SEPSIS-INDUCED RED CELL DYSFUNCTION (SIRD)

描述(由申请人提供)：本项目的目标是：(1)充分描述SIRD是一种截然不同的器官衰竭形式，损害脓毒症患者的氧气输送；(2)阐明SIRD的作用 在多器官衰竭(MOF)进展中，以及(3)评估针对SIRD病理生物学的基于机制的治疗。在脓毒症中，已经(单独)描述了许多红细胞缺陷：氧亲和力改变，膜变形，红细胞聚集和黏附，以及以红细胞为基础的一氧化氮(NO)处理失调。我们认为，这些缺陷包括一种独特的器官衰竭(我们称之为SIRD)，它使O2无法从肺转移到组织。基于我们的初步发现，我们提出了一种新的假说，即在脓毒症中，RBC抗氧化系统的能量支持失败，SRD的发生是由于在血红蛋白O2结合/释放过程中产生未猝灭的活性氧物种(ROS)。因此，通过严重损害氧气的输送(通过将红细胞的输送限制为 组织[如流动]和从输送的红细胞中释放氧气)，SERD加剧了缺氧和多器官功能衰竭的进展。我们提出了一种机械性的“反向转换”方法，在严重脓毒症儿童的全面表型队列中测试这一假设(使我们能够研究缺乏共病的受试者，这些合并症也损害红细胞功能，例如糖尿病、肾功能衰竭等)。 我们将在NICHD协作性儿科重症监护研究网络[CPCCRN]进行的《儿科脓毒症所致多器官衰竭的炎症表型研究》(PHEROMS试验[GM108618])中对儿童进行研究。这将使我们能够利用已建立的CPCCRN基础设施以及对表现型队列的详细表型和结果评估，以便我们可以将SRD与脓毒症综合征的进展、多器官功能衰竭的演变以及结果联系起来。我们将通过追求以下具体目标来构建我们的方法：SA1。定义败血症引起的影响氧气输送的红细胞生化改变。我们将在体外测试平台、器官生物测试和体内模型中对PEROMS受试者的红细胞进行研究，以量化O2结合/传递以及血管张力和血流控制方面的缺陷(以及超氧化物歧化酶模拟物在恢复方面的有效性)。SA2定义了败血症引起的影响氧气输送的红细胞生物物理变化。如上所述，受试者的红细胞将使用最先进的生物物理分析(膜变形性、红细胞聚集性和内皮黏附)和活体显微镜进行研究，以量化红细胞通过血管通道的运输和与激活的内皮细胞的黏附方面的缺陷(以及超氧化物歧化酶模拟物在修复中的有效性)。SA3是脓毒症引起的红细胞能量代谢、抗氧化系统和氧化损伤的改变。研究对象的红细胞将受到控制的氧化负荷，以量化动态范围(以及超氧化物歧化酶模拟物在修复中的有效性)：糖酵解通量(乳酸同位素异构体的1H核磁共振分析)，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.