Mechanisms of Toxoplasma gondii dissemination and transmigration

弓形虫传播和迁移机制

• 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
• 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

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）：T.弓形虫从血液循环进入组织是寄生虫进入器官致病的关键步骤。当寄生虫穿过胎盘并感染发育中的胎儿时，就会出现先天性感染，导致严重的眼部和神经系统疾病。T.弓形虫进入大脑可导致危及生命的脑炎。证据表明T.弓形虫可以利用活动的免疫细胞，如树突细胞和单核细胞，作为“特洛伊木马”进入新的组织。游离速殖子也可直接感染内皮细胞并穿越生物屏障。 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，我们已经调查了粘附分子感染的人单核细胞和细胞外速殖子介导的相互作用与内皮细胞通过使用流体和延时显微镜系统。本提案的目的是确定寄生虫粘附和穿越血管内皮的机制。中心假设是T.刚地虫通过细胞内和细胞外的迁移方式穿过内皮屏障。提出了三个具体的目标来验证这一假设：1）确定感染的人单核细胞的迁移的分子事件，2）确定游离寄生虫粘附和迁移的机制，3）确定T。体内弓形虫跨内皮迁移。在第一个目标中，将研究感染的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和它们在调节迁移连接中的作用。在第二个目标中，将测试寄生虫表面粘附素MIC 2与内皮细胞ICAM-1的相互作用在剪切应力条件下介导速殖子粘附和在内皮细胞上滑动中的作用。第三个目标是研究T.通过使用双光子显微镜、转基因和基因缺陷小鼠的组合，在小鼠中观察弓形虫穿过血脑屏障的迁移。所有拟议的试剂和工具目前都在实验室或市售，拟议的方法目前都由研究小组执行。本研究具有重要的意义，因为了解T细胞粘附和外渗的分子介导。将弓形虫感染到组织中可能允许有针对性的方法来限制传播。该方法是创新的，因为它整合了T.弓形虫和免疫学研究与工程技术和系统，以确定发病机制，以前不能解决。这项研究的成功完成有望为T。弓形虫速殖子穿过人内皮屏障和小鼠血脑屏障。