During fiscal year 2007 we accomplished the following:? ? (1) We developed a working protocol to minimize sample-to-sample variations. Our most important consideration was to develop a scheme that minimized experimenter-dependent variables that could potentially confound long-term longitudinal studies. The protocol includes a roadmap for cell purification, accounts for cell number variations by identifying 2-3 activation time points per sample, identifies anti-TCR concentrations for optimal induction and standardizes quantitative RT-PCR conditions to study inducible gene expression of cytokine genes.? ? (2) We developed a negative selection protocol to purify naive (CD45RA+) and memory (CD45RO+) CD4 + T cells from apheresis packs. These populations were activated with anti TCR antibodies to study the kinetics of NF-kB induction and NF-kB-dependent gene transcription. ? ? (3) We analyzed RNA purified from unactivated CD4+ T cells and from cells treated with anti-TCR for 2 and 4 hours by Illumina microarrays. The patterns from the 93 RNA samples were separated based on age and intracellular activation pathways.? ? (4) We initiated studies of NF-kB activation and gene expression with purified human B lymphocytes from apheresis samples.
| Ruggiero, Carmelinda; Metter, E Jeffrey; Cherubini, Antonio et al. (2007) White blood cell count and mortality in the Baltimore Longitudinal Study of Aging. J Am Coll Cardiol 49:1841-50 |
