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Sepsis is the major cause of morbidity and mortality in critical care units in the USA. During sepsis, expression of pro-inflammatory genes is repressed when blood leukocytes are stimulated ex vivo with bacterial endotoxin. Endotoxin tolerance may reflect a state of immunosuppression that contributes to the high mortality rate observed in sepsis. The objective of this proposal is to define the molecular basis of endotoxin tolerance, using three models: blood leukocytes of patients with sepsis, blood monocytes, and THP-1 pro-monocytic cells. We will test the hypothesis that a labile factor(s) mediate the tolerant phenotype by repressing the transcription of pro-inflammatory genes such as IL-1beta and PGHS-2. Aim 1 using a biochemical approach and an in vitro transcription assay to identify, characterize, and purify the transcription repressor(s). An alternative genetic approach seeks to identify the transcription repressor in endotoxin tolerant THP-1 cells using differential display RT-PCR. Aim 2 investigates the intracellular signaling pathways utilized by endotoxin to modulate pro-inflammatory gene expression. Constitutively active or dominant-negative mutants of kinase mediators are employed in co-transfection assays to test the impact of various kinase activities on IL-1beta transcription. Immunologic approaches are used to identify activated/inactivated kinases in the normal and tolerant phenotypes. Aim 3 proposes to translate our understanding of the in vitro endotoxin tolerant THP-1 cell model to the in vivo tolerant phenotype of septic patients. A genetic approach, using differential display RT-PCR, and a biochemical approach, using the purified repressor, will compare the two phenotypes. Immunological techniques also will be applied to the septic patient model to identify kinases that regulate endotoxin responses and they be activated/inactivated in the tolerant phenotype. These investigations will increase our understanding of the mechanisms that regulate pro- inflammatory gene expression and contribute to improving the management of patients with sepsis.
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