During FY13, we accomplished the following: 1) Continued to purify CD4+ T cells from IDEAL participants. Nuclear and cytoplasmic extracts, and RNA, was prepared from cells activated by anti-CD3 treatment for 2 and 4 hours. Combined analyses of this cohort will be compared to healthy old participants in the Baltimore Longitudinal Studies on Aging (BLSA). 2) Continued collection of RNA from CD4+ T cells treated with anti-CD3 for longer times (6,8,12h). 3) Continued ITRAQ analysis of cytoplasmic extracts from CD4+ T cells obtained from young and old individuals. These studies were carried out at the NHLBI facility directed by Dr. Marjan Gucek. 4) Studied the mechanism of metabolic activity-induced up-regulation of a subset of putative NF-κB target genes in humans peripheral blood CD4+ T cells. We tested the hypothesis that these genes were activated by mitochondria-generated reactive oxygen species (ROS). Our experiments conclusively ruled out a role of mitochondrial ROS. Therefore, we used pharmacologic inhibitors to block other metabolically-induced signaling pathways. We found that rapamycin had no effect on the subset of genes induced by 37⁰C incubation of CD4+ T cells. However, the PI3K inhibitor LY294002 blocked induction of a subject of these genes. We infer that a PI3K-dependent pathway is up-regulated in CD4+ T cells from the elderly. 5) Continued analysis of B lymphocytes obtained from PBMC. Established condition to purify nave and memory B cell subsets from PBMC. These cells were activated by treatment with PMA and calcium ionosphore for different times. RNA and protein extracts were prepared, and chromatin immunoprecipitation carried out with anti-RelA antibodies. B cell activation was also carried out in the presence of dexamethasone to evaluate the role of corticosteroids in inflammatory responses of B lymphocytes.
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