INFECTION MEDIATED OXIDANT DAMAGE IN SICKLE CELL ANEMIA

这项提案描述了一项研究感染中介作用的计划。 镰状细胞性贫血(SCA)的氧化损伤。感染是已知的 诱发SCA的溶血亢进和血管闭塞危象。我计划这样做 确定感染期间中性粒细胞释放的氧化剂是否会改变 红细胞膜，从而促成这些临床事件。这个 我研究的第一阶段将利用敏感技术在体内检测 感染期间红细胞(RBC)氧化剂受损的证据。这些 技术包括高效液相色谱检测血脂 过氧化氢，检测丙二醛的荧光光谱分析 用于检测过氧化的交联脂或蛋白质和双烯偶联 脂类。细胞密度和氧化剂损伤之间的关系将是 探索过了。我接下来将进行体外研究，利用激活的 中性粒细胞(AN)与红细胞孵育以确定结构变化是否 在镰状细胞膜中由氧自由基诱导。在……里面 除了标准技术外，还可以测量脂类和 蛋白质，我将使用电子自旋共振和自旋标记技术 研究脂质/蛋白质变化和硫醇-二磺酸盐交换 用层析法测定巯基的氧化。这些研究将 还结合使用不同的自由基清除剂来识别产品 参与氧化的一种。功能改变(AN对膜的影响 酶活性、阳离子通量、细胞密度和变形性)，这可能 对AN引起的伴随结构损伤进行了研究。相互关联 氧化剂损伤随着细胞表面性质的可能改变， 将测量AN对RBC与单核细胞黏附的影响。这个 这项研究的最后阶段将调查诱导的 氧化剂损伤。我将使用两个模型系统：血小体[血红蛋白 (Hb)S或A被包裹在脂质体中]和杂交红细胞(正常 膜/镰状Hb；镰状膜/正常Hb)。这些系统将 允许操纵Hb、脂质含量、自由基清除剂和 为了阐明各膜组分在细胞周期中的作用 镰刀状红细胞对介导性损伤的敏感性。总而言之，这是 该项目将1)在体内寻找感染患者的红细胞氧化剂损伤的证据 SS患者，2)定义由An和And引起的膜结构损害 结构损伤与由此产生的功能性细胞变化相关 这可能导致SCA的恶化，以及3)调查 一种诱导氧化剂损伤的机制。

This proposal describes a plan to study the role of infection mediated oxidant damage in sickle cell anemia (SCA). Infections are known to precipitate hyperhemolysis and vaso-occlusive crisis in SCA. I plan to determine if oxidants released from neutrophils during infection alter the red cell membrane and thereby contribute to these clinical events. The first phase of my study will utilize sensitive techniques to detect in vivo evidence for red blood cell (RBC) oxidant damage during infection. These techniques include high performance liquid chromatography to detec lipid hydroperoxides, fluorescent spectroscopy to detect malondialdehyde cross-linked lipids or proteins and diene conjugation to detect peroxidized lipids. Relationships between cell density and oxidant damage will be explored. I will next perform in vitro studies which utilize activated neutrophils (AN) incubated with RBCs to determine if structural changes are induced in sickle cell membranes by oxy radicals elaborated from AN. In addition to standard techniques to measure oxidant damage of lipids and proteins, I will use electron spin resonance and spin labeling techniques to study lipid/protein alterations and thiol-disulfine exchange chromatography to measure sulfhydryl group oxidation. These studies will also incorporate use of different radical scavengers to identify products of AN involved in oxidation. Functional changes (effects of AN on membrane enzyme activities, cation flux, cell density and deformability) which may accompany structural damage induced by AN will be studied. To correlate oxidant damage with possible alterations in cell surface properties, the effects of AN on adherence of RBC's to monocytes will be measured. The last phase of this study will investigate the mechanisms of AN induced oxidant damage. I will employ two model systems: hemosomes [hemoglobin (Hb) S or A encapsulated into liposomes] and hybrid erythrocytes (normal membranes/sickle Hb; sickle membranes/normal Hb). These systems will permit manipulations of Hb, lipid content, free radical scavengers and membrane components in order to elucidate the role of each in susceptibility of sickle RBC to AN mediated damage. In summary, this project will 1) look for in vivo evidence of RBC oxidant damage in infected SS patients, 2) define membrane structural damage caused by AN and correlate structural damage with resultant functional cellular changes which may predispose to exacerbations of SCA, and 3) investigate the mechanisms of AN induced oxidant damage.