This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.While initial survival following severe trauma has improved, long term recovery is frequently impaired by the development of multiple organ dysfunction syndrome (MODS). Following trauma, MODS is usually in response to infection and associated factors, such as lipopolysaccharide (LPS), which is characteristic of sepsis. Following trauma and during sepsis, the circulating monocyte and tissue-fixed macrophage undergo phenotypic differentiation and reprogramming that results in organ injury. In our background studies, we found that this state can be mimicked in vitro by factors induced by severe trauma, such as platelet activating factor (PAF), oxidant stress and complement 5a (C5a) and by tolerance induction.Cellular reprogramming is not associated with significant liberation of inflammatory mediators. Rather, cellular reprogramming leads to dysregulated inflammatory mediator production in response to subsequent infectious stimuli, thus increasing the risk for organ dysfunction. The mechanism responsible is associated with initial ceramide generation by sphingomyelinase, resulting in altered fluidity within specialized membrane components termed lipid rafts. Due to this gel-phase fluidity, altered kinetics occur leading to re-organization of raft proteins. This alteration in protein content is associated with the pre-assembly of Toll-like receptor (TLR) components that results in accelerated TLR complex assembly and activation in response to subsequent stimuli. To explore the molecular mechanisms responsible, we will test the hypothesis that formation of these complexes occurs during cellular reprogramming and activation using proteomic techniques on raft and non-raft protein isolates.
The specific aims of this project are to:1) Determine the lipid raft characteristics of mononuclear cells following LPS stimulation2) Determine the lipid raft characteristics of mononuclear cells following reprogramming by factors induced by severe injury
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