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.

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.

(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%.

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.

Celiac disease (CD) is an immune-mediated enteropathy of the small intestine triggered by gluten ingestion. Although the small bowel is the main organ affected, peripheral blood cell alterations have also been described in CD. We aimed to investigate immunological cell patterns in the blood of treated CD patients and in response to a 3-day gluten challenge (GC). Blood samples were collected from 10 patients with CD and 8 healthy controls on a gluten-free diet at baseline and 6 days after initiating the GC. All the samples were analyzed by spectral flow cytometry using a 34-marker panel. We found that patients with CD displayed a lower proportion of memory B cells compared to healthy controls, both at baseline and post-GC. Additionally, we observed the previously reported activated gut-homing CD4⁺, CD8⁺, and TCRγδ⁺ T lymphocytes on day 6 post-GC, and found the CD8⁺ subpopulation to be the most readily identifiable by flow cytometry. Importantly, the CCR9 marker proved effective in enhancing the selection of these gluten-responsive T cells, offering the potential for increased diagnostic accuracy. Spectral flow cytometry involves a complex data analysis, but it offers valuable insights into previously unexplored immunological responses and enables in-depth cell characterization.

We present the case of a 62-year-old patient who developed melenas and in whom conventional endoscopic tests could not detect any bleeding lesion. In our case, capsule endoscopy and enteroscopy were the pivotal elements in establishing the diagnosis of a neuroendocrine tumour with an atypical location. As a result, it was possible to surgically remove the lesions at an early stage of the malignancy without metastatic disease and without the need for adjuvant therapy. Our case demonstrates the need for these new techniques in tumours of atypical location and aggressive course. Otherwise, this malignancy may be underdiagnosed until an advanced stage.