Fernando G. Chirdo

Celiac disease pathogenesis: the proinflammatory cytokine network.

In susceptible individuals, the adaptive response, mediated by the activation of antigen-specific T lymphocytes, drives a proinflammatory response, which ends in an immune-mediated enteropathy characterized by villous atrophy, crypt hyperplasia, and recruitment of intraepithelial lymphocytes. In addition, some gluten peptides are able to induce an innate immune response in intestinal mucosa. The molecular mechanisms and the cells involved in the initial stages of the gluten–intestinal mucosa interaction are poorly understood to date. There is evidence of a direct toxic effect of gluten peptides in several biological models. However, the failure to control the inflammatory response may be one of the factors underlying gluten intolerance in these individuals. The cytokine network involved in celiac disease is characterized by abundant interferon-γ in the intestinal mucosa. In addition, the production of interleukin (IL)-15, IL-18, and IL-21 is linked to gluten intake, which can drive the inflammatory response probably sustained by IL-18, IL-21, and perhaps IL-27 through STAT1 and STAT5 pathways, whereas neither IL-12 nor IL-23 plays a significant role in pathogenic mechanisms. Herein we describe the involvement of these activation pathways in the context of the pathogenesis of celiac disease.

Deamidated gliadin peptides form epitopes that transglutaminase antibodies recognize.

Objective: Deamidated gliadin peptides are efficient antigens in diagnostic tests for celiac disease, and results correlate better with transglutaminase 2–based assays than those with native gliadin. We investigated whether deamidated gliadin antigens are structurally similar to transglutaminase 2 or could mimic transglutaminase epitopes. Patients and Methods: Serum samples from 74 celiac and 65 control patients, and 13 different transglutaminase 2–specific monoclonal mouse antibodies were investigated for their binding to commercially available deamidated gliadin peptides using enzyme-linked immunosorbent assay, competition studies, and molecular modelling. Results: The enzyme-linked immunosorbent assay with deamidated gliadin peptides had 100% sensitivity and 98.5% specificity in patients. Deamidated gliadin epitopes also were recognized by 3 transglutaminase-specific monoclonal antibodies, and antibodies affinity-purified with deamidated gliadin peptides from celiac patient sera reacted with transglutaminase but did not show endomysial binding. The binding of the monoclonal antibodies to deamidated gliadin was inhibited dose dependently by full-length recombinant human transglutaminase, its fragments containing the binding sites of these monoclonal antibodies, or by celiac patient antibodies. Deamidated gliadin peptides decreased the binding of transglutaminase-specific monoclonal antibodies to transglutaminase. Three different cross-reacting transglutaminase epitopes were found, of which 2 are located in the C-terminal domain and 1 is conformational. The binding of celiac serum samples to deamidated gliadin peptides could not be abolished by transglutaminase or by any of the transglutaminase-specific monoclonals, indicating that celiac sera also contain additional antibodies to gliadin epitopes different from transglutaminase. Conclusions: Certain deamidated gliadin–derived peptides and transglutaminase 2 epitopes have similar 3-dimensional appearance. This homology may contribute to the induction of transglutaminase autoantibodies by molecular mimicry.

Analysis of anti-gliadin antibodies by immunoblot analysis and enzyme-linked immunosorbent assay using gliadin fractions as antigens.

BACKGROUND Anti-gliadin antibody (AGA) determination has been widely used in the screening test to detect celiac patients in the general population and in risk groups. Serological assays present variable efficiency, probably caused by differences in the antigenic mixtures employed as antigen. The objective of this work is to evaluate the use of purified gliadin fractions in an enzyme-linked immunosorbent assay (ELISA) test. METHODS Anti-gliadin antibody reactivity was characterized in the sera of patients with celiac disease, and AGA levels were determined by immunoblot analysis using purified gliadin fractions after separation of wheat proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE and after indirect ELISA. Seven antigenic mixtures were tested: commercial gliadin, ethanolic wheat extract, and five fast protein liquid chromatography-purified fractions (omega-gliadins, two mixtures of alpha-/beta- and beta-/gamma-gliadins). Immunoblot analysis after A-PAGE separation showed that immunoglobulin (Ig)A reactivity was frequently more restricted than that of IgG. Serum IgA in 15 of 23 patients showed intense reactivity against omega-gliadins. RESULTS In seven cases, only omega-gliadins were detected. To compare the efficiency of ELISA tests, serum samples of 28 patients with celiac disease and 31 control subjects were tested against the seven gliadin fractions. Immunoglobulin G AGAs demonstrated similar levels against the different gliadin fractions, whereas IgA AGAs showed a heterogeneous reactivity that depended on the fraction tested. The lowest number of false-positive and false-negative results was obtained when the omega-gliadin fraction was used. Parameters for ELISA showed that the omega-gliadin fraction elicited the highest assay efficiency for determinations of both IgA and IgG AGAs. A good correlation was found between IgG and IgA anti-omega-gliadin and antiendomysial antibody determinations. Of the 28 biopsy-confirmed patients with celiac disease, 26 samples (23 positive and 3 negative) were found to have concordant results among the three determinations. CONCLUSIONS In this study, an intense and, in many cases, selective recognition of omega-gliadins was observed. Results suggest that a higher performance in AGA determination could be achieved using omega-gliadin as an antigen in indirect ELISA.

A galectin‐specific signature in the gut delineates Crohn's disease and ulcerative colitis from other human inflammatory intestinal disorders

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohns disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for β-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosal-associated galectins (Gal-1, -3, -4, -9) is dysregulated in inflamed tissues of IBD patients compared with non-inflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.

Analysis of Anti-Prolamin Monoclonal Antibody Reactivity Using Prolamin Fractions Purified by Preparative Electrophoresis

Coeliac disease (CD) is a gastrointestinal afliction triggered by the ingestion of prolamins from wheat, barley, rye and possibly oats. The only treatment for CD is a strict diet, free of the toxic components. Immunochemical methods are usually applied to certify foods aimed at coeliac patients. The characterization of a panel of four anti-prolamin monoclonal antibodies (MAbs) to be used to certify gluten-free products is described here. To this aim, purified gliadin, secalin and hordein fractions were obtained by preparative electrophoresis at acid pH. This procedure provides purified fractions not exposed to denaturing conditions. The specificity of the MAbs was tested by ELISA against purified fractions and ethanol extracts of wheat, barley, rye, oats, rice, maize, buckwheat, sorghum and soy. The four MAbs recognized only coeliac-toxic cereals. Each MAb reacted strongly with gliadins and showed differential reactivity against the different prolamin purified fractions. Some MAbs showed a broad pattern of recognition whereas others presented a more restricted one. The reactivity observed corresponded to structural homologies among gliadin, secalin and hordein fractions. It is remarkable that some fractions obtained by electrophoresis in the presence of sodium dodecylsulphate were not recognized by some MAbs, whereas the same components obtained by preparative A-PAGE showed high reactivity. This reinforces the suitability of the purification method employed in this study to isolate prolamin fractions. Using these purified prolamins, characterization of anti-prolamin MAb reactivity was achieved.

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.