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Celiac disease (CD) is an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals by genetically predisposed individuals. Constitutive differences between cells from CD patients and control subjects, including levels of protein phosphorylation, alterations of vesicular trafficking, and regulation of type 2 transglutaminase (TG2), have been reported. In the present work, we investigated how skin-derived fibroblasts from CD and control subjects responded to thapsigargin, an endoplasmic reticulum ER stress inducer, in an attempt to contribute to the comprehension of molecular features of the CD cellular phenotype. We analyzed Ca2+ levels by single-cell video-imaging and TG2 activity by a microplate assay. Western blots and PCR analyses were employed to monitor TG2 levels and markers of ER stress and autophagy. We found that the cytosolic and ER Ca2+ level of CD cells was lower than in control cells. Treatments with thapsigargin differently activated TG2 in control and CD cells, as well as caused slightly different responses regarding the activation of ER stress and the expression of autophagic markers. On the whole, our findings identified further molecular features of the celiac cellular phenotype and highlighted that CD cells appeared less capable of adapting to a stress condition and responding in a physiological way.

Mediterranean Diet (Med Diet) is one of the healthiest dietary patterns. We aimed to verify the effects of weaning (i.e., the introduction of solid foods in infants previously fed only with milk) using adult foods typical of Med Diet on children eating habits, and on the microbiota composition. A randomized controlled clinical trial on 394 healthy infants randomized in a 1:1 ratio in a Med Diet group weaned with fresh; seasonal and tasty foods of Med Diet and control group predominantly weaned with industrial baby foods. The primary end point was the percentage of children showing a good adherence to Med Diet at 36 months. Secondary end points were mother’s changes in adherence to Med Diet and differences in children gut microbiota. At 36 months, children showing a good adherence to Med Diet were 59.3% in the Med Diet group and 34.3% in the control group (p < 0.001). An increase in adherence to the Med Diet was observed in the mothers of the Med Diet group children (p < 0.001). At 4 years of age children in the Med Diet group had a higher gut microbial diversity and a higher abundance of beneficial taxa. A Mediterranean weaning with adult food may become a strategy for early nutritional education, to develop a healthy microbiota, to prevent inflammatory chronic diseases and to ameliorate eating habits in children and their families.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1β, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFβ, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.

Celiac disease (CD) is an immune-mediated enteropathy triggered in genetically susceptible individuals by gluten-containing cereals. A central role in the pathogenesis of CD is played by the HLA-restricted gliadin-specific intestinal T cell response generated in a pro-inflammatory environment. The mechanisms that generate this pro-inflammatory environment in CD is now starting to be addressed. In vitro study on CD cells and organoids, shows that constant low-grade inflammation is present also in the absence of gluten. In vivo studies on a population at risk, show before the onset of the disease and before the introduction of gluten in the diet, cellular and metabolic alterations in the absence of a T cell-mediated response. Gluten exacerbates these constitutive alterations in vitro and in vivo. Inflammation, may have a main role in CD, adding this disease tout court to the big family of chronic inflammatory diseases. Nutrients can have pro-inflammatory or anti-inflammatory effects, also mediated by intestinal microbiota. The intestine function as a crossroad for the control of inflammation both locally and at distance. The aim of this review is to discuss the recent literature on the main role of inflammation in the natural history of CD, supported by cellular fragility with increased sensitivity to gluten and other pro-inflammatory agents.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1β and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1β by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1β and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1β, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

Coeliac disease (CD) is the prototype of an inflammatory chronic disease induced by food. In this context, gliadin p31-43 peptide comes into the spotlight as an important player of the inflammatory/innate immune response to gliadin in CD. The p31-43 peptide is part of the p31-55 peptide from α-gliadins that remains undigested for a long time, and can be present in the small intestine after ingestion of a gluten-containing diet. Different biophysical methods and molecular dynamic simulations have shown that p31-43 spontaneously forms oligomeric nanostructures, whereas experimental approaches using in vitro assays, mouse models, and human duodenal tissues have shown that p31-43 is able to induce different forms of cellular stress by driving multiple inflammatory pathways. Increased proliferative activity of the epithelial cells in the crypts, enterocyte stress, activation of TG2, induction of Ca

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls’ DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.

Celiac disease (CD) is a type of inflammatory chronic disease caused by nutrients such as gliadin that induce a TC (T cell)-mediated response in a partially known genetical background in an environment predisposed to inflammation, including viruses and food. Various experimental and clinical observations suggest that multiple agents such as viruses and bacteria have some common, inflammatory pathways predisposing individuals to chronic inflammatory diseases including celiac disease (CD). More recently, a Western diet and lifestyle have been linked to tissue inflammation and increase in chronic inflammatory diseases. In CD, the gliadin protein itself has been shown to be able to induce inflammation. A cooperation between viruses and gliadin is present in vitro and in vivo with common mechanisms to induce inflammation. Nutrients could have also a protective effect on CD, and in fact the anti-inflammatory Mediterranean diet has a protective effect on the development of CD in children. The possible impact of these observations on clinical practice is discussed.

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.

Thirty-six founding members from Europe were present in 1968, when the European Society of Paediatric Gastroenterology (ESGA) had its first meeting in Paris. The aim was to create a forum for presentations and discussions of research activities in paediatric gastroenterology in Europe. At the second meeting of ESGA 1969 in Interlaken, an important landmark was set for all gastroenterologists in the world. In this conference, the first ever criteria for "the Diagnosis of Coeliac Disease" (CD) were established. In 1990, the revised criteria for the diagnosis of CD were published. After the introduction of new noninvasive techniques, like tissue transglutaminase 2-antibodies and the HLADQ2/HLADQ8 determinations in blood, "new" criteria for the diagnosis of CD were published in 2012 by the European society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN). Close collaboration of ESPGHAN and the North American Society of Paediatric Gastroenterology, Hepatology and Nutrition led to mutual meetings. The first combined meeting was 1978 in Paris, followed by meetings in New York, Amsterdam, Houston, and last in Toulouse. The first World Congress of Paediatric Gastroenterology took place in Boston 2000 followed by congresses in Paris, Iguazu, Taipeh, and Toronto. The creation of specialised committees (Nutrition Committees, GI-Committee, and Hepatology-Committee) enabled the society to elaborate numerous guidelines for standards in the diagnosis and treatment of diseases within the subspecialties. The Journal of Paediatric Gastroenterology and Nutrition, as organ of ESPGHAN and the North American Society of Paediatric Gastroenterology, Hepatology and Nutrition since 1991, serves as the voice for these worldwide accepted guidelines. Growing educational activities with summer schools, the Young Investigator Forum and the creation of working groups have distributed our current knowledge among the younger generation and led to fruitful reports, guidelines, and syllabus. In 1992, ESPGHAN was 1 of the founding 7 members of the United European Gastroenterology Federation (UEGF), which developed into a successful organisation for gastroenterology in Europe. This year we celebrate the 50th anniversary of ESPGHAN at our annual Meeting in Geneva.

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the &alpha, gliadin peptide 31&ndash, 43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31&ndash, 43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31&ndash, 43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31&ndash, 43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 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.

Since the conception of an idea of a few paediatric gastroenterologists in Europe to create a society for Paediatric Gastroenterology in 1967, and its foundation in 1968, half a century has passed. The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) now celebrates its 50th anniversary and its utmost success in combining clinical and scientific expertise in the fields of paediatric gastroenterology, haepatology, and nutrition. To describe this success story 14 of the still living presidents of ESPGHAN recount their impressions of the steady growth of ESPGHAN with all the historical facets from their own points of view. This historical view of ESPGHAN over the last 5 decades provides personal accounts of the development of all activities and creations of this great European Society. The Society started as a small family of experts in the field into a global working open society involved in a large variety of activities within the subspecialties, becoming a leading organisation in Europe and beyond. This unique view gives also a wonderful insight into the famous clinicians and researchers from all over Europe who have helped in the growth and development of ESPGHAN. By describing all these activities and collaborations it becomes clear that this astonishing pan-European enterprise was achieved by people who put considerable effort and time into the development of this society. Their statements serve as a historical source and reference for future evaluation of the first 50 years of ESPGHAN. In depicting different time episodes, and by assembling all the historical pieces of a puzzle together, the statements help to illustrate how a highly structured society such as ESPGHAN has evolved over the last 50 years, for what it stands for today and what is to be expected in the future.

Laser capture microdissection (LCM) is a powerful tool for selection and isolation of single cells or compartments from complex primary tissues to perform molecular analyses. Celiac disease is a genetic autoimmune disorder where the ingestion of gluten leads to damage in the small intestine. Increased intraepithelial lymphocytes and the presence of the lamina propria inflammatory infiltrate of the duodenal mucosa is a common part of the disease. These cells promote inflammatory processes through the release of cytokines. Here, we describe the use of LCM and real-time quantitative PCR (RT-qPCR) to analyze cytokine profile information in distinct duodenal mucosa tissue compartments of celiac patients.

Laser capture microdissection (LCM) is a powerful tool for selection and isolation of single cells or compartments from complex primary tissues to perform molecular analyses. Celiac disease is a genetic autoimmune disorder where the ingestion of gluten leads to damage in the small intestine. Increased intraepithelial lymphocytes and the presence of the lamina propria inflammatory infiltrate of the duodenal mucosa is a common part of the disease. These cells promote inflammatory processes through the release of cytokines. Here, we describe the use of LCM and real-time quantitative PCR (RT-qPCR) to analyze cytokine profile information in distinct duodenal mucosa tissue compartments of celiac patients.

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.

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.

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.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

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.

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 (

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)

Coeliac disease (CD) is a very common food-sensitive enteropathy, which is triggered by gluten ingestion and is mediated by CD4 + T cells. In addition, alterations in the intestinal microbiota that is normally involved in the homeostasis of GALT (gut-associated lymphoid tissue) seem to play a role in CD. In accordance with these findings, we previously reported that Lactobacillus casei can induce a strong enhancement of the T cell-mediated response to gliadin without inducing enteropathy. In this study, we analysed the effects of L. casei administration in a mouse model of gliadin-induced villous damage that was recently developed and involves the inhibition of cyclo-oxygenase (COX) activities in gliadin-sensitized HLA-DQ8 transgenic mice. To address the issue, we assessed the weight loss, the intestinal cytokine pattern, the density of CD25 + cells and morphometry of the gut mucosa. We confirmed that COX inhibition in sensitized mice caused villus blunting, dysregulated expression of tumour necrosis factor (TNF)-a and reduced gliadin-specific IL-2 production. Notably, the administration of probiotic strain induced a complete recovery of villus blunting. This finding was associated with a delay in weight decrease and a recovery of basal TNF-a levels, whereas the numbers of CD25 + cells and the levels of IL-2 remained unchanged. In conclusion, our data suggest that the administration of L. casei can be effective in rescuing the normal mucosal architecture and GALT homeostasis in a mouse model of gliadin-induced enteropathy.

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.

Celiac disease (CD) is an enteropathy triggered by gluten and mediated by CD4+T cells. A complete understanding of CD immunopathogenesis has been hindered due to the lack of adequate in vivo models. Here, we explored the effect of the inhibition of COX by indomethacin in wheat gliadin-sensitized transgenic mice expressing the HLA-DQ8 heterodimer, a molecule associated with CD. Treated mice showed a gliadin-specific immune response with a significant reduction of villus height, not linked to crypt hyperplasia and to expansion of intraepithelial T cells. Notably, treated mice showed increased numbers of CD25+ and apoptotic cells in the lamina propria, whereas high basal levels of IFN-γ secretion, along with a reduced gliadin-specific IL-2 expression were detected in MLN. Biochemical assessment of the lesion revealed increased mRNA of Lamb3 and Adamts2, encoding for ECM proteins, and enhanced activities of metalloproteinases MMP1, 2 and 7. We conclude that an intestinal sensitivity to gliadin, in connection with COX inhibition, caused a decreased villus height in DQ8 tg mice. The lesion was induced by a deregulated mucosal cell immunity to gliadin, thus triggering activation of a specific ECM protein pathway responsible for lamina propria remodeling.

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.

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.

A single-nucleotide variant, C/T(-13910), located 14 kb upstream of the lactase gene (LCT), has been shown to be completely correlated with lactase persistence (LP) in northern Europeans. Here, we analyzed the background of the alleles carrying the critical variant in 1,611 DNA samples from 37 populations. Our data show that the T(-13910) variant is found on two different, highly divergent haplotype backgrounds in the global populations. The first is the most common LP haplotype (LP H98) present in all populations analyzed, whereas the others (LP H8-H12), which originate from the same ancestral allelic haplotype, are found in geographically restricted populations living west of the Urals and north of the Caucasus. The global distribution pattern of LP T(-13910) H98 supports the Caucasian origin of this allele. Age estimates based on different mathematical models show that the common LP T(-13910) H98 allele (approximately 5,000-12,000 years old) is relatively older than the other geographically restricted LP alleles (approximately 1,400-3,000 years old). Our data about global allelic haplotypes of the lactose-tolerance variant imply that the T(-13910) allele has been independently introduced more than once and that there is a still-ongoing process of convergent evolution of the LP alleles in humans.

Background: Gliadins, a family of wheat proteins, are central to the pathogenesis of celiac disease (CD). In addition to ‘immunogenic’ effects, gliadin directly affects cultured cells and intestine preparations, and produces damage in vivo, via a separate ‘toxic’ peptide, such as A-gliadin p31–43 (P31–43). Aims: Understanding the molecular mechanisms underlying direct non T-cell mediated effects of gliadin peptides, and assessing their potential role in promoting CD. Method: Gliadin effects were tested on a number of cell lines and on cultured mucosa samples by evaluating cytoskeleton rearrangements, endocytosis, proliferation and apoptosis. Standard biochemical methods were used to assess prolonged epidermal growth factor receptor (EGFR) activation. Results: Crude gliadin peptic-tryptic peptides (PTG], or P31–43 alone, fully reproduce the effects of epidermal growth factor (EGF] on actin cytosketon, cell cycle and cell proliferation of various cell lines. Inhibitor studies demonstrate the role of EGFR in the early response to gliadin exposure, pointing to activation of the EGFR pathway. Peptide P31–43 is not similar to any EGFR ligand, but can delay inactivation of the EGFR interfering with its endocytosis. Gliadin-induced delay of EGFR endocytosis in cultured intestinal biopsies, together with S-phase entry of epithelial intestinal cells, confirm a role for EGFR activation in CD. Conclusion: The ability of gliadin peptides to delay EGFR inactivation through interference with the endocytic pathway suggests a model where gliadin fragments amplify the effects of trace amounts of EGF, and possibly of other growth factors, by prolonging receptor activation. The results, using cultures of coeliac intestinal biopsies, highlight the role of the EGF pathway in establishing and maintaining the typical atrophic and proliferative alterations of the small intestine in CD.

With increasing affluence, the traditional food choices of Mediterranean populations are changing. The changes appear to begin in early infancy, with increased consumption of processed foods. To determine current consumption patterns of the diets of 450 toddlers, 6-32 months old, enrolled from 17 paediatric practices in the Campania region of Italy, quantitative data were collected for 7 d, using calibrated feeding bottles, cups and dishes. Automated food analysis was employed and quality control was performed on a sub-sample. The average daily intakc was 373 kJ/kg. Forty-three percent of energy was provided by carbohydrates, while fats supplied 39%, 45% of fats were saturated and 46.7% were monosatutated (primarily olive oil). Proteins provided 18% of daily energy; most (81.4%) were of animal origin. Fibre was virtually absent from the study children's diet. Total daily cholesterol reached an average value of 191 mg/d, corresponding to 201 mg/4.2 mJ. The intake per kg of cholesterol increased with age in the first year, while proteins and fats decreased. Dairy products were the main source of energy, fats and cholesterol. Vegetables and legumes were a minor source of energy at all ages: the consumption of fruit decreased with age. Although the overall pattern of feeding of this cohort remains favourable compared with other children groups in developed countries, consumption of whole milk and full fat dairy products may be reduced to equilibrate the diet.

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.