Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet.

BACKGROUND: The gluten-free diet (GFD) has limitations, and there is intense research in the development of adjuvant therapies. AIM: To examine the effects of orally administered Aspergillus niger prolyl endopeptidase protease (AN-PEP) on inadvertent gluten exposure and symptom prevention in adult celiac disease (CeD) patients following their usual GFD. METHODS: This was an exploratory, double-blind, randomized, placebo-controlled trial that enrolled CeD patients on a long-term GFD. After a 4-wk run-in period, patients were randomized to 4 wk of two AN-PEP capsules (GliadinX; AVI Research, LLC, United States) at each of three meals per day or placebo. Outcome endpoints were: (1) Average weekly stool gluten immunogenic peptides (GIP) between the run-in and end of treatments and between AN-PEP and placebo; (2) celiac symptom index (CSI); (3) CeD-specific serology; and (4) quality of life. Stool samples were collected for GIP testing by ELISA every Tuesday and Friday during run-ins and treatments. RESULTS: Forty patients were randomized for the intention-to-treat analysis, and three were excluded from the per-protocol assessment. Overall, 628/640 (98.1%) stool samples were collected. GIP was undetectable (< 0.08 µg/g) in 65.6% of samples, and no differences between treatment arms were detected. Only 0.5% of samples had GIP concentrations sufficiently high (> 0.32 µg/g) to potentially cause mucosal damage. Median GIP concentration in the AN-PEP arm was 44.7% lower than in the run-in period. One-third of patients exhibiting GIP > 0.08 µg/g during run-in had lower or undetectable GIP after AN-PEP treatment. Compared with the run- in period, the proportion of symptomatic patients (CSI > 38) in the AN-PEP arm was significantly lower (P < 0.03). AN-PEP did not result in changes in specific serologies. CONCLUSION: This exploratory study conducted in a real-life setting revealed high adherence to the GFD. The AN-PEP treatment did not significantly reduce the overall GIP stool concentration. However, given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm, further clinical research is warranted.

Oral tryptophan activates duodenal aryl hydrocarbon receptor in healthy subjects: a crossover randomized controlled trial.

Background and Aims: Tryptophan is an essential amino acid transformed by host and gut microbial enzymes into metabolites that regulate mucosal homeostasis through Aryl hydrocarbon receptor (AhR) activation. Alteration of tryptophan metabolism has been associated with chronic inflammation, however whether tryptophan supplementation affects the metabolite repertoire and AhR activation under physiologic conditions in humans, is unknown. Methods: We performed a randomized, double blind, placebo-controlled, crossover study in 20 healthy volunteers. Subjects on a low tryptophan background diet were randomly assigned to a 3-week L-tryptophan supplementation (3 g/day) or placebo, and after a 2-week washout switched to opposite interventions. We assessed gastrointestinal and psychological symptoms by validated questionnaires, AhR activation by cell reporter assay, tryptophan metabolites by liquid chromatography and high-resolution mass spectrometry, cytokine production in isolated monocytes by ELISA and microbiota profile by 16S rRNA Illumina technique. Results: Oral tryptophan supplementation was well tolerated, with no changes in gastrointestinal or psychological scores. Compared with placebo, tryptophan increased AhR activation capacity by duodenal contents, but not by feces. This was paralleled by higher urinary and plasma kynurenine metabolites and indoles. Tryptophan had a modest impact on fecal microbiome profiles, and no significant effect on cytokine production. Conclusions: At the doses used in this study, oral tryptophan supplementation in humans induces microbial indole and host kynurenine metabolic pathways in the small intestine, known to be immunomodulatory. The results should prompt tryptophan intervention strategies in inflammatory conditions of the small intestine where the AhR pathway is impaired.

Evaluating the Effects of Chronic Oral Exposure to the Food Additive Silicon Dioxide on Oral Tolerance Induction and Food Sensitivities in Mice.

BACKGROUND: The increasing prevalence of food sensitivities has been attributed to changes in gut microenvironment; however, ubiquitous environmental triggers such as inorganic nanoparticles (NPs) used as food additives have not been thoroughly investigated. OBJECTIVES: We explored the impact of the NP-structured food-grade silicon dioxide (fg-SiO2) on intestinal immune response involved in oral tolerance (OT) induction and evaluated the consequences of oral chronic exposure to this food-additive using a mouse model of OT to ovalbumin (OVA) and on gluten immunopathology in mice expressing the celiac disease risk gene, HLA-DQ8. METHODS: Viability, proliferation, and cytokine production of mesenteric lymph node (MLN) cells were evaluated after exposure to fg-SiO2. C57BL/6J mice and a mouse model of OT to OVA were orally exposed to fg-SiO2 or vehicle for 60 d. Fecal lipocalin-2 (Lcn-2), anti-OVA IgG, cytokine production, and immune cell populations were analyzed. Nonobese diabetic (NOD) mice expressing HLA-DQ8 (NOD/DQ8), exposed to fg-SiO2 or vehicle, were immunized with gluten and immunopathology was investigated. RESULTS: MLN cells exposed to fg-SiO2 presented less proliferative T cells and lower secretion of interleukin 10 (IL-10) and transforming growth factor beta (TGF-ß) by T regulatory and CD45+ CD11b+ CD103+ cells compared to control, two factors mediating OT. Mice given fg-SiO2 exhibited intestinal Lcn-2 level and interferon gamma (IFN-γ) secretion, showing inflammation and less production of IL-10 and TGF-ß. These effects were also observed in OVA-tolerized mice exposed to fg-SiO2, in addition to a breakdown of OT and a lower intestinal frequency of T cells. In NOD/DQ8 mice immunized with gluten, the villus-to-crypt ratio was decreased while the CD3+ intraepithelial lymphocyte counts and the Th1 inflammatory response were aggravated after fg-SiO2 treatment. DISCUSSION: Our results suggest that chronic oral exposure to fg-SiO2 blocked oral tolerance induction to OVA, and worsened gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between SiO2 exposure and food sensitivities in humans. https://doi.org/10.1289/EHP12758.

Vascular dysfunction and arterial hypertension in experimental celiac disease are mediated by gut-derived inflammation and oxidative stress.

AIMS: We examined the cardiovascular effects of celiac disease (CeD) in a humanized mouse model, with a focus on vascular inflammation, endothelial dysfunction, and oxidative stress. METHODS AND RESULTS: NOD.DQ8 mice genetically predisposed to CeD were subjected to a diet regime and oral gavage to induce the disease (gluten group vs. control). We tested vascular function, confirmed disease indicators, and evaluated inflammation and oxidative stress in various tissues. Plasma proteome profiling was also performed. CeD markers were confirmed in the gluten group, indicating increased blood pressure and impaired vascular relaxation. Pro-inflammatory genes were upregulated in this group, with increased CD11b+ myeloid cell infiltration and oxidative stress parameters observed in aortic and heart tissue. However, heart function remained unaffected. Plasma proteomics suggested the cytokine interleukin-17A (IL-17A) as a link between gut and vascular inflammation. Cardiovascular complications were reversed by adopting a gluten-free diet. CONCLUSION: Our study sheds light in the heightened cardiovascular risk associated with active CeD, revealing a gut-to-cardiovascular inflammatory axis potentially mediated by immune cell infiltration and IL-17A. These findings augment our understanding of the link between CeD and cardiovascular disease providing clinically relevant insight into the underlying mechanism. Furthermore, our discovery that cardiovascular complications can be reversed by a gluten-free diet underscores a critical role for dietary interventions in mitigating cardiovascular risks associated 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.

Guidelines for best practices in monitoring established coeliac disease in adult patients.

Coeliac disease (CeD) is an immunological disease triggered by the consumption of gluten contained in food in individuals with a genetic predisposition. Diagnosis is based on the presence of small bowel mucosal atrophy and circulating autoantibodies (anti-type 2 transglutaminase antibodies). After diagnosis, patients follow a strict, life-long gluten-free diet. Although the criteria for diagnosis of this disease are well defined, the monitoring phase has been studied less and there is a lack of specific guidelines for this phase. To develop a set of clinical guidelines for CeD monitoring, we followed the Grading of Recommendations Assessment, Development and Evaluation methodology. Statements and recommendations with the level of evidence were developed and approved by the working group, which comprised gastroenterologists, pathologists, dieticians and biostatisticians. The proposed guidelines, endorsed by the North American and European coeliac disease scientific societies, make recommendations for best practices in monitoring patients with CeD based on the available evidence. The evidence level is low for many topics, suggesting that further research in specific aspects of CeD would be valuable. In conclusion, the present guidelines support clinicians in improving CeD treatment and follow-up and highlight novel issues that should be considered in future studies.

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.

Elucidating the role of microbes in celiac disease through gnotobiotic modeling.

Celiac disease (CeD) is a common immune-mediated disease triggered by the ingestion of gluten in genetically predisposed individuals. CeD is unique in that the trigger (gluten), necessary genes (HLA-DQ2 and DQ8), and the autoantigen (tissue transglutaminase) have been identified, allowing additional environmental co-factors, like the intestinal microbiota, to be studied through relevant in vivo models. Murine models for CeD have come a long way in the past decade and there are now in vitro and in vivo tools available that mimic certain aspects of clinical disease. These models, many of which express the CeD risk genes, have recently been used to study the mechanisms through which the microbiota play a role in CeD pathogenesis through a gnotobiotic approach. Historically, the generation of gnotobiology technology in mid-20th century allowed for the study of immunity and physiology under a complete absence of microbes (axenic) or known colonized status (gnotobiotic). This enabled understanding of mechanisms by which certain bacteria contribute to health and disease. With this perspective, here, we will discuss the various murine models currently being used to study CeD. We will then describe how utilizing axenic and gnotobiotic CeD models has increased our understanding of how microbes influence relevant steps of CeD pathogenesis, and explain key methodology involved in axenic and gnotobiotic modeling.

The gut protist Tritrichomonas arnold restrains virus-mediated loss of oral tolerance by modulating dietary antigen-presenting dendritic cells.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.

Crohn's disease proteolytic microbiota enhances inflammation through PAR2 pathway in gnotobiotic mice.

Emerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn's disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2-/-; R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2-/- mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway.

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.