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cope The ancient diploid Triticum monococcum is of special interest as a candidate low-toxic wheat species for celiac disease patients. Here, we investigated how an in vitro gastro-intestinal digestion, affected the immune toxic properties of gliadin from diploid compared to hexaploid wheat. Methods and results Gliadins from Triticum monococcum, and Triticum aestivum cultivars were digested using either a partial proteolysis with pepsin-chymotrypsin, or an extensive degradation that used gastrointestinal enzymes including the brush border membrane enzymes. The immune stimulatory properties of the digested samples were investigated on T-cell lines and jejunal biopsies from celiac disease patients. The T-cell response profile to the Triticum monococcum gliadin was comparable to that obtained with Triticum aestivum gliadin after the partial pepsin-chymotrypsin digestion. In contrast, the extensive gastrointestinal hydrolysis drastically reduced the immune stimulatory properties of Triticum monococcum gliadin. MS-based analysis showed that several Triticum monococcum peptides, including known T-cell epitopes, were degraded during the gastrointestinal treatment, whereas many of Triticum aestivum gliadin survived the gastrointestinal digestion. Conclusion The pattern of Triticum monococcum gliadin proteins is sufficiently different from those of common hexaploid wheat to determine a lower toxicity in celiac disease patients following in vitro simulation of human digestion.

Objectives To prospectively evaluate the effect of different dietary management strategies on the rate of acquisition of tolerance in children with cow’s milk allergy (CMA). Study design Otherwise healthy children (aged 1-12 months) diagnosed with CMA were prospectively evaluated. The study population was divided into 5 groups based upon the formula used for management: (1) extensively hydrolyzed casein formula ([EHCF], n = 55); (2) EHCF + Lactobacillus rhamnosus GG [LGG], n = 71); (3) hydrolyzed rice formula (RHF, n = 46); (4) soy formula (n = 55); and (5) amino acid based formula (n = 33). A food challenge was performed after 12 months to assess acquisition of tolerance. Results Two hundred sixty children were evaluated (167 male, 64.2%; age 5.92 months, 95% CI 5.48-6.37; body weight 6.66 kg, 95% CI 6.41-6.91; IgE-mediated CMA 111, 42.7%). The rate of children acquiring oral tolerance after 12 months was significantly higher (P < .05) in the groups receiving EHCF (43.6%) or EHCF + LGG (78.9%) compared with the other groups: RHF (32.6%), soy formula (23.6%), and amino acid based formula (18.2%). Binary regression analysis coefficient (B) revealed that the rate of patients acquiring tolerance at the end of the study was influenced by 2 factors: (1) IgE-mediated mechanism (B 2.05, OR 0.12, 95% CI 0.06-0.26; P < .001); and (2) formula choice, such that those receiving either EHCF (B 1.48, OR 4.41, 95% CI 1.44-13.48; P = .009) or EHCF + LGG (B 3.35, OR 28.62, 95% CI 8.72-93.93; P < .001). Conclusions EHCF accelerates tolerance acquisition in children with CMA if compared with other dietetic choices. This effect is augmented by LGG. (J Pediatr 2013;163:771-7).

Objective The aim of this study was to summarise the evidence from 2004 to September 2009 on the performance of laboratory-based serological and point of care (POC) tests for diagnosing coeliac disease (CD) in children using histology as reference standard. Patients and methods We searched MEDLINE and EMBASE for studies reporting on children for tests based on IgA and IgG anti-gliadin (AGA), endomysial (EmA), anti-transglutaminase-2 (TG2), and anti-deamidated gliadin peptides (DGP) antibodies or POC tests. For inclusion, histological analysis of duodenal biopsies and sensitivity and specificity for index tests had to be reported. Data were pooled and summary measures calculated for sensitivity, specificity, positive and negative likelihood ratios ("LR+", "LR-"), and diagnostic odds ratios (DOR). In case of elevated statistical heterogeneity, studies reaching 90% sensitivity or specificity were reported. Results A total of 2510 articles were reviewed; 16 entered meta-analysis, reporting on 3110 patients (1876 with CD, 1234 without CD). For IgA-EmA, sensitivity was ≥90% in 7/11 studies and pooled specificity 98.2%. For IgA-anti-TG2, 11/15 studies yielded sensitivities ≥90% and 13/15 specificities ≥90%. For IgA-DGP, sensitivity ranged between 80.7% and 95.1% (specificity 86.3%-93.1%); for IgG-DGP between 80.1% and 98.6% (specificity 86.0-96.9%). IgA-EmA had the highest pooled DOR (554) and LR+ (31.8) for a laboratory test, followed by IgA-anti-TG2, IgG-DGP, IgA-DGP and IgA-AGA. POC tests showed a pooled sensitivity of 96.4% for IgA-TG2 (specificity 97.7%). Conclusions IgA-EmA and IgA-anti-TG2 tests appear highly accurate to diagnose CD. IgG-anti-DGP tests may help in excluding CD. IgA-AGA and IgA-DGP tests show inferior accuracy. POC tests may achieve high accuracy in the hands of experienced readers, but IgA-anti-TG2/EmA were superior.

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the coexpression of CD3 and CD56 molecules (TR3-56), is reduced in individuals with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating TR3-56 cells is associated with reduced beta-cell function and with the presence of diabetic ketoacidosis. Since autoreactive CD8+ T cells mediate disruption of insulin-producing beta cells1–3, we demonstrate that TR3-56 cells can suppress CD8+ T cell functions in vitro by reducing the levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of TR3-56 cells are also altered in children with T1D. Together, our findings indicate that TR3-56 cells constitute a regulatory cell population that controls CD8+ effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D. Low numbers of circulating CD3+CD56+ regulatory T cells are associated with reduced beta-cell function and diabetic ketoacidosis in three separate cohorts of children with type 1 diabetes.

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt-CD), while others have a morphologically normal mucosa, despite the presence of CD-specific autoantibodies (potential-CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt- or potential-CD and in healthy controls. Density of TCRγδ+ T cells was found markedly enhanced in intestinal mucosa of children with overt-CD compared to potential-CD or controls. By contrast, very few IL4+ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4+ T cells were classical CD4+ T-helper cells (CD161- ), producing or not IFN-γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ+ T cells, and concomitant disappearance of IL4+ cells. These findings suggest that immunomodulatory mechanisms are active in potential-CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4+ and TCRγδ+ T cells as biomarkers of the different CD forms.

Objectives Use of deamidated gliadin peptide (DGP) test kits as adjunctive to Tissue-transglutaminase IgA (TTG-IgA) for the diagnosis of celiac disease (CD) has been a controversial issue. The objectives of our study were to evaluate the diagnostic performance of DGP antibodies compared to TTG-IgA and to evaluate the correlation between DGP-antibodies titers and degree of enteropathy. Methods We included children who underwent endoscopy and biopsies because of positivity of any of the serology tests in the "celiac profile" (TTG-IgA, DGP-IgA, and DGP-IgG), from 2012 to 2019. We divided children into clinically suspected cases of CD (Group 1) and asymptomatic cases screened because they were from a high risk-group (Group 2). Results Group 1 constituted 52 children and group 2 included 81 children [76 type1-diabetes (T1D)]. The sensitivity and positive predictive value (PPV) of DGP-IgG in group 1 (90%, 98%) and group 2 (91%, 85.5%) were comparable to TTG-IgA (98%, 92% in group 1; 100%, 80% in group 2). By adding DGP-IgG to TTG-IgA, the performance of TTG-IgA has improved marginally in group 1 (sensitivity 100%, PPV 92.3%). All cases with DGP-IgG titer 2 times ULN in group 1, and > 4 times ULN in group 2 had villous atrophy. All T1D patients with TTG IgA > 10 times ULN had villous atrophy. Conclusion DGP-IgG assay did not add to the performance of TTG-IgA. DGP-IgG titer correlated with enteropathy. The diagnosis of CD can be made in asymptomatic T1D child with TTG-IgA titer > 10 times ULN and positive endomyseal antibodies.

SCOPE: Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. We analysed gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge. METHODS AND RESULTS: Seventeen patients with CD (median age 13 years) consumed for 3-days sandwiches made with TM (cultivar Norberto-ID331, N = 11), or TA (cultivar Sagittario, N = 11) flours, corresponding to 12 gr of gluten/die. Immunostimulatory properties were assessed in blood by measuring the IFN-?-secreting T cells by EliSpot and the expression of inflammatory cytokines/receptors (IL-12A, IL-15, IL-18RAP, IFN-?) by qPCR. TA mobilized a remarkable number of gliadin specific, IFN-?-secreting T cells (p < 0.05), whilst no significant cell mobilization was induced by TM (p = ns). Similar results were obtained in response to five immunogenic peptides from ?-, ?-, and ?-gliadins, although with a large individual variability. An increased mRNA expression for IL-12A and IFN-? was detected in group eating TA compared to those consuming TM (p < 0.05). CONCLUSIONS: Although Triticum monococcum is a cereal not suitable for the diet of celiacs, we demonstrated that this diploid wheat elicits a reduced in vivo T-cell response compared to Triticum aestivum in celiac patients. This article is protected by copyright. All rights reserved.

Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

To analyze the presence of total IgA and anti-gliadin antibodies (AGA) in BM from CD mothers who follow a gluten-free diet (GFD) and from mothers on a normal gluten-containing diet (ND). 218 samples of mature milk were obtained at different months of lactation (1–6) from 83 mothers (2 or more samples per mother) from Italy (Naples), The Netherlands (Leiden) and Spain (Madrid, Valencia and Reus): 42 CD mothers on GFD for more than 2 years and 41 non-CD mothers on a ND. Whey samples were analyzed for AGA-IgA by an indirect homemade ELISA and for total IgA (g/L) by a commercial ELISA kit. AGA-IgA was detected in BM, both in mothers on a GFD and mothers on a ND. AGA-IgA levels in both groups of mothers, CD and non-CD, show the same trend towards decreasing slightly along the months of lactation (p = 0.91). A different trend is observed for total IgA levels, decreasing markedly in CD mothers from the first month of lactation onwards but remaining stable in non-CD mothers (p = 0.048). A statistically significant association was found between the means of total IgA and AGA-IgA (p

Celiac Disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides (e.g., A-gliadin P57-68) induce an adaptive Th1 pro-inflammatory response. Other gliadin peptides (e.g., A-gliadin P31-43) induce a stress/innate immune response involving interleukin 15 (IL15) and interferon α (IFN-α). In the present study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. This constitutive lesion might mediate the stress/innate immune response to gliadin, which can be one of the triggers of the gliadin-specific T-cell response., Giuliana Lania, Merlin Nanayakkara et al. show that cells from celiac disease patients have delays in early endocytic trafficking and an increase sensitivity to gliadin peptide P31-43. Delaying early vesicular trafficking in control cells induces a celiac-like cellular phenotype, highlighting a role for the early-recycling endosomal system in celiac disease.

Celiac disease (CD) is a common lifelong food intolerance triggered by dietary gluten affecting 1% of the general population. Gliadin-specific T-cell lines and T-cell clones obtained from intestinal biopsies have provided great support in the investigation of immuno-pathogenesis of CD. In the early 2000 a new in vivo, less invasive, approach was established aimed to evaluate the adaptive gliadin-specific T-cell response in peripheral blood of celiac patients on a gluten free diet. In fact, it has been demonstrated that three days of ingestion of wheat-containing food induces the mobilization of memory T lymphocytes reactive against gliadin from gut-associated lymphoid tissue into peripheral blood of CD patients. Such antigen-specific T-cells releasing interferon-γ can be transiently detected by using the enzyme-linked immunospot (ELISPOT) assays or by flow cytometry tetramer technology. This paper discusses the suitability of this in vivo tool to investigate the repertoire of gluten pathogenic peptides, to support CD diagnosis, and to assess the efficacy of novel therapeutic strategies. A systematic review of all potential applications of short oral gluten challenge is provided.

Background & Aims Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. Methods We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. Results Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. Conclusions We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell–mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

Celiac disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides are resistant to intestinal digestion (e.g., A-gliadin P31–43) and induce a stress/innate immune response, but the reason why they are dangerous in the intestines of patients with CD is unknown. In the present study, P31–43 activated IFN-α, a mediator of the innate immune response in CD, in the intestine of subjects with CD and an enterocyte cell line, CaCo-2. P31–43 cooperated with a viral ligand to activate the TLR7 pathway by interfering with endocytic trafficking. Based on these results, the vesicular pathway regulates the innate/inflammatory response to viral ligands and bioactive dietary peptides. Suggesting that together with viral infections, alimentary proteins able to mimic and potentiate the innate immune response to viruses, can trigger an autoimmune disease such as CD.

Coeliac disease has for a long time simply been regarded as a gluten-dependent enteropathy and a duodenal biopsy was required in all patients for the diagnosis. It is now accepted that autoimmunity against transglutaminase 2 is an earlier, more universal and more specific feature of coeliac disease than histologic lesions. Moreover, high serum levels of combined anti-transglutaminase 2 and anti-endomysium antibody positivity have excellent predictive value for the presence of enteropathy with villous atrophy. This makes the histology evaluation of the gut no longer necessary in well defined symptomatic paediatric patients with compatible HLA-DQ2 and/or DQ8 background. The biopsy-sparing diagnostic route is not yet recommended by gastroenterologists for adults, and certain clinical circumstances (immunodeficiency conditions, extraintestinal manifestations, type-1 diabetes mellitus, age less than 2 years) may require modified diagnostic approaches. Coeliac patients with preserved duodenal villous structure do exist and these need a more extended evaluation by immunologic and molecular biology tools.

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides induce innate and adaptive T cell-mediated immune responses. The major mediator of the stress and innate immune response to gliadin peptides (i.e., peptide 31–43, P31–43) is the cytokine interleukin-15 (IL-15). The role of epithelial growth factor (EGF) as a mediator of enterocyte proliferation and the innate immune response has been described. In this paper, we review the most recent literature on the mechanisms responsible for triggering the up-regulation of these mediators in CD by gliadin peptides. We will discuss the role of P31–43 in enterocyte proliferation, structural changes and the innate immune response in CD mucosa in cooperation with EGF and IL-15, and the mechanism of up-regulation of these mediators related to vesicular trafficking. We will also review the literature that focuses on constitutive alterations of the structure, signalling/proliferation and stress/innate immunity pathways of CD cells. Finally, we will discuss how these pathways can be triggered by gliadin peptide P31–43 in controls, mimicking the celiac cellular phenotype.

Initial studies associated the HLA class I A*01 and B*08 alleles with celiac disease (CD) susceptibility. Subsequent analyses showed a primary association with HLA class II alleles encoding for the HLA DQ2.5 molecule. Because of the strong linkage disequilibrium of A*01 and B*08 alleles with the DR3-DQ2.5 haplotype and a recent genome-wide association study indicating that B*08 and B*39 are predisposing genes, the etiologic role of HLA class I in CD pathogenesis needs to be addressed. We screened gliadin proteins (2α-, 2ω-, and 2γ-gliadin) using bioinformatic algorithms for the presence of peptides predicted to bind A*0101 and B*0801 molecules. The top 1% scoring 9- and 10-mer peptides (N = 97, total) were synthesized and tested in binding assays using purified A*0101 and B*0801 molecules. Twenty of ninety-seven peptides bound B*0801 and only 3 of 97 bound A*0101 with high affinity (IC50 < 500 nM). These 23 gliadin peptides were next assayed by IFN-γ ELISPOT for recognition in peripheral blood cells of CD patients and healthy controls carrying the A*0101 and/or B*0801 genes and in A*0101/B*0801− CD patients. Ten of the twenty-three peptides assayed recalled IFN-γ responses mediated by CD8+ T cells in A*0101/B*0801+ patients with CD. Two peptides were restricted by A*0101, and eight were restricted by B*0801. Of note, 50% (5/10) of CD8+ T cell epitopes mapped within the γ-gliadins. Our results highlight the value of predicted binding to HLA molecules for identifying gliadin epitopes and demonstrate that HLA class I molecules restrict the anti-gluten T cell response in CD patients.

Background Newborns at high risk of celiac disease (CD) were recruited in Italy in the context of the PreventCD study and closely monitored for CD, from 4 months up to a mean age of 8 years at follow-up. The aim of our study was to investigate intestinal T-cell reactivity to gliadin at the first clinical and/or serological signs of CD. Methods Gliadin-reactive T-cell lines were generated from intestinal biopsies of 19 HLA-DQ2-or HLA-DQ8-positive children. At biopsy, 11 children had a diagnosis of acute CD, two of potential CD, and six were non-celiac controls. Immune reactivity was evaluated against gliadin and known immunogenic peptides from α-, γ-, or ω-gliadins. The role of deamidation by transglutaminase (tTG) in determining the immunogenicity of gliadin was also investigated. Results Most of the children with CD (either acute or potential) had an inflammatory response to gliadin. Notably, signs of T-cell reactivity to gliadin were also found in some non-celiac subjects, in which IFN-γ responses occurred mainly when regulatory IL-10 and TGF-β cytokines were blocked. Interestingly, PreventCD children reacted to gliadin peptides found active in adult CD patients, and tTG deamidation markedly enhanced gliadin recognition. Conclusions T cells reactive to gliadin can be detected in the intestine of children at high risk of developing CD, in some cases also in the presence of a normal mucosa and negative CD-associated antibodies. Furthermore, children at a very early stage of CD recognize the same gliadin epitopes that are active in adult CD patients. Tissue transglutaminase strongly enhances gluten T-cell immunogenicity in early CD.

SummaryBackground A gluten-free diet is currently the only reliable therapeutic strategy that is approved for coeliac disease (CD). For many patients, however, compliance remains inadequate. Aim To investigate the immunogenicity of wheat flour that was pre-treated with selected lactobacilli and fungal proteases (hydrolysed wheat gluten) in coeliac patients. Methods The immunogenicity of hydrolysed wheat gluten was evaluated both in vitro in intestinal T cell lines (TCLs) and in vivo in treated CD patients after a short-term gluten challenge. Twenty treated CD patients were enrolled and equally randomised into two groups. The patients ate bread that was prepared with hydrolysed wheat flour or natural wheat flour (10 g of gluten/d for 3 days). The interferon (INF)-γ responses to natural gliadin and a 33-mer peptide were assessed by the enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) both before and 6 days after the start of the challenge. Results Hydrolysed wheat was not able to activate the TCLs from the coeliac intestinal mucosa. Consistent with the in vitro results, no significant increase in INF-γ secretion was observed in patients who consumed hydrolysed wheat flour. Conversely, the consumption of natural wheat gluten mobilised INF-γ secreting cells in the blood (P

Celiac disease (CD) is an enteropathy caused by the ingestion of wheat gluten in genetically susceptible individuals. A complete understanding of the pathogenic mechanisms in CD has been hindered because of the lack of adequate in vivo models. In the present study, we explored the events after the intragastric administration of gliadin and of the albumin/ globulin fraction from wheat in human leukocyte antigen-DQ8 transgenic mice (DQ8 mice) treated with indomethacin, an inhibitor of cyclooxygenases (COXs). After 10 days of treatment, mice showed a significant reduction of villus height, increased crypt depth, increased number of lamina propria-activated macrophages, and high basal interferon-? secretion in mesenteric lymph nodes, all of which were specifically related to gliadin intake, whereas the albumin/globulin fraction of wheat was unable to induce similar changes. Cotreatment with NS-398, a specific inhibitor of COX-2, also induced the intestinal lesion. Enteropathy onset was further characterized by high levels of oxidative stress markers, similar to CD. Biochemical assessment of the small intestine revealed the specific activation of matrix metalloproteinases 2 and 9, high caspase-3 activity, and a significant increase of tissue transglutaminase protein levels associated with the intestinal lesion. Notably, after 30 days of treatment, enteropathic mice developed serum antibodies toward gliadin (IgA) and tissue transglutaminase (IgG). We concluded that gliadin intake in combination with COX inhibition caused a basal inflammatory status and an oxidative stress condition in the small intestine of DQ8 mice, thus triggering the mucosal lesion and, subsequently, an antigen-specific immunity. © 2014 the American Physiological Society.

Several recent reports describe a role of probiotics as a therapeutic approach for celiac disease (CD). Two undigested A-gliadin peptides, P31-43 and P57-68, are central to CD pathogenesis, inducing an innate and an adaptive immune response, respectively. They enter enterocytes and localize to vesicular compartment to induce their toxic/immunogenics effects. In this article, we tested the effect of probiotic Lactobacillus paracasei (LP) CBA L74 (International Depository Accession Number LMG P-24778), its supernatant and LP-fermented cereals on gliadin peptides, P31-43 and P57-68, entrance in Caco-2 cells. Both LP CBA L74 and its supernatant inhibit P31-43 (intensity of fluorescence; FI: 75%) and P57-68 (FI: 50%) entrance in Caco2 cells, indicating that this biological effect is due to some product included in LP CBA L74 supernatant. This effect was present also after fermentation of cereals. This study describes a novel effect of probiotics in the prevention of undigested gliadin peptides toxic effects.

Background: On ingestion of gliadin, the major protein component of wheat and other cereals, the celiac intestine is characterized by the proliferation of crypt enterocytes with an inversion of the differentiation/proliferation program. Gliadins and A-gliadin peptide P31-43, in particular, act as growth factors for crypt enterocytes in patients with celiac disease (CD). The effects of gliadin on crypt enterocyte proliferation and activation of innate immunity are mediated by epidermal growth factors (EGFs) and innate immunity mediators [interleukin 15 (IL15)]. Objective: The aim of this study was to determine the molecular basis of proliferation and innate immune response to gliadin peptides in enterocytes. Design: The CaCo-2 cell line was used to study EGF-, IL15-, and P31-43–induced proliferation. Silencing messenger RNAs and blocking EGF receptor and IL15 antibodies have been used to study proliferation in CaCo-2 cells and intestinal biopsy samples from patients with CD and control subjects. Results: In the CaCo-2 cell model, IL15 and EGF cooperated to induce proliferation in intestinal epithelial cells at both the transcriptional and posttranscriptional levels, and the respective receptors interacted to activate each other’s signaling. In addition, the effects of the P31-43 peptide on CaCo-2 cell proliferation and downstream signaling were mediated by cooperation between EGF and IL15. The increased crypt enterocyte proliferation in intestinal biopsy samples from patients with CD was reduced by EGF receptor and IL15 blocking antibodies only when used in combination. Conclusions: EGF receptor/IL15R-a cooperation regulates intestinal epithelial cell proliferation induced by EGF, IL15, and the gliadin peptide P31-43. Increased proliferation of crypt enterocytes in the intestine of CD patients is mediated by EGF/IL15 cooperation. Am J Clin Nutr 2013;98:1123–35.

Tissue transglutaminase (TG2) plays a central role in celiac disease (CD) pathogenesis by strongly enhancing the immunogenicity of gluten, the CD-triggering antigen. By deamidating specific glutamine (Q) residues, TG2 favors the binding of gluten peptides to DQ2/8 molecules and, subsequently, their recognition by cognate T cells. Six peptides were previously identified within wheat gliadin whole extracts by tagging the TG2-susceptible Q residues with monodansylcadaverine (MDC) and nanospray tandem mass spectrometry (nanoESI-MS/MS). The immunogenicity of these peptides was next tested in gliadin-specific T-cell lines established from CD intestinal mucosa. Four peptides, corresponding to known epitopes of ?- and ?-gliadins, induced cell proliferation and interferon (IFN)-? production. Interestingly, one of the two non-T-cell stimulatory peptides corresponded to the 31-49 ?-gliadin peptide implicated in the innate immune activation in CD mucosa. This study describes a strategy for identifying immunogenic gluten peptides potentially relevant for CD pathogenesis in protein extracts from wheat and other edible cereals.

Different approaches have been used to study the pattern of cytokines in celiac disease (CD). Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of CD patients, dissected by LCM, analyzing cytokine expression profile. Frozen section of jejunum was obtained from 15 untreated CD and 15 control. Surface epithelium and lamina propria compartment were isolated by LCM. RNA from each LCM sample was extracted and, after a retrotranscription step, messenger RNA levels for MxA, IL-15, TNF-α, IFN-γ, IL-17α, IL-21, IL-10, and TGF-β were determined by quantitative reverse transcriptase-PCR. Increased gene expression levels of MxA, IL-15, TNF-α, IL-10, and TGF-β was observed in the surface epithelium of untreated CD with respect to control. Furthermore, all the cytokines investigated were upregulated in the lamina propria of untreated CD as compared to control. Within the untreated CD group the expression of IL-15 was higher, in the surface epithelium than in the lamina propria, whereas the expression levels of IL-17 and IL-21 were higher in the lamina propria than in the surface epithelium. Finally, high levels of IL-10 and TGF-β were detected in both compartments of untreated CD biopsies. In CD, surface epithelium and lamina propria compartments, play a prominent role in determining innate and adaptive immunity, respectively. Conversely, surface epithelium and lamina propria produce high levels of anti-inflammatory cytokines, suggesting that both compartments are involved in the immunoregulatory response.

Celiac disease (CD) is caused by loss of tolerance toward gluten and related cereal products. The delivery of gliadin peptides (GP) to HLA-DR-positive late endosomes (LE) of enterocytes is required for antigen presentation and tolerance generation. We hypothesized that anti-gliadin antibodies in CD serum modify gliadin transport into LE within enterocytes. CD and control duodenal biopsies were incubated with digests of gluten as well as with serum of CD patients. Lissamin-labeled GP AA31-43 and AA56-68 were endocytozed by Caco-2 cells with serum of CD- or control patients. Colocalization of gliadin with the LE marker LAMP-2 and cathepsin D was determined and quantified on immunofluorescence and immunoelectron microscopical level. Up to 13% of internalized gliadin was located in LE of CD biopsies incubated with CD serum compared with less than 4% in CD biopsies without CD serum as well as in control biopsies. In Caco-2 cells, the colocalization coefficient of GP AA31-43 and LE was 0.82 with CD serum, 0.42 with control serum, and 0.48 with culture medium. Incubation with CD serum can direct GP AA31-43 into LE of enterocytes which is required for antigen presentation. (Pediatr Res 70: 357-362, 2011)

Objective. Most of the recent studies suggest that oats are well tolerated by celiac disease (CD) patients. However, it is still possible that different oat cultivars may display different biological properties relevant for CD pathogenesis. We aimed to investigate biological and immunological properties of two oat varieties, Avena genziana and Avena potenza, in relation to their safety for CD patients. Material and Methods. Phosphorylation of extracellular signal-regulated kinase (ERK) and transepithelial electrical resistance (TEER) were evaluated in CaCo-2 cells treated with peptic–tryptic (PT) digests from the two oats and from gliadin (PTG). With the same PT-digests, duodenal biopsies from 22 CD patients were treated in vitro for 24 h and density of CD25 + cells in lamina propria and of intraepithelial CD3 + T cells was measured, as well as crypt cell proliferation and epithelial expression of interleukin 15. Finally, interferon g (IFN-g) production was measured as evidence of gliadin-specific T-cell activation by PT-digests. Results. In contrast to PTG, oats PT-digests were not able to induce significant increase in ERK phosphorylation and decrease in TEER in CaCo-2 cells. In the organ culture system, oats PTdigests, unlike PTG, did not induce significant increase in crypt enterocyte proliferation, increase in interleukin 15 expression or in lamina propria CD25 + cells. Nevertheless Avena potenza increased intraepithelial T-cell density, while Avena genzianainduced IFN-g production in 3/8 CD intestinal T cell lines. Conclusions. Our data show that Avena genziana and Avena potenza do not display in vitro activities related to CD pathogenesis. Some T-cell reactivity could be below the threshold for clinical relevance.

See editorial on page 284. BACKGROUND & AIMS: The presence of celiac diseaseassociated autoantibodies (antiendomysium and antitissue transglutaminase [anti-TG2]) with normal jejunal mucosa indicate potential celiac disease. We performed a prospective, 3-year cohort study to determine the natural history of potential celiac disease in children. METHODS: The study included 106 children with potential celiac disease, based on serology analysis and normal duodenal architecture. All but 2 carried the HLA-DQ2 and/or DQ8 haplotype. In all children, every 6 months, growth, nutritional parameters, celiac disease serology, and autoimmunity were investigated. In biopsies, intraepithelial-, CD3-, and lamina propria CD25-positive cells were counted; duodenal deposits of anti-TG2 immunoglobulin A were detected. Biopsy analysis was repeated after 2 years on patients with persistent positive serology and/or symptoms. RESULTS: Celiac disease was detected primarily in firstdegree relatives and patients with autoimmune disorders (40.6%). A gluten-free diet was prescribed to 20/106 patients because of symptoms, which were relieved in only 11. Eightynine of the 106 patients entered the follow-up study, with normal daily consumption of gluten. During the follow-up antibodies disappeared in 14.6% and fluctuated in 32.6%. Villous atrophy was observed in 12/39 patients (30.8%) who underwent a repeat biopsy. CONCLUSIONS: Most children with potential celiac disease remain healthy. After 3 years, approximately 33% of patients develop villous atrophy. Intestinal deposits of anti-TG2 IgA identify children at risk for villous atrophy.

The identification of gluten peptides eliciting intestinal T cell responses is crucial for the design of a peptide-based immunotherapy in celiac disease (CD). To date, several gluten peptides have been identified to be active in CD. In the present study, we investigated the recognition profile of gluten immunogenic peptides in adult HLA-DQ2(+) celiac patients. Polyclonal, gliadin-reactive T cell lines were generated from jejunal mucosa and assayed for both proliferation and IFN-gamma production in response to 21 peptides from wheat glutenins and alpha-, gamma-, and omega-gliadins. A magnitude analysis of the IFN-gamma responses was performed to assess the hierarchy of peptide potency. Remarkably, 12 of the 14 patients recognized a different array of peptides. All alpha-gliadin stimulatory peptides mapped the 57-89 N-terminal region, thus confirming the relevance of the known polyepitope 33-mer, although it was recognized by only 50% of the patients. By contrast, gamma-gliadin peptides were collectively recognized by the great majority (11 of 14, 78%) of CD volunteers. A 17-mer variant of 33-mer, QLQPFPQPQLPYPQPQP, containing only one copy of DQ2-alpha-I and DQ2-alpha-II epitopes, was as potent as 33-mer in stimulating intestinal T cell responses. A peptide from omega-gliadin, QPQQPFPQPQQPFPWQP, although structurally related to the alpha-gliadin 17-mer, is a distinct epitope and was active in 5 out of 14 patients. In conclusion, these results showed that there is a substantial heterogeneity in intestinal T cell responses to gluten and highlighted the relevance of gamma- and omega-gliadin peptides for CD pathogenesis. Our findings indicated that alpha-gliadin (57-73), gamma-gliadin (139-153), and omega-gliadin (102-118) are the most active gluten peptides in DQ2(+) celiac patients.

Background & Aims The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy. Methods We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy. Results IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors. Conclusions A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

Objective To assess the effectiveness of the combined use of fecal calprotectin (FC), anti-Saccharomyces cerevisiae antibody (ASCA), perinuclear staining antineutrophil antibody (pANCA), small intestinal permeability test (IP), and bowel wall ultrasonography measurement (BWUS) in the diagnostic work-up of children with suspected inflammatory bowel disease (IBD). Methods All children referred for initial assessment of possible IBD were eligible. Patients with symptoms or signs (right-lower quadrant mass, perianal disease, or hematochezia) mandating a complete work-up for IBD were excluded. All enrolled patients underwent a clinical, laboratory, radiographic, and endoscopic evaluation including biopsy examinations. The immunoglobulin (Ig)G and IgA ASCA, IgG pANCA, FC, IP, and BWUS were tested in all patients at the initial assessment. Results A final diagnosis of IBD was made in 27 patients: 17 Crohn disease and 10 ulcerative colitis. Eighteen children had other gastrointestinal diagnoses (8 functional bowel disorders, 5 food allergy-mediated diseases, 4 infectious enterocolitis, 1 familial Mediterranean fever). In patients with simultaneous abnormal values of FC, BWUS, and ASCA/pANCA, the estimated probability of having IBD was 99.47%. Patients with negative results on all tests had a 0.69% of probability of IBD. Conclusions The incorporation of noninvasive diagnostic tests into the initial diagnostic approach may avoid unnecessary invasive procedures and facilitate clinical decision-making when the diagnosis of IBD in children is initially uncertain.

Celiac disease (CD) is a complex small intestinal disorder due to a dysregulated immune response to wheat gliadin and related proteins which leads to a small intestinal enteropathy. It is generally accepted that CD is a T-cell mediated disease, in which, gliadin derived peptides, either in native form or deamidated by tissue transglutaminase, activate lamina propria infiltrating T lymphocytes which release proinflammatory cytokines. Recent studies indicate that gliadin contains also peptides able to activate an innate immune response. In particular, they induce a selective expansion of IEL, particularly TCRgamma/delta+ and CD8+TCR alpha/beta+ lymphocytes bearing the CD94 NK receptor, as well as a strong epithelial expression of MICA molecules which interact with NKG2D receptor expressed on TCRgamma/delta+ and NK cells. Most of the events of innate immune activation events are inhibited by antibodies neutralizing IL-15, thus confirming the key role of this cytokine as a mediator of intestinal mucosa damage induced by ingestion of gliadin. It remains to be established to what extent the ability of gliadin peptides to activate innate immunity relates to other biological properties exerted not only on celiac cells and tissues; the specificity of celiac patients is probably related to their genetic make up.

BACKGROUND: Enteropathy in coeliac disease (CD) is sustained by a gliadin specific Th1 response. Interleukin (IL)-10 can downregulate Th1 immune responses. AIM: We investigated the ability of recombinant human (rh) IL-10 to suppress gliadin induced Th1 response. PATIENTS AND METHODS: IL-10 RNA transcripts were analysed by competitive reverse transcription-polymerase chain reaction in duodenal biopsies from untreated and treated CD patients, non-coeliac enteropathies (NCE), and controls. CD biopsies were cultured with a peptic-tryptic digest of gliadin with or without rhIL-10. The proportion of CD80+ and CD25+ cells in the lamina propria, epithelial expression of Fas, intraepithelial infiltration of CD3+ cells, as well as cytokine synthesis (interferon gamma (IFN-gamma) and IL-2) were measured. Short term T cell lines (TCLs) obtained from treated CD biopsies cultured with gliadin with or without rhIL-10 were analysed by ELISPOT for gliadin specific production of IFN-gamma. RESULTS: In untreated CD and NCE, IL-10 RNA transcripts were significantly upregulated. In ex vivo organ cultures, rhIL-10 downregulated gliadin induced cytokine synthesis, inhibited intraepithelial migration of CD3+ cells, and reduced the proportion of lamina propria CD25+ and CD80+ cells whereas it did not interfere with epithelial Fas expression. In short term TCLs, rhIL-10 abrogated the IFN-gamma response to gliadin. CONCLUSIONS: rhIL-10 suppresses gliadin specific T cell activation. It may interfere with the antigen presenting capacity of lamina propria mononuclear cells as it reduces the expression of CD80. Interestingly, rhIL-10 also induces a long term hyporesponsiveness of gliadin specific mucosal T cells. These results offer new perspectives for therapeutic strategies in coeliac patients based on immune modulation by IL-10.

Celiac disease (CD) is an enteropathy characterized by a Th1-type immune response to the dietary gluten. The transcriptional mechanisms or factors that control Th1 cell development in this condition remain to be elucidated. The aim of this study was to analyze in CD the expression of interferon (IFN) regulatory factor (IRF)-1, a transcription factor that regulates the differentiation and function of Th1 cells. Duodenal biopsies were taken from children with untreated CD and control children, and analyzed for IRF-1 by Southern blotting of reverse-transcriptase PCR products and Western blotting. IRF-1 DNA-binding activity was assessed by electrophoretic shift mobility assay. The effect of gliadin stimulation on IRF-1 induction was investigated in an ex vivo organ culture of treated CD biopsies. Enhanced IRF-1 was seen in untreated CD in comparison with controls. This was evident at both the RNA and protein level. Furthermore, untreated CD samples exhibited stronger nuclear accumulation and DNA-binding activity of IRF-1 than controls. In contrast, IRF-2, a transcriptional repressor that binds the same DNA element and competes with IRF-1, was expressed at the same level in nuclear proteins extracted from both untreated CD and control patients. In explant cultures of treated CD biopsies, gliadin enhanced both IRF-1 RNA and protein. This effect was prevented by a neutralizing IFN-gamma antibody. Furthermore, stimulation of normal duodenal biopsies with IFN-gamma enhanced IRF-1. These data indicate that IRF-1 is a hallmark of the gliadin-mediated inflammation in CD and suggest that IFN-gamma/IRF-1 signaling pathway can play a key role in maintaining and expanding the local Th1 inflammatory response in this disease.

OBJECTIVES: The molecular and functional properties of small intestinal tissue transglutaminase are largely unknown despite growing interest because of its role in celiac disease (CD). In this study, we aimed to evaluate tissue transglutaminase expression and enzymatic activity in bioptic fragments obtained from the duodenum of untreated individuals with CD and from control subjects. METHODS: Analysis of tissue transglutaminase mRNA expression was performed by reverse transcription-polymerase chain reaction (RT-PCR). The presence of the enzyme in bioptic fragments as well as in homogenates from CD patients and controls was revealed by immunohistochemistry and Western blot, respectively, using the antitissue transglutaminase CUB 7402 clone. To evaluate in situ transglutaminase activity, sections of bioptic fragments were incubated in the presence of 5 mmol/L CaCl(2) with 5-(biotinamido)pentylamine or, alternatively, with a biotinylated glutamine-containing hexapeptide (TVQQEL) and the biotinylated 31-43 A-gliadin-derived peptide. RESULTS: Tissue transglutaminase mRNA levels were 1.0-fold higher (p < 0.05) in CD patients than in controls. Immunohistochemistry and in situ demonstration of enzymatic activity in celiac mucosa clearly showed an increased expression of active tissue transglutaminase in the extracellular matrix of the subepithelial region and in the enterocytes. Staining of the biotinylated 31-43 A-gliadin peptide in the same area of tissue transglutaminase suggested the presence of lysine-donor substrates in intestinal mucosa. CONCLUSIONS: Tissue transglutaminase is more expressed and active in defined areas of the small intestinal mucosa from patients with CD. The presence in the celiac mucosa of proteins able to act as amine-donor substrates suggests that tissue transglutaminase-mediated post-translational modification of proteins cross-linked with gliadin peptides may represent a pathogenic mechanism of CD.

It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25+, CD3+, and TCR- + was assessed in subjects with positive (n = 32) and negative (n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all (n = 8) positive subjects. Lamina propria CD25+ cell count was significantly (p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR- +/CD3+ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten.

In the recent years, the incidence of inflammatory bowel disease (IBD) has dramatically increased in young subjects, however, the pathogenesis of paediatric IBD is poorly investigated. In this study we aimed to evaluate the cytokine pattern and the phenotype of cytokine producing cells in the intestinal mucosa of paediatric patients affected by Crohn’s disease (CD) or ulcerative colitis (UC) and of non-IBD healthy controls (HC). Cytokine (IL-15, TNF-α, INF-γ) production was analyzed at basal condition and after mitogen stimulation either intra-cellularly by flow cytometry or in intestinal cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). A higher frequency of enterocytes (EpCam+ cells) was observed in UC patients compared to CD or HC. An expansion of enterocytes producing IL-15 and TNF-α were found in IBD patients compared to HC. A marked expression of IL-15 in the intestinal epithelium of IBD patients was further confirmed by immunohistochemistry. Myeloid dendritic (CD11c+) cells producing TNF-α and INF-γ were increased in IBD biopsies. Unexpectedly, only after a strong mitogen stimulus, as phytohaemagglutinin, the frequency of CD3+ cells producing IFN-γ was increased in IBD compared to control intestinal mucosa. Interestingly, functional studies performed on organ cultures of intestinal biopsies with neutralizing anti-IL-15 monoclonal antibody showed a marked reduction of mononuclear cell activation, proliferation of crypt enterocytes, as well as a reduction of TNF-α release in organ culture supernatants. In conclusion, we found that in the gut mucosa of IBD children both enterocytes and dendritic cells produce proinflammatory cytokines. The over-expression of IL-15 by enterocytes in IBD intestine and the reduced IBD inflammation by IL-15 blockage suggests that this cytokine could be a therapeutic target in IBD.

Summary It has always been known that anti-tissue transglutaminase 2 (anti-TG2) antibodies are produced in the small intestine. Their serum titres correlate with mucosal damage degree and decrease on a gluten-free diet (GFD). We aimed to correlate intestinal anti-TG2 antibodies levels with degree of mucosal damage and GFD duration. Thirty-four active, 71 potential and 24 CD patients on GFD for at least 2 years were enrolled. Anti-TG2 deposits were detected in intestinal biopsies by double immunofluorescence. Biopsies were cultured for 24 h with medium, and with gliadin peptic tryptic digest (PTG) or A-gliadin peptide 31–43 (P31-43). Anti-TG2 antibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All active CD patients secreted high titres of anti-TG2 antibodies into culture medium that increased with the worsening of mucosal injury (Spearman's r = 0·71; P < 0·0001). Seventy of 71 potential CD patients and 15 of 24 treated CD patients secreted low titres of anti-TG2 antibodies into supernatants, eight of nine negative treated patients being on GFD for more than 10 years. An inverse correlation between antibody titres and duration of GFD was found, (Spearman's r = −0·52; P < 0·01). All active, 53 of 71 potential and six of 24 treated, CD patients showed anti-TG2 mucosal deposits. Five of six positive treated CD patients had been on GFD for fewer than 6 years and were also positive for secreted anti-TG2. In treated patients, PTG/P31-43 was not able to induce secretion of anti-TG2 antibodies into culture medium. Measurement of anti-TG2 antibodies in biopsy supernatants proved to be more sensitive than detection by immunofluorescence to reveal their intestinal production. Intestinal antiTG2 antibodies titres correlated positively with the degree of mucosal damage and inversely with the duration of GFD.

Celiac disease (CD) has a multifactorial etiology with complex genetics and frequently occurs in association with other autoimmune disorders. Even though triggered by a dietary antigen, it shows many autoimmune features, the most peculiar being the presence of high titers of anti-tissue transglutaminase 2 autoantibodies, produced in the small intestinal mucosa since the early stages of the disease. More than 60% of CD-associated susceptibility loci are shared with at least another autoimmune condition, suggesting common pathogenic mechanisms. In particular, recognition of peptides by HLA molecules, posttranslational modifications required for optimal peptide binding and immune mechanisms leading to tissue damage have been found in CD as well as in other autoimmune diseases. This review briefly summarizes the main autoimmune features of CD, underlining the similarities with other autoimmune disorders, in particular with type 1 diabetes mellitus. Furthermore, the role of gluten and microbiome in driving autoimmunity is discussed.

Background—CD3 and γδ cells in the rectal mucosa increase after local instillation of gluten in children with coeliac disease and in half of their siblings.Aim—To establish an in vitro system for assessing immunological changes induced by gluten in the rectum.Patients and Methods—Rectal biopsy specimens obtained from 13 treated coeliac children, nine of their siblings, and nine controls were cultured in vitro with a peptic-tryptic digest of gliadin or ovalbumin. CD3 and CD25 cells were counted, and the expression of adhesion molecules evaluated.Results—In the lamina propria of coeliac biopsy samples cultured with gliadin, but not in those from controls, the expression of vascular cell adhesion molecule 1 (VCAM-1) was enhanced, and the number of CD25 cells was significantly higher than in those cultured in medium alone; the density of intraepithelial CD3 cells was also significantly higher. No differences were noted in coeliac biopsy specimens cultured with ovalbumin. A discriminant analysis allowed correct classification of all controls and all coeliacs but one, but three of nine siblings were allocated to the coeliac group.Conclusions—Our data confirm that gliadin is able to activate cell mediated immunity in the rectal mucosa in coeliac patients and in a subset of their first degree relatives.

Celiac disease (CD) occurs frequently, and is caused by ingestion of prolamins from cereals in subjects with a genetic predisposition. The small intestinal damage depends on an intestinal stress/innate immune response to certain gliadin peptides (e.g., A-gliadin P31-43) in association with an adaptive immune response to other gliadin peptides (e.g., A-gliadin P57-68). Gliadin and peptide P31-43 affect epithelial growth factor receptor (EGFR) signaling and CD enterocyte proliferation. The reason why the stress/innate immune and proliferative responses to certain gliadin peptides are present in CD and not in control intestine is so far unknown. The aim of this work is to investigate if, in CD, a constitutive alteration of enterocyte proliferation and signaling exists that may represent a predisposing condition to the damaging effects of gliadin. Immunofluorescence and immunohistochemistry were used to study signaling in CD fibroblasts and intestinal biopsies. Western blot (WB) analysis, immunoprecipitation, and quantitative PCR were also used. We found in CD enterocytes enhancement of both proliferation and Epidermal Growth Factor Receptor (EGFR)/ligand system. In CD enterocytes and fibroblasts we found increase of the phosphorylated downstream signaling molecule Extracellular Signal Regulated Kinase (ERK); block of the ERK activation normalizes enterocytes proliferation in CD mucosa. In conclusion the same pathway, which gliadin and gliadin peptide P31-43 can interfere with, is constitutively altered in CD cells. This observation potentially explains the specificity of the damaging effects of certain gliadin peptides on CD intestine.

Background: Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by life-long, severe diarrhea with intestinal Cl - malabsorption. It results from a reduced activity of the down regulated in adenoma exchanger (DRA), due to mutations in the solute carrier family 26, member 3 (SLC26A3) gene. Currently available therapies are not able to limit the severity of diarrhea in CLD. Conflicting results have been reported on the therapeutic efficacy of oral butyrate. Methods. We investigated the effect of oral butyrate (100 mg/kg/day) in seven CLD children with different SLC26A3 genotypes. Nasal epithelial cells were obtained to assess the effect of butyrate on the expression of the two main Cl- transporters: DRA and putative anion transporter-1 (PAT-1). Results: A variable clinical response to butyrate was observed regarding the stool pattern and fecal ion loss. The best response was observed in subjects with missense and deletion mutations. Variable response to butyrate was also observed on SLC26A3 (DRA) and SLC26A6 (PAT1) gene expression in nasal epithelial cells of CLD patients. Conclusions: We demonstrate a genotype-dependency for butyrate therapeutic efficacy in CLD. The effect of butyrate is related in part on a different modulation of the expression of the two main apical membrane Cl- exchangers of epithelial cells, members of the SLC26 anion family. Trial registration. Australian New Zealand Clinical trial Registry ACTRN12613000450718

BACKGROUND: The single-nucleotide polymorphism T300A of ATG16L1, a Crohn's disease (CD)-associated gene, is responsible for decreased autophagy. This study aimed to investigate the effects of this single-nucleotide polymorphism on the uptake and processing of antigens by dendritic cells (DCs) and the interaction between DC and intestinal epithelium in pediatric patients with CD. METHODS: Pediatric patients who homozygously carry either the protective (wild type, n = 7) or risk allele (risk, n = 13) of ATG16L1, as well as heterozygous patients (het, n = 13) were enrolled. The monocyte-derived DC were analyzed for phenotype, antigen sampling, and processing by flow cytometry, whereas the capability of DC to form transepithelial protrusions was determined by confocal microscopy. RESULTS: DC generated from wild type patients showed higher bacteria sampling and antigen processing compared with risk patients. Additionally, after exposure to either bacteria particles or the antigen DQ-ovalbumin, wild type DC showed a significant increase in the expression of the HLA-DR and CD86 when compared with risk DC. Interestingly, also het patients showed an impairment in bacteria uptake and expression of activation marker when compared with the wild type. In the Caco2/DC coculture, the formation of transepithelial protrusions were less numerous in risk DC compared with wild type and the antigen uptake decreased. CONCLUSIONS: DC of pediatric patients with CD carrying the T300A allele showed a marked impairment of antigen uptake and processing and defective interactions between DC and intestinal epithelium. Collectively, our results suggest that an autophagy defect is associated with an impairment of intestinal innate immunity in pediatric CD.

Atopy patch tests (APTs) have been proposed for the diagnostic approach in children with non-IgE-mediated cow's milk allergy and gastrointestinal symptoms. We aimed to investigate the benefit of APTs in predicting oral tolerance in these patients. We prospectively evaluated 172 subjects with a sure diagnosis of non-IgE-mediated CMA and gastrointestinal symptoms (97 boys, 56.4%; age, 6.37 m; range, 212 m). At diagnosis, 113/172 (65.7%) children had positive APTs to cow's milk proteins (CMP). After 12 months of exclusion, diet APTs were repeated immediately before OFC. APTs significantly correlated (P < 0.001) with the OFC outcome (r 0.579). Diagnostic accuracy was sensitivity of 67.95%, specificity of 88.3%, PPV of 82.81%, NPV of 76.85%, and a +LR of 5.80. APTs are a valuable tool in the follow-up of children with non-IgE-mediated CMA-related gastrointestinal symptoms by contributing in determining whether an OFC can safely be undertaken.

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.

Gluten-sensitive enteropathy is induced by dietary wheat gliadin and related proteins in genetically susceptible individuals. Most evidence suggests that the mucosal lesion represents an immunologically mediated injury triggered by gluten in the context of a particular assortment of major histocompatibility complex genes. The amino acid residues of gliadin and related proteins responsible for toxicity have not been identified; in vitro systems are available, but definitive conclusions must rely on in vivo jejunal challenges. At a conservative estimate, symptomatic gluten-sensitive enteropathy affects approximately 1 in 1000 individuals in Europe; however, it is now becoming clear that a greater proportion of individuals has clinically silent disease, and probably many others have a minor form of the the enteropathy. In most countries, the clinical presentation has changed over the past few years coming closer to the adult type of the disease, and the age of onset of symptoms is shifting upward. Liver, joint, hematologic, dental, and neurologic symptoms are increasingly being recognized. Several diseases are associated the gluten-sensitive enteropathy, such as IgA deficiency, insulin-dependent diabetes mellitus, and a range of other autoimmune diseases. Tests based on the measurement of antigliadin and antiendomysium antibodies have gained success as noninvasive screening tests; however, the ultimate diagnosis still is based on the finding of a severe histologic lesion of the jejunum while the patient is on a gluten-containing diet and on its disappearance once the gluten is excluded from the diet. A lifelong, strict GFD is mandatory for celiac children. Among other long-term problems, an increased risk of intestinal lymphoma has been reported in patients on a normal gluten-containing diet.

Summary It has been reported that interferon (IFN)-γ-secreting T cells reactive to gluten can be detected in the peripheral blood of individuals with treated coeliac disease (CD) after a short consumption of wheat-containing food. By contrast, very little is known about the reproducibility of this in-vivo procedure in the same patient cohort which underwent two, or more, gluten consumptions. Fourteen coeliac patients in remission consumed wheat bread for 3 days; 13 underwent a second gluten challenge after a wash-out of 3–10 months on a strict gluten-free diet. Immune reactivity to gluten was analysed in peripheral blood by detecting IFN-γ before and 6 days after commencing a gluten diet. Gliadin-specific IFN-γ-secreting CD4+ T cells increased significantly on day 6 of the first challenge. These cells resulted as prevalently human leucocyte antigen (HLA)-DQ restricted and with a phenotype of gut homing, as suggested by the expression of β7-integrin. Similarly, reactiveness to gliadin was observed after the second wheat consumption, although with an individual variability of responses at each challenge. Our findings confirmed that the short wheat challenge is a non-invasive approach to investigate the gluten-related immune response in peripheral blood of subjects intolerant to gluten. Furthermore, we demonstrated that the in-vivo procedure can be reproduced in the same subject cohort after a gluten wash-out of at least 3 months. Our study has important implications for the application of this procedure to clinical practice.

Background: Research is intense to find wheat of low or null toxicity for patients with celiac disease (CD). Among candidates, there are diploid wheat species. Objective: We compared the immunological properties of 2 lines of diploid monococcum wheat (Triticum monococcum ssp. monococcum), Monlis and ID331, with those of common wheat (Triticum aestivum). Design: Interferon-g production and the proliferation of intestinal gliadin-specific T cell lines and clones were measured as evidence of T cell activation by peptic and tryptic (PT) digests of gliadins from 2 monococcum lines. Furthermore, organ cultures of jejunal biopsies from 28 CD patients were set up to assess the effects of PT gliadin on innate and adaptive immune response by using immunohistochemistry. Results: Monlis and ID331 induced interferon-g production and proliferation in celiac mucosal T cells. In organ cultures, Monlis PT digest induced a significant increase of IL-15 epithelial expression and crypt enterocyte proliferation, whereas ID331 had no effect. Both monococcum lines caused intraepithelial T cell infiltration and lamina propria T cell activation. Conclusions: Our data show that the monococcum lines Monlis and ID331 activate the CD T cell response and suggest that these lines are toxic for celiac patients. However, ID331 is likely to be less effective in inducing CD because of its inability to activate the innate immune pathways. Am J Clin Nutr 2012;96:1339‐45.

Several studies analyzing the immune responses in patients with cow's milk allergy (CMA) have used T-cell lines or T-cell clones that require prolonged in vitro cell culturing and may result in a switched cell phenotype and function. We investigated immune responses to beta-lactoglobulin (b-LG) in peripheral blood mononuclear cells after a short in vitro antigen stimulation in children with acute CMA (both IgE-mediated and non-IgE-mediated forms) and in those who outgrew an IgE-mediated CMA. Healthy controls were also investigated. Peripheral blood mononuclear cells were assayed for IL-13, IFN-γ, IL-4, and IL-10. Although b-LG induced a cytokine production and/or cell proliferation almost in all children, included healthy controls, differences were observed among the four groups. Children with IgE-mediated CMA had a marked Th2-response, with high IL-13 production and proliferation, but low IFN-γ; by contrast, children with non-IgE-mediated CMA produced no, or very low, IL-13 and cell proliferation. Children, who outgrew CMA, showed a shift to a Th1-response, with reduced IL-13 and increased IFN-γ. IL-10-responses were high in all groups, with the highest level in healthy children; by contrast, IL-4 was undetectable in all children. This study highlights the use of shortly stimulated peripheral blood cells to investigate the food-induced immune responses.

Background & Aims: Celiac disease (CD) is characterized by an inflammatory response to wheat gluten, rye, and barley proteins. Fermentation of wheat flour with sourdough lactobacilli and fungal proteases decreases the concentration of gluten. We evaluated the safety of daily administration of baked goods made from this hydrolyzed form of wheat flour to patients with CD. Methods: Patients were randomly assigned to consumption of 200 g per day of natural flour baked goods (NFBG) (80,127 ppm gluten; n = 6), extensively hydrolyzed flour baked goods (S1BG) (2480 ppm residual gluten; n = 2), or fully hydrolyzed baked goods (S2BG) (8 ppm residual gluten; n = 5) for 60 days. Results: Two of the 6 patients who consumed NFBG discontinued the challenge because of symptoms; all had increased levels of anti-tissue transglutaminase (tTG) antibodies and small bowel deterioration. The 2 patients who ate the S1BG goods had no clinical complaints but developed subtotal atrophy. The 5 patients who ate the S2BG had no clinical complaints; their levels of anti-tTG antibodies did not increase, and their Marsh grades of small intestinal mucosa did not change. Conclusions: A 60-day diet of baked goods made from hydrolyzed wheat flour, manufactured with sourdough lactobacilli and fungal proteases, was not toxic to patients with CD. A combined analysis of serologic, morphometric, and immunohistochemical parameters is the most accurate method to assess new therapies for this disorder.

Background High serum levels of anti-tissue-transglutaminase-2 IgA antibodies (anti-TG2), which are produced and deposited in the intestine, characterize celiac disease. Aim To assess the diagnostic value of intestinal deposits of anti-TG2 IgA for celiac disease in a paediatric population. Methods 344 children underwent duodenal biopsy for the suspicion of CD, and were divided into 3 groups: group A, 144 celiac subjects with villous atrophy (Marsh 3b–c); group B, 109 subjects with high serum levels of anti-TG2 but normal intestinal mucosa (Marsh 0–1) (potential celiac disease patients); group C, 91 subjects with normal levels of serum anti-TG2: 70 with Marsh 0–1 and 21 with Marsh 3a mucosa. All duodenal sections were evaluated for the presence of intestinal deposits of anti-TG2 IgA by double immunofluorescence. Results Deposits of anti-TG2 IgA were present in 96%, 68%, 12% of patients from groups A, B, C, respectively. Diagnostic sensitivity and specificity for celiac disease were 96% and 88% vs. 97% and 100% for serum anti-TG2, respectively. The degree of concordance with serum anti-TG2 was 85%. Conclusion Detection of intestinal deposits of anti-TG2 IgA is a useful diagnostic tool. Further research is needed regarding their ability to predict evolution to villous atrophy in potential celiac disease.

International audience; Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31-43 (but not peptide 57-68) uptake by cells, thereby impairing the ability of p31-43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31-43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.