Impaired caspase-3 expression by peripheral T cells in chronic autoimmune thyroiditis and in autoimmune polyendocrine syndrome-2.

CONTEXT: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance, and caspase-3 represents its major executioner. AICD impairment contributes to the persistence of autoreactive T cells, and defective AICD has been reported in autoimmune thyroiditis as well as in type 1 diabetes mellitus. OBJECTIVE: The objective of this study was to evaluate the involvement of caspase-3 in the regulation of AICD resistance in thyroid and polyendocrine autoimmunity. DESIGN/SETTINGS/PATIENTS/INTERVENTION: Caspase-3 expression was analyzed in peripheral blood lymphocytes from 26 adults (A-AT) and 25 children (Y-AT) affected by autoimmune thyroiditis and 13 individuals affected by chronic autoimmune thyroiditis plus Addison's disease [autoimmune polyendocrine syndrome-2 (APS-2)] in comparison with 32 age-matched normal control subjects (NC). OUTCOME MEASURES: Caspase-3 mRNA expression in peripheral T cells was evaluated by quantitative real-time PCR; protein expression of both procaspase-3 and activated caspase-3 by Western blot analysis was followed by scanning densitometry. RESULTS: Caspase-3 mRNA expression was significantly reduced in resting lymphocytes from both A-AT (P = 0.001) and Y-AT (P = 0.016) compared with NC. After lymphocyte activation, protein levels of caspase-3 active form were significantly reduced in A-AT (P = 0.023) and Y-AT (P = 0.001) compared with NC. The APS-2 group displayed characteristics similar to the A-AT group because both caspase-3 mRNA and protein active form levels were significantly reduced compared with NC (P = 0.004 and 0.002, respectively). CONCLUSION: Our data show that peripheral lymphocytes of subjects affected by thyroid autoimmunity or APS-2 show defective expression of the major executioner of AICD, thus potentially contributing to AICD resistance and to the development of autoimmunity

Protein synthesis inEscherichia coli at 4°C

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Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes