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The objective of this study is several-fold. We wish to determine the response of white blood cells (neutrophils, monocytes, eosinophils, lymphocytes) to various signals in the test tube, to test how they sense the signals in the body, and what substances they produce. We wish to determine how these cells may, under certain circumstances, contribute to inflammation. We wish to measure the substances in the blood plasma that might contribute to stimulation of white blood cells to understand the response of the whole blood to potential disease-associated conditions.

The objective is to define the signaling pathways activated by lipopolysaccharide (LPS) and other selected innate immunity stimuli, and the downstream inflammatory functional consequences, in human leukocytes in vitro. Adult (18-65 yrs. old), nonpregnant, healthy volunteers will have 320 ml of whole blood collected by venipuncture in a monitored setting no more frequently than once every 8 weeks. No further interventions will be exercised upon the subjects. The whole blood will be fractionated into neutrophil, red blood cell, mononuclear cell, and plasma fractions using plasma-Percoll discontinuous centrifugation. Leukocytes will be subjected in vitro to inflammatory stimuli (e.g., LPS), and selected signaling outcomes (e.g., mitogen-activated protein kinase activation, Rho GTPase activation, protein-protein interactions) and functional measures (e.g., chemotaxis, superoxide anion and cytokine production) quantified in the absence and presence of relevant chemical inhibitors (e.g., SB203580, a p38 MAPK inhibitor). In each such experiment, cells from the daily donor will be used as paired controls to the in vitro experimental intervention (e.g., SB203580 inhibitor vs. DMSO vehicle). Three or more repetitions (on different donors) of each specific experimental outcome, as necessary, will be performed to establish statistical significance of findings.

A specific focus of the studies planned will be to define the role of lipid raft membrane microdomains in transduction of the LPS signal in human leukocytes. Lipid rafts are cholesterol-rich microdomains in the plasma membrane, within which the LPS receptor, Toll-like Receptor 4 (TLR4), has been described to reside. LPS signaling has been reported to be sensitive to raft cholesterol content, presumably because the specific repertoire of proteins in rafts is sensitive to raft cholesterol content. Rafts are thought to act as dynamic signaling platforms for co-segregation of proximal adaptor proteins, kinases, and other signaling proteins. Of interest, while LPS has been described to modulate the activity of proteins that determine raft cholesterol content (egg, Liver X Receptor, ABCA1), virtually no work has been done to clarify: 1) the mechanisms underlying LPS-induced intracellular cholesterol redistribution, and, more importantly, 2) whether such intracellular redistribution of cholesterol is causal to the signaling events triggered by LPS, or 3) whether innate immunity signaling is dependent upon inter-subject variations in raft cholesterol content.

Furthermore, we will investigate the role of the tumor suppressor gene p53 in the regulation of inflammation. It is now widely accepted that inflammation and cancer development are interconnected. Dr. Menendez is one of the international leaders in the study of the tumor suppressor gene p53. His group has discovered that activation of p53 through exposure to carcinogenic stimuli leads to differential expression of genes that have a direct effect on the inflammatory response, such as several toll-like-receptor genes. Dr. Menendez will use human leukocytes that will be isolated from whole blood. He will expose these cells to stimuli that activate p53, such as doxorubicin (a chemotherapy agent) or radiation, and examine the expression of toll-like-receptor genes as well as the response to LPS and other inflammatory agents in vitro.

Finally, we will investigate the role of zinc-finger proteins in gene expression in inflammatory cells. ZFP36 is an RNA binding zinc finger protein that is involved in the turnover of mRNAs encoding several clinical important cytokines, including TNFa. Several missense, promoter and 3'-untranslated variants have been identified in humans; in some cases, these variants and their haplotypes have been associated with human inflammatory disease. We will isolate peripheral blood macrophages and stimulate them with LPS and other cytokines, then evaluate the behavior of cytokine mRNA and gene expression.