Gliadin Challenge Research Articles

High Fat Diet-Wheat Gliadin Interaction and its Implication for Obesity and Celiac Disease Onset: In Vivo Studies.

The intestinal immune system plays a crucial role in obesity and insulin resistance. An altered intestinal immunity is associated with changes to the gut microbiota, barrier function, and tolerance to luminal antigens. Lipid metabolism and its unbalance can also contribute to acute and chronic inflammation in different conditions. In celiac disease (CD), the serum phospholipid profile in infants who developed CD is dramatically different when compared to that of infants at risk of CD not developing the disease. In a mouse model of gluten sensitivity, oral wheat gliadin challenge in connection with inhibition of the metabolism of arachidonic acid, an omega-6 polyunsaturated fatty acid, specifically induces the enteropathy. Recent evidence suggests that gluten may play a role also for development of life-style related diseases in populations on a high fat diet (HFD). However, the mechanisms behind these effects are not yet understood. Exploratory studies in mice feed HFD show that wheat gliadin consumption affects glucose and lipid metabolic homeostasis, alters the gut microbiota, and the immune cell profile in liver.

In vitro differentiation of macrophages from peripheral blood cells of celiac patients.

Macrophages have both a protective and pathological role in many autoimmune and inflammatory diseases. Macrophages phenotype is regulated by the environment that affects their polarization toward a pro- or anti-inflammatory phenotype. We describe a protocol for in vitro differentiation of macrophages from blood peripheral monocytes, that may be adopted to study different pathologies. Here, we are interested to study the phenotype of macrophages differentiated from patients affected by acute celiac disease (CD) or subjects following a gluten free diet (GFD), after in vitro gliadin challenge. We assess the pro-inflammatory phenotype of these macrophages by cytokines quantization on the cell supernatant. Moreover, our proposed protocol allows the preparation of total RNA to analyze the expression profile of many genes.

Effect of Gliadin Stimulation on HLA-DQ2.5 Gene Expression in Macrophages from Adult Celiac Disease Patients.

Macrophages play an important role in the pathogenesis of celiac disease (CD) because they are involved in both inflammatory reaction and antigen presentation. We analyzed the expression of CD-associated HLA-DQ2.5 risk alleles on macrophages isolated by two cohorts of adult patients, from the U.S. and Italy, at different stages of disease and with different genotypes. After isolating and differentiating macrophages from PBMC, we assessed the HLA genotype and quantified the HLA-DQ2.5 mRNAs by qPCR, before and after gliadin stimulation. The results confirmed the differences in expression between DQA1*05:01 and DQB1*02:01 predisposing alleles and the non-CD associated alleles, as previously shown on other types of APCs. The gliadin challenge confirmed the differentiation of macrophages toward a proinflammatory phenotype, but above all, it triggered an increase of DQA1*05:01 mRNA, as well as a decrease of the DQB1*02:01 transcript. Furthermore, we observed a decrease in the DRB1 genes expression and a downregulation of the CIITA transactivator. In conclusion, our findings provide new evidences on the non-coordinated regulation of celiac disease DQ2.5 risk genes and support the hypothesis that gliadin could interfere in the three-dimensional arrangement of chromatin at the HLA locus.

L-Arabinose Attenuates Gliadin-Induced Food Allergy via Regulation of Th1/Th2 Balance and Upregulation of Regulatory T Cells in Mice.

Gliadins are the main cause of wheat allergies, and the prevalence of gliadin allergy has increased in many countries. l-Arabinose, a kind of plant-specific five-carbon aldose, possesses beneficial effects on food allergy to gliadins. This study investigated the antiallergic activities and underlying mechanisms of l-arabinose in a wheat gliadin-sensitized mouse model. BALB/c mice were sensitized to gliadin by intraperitoneal injections with gliadin followed by being given a gliadin challenge. l-arabinose-treated mice exhibited a marked reduction in the productions of total immunoglobulin E (IgE), gliadin-specific IgE, gliadin-specific IgG1, and histamine, with an increase in IgG2a level as compared with gliadin-sensitized mice. Beside that, a significant decrease in Th2-related cytokine level, IL-4, and an increase in Th1-related cytokine level, IFN-γ, in the serum and splenocytes were observed after treatment with l-arabinose. l-Arabinose treatment also improved the imbalance of Th1/Th2 immune response on the basis of the expression levels of related cytokines and key transcription factors in the small intestine and spleen of sensitized mice. In addition, gliadin-induced intestinal barrier impairment was blocked by l-arabinose treatment via regulation of TJ proteins and suppression of p38 MAPK and p65 NF-κB inflammation signaling pathways. Notably, the results confirmed that l-arabinose treatment increased CD4+ Foxp3+ T cell populations and Treg-related factors associated with increased expression of IL-2 and activation of STAT5 in gliadin-sensitized mice. In conclusion, l-arabinose attenuated the gliadin-induced allergic symptoms via maintenance of Th1/Th2 immune balance and regulation of Treg cells in a gliadin-induced mouse model, suggesting l-arabinose could be used as a promising agent to alleviate gliadin allergy.

Induction of Oral Tolerance by Pepsin-Digested Gliadin Retaining T Cell Reactivity in a Mouse Model of Wheat Allergy

Background: Wheat is known as the most widely consumed food all over the world. Although many types of wheat allergy have been recognized, their treatment still has a long way to go due to the complex pathogenesis. Oral immunotherapy (OIT) is under investigation for the treatment of wheat allergies. Previous studies have demonstrated that OIT using intact wheat allergens can induce tolerance, but is accompanied by a high risk of anaphylactic reactions. Objectives: Our objective was to prepare modified wheat allergens with hypoallergenic and tolerance-inducing properties to reduce adverse effects during immunotherapy. Methods: Wheat gliadin was degraded by hydrolysis with pepsin and trypsin, and then the hydrolysate was deamidated with hydrochloric acid. The IgE-binding capacity and T cell reactivity of the degraded gliadins were evaluated in vitro. Pepsin-digested gliadin (peptic-GLI) was applied in a mouse model to investigate whether it would induce oral tolerance. Results: Degradation with pepsin decreased IgE-binding capacity and maintained T cell reactivity. Oral administration of peptic-GLI to mice before sensitization and challenge with gliadin could significantly suppress the production of IgE, IgG1, and type 2 T helper cytokines. Moreover, the development of anaphylactic reactions and allergic responses of the small intestine induced by gliadin challenge were inhibited by oral administration of peptic-GLI. Conclusions: The findings of this study indicate that peptic-GLI with low allergenicity and potential for tolerance induction may become useful in wheat immunotherapy with less adverse effects.

Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease.

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Coregulation and modulation of NF B-related genes in celiac disease: uncovered aspects of gut mucosal inflammation

It is known that the NFκB route is constitutively upregulated in celiac disease (CD), an immune-mediated disorder of the gut caused by intolerance to ingested gluten. Our aim was to scrutinize the expression patterns of several of the most biologically relevant components of the NFκB route in intestinal biopsies from active and treated patients and after in vitro gliadin challenge, and to assess normalization of the expression using an inhibitor of the MALT1 paracaspase. The expression of 93 NFκB genes was measured by RT-PCR in a set of uncultured active and treated CD and control biopsies, and in cultured biopsy series challenged with gliadin, the NFκB modulator, both compounds and none. Methylation of eight genes involved in NFκB signaling was analyzed by conventional pyrosequencing. Groups were compared and Pearson's correlation matrixes were constructed to check for coexpression and co-methylation. Our results confirm the upregulation of the NFκB pathway and show that constitutively altered genes usually belong to the core of the pathway and have central roles, whereas genes overexpressed only in active CD are more peripheral. Additionally, this is the first work to detect methylation level changes in celiac intestinal mucosa. Coexpression is very common in controls, whereas gliadin challenge and especially chronic inflammation present in untreated CD result in the disruption of the regulatory equilibrium. In contrast, co-methylation occurs more often in active CD. Importantly, NFκB modulation partially restores coregulation, opening the door to future therapeutic possibilities and targets.

THEMIS and PTPRK in celiac intestinal mucosa: coexpression in disease and after in vitro gliadin challenge

Celiac disease (CD) is an immune mediated, polygenic disorder, where HLA-DQ2/DQ8 alleles contribute around 35% to genetic risk, but several other genes are also involved. Genome-wide association studies (GWASs) and the more recent immunochip genotyping projects have fine-mapped 39 regions of genetic susceptibility to the disease, most of which harbor candidate genes that could participate in this disease process. We focused our attention to the GWAS peak on chr6: 127.99-128.38 Mb, a region including two genes, thymocyte-expressed molecule involved in selection (THEMIS) and protein tyrosine phosphatase, receptor type, kappa (PTPRK), both of which have immune-related functions. The aim of this work was to evaluate the expression levels of these two genes in duodenal mucosa of active and treated CD patients and in controls, and to determine whether SNPs (rs802734, rs55743914, rs72975916, rs10484718 and rs9491896) associated with CD have any influence on gene expression. THEMIS showed higher expression in active CD compared with treated patients and controls, whereas PTPRK showed lower expression. Our study confirmed the association of this region with CD in our population, but only the genotype of rs802734 showed some influence in the expression of THEMIS. On the other hand, we found a significant positive correlation between THEMIS and PTPRK mRNA levels in CD patients but not in controls. Our results suggest a possible role for both candidate genes in CD pathogenesis and the existence of complex, regulatory relationships that reside in the vast non-coding, functional intergenic regions of the genome. Further investigation is needed to clarify the impact of the disease-associated SNPs on gene function.

Gliadin Does Not Induce Mucosal Inflammation or Basophil Activation in Patients With Nonceliac Gluten Sensitivity

Nonceliac gluten-sensitive (NCGS) patients report intestinal and extra-intestinal symptoms shortly after ingesting gluten; these symptoms disappear on gluten-free diets, although these patients have no serologic markers of celiac disease or intestinal damage. In fact, there is no evidence for mucosal or serologic modifications in those individuals. We investigated immunologic responses of duodenal mucosa samples and peripheral blood basophils, isolated from NCGS patients, after exposure to gliadin. Participants underwent a complete clinical evaluation to exclude celiac disease while on a gluten-containing diet, a skin prick test to exclude wheat allergy, and upper endoscopy (n= 119) at 2 tertiary medical centers in Italy. Patients were considered to have NCGS based on their symptoms and the current definition of the disorder. Subjects were assigned to the following groups: patients with celiac disease on gluten-free diets (n= 34), untreated patients with celiac disease (n= 35), patients with NCGS (n= 16), or controls (n= 34). Duodenal biopsy samples collected during endoscopy were incubated with gliadin peptides, and levels of inflammatory markers were assessed. Peripheral blood basophils were extracted and incubated with gliadin peptides or a mix of wheat proteins; activation was assessed based on levels of CD203c, CD63, and CD45. Duodenal mucosa samples collected from 69 patients with celiac disease showed markers of inflammation after incubation with gliadin. Some, but not all, markers of inflammation were detected weakly in biopsy samples from 3 controls and 3 NCGS patients (P= .00 for all markers). There were no significant increases in the levels of CD63 and CD203c in NCGS patients. Unlike the duodenal mucosa from patients with celiac disease, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin. The invitro gliadin challenge therefore should not beused to diagnose NCGS.

Sa1289 Non Celiac Gluten Sensitivity: Lack of Response to In Vitro Gliadin Challenge and Basophils Activation Assay

In recent years it has become more frequent for physicians to come across types of gluten sensitivity other than celiac disease (CD). Regardless of the absence of any serological CD markers or intestinal damage, gluten sensitive (GS) patients often report intestinal and extraintestinal symptoms shortly after the ingestion of gluten and the disappearance of such symptoms on a gluten free diet (GFD). Regrettably, at present, there is no evidence of any mucosal or serological modifications in GS patients, or that gluten is really the trigger factor in gluten sensitivity. We aimed at evaluating the impact of gliadin challenge on two experimental settings, i.e. in vitro gliadin challenge of duodenal mucosa and peripheral blood basophils in GS patients compared to CD patients and healthy controls. We consecutively enrolled patients referred to tertiary centers for food intolerance and celiac disease. Participants underwent a complete clinical screening in order to rule out CD (serum a-tTG, EMA, total IgA and HLA status, where appropriate), an interview for evaluation of diet and symptoms, a skin prick test to exclude wheat allergy, upper endoscopy for duodenal biopsies used for both histological assessment and for the in vitro evaluation of the gliadin-induced mucosal expression of early and late inflammatory markers: PY99, epithelial HLA-DR, ICAM1, crypt HLA-DR, CD3, CD25 and CD69. In addition, a basophils activation assay, in which patients' basophils were challenged with a gliadin extract, was performed. Basophils were analyzed for the expression of hematopoietic membrane antigens CD203c, CD63 and CD45 by flow cytometry looking at differences in antigen expression. 119 subjects were screened for CD and GS and, according to the results, four groups were obtained: GS patients (no.16), CD on gluten free diet (no.34), CD on gluten containing diet (no.35) and healthy controls (no.34). As for the in vitro gliadin challenge, all CD patients, regardless of their dietetic status, showed a clear mucosal activation (increased immunofluorescence intensity both for early and delayed inflammation markers) when stimulated with gliadin, while only 3 controls and 3 GS patients showed an weak response for some, not all, inflammation markers. Also, there were no significant differences in the skin prick test results or in the level of CD63 and CD203c basophils expression, used as a marker of cell activation, among the groups. The present study shows that in GS patients, gliadin challenge test did not show any significant modifications in the expression of those mucosal inflammation markers found increased in CD patients. Therefore, this tool should not be used as a diagnostic instrument in case of GS. Moreover, the lack of differences in basophils activation and skin prick test seems to exclude this form of cellular sensitivity in our GS series also.

Difficult diagnosis of celiac disease: diagnostic accuracy and utility of chromo-zoom endoscopy

Chromo-zoom endoscopy has been demonstrated to be valuable in assessing the degree of intestinal villous atrophy in patients with suspected celiac disease. To evaluate the diagnostic accuracy of chromo-zoom endoscopy in patients with difficult diagnosis because of nonconcordant test results and/or the confounding of a gluten-free diet initiated before an appropriate diagnosis of celiac disease and to compare the findings to a recent reference standard, the in vitro gliadin challenge test. Prospective, case-control study. Tertiary-care referral hospital. Patients without celiac disease (negative control group, n = 9), patients with celiac disease (positive control group, n = 41), and patients with difficult diagnosis (n = 27). Chromo-endoscopy with indigo carmine and endoscopic zoom-magnification were performed. Duodenal fragments were collected for the in vitro gliadin challenge test. The area under the receiver operating characteristic curve (ROC) was used for statistical analyses on accuracy. Diagnostic accuracy of chromo-zoom endoscopy for detection of mucosal abnormalities in patients with difficult diagnosis. Chromo-zoom endoscopy had a high accuracy for celiac disease diagnosis in analyses on negative controls and positive controls (area under roc = 0.99). In the difficult diagnosis group, the accuracy of chromo-zoom endoscopy was lower (area under roc = 0.83), but it increased after exclusion of patients with celiac disease on gluten-free diet (area under roc = 0.88). There was a 4% failure rate in the ability to cultivate biopsies. Also, the study was done at an academic medical center. Chromo-zoom endoscopy has high accuracy for cases of difficult diagnosis of celiac disease but only in untreated patients with celiac disease.

In Vitro Gliadin Challenge: Diagnostic Accuracy and Utility for the Difficult Diagnosis of Celiac Disease

Diagnosis of celiac disease is difficult when treatment with gluten-free diet (GFD) is started before diagnosis and/or when the results of tests are inconsistent. The objective of this study was to evaluate the in vitro gliadin challenge. The study cohort included patients without celiac disease (negative controls, n=57), patients with celiac disease (positive controls, n=166 untreated and n=55 on GFD), and patients with difficult diagnosis (n=59). All patients underwent endoscopy for collection of duodenal samples, which served for the diagnosis of celiac disease and for the in vitro evaluation of the gliadin-induced mucosal expression of seven inflammatory markers: PY99, ICAM-1 (intercellular cell adhesion molecule), HLA-DR, CD3, CD25, CD69, and transglutaminase 2 IgA. Diagnostic work-up for celiac disease included the search of specific serum antibodies. Patients of the difficult diagnosis group were asked to stop GFD for repeated search of these antibodies under untreated conditions. The area under the receptor-operated curve (ROC) was used for statistical analyses on accuracy. HLA-DR had the highest accuracy for celiac disease diagnosis in analyses on negative controls and positive controls also excluding patients on GFD (area under ROC=0.99). Accuracy of test did not increase combining data of HLA-DR with data of other markers. Findings were similar in the 39 patients of the difficult diagnosis group undergoing the search celiac disease-specific antibodies under untreated conditions. The in vitro response of mucosal HLA-DR to gliadin is an accurate tool for the diagnosis of celiac disease also in patients with difficult diagnosis.

Influence of Bifidobacterium longum CECT 7347 and Gliadin Peptides on Intestinal Epithelial Cell Proteome

Celiac disease is an enteropathy caused by an abnormal immune response to cereal gluten proteins (gliadin). To unravel the possible role of the interactions between gliadin peptides and specific intestinal bacteria, the response of intestinal epithelial (Caco-2) cells to gliadin subjected to gastrointestinal digestion in the presence or absence of Bifidobacterium longum CECT 7347 has been studied. Changes in the proteome of Caco-2 cells were determined by 2DE and MALDI-TOF. Gliadins digested without B. longum altered the expression of a higher number of proteins than in the presence of the bacterium (21 versus 9), and these proteins were involved in disorganization of cell cytoskeleton, inflammation, and apoptosis. Gliadins digested in the presence of the bacterium influenced the production of proteins involved in calcium homeostasis and cell survival and function. Therefore, B. longum CECT 7347 might ameliorate gliadin toxicity and modify the responses of intestinal epithelial cells to the gliadin challenge.

Altered expression of the lymphocyte activation antigen CD30 in active celiac disease

Interleukin (IL)-15 and CD30 may be associated with the ongoing intestinal immunologic activation in celiac disease (CD). We studied duodenal biopsies and blood samples of patients with active CD (Cel) and controls in order to determine the regulatory role proposed for CD30+ T cells in this Th1-driven disease and the potential influences of IL-15 on CD30 expression. We detected that a CD30+ T-cell subpopulation persists longer in Cel after a 5 day incubation with anti-CD3 antibody than in controls (p = 0.0063). CD30 upregulation by IL-15 in T blasts was greater in Cel than in controls (p = 0.0062). At the mucosal compartment, the CD30 antigen was examined by immunohistochemistry and quantified on isolated lamina propria (LP) and epithelial T cells by flow cytometry. For Cel and controls, similar mean percentages of CD3+CD30+ intraepithelial T cells (5.88 vs. 5.51, p = ns) and LP T cells (7.38 vs. 7.49, p = ns) were observed at baseline and after in vitro gliadin challenge of duodenal biopsy samples. Our study demonstrates the occurrence of potentially important alterations of the immune response at the peripheral compartment. Our findings also allow us to speculate that a negative effect of soluble mediators at the mucosal compartment might counteract the latent influence of IL-15 on CD30 expression precluding a more severe course of active CD.

Efficacy of budesonide therapy in the early phase of treatment of adult coeliac disease patients with malabsorption: An in vivo/in vitro pilot study

1. Budesonide is a glucocorticosteroid with a local anti-inflammatory effect. Coeliac disease is an immune-mediated disease caused by gluten ingestion in intolerant patients. The aim of the present study was to investigate the efficacy of budesonide in malabsorptive coeliac patients and its effect in an in vitro gliadin challenge. 2. Twenty coeliac patients with malabsorption were enrolled in the present study and were randomly assigned to one of two 4 week treatments: (i) a gluten-free diet alone; or (ii) a gluten-free diet plus 6 mg budesonide daily. At the end of 4 weeks treatment, all patients underwent clinical evaluation, laboratory tests and self-evaluation of well-being using a visual analogue scale. Intestinal biopsies from five coeliac patients (selected randomly) and four non-coeliac disease controls who underwent upper endoscopy for intestinal bleeding were challenged with gliadin (0.5 mg/mL) and budesonide (10-30 microg/mL) for 3 and 24 h. Biopsies were tested by immunohistochemistry and immunofluorescence for known markers of inflammation. 3. Treatment of patients with 6 mg budesonide daily for 4 weeks resulted in increased bodyweight, a decreased number of evacuations and decreased stool weight compared with patients on a gluten-free diet alone for 4 weeks. Well-being scores were higher in patients treated with both a gluten-free diet and budesonide compared with those receiving a gluten-free diet alone. 4. In vitro studies showed that budesonide reduced epithelial tyrosine phosphorylation and expression of histocompatibility leucocyte antigen complex DR (HLA-DR) elicited by gliadin-derived peptides. In addition, the expression of cyclo-oxygenase (COX)-2 and intercellular adhesion molecule (ICAM)-1 in the lamina propria was reduced in patients treated with both gliadin and budesonide compared with patients treated with gliadin alone. Budesonide alone decreased HLA-DR in crypt enterocytes, as well as ICAM-1 and COX-2 expression in the lamina propria of biopsy specimen of coeliac patients. Budesonide had no effect in control samples. 5. In conclusion, the results of the present study indicate that budesonide shows efficacy in the treatment of symptoms in adult coeliac patients with overt malabsorption. The mechanism underlying the effects of budesonide in reducing symptoms was elucidated by in vitro studies involving a gliadin challenge.

Increased expression of mucosal addressin cell adhesion molecule 1 in the duodenum of patients with active celiac disease is associated with depletion of integrin α4β7-positive T cells in blood

Mucosal addressin cell adhesion molecule 1, expressed on gut endothelial cells, in conjunction with integrin alpha(4)beta(7), expressed on lymphocytes, is critical in lymphocyte homing to the gut. The mucosal addressin cell adhesion molecule 1/integrin alpha(4)beta(7) pathway is involved in the pathogenesis of chronic intestinal inflammation by recruiting lymphocytes into inflamed gut. We explored the duodenal expression of mucosal addressin cell adhesion molecule 1 and the peripheral T-cell expression of integrin alpha(4)beta(7) in patients with celiac disease. Duodenal biopsies and a peripheral blood sample were collected from 15 celiac patients, before and after 12 months of gluten-free diet, and from 12 control subjects. Treated celiac biopsies were cultured with peptic-tryptic digest of gliadin and/or an anti-interferon alpha neutralizing antibody. Mucosal addressin cell adhesion molecule 1 was determined by confocal immunofluorescence microscopy and immunoblotting. Integrin beta(7)-positive T cells were analyzed by flow cytometry. Mucosal addressin cell adhesion molecule 1 expression was significantly higher in active celiac disease than in normal mucosa. After gluten-free diet, a dramatic reduction of mucosal addressin cell adhesion molecule 1 was also observed. No difference was seen between patients with celiac disease after treatment and controls. Ex vivo peptic-tryptic digest of gliadin challenge induced a marked increase of mucosal addressin cell adhesion molecule 1 expression. Blocking interferon alpha inhibited the peptic-tryptic digest of gliadin-induced mucosal addressin cell adhesion molecule 1 overexpression. The percentage of circulating beta(7)-positive T cells was significantly lower in untreated celiac disease in comparison to controls but normalized after gluten-free diet. Mucosal addressin cell adhesion molecule 1 is strongly up-regulated in active celiac disease dependent on interferon alpha and is associated with peripheral depletion of integrin alpha(4)beta(7)-expressing T cells. We conclude that mucosal addressin cell adhesion molecule 1 may represent an important determinant for the generation of mucosal damage in celiac disease.

Distribution, Proliferation, and Function of Paneth Cells in Uncomplicated and Complicated Adult Celiac Disease

Paneth cells, granulated epithelial cells located at the base of small bowel crypts, have a crucial role in innate immunity. Because controversies remain concerning Paneth cell numbers and function in celiac disease (CD), we quantified Paneth cells and human alpha-defensin (HD)-5 and HD-6 in 28 patients with uncomplicated CD, 8 patients with complicated CD (3 with ulcerative jejunoileitis, 2 with refractory sprue, and 3 with enteropathy-associated T-cell lymphoma), and 14 control subjects. Paneth cell numbers and proliferation did not differ in uncomplicated untreated and treated CD and control cases. However, the number of Paneth cells was significantly reduced in complicated CD. Mucosal HD-5 and HD-6 were comparable in uncomplicated untreated and treated CD and control cases. Ex vivo gliadin challenge of treated CD biopsy specimens had no effect on mucosal HD-5 and HD-6 transcripts. Paneth cell numbers and alpha-defensins are unchanged in the mucosa in uncomplicated CD. Further studies are needed to clarify the implications of reduction of numbers of Paneth cells in complicated CD.

Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits

BackgroundIn celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase 2 (TG2). These autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. In addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. Since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal TG2-specific IgA deposits in the cultured biopsy samples.ResultsIn the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial CD25+ cells. However, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal TG2-specific IgA autoantibody deposits were still present. Furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge.ConclusionOur results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal IgA deposits present. In addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method.

Enteric Glial-Derived S100B Protein Stimulates Nitric Oxide Production in Celiac Disease

Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production.

Unexpected Role of Surface Transglutaminase Type II in Celiac Disease

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. 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, palpha-9, and deamidated palpha-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. 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. 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.