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.

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.

Rapid Anti-tTG-IgA Screening Test for Early Diagnosis of Celiac Disease in Pediatric Populations.

A large number of patients with celiac disease (CD) remain undiagnosed because they do not fulfill the criteria for entry into the conventional diagnostic workflow. This study evaluated the clinical utility of anti-tissue transglutaminase IgA antibody lateral flow immunoassays (anti-tTG-IgA LFIA) in the undiagnosed-CD-based pediatric population and the impact of a gluten-free diet (GFD) on screening-detected CD. A total of 576 volunteers were tested for anti-tTG-IgA. Gluten consumption habits, CD related symptoms, and risk factors for CD development were evaluated. Volunteers testing positive for anti-tTG-IgA were referred to the conventional CD diagnostic workflow, and the impact of the GFD on health-related quality of life (HR-QoL) was measured. Among them, 13 had a positive anti-tTG-IgA LFIA test result: 11 had confirmed CD (1.91%), one refused confirmatory tests, and another is undergoing diagnosis. Regarding the CD prevalence, no significant differences were observed among risk (1.89%) and symptomatic (2.65%) groups and the entire tested population (1.55%). Rapid anti-tTG-IgA LFIAs could be of clinical utility in primary care for the early identification of children with CD unidentified by the conventional diagnostic workflow. It could potentially reduce the costs of undiagnosed CD, avoiding unnecessary referrals to gastroenterologists, reducing diagnosis delays and long-term problems, and improving patients' HR-QoL.

Evaluation of the Usefulness of an Automatable Immunoassay for Monitoring Celiac Disease by Quantification of Immunogenic Gluten Peptides in Urine.

A gluten-free diet (GFD) is currently the only treatment available for patients with celiac disease (CD). However, adherence to a GFD can be challenging because gluten is present in many foods. A lifelong follow-up of patients with CD must be performed to promote adherence to a GFD and to identify the appearance of symptoms and the associated diseases. Therefore, the development of tools to analyze gluten exposure in these patients is important. This study proposes the development of the first automatable ELISA to monitor adherence to a GFD through the quantification of urine gluten immunogenic peptides (u-GIP). Seven healthy volunteers without suspicion of CD and 23 patients with CD were monitored as part of this study to optimize, validate, and apply this assay. Non-interference was found in the urine matrix, and the recovery percentage for spiked samples was 81-101%. The u-GIP was stable for up to 16 days when the samples were stored at different temperatures. Overall, 100% of the patients had detectable u-GIP at diagnosis (range of 0.39-2.14 ng GIP/mL), which reduced to 27% after 12 months on a GFD. Therefore, this highly sensitive immunoassay would allow the analysis of u-GIP from a large battery of samples in clinical laboratories of specialized healthcare centers.

Clinical utility of urinary gluten immunogenic peptides in the follow-up of patients with coeliac disease.

BACKGROUND: Gluten-free diet (GFD) is the only treatment for patients with coeliac disease (CD) and its compliance should be monitored to avoid cumulative damage. AIMS: To analyse gluten exposures of coeliac patients on GFD for at least 24 months using different monitoring tools and its impact on duodenal histology at 12-month follow-up and evaluate the interval of determination of urinary gluten immunogenic peptides (u-GIP) for the monitoring of GFD adherence. METHODS: Ninety-four patients with CD on a GFD for at least 24 months were prospectively included. Symptoms, serology, CDAT questionnaire, and u-GIP (three samples/visit) were analysed at inclusion, 3, 6, and 12 months. Duodenal biopsy was performed at inclusion and 12 months. RESULTS: At inclusion, 25.8% presented duodenal mucosal damage; at 12 months, this percentage reduced by half. This histological improvement was indicated by a reduction in u-GIP but did not correlate with the remaining tools. The determination of u-GIP detected a higher number of transgressions than serology, regardless of histological evolution type. The presence of >4 u-GIP-positive samples out of 12 collected during 12 months predicted histological lesion with a specificity of 93%. Most patients (94%) with negative u-GIP in ≥2 follow-up visits showed the absence of histological lesions (p < 0.05). CONCLUSION: This study suggests that the frequency of recurrent gluten exposures, according to serial determination of u-GIP, could be related to the persistence of villous atrophy and that a more regular follow-up every 6 months, instead of annually, provides more useful data about the adequate adherence to GFD and mucosal healing.

Consumption of Tritordeum Bread Reduces Immunogenic Gluten Intake without Altering the Gut Microbiota.

Gluten proteins are responsible for the wheat breadmaking quality. However, gluten is also related to human pathologies for which the only treatment is a gluten-free diet (GFD). GFD has gained popularity among individuals who want to reduce their gluten intake. Tritordeum is a cereal species that originated after crossing durum wheat with wild barley and differs from bread wheat in its gluten composition. In this work, we have characterized the immunogenic epitopes of tritordeum bread and results from a four-phase study with healthy adults for preferences of bread and alterations in the gut microbiota after consuming wheat bread, gluten-free bread, and tritordeum bread are reported. Tritordeum presented fewer peptides related to gluten proteins, CD-epitopes, and IgE binding sites than bread wheat. Participants rated tritordeum bread higher than gluten-free bread. Gut microbiota analysis revealed that the adherence to a strict GFD involves some minor changes, especially altering the species producing short-chain fatty acids. However, the short-term consumption of tritordeum bread does not induce significant changes in the diversity or community composition of the intestinal microbiota in healthy individuals. Therefore, tritordeum bread could be an alternative for healthy individuals without wheat-related pathologies who want to reduce their gluten consumption without harming their gut health.

Advances in Celiac Disease and Gluten-Free Diet.

Celiac disease (CD) is a systemic disease that causes chronic enteropathy of the small intestine and develops through an inadequate immune response to gluten in genetically predisposed individuals [...].

A Highly Sensitive Method for the Detection of Hydrolyzed Gluten in Beer Samples Using LFIA.

Most gluten analysis methods have been developed to detect intact gluten, but they have shown limitations in certain foods and beverages in which gluten proteins are hydrolyzed. Methods based on G12/A1 moAbs detect the sequences of gluten immunogenic peptides (GIP), which are the main contributors to the immune response of celiac disease (CD). Immunogenic sequences with tandem epitopes for G12/A1 have been found in beers with <20 mg/kg gluten, which could be consumed by CD patients according to the Codex Alimentarius. Therefore, an accurate method for the estimation of the immunogenicity of a beer is to use two moAbs that can recognize celiac T cell epitopes comprising most of the immunogenic response. Here, a specific and sensitive method based on G12/A1 LFIA was developed to detect GIP in beers labeled gluten-free or with low gluten content, with an LOD of 0.5 mg/kg. A total of 107 beers were analyzed, of those 6.5% showed levels higher than 20 mg/kg gluten and 29% showed levels above the LOD. In addition, G12/A1 LFIA detected gluten in 15 more beer samples than competitive ELISA with another antibody. Despite their labeling, these beers contained GIP which may cause symptoms and/or intestinal damage in CD patients.

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients.

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.

Food Safety and Cross-Contamination of Gluten-Free Products: A Narrative Review.

A gluten-free diet (GFD) is currently the only effective treatment for celiac disease (CD); an individual's daily intake of gluten should not exceed 10 mg. However, it is difficult to maintain a strict oral diet for life and at least one-third of patients with CD are exposed to gluten, despite their best efforts at dietary modifications. It has been demonstrated that both natural and certified gluten-free foods can be heavily contaminated with gluten well above the commonly accepted threshold of 20 mg/kg. Moreover, meals from food services such as restaurants, workplaces, and schools remain a significant risk for inadvertent gluten exposure. Other possible sources of gluten are non-certified oat products, numerous composite foods, medications, and cosmetics that unexpectedly contain "hidden" vital gluten, a proteinaceous by-product of wheat starch production. A number of immunochemical assays are commercially available worldwide to detect gluten. Each method has specific features, such as format, sample extraction buffers, extraction time and temperature, characteristics of the antibodies, recognition epitope, and the reference material used for calibration. Due to these differences and a lack of official reference material, the results of gluten quantitation may deviate systematically. In conclusion, incorrect gluten quantitation, improper product labeling, and poor consumer awareness, which results in the inadvertent intake of relatively high amounts of gluten, can be factors that compromise the health of patients with CD.

Challenges of Monitoring the Gluten-Free Diet Adherence in the Management and Follow-Up of Patients with Celiac Disease.

Celiac disease (CD) is a chronic gluten-responsive immune mediated enteropathy and is treated with a gluten-free diet (GFD). However, a strict diet for life is not easy due to the ubiquitous nature of gluten. This review aims at examining available evidence on the degree of adherence to a GFD, the methods to assess it, and the barriers to its implementation. The methods for monitoring the adherence to a GFD are comprised of a dietary questionnaire, celiac serology, or clinical symptoms; however, none of these methods generate either a direct or an accurate measure of dietary adherence. A promising advancement is the development of tests that measure gluten immunogenic peptides in stools and urine. Causes of adherence/non-adherence to a GFD are numerous and multifactorial. Inadvertent dietary non-adherence is more frequent than intentional non-adherence. Cross-contamination of gluten-free products with gluten is a major cause of inadvertent non-adherence, while the limited availability, high costs, and poor quality of certified gluten-free products are responsible for intentionally breaking a GFD. Therefore, several studies in the last decade have indicated that many patients with CD who follow a GFD still have difficulty controlling their diet and, therefore, regularly consume enough gluten to trigger symptoms and damage the small intestine.

Rapid, Effective, and Versatile Extraction of Gluten in Food with Application on Different Immunological Methods.

One of the main concerns in gluten analysis is to achieve efficient extraction of gluten proteins. Conventional ethanol-based extraction solutions are inefficient and, because of this, it is necessary to use reducing agents or acids for proper solubilization. The extraction recommended by CODEX Standard 118-1979 (revised 2008) utilizes Cocktail solution (patent WO 02/092633 A1). However, it is harmful with a disgusting odor and is not compatible with some immunological techniques. Here, the versatility and extraction capacity of a new Universal Gluten Extraction Solution (UGES) (patent ES 2 392 412 A1) were evaluated using different methodological conditions, food matrices, and various immunological methods. UGES includes safer compounds for both the user and the environment, and it displayed similar extraction efficiency to that of the extraction method recommended for sandwich enzyme-linked immunosorbent assay (ELISA). The extraction time was significantly reduced from 100 to 40 min, depending on the type of the sample. Furthermore, unlike the currently used solution, UGES is compatible with competitive ELISA.

Persistent Villous Atrophy in De Novo Adult Patients With Celiac Disease and Strict Control of Gluten-Free Diet Adherence: A Multicenter Prospective Study (CADER Study).

INTRODUCTION: A substantial proportion of adult patients with celiac disease on a gluten-free diet exhibit persistent villous atrophy, and inadvertent gluten exposure may be one of the causes. The aim of the present study was to evaluate villous atrophy persistence after 2 years on a gluten-free diet in de novo adult patients with celiac disease with strict control of gluten exposure. METHODS: Symptomatic de novo adult patients with celiac disease were prospectively included. Clinical visits and dietary surveillance were scheduled every 6 months during a 2-year follow-up period. At each visit, fecal samples were collected and stored at -20 °C until analysis for gluten immunogenic peptides (f-GIPs). A follow-up duodenal biopsy was performed at 2 years. We evaluated the variables associated with persistent villous atrophy. RESULTS: Seventy-six patients completed the study (36.5 ± 1.6 years, 73% women); persistent villous atrophy was observed in 40 (53%), whereas 72.5% were asymptomatic and 75% had negative serology. Detectable f-GIP >0.08 µg/g in at least 1 fecal sample was seen in 69% of patients. There were no significant differences in the median f-GIP at each visit and median area under the curve over the serial measurements between patients with persistent villous atrophy and those who recovered. On multivariate analysis, only older age was associated with persistent villous atrophy (32% for 16-30 years; 67% for >30 years; P = 0.016). DISCUSSION: The rate of persistent villous atrophy after 2 years was high in adult patients with celiac disease on an intentionally strict gluten-free diet. Low-level ongoing inadvertent gluten exposure could be a contributing factor to persistent villous atrophy.

Tritordeum breads are well tolerated with preference over gluten-free breads in non-celiac wheat-sensitive patients and its consumption induce changes in gut bacteria.

BACKGROUND: The ingestion of wheat and other cereals are related to several gut disorders. The specific components responsible for non-celiac wheat-sensitivity (NCWS) may include gluten and other compounds. Tritordeum is a new cereal derived from crossing durum wheat with a wild barley species, which differs from bread wheat in its gluten composition. In the present work, we examined the response of NCWS patients to tritordeum bread Gastrointestinal symptoms as well as tritordeum acceptability, gluten immunogenic peptides excretion, and the composition and structure of the intestinal microbiota were evaluated. RESULTS: Gastrointestinal symptoms of the subjects showed no significant change between the gluten-free bread and the tritordeum bread. Participating subjects rated tritordeum bread higher than the gluten-free bread. Analysis of the bacterial gut microbiota indicated that tritordeum consumption does not alter the global structure and composition of the intestinal microbiota, and only a few changes in some butyrate-producing bacteria were observed. CONCLUSIONS: All the results derived from acceptability, biochemical and microbiological tests suggest that tritordeum may be tolerated by a sub-set of NCWS sufferers who do not require strict exclusion of gluten from their diet. © 2020 Society of Chemical Industry.

Exposure sources, amounts and time course of gluten ingestion and excretion in patients with coeliac disease on a gluten-free diet.

BACKGROUND: A major deficit in understanding and improving treatment in coeliac disease (CD) is the lack of empiric data on real world gluten exposure. AIMS: To estimate gluten exposure on a gluten-free diet (GFD) using immunoassays for gluten immunogenic peptides (GIP) and to examine relationships among GIP detection, symptoms and suspected gluten exposures METHODS: Adults with biopsy-confirmed CD on a GFD for 24 months were recruited from a population-based inception cohort. Participants kept a diary and collected urine samples for 10 days and stools on days 4-10. 'Doggie bags' containing » portions of foods consumed were saved during the first 7 days. Gluten in food, stool and urine was quantified using A1/G12 ELISA. RESULTS: Eighteen participants with CD (12 female; age 21-70 years) and three participants on a gluten-containing diet enrolled and completed the study. Twelve out of 18 CD participants had a median 2.1 mg gluten per exposure (range 0.2 to >80 mg). Most exposures were asymptomatic and unsuspected. There was high intra-individual variability in the interval between gluten ingestion and excretion. Participants were generally unable to identify the food. CONCLUSIONS: Gluten exposure on a GFD is common, intermittent, and usually silent. Excretion kinetics are highly variable among individuals. The amount of gluten varied widely, but was typically in the milligram range, which was 10-100 times less than consumed by those on an unrestricted diet. These findings suggest that a strict GFD is difficult to attain, and specific exposures are difficult to detect due to variable time course of excretion.

Negative predictive value of the repeated absence of gluten immunogenic peptides in the urine of treated celiac patients in predicting mucosal healing: new proposals for follow-up in celiac disease.

BACKGROUND: The treatment of celiac disease (CD) is a lifelong gluten-free diet (GFD). The current methods for monitoring GFD conformance, such as a dietary questionnaire or serology tests, may be inaccurate in detecting dietary transgressions, and duodenal biopsies are invasive, expensive, and not a routine monitoring technique. OBJECTIVES: Our aim was to determine the clinical usefulness of urine gluten immunogenic peptides (GIP) as a biomarker monitoring GFD adherence in celiac patients and to evaluate the concordance of the results with the degree of mucosal damage. METHODS: A prospective observational study was conducted involving 22 de novo CD patients, 77 celiac patients consuming a GFD, and 13 nonceliac subjects. On 3 d of the week, urine samples were collected and the GIP concentrations were tested. Simultaneously, anti-tissue transglutaminase antibodies, questionnaire results, clinical manifestations, and histological findings were analyzed. RESULTS: Approximately 24% (18 of 76) of the celiac patients consuming a GFD exhibited Marsh II-III mucosal damage. Among this population, 94% (17 of 18) had detectable urine GIP; however, between 60% and 80% were asymptomatic and exhibited negative serology and appropriate GFD adherence based on the questionnaire. In contrast, 97% (31 of 32) of the celiac patients without duodenal damage had no detectable GIP. These results demonstrated the high sensitivity (94%) and negative predictive value (97%) of GIP measurements in relation to duodenal biopsy findings. In the de novo CD-diagnosed cohort, 82% (18 of 22) of patients had measurable amounts of GIP in the urine. CONCLUSIONS: Determining GIP concentrations in several urine samples may be an especially convenient approach to assess recent gluten exposure in celiac patients and appears to accurately predict the absence of histological lesions. The introduction of GIP testing as an assessment technique for GFD adherence may help in ascertaining dietary compliance and to target the most suitable intervention during follow-up.

Stimulatory Response of Celiac Disease Peripheral Blood Mononuclear Cells Induced by RNAi Wheat Lines Differing in Grain Protein Composition.

Wheat gluten proteins are responsible for the bread-making properties of the dough but also for triggering important gastrointestinal disorders. Celiac disease (CD) affects approximately 1% of the population in Western countries. The only treatment available is the strict avoidance of gluten in the diet. Interference RNA (RNAi) is an excellent approach for the down-regulation of genes coding for immunogenic proteins related to celiac disease, providing an alternative for the development of cereals suitable for CD patients. In the present work, we report a comparative study of the stimulatory capacity of seven low-gluten RNAi lines differing in grain gluten and non-gluten protein composition, relevant for CD and other gluten pathologies. Peripheral blood mononuclear cells (PBMCs) of 35 patients with active CD were included in this study to assess the stimulatory response induced by protein extracts from the RNAi lines. Analysis of the proliferative response and interferon-gamma (INF-γ) release of PBMCs demonstrated impaired stimulation in response to all RNAi lines. The lower response was provided by lines with a very low content of α- and γ-gliadins, and low or almost devoid of DQ2.5 and p31-43 α-gliadin epitopes. The non-gluten protein seems not to play a key role in PBMC stimulation.

Prospective longitudinal study: use of faecal gluten immunogenic peptides to monitor children diagnosed with coeliac disease during transition to a gluten-free diet.

BACKGROUND: Treatment for coeliac disease is a lifelong strict gluten-free diet. Although guidelines recommend regular follow-up with dietary interviews and coeliac serology, these methods may be inaccurate. AIM: To evaluate the usefulness of faecal gluten immunogenic peptides to support the diagnosis and to determine the adherence to the gluten-free diet in coeliac children. METHODS: Multicentre prospective observational study including 64 coeliac children. Faecal gluten peptides, and tissue transglutaminase and deamidated gliadin peptide antibodies were analyzed at diagnosis, and 6, 12 and 24 months thereafter. Gluten consumption was estimated from gluten peptide levels. RESULTS: Most children (97%) had detectable gluten peptides at diagnosis. On a gluten-free diet, the rate of gluten peptides increased from 13% at 6 months to 25% at 24 months. Mean estimated gluten exposure dropped from 5543 mg/d at diagnosis to 144 mg/d at 6 months, then increased to 606 mg/d by 24 months. In contrast, deamidated gliadin peptide antibodies normalised and only 20% had elevated tissue transglutaminase antibody by 24 months. The elevation of tissue transglutaminase antibody was more prolonged in patients with detectable gluten peptides (P < 0.05). Nevertheless, absolute levels of tissue transglutaminase antibody had low sensitivity to identify patients with detectable gluten peptides (P > 0.1). Dietitian assessment was only moderately correlated with gluten peptide detection (κ = 0.5). CONCLUSIONS: Faecal gluten peptides testing may guide treatment of coeliac disease prior to diagnosis and during the assessment diet adherence. Further studies could determine if early identification of gluten exposure reduces the need for expensive/invasive investigations for non-responsive coeliac disease. ClinicalTrials.gov Number: NCT02711397.