LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

利什曼原虫-巨噬细胞体外相互作用

• 批准号: 3130188
• 负责人: Kwang Poo Chang
• 金额: $ 24.18万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3130188
• 关键词: Leishmania donovani; antiprotozoal agents; cell cell interaction; chromatography; electron microscopy; electrophoresis; genetic translation; host organism interaction; hydrolase; immunologic techniques; leishmaniasis; lysosomes; macrophage; membrane activity; messenger RNA; microorganism growth; microorganism immunology; monoclonal antibody; phagocytes; radioassay; surface antigens; tissue /cell culture; virulence

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.

我们的长期目标是阐明利什曼尼亚的问题 感染巨噬细胞，以及这些寄生虫如何区分， 在这些吞噬细胞中生存并繁殖。 了解细胞和 这种宿主 - 寄生虫相互作用的分子机制将提供铅 用于开发更有效的化学疗法和/或免疫疗法以及预防 对于利什曼病。 这里提出的实验代表了以前的工作的延续 Leishmania Mexicana Amazonensis-J774 G8巨噬细胞在体外系统研究 利什曼原虫巨噬细胞膜相互作用和调节机制 膜蛋白生物合成在利什曼族分化过程中的生物合成 巨噬细胞。 我们以前已经准备了一组单克隆抗体 特定于寄生虫的表面分子。 一个特殊的单克隆 6H12，识别由糖肽组成的主要表面抗原 SDS-PAGE后的亚基（65-68千洛达尔顿）。 膜抗原出现 在巨噬细胞中利什曼尼斯的生存中发挥作用。 在其他 实验，我们发现蛋白质的调节机制 利什曼语分化过程中的生物合成甚至不同 密切相关的蛋白质，即α-和β-微管蛋白。 根据上述发现，我们现在建议：1）尝试 使用单克隆抗体亲和力纯化主要的表面抗原 色谱； 2）探测抗原在其中的功能 脱糖基化后与巨噬细胞和水解酶相互作用， 例如，穿衣霉素和单克隆抗体介导的调节 利什曼原虫膜糖蛋白（S）的3）研究生物合成， 主要表面抗原的表达，加工和降解， 尤其是在巨噬细胞中的利什曼语分化期间； 4）研究 利什曼膜的分化相关调节机制 使用mRNA中的体外蛋白质翻译系统中的糖蛋白合成。 单克隆抗体的特异性已经制成 可以识别并定量特定的抗原 提出的实验。 这些研究的结果不仅会帮助我们 了解利什曼语的生物化学，细胞和分子生物学 膜糖蛋白，以及它们在宿主相互作用中的作用 在利什曼病。