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Background: The differential diagnosis between patients with celiac disease (CD) and non-celiac gluten sensitivity (NCGS) is difficult when a gluten-free diet (GFD) has been initiated before the diagnostic work-up. Isolated increases in TCRγδ+ and celiac lymphogram (increased TCRγδ+ plus decreased CD3−) may enable differential diagnosis in this challenging clinical setting. This study evaluated: (1) the accuracy of %TCRγδ+ and celiac lymphogram for diagnosing CD before and after GFD and for differentiation with NCGS; (2) TCRγδ+ kinetics at baseline and after starting GFD in both CD and NCGS. Methods: The inclusion criteria were patients with CD (n = 104), NCGS (n = 37), and healthy volunteers (n = 18). An intestinal biopsy for intraepithelial lymphogram by flow cytometry was performed at baseline and after GFD. The optimal cutoff for CD diagnostic accuracy was established by maximizing the Youden index and via logistic regression. Results: %TCRγδ+ showed better diagnostic accuracy than celiac lymphogram for identifying CD before and after GFD initiation. With a cutoff > 13.31, the accuracy for diagnosing CD in patients under GFD was 0.88 [0.80–0.93], whereas the accuracy for diagnosing NCGS (%TCRγδ+ ≤ 13.31) was 0.84 [0.76–0.89]. The percentage of TCRγδ+ cells showed differential kinetics between CD (baseline 22.7% [IQR, 16.4–33.6] vs. after GFD 26.4% [IQR, 17.8–36.8]; p = 0.026) and NCGS (baseline 9.4% [IQR, 4.1–14.6] vs. after GFD 6.4% [IQR, 3.2–11]; p = 0.022). Conclusion: TCRγδ+ T cell assessment accurately diagnoses CD before and after a GFD. Increased TCRγδ+ was maintained in the long term after GFD in CD but not in NCGS. Altogether, this suggests the potential usefulness of this marker for the differential diagnosis of these two entities in patients on a GFD.

Abstract 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+ β7hi CD38+/total CD8+ and TCRγδ+ CD103+ β7hi 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.

Abstract Background Previous studies suggested that Interleukin‐10 (IL‐10) depletion in Crohn's disease (CD) could predict outcome. Aim: To determine IL‐10 in blood and at different intestinal locations in patients with active CD and to assess its potential prognostic capacity to identify aggressive CD. Methods Twenty‐three patients with CD were included. Ulcerative colitis (UC), infectious colitis and healthy individuals acted as controls. Serum and mucosal samples were taken at baseline and 1 month after steroid initiation in CD patients. Patients were classified according to steroid response. Control samples were obtained from different intestinal locations. IL‐10 expression was measured with real‐time polymerase chain reaction, immunofluorescence (intestine) and ELISA (serum, biopsy cultures' supernatants and tissue homogenates). Results CD and UC showed an increase in IL‐10 messenger RNA (mRNA) versus controls (p

BackgroundPersonalized medicine requires finding relationships between variables that influence a patient's phenotype and predicting an outcome. Sparse generalized canonical correlation analysis identifies relationships between different groups of variables. This method requires establishing a model of the expected interaction between those variables. Describing these interactions is challenging when the relationship is unknown or when there is no pre-established hypothesis. Thus, our aim was to develop a method to find the relationships between microbiome and host transcriptome data and the relevant clinical variables in a complex disease, such as Crohn's disease.ResultsWe present here a method to identify interactions based on canonical correlation analysis. We show that the model is the most important factor to identify relationships between blocks using a dataset of Crohn's disease patients with longitudinal sampling. First the analysis was tested in two previously published datasets: a glioma and a Crohn's disease and ulcerative colitis dataset where we describe how to select the optimum parameters. Using such parameters, we analyzed our Crohn's disease data set. We selected the model with the highest inner average variance explained to identify relationships between transcriptome, gut microbiome and clinically relevant variables. Adding the clinically relevant variables improved the average variance explained by the model compared to multiple co-inertia analysis.ConclusionsThe methodology described herein provides a general framework for identifying interactions between sets of omic data and clinically relevant variables. Following this method, we found genes and microorganisms that were related to each other independently of the model, while others were specific to the model used. Thus, model selection proved crucial to finding the existing relationships in multi-omics datasets.

Introduction: The study of intraepithelial lymphocytes (IEL) by flow cytometry is a useful tool in the diagnosis of coeliac disease (CD). Previous data showed that an increase in %TCRγδ+ and decrease of %CD3− IEL constitute a typical CD cytometric pattern with a specificity of 100%. However, there are no data regarding whether there are differences in the %TCRγδ+ related to sex, age, titers of serology, and degree of histological lesion. Study aims: To confirm the high diagnostic accuracy of the coeliac cytometric patterns. To determine if there are differences between sex, age, serology titers, and histological lesion grade. Results: We selected all patients who fulfilled “4 of 5” rule for CD diagnosis (n = 169). There were no differences in %TCRγδ+ between sexes (p = 0.909), age groups (p = 0.986), serology titers (p = 0.53) and histological lesion grades (p = 0.41). The diagnostic accuracy of complete CD cytometric pattern was: specificity 100%, sensitivity 82%, PPV 100%, NPV 47%. Conclusion: We confirmed, in a validation cohort, the high diagnostic accuracy of complete CD pattern irrespective of sex, age, serology titers, and grade of mucosal lesion.

1. Background: The long-term effect of a gluten-free diet (GFD) on functional bowel disorders (FBDs) has been scarcely studied. The aim was to assess the effect of a GFD on FBD patients, and to assess the role of both the low-grade coeliac score and coeliac lymphogram in the probability of response to a GFD. 2. Methods: 116 adult patients with either predominant diarrhoea or abdominal bloating, fulfilling Rome IV criteria of FBD, were treated with a GFD. Duodenum biopsies were performed for both pathology studies and intraepithelial lymphocyte subpopulation patterns. Coeliac lymphogram was defined as an increase in TCRγδ+ cells plus a decrease in CD3− cells. A low-grade coeliac score >10 was considered positive. 3. Results: Sustained response to GFD was observed in 72 patients (62%) after a median of 21 months of follow-up, who presented more often with coeliac lymphogram (37.5 vs. 11.4%; p = 0.02) and a score >10 (32 vs. 11.4%; p = 0.027) compared to non-responders. The frequency of low-grade coeliac enteropathy was 19.8%. 4. Conclusion: A GFD is effective in the long-term treatment of patients with previously unexplained chronic watery diarrhoea- or bloating-predominant symptoms fulfilling the criteria of FBD. The response rate was much higher in the subgroup of patients defined by the presence of both a positive low-grade coeliac score and coeliac lymphogram.

Background & Aims: Determining whether patients with lymphocytic enteritis (LE) have coeliac disease is a challenge. We analysed the variables associated with a low-grade coeliac enteropathy diagnosis in patients with suspected coeliac disease but without villous atrophy, and developed a scoring system to identify them. Methods: We collected data from 2010 through to 2016 on patients with lymphocytic enteritis and persistent symptoms compatible with the clinical spectrum of coeliac disease. One hundred and four patients starting on a gluten-free diet (GFD) were included. Duodenal biopsies were collected before the GFD and analysed for numbers of CD3+ T-cell receptor gamma delta+ (TCRγδ+), and CD3− intraepithelial lymphocytes. We performed a logistic regression analysis to identify factors associated with a low-grade coeliac enteropathy diagnosis. Results: Sixty-two patients achieved clinical remission after the GFD. Fifty of these 62 patients were diagnosed with low-grade coeliac enteropathy. Multivariate analysis identified the presence of >25% intraepithelial lymphocytosis, HLA-DQ2.5, positive serology, and increased numbers of TCRγδ+ cells with a low-grade coeliac enteropathy diagnosis. We developed a scoring system that identified patients with an area under the ROC curve (AUC) of 0.91. Scores of >10 had 86% sensitivity and 85% specificity. Conclusion: We developed a scoring system that identifies patients likely to be diagnosed with low-grade coeliac enteropathy with an AUC value of 0.91.