ERYTHROCYTE ADHESION IN FALCIPARUM MALARIA

恶性疟疾中的红细胞粘附

• 批准号: 6497113
• 负责人: Irwin W Sherman
• 金额: $ 25.53万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497113
• 关键词: Plasmodium falciparum; SDS polyacrylamide gel electrophoresis; affinity chromatography; antibody titering; antigen antibody reaction; cell adhesion; clinical research; conformation; electron microscopy; erythrocyte membrane; erythrocytes; freeze etching; high performance liquid chromatography; human subject; immunoprecipitation; intracellular parasitism; malaria; matrix assisted laser desorption ionization; membrane proteins; protein binding; protein structure function; proteolysis; vascular endothelium; western blottings

Plasmodium falciparum contributes to 1.5-2.7 million deaths annually. Cerebral malaria, a complication of P. falciparum infections, is associated with sequestration of erythrocytes in the blood vessels of the brain and is due to the adhesion of malaria-infected erythrocytes (PE) to the endothelial cells (EC) lining the microcirculation. Sequestration of PE in the placenta of pregnant women leads to complications that include low fetal birth weight and an increased risk of death. At present none of the therapies for malaria specifically target the adhesive phenomena associated with sequestration. Describing the molecular architecture of the PE and EC surfaces is critical to understanding the mechanisms that lead to sequestration and for developing anti-adhesive treatments. It is known that increased adhesiveness of the PE is due to parasite-induced changes in the erythrocyte membrane protein, band 3, as well as the surface exposure of parasite-encoded proteins. Though the amino acid sequences of band 3 critical to adhesion have been identified it is not known how this adhesin is formed. To define the possible role of band 3 aggregation and/or proteolysis in inducing conformational change, the lateral distribution of band 3 within the bilayer will be evaluated by freeze fracture microscopy and coordinated with electron micrographic immunolocalization of adhesive sites, as well as by the use of fluorescence photobleaching recovery, polarized fluorescence depletion and luminescence quenching. In addition, to biochemically characterize the structural changes in band 3 that could influence the disposition of adhesive sequences, attempts will be made to isolate truncated forms of band 3 and cleavage sites identified. Immunoprecipitation, immunoblotting as well as competitive inhibition of antibody binding will be used to identify common adhesive motifs in the band 3-related and the parasite-encoded adhesin Plasmodium falciparum erythrocyte membrane protein-1; estimates of their contribution to cytoadherence will be determined. Other PE adhesins as well as EC ligands of microvessels in the human brain, dermis and bone marrow that bind PE will be identified and characterized through the use of monoclonal antibodies, phage display and synthetic combinatorial library technologies, and peptide affinity chromatography. Once identified, it should be possible to design drugs (i.e. peptidomimetics, small molecules, antibodies) that could inhibit or reverse sequestration thus allowing for better and safer therapeutic modalities for individuals suffering from falciparum malaria.

恶性疟原虫每年造成1.5-270万人死亡。 大脑疟疾是恶性疟原虫感染的并发症，与脑血管中的红细胞隔离有关，这是由于疟疾感染的红细胞（PE）粘附于微循环层衬里的内皮细胞（EC）。 PE在孕妇的胎盘中的隔离会导致并发症，包括胎儿出生体重低和死亡风险增加。 目前，疟疾的疗法均未针对与隔离相关的粘合剂现象。 描述PE和EC表面的分子结构对于理解导致隔离和发展抗粘附治疗的机制至关重要。 众所周知，PE的粘附性提高是由于寄生虫诱导的红细胞膜蛋白（条带3）以及寄生虫编码蛋白的表面暴露。 尽管已经鉴定出对粘附至关重要的条带3的氨基酸序列，但尚不知道该粘附素是如何形成的。 为了确定频带3聚集和/或蛋白解析在诱导构象变化中的可能作用，将通过冻结显微镜进行评估双层中带3的横向分布，并通过胶水显微镜的粘附部位进行了微观的免疫定位，并通过使用荧光光片恢复，荧光恢复，并使用胶片的胶水粘合剂进行了微观化。 此外，为了表征频带3中可能影响粘合剂序列处置的结构变化，将尝试隔离符号3的截短形式和识别的裂解位点。 免疫沉淀，免疫印迹以及抗体结合的竞争抑制作用将用于鉴定带3相关的带和寄生虫编码的粘合剂粘附质质疟原虫红质细胞红细胞膜膜蛋白-1；将确定它们对细胞加容的贡献的估计。 通过使用单克隆抗体，噬菌体显示和合成组合图书馆技术以及肽亲和色谱法，将鉴定并表征其他PE的PE粘附素以及与PE结合的PE的微分子，真皮和骨髓的EC配体。 一旦确定，就应该可以设计药物（即肽仪，小分子，抗体），这些药物可以抑制或反向隔离，从而可以为患有恶性疟疾的患者提供更好，更安全的治疗方式。