LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

我们的长期目标是阐明利什马尼亚病毒如何 感染巨噬细胞，以及这些寄生虫随后如何分化， 在这些吞噬细胞中生存和繁殖。了解蜂窝和 这种寄主-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/或免疫疗法和预防 治疗利什曼病。 这里提出的实验是先前工作的延续，使用 亚马逊利什曼原虫-J774 G8巨噬细胞体外系统的研究 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。我们之前已经准备了一组单抗 对寄生虫的表面分子具有特异性。一种特殊的单克隆体， 6H12，识别一种主要的表面抗原，由糖肽组成 SDS-PAGE后亚基(65-68kodalton)。膜抗原出现 在巨噬细胞中利什曼原虫的生存中发挥作用。在其他 实验中，我们发现了蛋白质的调节机制 利什曼分化过程中的生物合成甚至是不同的 密切相关的蛋白质，即α-微管蛋白和β-微管蛋白。 基于上述发现，我们现在建议：1)尝试 利用单抗亲和力纯化主要表面抗原 层析；2)探讨抗原(S)在小鼠体内的作用 脱糖后与巨噬细胞和水解酶的相互作用 例如，使用衣霉素和单抗介导的调节 利什曼原虫膜糖蛋白(S)；3)生物合成研究， 主要表面抗原的表达、加工和降解， 尤其是在巨噬细胞利什曼分化过程中；4)研究 利什曼氏膜的分化相关调控机制 利用mRNAs在体外蛋白质翻译系统中合成糖蛋白。 已经制备的单抗的特异性使它 可能鉴定和定量这些特定的抗原(S) 拟议的实验。这些研究的结果不仅对我们的 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.