SEPSIS-INDUCED RED CELL DYSFUNCTION (SIRD)

描述（由申请人提供）：本项目的目标是：（1）充分表征SIRD作为脓毒症中器官衰竭损害O2输送的独特形式，（2）阐明SIRD的作用 在多器官衰竭（MOF）进展中，和（3）评价靶向SIRD病理生物学的基于机制的治疗。在脓毒症中，已经（单独）描述了许多RBC缺陷：改变的O2亲和力，膜变形性，RBC聚集和粘附，以及基于RBC的一氧化氮（NO）处理失调。我们认为，这些缺陷包括一类独特的器官衰竭（我们称之为SiRD），使O2从肺部运输到组织。基于我们的初步研究结果，我们提出了一个新的假设，即在败血症，红细胞抗氧化系统的有力支持失败，与SiRD随之产生的未淬灭的活性氧（ROS）在血红蛋白O2结合/释放过程中产生的。因此，通过严重损害O2的输送（通过限制RBC的输送， 组织[例如流动]和从递送的RBC释放O2），SiRD加剧了缺氧和MOF进展。我们提出了一种机械的“反向翻译”方法，以测试这一假设在一个全面的表型队列的儿童严重脓毒症（使我们能够研究受试者缺乏合并症，也损害红细胞功能，如糖尿病，肾功能衰竭等）。 我们将在“儿科脓毒症诱导的多器官衰竭中的炎症表型”（PHENOMS试验[GM 108618]，将由NICHD协作儿科重症监护研究网络[CPCCRN]进行）中研究儿童。这将使我们能够利用已建立的CPCCRN基础设施和PHENOMS队列的详细表型和结局评价，以便我们可以将SiRD与脓毒症综合征的进展、MOF演变和结局联系起来。我们将通过追求以下具体目标来构建我们的方法：SA 1。定义败血症诱导的红细胞生化改变，影响O2输送。将在离体测定平台、器官生物测定和体内模型中研究PHENOMS受试者的RBC，以定量以下方面的缺陷（和SOD模拟物在恢复中的功效）：O2结合/递送以及血管张力和血流的控制。SA 2定义脓毒症诱导的红细胞生物物理学改变，影响O2输送。如上所述，将采用最新技术水平的生物物理分析（用于膜变形性、RBC聚集和内皮粘附）和活体显微镜检查来研究受试者的RBC，以定量以下缺陷（和SOD模拟物在恢复中的功效）：RBC通过血管通道的转运和与活化内皮的粘附。SA 3表征败血症诱导的RBC能量代谢、抗氧化系统和氧化损伤的改变。将对研究受试者的RBC进行受控的氧化负荷，以定量以下方面的动态范围（以及SOD模拟物在恢复中的功效）：糖酵解通量（乳酸盐同位素异构体的1H NMR分析）、抗氧化系统中的氧化还原平衡，以及（c）对膜和蛋白质的氧化损伤。

DESCRIPTION (provided by applicant): The goals of this project are to: (1) fully characterize SIRD as a distinct form of organ failure impairing O2 delivery in sepsis, (2) elucidate SIRD's role in multiple organ failure (MOF) progression, and (3) evaluate a mechanism-based therapy targeted to SIRD pathobiology. In sepsis, a number of RBC defects have been (individually) described: altered O2 affinity, membrane deformability, RBC aggregation and adhesion, as well as dysregulated RBC-based nitric oxide (NO) processing. We suggest that these defects comprise a unique class of organ failure (which we term SiRD) that disables transport of O2 from lungs to tissue. Based upon our preliminary findings, we propose the novel hypothesis that in sepsis, energetic support of RBC antioxidant systems fails, with SiRD arising consequent to unquenched reactive oxygen species (ROS) generated in the course of hemoglobin O2 binding/release. As such, by critically impairing O2 delivery (by limiting both delivery of RBCs to tissue [e.g. flow] and release of O2 from delivered RBCs), SiRD exacerbates dysoxia and MOF progression. We propose a mechanistic 'reverse translational' approach to test this hypothesis in a comprehensively phenotyped cohort of children with severe sepsis (enabling us to study subjects lacking comorbidities which also impair RBC function, e.g. diabetes, renal failure, etc.). We will study children in the "Inflammation Phenotypes in Pediatric Sepsis Induced Multiple Organ Failure" (PHENOMS Trial [GM108618], which will be conducted by the NICHD Collaborative Pediatric Critical Care Research Network [CPCCRN]). This will enable us to leverage the established CPCCRN infrastructure and the detailed phenotype and outcome evaluation of the PHENOMS cohort so that we may link SiRD to progression of sepsis syndromes, MOF evolution, and to outcome. We will structure our approach by pursuing the following Specific Aims: SA1. Define sepsis-induced biochemical alterations to RBCs that influence O2 delivery. PHENOMS subjects' RBCs will be studied in ex vivo assay platforms, organ bioassays and in vivo models to quantitate defects in (and efficacy of SOD mimetics in restoring): O2 binding/delivery as well as control of vascular tone and blood flow. SA2 Define sepsis-induced biophysical alterations to RBCs that influence O2 delivery. As above, subjects' RBCs will be studied employing state of the art biophysical analysis (for membrane deformability, RBC aggregation and endothelial adhesion) and intravital microscopy to quantitate defects in (and efficacy of SOD mimetics in restoring): RBC transit through vascular channels and adhesion to activated endothelium. SA3 Characterize sepsis-induced alterations in RBC energy metabolism, antioxidant systems and oxidative injury. Study subjects' RBCs will be subjected to controlled oxidative loading to quantitate the dynamic range in (and efficacy of SOD mimetics in restoring): glycolytic flux (1H NMR analysis of lactate isotopomers), redox poise in antioxidant systems, and (c) oxidative injury to membrane and proteins.