INFECTION MEDIATED OXIDANT DAMAGE IN SICKLE CELL ANEMIA

镰状细胞性贫血中感染介导的氧化损伤

• 批准号: 3448656
• 负责人: SUSAN CLASTER
• 金额: $ 5.31万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3448656
• 关键词: antioxidants; autooxidation; bactericidal immunity; cellular pathology; density gradient ultracentrifugation; electron spin resonance spectroscopy; erythrocyte membrane; fluorescence spectrometry; free radicals; hemoglobin As; hemoglobin Ss; hemolysis; high performance liquid chromatography; human subject; hydroxyl group; lipid peroxides; liposomes; malonaldehyde; membrane activity; membrane lipids; membrane permeability; membrane structure; neutrophil; oxidizing agents; sickle cell anemia; sickle cell crisis; superoxides; thin layer chromatography

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.

该提案描述了一个计划，研究感染介导的作用， 镰状细胞性贫血（SCA）中的氧化损伤。 已知感染 在SCA中沉淀高溶血和血管闭塞危象。 我计划 确定感染期间中性粒细胞释放的氧化剂是否改变了 红细胞膜，从而促成这些临床事件。 的 我的研究的第一阶段将利用敏感的技术来检测体内 感染期间红细胞（RBC）氧化损伤的证据。 这些 技术包括高效液相色谱法检测脂质 氢过氧化物，荧光光谱法检测丙二醛 交联的脂质或蛋白质和二烯共轭以检测过氧化的 脂质。 细胞密度和氧化损伤之间的关系将是 探讨了 我接下来将进行体外研究， 中性粒细胞（AN）与红细胞孵育，以确定结构变化是否 诱导镰状细胞膜的氧自由基阐述从AN。 在 除了测量脂质氧化损伤的标准技术， 蛋白质，我将使用电子自旋共振和自旋标记技术 研究脂质/蛋白质改变和硫醇-二硫醚交换 色谱法测量巯基氧化。 这些研究将 还包括使用不同的自由基清除剂来识别产品 参与氧化反应。 功能变化（AN对膜的影响 酶活性、阳离子通量、细胞密度和可变形性），其可以 将研究AN引起的伴随结构损伤。 关联 氧化损伤与细胞表面性质的可能改变， 将测量AN对RBC与单核细胞粘附的影响。 的 本研究的最后一个阶段将探讨AN诱导的机制， 氧化损伤 我将使用两个模型系统：血色素[血红蛋白 (Hb)S或A包封在脂质体中]和杂交红细胞（正常 膜/镰状Hb;镰状膜/正常Hb）。 这些系统将 允许操作Hb、脂质含量、自由基清除剂和 膜组件，以阐明每一个在 镰状红细胞对AN介导的损伤的易感性。 总之，这 该项目将1）在受感染的人中寻找RBC氧化损伤的体内证据， SS患者，2）定义由AN引起的膜结构损伤， 将结构损伤与由此产生的功能性细胞变化相关联 这可能使SCA恶化，3）调查 AN诱导的氧化损伤机制。