Gluten Challenge Research Articles

Pathogenic T Cells in Celiac Disease Change Phenotype on Gluten Challenge: Implications for T-Cell-Directed Therapies.

Gluten‐specific CD4+ T cells being drivers of celiac disease (CeD) are obvious targets for immunotherapy. Little is known about how cell markers harnessed for T‐cell‐directed therapy can change with time and upon activation in CeD and other autoimmune conditions. In‐depth characterization of gluten‐specific CD4+ T cells and CeD‐associated (CD38+ and CD103+) CD8+ and γδ + T cells in blood of treated CeD patients undergoing a 3 day gluten challenge is reported. The phenotypic profile of gluten‐specific cells changes profoundly with gluten exposure and the cells adopt the profile of gluten‐specific cells in untreated disease (CD147+, CD70+, programmed cell death protein 1 (PD‐1)+, inducible T‐cell costimulator (ICOS)+, CD28+, CD95+, CD38+, and CD161+), yet with some markers being unique for day 6 cells (C‐X‐C chemokine receptor type 6 (CXCR6), CD132, and CD147) and with integrin α4β7, C‐C motif chemokine receptor 9 (CCR9), and CXCR3 being expressed stably at baseline and day 6. Among gluten‐specific CD4+ T cells, 52% are CXCR5+ at baseline, perhaps indicative of germinal‐center reactions, while on day 6 all are CXCR5−. Strikingly, the phenotypic profile of gluten‐specific CD4+ T cells on day 6 largely overlaps with that of CeD‐associated (CD38+ and CD103+) CD8+ and γδ + T cells. The antigen‐induced shift in phenotype of CD4+ T cells being shared with other disease‐associated T cells is relevant for development of T‐cell‐directed therapies.

Larazotide acetate for treatment of celiac disease: A systematic review and meta-analysis of randomized controlled trials

PurposeThe standard of care for treatment of celiac disease (CD) is a stringent lifetime gluten-free diet (GFD). Larazotide acetate (AT-1001) is an anti-zonulin which functions as a gut permeability regulator for treatment of CD. We endeavored to conduct a systematic review and meta-analysis of all randomized controlled trials (RCTs) which studied the efficacy and safety of AT-1001 in patients with CD. MethodsWe examined four databases from inception to 20-August-2020. We pooled continuous outcomes as mean difference and dichotomous outcomes as risk ratio with 95% confidence interval under the fixed-effects meta-analysis model. ResultsFour RCTs met our eligibility criteria, comprising 626 patients (AT-1001, n=465, placebo, n=161). Three and two RCTs reported outcomes of patients undergoing gluten challenge (intake of 2.4–2.7 grams of gluten/day) and GFD, respectively. For change in lactulose-to-mannitol ratio, the endpoint did not significantly differ between AT-1001 and placebo groups, irrespective of the gluten status. Subgroup analysis of patients undergoing gluten challenge showed AT-1001 treatment (compared with placebo) significantly correlated with better symptomatic improvement in the two endpoints of change in total gastrointestinal symptom rating scale (total GSRS) and CD-specific GSRS (CD-GSRS). However, no significant difference was noted among patients undergoing GFD for the abovementioned two efficacy endpoints. Compared with placebo, AT-1001 favorably reduced the adverse event (AE) of gluten-related diarrhea in patients who underwent gluten challenge. Other AEs were comparable between both AT-1001 and placebo groups. ConclusionsAT-1001 is largely well-endured and seems somehow superior to placebo in alleviating gastrointestinal symptoms among CD patients undergoing gluten challenge. Nevertheless, additional RCTs are warranted to validate these findings.

Non-Celiac Gluten Sensitivity in Children: Controversial Role of Gluten and Diagnostic Enigma

Non-celiac gluten sensitivity (NCGS) is a debatable condition that affects less than 6% of children. The absence of specific diagnostic markers and standardized diagnostic procedures make the diagnosis of NCGS challenging, covering patients with different and varied symptoms. Generally, the parents of small and younger children introduce a gluten-free diet (GFD) based on their personal experiences and expectations. Additionally, a “fad component” exists, contributing to the recent rise in the popularity of GFD. Thus, celiac disease (CD) and wheat allergy (WA) must be excluded as these also appear in individuals experiencing symptoms similar to those of NCGS, improving and worsening with gluten withdrawal and consumption, respectively. The role of gluten inducing gastrointestinal symptoms in individuals with self-reported NCGS has been skeptically assessed based on evidence in recent years. However, currently, it is unknown whether a strict GFD is necessary for patients with NCGS. Thus, the placebo-controlled gluten challenge remains the gold standard for a challenging diagnosis like NCGS. The present review evaluates the published studies based largely on the adult population and describes the key elements in diagnosing NCGS and differential diagnosis with CD and WA.

A Randomized Trial of a Transglutaminase 2 Inhibitor for Celiac Disease

BackgroundIn celiac disease, small intestinal transglutaminase 2 causes deamidation of glutamine residues in gluten peptides, which enhances stimulation of T cells and leads to mucosal injury. Inhibition of transglutaminase 2 is a potential treatment for celiac disease.MethodsIn a proof-of-concept trial, we assessed the efficacy and safety of a 6-week treatment with ZED1227, a selective oral transglutaminase 2 inhibitor, at three dose levels as compared with placebo, in adults with well-controlled celiac disease who underwent a daily gluten challenge. The primary end point was the attenuation of gluten-induced mucosal damage, as measured by the ratio of villus height to crypt depth. Secondary end points included intraepithelial lymphocyte density, the Celiac Symptom Index score, and the Celiac Disease Questionnaire score (for assessment of health-related quality of life).ResultsOf the 41 patients assigned to the 10-mg ZED1227 group, the 41 assigned to the 50-mg group, the 41 assigned to the 100-mg group, and the 40 assigned to the placebo group, 35, 39, 38, and 30 patients, respectively, had adequate duodenal-biopsy samples for the assessment of the primary end point. Treatment with ZED1227 at all three dose levels attenuated gluten-induced duodenal mucosal injury. The estimated difference from placebo in the change in the mean ratio of villus height to crypt depth from baseline to week 6 was 0.44 (95% confidence interval [CI], 0.15 to 0.73) in the 10-mg group (P=0.001), 0.49 (95% CI, 0.20 to 0.77) in the 50-mg group (P<0.001), and 0.48 (95% CI, 0.20 to 0.77) in the 100-mg group (P<0.001). The estimated differences from placebo in the change in intraepithelial lymphocyte density were −2.7 cells per 100 epithelial cells (95% CI, −7.6 to 2.2) in the 10-mg group, −4.2 cells per 100 epithelial cells (95% CI, −8.9 to 0.6) in the 50-mg group, and −9.6 cells per 100 epithelial cells (95% CI, −14.4 to −4.8) in the 100-mg group. Use of the 100-mg dose may have improved symptom and quality-of-life scores. The most common adverse events, the incidences of which were similar across all groups, were headache, nausea, diarrhea, vomiting, and abdominal pain. Rash developed in 3 of 40 patients (8%) in the 100-mg group.ConclusionsIn this preliminary trial, treatment with ZED1227 attenuated gluten-induced duodenal mucosal damage in patients with celiac disease. (Funded by Dr. Falk Pharma; CEC-3 EudraCT number, 2017-002241-30.)

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Non-Celiac Gluten Sensitivity: An Update.

Non-celiac gluten sensitivity (NCGS) is a clinical entity characterized by the absence of celiac disease and wheat allergy in patients that trigger reproducible symptomatic responses to gluten-containing foods consumption. Due to the lack of sensitive and reproducible biomarkers for NCGS diagnosis, placebo-controlled gluten challenges must be carried out for its diagnosis. The gluten challenges can be either double- or single-blind, for research or clinical practice purposes, respectively. For improving our understanding about the magnitude and relevance of NCGS in different populations, epidemiological studies based on self-report have been carried out. However, the gluten challenge-based prevalence of NCGS remains to be estimated. Since NCGS was recently recognized as a clinical entity, more studies are needed to delve into NCGS pathogenesis, for instance, the molecular interactions between the suspected cereal grain components that trigger NCGS, such as fermentable oligo-, di-, monosaccharides, and polyols (FODMAPs) and amylase and trypsin inhibitors, and the immune system remains to be elucidated. Although still under debate, NCGS patients can be susceptible to only one or more than one of the NCGS triggers. The treatment of NCGS involves the dietary restriction of the suspected triggers of the disease, but there is controversial data about the effectiveness of different dietary interventions such as the gluten-free diet and low-FODMAP diet. Certainly, our understanding of NCGS is improving quickly due to the constant availability of new scientific information on this topic. Thus, the aim of the present narrative review is to present an up-to-date overview on NCGS from epidemiology to current therapy.

Non-Celiac Gluten/Wheat Sensitivity: Clinical Characteristics and Microbiota and Mycobiota Composition by Response to the Gluten Challenge Test.

The aims of this observational “proof-of-concept” study were to analyze the clinical/psychological characteristics and gut microbiota/mycobiota composition of individuals with suspected non-celiac gluten/wheat sensitivity (NCGS/WS) according to responses to the double-blind-placebo-controlled (DBPC) crossover gluten challenge test. Fifty individuals with suspected NCGS/WS were subjected to the DBPC challenge test; anthropometric measurements, psychometric questionnaires, and fecal samples were collected. Twenty-seven (54%) participants were gluten responsive (NCGS), and 23 were placebo responsive, with an order effect. NCGS individuals displayed a significantly lower risk of eating disorders and a higher mental health score when compared to placebo-responsive participants, confirmed by multiple logistic regression analyses (OR = 0.87; 95% CI 0.76–0.98, p = 0.021, and OR = 1.30; 95% CI 1.06–1.59, p = 0.009, respectively). Principal coordinate analyses based on microbiota composition showed a separation by the DBPC response (p = 0.039). For Bacteroides (p = 0.05) and Parabacteroides (p = 0.007), the frequency of amplicon sequence variants was lower, and that for Blautia (p = 0.009) and Streptococcus (p = 0.004) was higher in NCGS individuals at multiple regression analyses. No difference in the mycobiota composition was detected between the groups. In conclusion, almost half of the individuals with suspected gluten sensitivity reported symptoms with placebo; they showed lower mental health scores, increased risk for eating disorders, and a different gut microbiota composition.

The Evolving Landscape of Biomarkers in Celiac Disease: Leading the Way to Clinical Development.

Celiac disease is a common immune-mediated disease characterized by abnormal T-cell responses to gluten. For many patients, symptoms and intestinal damage can be controlled by a gluten-free diet, but, for some, this approach is not enough, and celiac disease progresses, with serious medical consequences. Multiple therapies are now under development, increasing the need for biomarkers that allow identification of specific patient populations and monitoring of therapeutic activity and durability. The advantage of identifying biomarkers in celiac disease is that the underlying pathways driving disease are well characterized and the histological, cellular, and serological changes with gluten response have been defined in gluten challenge studies. However, there is room for improvement. Biomarkers that measure histological changes require duodenal biopsies and are invasive. Less invasive peripheral blood cell and cytokine biomarkers are transient and dependent upon gluten challenge. Here, we discuss established biomarkers and new approaches for biomarkers that may overcome current limitations.

TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac Disease: A Randomized, Double-Blind, Placebo-Controlled Study

In celiac disease (CeD), gluten induces immune activation, leading to enteropathy. TAK-101, gluten protein (gliadin) encapsulated in negatively charged poly(dl-lactide-co-glycolic acid) nanoparticles, is designed to induce gluten-specific tolerance. TAK-101 was evaluated in phase 1 dose escalation safety and phase 2a double-blind, randomized, placebo-controlled studies. Primary endpoints included pharmacokinetics, safety, and tolerability of TAK-101 (phase 1) and change from baseline in circulating gliadin-specific interferon-γ-producing cells at day 6 of gluten challenge, in patients with CeD (phase 2a). Secondary endpoints in the phase 2a study included changes from baseline in enteropathy (villus height to crypt depth ratio [Vh:Cd]), and frequency of intestinal intraepithelial lymphocytes and peripheral gut-homing T cells. In phase 2a, 33 randomized patients completed the 14-day gluten challenge. TAK-101 induced an 88% reduction in change from baseline in interferon-γ spot-forming units vs placebo (2.01 vs 17.58, P= .006). Vh:Cd deteriorated in the placebo group (-0.63, P= .002), but not in the TAK-101 group (-0.18, P= .110), although the intergroup change from baseline was not significant (P= .08). Intraepithelial lymphocyte numbers remained equal. TAK-101 reduced changes in circulating α4β7+CD4+ (0.26 vs 1.05, P= .032), αEβ7+CD8+ (0.69 vs 3.64, P= .003), and γδ (0.15 vs 1.59, P= .010) effector memory T cells. TAK-101 (up to 8 mg/kg) induced no clinically meaningful changes in vital signs or routine clinical laboratory evaluations. No serious adverse events occurred. TAK-101 was well tolerated and prevented gluten-induced immune activation in CeD. The findings from the present clinical trial suggest that antigen-specific tolerance was induced and represent a novel approach translatable to other immune-mediated diseases. ClinicalTrials.gov identifiers: NCT03486990 and NCT03738475.

Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease.

Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n=6; cohort 2, n=12) and unaffected controls (cohort 3, n=9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3h, 6h and 6days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

In Well-Treated Celiac Patients Low-Level Mucosal Inflammation Predicts Response to 14-day Gluten Challenge.

In celiac disease (CeD), gluten activates adaptive immune cells that cause damage to the small intestinal mucosa. Histological evaluation of intestinal biopsies allows for grading of disease severity. CeD can effectively be treated with a life‐long gluten‐free diet. Gluten challenge of treated CeD patients is used to confirm diagnosis and to test drug efficacy in clinical trials, but patients respond with different magnitudes to the same gluten challenge. In this study of 19 well‐treated CeD patients, proteome analysis of total tissue or isolated epithelial cell compartment from formalin‐fixed paraffin embedded biopsies collected before and after 14‐day gluten challenge demonstrates that patients with strong mucosal response to challenge have signs of ongoing tissue inflammation already before challenge. This low‐level tissue inflammation at baseline is paralleled by increased gluten specific CD4+ T‐cell frequencies in the gut and presence of a low‐level blood inflammatory profile. Thus, apparently well‐treated CeD is frequently not entirely quiescent, with presence of low‐grade inflammation and antigluten immunity in the gut mucosa. Histology assessment alone appears insufficient to judge full recovery and gut mucosal healing of CeD patients. The findings raise a concern whether a seemingly proper gluten‐free diet is able to curb gut inflammation in all CeD patients.

Effects of In Vivo Gluten Challenge on PBMC Gene Expression Profiles in Diet Treated Celiac Disease.

The pathological mechanisms that lead to the onset and reactivation of celiac disease (CD) remain largely unknown. While gluten free diet (GFD) improves the intestinal damage and associated clinical symptoms in majority of cases, it falls short of providing full recovery. Additionally, late or misdiagnosis is also common as CD presents with a wide range of symptoms. Clear understanding of CD pathogenesis is thus critical to address both diagnostic and treatment concerns. We aimed to study the molecular impact of short gluten exposure in GFD treated CD patients, as well as identify biological pathways that remain altered constitutively in CD regardless of treatment. Using RNAseq profiling of PBMC samples collected from treated CD patients and gluten challenged patient and healthy controls, we explored the peripheral transcriptome in CD patients following a short gluten exposure. Short gluten exposure of just three days was enough to alter the genome-wide PBMC transcriptome of patients. Pathway analysis revealed gluten-induced upregulation of mainly immune response related pathways, both innate and adaptive, in CD patients. We evaluated the perturbation of biological pathways in sample-specific manner. Compared to gluten exposed healthy controls, pathways related to tight junction, olfactory transduction, metabolism of unsaturated fatty acids (such as arachidonic acid), metabolism of amino acids (such as cysteine and glutamate), and microbial infection were constitutively altered in CD patients regardless of treatment, while GFD treatment appears to mostly normalize immune response pathways to “healthy” state. Upstream regulator prediction analysis using differentially expressed genes identified constitutively activated regulators relatively proximal to previously reported CD associated loci, particularly SMARCA4 on 19p13.2 and CSF2 on 5q31. We also found constitutively upregulated genes in CD that are in CD associated genetic loci such as MEF2BNB-MEF2B (BORCS8-MEF2B) on 19p13.11 and CSTB on 21q22.3. RNAseq revealed strong effects of short oral gluten challenge on whole PBMC fraction and constitutively altered pathways in CD PBMC suggesting important factors other than gluten in CD pathogenesis.

T-Cell Response in Dermatitis Herpetiformis: May Epidermal Transglutaminase Play a Role in Predicting Clinical Relapse?

We read with interest the paper by Kalliokoski et al. (2020) investigating whether PBMCs collected from patients with dermatitis herpetiformis (DH) undergoing a 3-day oral gluten challenge react to gluten-derived gliadin in the enzyme-linked immunospot (ELISpot) assay similarly to those from patients with celiac disease (CD). Interestingly, the authors found that on stimulation with tissue transglutaminase (TG)-gliadin or either of the gliadin peptides, 7 of the 15 patients (47%) with DH and 15 of the 18 patients (83%) with CD were considered responders in the IFN-γ ELISpot assay.

Randomized, Placebo Controlled Trial of Experimental Hookworm Infection for Improving Gluten Tolerance in Celiac Disease

INTRODUCTION:Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled.METHODS:We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year).RESULTS:Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1–9); L3-20, 1 (0–9); and L3-40, 0 (0–3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, −0.6 [−1.3 to 0.2]; L3-20, −0.5 [−0.8 to 0.2]; and L3-40, −1.1 [−1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9–44] vs 45.9 [39.2–52.6]).DISCUSSION:Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Patient factors influencing acute gluten reactions and cytokine release in treated coeliac disease

BackgroundPatients with coeliac disease (CD) commonly report a variety of adverse symptoms to gluten, but descriptions of the symptomatic response in the literature may have been confounded by the presence of food components such as fermentable carbohydrates (FODMAPs) causing symptoms of irritable bowel syndrome independent of gluten. In recent unmasked and masked low FODMAP gluten challenge studies in small groups of treated CD patients, nausea and vomiting were shown to be the key symptoms associated with serum interleukin (IL)-2 release. Our objective was to utilise a large and diverse cohort of people with CD undertaking a standardised gluten food challenge to characterise the demographic, genetic and clinical factors influencing the severity and timing of acute gluten reactions and IL-2 production.MethodsA total of 295 adults treated for CD were observed for 6 h after an unmasked food challenge consisting of 10 g vital wheat gluten (low in FODMAPs) in 100 ml water. Assessments included patient-reported outcomes, serum IL-2 and adverse events. Responses were analysed according to patient characteristics, HLA-DQ genotype, duodenal histology and response to a second gluten challenge.ResultsPeak symptom severity was at 3 h (median severity 5/10). Peak IL-2 was at 4 h (median 4 pg/ml, range undetectable to 1028 pg/ml). Older age, older age at diagnosis, HLA-DQ2.5 positivity and homozygosity for HLA-DQB1*02 were each significantly associated with IL-2 elevations after gluten. Patients positive for HLA-DQ2.5, DQ8, DQ2.2 or DQ7 showed elevated IL-2 after gluten. Patient factors were not significantly associated with severity of digestive symptoms, but symptoms were correlated to one another and serum IL-2. Gluten challenge after 5 months caused more vomiting and higher IL-2 levels, but responses correlated with the first.ConclusionsGluten-induced symptoms and cytokine release is common in adults with treated CD. Age, genetics and previous response to gluten predict these acute reactions to gluten challenge. Structured symptom assessment and serum IL-2 after standardised gluten challenge may inform on patient diagnosis, the role of gluten in symptomatology and the need for adjunctive treatment.Trial registrationClinicalTrials.gov, NCT03644069 Registered 21 May 2018.

Evaluating Responses to Gluten Challenge: A Randomized, Double-Blind, 2-Dose Gluten Challenge Trial

Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology. In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence. All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot,gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or nearsignificantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure. Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.

The gluten challenge is still the best option for children with a difficult diagnosis like non-coeliac gluten sensitivity.

Gluten-related diseases, such as coeliac disease, wheat allergy and non-coeliac gluten sensitivity, are increasingly being diagnosed in children. Non-coeliac gluten sensitivity is an unclear and controversial entity, unlike the other two conditions. The recommended diagnosis is a double-blind, placebo-controlled gluten provocation test. Similarly, the test may be performed on children with suspected gluten-related diseases in very specific circumstances and under medical supervision. One example is patients who have started a gluten-free diet without undergoing a duodenal biopsy.

Brain fog and non-coeliac gluten sensitivity: Proof of concept brain MRI pilot study.

AimsNon-Coeliac Gluten Sensitivity (NCGS) is poorly understood, particularly in terms of its neurological outcomes. We initially conducted a prospective postal survey to investigate its neurological presentation and symptom course. Results from this then motivated a follow-up pilot study utilising brain MRI to characterise potential diagnostic biomarkers for future research.MethodsPatients with NCGS were recruited from a specialist centre and completed a prospective postal questionnaire (N = 125). This summarised symptoms experienced, their severity and their course. Onset time was compared by Chi-squared analysis to data from the same centre concerning coeliac disease patients (N = 224). Five respondents on a strict gluten-free diet who self-reported brain fog then attended a pilot study, completing MR brain imaging/questionnaires before/after a gluten challenge. “Baseline” data were assessed for abnormalities, while symptom severity and cerebral blood flow (CBF) were compared before/after challenge.ResultsSurvey participants were aged 47 (85% female). Prevalence of neurological symptoms were: headaches (51%), brain fog (48%), balance issues (31%), tingling (19%). Median symptom resolution time was 48 hours, while onset was 90 minutes; onset pattern was not significantly different compared to CD patients (p = 0.322). Extra-intestinal symptoms worsened by 37%(±28) during a typical reaction. Predominantly non-statistical observations from the brain imaging study are discussed.ConclusionsNeurological symptoms in NCGS are common, and onset time is comparable to that in CD. Brain imaging may be a useful future means of investigating physiological injury and responses to gluten in further study.

Genome-Wide Transcriptomic Analysis of Intestinal Mucosa in Celiac Disease Patients on a Gluten-Free Diet and Postgluten Challenge

Background & AimsGluten challenge studies are instrumental in understanding the pathophysiology of celiac disease. Our aims in this study were to reveal early gluten-induced transcriptomic changes in duodenal biopsies and to find tools for clinics.MethodsDuodenal biopsies were collected from 15 celiac disease patients on a strict long-term gluten-free diet (GFD) prior to and post a gluten challenge (PGC) and from 6 healthy control individuals (DC). Biopsy RNA was subjected to genome-wide 3’ RNA-Seq. Sequencing data was used to determine the differences between the three groups and was compared to sequencing data from the public repositories. The biopsies underwent morphometric analyses.ResultsIn DC vs. GFD group comparisons, 167 differentially expressed genes were identified with 117 genes downregulated and 50 genes upregulated. In PGC vs. GFD group comparisons, 417 differentially expressed genes were identified with 195 genes downregulated and 222 genes upregulated. Celiac disease patients on a GFD were not “healthy”. In particular, genes encoding proteins for transporting small molecules were expressed less. In addition to the activation of immune response genes, a gluten challenge induced hyperactive intestinal wnt-signaling and consequent immature crypt gene expression resulting in less differentiated epithelium. Biopsy gene expression in response to a gluten challenge correlated with the extent of the histological damage. Regression models using only four gene transcripts described 97.2% of the mucosal morphology and 98.0% of the inflammatory changes observed.ConclusionsOur gluten challenge trial design provided an opportunity to study the transition from health to disease. The results show that even on a strict GFD, despite being deemed healthy, patients reveal patterns of ongoing disease. Here, a transcriptomic regression model estimating the extent of gluten-induced duodenal mucosal injury is presented.