Changes upon the gluten-free diet of HLA-DQ2 and TRAFD1 gene expression in peripheral blood of celiac disease patients.

Background: Celiac disease (CD) is a chronic immuno-mediated enteropathy caused by dietary gluten in genetically susceptible individuals carrying HLA (Human Leukocytes Antigen) genes that encode for DQ2.5 and DQ8 molecules. TRAFD1 (TRAF-type zinc finger domain 1) is a gene recently found associated with CD and defined as a master regulator of IFNγ signalling and of MHC class I antigen processing/presentation. There is no specific drug therapy and the only effective treatment is the gluten-free diet (GFD). The great majority of celiac patients when compliant with GFD have a complete remission of symptoms and recovery of gut mucosa architecture and function. Until now, very few studies have investigated molecular differences occurring in CD patients upon the GFD therapy. Methods: We looked at the expression of both HLA DQ2.5 and TRAFD1 risk genes in adult patients with acute CD at the time of and in treated patients on GFD. Specifically, we measured by qPCR the HLA-DQ2.5 and TRAFD1 mRNAs on peripheral blood mononuclear cells (PBMC) from the two groups of patients. Results: When we compared the HLA-DQ mRNA expression, we didn't find significant variation between the two groups of patients, thus indicating that GFD patients have the same capability to present gliadin antigens to cognate T cells as patients with active disease. Conversely, TRAFD1 was more expressed in PBMC from treated CD subjects. Notably, TRAFD1 transcripts significantly increased in the patients analyzed longitudinally during the GFD, indicating a role in the downregulation of gluten-induced inflammatory pathways. Conclusion: Our study demonstrated that HLA-DQ2.5 and TRAFD1 molecules are two important mediators of anti-gluten immune response and inflammatory process.

IL-10-producing regulatory cells impact on celiac disease evolution.

Celiac Disease (CD) is a T-cell mediated disorder caused by immune response to gluten, although the mechanisms underlying CD progression are still elusive. We analyzed immune cell composition, plasma cytokines, and gliadin-specific T-cell responses in patients with positive serology and normal intestinal mucosa (potential-CD) or villous atrophy (acute-CD), and after gluten-free diet (GFD). We found: an inflammatory signature and the presence of circulating gliadin-specific IFN-γ+ T cells in CD patients regardless of mucosal damage; an increased frequency of IL-10-secreting dendritic cells (DC-10) in the gut and of circulating gliadin-specific IL-10-secreting T cells in potential-CD; IL-10 inhibition increased IFN-γ secretion by gliadin-specific intestinal T cells from acute- and potential-CD. On GFD, inflammatory cytokines normalized, while IL-10-producing T cells accumulated in the gut. We show that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten: DC-10 protect the intestinal mucosa from damage and represent a marker of potential-CD.

Mass spectrometry-based quantification of immunostimulatory gliadin proteins and peptides in coloured wheat varieties: Implications for Celiac Disease.

Pigmented wheat varieties (Triticum aestivum spp.) are getting increasingly popular in modern nutrition and thoroughly researched for their functional and nutraceutical value. The colour of these wheat grains is caused by the expression of natural pigments, including carotenoids and anthocyanins, that can be restricted to either the endosperm, pericarp and/or aleurone layers. While contrasts in phytochemical synthesis give rise to variations among purple, blue, dark and yellow grain's antioxidant and radical scavenging capacities, little is known about their influence on gluten proteins expression, digestibility and immunogenic potential in a Celiac Disease (CD) framework. Herein, it has been found that the expression profile and immunogenic properties of gliadin proteins in pigmented wheat grains might be affected by anthocyanins and carotenoids upregulation, and that the spectra of peptide released upon simulated gastrointestinal digestion is also significantly different. Interestingly, anthocyanin accumulation, as opposed to carotenoids, correlated with a lower immunogenicity and toxicity of gliadins at both protein and peptide levels. Altogether, this study provides first-level evidence on the impact modern breeding practices, seeking higher expression levels of health promoting phytochemicals at the grain level, may have on wheat crops functionality and CD tolerability.

Booster Dose of SARS-CoV-2 mRNA Vaccine in Kidney Transplanted Patients Induces Wuhan-Hu-1 Specific Neutralizing Antibodies and T Cell Activation but Lower Response against Omicron Variant.

Kidney transplanted recipients (KTR) are at high risk of severe SARS-CoV-2 infection due to immunosuppressive therapy. Although several studies reported antibody production in KTR after vaccination, data related to immunity to the Omicron (B.1.1.529) variant are sparse. Herein, we analyzed anti-SARS-CoV-2 immune response in seven KTR and eight healthy controls after the second and third dose of the mRNA vaccine (BNT162b2). A significant increase in neutralizing antibody (nAb) titers were detected against pseudoviruses expressing the Wuhan-Hu-1 spike (S) protein after the third dose in both groups, although nAbs in KTR were lower than controls. nAbs against pseudoviruses expressing the Omicron S protein were low in both groups, with no increase after the 3rd dose in KTR. Reactivity of CD4+ T cells after boosting was observed when cells were challenged with Wuhan-Hu-1 S peptides, while Omicron S peptides were less effective in both groups. IFN-γ production was detected in KTR in response to ancestral S peptides, confirming antigen-specific T cell activation. Our study demonstrates that the 3rd mRNA dose induces T cell response against Wuhan-Hu-1 spike peptides in KTR, and an increment in the humoral immunity. Instead, humoral and cellular immunity to Omicron variant immunogenic peptides were low in both KTR and healthy vaccinated subjects.

Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases.

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases.

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

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, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, 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.

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

BACKGROUND: In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. METHODS: CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). RESULTS: We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. CONCLUSIONS: The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. TRIAL REGISTRATION: ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . IMPACT: Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

E40 glutenase detoxification capabilities of residual gluten immunogenic peptides in in vitro gastrointestinal digesta of food matrices made of soft and durum wheat.

Gluten degrading enzymes, which are commonly referred to as "glutenases," represent attractive candidates for the development of a pharmacological treatment of gluten related disorders, such as coeliac disease (CeD). Endoprotease-40 (E40), a novel glutenase secreted by the actinomycete Actinoallomurus A8 and recombinantly produced in S. lividans TK24, was shown to be active at pH 3 to 6 (optimum pH 5), resistant to pepsin and trypsin degradation, able to destroy immunotoxicity of both gliadin 33-mer peptide and whole proteins and to strongly reduce the response of specific T cells when added to gliadin in in vitro gastrointestinal digestion. This study aims to functionally assess the capabilities of Endoprotease-40 (E40) to detoxify residual gluten immunogenic peptides in gastrointestinal digesta of food matrices made of soft and durum wheat. The INFOGEST harmonized protocols were applied to the multicompartmental model of simulated human gastrointestinal digestion, for the quantitative assessment of residual gluten in liquid (beer) and solid (bread and pasta) foods, made of either soft or durum wheat. Proteomic and immunological techniques, and functional assays on intestinal T cell lines from celiac disease patients were used to identify gluten-derived immunogenic peptide sequences surviving in gastric and gastrointestinal digesta after the addition of E40 at increasing enzyme: wheat proteins ratios. During the gastric phase (2 h incubation time), the addition of E40 demonstrated an extensive (≥ 95%) dose-dependent detoxification of whole gluten in real food matrices. Overall, the residual gluten content was found at, or even below, the 20 ppm gluten-free threshold for soft and durum wheat-based food. Furthermore, unlike in untreated gastrointestinal digesta, none of the immunodominant α-gliadin peptides survived in E40-treated digesta. Traces of ω- and γ-gliadin derived immunogenic peptides were still detected in E40-treated digesta, but unable to stimulate celiac-intestinal T cells. In conclusion, E40 is a promising candidate for the oral enzymatic therapy of CeD, as a stand-alone enzyme being efficient along the complete gastrointestinal digestion of gluten.

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.

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 < 0.005) and with the serum levels of anti-tissue transglutaminase antibodies (p < 0.01). The changes of these two cell subsets strongly correlated with mucosal lesions, according to the histological Marsh classification, as the transition from M0 to M3 lesions was associated with a significant reduction of IL4+ T cells (M0 vs. M1 p < 0.04, M0 vs. M3 p < 0.007) and an increase of TCRγδ+ T cells (M0 vs. M1 p < 0.05, M0 vs. M3 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.

Triticum monococcum amylase trypsin inhibitors possess a reduced potential to elicit innate immune response in celiac patients compared to Triticum aestivum.

SCOPE: Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. METHODS AND RESULTS: ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. CONCLUSION: TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

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

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

HLA class II genes in precision-based care of childhood diseases: what we can learn from celiac disease.

Celiac disease (CeD) is a chronic immuno-mediated enteropathy caused by dietary gluten with marked autoimmunity traits. The human leukocyte antigen (HLA) class II heterodimers represent the main predisposing factor, although environmental agents, as viral infection, gut microbiota, and dietary regimen, also contribute to CeD risk. These molecules are involved in autoimmunity as they present self-antigens to autoreactive T cells that have escaped the thymic negative selection. In CeD, the HLA class II risk alleles, DQA1*05-DQB1*02 and DQA1*03-DQB1*03, encode for DQ2.5 and DQ8 heterodimers, and, furthermore, disease susceptibility was found strictly dependent on the dose of these genes. This finding questioned how the expression of HLA-DQ risk genes, and of relative surface protein on antigen-presenting cells, might be relevant for the magnitude of anti-gluten inflammatory response in CeD patients, and impact the natural history of disease, its pathomechanisms, and compliance to dietary treatment. In this scenario, new personalized medical approaches will be desirable to support an early, accurate, and non-invasive diagnosis, and to define genotype-guided preventive and therapeutic strategies for CeD. To reach this goal, a stratification of genetic risk, disease outcome, and therapy compliance based on HLA genotypes, DQ2.5/DQ8 expression measurement and magnitude of T cell response to gluten is mandatory. IMPACT: This article revises the current knowledge on how different HLA haplotypes, carrying the DQ2.5/DQ8 risk alleles, impact the onset of CeD. This review discusses how the expression of susceptibility HLA-DQ genes can determine the risk assessment, outcome, and prevention of CeD. The recent insights on the environmental factors contributing to CeD in childhood are reviewed. This review discusses the use of HLA risk gene expression as a tool to support medical precision approaches for an early and non-invasive diagnosis of CeD, and to define genotype-guided preventive and therapeutic strategies.

Differential expression of predisposing HLA-DQ2.5 alleles in DR5/DR7 celiac disease patients affects the pathological immune response to gluten.

The DR5-DQ7/DR7-DQ2 genotype is very frequent among patients affected by celiac disease (CD), in Europe. This genotype, associated to high risk of CD, carries the HLA-DQA1*05 and HLA-DQB1*02 predisposing alleles, in trans configuration. The alleles encode the DQ2.5 heterodimer responsible of gluten peptide presentation on the surface of antigen-presenting cells (APCs), and consequent pathogenic CD4+ T cell activation. We demonstrated that DR5/DR7 APCs induce an anti-gluten CD4+ T cell response, of comparable intensity to that observed with APCs carrying DR1/DR3 genotype, which risk alleles are in cis configuration. In addition, we showed that DR5/DR7 APCs from celiac patients stimulated an effector CD4+ T cell response higher with respect to that induced by DR5/DR7 APCs from healthy subjects. To explain these findings, we assessed the DQ2.5 RNA and protein quantity. We showed that the expression of DQA1*05 and DQB1*02 risk alleles is much higher than the expression of non-CD-associated alleles, in agreement with the previous results obtained with DR1/DR3 genotype. The differential expression of transcripts influences the quantity of DQα1*05 and DQß1*02 chains and, as consequence, the cell surface density of DQ2.5 heterodimers. Moreover, both RNA and proteins, are more abundant in APCs from celiac patients than controls. Finally, to unravel the mechanism regulating the expression of predisposing DQA1*05 and DQB1*02 alleles, we quantified the new synthetized RNA and found that the differential expression is explained by their transcription rate. Our results confirmed that the strength of antigen-specific CD4+ T cell response is mainly determined by the amount of gluten in the diet and provided a new possible approach for a personalized diagnosis and for risk stratification.

Immunogenic Potential of Beer Types Brewed With Hordeum and Triticum spp. Malt Disclosed by Proteomics.

The protein/peptide composition of five beer kinds, including two experimental beer-like products brewed with einkorn (Triticum monococcum), a beer labeled as "gluten-free," a traditional all-barley malt and a wheat (T. aestivum) containing beer, was characterized with HPLC-ESI MS/MS-based proteomics. To enlarge the characterization of the components, the polypeptides were fractionated according to their molecular size (cut-off 6 kDa). All the beer types contained a variety of polypeptides arising from all the gliadin subfamilies (α-/ß-, γ-, and ω-gliadins) able to induce an immune response in celiac disease (CD) patients in addition to a panel of IgE-reactive food allergens. Wheat storage proteins were heavily hydrolyzed in the beer samples brewed with einkorn. The presence of gluten-like fragments, also including the 25-mer and 33-mer-like of α-gliadin, was confirmed in beer brewed with barley and wheat malt as well as in the gluten-free beer. Both CD-toxic and allergenic peptides of all beer samples were drastically degraded when subjected to a simulated gastroduodenal (GD) digestion. After in vitro digestion, the level of gluten-like peptides assayed with the G12 competitive ELISA, was below the threshold (20 ppm) for a food to be considered as "gluten-free." A few gliadin-derived epitopes occurred in the digests of beers crafted with wheat or Norberto-ID331 line of einkorn. In contrast, digests of all barley malt and gluten-free beers did not contain detectable gluten-like epitopes, but only minor fragments of hordeins and IgE-reactive food allergens. All beer samples evoked a weak immune response on gliadin-reactive celiac T cells isolated from intestinal biopsies of celiac patients. Compared to undigested polypeptides, the response was markedly reduced by GD digestion. Although the consumption of a moderate amount of beer brewed with barley or einkorn could deliver a relatively low amount of CD-toxic epitopes, the findings of this study emphasize the urgent need of a reliable and accurate quantification of gluten epitopes in all types of beer, also including the gluten-free one, to compute realistically the contribution of beer to the overall gluten intake, which can be responsible of intestinal tissue damages in celiacs.

Comparative Analysis of in vitro Digestibility and Immunogenicity of Gliadin Proteins From Durum and Einkorn Wheat.

Recent studies suggested that gliadin proteins from the ancient diploid einkorn wheat Triticum monococcum retained a reduced number of immunogenic peptides for celiac disease patients because of a high in vitro digestibility with respect to hexaploid common wheat. In this study, we compared the immunological properties of gliadins from two Triticum monococcum cultivars (Hammurabi and Norberto-ID331) with those of a Triticum durum cultivar (Adamello). Gliadins were digested by mimicking the in vitro gastrointestinal digestion process that includes the brush border membrane peptidases. Competitive ELISA, based on R5 monoclonal antibody, showed that gastrointestinal digestion reduced the immunogenicity of Triticum monococcum gliadins; conversely, the immunogenic potential of Triticum durum gliadins remained almost unchanged by the in vitro digestion. The immune stimulatory activity was also evaluated by detecting the IFN-γ production in gliadin-reactive T-cell lines obtained from the small intestinal mucosa of HLA-DQ2+ celiac disease patients. Interestingly, gastrointestinal digestion markedly reduced the capability of Triticum monococcum gliadins (p <0.05) of both cultivars to activate T cells, while it slightly affected the activity of Triticum durum. In conclusion, our results showed that Triticum durum was almost unaffected by the in vitro gastrointestinal digestion, while Triticum monococcum had a marked sensibility to digestion, thus determining a lower toxicity for celiac disease patients.

In Celiac Disease Patients the In Vivo Challenge with the Diploid Triticum monococcum Elicits a Reduced Immune Response Compared to Hexaploid Wheat.

SCOPE: Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. Gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge are analyzed. 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 are assessed in blood by measuring the IFN-γ-secreting T cells by EliSpot and the expression of inflammatory cytokines/receptors (IL-12A, IL-15, IL-18RAP, IFN-γ) by qPCR. TA mobilizes a remarkable number of gliadin-specific, IFN-γ-secreting T cells (p<0.05), while no significant cell mobilization is induced by TM (p=ns). Similar results are obtained in response to five immunogenic peptides from α-, ω-, and γ-gliadins, although with a large individual variability. An increased mRNA expression for IL-12A and IFN-γ is detected in the group eating TA compared to those consuming TM (p<0.05). CONCLUSIONS: Although T. monococcum is a cereal not suitable for the diet of celiacs, this diploid wheat elicits a reduced in vivo T-cell response compared to T. aestivum in celiac patients.

Reinventing the nutraceutical value of gluten: The case of l-theanine-gluten as a potential alternative to the gluten exclusion diet in celiac disease.

Functional foods have created an open environment for the development of new solutions to health-related issues. In celiac disease, there is still no therapeutic alternative other than the observance of a gluten-free diet. In this context, we developed a wheat flour enriched in l-theanine aimed to be a potential alternative to the gluten-free diet. Through microbial transglutaminase-catalysed transamidation of gluten proteins using ethylamine as amine nucleophile, substantial amounts of glutamine residues were converted in theanine residues. Furthermore, using T-cell lines generated from intestinal biopsy specimens of celiac disease patients, this treatment showed the potential to strongly reduce the ability of gluten proteins to stimulate a T-cell-mediated immune response. From a rheological point of view, the functionality of gluten was retained. Considering L-theanine's evidence-based health benefits, a novel functional food is presented here and for celiac disease can be a path towards the development of an alternative to the gluten-free diet.

Recent Progress and Recommendations on Celiac Disease From the Working Group on Prolamin Analysis and Toxicity.

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis.