ERYTHROCYTE ADHESION IN FALCIPARUM MALARIA

恶性疟原虫每年造成150-270万人死亡。脑型疟疾是恶性疟原虫感染的一种并发症，与脑血管中红细胞的隔离有关，是由于感染疟疾的红细胞(PE)与微循环衬里的内皮细胞(EC)的黏附。将PE隔离在孕妇胎盘中会导致并发症，包括胎儿出生体重低和死亡风险增加。目前，没有一种疟疾疗法专门针对与隔离有关的粘连现象。描述PE和EC表面的分子结构对于理解导致隔离的机制和开发防粘连处理至关重要。众所周知，PE的粘附性增加是由于寄生虫引起红细胞膜蛋白带3的变化，以及寄生虫编码蛋白的表面暴露。虽然对粘附性至关重要的第3带的氨基酸序列已经确定，但这种粘附素是如何形成的还不清楚。为了确定带3聚集和/或蛋白分解在诱导构象变化中的可能作用，将用冷冻断裂显微镜评估带3在双层内的横向分布，并与电子显微镜结合的粘附点的免疫定位，以及使用荧光光漂白恢复、偏振荧光耗尽和发光猝灭。此外，为了对可能影响粘连序列处理的带3的结构变化进行生化表征，将尝试分离带3的截短形式和已确定的裂解位点。免疫沉淀、免疫印迹以及抗体结合的竞争性抑制将被用来鉴定带3相关的和寄生虫编码的粘附素恶性疟原虫红细胞膜蛋白-1中的共同黏附基序；将确定它们对细胞黏附的贡献。其他PE粘附素以及人脑、真皮和骨髓中与PE结合的微血管的EC配体将通过使用单抗、噬菌体展示和合成组合库技术以及多肽亲和层析来鉴定和表征。一旦确定，应有可能设计能够抑制或逆转隔离的药物(即模拟肽类药物、小分子、抗体)，从而为患有恶性疟疾的个人提供更好和更安全的治疗方式。

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.