The focus of this application is molecular mechanisms of septic shock with attendant Disseminated Intravascular Coagulation (DIC) affecting annually 400,000 hospitalized patients in the US. Septic shock along with acute respiratory distress syndrome and multiple organ failure constitutes the extreme form of systemic inflammatory response syndrome. The prototypical inducers of septic shock are endotoxin (lipopolyscacharide, LPS), a product of Gram-negative bacteria and superantigens, a product of Gram- positive bacteria. In many cases these bacterial infections occur in the setting of indwelling catheters coated with bacterial biofilms following their insertion for neonatal parenteral nutrition, accident- and surgery-associated trauma, cancer chemotherapy, and immunosuppression for bone marrow and other transplants. The central and unifying hypothesis of the molecular mechanism of septic shock is based on intracellular signaling to the nucleus in response to inflammatory, oxidant, and immune stress. This signaling is mediated by transcription factors NFkappaB, AP-1, and others controlling expression of multiple genes encoding cytokines and chemokines, procoagulant and cell adhesion molecules, inducible NO synthase and cyclooxygenase 2, and apoptosis regulators. Activation of NF- kappaB requires signal-dependent phosphorylation and intracellular proteolysis of inhibitory molecules, IkappaBalpha, and epsilon, sequestering NF-kappaB in cytoplasm. The key signal transducers responding to inducers (LPS) and mediators (cytokines) of septic shock are recently discovered IkappaB kinase (IKK) complex. To establish their pivotal role in the molecular mechanism of cellular responses to LPS and toxic superantigens we will analyze regulation of I kappa B kinases in monocytic and T cells targeted by LPS and superantigens. The first level of analysis will involve the role of Toll-like receptors, in activation of IKK complex by LPS. The second level of analysis will be activation of IKK complex by superantigen-T cell receptor CD3 signalling. The third level of analysis will be signaling to the nucleus by NF-kappaB and other transcription factors based on Nuclear Localization Sequence (NLS) recognition by importins/karyopherins involved in nuclear import of transcription factors. Novel technologies of non-invasive delivery of peptides, protein and recombinant fragments to cells involved in septic shock will be developed and tested in vitro and in vivo to attenuate activation of genes regulated by NFkappaB and other transactivators to prevent monocytic, endothelial and T cells from acquiring a """"""""septic shock activated"""""""" phenotype. Implicit in this analysis of the molecular mechanism of septic shock and DIC is the development of new therapies targeted at intracellular signaling to the nucleus. The ultimate goal of the proposed studies is to provide a new conceptual framework for reduction of high mortality due to septic shock.
