New insights into non-dietary treatment in celiac disease: Emerging therapeutic options

To date, the only treatment for celiac disease (CD) consists of a strict lifelong gluten-free diet (GFD), which has numerous limitations in patients with CD. For this reason, dietary transgressions are frequent, implying intestinal damage and possible long-term complications. There is an unquestionable need for non-dietary alternatives to avoid damage by involuntary contamination or voluntary dietary transgressions. In recent years, different therapies and treatments for CD have been developed and studied based on the degradation of gluten in the intestinal lumen, regulation of the immune response, modulation of intestinal permeability, and induction of immunological tolerance. In this review, therapeutic lines for CD are evaluated with special emphasis on phase III and II clinical trials, some of which have promising results.Federación de Asociaciones de Celíacos de España (FACE) SUBN/2019/00

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 nu-merous 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.Junta de Andalucía AT17_5489_USE, PI-0053-201

Celiac Immunogenic Potential of α-Gliadin Epitope Variants from Triticum and Aegilops Species

The high global demand of wheat and its subsequent consumption arise from the physicochemical properties of bread dough and its contribution to the protein intake in the human diet. Gluten is the main structural complex of wheat proteins and subjects affected by celiac disease (CD) cannot tolerate gluten protein. Within gluten proteins, α-gliadins constitute the most immunogenic fraction since they contain the main T-cell stimulating epitopes (DQ2.5-glia-α1, DQ2.5-glia-α2, and DQ2.5-glia-α3). In this work, the celiac immunotoxic potential of α-gliadins was studied within Triticeae: diploid, tetraploid, and hexaploid species. The abundance and immunostimulatory capacity of CD canonical epitopes and variants (with one or two mismatches) in all α-gliadin sequences were determined. The results showed that the canonical epitopes DQ2.5-glia-α1 and DQ2.5-glia-α3 were more frequent than DQ2.5-glia-α2. A higher abundance of canonical DQ2.5-glia-α1 epitope was found to be associated with genomes of the BBAADD, AA, and DD types; however, the abundance of DQ2.5-glia-α3 epitope variants was very high in BBAADD and BBAA wheat despite their low abundance in the canonical epitope. The most abundant substitution was that of proline to serine, which was disposed mainly on the three canonical DQ2.5 domains on position 8. Interestingly, our results demonstrated that the natural introduction of Q to H at any position eliminates the toxicity of the three T-cell epitopes in the α-gliadins. The results provided a rational approach for the introduction of natural amino acid substitutions to eliminate the toxicity of three T-cell epitopes, while maintaining the technological properties of commercial wheats

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.This study was funded in part by Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (PI-0427-2017 and PI-0053-2018).Peer reviewe

Nuevas estrategias para el control de la enfermedad celíaca: caracterización de los péptidos imunogénicos del gluten y validación clínica de un biomarcador para el seguimiento del paciente celíaco

La enfermedad celiaca (EC) se define como una enteropatía inflamatoria crónica del intestino delgado causada por la ingesta de gluten en individuos genéticamente predispuestos. El término gluten hace referencia a las proteínas responsables de la cohesividad, viscosidad y elasticidad de la masa del trigo, la cebada, el centeno, la avena, y sus derivados. El gluten es una mezcla de dos grupos de proteínas las prolaminas (denominadas gliadinas en el trigo, hordeínas en la cebada, secalinas en el centeno y aveninas en la avena) y las glutelinas (gluteninas para el trigo, y sus homólogas para cebada, centeno y avena). Dichas proteínas son resistentes a las enzimas digestivas y, por lo tanto, posterior a la ingestión de alimentos que contienen gluten, se generan péptidos inmunogénicos capaces de producir daño en la mucosa intestinal de los pacientes celíacos. Esta lesión se caracteriza, histológicamente, por un infiltrado de linfocitos a nivel intraepitelial y una hiperplasia de criptas con distintos grados de atrofia de las vellosidades intestinales. Clínicamente la EC presenta una gran variedad de síntomas, tanto gastrointestinales como extra-intestinales. Los síntomas clásicos incluyen diarrea crónica, esteatorrea, distensión abdominal, dolor, pérdida de peso y anemia. En niños además es común que presenten retraso en el crecimiento y baja estatura. Sin embargo, existen situaciones en las que las manifestaciones digestivas están ausentes u ocupan un segundo lugar. Estas formas atípicas incluyen manifestaciones que pueden ser orales, cutáneas, cardíaca, dermatológicas, neurológicas, articulares, hepáticas, endocrinas, ginecológicas, psiquiátricas y hematológicas. También es frecuente que aparezcan otras complicaciones graves como adenocarcinomas intestinales. En la actualidad, el único tratamiento disponible para los celíacos es el seguimiento de dieta sin gluten (DSG). Se ha demostrado que una estricta adherencia a la DSG es fundamental para eliminar los síntomas de la enfermedad, evitar deficiencias nutricionales y mejorar la calidad de vida de los enfermos celíacos evitando complicaciones a corto-medio plazo de gran morbilidad y coste socio-sanitario (osteopenia-osteoporosis, enfermedades autoinmunes, desnutriciones intrauterinas, etc.). No obstante, numerosos estudios han sugerido que las transgresiones de la dieta tanto deliberadas como involuntarias son relativamente frecuentes. Entre las principales causas del incumplimiento se encuentran los condicionamientos de la vida social, el elevado coste de los productos dietéticos especiales o las dificultades tecnológicas para garantizar la ausencia de gluten en los alimentos complejos. La cantidad de gluten capaz de causar daño en la salud del celíaco es muy baja se ha descrito que el límite es inferior de 10 mg por día. Por lo tanto, es imposible evaluar la suma de todas las contaminaciones diarias individuales para saber si el celíaco ha excedido este umbral. Así pues, mientras que algunos pacientes logran una recuperación total de su enfermedad mediante el seguimiento de una DSG; hay celíacos que requieren un control periódico para conocer la eficacia del tratamiento; y otros muchos que darían la bienvenida a nuevos productos alimenticios que permitan una mayor flexibilidad en su dieta. En base a ello, el trabajo realizado en esta Tesis Doctoral se ha centrado en el estudio de nuevas estrategias de control de la enfermedad celíaca, por un lado, mediante la caracterización de péptidos y epítopos inmunogénicos del gluten en distintas variedades de trigos comerciales; y por el otro, mediante la validación clínica de un biomarcador para el seguimiento de la dieta del paciente celíaco. El Capítulo 1 es una introducción general sobre la enfermedad celíaca. En el Capítulo 2 se exponen los antecedentes del tema y los objetivos principales de esta Tesis Doctoral. El Capítulo 3 comprende una revisión bibliográfica global sobre las patologías relacionadas con la ingesta de gluten, que son un grupo de desórdenes inmunológicos que se clasifican según los síntomas clínicos que producen y la respuesta inmune generada. La elevada prevalencia de estas enfermedades y los efectos nocivos de las proteínas del gluten para la salud representan un reto clínico y científico importante. En el capítulo 4, se caracterizan variantes de epítopos inmunogénicos del gluten en las α-gliadinas de la tribu Triticeae, tanto en especies diploides, como tetraploides y hexaploides de trigo. Estas gliadinas representan la fracción con mayor inmunogenicidad del gluten. Por ello, el estudio de la capacidad inmunoestimuladora de variantes de epítopos canónicos con sustituciones de aminoácidos, de manera que se elimine la toxicidad de estos epítopos, ofrece nuevas posibilidades en la generación de trigos con una toxicidad reducida. Dichas variedades ayudarían a minimizar la presencia de epítopos inmunogénicos en la harina de trigo, manteniendo las propiedades nutricionales y tecnológicas de este cereal. En el Capítulo 5 se ha llevado a cabo un ensayo clínico prospectivo con pacientes celíacos, que llevan más dos años a DSG, en el que se ha determinado la utilidad clínica de los péptidos inmunogénicos del gluten (GIP) en orina como nuevo biomarcador para controlar la adherencia a la DSG correlacionándolo con parámetros clínicos e histológicos característicos de la enfermedad. Mediante este estudio, se ha demostrado que la ausencia repetida de GIP en orina permite verificar el correcto cumplimiento de la DSG a la vista de su relación con la ausencia de atrofia vellositaria, evitando la necesidad del uso de técnicas más invasivas para estudiar las posibles lesiones histológicas en el intestino del enfermo celíaco. En el Capítulo 6, se discuten los resultados obtenidos en esta Tesis Doctoral, abordando su aplicación para el control y seguimiento de la DSG. Por último, en el Capítulo 7, se incluyen las conclusiones alcanzadas en esta Tesis Doctoral

Celiac Disease, Management, and Follow-Up

Celiac disease (CD) is a systemic immune-mediated disorder characterized by a specific serological and histological profile triggered by gluten ingestion, which is given in genetically predisposed subjects. Heterogeneous clinical presentation is characteristic in CD, affecting any organ or tissue with gastrointestinal, extraintestinal, seronegative, or nonresponsive manifestations. CD diagnosis is based on several criteria, including genetic and serological tests, clinical symptoms and/or risk conditions, and duodenal biopsy. Currently, the available treatment for CD is a strict gluten-free diet (GFD) that essentially relies on the consumption of naturally gluten-free foods, such as animal-based products, fruits, vegetables, legumes, and nuts, as well as gluten-free dietary products that may not contain more than 20 mg of gluten per kg of food according to Codex Alimentarius. 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. Difficulties adhering to a GFD have led to new tools to monitor the correct adherence to GFD and alternative forms of treatment

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.Ministerio de Economía, Conocimiento, Empresa y Universidad (project RTC-2016-5441-1

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

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