During FY12 we accomplished the following: 1. Continued NF-κB induction and mRNA studies with Ideal participants. 2. Initiated studies to examine inducible gene expression in human CD4+ T cells after longer activation times compared to ongoing studies. 3. Carried out protein analysis of cytoplasmic extracts using iTRAQ methodology. For this we used 50 g extracts from 2 healthy young individuals (age 21, 30 years), three healthy old individuals (age 74-77 years) and three healthy ideal individuals (age 74-82). Data analysis and validation of iTRAQ results are underway. 4. We initiated studies of the human B cell response to activation. Total, or purified nave and memory, B cells obtained from cytapheresis packs were activated with phorbol ester and calcium ionophore for varying time points followed by preparation of nuclear and cytosolic protein extracts, and isolation of mRNA. To determine the effects of corticosteroids on inducible gene activation, B cells were also activated in the presence of dexamethasone followed by protein and RNA isolation. Analysis of RNA by microarrays is underway. 5. Several years ago we had initiated studies to examine the lipopolysaccharide (LPS) response of human monocytes. These studies were terminated when the post-doctoral fellow doing these experiments left prematurely. We have re-initiated these studies during FY12. Purified monocytes were incubated with LPS for varying times followed by preparation of nuclear and cytosolic extracts, and RNA isolation. Several young donors have been analyzed and we are awaiting availability of 4-5 older donors to complete the preliminary phase of analysis.
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