Pathogenesis of Lyme Borreliosis in the Rhesus Monkey

恒河猴莱姆疏螺旋体病的发病机制

• 批准号: 6828736
• 负责人: MARIO TOMAS PHILIPP
• 金额: $ 26.29万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6828736
• 关键词: Borrelia; Lyme disease; Macaca mulatta; animal tissue; apoptosis; arthropod borne communicable disease; blood proteins; brain cell; central nervous system disorders; computer program /software; confocal scanning microscopy; cooperative study; cytokine; glia; image processing; neural degeneration; neurons; neuropathology; pathologic process; polymerase chain reaction; spirochetes disease; terminal nick end labeling; tissue /cell culture; toll like receptor

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to understand the pathogenesis of Lyme neuroborreliosis of the central nervous system (CNS). Inflammation in the CNS is thought to play a primary role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and the AIDS-dementia complex. Lyme borreliosis (LB), caused by the spirochete Borrelia burgdorferi, is primarily an inflammatory disease, and neuroborreliosis, i.e. LB of the central and peripheral nervous systems, may manifest as a mild neurodegenerative disease. The central hypothesis to be explored in this grant proposal is that 1) B. burgdorferi spirochetes can cause inflammatory responses in CNS cells and tissues, and 2) these inflammatory responses may lead to neural injury, and ultimately to loss of glial and neuronal cells. Limited loss of these cells is viewed as a determinant of the neurologic impairment seen in Lyme neuroborreliosis. The secondary hypothesis is that spirochetal lipoproteins can induce, in part or in toto, the inflammatory effects of B. burgdorferi. We will use tissues and cells from the brain of rhesus macaques, a model developed under this Cooperative Agreement. Specific Aims; SA-I: Experiments ex vivo. SA-la: Assessment of inflammation and glial/neuronal loss in freshly obtained brain tissues fr6m rhesus macaques after exposure to lipoprotein and spirochetes. Local cytokine secretion (IL-6, TNF-alpha, and IL-1 beta) as well as expression of iNOS and COX-2 will be assessed by confocal microscopy using a novel procedure whereby cytokines and other inflammatory mediators are stained intracellularly with appropriate antibodies in fixed brain sections. Freshly obtained rhesus brain slices from normal animals will be stimulated ex vivo with lipoprotein or live spirochetes in the presence of the protein-secretion blocker Brefeldin A. After fixation and cutting of frozen sections, the cytokine/mediator-producing cell phenotype will be identified with appropriate antibodies, Toll-like receptor (TLR) expression determined, and glial/neuronal apoptosis verified in situ by the TUNEL assay. Spirochetes will be localized with an anti-lipoprotein antibody. Unstimulated rhesus brain slices will be used as negative controls. SA-lb: Effect of mitogen-activated protein-kinase inhibitors upon inflammation and glial/neuronal loss induced by lipoprotein in freshly obtained brain tissues from rhesus macaques. SA-lc: Assessment of inflammation and glial/neuronal loss in archival fixed samples. Archival samples of B. burgdorferi-infected rhesus meninges and brain parenchyma will be assessed for presence of inflammatory infiltrate (meninges), association of similar infiltrates present in brain specimens or areas of gliosis with foci of glial/neuronal apoptosis, and the overall correlation of such findings with presence of B. burgdorferi lipoprotein. Cumulative neuronal loss will be quantified relative to equivalent tissue sections from uninfected control animals using image analysis software. SA-2: Experiments in vitro. SA-2a will assess the production of inflammatory cytokines and mediators in single-cell-type and mixed primary cultures of rhesus glia stimulated with live B. burgdorferi and lipoprotein. The human neuroblastoma cell lines SK-N-SH and SH-SY5Y also will be employed, alone and in combinations with rhesus glia, as well as rhesus neuronal-glial primary cultures. SA-2b will assess the role of glial cells and cytokines in eliciting glial/neuronal apoptosis. SA-2c will assess the role of innate CNS responses to B. burgdorferi and lipoprotein in inflammation and neurodegeneration, specifically, the expression and involvement of pertinent TLR in mediating cytokine production and apoptosis in the different cell combinations.

描述（由申请人提供）：该提案的广泛、长期目标是了解中枢神经系统（CNS）莱姆神经疏螺旋体病的发病机制。中枢神经系统炎症被认为在神经退行性疾病（例如阿尔茨海默病和艾滋病痴呆症）的发病机制中发挥着主要作用。莱姆疏螺旋体病 (LB) 由螺旋体伯氏疏螺旋体引起，主要是一种炎症性疾病，而神经疏螺旋体病，即中枢和周围神经系统的 LB，可能表现为轻度神经退行性疾病。本拨款提案要探讨的中心假设是：1）伯氏疏螺旋体可引起中枢神经系统细胞和组织的炎症反应，2）这些炎症反应可能导致神经损伤，并最终导致神经胶质细胞和神经元细胞的损失。这些细胞的有限损失被认为是莱姆神经疏螺旋体病神经损伤的决定因素。第二个假设是螺旋体脂蛋白可以部分或全部诱导伯氏疏螺旋体的炎症作用。我们将使用恒河猴大脑的组织和细胞，这是根据本合作协议开发的模型。具体目标； SA-I：离体实验。 SA-la：暴露于脂蛋白和螺旋体后新鲜获得的恒河猴脑组织中炎症和神经胶质/神经元损失的评估。局部细胞因子分泌（IL-6、TNF-α 和 IL-1 β）以及 iNOS 和 COX-2 的表达将通过共聚焦显微镜进行评估，使用一种新方法，用适当的抗体对细胞因子和其他炎症介质进行细胞内染色在固定的大脑切片中。在蛋白质分泌阻断剂 Brefeldin A 存在的情况下，用脂蛋白或活螺旋体离体刺激从正常动物新鲜获得的恒河猴脑切片。固定和切割冷冻切片后，将通过以下方法鉴定产生细胞因子/介质的细胞表型：合适的抗体、Toll 样受体 (TLR) 表达的测定以及通过 TUNEL 测定原位验证的胶质细胞/神经元凋亡。螺旋体将被抗脂蛋白抗体定位。未刺激的恒河猴脑切片将用作阴性对照。 SA-1b：丝裂原激活的蛋白激酶抑制剂对来自恒河猴的新鲜获得的脑组织中脂蛋白诱导的炎症和神经胶质/神经元损失的作用。 SA-lc：评估档案固定样本中的炎症和神经胶质/神经元损失。将评估伯氏疏螺旋体感染的恒河猴脑膜和脑实质的档案样本是否存在炎症浸润（脑膜）、脑样本或神经胶质细胞增生区域中存在的类似浸润与神经胶质/神经元凋亡病灶的关联，以及炎症浸润的总体相关性。这些发现与伯氏疏螺旋体脂蛋白的存在有关。使用图像分析软件，相对于未感染对照动物的等效组织切片，对累积神经元损失进行量化。 SA-2：体外实验。 SA-2a将评估生产 用活伯氏疏螺旋体和脂蛋白刺激的恒河猴神经胶质细胞的单细胞型和混合原代培养物中的炎症细胞因子和介质。人神经母细胞瘤细胞系 SK-N-SH 和 SH-SY5Y 也将单独使用，或与恒河猴神经胶质细胞以及恒河猴神经元-神经胶质原代培养物组合使用。 SA-2b 将评估神经胶质细胞和细胞因子在引发神经胶质/神经元凋亡中的作用。 SA-2c 将评估先天 CNS 对伯氏疏螺旋体和脂蛋白的反应在炎症和神经变性中的作用，特别是相关 TLR 的表达和参与介导不同细胞组合中细胞因子的产生和凋亡。