Supplementary MaterialsData_Sheet_1

Supplementary MaterialsData_Sheet_1. the next year of progression. Secondly, to study the tolerogenic functionality of DCs, liposomes with phosphatidylserine (PS) were designed to mimic apoptotic beta cells, which are able to induce tolerance, as previously demonstrated by our group in DCs from adult patients with T1D. In this study, monocyte-derived DCs from pediatric patients with T1D and control subjects were assessed in terms of PS-liposomes capture kinetics, and transcriptional and phenotypic changes. DCs from pediatric Withaferin A patients with T1D were found to phagocyte PS-liposomes more slowly and less efficiently than DCs from control subjects, inversely correlating with disease evolution. Nonetheless, the transcription of PS receptors and immunoregulatory genes, cytokine profile, and membrane expression of immunological markers in DCs was consistent with tolerogenic potential after PS-liposomes phagocytosis. In conclusion, T1D progression in childhood entails altered peripheral blood DCs subsets, as well as impaired DCs phagocytosis, although tolerance induction could still function optimally. Therefore, this study provides useful data for patient follow-up and stratification in immunotherapy clinical trials. gene, which encodes for the low-affinity IgG Fc region receptor II-a involved in Withaferin A phagocytosis, contribute to its pathogenesis (16). However, no data regarding the phagocytic function of DCs in human T1D are available. In patients with type 2 diabetes, there is growing evidence that impaired phagocytosis in neutrophils (17, 18) and macrophages (19) is related to glycemic control, albeit it can be alleviated with improved metabolic regulation. These data suggest that changes in glucose homeostasis can alter phagocytic processes, with the particularity that impairment of efferocytosis can contribute to the perpetuation of the autoreactivity in T1D. By exploiting the inherent ability of apoptotic cell clearance to induce tolerance, a synthetic lipid-vesicle strategy that mimics apoptotic beta cells by being enriched in PS and encapsulating insulin was designed (20). This immunotherapy arrested autoimmunity upon administration to NOD mice after PS-liposomes were phagocyted by DCs, thus eliciting tolerogenic features. This finding was replicated in human DCs from adult patients with T1D (21). Therefore, this strategy achieves successful apoptotic mimicry and constitutes a promising strategy for tackling autoimmune diseases. Unfortunately, T1D incidence is dramatically increasing in children (22, 23), in whom the disease is more aggressive and entails extra administration problems. Since severe dysglycemia could impair DCs functionality, the aim of this study was to analyze circulating DCs subpopulations in pediatric T1D at different stages, as well as to characterize phagocytosis in T1D at the onsethyperglycemic phase and at established diseasewhen glucose levels are better controlled, and to evaluate the role of phagocytosis in tolerance induction. Materials and Methods Withaferin A Patients and Control Individuals Pediatric patients with T1D (= 61) were visited as outpatients or hospitalized in the Pediatric Section at Germans Trias i Pujol Hospital. Patients were recruited at the onset and with a longer evolution (more than 6 months). Pediatric control individuals (= 21) were also recruited. Inclusion criteria were 1C18 years of age and normal body mass index (BMI). Exclusion criteria were being under immunosuppressive or anti-inflammatory treatment, the presence of other autoimmune Withaferin A diseases, type 2 diabetes, pregnancy, and compromised kidney or liver function. All the experiments were carried out in strict accordance with the principles outlined in the Declaration of Helsinki for human research and after the approval of the Committee on Rabbit Polyclonal to HP1alpha the Ethics of Research of the Germans Trias i Pujol Hospital (PI-16-083). Signed informed consent was given by the parents of all subjects, and directly by children older than 12 years old. Analysis of Peripheral Blood DC Subsets In order to analyze DCs subsets, 2 mL blood samples were collected in EDTA tubes from control subjects and patients with T1D at diagnosis, and at first and second year of disease.

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