Society: AGA

Background: Contamination-free oats are considered safe for most patients with celiac disease (CeD). However, we and others have isolated pro-inflammatory oat protein (avenin)-specific CD4+ T cells from the blood and duodenum of some people with CeD, a finding that carries troubling implications for safe oats ingestion in CeD. Does the presence of avenin-specific T cells identify CeD patients susceptible to harm from oats?
Aim: To determine the frequency of symptoms and immune activation in patients with CeD after single-bolus ingestion of purified avenin and then assess symptomatic, immune and clinical (safety) effects after extended avenin ingestion.
Methods: For the first time, food-grade avenin was purified from contamination-free oats to enable feeding studies at high doses not achievable using oats. We employed a series of single-bolus avenin challenges in HLA-DQ2.5+ CeD adults with assessment of symptoms and serum interleukin (IL)-2 at 4 hrs, a highly sensitive marker of gluten-specific T cell activation. Avenin was given in increasing amounts (0.05, 0.1, 0.5, 1, 4, and 6 g) with 4-wk washout periods. Subsequently, in patients with IL-2 responses, avenin was consumed daily for 6 wks at the highest tolerated dose that triggered IL-2. T-cells were assessed using avenin-specific tetramers, serum cytokines (MSD and O-link Inflammation 96-panel) and duodenal histology was examined pre- and post- challenge. A CeD patient undertook a 6 wk wheat gluten challenge as a positive control.
Results: Surprisingly, avenin induced a significant acute IL-2 elevation in 11/29 (38%) CeD patients (mean 16-fold elevation) and adverse symptoms such as pain, diarrhea and vomiting were induced in 60%, with severity correlated to higher IL-2 elevation. Five IL-2 responders then undertook 6-wk avenin challenge. Activated avenin-specific tetramer+ effector memory CD4+ T cells were increased on day 6. Interestingly, after 6 wks of avenin, these had returned to baseline and IL-2 responses after avenin were undetectable; all patients were tolerating avenin without the acute initial symptoms. In the duodenum, a similar frequency of tetramer+ cells were seen at baseline and at 6 wks. Notably, duodenal histology after 6 wks avenin remained normal, in contrast to significant deterioration in the wheat challenged patient. Serum inflammatory cytokines were not elevated by avenin except in one highly symptomatic CeD patient to a similar degree as the wheat challenged patient.
Conclusion: Purified avenin induces acute symptoms and T-cell responses in a subset of “sensitive” CeD patients. Reassuringly, our findings suggest that in most CeD patients oats is unable to sustain a pathogenic immune response above the threshold required for mucosal deterioration, in contrast to wheat gluten. These findings help resolve the discrepancy between clinical oats safety and oats immunity in CeD.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complex of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52).
Aim: To assess the ability of DONQ52 to inhibit CeD patient-derived T cell responses to the most immunogenic wheat gluten peptides encompassing immunodominant T cell epitopes.
Methods: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T cell responses. HLA-DQ2.5+ CeD patients undertook a 3-day wheat bread challenge (10g/d gluten) with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon-γ enzyme-linked immunosorbent spot assay (ELISpot) testing for responses to gluten peptides encompassing DQ2.5-glia-α1a/α2, DQ2.5-glia-ω1/ω2 and DQ2.5-glia-γ1 and deamidated gliadin protein. The effect of addition of DONQ52 (4 or 40 μg/mL) was assessed and compared to pan-DQ blockade (SPVL3 antibody).
Results: 20 CeD patients completed 3-day wheat challenge and 15/20 (75%) had robust gluten-specific T cell responses to the α- and ω-gliadin peptides and the 33mer on D6. Notably, DONQ52 at both concentrations was highly efficient at inhibiting IFN-γ responses to all stimulatory gluten peptides in all responsive patients, with a mean reduction in response of 88% (33-mer), 89% (DQ2.5-glia-α1a/α2), 63% (DQ2.5-glia-ω1/ω2) and 84% (cocktail of peptides containing DQ2.5-glia-α1a/α2, DQ2.5-glia-ω1/ω2 and DQ2.5-glia-γ1), with DONQ52 at 40 μg/mL. DONQ52 was generally better than Pan-DQ antibody blockade in reducing gluten peptide responses. T cell responses to whole protein (gliadin) were less consistent, but nevertheless, blocking by DONQ52 was seen in 67% of gliadin responders. DONQ52 had no effect on activating non-specific T cell responses when tested using PBMC isolated on D1 prior to gluten challenge.
Conclusion: DONQ52 is highly effective at blocking gluten-specific T cell responses to dominant wheat gluten epitopes. Studies to assess DONQ52 blockade of barley hordein and rye secalin-specific T cells induced by barley and rye challenge, respectively, are underway. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against HLA-DQ2.5/gluten peptides and provides proof-of-concept that such an approach has the potential to meaningfully inhibit pathogenic gluten-specific T cell responses in CeD.

Background and Aim: The worldwide prevalence of food sensitivities, including food allergies and autoimmune conditions, such as celiac disease (CeD), has increased in the past decades. This suggests that co-factors, linked to industrialization, are involved. One understudied factor typically used in processed foods and considered harmless, are inorganic nanoparticles (NPs). Our aim was to investigate whether daily exposure to the NP-structured silicon dioxide (SiO₂), employed as an anticaking agent in powdered foods, including infant formula, alters oral tolerance (OT) induction to ovalbumin (OVA), and increases severity of gluten sensitivity in mice.
Methods: An OT induction model to OVA was used in C57BL/6J mice that were orally exposed to food-grade (fg) SiO₂ for 60 days (10mg/kg body weight/day) in water suspension (gastric gavage) or incorporated into food pellets (solid matrix). Controls were gavaged with water. The gut immune response and immune cell populations induced were analysed by flow cytometry. Non-obese diabetic (NOD) mice expressing the CeD susceptibility gene HLA-DQ8 (NOD/DQ8) were exposed to fg-SiO₂, and subsequently immunized with gluten, the main environmental trigger of CeD. Small intestinal immunopathology was investigated at endpoint through assessment of villus-to-crypt ratios and intraepithelial lymphocyte (IEL) counts.
Results: fg-SiO₂ at human dietary levels led to low-grade intestinal inflammation evidenced by higher pro-inflammatory cytokines, such as IFN-γ, and lower anti-inflammatory cytokines, including IL-10 and TGF-β, known mediators in OT. After OT induction to OVA, fg-SiO₂ exposure was associated with decreased expansion of IL-10 and TGF-β-producing T cells and the development of a pro-inflammatory Th1 inflammatory response. fg-SiO₂ exposure through gastric gavage, but not through food pellets, decreased the frequency of CD103⁺ dendritic cells. In NOD/DQ8 mice, fg-SiO₂ decreased villus-to-crypt ratios, increased IELs counts, and enhanced the Th1 inflammatory response induced by immunization to gluten.
Conclusions: Chronic oral exposure to the food additive SiO₂ blocked the establishment of OT in the gut leading to intestinal inflammation and worsening of gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between SiO₂exposure and food sensitivities in humans.

Background: The incidence of celiac disease (CD) has increased over the last decades, especially in females and children. It’s not clear if this is a true increase or due to the availability of highly sensitive testing and increased awareness. Recent reports suggest that CD incidence reached its peak and starting to plateau in many countries over the world. Our study aims to update the CD incidence trend over the last two decades in a well-defined US population.
Methods: Using the Rochester Epidemiology Project (REP) data, we reviewed the electronic health records of both Mayo Clinic and Olmsted Medical Center between January 01, 2000, and December 31, 2021. We identified all CD patients who were diagnosed during the study periods and resided in Olmsted County. Incidence rates were calculated by age, sex, and calendar year and standardized to the 2020 US population.
Results: We identified a total of 662 patients who were diagnosed with CD during the study period. Of those 182 (27%) were children and 424 (64%) were females. The CD incidence rate was higher at the end of the study period at 16.7 (2000-2002) vs 10.3 (2018-2021) per 100.000 person-year. The overall CD incidence rate peaked at 26.4 per 100.000 person-year between 2009 and 2011 and seems to have plateaued and trended down for adults after 2011 especially patients who were > 65 years at diagnosis. On the other hand, the CD incidence rate continued to increase in children. The CD incidence rate in younger children aged (0-10) years at diagnosis is continuing to increase over the last two decades from 11 (2000-2002) to 30 (2018-2021) per 100.000 person-year. There was no significant difference in incidence trend over the last two decades based on gender although females consistently had higher incidence. Figure1
Conclusion: Even though the overall CD incidence rate seems to be plateauing in Olmsted County. CD incidence rate continues to increase in children, which can reflect a shift to earlier diagnosis, or due to a time-based environmental condition(s), that is losing its effect on older patients but affects children as new arrivals to the risk environment.

Sex Adjusted Incidence Rate of Celiac Disease by Age groups between 2000-2021

Sex Adjusted Incidence Rate of Celiac Disease by Age groups between 2000-2021

INTRODUCTION: Alpha-gal syndrome (AGS) is an immunoglobulin E (IgE) mediated delayed hypersensitivity reaction to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate on the cells of non-primate mammals. Sensitization is thought to be secondary to ectoparasitic bites, primarily the Lone Star tick endemic to the Southeastern and Eastern United States. Angioedema, anaphylaxis, urticaria, and/or gastrointestinal symptoms occurring 2 to 6 hours following ingestion of non-primate mammalian derived products is the classic presentation. There are reports of a primarily gastrointestinal phenotype of AGS in adults. We sought to determine clinical presentation and response to dietary management of patients with alpha-gal sensitization who presented to a pediatric gastroenterology clinic in an endemic area.
METHODS: We performed a retrospective chart review on patients ages 0-19 from January 1st, 2017, through April 1st, 2022, that were tested for alpha-gal specific IgE (sIgE) at Monroe Carell Jr. Children’s Hospital at Vanderbilt in Nashville, TN. We assessed charts of patients tested through the pediatric gastroenterology clinic to identify demographic data, clinical presentation, and response to elimination diet. Positive alpha-gal was defined as > 0.10 kU/L (Viracor). sIgE for beef, lamb/mutton, and pork (positive > 0.35 kU/L) were reported. Chi-squared and Fisher Exact tests were used for categorical data and Wilcoxon rank-sum test for continuous variables. One way ANOVA was used when comparing response to dietary elimination.
RESULTS: We identified 199 patients who had testing for AGS of which 20.1% (n=40) had a positive alpha-gal sIgE. Positive patients were more likely to be male (67.5% vs 40.9%, p=0.003) and have a known tick exposure (40% vs 11.9%, p<0.001). Positive patients were more likely to have additional symptoms of classic AGS (urticaria, angioedema, etc.) beyond GI symptoms, but 31/40 (77.5%) reported GI symptoms alone. Of 40 patients with positive testing, 30 attempted dietary elimination. Eight (27%) saw complete improvement in their symptoms, 8 (27%) saw partial improvement and 14 (47%) saw no improvement. Patients who improved with dietary elimination were more likely to be male and had a non-significant trend for higher overall specific IgE against alpha-gal, but it was higher sIgE for beef, lamb, and pork that was statistically significant as a predictor of elimination response, compared to those that did not experience improvement.
CONCLUSIONS: In an area endemic for alpha-gal, 20.1% of pediatric patients tested positive for alpha-gal sIgE after evaluation in a pediatric gastroenterology clinic and the majority presented with GI symptoms alone. At least 40% of these patients responded to dietary elimination. Responders were more likely to be male and have higher levels of sIgE for beef, lamb, and pork.

Objectives and Study
The pathogenesis of celiac disease (CD) involves a triad of predisposing genes (HLA DQ2/8), gluten and environmental factors. The environmental factors remain not well defined. Since epigenetic mechanisms operate at the interface between genetic predisposition and environment, the intestinal epithelial cells (IECs), forming a critical barrier to the environment, are the disease-relevant cell type to identify epigenetic alterations involved in the pathogenesis of CD.
Our aims are:
1) To identify genes and pathways epigenetically and transcriptionally altered in IECs from CD patients;
2) Generation of patients-derived intestinal epithelial organoids (IEOs) to demonstrate that CD-associated epigenetic alterations are retained in this in vitro model.
Methods
CD patients in gluten containing diet (GCD) and in gluten-free diet (GFD) have been consecutively enrolled together with a matched cohort of controls suffering from other inflammatory gastrointestinal disorders (IGD) (e.g. inflammatory bowel diseases, gastritis, esophagitis).
IECs, highly purified from intestinal biopsies by using enzyme digestion and magnetic beads sorting for the CD326, have been analyzed for genome-wide DNA methylation patterns and gene expression profiles.
IEOs have been generated from a subset of patients and epigenetic results have been compared with the alterations observed in IECs.
Results
We enrolled 181 patients: 96 CD patients in GCD, 20 CD patients in GFD and 65 controls.
Comparing CD patients in GCD and CD patients in GFD versus controls more than 700 and 200 differentially methylated genes (DMGs) have been found, respectively. DMGs have been ordered by significant p-value and the first 17 DMGs are shared. Among these 17 DMGs the main biological processes affected are related to antigen processing and presentation pathway. In particular: PSMB8, PSMB9, TAP1, TAP2 and TAPBP are involved in antigen processing pathway and MHC class I molecules such as HLA-E, HLA-F and HLA-DPB1 are involved in antigen presentation.
All the genes involved in these pathways have been found hypomethylated and overexpressed in CD.
IEOs have been generated from 5 controls and 5 CD patients. The methylation analysis showed that the DMGs in IELs are retained in IEOs.
Conclusions
The intestinal epithelium of CD patients in GCD or GFD is altered showing antigen processing and presentation pathways constitutively activated only in CD patients and not in patients suffering from other IGD. In IECs from patients with CD high amounts of endogenous peptides (gliadin) are pumped to the endoplasmic reticulum, loaded in MHC class I molecules and transported to the cell surface to be recognized by T-cells.
The environment-induced epigenetic modifications of IECs are retained in IEOs suggesting it is a good in vitro model to further investigate the role of these pathways in the pathogenesis of CD.

Giuseppe Molinario

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