Mechanisms of Toxoplasma gondii dissemination and transmigration

弓形虫传播和迁移机制

基本信息

批准号：
8893191
负责人：
Melissa Bruckner Lodoen
金额：
$ 36.89万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-06 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8893191
关键词：
Address Adhesions Antibodies Bacterial Adhesins Binding Biological Biological Assay Blood Blood - brain barrier anatomy Blood Circulation Brain Cell Adhesion Molecules Cells Data Dendritic Cells Disease Encephalitis Endothelial Cells Endothelium Engineering Equus caballus Event Extravasation Fetus Genes Goals Hand Health Homing Human Image Immune Immunology In Vitro Individual Infection Integrins Intercellular Junctions Intercellular adhesion molecule 1 Intervention Knockout Mice Label Lateral Left Life Macrophage-1 Antigen Mediating Methodology Microscopy Molecular Molecular Genetics Mus Organ Outcome Parasite Control Parasites Pathogenesis Patients Placenta Process Proteins Proteolytic Processing Public Health Reagent Regulation Research Role Route Severities Small Interfering RNA Stress Surface System Technology Testing Tight Junctions Time Tissues Toxoplasma gondii Transgenic Mice Transgenic Organisms Vascular Endothelium Wild Type Mouse base cadherin 5 congenital infection extracellular human disease in vivo innovation intravital imaging knock-down migration monocyte nervous system disorder pathogen receptor research study shear stress therapeutic target tool trafficking transcytosis two-photon

项目摘要

DESCRIPTION (provided by applicant): The dissemination of T. gondii from the circulation into tissues is a critical step in the parasite's ability to enter organs where it causes disease. Congenital infection arises when the parasite crosses the placenta and infects the developing fetus, leading to severe ocular and neurological disease. T. gondii entry into the brain can cause life-threatening encephalitis. Evidence suggests that T. gondii can use motile immune cells, such as dendritic cells and monocytes, as "Trojan horses" to gain access to new tissues. Free tachyzoites can also directly infect endothelial cells and transmigrate across biological barriers. What remain unknown are the mechanisms by which infected immune cells or free parasites adhere to and migrate across endothelial barriers under the conditions of shear flow found in the vasculature. To address these questions, we have investigated the adhesion molecules on infected human monocytes and extracellular tachyzoites that mediate interactions with endothelium by using a fluidic and time-lapse microscopy system. The objective of this proposal is to define mechanisms of parasite adhesion and transmigration across vascular endothelium. The central hypothesis is that T. gondii crosses endothelial barriers using both intracellular and extracellular modes of transmigration. Three specific aims are proposed to test this hypothesis: 1) Define the molecular events in the transmigration of infected human monocytes, 2) Determine the mechanisms of free parasite adhesion and transmigration, and 3) Define the routes of T. gondii transendothelial migration in vivo. In the first aim, the integrins LFA-1 and Mac-1 on infected monocytes will be investigated to determine their function in monocyte migration and their role in regulation of the transmigration junction. In the second aim, interaction of the parasite surface adhesin MIC2 with endothelial ICAM-1 will be tested for its role in mediating tachyzoite adhesion and gliding on endothelium in shear stress conditions. The third aim will examine mechanisms of T. gondii transmigration across the blood-brain barrier in mice by using a combination of two-photon microscopy, transgenic, and gene-deficient mice. All of the proposed reagents and tools are currently on-hand in the lab or commercially available, and the proposed methodologies are all currently being performed by the research team. This research is significant because understanding the molecules that mediate the adhesion and extravasation of T. gondii into tissues may allow for targeted approaches to limit dissemination. The approach is innovative because it integrates molecular and genetic tools in T. gondii and immunology research with technologies and systems from engineering to define mechanisms of pathogenesis that could not previously be addressed. The successful completion of the proposed research is expected to provide a molecular understanding of how T. gondii tachyzoites cross human endothelial barriers and the blood-brain barrier in mice.

描述（由申请人提供）：弓形虫从循环系统传播到组织中是寄生虫进入引起疾病的器官能力的关键步骤。当寄生虫穿过胎盘并感染发育中的胎儿时，就会发生先天性感染，导致严重的眼部和神经系统疾病。弓形虫进入大脑可引起危及生命的脑炎。有证据表明弓形虫可以利用树突状细胞和单核细胞等运动免疫细胞作为“特洛伊木马”来进入新组织。游离的速殖子还可以直接感染内皮细胞并跨越生物屏障。目前尚不清楚的是，在脉管系统中发现的剪切流条件下，受感染的免疫细胞或游离寄生虫粘附并迁移穿过内皮屏障的机制。为了解决这些问题，我们通过使用流体和延时显微镜系统研究了受感染的人类单核细胞和细胞外速殖子上介导与内皮细胞相互作用的粘附分子。该提案的目的是确定寄生虫粘附和跨血管内皮迁移的机制。中心假设是弓形虫利用细胞内和细胞外的迁移模式穿过内皮屏障。提出了三个具体目标来检验这一假设：1）定义受感染的人类单核细胞迁移的分子事件，2）确定游离寄生虫粘附和迁移的机制，3）定义体内弓形虫跨内皮迁移的途径。第一个目标是研究受感染单核细胞上的整合素 LFA-1 和 Mac-1，以确定它们在单核细胞迁移中的功能及其在调节轮回连接中的作用。在第二个目标中，将测试寄生虫表面粘附素 MIC2 与内皮 ICAM-1 的相互作用，以了解其在剪切应力条件下介导速殖子粘附和在内皮上滑动的作用。第三个目标是通过结合使用双光子显微镜、转基因小鼠和基因缺陷小鼠来研究弓形虫跨过小鼠血脑屏障的迁移机制。所有提议的试剂和工具目前都在实验室或市售，并且提议的方法目前均由研究团队执行。这项研究意义重大，因为了解介导弓形虫粘附和外渗到组织中的分子可能有助于采取有针对性的方法来限制传播。该方法具有创新性，因为它将弓形虫中的分子和遗传工具以及免疫学研究与工程技术和系统相结合，以定义以前无法解决的发病机制。该研究的成功完成预计将为弓形虫速殖子如何穿越人类内皮屏障和小鼠血脑屏障提供分子理解。