Cryptococcus traversal of Brain Endothelial cells

描述(申请人提供)：新生隐球菌(C.neformans)是一种被包裹的酵母菌，主要对感染艾滋病毒的患者的脑膜炎/脑膜炎负责。对于世界上艾滋病毒流行的地区，新生隐球菌属是培养阳性脑膜炎的最常见原因(例如津巴布韦、马拉维和柬埔寨)。新生葡萄球菌通常是通过吸入获得的。肺外扩散可导致血液感染和随后的血源性扩散到靶器官，最常见的结果是脑膜脑炎。来自实验性隐球菌性脑膜炎小鼠模型的几条证据表明，新生隐球菌在真菌血症之后入侵大脑，而大脑毛细血管，而不是脉络丛，是进入的门户。然而，目前尚不清楚新生葡萄球菌是如何穿越血脑屏障的。我们通过分离培养人脑微血管内皮细胞(HBMEC)建立了血脑屏障的体外模型。我们的原代HBMEC在胶原涂层的Transwell插入物上培养后，形成连续的内皮细胞衬里，并显示出300-600欧姆/cm2的跨内皮电阻，这是脑微血管内皮单分子层的独特特性。我们的初步研究结果表明，新生葡萄球菌能够穿过HBMEC原代单层，而HBMEC单层的完整性没有任何变化。基于这一新的发现，我们希望研究以下具体目标：1.研究宿主细胞肌动蛋白细胞骨架重排和相关信号转导通路在新生葡萄球菌穿越HBMEC原代单层中的作用。2.评估新生葡萄球菌如何穿透原代HBMEC单层。从这一探索性应用中获得的信息应该提供关于新生芽孢杆菌穿越HBMEC单层所涉及的机制的信息，并将成为未来研究的基础，以表征与隐球菌性脑膜炎/脑膜脑炎发病机制相关的微生物-宿主相互作用。

DESCRIPTION (provided by applicant): Cryptococcus neoformans (C. neoformans) is an encapsulated yeast mainly responsible for meningitis/meningocephalitis in patients infected with HIV. For areas of the world where HIV is endemic, C. neoformans is the most common cause of culture-positive meningitis (e.g., Zimbabwe, Malawi, and Cambodia). C. neoformans is commonly acquired by inhalation. Extrapulmonary dissemination can lead to infection of the bloodstream and subsequent hematogenous dissemination to target organs, most commonly resulting in meningoencephalitis. Several lines of evidence from experimental mouse models of cryptococcal meningitis indicate that C. neoformans invasion into the brain follows fungemia, and cerebral capillaries, not the choroid plexus, are the portal of entry. It is, however, unclear how C. neoformans traverses the blood-brain barrier. We have developed the in vitro model of the blood-brain barrier by isolation and cultivation of human brain microvascular endothelial cells (HBMEC). Our primary HBMEC, upon cultivation on collagen-coated Transwell inserts, form a continuous lining of endothelial cells and exhibit transendothelial electrical resistance of 300-600 ohms/cm2, a unique property of the brain microvascular endothelial monolayer. Our preliminary findings revealed that C. neoformans strains were able to traverse primary HBMEC monolayers without any change in the integrity of HBMEC monolayers. Based on this novel finding, we would like to examine the following specific aims: 1. To examine the role of host cell actin cytoskeleton rearrangements and related signal transduction pathways that are involved in C. neoformans traversal of primary HBMEC monolayers. 2. To assess how C. neoformans traverses primary HBMEC monolayers. The information derived from this exploratory application should provide the information on the mechanisms involved in C. neoformans traversal of HBMEC monolayers and will be the basis of future investigations to characterize microbial-host interactions that are relevant to the pathogenesis of cryptococcal meningitis/meningoencephalitis.