During the previous grant period our research focused on understanding the early intracellular signaling events which are involved in the initiation and propagation of sepsis syndrome. We found that suppressing pro-inflammatory intracellular signaling events during the early phase of sepsis strongly correlated with improved outcome. Specifically, we investigated the protective efficacy of glucan ligands in polymicrobial sepsis and determined that receptor recognition of glucan modulates intracellular signaling pathways such that the inflammatory response to sepsis is """"""""blunted"""""""". We established that glucan pre- or post-treatment would blunt sepsis induced tissue NFkappaB and NF-IL6 activation as well as decrease pro-inflammatory cytokine gene transcription. Blunting early increases in transcription factor activity and cytokine gene expression strongly correlated with decreased morbidity and mortality. We determined that glucan treatment blunted LPS induced NFkappaB activity through decreased MEKK1, NIK and IKKalpha/beta kinase activity as well as decreased Ikappa-Balpha phosphorylation and degradation. We confirmed the existence of multiple glucan binding sites on macrophages, defined the molecular structure of a glucan and confirmed that a heptaose (7 glucose subunit) polymer was the minimum binding unit. Preliminary data suggest that Toll receptor (TLR) 2, and perhaps CR3, confers responsiveness to glucan. The hypothesis for this continuation proposal is that lucan ligands ameliorate septic sequelae in polymicrobial sepsis by modulating inflammatory responses via interactions with Toll-like receptors (TLR) and/or CR3 (CD11b/CD18) binding sites. There are four specific aims. 1. We will characterize the receptor mediated interaction of glucan ligands with TLR 1, 2 and 4, the type 3 complement receptor (CR3) and CD 14 using a surface plasmon resonance approach. 2. We will establish the role of TLR2 and TLR4 in the anti-sepsis effect of glucan by studying CLP sepsis in TLR2 and TLR4 knockout mice. 3. We will investigate the role of CR3 (CD11b/CD18) in the anti-sepsis effect of glucan by studying CLP sepsis in CR3 knockout mice. 4. During the last grant period we made great strides in understanding the basic chemistry of (1-3)-beta-D-glucans. Using this knowledge, we will synthesize chemically pure, highly uniform, water soluble (1-3)-beta-D-glucan ligands which have specific structural characteristics. We will prepare a library of small molecular weight (1-3)-beta-D-glucans which will be used to prepare larger polymers. The synthetic polymers will be evaluated in receptor binding studies, in vitro intracellular signaling studies and in vivo protection studies using the CLP model. The successful completion of these aims will advance our understanding of the cellular and molecular events associated with sepsis syndrome and may lead to the development of new therapeutics.
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