Gram-negative bacterial septicemia remains a common cause of death in patients who survive traumatic and thermal injury. Bacterial lipopolysaccharide (LPS, endotoxin) is an important cause of the sepsis syndrome. LPS binds CD14, a glycosyl phosphatidylinositol-linked protein on monocytic cells, to initiate inflammatory mediator release from phagocytes. The LPS-signal transduction system has been partially characterized using both natural and synthetic analogs of lipid A as antagonists or mimetics of LPS stimulation. In addition, components of the CD14-signaling pathway have been constructed in transfectabie cell lines and studied as a model of monocytic function. Recent studies combining both approaches to LPS signaling have indicated that the target of the LPS antagonists was not CD14, but may be an associated signal transducing molecule. The central hypothesis of this proposal is that stimulation ofphagocytes during sepsis begins with the binding of LPS to a receptor complex consisting of CD14 and a yet to be defined signal transduction molecule. LPS binding activates a signal transduction cascade which leads to the translocation of nuclear factor-kB and then to the activation of genes encoding inflammatory mediators. The focus of this proposal is to identify elements of LPS-signaling pathways in CD14-transfected fibroblast and glial cell lines. Unlike monocytic lines, these lines can be genetically manipulated in order to identify genes involved in LPS.
Five Specific Aims are proposed.
Aim 1 is designed to define the pharmacology of LPS signaling, because ultimately the pharmacology and genetics of LPS signaling must conform with one another in any valid model of LPS signaling.
Aim 2 is designed to identify the genetic defect for LPS signaling in CD14-transfected HeLa cells.
Aim 3 describes somatic cell mutagenesis studies designed to exploit the availability of stable CD14- expressing lines which """"""""report"""""""" in response to LPS in order to clone genes involved in CD14-mediated NF-kB translocation.
In Aim 4, genes which are identified in engineered cell lines as critical components of an LPS signaling pathway will be examined in monocytes in order to determine their role in the induction of cytokine release.
In Aim 5, we propose to define pathways of cytokine gene expression in CD14-transfected HT 1080 fibroblasts. The completion of these aims should lead to a greater understanding of how LPS receptors function. Such an understanding is a clear requisite for the design of effective anti-endotoxin therapies.
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