Gluten-Dependent Activation of CD4+ T Cells by MHC Class II-Expressing Epithelium.

BACKGROUND & AIMS: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T-cell interactions in organoid monolayers expressing human major histocompatibility complex class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD. METHODS: Epithelial major histocompatibility complex class II (MHCII) was determined in active and treated CeD, and in nonimmunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without interferon (IFN)-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T-cell co-cultures were incubated with gluten, predigested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T-cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants. RESULTS: Patients with active CeD and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer T-cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T-cell activation markers, and the release of interleukin-2, IFN-γ, and interleukin-15 in co-culture supernatants. Gluten metabolized by P aeruginosa, but not the lasB mutant, enhanced CD4+ T-cell proliferation and activation. CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten predigestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in patients with CeD.

Standardizing Randomized Controlled Trials in Celiac Disease: An International Multidisciplinary Appropriateness Study.

BACKGROUND & AIMS: There is a need to develop safe and effective pharmacologic options for the treatment of celiac disease (CeD); however, consensus on the appropriate design and configuration of randomized controlled trials (RCTs) in this population is lacking. METHODS: A 2-round modified Research and Development/University of California Los Angeles Appropriateness Method study was conducted. Eighteen gastroenterologists (adult and pediatric) and gastrointestinal pathologists voted on statements pertaining to the configuration of CeD RCTs, inclusion and exclusion criteria, gluten challenge, and trial outcomes. Two RCT designs were considered, representing the following distinct clinical scenarios for which pharmacotherapy may be used: trials incorporating a gluten challenge to simulate exposure; and trials evaluating reversal of histologic changes, despite attempted adherence to a gluten-free diet. Each statement was rated as appropriate, uncertain, or inappropriate, using a 9-point Likert scale. RESULTS: For trials evaluating prevention of relapse after gluten challenge, participants adherent to a gluten-free diet for 12 months or more with normal or near-normal-sized villi should be enrolled. Gluten challenge should be FODMAPS (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) free, and efficacy evaluated using histology with a secondary patient-reported outcome measure. For trials evaluating reversal of villus atrophy, the panel voted it appropriate to enroll participants with a baseline villus height to crypt depth ratio ≤2 and measure efficacy using a primary histologic end point. Guidance for measuring histologic, endoscopic, and patient-reported outcomes in adult and pediatric patients with CeD are provided, along with recommendations regarding the merits and limitations of different end points. CONCLUSIONS: We developed standardized recommendations for clinical trial design, eligibility criteria, outcome measures, gluten challenge, and disease evaluations for RCTs in patients with CeD.

Vasoactive Intestinal Polypeptide Plays a Key Role in the Microbial-Neuroimmune Control of Intestinal Motility.

BACKGROUND & AIMS: Although chronic diarrhea and constipation are common, the treatment is symptomatic because their pathophysiology is poorly understood. Accumulating evidence suggests that the microbiota modulates gut function, but the underlying mechanisms are unknown. We therefore investigated the pathways by which microbiota modulates gastrointestinal motility in different sections of the alimentary tract. METHODS: Gastric emptying, intestinal transit, muscle contractility, acetylcholine release, gene expression, and vasoactive intestinal polypeptide (VIP) immunoreactivity were assessed in wild-type and Myd88-/-Trif-/- mice in germ-free, gnotobiotic, and specific pathogen-free conditions. Effects of transient colonization and antimicrobials as well as immune cell blockade were investigated. VIP levels were assessed in human full-thickness biopsies by Western blot. RESULTS: Germ-free mice had similar gastric emptying but slower intestinal transit compared with specific pathogen-free mice or mice monocolonized with Lactobacillus rhamnosus or Escherichia coli, the latter having stronger effects. Although muscle contractility was unaffected, its neural control was modulated by microbiota by up-regulating jejunal VIP, which co-localized with and controlled cholinergic nerve function. This process was responsive to changes in the microbial composition and load and mediated through toll-like receptor signaling, with enteric glia cells playing a key role. Jejunal VIP was lower in patients with chronic intestinal pseudo-obstruction compared with control subjects. CONCLUSIONS: Microbial control of gastrointestinal motility is both region- and bacteria-specific; it reacts to environmental changes and is mediated by innate immunity-neural system interactions. By regulating cholinergic nerves, small intestinal VIP plays a key role in this process, thus providing a new therapeutic target for patients with motility disorders.

Comparison of weekly gluten immunogenic peptide measurement and conventional tools to assess adherence to the gluten-free diet in celiac disease: An observational prospective study.

BACKGROUND: Adherence to the gluten-free diet (GFD) is critical to achieving symptom control and mucosal healing in celiac disease (CeD), but its assessment is difficult. OBJECTIVES: We sought to compare stool gluten immunogenic peptides (GIPs) measurements over a 4-wk period with conventional tools commonly used to monitor compliance with a GFD. METHODS: Consecutive adult patients with CeD attending the Small Bowel Section of the Buenos Aires Gastroenterology Hospital were invited to this observational study and were instructed to collect stool samples on Fridays for 4 consecutive weeks. Weekly mean stool GIP concentration was measured was estimated. GIP results were compared with a self-assessment scale of adherence, specific CeD serology, the celiac symptom index, and the assessment by an expert dietitian. RESULTS: Fifty-three CeD patients were enrolled and those with stool GIP ≥0.65 µg/g/wk (n = 13; 24.5%) had higher serum concentrations of IgA deamidated gliadin peptides (DGPs) antibodies [69 (29-109) compared with 14 (13-29); P = 0.0005] and IgA tissue transglutaminase [42 (14-200) compared with 10 (7-16); P = 0.02], higher proportion of cases with IgA DGP antibodies >20 AU/mL (84.6% compared with 33.3%; P = 0.002), and a higher self-estimated adherence score [5 (4-9) compared with 9 (7-10); P = 0.003]. GIP did not correlate with celiac symptom index scores (55.6% compared with 30.8%; P = 0.9). Expert dietitian assessment identified 69% [odds ratio (OR): 5.25; 95% CI: 1.1-27.2; P = 0.01] of nonadherent cases when high stool GIP. Logistic regression analysis determined that IgA DGP (adjusted OR: 1.1; 95% CI: 1.01-1.11; P = 0.02) and males (adjusted OR: 28.3; 95% CI: 1.1-722.6; P = 0.04) were independently associated with excessive gluten exposure. CONCLUSIONS: Weekly stool GIP identifies gluten exposure that is not always detected by commonly used GFD adherence assessment methods. The higher the concentration of stool GIP, the better the predictive value of serology and dietitian interviews. Stool GIP is a useful and practical test for GFD monitoring, particularly for risky gluten exposure in real-life scenarios.

Protection from inflammatory bowel disease.

Immunomodulation of lymphocytes by intestinal epithelial cells could lead to new therapies.

Diet-induced obesity alters intestinal monocyte-derived and tissue-resident macrophages and increases intestinal permeability in female mice independent of tumor necrosis factor.

Macrophages are essential for homeostatic maintenance of the anti-inflammatory and tolerogenic intestinal environment, yet monocyte-derived macrophages can promote local inflammation. Proinflammatory macrophage accumulation within the intestines may contribute to the development of systemic chronic inflammation and immunometabolic dysfunction in obesity. Using a model of high-fat diet-induced obesity in C57BL/6J female mice, we assessed intestinal paracellular permeability by in vivo and ex vivo assays and quantitated intestinal macrophages in ileum and colon tissues by multicolor flow cytometry after short (6 wk), intermediate (12 wk), and prolonged (18 wk) diet allocation. We characterized monocyte-derived CD4-TIM4- and CD4+TIM4- macrophages, as well as tissue-resident CD4+TIM4+ macrophages. Diet-induced obesity had tissue- and time-dependent effects on intestinal permeability, as well as monocyte and macrophage numbers, surface marker phenotype, and intracellular production of the cytokines IL-10 and tumor necrosis factor (TNF). We found that obese mice had increased paracellular permeability, in particular within the ileum, but this did not elicit recruitment of monocytes nor a local proinflammatory response by monocyte-derived or tissue-resident macrophages in either the ileum or colon. Proliferation of monocyte-derived and tissue-resident macrophages was also unchanged. Wild-type and TNF-/- littermate mice had similar intestinal permeability and macrophage population characteristics in response to diet-induced obesity. These data are unique from reported effects of diet-induced obesity on macrophages in metabolic tissues, as well as outcomes of acute inflammation within the intestines. These experiments also collectively indicate that TNF does not mediate effects of diet-induced obesity on paracellular permeability or intestinal monocyte-derived and tissue-resident intestinal macrophages in young female mice.NEW & NOTEWORTHY We found that diet-induced obesity in female mice has tissue- and time-dependent effects on intestinal paracellular permeability as well as monocyte-derived and tissue-resident macrophage numbers, surface marker phenotype, and intracellular production of the cytokines IL-10 and TNF. These changes were not mediated by TNF.

Upper gastrointestinal endoscopic findings in celiac disease at diagnosis: A multicenter international retrospective study.

BACKGROUND: Gastroduodenal endoscopy and biopsy following positive specific serology is considered the gold standard to diagnose celiac disease (CeD) in adults. Whether upper endoscopy helps detect comorbid conditions is unknown. AIM: To investigate the prevalence of non-celiac endoscopic findings in patients in whom endoscopy was performed to confirm CeD diagnosis. METHODS: This is an observational, descriptive, multicenter, retrospective study that reports endoscopic findings obtained in adult patients enrolled in local registries from four tertiary centers. We collected data reported on first endoscopy, indicated for investigation of CeD. Diagnosis of CeD was performed by histology (≥ Marsh 2 type mucosal damage) and specific serology. Two European and one North American center included biopsy-confirmed CeD following positive serology. A fourth center (South America) included symptomatic patients undergoing endoscopy, irrespective of CeD serology. The latter cohort included a non-CeD control group. RESULTS: A total of 1328 patients (80% female; 35 years median age) were enrolled, of whom 95.6% had positive specific serology. In 135 patients, endoscopy revealed 163 abnormalities unrelated to CeD (prevalence: 10.1%). Erosive reflux esophagitis (6.4%), gastric erosions (2.0%), and suspicion of esophageal metaplasia (1.2%) were the most common findings. Biopsy-confirmed Barrett's esophagus was infrequent (0.2%). No endoscopic cancer was detected. Older patients (≥ 51 years of age) had a higher prevalence of endoscopic findings than those ≤ 50 (P < 0.01). Within the South American cohort, CeD was associated with a lower rate (8.2%) of comorbid endoscopic findings compared with controls (29.1%; P < 0.001). In the adjusted multivariate analysis of this cohort, having CeD was associated with a 72% reduction in the risk of any endoscopic abnormality (P < 0.0001), and having alarm symptoms was associated with a 37% reduction in the risk of finding at least one endoscopic lesion (P < 0.02). CONCLUSION: In this large multicenter study, young adults with positive CeD serology had few comorbid endoscopic findings. Although patients over 51 years had a high prevalence of non-CeD gastroduodenal mucosal damage, no malignancy or premalignant lesions were found.

Tet2 deficiency drives liver microbiome dysbiosis triggering Tc1 cell autoimmune hepatitis.

The triggers that drive interferon-γ (IFNγ)-producing CD8 T cell (Tc1 cell)-mediated autoimmune hepatitis (AIH) remain obscure. Here, we show that lack of hematopoietic Tet methylcytosine dioxygenase 2 (Tet2), an epigenetic regulator associated with autoimmunity, results in the development of microbiota-dependent AIH-like pathology, accompanied by hepatic enrichment of aryl hydrocarbon receptor (AhR) ligand-producing pathobionts and rampant Tc1 cell immunity. We report that AIH-like disease development is dependent on both IFNγ and AhR signaling, as blocking either reverts ongoing AIH-like pathology. Illustrating the critical role of AhR-ligand-producing pathobionts in this condition, hepatic translocation of the AhR ligand indole-3-aldehyde (I3A)-releasing Lactobacillus reuteri is sufficient to trigger AIH-like pathology. Finally, we demonstrate that I3A is required for L. reuteri-induced Tc1 cell differentiation in vitro and AIH-like pathology in vivo, both of which are restrained by Tet2 within CD8 T cells. This AIH-disease model may contribute to the development of therapeutics to alleviate AIH.

Coeliac disease.

Coeliac disease is an autoimmune disorder that primarily affects the small intestine, and is caused by the ingestion of gluten in genetically susceptible individuals. Prevalence in the general population ranges from 0·5% to 2%, with an average of about 1%. The development of the coeliac enteropathy depends on a complex immune response to gluten proteins, including both adaptive and innate mechanisms. Clinical presentation of coeliac disease is highly variable and includes classical and non-classical gastrointestinal symptoms, extraintestinal manifestations, and subclinical cases. The disease is associated with a risk of complications, such as osteoporosis and intestinal lymphoma. Diagnosis of coeliac disease requires a positive serology (IgA anti-transglutaminase 2 and anti-endomysial antibodies) and villous atrophy on small-intestinal biopsy. Treatment involves a gluten-free diet; however, owing to the high psychosocial burden of such a diet, research into alternative pharmacological treatments is currently very active.

Gluten-induced RNA methylation changes regulate intestinal inflammation via allele-specific XPO1 translation in epithelial cells.

OBJECTIVES: Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nucleotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. DESIGN: The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m6A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. RESULTS: Individuals harbouring the risk allele had higher m6A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m6A methylation in humans as well as in in vitro and in vivo models. CONCLUSION: We identify a novel m6A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m6A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

Real-World Gluten Exposure in Patients With Celiac Disease on Gluten-Free Diets, Determined From Gliadin Immunogenic Peptides in Urine and Fecal Samples.

BACKGROUND & AIMS: It is not clear how often patients who are on gluten-free diets (GFDs) for treatment of celiac disease still are exposed to gluten. We studied levels of gluten immunogenic peptides (GIP) in fecal and urine samples, collected over 4 weeks, from patients with celiac disease on a long-term GFD. METHODS: We performed a prospective study of 53 adults with celiac disease who had been on a GFD for more than 2 years (median duration, 8 y; interquartile range, 5-12 y) in Argentina. At baseline, symptoms were assessed by the celiac symptom index questionnaire. Patients collected stool each Friday and Saturday and urine samples each Sunday for 4 weeks. We used a commercial enzyme-linked immunosorbent assay to measure GIP in stool and point-of-care tests to measure GIP in urine samples. RESULTS: Overall, 159 of 420 stool and urine samples (37.9%) were positive for GIP; 88.7% of patients had at least 1 fecal or urine sample that was positive for GIP (median, 3 excretions). On weekends (urine samples), 69.8% of patients excreted GIP at least once, compared with 62.3% during weekdays (stool). The number of patients with a sample that was positive for GIP increased over the 4-week study period (urine samples in week 1 vs week 4: P < .05). Patients with symptoms had more weeks in which GIP was detected in stool than patients without symptoms (P < .05). The number of samples that were positive for GIP correlated with titers of deamidated gliadin peptide IgA in patients' blood samples, but not with levels of tissue transglutaminase. CONCLUSIONS: Patients with celiac disease on a long-term GFD still frequently are exposed to gluten. Assays to detect GIP in stool and urine might be used to assist dietitians in assessment of GFD compliance.

Probiotics for Celiac Disease: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

INTRODUCTION: Many patients with celiac disease (CD) experience persistent symptoms despite adhering to the gluten-free diet. Different studies have assessed the use of probiotics as an adjuvant treatment for CD. We performed a systematic review and meta-analysis to evaluate the efficacy of probiotics in improving gastrointestinal (GI) symptoms and quality of life (QOL) in patients with CD. METHODS: We searched EMBASE, MEDLINE, CINAHL, Web of Science, CENTRAL, and DARE databases up to February 2019 for randomized controlled trials (RCTs) evaluating probiotics compared with placebo for treating CD. We collected data on GI symptoms, QOL, adverse events, serum tumor necrosis factor-α, intestinal permeability, and microbiota composition. RESULTS: We screened 2,831 records and found that 7 articles describing 6 RCTs (n = 279 participants) were eligible for quantitative analysis. Probiotics improved GI symptoms when assessed by the GI Symptoms Rating Scale (mean difference symptom reduction: -28.7%; 95% confidence interval [CI] -43.96 to -13.52; P = 0.0002). There was no difference in GI symptoms after probiotics when different questionnaires were pooled. The levels of Bifidobacteria increased after probiotics (mean difference: 0.85 log colony-forming units (CFU) per gram; 95% CI 0.38-1.32 log CFU per gram; P = 0.0003). There were insufficient data on tumor necrosis factor-a levels or QOL for probiotics compared with placebo. No difference in adverse events was observed between probiotics and placebo. The overall certainty of the evidence ranged from very low to low. DISCUSSION: Probiotics may improve GI symptoms in patients with CD. High-quality clinical trials are needed to improve the certainty in the evidence (see Visual abstract, Supplementary Digital Content 2, http://links.lww.com/AJG/B595).

Association Between Inflammatory Bowel Diseases and Celiac Disease: A Systematic Review and Meta-Analysis.

BACKGROUND & AIMS: There is controversy over the association between celiac disease (CeD) and inflammatory bowel diseases (IBD). We performed a systematic review and meta-analysis to assess evidence for an association between CeD and IBD. METHODS: We searched databases including MEDLINE, EMBASE, CENTRAL, Web of Science, CINAHL, DARE, and SIGLE through June 25, 2019 for studies assessing the risk of CeD in patients with IBD, and IBD in patients with CeD, compared with controls of any type. We used the Newcastle-Ottawa Scale to evaluate the risk of bias and GRADE to assess the certainty of the evidence. RESULTS: We identified 9791 studies and included 65 studies in our analysis. Moderate certainty evidence found an increased risk of CeD in patients with IBD vs controls (risk ratio [RR] 3.96; 95% confidence interval [CI] 2.23-7.02) and increased risk of IBD in patients with CeD vs controls (RR 9.88; 95% CI 4.03-24.21). There was low-certainty evidence for the risk of anti-Saccharomyces antibodies, a serologic marker of IBD, in patients with CeD vs controls (RR 6.22; 95% CI 2.44-15.84). There was low-certainty evidence for no difference in risk of HLA-DQ2 or DQ8 in patients with IBD vs controls (RR 1.04; 95% CI 0.42-2.56), and very low-certainty evidence for an increased risk of anti-tissue transglutaminase in patients with IBD vs controls (RR 1.52; 95% CI 0.52-4.40). Patients with IBD had a slight decrease in risk of anti-endomysial antibodies vs controls (RR 0.70; 95% CI 0.18-2.74), but these results are uncertain. CONCLUSIONS: In a systematic review and meta-analysis, we found an increased risk of IBD in patients with CeD and increased risk of CeD in patients with IBD, compared with other patient populations. High-quality prospective cohort studies are needed to assess the risk of CeD-specific and IBD-specific biomarkers in patients with IBD and CeD.

Investigation of the Gut Microbiome in Patients with Schizophrenia and Clozapine-Induced Weight Gain: Protocol and Clinical Characteristics of First Patient Cohorts.

BACKGROUND: Emerging evidence suggests an important role of the human gut microbiome in psychiatry and neurodevelopmental disorders. An increasing body of literature based on animal studies has reported that the gut microbiome influences brain development and behavior by interacting with the gut-brain axis. Furthermore, as the gut microbiome has an important role in metabolism and is known to interact with pharmaceuticals, recent evidence suggests a role for the microbiome in antipsychotic-induced metabolic side effects in animals and humans. PURPOSE: Here we present the protocol for a two-phase study investigating the gut microbiome in healthy controls and in patients with schizophrenia treated with antipsychotics. METHODS: Phase I of our study involves humans exclusively. We recruit 25 patients who are chronically treated with clozapine and compare them with 25 healthy controls matched for age, sex, BMI, and smoking status. A second cohort consists of 25 patients newly starting on clozapine, and a third cohort includes 25 antipsychotic-naive patients. The patients in the second cohort and third cohort are prospectively assessed for up to 6 and 12 weeks, respectively. Phase II of this study will incorporate microbiota humanized mouse models to examine the influence of human fecal transplant on metabolic parameters and the gut-brain axis. Progress and Future Directions: We are underway with the first participants enrolled in all phase I treatment cohorts. This study will contribute to elucidating the role of the gut microbiome in schizophrenia and metabolic side effects. In addition, its results may help to explore potential therapeutic targets for antipsychotic-induced metabolic side effects.

Active thrombin produced by the intestinal epithelium controls mucosal biofilms.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Gluten immunogenic peptide excretion detects dietary transgressions in treated celiac disease patients.

BACKGROUND: Life-long removal of gluten from the diet is currently the only way to manage celiac disease (CeD). Until now, no objective test has proven useful to objectively detect ingested gluten in clinical practice. Recently, tests that determine consumption of gluten by assessing excretion of gluten immunogenic peptides (GIP) in stool and urine have been developed. Their utility, in comparison with conventional dietary and analytical follow-up strategies, has not been fully established. AIM: To assess the performance of enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (PoCTs) for GIP excretion in CeD patients on gluten-free diet (GFD). METHODS: We conducted an observational, prospective, cross-sectional study in patients following a GFD for at least two years. Using the Gastrointestinal Symptom Rating Scale questionnaire, patients were classified at enrollment as asymptomatic or symptomatic. Gluten consumption was assessed twice by 3-d dietary recall and GIP excretion (by ELISA in stool and PoCTs (commercial kits for stool and urine) in two consecutive samples. These samples and dietary reports were obtained 10 day apart one from the other. Patients were encouraged to follow their usual GFD during the study period. RESULTS: Forty-four patients were enrolled, of which 19 (43.2%) were symptomatic despite being on a GFD. Overall, 83 sets of stool and/or urine samples were collected. Eleven out of 44 patients (25.0%) had at least one positive GIP test. The occurrence of at least one positive test was 32% in asymptomatic patients compared with 15.8% in symptomatic patients. GIP was concordant with dietary reports in 65.9% of cases (Cohen´s kappa: 0.317). PoCT detected dietary indiscretions. Both ELISA and PoCT in stool were concordant (concomitantly positive or negative) in 67 out of 74 (90.5%) samples. Excretion of GIP was detected in 7 (8.4%) stool and/or urine samples from patients considered to be strictly compliant with the GFD by dietary reports. CONCLUSION: GIP detects dietary transgressions in patients on long-term GFD, irrespective of the presence of symptoms. PoCT for GIP detection constitutes a simple home-based method for self-assessment of dietary indiscretions.

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Inflammation-related differences in mucosa-associated microbiota and intestinal barrier function in colonic Crohn's disease.

Crohn's disease (CD), characterized by discontinuous intestinal injury and inflammation, has been associated with changes in luminal microbial composition and impaired barrier function. The relationships between visual features of intestinal injury, permeability, and the mucosa-associated microbiota are unclear. Individuals undergoing routine colonoscopy (controls) and patients with CD were evaluated by clinical parameters and confocal laser scanning endomicroscopic colonoscopy (CLE). Patients with CD were categorized as either CD with no injury (CD-NI) or CD with injury (CD-I). Colonic biopsies were taken from adjacent matched sites in all individuals, and CLE images from these sites were analyzed for vascular permeability. Microbial composition was evaluated by 16S rRNA gene sequencing of the V3 region, and the mycome was identified through internal transcribed spacer 2 sequencing. Subgroup analyses were performed for histology, paracellular permeability (Ussing chamber), and encroachment of bacteria (fluorescent in situ hybridization). CD-I patients showed an altered microbial community compared with both controls and CD-NI patients, with enrichment in Escherichia and a decrease in Firmicutes, including Lachnospira, Faecalibacterium, and Blautia. In CD-I patients, bacterial encroachment to host epithelial cells was greater in sites of injury than in matched biopsy sites. Biopsies from sites of injury also demonstrated greater vascular and paracellular permeability. Overall, CD-I patients showed an altered mucosal microbial community compared with CD-NI patients and controls. Matched biopsy samples in CD-I patients revealed that sites of injury, identified endoscopically, are characterized by increased encroachment of bacteria to host epithelial cells, associated with increased paracellular and vascular permeability, which may drive inflammation in CD. NEW & NOTEWORTHY Patients with Crohn's disease (CD) with areas of colonic injury have an altered microbial community compared with patients who have no endoscopic evidence of injury or active disease. Although matched biopsies from patients with colonic injury show no differences in the mucosa-associated microbiota, injured sites are associated with increased permeability and increased encroachment. Our results support the notion that dysbiotic communities within patients with colonic injury cause or permit disruption of the mucosal and endothelial layers in CD.