Biobank of small extracellular vesicles for pediatric sepsis

项目总结 脓毒症是一种宿主对感染的失调反应，可导致多器官功能障碍综合征 (MODS)。对于脓毒症引起的多器官功能障碍，目前还没有有效的治疗方法，很可能是因为这种异质性。 这种综合症的症状。描述器官衰竭的MODS内型和分子特征可能会带来更好的 了解脓毒症异质性所涉及的机制，并允许个体化治疗 战略。然而，很难从危重病人那里获得临床标本，这将使 对器官特异性机制变化的研究。胞外囊泡是一种球形微粒。 由双层磷脂膜包裹。Exosome或Small EV(SEV)是EV的一个亚型，由 内体生物发生。小型电动汽车可以从几乎任何类型的细胞释放到各种体液中，并 含有许多细胞成分。细胞特定的货物可以作为细胞间的通信器并被带走 被远端细胞上调，这会影响炎症状态。我们的初步数据显示，SEV从 与非脓毒症儿童相比，脓毒症患儿血清具有明显的促炎特性 脓毒症和脓毒症患者的体外SEV可在免疫细胞中诱导非典型炎症反应。自.以来 循环SEV表现出起源细胞的特征，它们已被用作液体活检。因此，SEV 具有作为器官特异性变化有用生物标志物的潜力。没有可供使用的SEV生物资源库 儿科败血症研究，部分原因是缺乏SEV分离的标准化方法学。整体而言 我们建议的目标是为SEV生物标记物的可靠生物储存库建立标准化程序 脓毒症危重患者的研究。这一提议将证明这样一种假设，即质量和一致性 血浆和血清样本的分离和纯化方案使建立可靠的生物储存库 脓毒症中SEV的未来研究。我们将利用两个基于重症监护部门的大型存储库 其中有来自儿科重症败血症和非败血症研究的生物标本。R21阶段目标1是 建立一种高产率和高纯度的SEV样本收集和分离方法，目标2是 证明储存的SEV适用于高通量的RNA货物剖面分析。曾经的里程碑 R21阶段已满足，我们将继续进行R33阶段，以回顾SEV内型的特征 有特定器官损伤的危重患者(目标3)，然后前瞻性地确定患者是否可以 根据它们的SEV特征进行分类。这些调查的结果将使这部小说 小儿败血症中SEV生物信息库的建立。这个生物仓库将使调查人员能够探索 用于SEV机制研究的器官特异性分子签名。

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.