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Rationale: Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity. Objectives: We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU). Methods: Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature. Results: A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation. Conclusions: Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.

Rationale: Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity. Objectives: We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU). Methods: Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature. Results: A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation. Conclusions: Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.

In DARPA-supported studies, we have shown that a family of novel synthetic glycolipids known as the aminoalkyl glucosaminide phosphates (AGPs) act as potent agonists of Toll-like receptor 4 (TLR4), and that administration of AGPs to the airways results in protection against lethal challenge with virulent bacterial and viral agents. Preliminary toxicological studies indicate that intranasal delivery of the compounds is safe, and human clinical trials with aerosolized TLR4 agonists are now anticipated. To monitor the effectiveness of immunoprophylaxis in human trials, it may become necessary to develop surrogate biomarkers of protection since experimental challenge endpoints are not readily available. Under the current contract, we have completed a series of in vitro and in vivo studies to identify candidate biomarkers via microarray-based-based transcriptional profiling, conventional multiplex cytokine and flow cytometric analyses. Through these studies we have identified biomarkers suitable for clinical monitoring of AGP mediated protection. Synthesis and scale- up requirements have been established and carried out for a single lead AGP candidate, CRX-527, for production of OMP grade material suitable for clinical trials. Formal GLP toxicology studies with CRX-527 will be initiated in early 2005 with a Phase I Clinical trial planned for the first quarter of 2006. Pre-IND documents for this phase I clinical trial are currently being assembled and will be submitted to the FDA for approval in mid-October.