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Non-celiac gluten sensitivity (NCGS) is still an undefined syndrome whose triggering mechanisms remain unsettled. This study aimed to clarify how cultured peripheral blood mononucleated cells (PBMC) obtained from NCGS patients responded to contact with wheat proteins. Results demonstrated that wheat protein induced an overactivation of the proinflammatory chemokine CXCL10 in PBMC from NCGS patients, and that the overactivation level depends on the cereal source from which proteins are obtained. CXCL10 is able to decrease the transepithelial resistance of monolayers of normal colonocytes (NCM 460) by diminishing the mRNA expression of cadherin-1 (CDH1) and tight junction protein 2 (TJP2), two primary components of the tight junction strands. Thus, CXCL10 overactivation is one of the mechanisms triggered by wheat proteins in PBMC obtained from NCGS patients. This mechanism is activated to a greater extent by proteins from modern with respect to those extracted from ancient wheat genotypes.

Introduction.Crohn’s disease (CD) is a chronic inflammatory auto-immune bowel disease (IBD) characterized by segmental transmural inflammation. In 1/3 of patients, CD is complicated by perianal fistulas which rarely heal spontaneously or after medical treatment. Disappointing results have been obtained in anti-inflammatory drug-based (TNF-inhibitors) clinical trials so that 2/3 of patients either do not respond or loose their response. Pre-clinical studies and preliminary clinical trials have suggested that, due to their immunomodulatory properties, mesenchymal stromal cell (MSC)-based therapy hold promising potential in the IBD treatment. Ongoing studies are aimed at evaluating the proper dose and mode of MSC administration needed for more effective results. Human platelet lysate (HPL) has been proposed as a valid FBS substitute to safely expand MSCs for therapeutic purpose. A recent paper demonstrates that in MSCs HPL triggers in the secretion of factors enhancing the initial inflammatory response to the injury, a key element in the wound healing process. We recently set up a novel murine model of IBD. This model, conversely to that adopted so far, does not lead to animal death, but it presents a mild intestinal damage which makes it the most accurate to study therapeutic agent effects. The aim of our study was to characterize the effects of MSCs isolated from adipose tissue of CD patients and resuspended in HPL (adCD-MSCs/HPL) in our IBD model. Methods.Colitis was induced in C57BL/6J mice by administration of 1,5% dextran sulphate sodium (DSS) in tap water. adCD-MSCs, w/wo HPL, were administrated via enema for 3 times (1x10^6 cells/mouse/time) every other day starting on day+7 from DSS induction. Results.We found that adCD-MSCs can be easily isolated and expanded from CD patients, showing typical MSC properties. We demonstrated that HPL potentiated the adCD-MSC efficacy in ameliorating DSS-induced colitis in our model. Indeed, DSS+adCD-MSC/HPL-treated mice group showed a reduced weight loss compared to DSS+PBS mice group. Disease Activity Index, including inflammation clinical parameters (weight loss, diarrohea, rectal bleeding), was lower in DSS+adCD-MSC/HPL-treated mice at each time point starting from the end of MSC treatment. Histopathological examination of the colon distal and proximal tracts showed that adCD-MSCs/HPL significantly reduced the extension and the severity of the inflamed area and the inflammatory cells infiltration due to DSS treatment. We also found that adCDMSCs/HLP decreased systemic inflammatory mediators levels (IL-1β, IL-6, IL-17, IFN-γ, TNF-α, IL-10) in the colitic mice plasma. Finally, we set up a novel method to label adCD-MSCs. We found that Nile red staining was very efficient and stable and preserved MSC biological properties. Thus, Nile red staining can be efficiently used to track injected MSCs. Conclusions.Overall, this study validate a novel promising adCD-MSC/HPL-based therapy for refractory CD treatment.