The proposed studies are focused on detailed characterization of the molecular mechanism of CD14-dependent activation of immunocompetent cells during sepsis and septic shock. CD14 is a membrane GPI-anchored """"""""pattern recognition receptor"""""""" that is found to be associated with sphingolipid-rich microdomains in monocytes and macrophages. Although CD14 lack of a transmembrane domain, it is capable of recognizing a variety of gram-negative and gram-positive bacterial components including but, perhaps, not limited, lipopolysaccharide (LPS), peptidoglycan, lipoarabinomannan (LAM), and lipoproteins. This CD14-mediated activation of monocytes/macrophages by bacterial products results in a potent host cytokine response. Evidently, specific binding of microbial constituents to CD14 initiates a signal transduction cascade in the target cells. The potential role of CD14 in the pathophysiologic sequelae of bacterial sepsis is supported by studies on CD14 knockout mice that strongly indicate the resistance of these mice to otherwise lethal LPS-mediated shock and to the effects of severe bacterial infection. However, the mechanism of CD14-dependent activation of phagocytes remains poor understood. A number of Toll-like receptors (TLRs), that are homologues to the Drosophila Toll receptor, have recently been characterized. It was shown that TLRs mediate signaling by LPS and other microbial constituents in mammalian cells. The PI and his colleagues have demonstrated a direct association between Toll-like receptors and LPS in the cell membrane. To define the functional role of CD14 and TLRs in signaling and activation of myeloid cells, it is proposed to use a combination of biochemical (delineation of CD14-TLR associated kinase cascade) and genetic approaches (selection of mutant macrophage cells and correction of their specific defects with a cDNA library). The relationship between CD14, scr kinases, G proteins and the TLRs in mechanism of signaling will be defined, as will cellular location. These studies will help to define how host invasion by microbes leads to septic shock and may provide novel targets for the design of therapeutic interventions against the life-threatening conditions associated with bacterial sepsis.
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