Raimondi F. Auricchio S. Quaratino S. Londei M. FAS engagement drives apoptosis of enterocytes of coeliac patients.Gut. 2001; 48: 418-424Crossref PubMed Scopus (76) Google Scholar in a blinded manner by 2 independent observers unaware of the culture conditions. Cells were detached by 0.5 g/L trypsin and 0.2 g/L EDTA solution (Gibco, Paisley, Scotland), washed in phosphate-buffered saline, and incubated for 1 hour at 4°C with 6B9 mAb, CUB 7402 mAb, or isotype control mAb at 10 μg/mL, followed with an FITC-conjugated rabbit anti-mouse secondary antibody (1:20; Dako) for 30 minutes at 4°C. Cells were analyzed using a fluorescence-activated cell sorter (Beckman Coulter, High Wycombe, UK). The data were analyzed using WinMDI software (WinMD, San Francisco, CA). Treated cells were fixed in 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.5% Triton X-100, and stained by incubation with 0.3 μmol/L phalloidin-FITC (Sigma) for 30 minutes at room temperature. PY99 mAb followed by a second layer (anti-mouse Ig FITC labeled) was also used as described by Maiuri et al.19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar Immunofluorescence was visualized by confocal microscopy (original magnification 200×; Leica TCS SP2; Leica Wetzlar, Bensheim, Germany). Seven-micrometer unfixed sections were collected on coated slides, preincubated with TG assay buffer (965 μL of 100 mmol/L Tris-HCl, pH 7.4, 25 μL of 200 mmol/L CaCl2) for 15 minutes, and then preincubated with the same TG assay buffer added with 10 μL of 10 mmol/L biotinylated monodansylcadaverine (bio-MDC) (Molecular Probes, Leiden, The Netherlands) for 1 hour at room temperature. The reaction was stopped with 25 mmol/L EDTA for 5 minutes; the slides were then fixed in 4% paraformaldehyde for 10 minutes. The incorporation of labeled substrate was visualized by incubation with RPE-conjugated streptavidin (1:50; Dako) for 30 minutes. Control experiments included the omission of bio-MDC and replacement of 200 mmol/L CaCl2 with 200 mmol/L EDTA. Alternatively, consecutive tissue sections were incubated under the same experimental conditions in TG assay buffer with 100 μg/mL of biotinylated p31-43 or biotinylated pα-9, pα-2, or their deamidated forms instead of bio-MDC for 1 hour at room temperature and the incorporation of the labeled substrate was visualized as previously described. Blocking experiments were also performed by incubating tissue section with anti-TG2 CUB 7402 mAb (20 μg/mL), 6B9 mAb (20 μg/mL), or the active site inhibitor R283 (250 μg/mL) for 1 hour before incubation with the substrates (bio-MDC or biotinylated peptides) and detection of TG enzymatic activity. Data were analyzed under fluorescence examination by a Zeiss Axioxop 2 Plus microscope (Zeiss, Jena, Germany). The simultaneous detection of TG protein and enzymatic activity on tissue sections was performed by incubating unfixed sections with bio-MDC for 1 hour at room temperature, according to the previously described procedure, and then with anti-TG2 CUB 7402 mAb (1:50) or 6B9 mAb (10 μg/mL) for 1 hour at room temperature. This was followed by simultaneous incubation with R-phycoerythrin (RPE)-conjugated streptavidin (1:50; Dako) and FITC-conjugated rabbit anti-mouse Ig F(ab)2 (1:20; Dako) for 1 hour. DNA fragmentation on tissue sections was assayed in all patients by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling as previously reported.23Maiuri L. Ciacci C. Auricchio S. Brown V. Quaratino S. Londei M. Interleukin 15 mediates epithelial changes in celiac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Apoptosis was determined using the annexin V/propidium iodide distribution (TACS Annexin-V FITC Apoptosis Detection Kit; R&D Systems, Minneapolis, MN). Analysis was performed on a Beckman Coulter fluorescence-activated cell sorter, and data were analyzed using WinMDI software. RNA was extracted from bowel biopsy specimens that had been tissue cultured for 6 hours and had been stimulated as described in the results section. Reagents were used at the following concentrations: peptides, 20 μg/mL; 6B9, 10 μg/mL; and R283, 250 μmol/L. Biopsy specimens had been frozen in RNAlater (Ambion Europe, Cambridgeshire, England). The RNeasy Kit was used to perform the extraction (Qiagen Ltd, West Sussex, England). One microgram of total RNA was reverse transcribed using oligo dT (Roche Diagnostics Ltd, East Sussex, England) ribonuclease inhibitor SUPERase (Ambion Europe) and Omniscript RT Kit (Qiagen Ltd). Polymerase chain reaction was performed using Quantitect SYBR Green PCR kit (Qiagen Ltd). Interferon gamma primers were prepared according to Forsberg et al28Forsberg G. Hernell O. Melgar S. Israelsson A. Hammarstrom S. Hammarstrom M.L. Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease.Gastroenterology. 2002; 123: 667-678Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar: sense primer, AGG TCA TTC AGA TGT AGC GGA TAA; anti-sense, AAG GAG ACA ATT TGG CTC TGC; glyceraldehyde-3-phosphate dehydrogenase primers29Lehmann M.H. Weber J. Gastmann O. Sigusch H.H. Pseudogene-free amplification of human GAPDH cDNA.Biotechniques. 2002; 33 (769–770): 766PubMed Google Scholar: sense primer, AGC CAC ATC GCT CAG ACA C; anti-sense, GAG GCA TTG CTG ATG ATC TTG. For interferon gamma and glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction, 60°C and 57°C annealing temperatures were used, respectively. The data are expressed as fold of increase compared with biopsy specimens cultured in medium alone. The standard curve was constructed of triplicates, and all results were analyzed using the 2-ΔΔCT method.30Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (135837) Google Scholar The level of interferon gamma was calculated as the ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase in each sample. Samples cultured with peptides were compared with those challenged with medium and with those cultured in the presence of p31-43 added with the tested inhibitors. Because some groups of variables showed a skewness >1 and in some experiments the sample size was small, nonparametric tests (Wilcoxon's sign test) for paired observations were applied throughout the study. The effect of p31-43 on patients with celiac disease was compared with that observed in controls by unpaired tests. Statistical analysis was performed using the SPSS package (SPSS Inc, Chicago, IL). We first explored whether the gliadin peptide, able to induce an innate immune response,19Maiuri L. Ciacci C. Ricciardelli I. Vacca L. Raia V. Auricchio S. Picard J. Osman M. Quaratino S. Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar could induce mucosal TG2 expression in organ culture of celiac disease intestinal explants. In 18 tested TCD samples, the peptide 31-43 (
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