Analytical and functional approaches to assess the immunogenicity of gluten proteins

Gluten proteins are the causative agents of celiac disease (CD), a lifelong and worldwide spread food intolerance, characterized by an autoimmune enteropathy. Gluten is a complex mixture of high homologous water-insoluble proteins, characterized by a high content of glutamine and proline amino acids that confers a marked resistance to degradation by gastrointestinal proteases. As a consequence of that, large peptides are released in the gut lumen with the potential to activate inflammatory T cells, in CD predisposed individuals. To date, several strategies aimed to detoxify gluten proteins or to develop immunomodulatory drugs to recover immune tolerance to gluten are under investigation. This review overviews the state of art of both analytical and functional methods currently used to assess the immunogenicity potential of gluten proteins from different cereal sources, including native raw seed flours and complex food products, as well as drug-treated samples. The analytical design to assess the content and profile of gluten immunogenic peptides, described herein, is based on the oral-gastro-intestinal digestion (INFOGEST model) followed by extensive characterization of residual gluten peptides by proteomic and immunochemical analyses. These approaches include liquid chromatography–high-resolution mass spectrometry (LC-MS/MS) and R5/G12 competitive ELISA. Functional studies to assess the immune stimulatory capabilities of digested gluten peptides are based on gut mucosa T cells or peripheral blood cells obtained from CD volunteers after a short oral gluten challenge

Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule

Consistency in Polyclonal T-cell Responses to Gluten between Children and Adults with Celiac Disease

BACKGROUND & AIMS: Developing antigen-specific approaches for diagnosis and treatment of celiac disease requires a detailed understanding of the specificity of T cells for gluten. The existing paradigm is that T-cell lines and clones from children differ from those of adults in the hierarchy and diversity of peptide recognition. We aimed to characterize the T-cell response to gluten in children vs adults with celiac disease. METHODS: Forty-one children with biopsy-proven celiac disease (median age, 9 years old; 17 male), who had been on strict gluten-free diets for at least 3 months, were given a 3-day challenge with wheat; blood samples were collected and gluten-specific T cells were measured. We analyzed responses of T cells from these children and from 4 adults with celiac disease to a peptide library and measured T-cell receptor bias. We isolated T-cell clones that recognized dominant peptides and assessed whether gluten peptide recognition was similar between T-cell clones from children and adults. RESULTS: We detected gluten-specific responses by T cells from 30 of the children with celiac disease (73%). T cells from the children recognized the same peptides that were immunogenic to adults with celiac disease; deamidation of peptides increased these responses. Age and time since diagnosis did not affect the magnitude of T-cell responses to dominant peptides. T-cell clones specific for dominant α- or ω-gliadin peptides from children with celiac disease had comparable levels of reactivity to wheat, rye, and barley peptides as T-cell clones from adults with celiac disease. The α-gliadin-specific T cells from children had biases in T-cell receptor usage similar to those in adults. CONCLUSIONS: T cells from children with celiac disease recognize similar gluten peptides as T cells from adults with celiac disease. The findings indicate that peptide-based diagnostics and therapeutics for adults may also be used for children. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved

Identification of gliadin peptides immunogenic for celiac patients

Celiac disease (CD) is a multifactorial disorders affecting susceptible individual following ingestion of gluten and related prolamines. It’s accepted that ingestion of gluten activates an immune cascade responsible for lymphocytic infiltration and destruction of the intestinal mucosa.The repertoire of gluten peptides activating the CD4+T cells restricted by CD-associated HLA DQ2 and DQ8 molecules, is well established, particularly in adult celiac patients. Conversely, very little is known about the repertoire of gluten peptides active in children celiacs, as well as the antigen specificity of CD8+T cells that massively infiltrate celiac intestinal mucosa. We aimed to investigate the repertoire of gluten peptides responsible of the inflammatory cascade both in acute disease, characterized by villous atrophy, as well as in the early and mild disorder, such as potential celiac disease. Furthermore we investigated the role of deamidation by tTGase in shaping the repertoire of gluten peptides. The comprehensive elucidation of pathogenic gluten peptidesmay have several translational applications, as the design of peptide-based therapies, alternative to the gluten exclusion diet, or the searching of grains with reduced or total absence of pathogenic sequences for prevention of the disease in at risk subjects. Although celiac disease is associated with HLA class II alleles coding for DQ2/8 heterodimers initial studies reported an increased frequency of HLA Class-I A*01 and B*08 alleles among CD subjects. Since A*01 and B*08 alleles are in strong linkage disequilibrium with the DR3-DQ2 haplotype, the role of A1 and B8 molecules in presenting gluten peptides to CD8 T cells should be revaluated. In order to identify potential HLA Class I-restricted epitopes, six gliadin proteins were screened and top 1% scoring peptides were synthesized and assayed for recognition in celiac patients grouped according to their positivity for A1 and/or B8 alleles. Ten peptides induced strong IFN-gamma responses in CD8+T cells from a subgroup of celiac patients, particularly in B8 positive CD volunteers. Importantly, these CTL epitopes are active in their native, non tTG-deamidated form. Immunogenicity of the selected peptides was further assessed on short-term CTL obtained from PBMC of responsive celiac patients. Several studies identified gluten peptides restricted by HLA class II immunodominant for adult celiacs. We investigated the contribution of gliadin-specific T cell response in childhood or adolescent CD by analyzing the T-cell reactivity to five peptides found immune-dominant in adult celiacs. Gliadin-specific T cell lines were generated by jejunal biopsies of young subjects and gliadin-specificity was assessed by IFN-gamma-ELISPOT and ELISA. Results shown a significant response (p<0.05) in IFN-gamma production in subjects with active or potential CD, but no in subjects without CD. Furthermore, children patients recognized the same pattern of gluten peptides found to be dominant in adult CD, such as DQ2.5-glia-alpha-1a/glia-alpha-2; DQ2.5-omega-1/glia-omega-2; DQ2.5-glia-γ-1; DQ2.5-glia-γ-2; DQ2.5-glia-γ-3/glia-γ-4b and 4c/ glia-γ5/glia-γ-2 in both naïve and deamidated forms. In conclusion, gliadin-specific T cell responses were found in intestinal mucosa of young subjects that presents autoantibodies (anti-tTG IgA) that are serology marker of disease, but a histologically and morphologically normal mucosa. Patients with potential CD had similar magnitude of IFN-γ responses of patients with mucosal atrophy, as well as similar pattern of active gliadin epitopes. Our results highlight the use of intestinal T cell response to gliadin as an additional diagnostic tool in the doubted cases of CD. Overall the present study offers new lights in the definition of the repertoire of gliadin peptides restricted by HLA class I & II recognized by celiac patients and involved in the pathogenesis of celiac disease

Gliadin-Specific T-Cells Mobilized in the Peripheral Blood of Coeliac Patients by Short Oral Gluten Challenge: Clinical Applications

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

Intestinal Cellular Biomarkers of Mucosal Lesion Progression in Pediatric Celiac Disease

Celiac disease (CD) is a chronic intestinal inflammation caused by gluten ingestion in genetically predisposed individuals. Overt-CD and potential-CD are the two main forms of gluten intolerance in pediatric patients with different grades of intestinal mucosa lesion and clinical management. For overt-CD patients the gluten-free diet is mandatory, while for potential-CD the dietary therapy is recommended only for those subjects becoming clinically symptomatic overtime. To date, specific early biomarkers of evolution to villous atrophy in potential-CD are lacking. We recently observed an expansion of TCRγδ+ T cells and a concomitant disappearance of IL4-producing T cells in the intestinal mucosa of overt-CD patients compared to potential-CD children, suggesting the involvement of these two cells subsets in the transition from potential-CD to overt-CD. In this study, we demonstrated that the intestinal densities of IL4+ T cells inversely correlated with TCRγδ+ T cell expansion (p p p p p p < 0.0006). These findings strongly suggest that the detection of TCRγδ+ and IL4+ T cells could serve as cellular biomarkers of mucosal lesion and targets of novel immunomodulatory therapies for CD

Effect of in situ gluten-chitosan interlocked self-assembled supramolecular architecture on rheological properties and functionality of reduced celiac-toxicity wheat flour

International audienceA new technology for reducing wheat flour toxicity for celiac disease patients through the in situ formation of gluten-chitosan interlocked self-assembled supramolecular architecture was developed. To have a deeper insight into the microstructure of this new molecular organization and its impact on the dough properties, its small and large deformation rheological properties and the macromolecular features of gluten-chitosan polymers were studied. The reduction of gluten proteins followed by spontaneous oxidation in the presence of the chitosan template in the range of 7.5:1 to 1.3:1 protein to chitosan weight ratio imposed a different reorganization of wheat flour proteins in the polymeric fraction changing conformation from homogeneous spherical molecules to polymer molecules with random-coil conformation. The polymeric fraction increased with decreasing protein to chitosan weight ratio attaining a maximum value at the 1.9:1 ratio. Moreover, the formation of the novel supramolecular architecture at this ratio allowed dough to maintain its ability to form a network after water addition and kneading showing a higher elastic and viscous moduli when compared to the control flour and the other studied formulations. It also presented a significantly higher resistance to extension, didn't inhibit the fermentation process, and retained the original dough ball shape while the dough made with the untreated flour presented a considerable extension during baking. Results show that it is possible to obtain a fully-functional wheat-based product when using a 1.9:1 protein to chitosan weight ratio with a reduced toxicity for celiac patients, opening in this way a new perspective concerning the quest for alternatives of gluten-exclusion diet

In situ gluten-chitosan interlocked self-assembled supramolecular architecture reduces T-Cell-mediated immune response to gluten in celiac disease

International audienceScope: The prevalence of celiac disease has increased since the last half of the 20th century and is now about 1% in most western populations. At present, people who suffer from celiac disease have to follow a gluten-exclusion diet throughout their lives. Compliance to this restrictive diet is demanding and the development of alternative strategies has become urgent. Methods and results: In this context, it is found that the biocompatible aminopolysaccharide chitosan imposes a different gluten reorganization after gluten redox reaction producing in situ mechanically interlocked supramolecular assemblies between gluten and chitosan. These new structures result in the decrease of gluten digestibility, tissue transglutaminase deamidation activity, and interferon-gamma production in intestinal T cell lines generated from biopsy specimens of celiac disease patients. Conclusion: Overall, the results demonstrate the potential of this research avenue to celiac disease is problematic, as the reorganization of gluten proteins to a novel supramolecular architecture shows a positive impact on known pathogenesis mechanisms of the disease. At present, the only therapy for celiac disease is adherence to a gluten-free diet. Here, it is shown that chitosan-imposed gluten reorganization to an interlocked self-assembled supramolecular architecture reduces gluten digestibility, R5-reactivity, tissue transglutaminase deamidation activity, and its capacity to stimulate a T-cell-mediated immune response in celiac disease

Gliadin-Specific CD8(+) T Cell Responses Restricted by HLA Class I A*0101 and B*0801 Molecules in Celiac Disease Patients

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