Severe sepsis is the leading cause of death in intensive care units and accounts for 9.3% of overall deaths in the United States annually (Angus, 2001). This complex disorder is difficult to diagnose and treat, and there is a great need for a test that can profile patients' immune dysfunction. In Phase I of this grant, we will demonstrate the feasibility of a novel rapid multiplex assay for biomarkers of sepsis. We have developed a proprietary method for using the real-time-polymerase chain reaction (PCR) technology to measure protein concentrations in a multiplexed fashion with very high sensitivity and reproducibility. The method, Multiplexed Real-time-Immuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary data have demonstrated that multiplexed cytokine measurements employing MRI-PCR assays provides ~2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase I studies, a panel of reagents to detect murine markers of sepsis will be prepared and evaluated in the MRI-PCR assay. The target proteins will then be spiked into normal mouse serum to validate the assay in a physiological sample. An ex vivo model of endotoxemia will be used to measure production of cytokines in response to lipopolysaccharide stimulation in whole mouse blood as well as in vivo in a mouse model of endotoxemia. A mouse model of sterile burn-induced systemic immunoinflammatory response syndrome (SIRS) as compared to burn injury plus bacterial infection-induced sepsis will be performed to determine if the MRI-PCR assay can distinguish between SIRS induced by bacterial as opposed to non-infectious causes in a clinically relevant setting. In Phase II, the assay will be validated in rodent models of sepsis (cecal ligation and puncture), and reagents to detect human biomarkers of sepsis will be prepared. The human sepsis MRI-PCR test will be validated and used to analyze serum from normal and septic humans. The results of these studies will lead to two products: the human sepsis test for clinical diagnosis, prognosis, and monitoring of therapy, and the rodent sepsis test for researchers. Currently it is very difficult to diagnose and monitor therapy in patients with sepsis. This proposal will develop a serological test that measures multiple biomarkers of sepsis, and will be useful for the diagnosis, prognosis and monitoring of therapy in septic patients. [unreadable] [unreadable] [unreadable]