Gram-negative bacterial sepsis and shock remains a significant cause of morbidity and mortality in surgical patients and it is now well established that gram-negative bacterial lipopolysaccharide (LPS, endotoxin) plays a critical role in the pathophysiology of this lethal disease process. Increasing evidence indicates that the lipid A portion of LPS is responsible for the majority of adverse effects that occur, including those mediated by cytokines such as tumor necrosis factor-alpha (TNF-alpha). Lipid A appears to be capable of interacting with the macrophage cell membrane to trigger the transcription and translation of TNF-alpha mRNA, following which secretion of this cytokine into the extracellular milieu occurs. The objective of this research program continues to be the development and testing of endotoxin antagonists. In this competitive renewal two specific aims are proposed:
Aim 1 : To develop, characterize, and compare and contrast the efficiency and nature of the in vitro inhibitory activity of three types of endotoxin antagonists: 1) anti-deep core/lipid A (DCLA) monoclonal antibodies (mAbs), 2) peptide derivatives of bactericidal permeability-increasing protein (BPI) and other closely related compounds, and 3) anti-idiotypic anti-DCLA mAbs, and Aim 2: To examine the protective capacity and to determine the mechanism(s) of action of each of these endotoxin antagonists in vivo in animal models of experimental gram-negative bacterial sepsis and endotoxemia in relation to: 1) cytokine transcription, translation, and secretion in the local tissue milieu and the systemic circulation, 2,) neutralization and clearance of endotoxin, and 3) bactericidal activity and bacterial clearance. The, capacity of each of these compounds to inhibit the effects of LPS will be examined in vitro, in several different systems: 1) LPS, neutralization 2) bactericidal activity, and 3) cytokine transcription, translation, and secretion. Protective efficacy will be tested in three types of animal models. 1) gram negative bacteremia, 2) gram negative, peritonitis and 3) endotoxemia. Cytokine transcription, translation, and secretion will be measured ex vivo to pinpoint events occurring in the local tissue milieu, and systemic cytokine,levels will be examined. Neutralization and clearance of endotoxin and bacterial clearance will be investigated as well. An attempt will be made to compare and contrast the in vitro and in vivo results obtained from testing each of the three types of compounds, thereby establishing precise mechanism(s) of action. Finally, comparisons at the molecular level of the specific components of each of these compounds that inhibit the effects of LPS will be undertaken using both sequencing and cloning techniques in order to identify those critical regions of the molecule that are responsible for anti LPS activity. These data will be used to develop additional potent LPS antagonists.
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