CWD: ROLE OF THE LYMPHOID TISSUE PHASE IN PRION DISEASE

候选人Christina J. Sigurdson， DVM的近期目标是完成朊病毒发病机制研究的指导培训计划，最终获得博士学位和博士后研究的机会。Sigurdson博士的长期目标是成为使用动物模型研究传染性海绵状脑病（tse）的诊断和发病机制的主要研究者。该研究培训项目将在赞助方科罗拉多州立大学Edward Hoover博士的指导下进行。培训环境在校外资助和培训dvm后科学家方面有着良好的记录。候选人的计划强调在应用于朊病毒发病机制动物模型的当代研究方法方面的初级实验室培训。提出了对淋巴组织中早期朊病毒蛋白积累的研究，这是几种tse的一个有趣的、局部的和重要的特征，包括人类的变异型克雅氏病（vCJD）、绵羊的痒病和鹿的慢性消耗性疾病（CWD）。了解朊病毒经黏膜进入和随后的神经侵袭的机制和途径对诊断分析和干预策略的发展至关重要；因此，我们建议研究鹿CWD作为vCJD/朊病毒发病机制的模型。CWD PrPres在感染鹿的淋巴组织中含量丰富，因此可能更容易在血液和循环白细胞中检测到。我们提出了三个目标来探索朊病毒生物学的基本问题：(1)第一个目标，我们将利用已建立的PrPres特异性双重免疫组织/细胞化学和双重免疫荧光法来鉴定淋巴组织中携带PrPres的细胞表型。我们将使用共聚焦和免疫金电镜在淋巴组织内定位PrPres的亚细胞。(2)对于朊病毒向淋巴组织或中枢神经系统的初始运输途径所知相对较少。在目标2中，我们将使用prpre特异性抗体、流式细胞术、双重免疫细胞化学和体内感染性来寻找朊病毒在cwd感染鹿血液和淋巴中传播的潜在细胞载体。在血液中检测到PrPres对接受血液的人具有重大意义。(3)树突状细胞（DC）被怀疑是淋巴组织中PrPres的主要蓄积者。在目标3中，我们将从血液中分离单核细胞来源的DC，用表位标记的鹿prpc表达载体转染这些细胞，将DC暴露于外源性CWD PrPres中，并使用免疫染色、共聚焦和电子显微镜来确定是否发生了原位PrPres转化。这些研究结果将有助于了解PrP在淋巴系统中的运输，并可为基于血液的诊断分析和干预策略的发展提供基础。

The immediate goal of the candidate, Christina J. Sigurdson, DVM, is to complete a mentored training program in prion pathogenesis research which will culminate in the PhD degree and the opportunity for postdoctoral study. Dr. Sigurdson's long term goal is to become a principal investigator employing animal models to investigate the diagnosis and pathogenesis of transmissible spongiform encephalopathies (TSEs). The research training program will be conducted under the guidance of the sponsor Dr. Edward Hoover at Colorado State University. The training environment has a strong record of extramural funding and post-DVM scientists trained. The candidate's plan emphasizes primary laboratory training in contemporary research methods applied to an animal model of prion pathogenesis. Proposed are studies of the early prion protein accumulation in lymphoid tissue, an intriguing, topical, and important feature of several TSEs, including variant Creutzfeldt-Jakob disease (vCJD) in humans, scrapie in sheep, and chronic wasting disease (CWD) in deer. Understanding mechanisms and pathways of prion transmucosal entry and subsequent neuroinvasion is critical for development of diagnostic assays and intervention strategies; thus, we propose to study CWD of deer as a model for vCJD/prion pathogenesis. CWD PrPres is abundant in lymphoid tissue of infected deer, therefore, may potentially be more readily detected in blood and circulating leukocytes. We propose three aims which explore basic issues in prion biology: (1) In the first aim, we will seek to identify PrPres-bearing cell phenotypes in lymphoid tissues by using established PrPres- specific dual immunohisto/cytochemistry and dual immunofluorescence assays. We will localize the PrPres subcellularly within lymphoid tissue using confocal and immunogold electron microscopy. (2) Relatively little is known of the initial prion trafficking pathways to lymphoid tissue or the CNS. In aim 2, we will use PrPres-specific antibodies, flow cytometry, dual immunocytochemistry, and in vivo infectivity to seek potential cell vectors for prion dissemination in blood and lymph of CWD-infected deer. Detection of PrPres in the blood has dramatic implications for blood recipients. (3) Dendritic cells (DC) are suspected to serve as the primary accumulator of PrPres in lymphoid tissue. In aim 3, we will isolate monocyte-derived DC from blood, transfect these cells with an epitope-tagged deer PrPc-expressing vector, expose the DC to exogenous CWD PrPres, and use immunostaining followed by confocal and electron microscopy to determine whether in situ PrPres conversion has occurred. The results of these studies will contribute to understanding PrP trafficking in the lymphoid system and could provide a basis for development of blood-based diagnostic assays and intervention strategies.