A comprehensive examination of small-bowel capsule endoscopy in Spanish centers to meet European Society of Gastrointestinal Endoscopy standards.

Background and study aims In 2019, the European Society of Gastrointestinal Endoscopy (ESGE) created a working group to develop technical and quality standards for small-bowel capsule endoscopy (SBCE) to improve the daily practice of endoscopy services. They developed 10 quality parameters, which have yet to be tested in a real-life setting. Our study aimed to evaluate the accomplishment of the quality standards in SBCE established by the ESGE in several Spanish centers. Materials and methods An online survey of 11 multiple-choice questions related to the ESGE performance measures was sent to Spanish centers with experience in SBCE. In order to participate and obtain reliable data, at least 100 questionnaires had to be answered per center because that is the minimum number established by ESGE. Results 20 centers participated in the study, compiling 2049 SBCEs for the analysis. Only one of 10 performance measures (cecal visualization) reached the minimum standard established by the ESGE. In five of 10 performance measures (Indication, lesion detection rate, terminology, and retention rate) the minimum standard was nearly achieved. Conclusions Our study is the first multicenter study regarding SBCE quality performance measures in a real setting. Our results show that the minimum standard is hardly reached in most procedures, which calls into question their clinical applicability in real life. We suggest performing similar studies in other countries to evaluate whether there is a need for quality improvement programs or a need to reevaluate the minimum and target values published so far.

Improving the Diagnosis of Dermatitis Herpetiformis Using the Intraepithelial Lymphogram.

Dermatitis herpetiformis is a cutaneous manifestation of celiac disease. Phenotyping of intraepithelial lymphocytes in the small bowel mucosa can strengthen the diagnosis of celiac disease when it is not clear-cut. We aim to evaluate the usefulness of the intraepithelial lymphogram to confirm dermatitis herpetiformis in equivocal cases. We performed a retrospective multicenter study on patients diagnosed with dermatitis herpetiformis and collected data from the intraepithelial lymphogram assessed by flow cytometry. A total of 36 patients were analyzed in relation to the severity of intestinal damage (18 had non-atrophic mucosa) at baseline (N = 28) and/or after the adoption of a gluten-free diet (median follow-up of three years, N = 16). We observed that patients with atrophy more often had positive celiac serology (p = 0.019), celiac clinical symptoms (p = 0.018), and iron-deficiency anemia (p = 0.018), but the severity of skin damage was similar in both groups (p = 0.79). At baseline, increased TCRγδ+ cells were present in 94% of patients with atrophy and 67% with non-atrophic lesions (p = 0.13). After a gluten-free diet, increased TCRγδ+ cells persisted in 100% and 63% of cases, respectively (p = 0.21). We concluded that increased TCRγδ+ cells may be helpful in confirming the diagnosis of dermatitis herpetiformis in equivocal cases, even in patients who were started on a gluten-free diet.

Development of a new recombinant antibody, selected by phage-display technology from a celiac patient library, for detection of gluten in foods.

Gluten, a group of ethanol-soluble proteins present in the endosperm of cereals, is extensively used in the food industry due to its ability to improve dough properties. However, gluten is also associated with a range of gluten-related diseases (GRDs), such as wheat allergies, celiac disease, and gluten intolerance. The recommended treatment for GRDs patients is a gluten-free diet. To monitor adherence to this diet, it is necessary to develop gluten-detection systems in food products. Among the available methods, immunodetection systems are the most popular due to their simplicity, reproducibility, and accuracy. The aim of this study was to generate novel high-affinity antibodies against gluten to be used as the primary reactant in an enzyme-linked immunosorbent assay (ELISA) test. These antibodies were developed by constructing an immune library from mRNA obtained from two celiac patients with a high humoral response to gluten-related proteins. The resulting library (composed by 1.1x107) was subjected to selection against gliadin using phage display technology. Following several rounds of selection, the Fab-C was selected, and demonstrated good functionality in ELISA tests, presenting a limit of detection of 15 mg/kg for detection of gluten in spiked mixtures and food products. The methodology can discriminate gluten-free products according to the current legislation.

Construction of a Fab Library Merging Chains from Semisynthetic and Immune Origin, Suitable for Developing New Tools for Gluten Immunodetection in Food.

The observed increase in the prevalence of gluten-related disorders has prompted the development of novel immunological systems for gluten detection in foodstuff. The innovation on these methods relies on the generation of new antibodies, which might alternatively be obtained by molecular evolution methods such as phage display. This work presents a novel approach for the generation of a Fab library by merging semi-synthetic heavy chains built-up from a pre-existent recombinant antibody fragment (dAb8E) with an immune light chain set derived from celiac donors. From the initial phage population (107 candidates) and after three rounds of selection and amplification, four different clones were isolated for further characterization. The phage Fab8E-4 presented the best features to be applied in an indirect ELISA for the detection of gluten in foods, resulting in improved specificity and sensitivity.

Activated gut-homing CD8+ T cells for coeliac disease diagnosis on a gluten-free diet.

BACKGROUND: The diagnosis of coeliac disease (CD) in individuals that have started a gluten-free diet (GFD) without an adequate previous diagnostic work-out is a challenge. Several immunological assays such as IFN-γ ELISPOT have been developed to avoid the need of prolonged gluten challenge to induce the intestinal damage. We aimed to evaluate the diagnostic accuracy of activated gut-homing CD8+ and TCRγδ+ T cells in blood after a 3-day gluten challenge and to compare it with the performance of IFN-γ ELISPOT in a HLA-DQ2.5 subsample. METHODS: A total of 22 CD patients and 48 non-CD subjects, all of them following a GFD, underwent a 3-day 10-g gluten challenge. The percentage of two T cell subsets (CD8+ CD103+ ß7hi CD38+/total CD8+ and TCRγδ+ CD103+ ß7hi CD38+/total TCRγδ+) in fresh peripheral blood drawn baseline and 6 days after the challenge was determined by flow cytometry. IFN-γ ELISPOT assays were also performed in HLA-DQ2.5 participants. ROC curve analysis was used to assess the diagnostic performance of the CD8+ T cell response and IFN-γ ELISPOT. RESULTS: Significant differences between the percentage of the two studied subsets of CD8+ and TCRγδ+ cells at days 0 and 6 were found only when considering CD patients (p < 10-3 vs. non-CD subjects). Measuring activated CD8+ T cells provided accurate CD diagnosis with 95% specificity and 97% sensitivity, offering similar results than IFN-γ ELISPOT. CONCLUSIONS: The results provide a highly accurate blood test for CD diagnosis in patients on a GFD of easy implementation in daily clinical practice.

Coeliac Disease in Elderly Patients: Value of Coeliac Lymphogram for Diagnosis.

(1) Background: Although a meta-analysis reported that the sensitivity of CD3+ TCRγδ+ cells for coeliac disease diagnosis was >93%, a recent study has suggested that sensitivity decreased to 65% in elderly patients. (2) Aim: To evaluate whether the sensitivity of intraepithelial lymphocyte cytometric patterns for coeliac disease diagnosis changes with advanced age. (3) Methods: We performed a multicentre study including 127 coeliac disease patients ≥ 50 years: 87 with baseline cytometry (45 aged 50-59 years; 23 aged 60-69 years; 19 aged ≥ 70 years), 16 also with a follow-up cytometry (on a gluten-free diet); and 40 with only follow-up cytometry. (4) Results: In Marsh 3 patients, a sensitivity of 94.7%, 88.9% and 86.7% was observed for each age group using a cut-off value of TCRγδ+ >10% (p = 0.27); and a sensitivity of 84.2%, 83.4% and 53.3% for a cut-off value >14% (p = 0.02; 50-69 vs. ≥70 years), with difference between applying a cut-off of 10% or 14% (p = 0.008). The TCRγδ+ count in the ≥70 years group was lower than in the other groups (p = 0.014). (5) Conclusion: In coeliac patients ≥ 70 years, the TCRγδ+ count decreases and the cut-off point of >10% is more accurate than >14%.

Gamma delta+ intraepithelial lymphocytes and coeliac lymphogram in a diagnostic approach to coeliac disease in patients with seronegative villous atrophy.

BACKGROUND: The causes of seronegative villous atrophy can be grouped as coeliac or noncoeliac related. There is no consensus on how to approach subjects with seronegative coeliac disease. AIM: To evaluate the accuracy of both an increase in CD3+ T-cell receptor gamma delta+ (TCRγδ+ ) intraepithelial lymphocytes and coeliac lymphogram for the diagnosis of coeliac disease in patients with seronegative villous atrophy. METHODS: Sixty-seven consecutive patients with seronegative villous atrophy were included. Duodenal biopsies to assess TCRγδ+ and CD3- by flow cytometry were performed at the index endoscopy. Coeliac lymphogram was defined as an increase in TCRγδ+ plus a decrease in CD3- intraepithelial lymphocytes. Sensitivity, specificity and Fagan's nomogram were calculated. RESULTS: Coeliac disease was diagnosed in 37 patients and noncoeliac villous atrophy in 30. Coeliac patients were younger (39 ± 3 vs 55 ± 3 years; P = 0.001), more often showed HLA-DQ2/8 (97.6% vs 61%; P = 0.002) and had a more severe histology (61% vs 32% Marsh 3b-c; P = 0.055), as compared to noncoeliac ones. Coeliac lymphogram was associated with a sensitivity of 87% (CI, 73.7-95) and specificity of 96.7% (82.7-99.9), whereas evaluating only TCRγδ+ yielded a sensitivity of 91.3% (79.2-97.6) and specificity of 83.3% (65.3-94.3). Among patients with a pre-test coeliac disease probability of 30%, post-test probabilities were 92% and 5% for positive and negative coeliac lymphogram, and 70% and 4% for positive and negative TCRγδ+ . CONCLUSIONS: Coeliac lymphogram was associated with a high level of diagnostic evidence either against or in favour of coeliac disease in patients with seronegative villous atrophy.

CX3CL1-CX3CR1 Axis: A New Player in Coeliac Disease Pathogenesis.

BACKGROUND: The CX3CL1-CX3CR1 axis has been related to numerous diseases. The aim of our study was to investigate its involvement in coeliac disease (CD) pathogenesis, particularly in the early phase of the disease. METHODS: We collected peripheral blood from CD patients and controls, enrolled in a 3-day gluten challenge, to study soluble CX3CL1, I-TAC and MIG by Luminex, CX3CL1 and CX3CR1 gene expression by qPCR, and CX3CR1 protein expression in monocytes and CD8+, CD4+ and γδ+ T cells, by flow cytometry. We also analysed the expression of the CX3CL1 and CX3CR1 mRNA and protein in the duodenal biopsies of CD patients with active and treated disease, and in non-CD control individuals, by qPCR and immunohistochemistry. RESULTS: After the gluten challenge, increased levels of CX3CL1, I-TAC and MIG proteins were observed in the peripheral blood of CD patients, with no changes in CX3CL1 mRNA, or CX3CR1 mRNA and protein. Regarding duodenal tissue, CX3CL1 was absent or barely present in the superficial and basal epithelium of CD patients, contrasting with the moderate to high levels present in controls. CONCLUSIONS: CX3CL1 seems to be involved in the appearance and progression of CD, and it appears to be a potential diagnostic biomarker. Its use as an alternative therapeutic target in CD deserves further research.