PROJECT SUMMARY Sepsis is a dysregulated host response to an infection and can lead to multiple organ dysfunction syndrome (MODS). There are no effective treatments for sepsis-induced MODS most likely because of the heterogeneity of the syndrome. Delineating MODS endotypes and molecular signatures of organ failures may lead to a better understanding of the mechanisms involved in sepsis heterogeneity and allow for personalized treatment strategies. It is difficult, however, to obtain clinical specimens from critically ill patients that would enable investigations into organ-specific mechanistic changes. Extracellular vesicles (EVs) are spherical microparticles enclosed by bilayer phospholipid membranes. Exosomes or small EVs (sEVs) are a subtype of EV formed by endosomal biogenesis. Small EVs can be released from almost any cell type into a variety of bodily fluids and contain many cellular components. The cell-specific cargo can serve as cell-to-cell communicators and be taken up by distant cells which can affect the inflammatory profile. Our preliminary data show that sEVs harvested from serum of pediatric patients with sepsis have a distinct pro-inflammatory trait compared to sEVs of children without sepsis and in vitro sEVs from septic patients can induce atypical inflammatory responses in immune cells. Since circulating sEVs manifest characteristics of the cell of origin, they have been used as liquid biopsy. Thus, sEVs hold potential as useful biomarker for organ-specific changes. There are no available biorepositories of sEVs for pediatric sepsis research, in part, because of the lack of standardized methodology for sEV isolation. The overall goal of our proposal is to establish standardized procedures for reliable biorepositories for sEV biomarker research in critically ill patients with sepsis. This proposal will prove the hypothesis that quality and consistency of isolation and purification protocols of plasma and serum samples enable setting-up reliable biorepositories for future research on sEVs in sepsis. We will take advantage of two large critical care-division based repositories which has biospecimens from pediatric critically ill septic and non-septic studies. The R21 phase Aim 1 is to develop a methodology for sample collection and isolation of sEVs with high yield and purity and Aim 2 is to demonstrate suitability of banked sEVs for high throughput analyses of RNA cargo profile. Once milestones for the R21 phase are met we will proceed to the R33 phase to retrospectively characterize sEV endotypes in critically ill patients with specific organ injuries (Aim 3) and then prospectively determine whether patients can be classified based on their sEV characteristics. Results from these investigations will allow for the novel development of a biorepository of sEVs in pediatric sepsis. This biorepository will enable investigators to explore organ-specific molecular signatures for mechanistic studies of sEVs.