Gram-negative bacterial sepsis and shock continues to be a significant problem for surgical patients. Despite improvements in antibiotic therapy and intensive care, mortality associated with gram-negative bacterial sepsis remains 40%. Increasing evidence indicates that the lipid A portion of the lipopolysaccharide (LPS, endotoxin) --an integral portion of the gram-negative bacterial membrane--interacts with a variety of host defenses to cause the release of cytokine mediators of sepsis such as tumor necrosis factor-alpha (TNFa), interleukin-1beta (IL-1b), interleukin-6 (IL-6), and interleukin-8 (IL-8). The objective of this research program continues to be to develop, characterize, and test LPS antagonists. In this competitive renewal, two specific aims are proposed:
Aim 1 : To develop and characterize in vitro the inhibitory activity of LPS antagonists including: 1) peptide and fusion protein derivatives of bactericidal/permeability-increasing protein (BPI) and Limulus anti-lipopolysaccharide factor (LALF), 2) anti-idiotypic anti-deep core/lipid a (DCLA) and anti-DCLA mAbs and 3) single-chain Fv constructs of these mAbs.
Aim 2 : To examine the protective capacity and to determine the mechanism(s) of action of each of these LPS antagonists in vivo in animal models of experimental gram-negative bacterial sepsis and endotoxemia. In addition to survival, the investigators will quantitate 1) neutralization and clearance of LPS, 2) bactericidal activity and bacterial clearance, and 3) cytokine transcription, translation, and secretion locally and systemically. They will compare and contrast the in vitro and in vivo results obtained from testing each type of LPS antagonist. Comparisons at the molecular level will be undertaken using sequencing and cloning techniques, in order to identify those critical regions of the molecule that are responsible for anti-LPS activity. This information will be used to delineate the molecular basis of endotoxin antagonism and to develop additional potent LPS antagonists. Use of transgenic mice devoid of either TNFR1, IL-1R1, or both in conjunction with the above mentioned reagents will be used to examine the relative roles of TNFa and IL-1 signalling during endotoxemia and gram-negative bacteremia and peritonitis.
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