LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

我们的长期目标是阐明利什曼病是如何 感染巨噬细胞，以及这些寄生虫随后如何分化， 在这些吞噬细胞中存活和繁殖。 了解细胞和 这种宿主-寄生虫相互作用的分子机制将提供线索 用于开发更有效的化疗和/或免疫疗法和化疗药物， 治疗利什曼病 这里提出的实验代表了以前工作的延续， 墨西哥利什曼原虫亚马逊亚种-J774 G8巨噬细胞体外体系研究 利什曼原虫-巨噬细胞膜相互作用及其调控机制 利什曼原虫分化过程中膜蛋白的生物合成 巨噬细胞 我们之前已经制备了一组单克隆抗体 寄生虫表面分子的特异性。 一种特殊的单克隆， 6 H12，识别由糖肽组成的主要表面抗原 SDS-PAGE后的亚基（65-68千道尔顿）。 膜抗原出现 在巨噬细胞中利什曼原虫的存活中发挥作用。 换句 实验中，我们发现蛋白质的调节机制 在利什曼原虫分化过程中的生物合成是不同的， 密切相关的蛋白质，即，α-和β-微管蛋白。 基于上述研究结果，我们现建议：（一）尝试 使用单克隆抗体亲和性纯化主要表面抗原 2）探测抗原在其细胞中的功能; 去糖基化后与巨噬细胞和水解酶的相互作用， 使用例如衣霉素和单克隆抗体介导的调节 利什曼原虫膜糖蛋白; 3）研究生物合成， 主要表面抗原的表达、加工和降解， 特别是在巨噬细胞的利什曼分化期间; 4）研究 利什曼膜分化相关的调节机制 使用mRNA的体外蛋白质翻译系统中的糖蛋白合成。 已经制备的单克隆抗体的特异性使其 可以鉴定和定量这些细胞中的特定抗原， 提出的实验。 这些研究的结果不仅有助于我们的 了解利什曼原虫的生物化学、细胞和分子生物学 膜糖蛋白，以及它们在宿主-寄生虫相互作用中的作用 利什曼病

Our long term objectives are to elucidate the questions of how leishmanias infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the cellular and molecular mechanisms of such host-parasite interactions will provide leads for developing more effective chemo- and/or immunotherapy and prophylaxes for leishmaniasis. Experiments proposed here represent a continuation of previous work using Leishmania mexicana amazonensis-J774 G8 macrophage in vitro system to study leishmania-macrophage membrane interactions and regulatory mechanism of membrane protein biosynthesis during leishmanial differentiation in macrophages. We have previously prepared a panel of monoclonal antibodies specific to surface molecules of the parasite. One particular monoclonal, 6H12, recognizes a major surface antigen consisting of glycopeptide subunits (65-68 kilodalton) after SDS-PAGE. The membrane antigen appears to play a role in the survival of leishmanias in the macrophages. In other experiments, we have found that the regulatory mechanism of protein biosynthesis during leishmanial differentiation is different even for closely related proteins, i.e., alpha- and beta-tubulins. On the basis of the above findings, we now propose to: 1) attempt to purify the major surface antigen using monoclonal antibody affinity chromatography; 2) probe the function of the antigen(s) in their interactions with macrophages and hydrolytic enzymes after deglycosylation, using, for example, tunicamycin and monoclonal antibody-mediated modulation of leishmania membrane glycoprotein(s); 3) study the biosynthesis, expression, processing and degradation of the major surface antigen, especially during leishmanial differentiation in macrophages; 4) study differentiation-related regulatory mechanisms of leishmanial membrane glycoprotein synthesis in in vitro protein translation sytem using mRNAs. The specificity of the monoclonal antibodies already prepared makes it possible to identify and to quantitate particular antigen(s) in these proposed experiments. The results of these studies will help not only our understanding the biochemistry, cell and molecular biology of leishmanial membrane glycoproteins, but also their roles in host-parasite interactions in leishmaniasis.