Endotoxic lipopolysaccharides (LPS), by virtue of their functional localization on the outer membrane of gram negative bacteria, play a pivotal role in the interaction of the bacterium with host defenses. One of the most important of these interactions is with serum proteins, particularly those of the complement system. Structural determinants of LPS have been recognized to be critical in defining sensitivity or resistance of gram negative bacteria to the lytic effects of serum complement. Equally important are the potential pathophysiological effects mediated by endotoxic LPS released from the bacterium following serum bacteriolysis, which include shock, hypotension and disseminated intravascular coagulation. In spite of the numerous serum factors reported to detoxify the endotoxic activities of LPS both the factors and their mechanism of action remain ill defined. The experiments outlined in this proposal have been designed to investigate the influence specific LPS polysaccharide structures have upon the interactions between LPS and both complement and non-complement serum proteins. For these studies the levels of specific LPS determinants contained within macromolecular LPS aggregates will be varied by manipulating LPS subunit composition. Specifically constructed LPS preparations will be used to investigate the mechanisms by which LPS structures regulate the activation of both the classical or alternative pathways of complement. For these experiments, purified complement components of both pathways will be used to assess directly the modulation of complement component interactions by specific LPS structures. These studies will increase the understanding of the structural parameters which allow components C1 and C3 to descriminate between self and non-self. Extensive use will be made of a radioiodinated photoactivable LPS derivitive to study serum components with an affinity for LPS which could bind LPS and alter its biological activities. Proteins of normal human serum identified by LPS crosslinking will be characterized by biochemical and immunological analysis. In addition, these proteins will be tested for their capacity to inhibit LPS mediated coagulation of Limulus ameobocyte lysates (LAL). Identification of LPS inactivating factors could allow for the development of potentially useful protocols for the diagnosis and treatment of endotoxemia.
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