Detection and Characterization of Blood-borne Prions

描述(申请人提供)：人类变异型克雅氏病(VCJD)、宫颈慢性衰弱病(CWD)和绵羊瘙痒病之间的共性非常显著。在人类和动物的传染性海绵状脑病(TSE)中，与淋巴网状系统(LRS)相关的血源性Prion被重述。特别是，vCJD、CWD和瘙痒病的特征都是疾病的长期无症状阶段，从这个阶段，传染性血液传播的普恩病毒有效地从一个宿主传播到另一个宿主。由于这些TSE在易感宿主中以类似的方式传播和传播，动物普恩疾病的使用提供了一个独特的机会来评估血液传播的普恩及其传播的生物学。我实验室这项研究的长期目标是开发干预策略，以检测、减轻和防止血源性普恩病毒的传播。作为实现这一目标的一步，我们建议使用动物TSE来更好地定义和了解与血源性普恩的贩运、传播和传播相关的机制。动物TSE的使用将允许对感染vCJD的人类做以前没有也不可能做的事情，即从感染后几分钟到晚期疾病收集连续的纵向血液样本。这项工作的统一假设是，血液传播的普鲁恩在感染后不久以特定的细胞表型在宿主内循环，并负责在宿主内的早期传播和宿主之间的隐蔽传播。为了验证这一假设，我们：1)采用快速、灵敏和经济高效的体外转换分析方法来检测血液传播的普恩病毒；2)建立了一个独特的纵向血液样本储存库，其中包括自然和实验感染TSE的宫颈、啮齿动物和灵长类动物；以及3)建立了能够在宫颈和啮齿动物体内模型中容纳和评估TSE的设施和合作者。我们建议解决两个特定的目标，1)确定感染TSE的宿主中蛋白血症的时间状态，2)定量确定哪些血液隔室和细胞表型含有蛋白。我们将使用来自自然和实验感染动物(CWD感染的颈鹿、实验感染的vCJD、BSE和CWD感染的灵长类动物以及TME感染的仓鼠)的血液来研究我们最近建立的PrP转换实验--全血实时颤动诱导转换(WBRT-QuIC)的有效性。这项工作的结果将确定普恩蛋白血症的时间状态，以及区分哪些隔室和特定的细胞表型含有普恩病毒。这些结果将在适当的情况下与我实验室现有的蛋白质错误折叠循环扩增和生物测定方法进行比较。这些目标将提供在普里恩领域长期寻求的东西；阐明普里恩在宿主中的贩运和传播机制。这将允许及时发现，从而为人类TSE和其他人类蛋白质错误折叠疾病提供更有效的治疗。

DESCRIPTION (provided by applicant): The commonalities between human variant Creutzfeldt-Jakob Disease (vCJD), cervid chronic wasting disease (CWD) and sheep scrapie are quite remarkable. Hematogenous prions exhibiting association with the lymphoreticular system (LRS) are recapitulated in both human and animal transmissible spongiform encephalopathies (TSEs). In particular, vCJD, CWD and scrapie are all characterized by a long asymptomatic phase of disease from which infectious blood-borne prions efficiently transmit from one host to another. Because these TSEs traffic and disseminate similarly within susceptible hosts, the use of animal prion diseases provides a unique opportunity to evaluate the biology of blood-borne prions and their spread. The long-term goal of this research in my laboratory is to develop intervention strategies to detect, mitigate and prevent hematogenous prion spread. As a step towards this goal, we propose the use of animal TSEs to better define and understand the mechanisms associated with trafficking, dissemination and transmission of blood-borne prions. The use of animal TSEs will allow what has not- and cannot- be done with humans infected with vCJD, namely collect serial longitudinal blood samples from minutes after infection to terminal disease. The unifying hypothesis for this work is that blood-borne prions circulate within the host on/in specific cell phenotypes soon after infection and are responsible for early dissemination within the host and covert transmission between hosts. To test this hypothesis, we: 1) adapted fast, sensitive and cost effective in vitro conversion assays for the detection of blood-borne prions; 2) assembled a unique repository of longitudinal blood samples from natural and experimental TSE-infected cervids, rodents and primates; and 3) established facilities and collaborators with the capacity to house and assess TSEs in cervid and rodent in vivo models. We propose to address two specific aims, to 1) determine the temporal status of prionemia in TSE- infected hosts and 2) quantitatively determine which blood compartments and cell phenotypes harbor prions. We will employ blood, from naturally and experimentally infected animals (CWD-infected cervids, experimentally vCJD, BSE and CWD-infected primates and TME-infected hamsters) to investigate the efficacy of our recently established PrP conversion assay, whole blood real-time quaking induced conversion (wbRT- QuIC). The outcome of this work will establish the temporal status of prionemia as well as discern which compartments and specific cell phenotypes harbor prions. These results will be compared, where appropriate, to protein misfolding cyclic amplification and bioassay methods in place in my laboratory. These aims will provide what has long been sought in the prion field; elucidating the mechanisms of prion trafficking and dissemination throughout the host. This will permit timely detection, and thus, more effective therapies for human TSEs and other human protein misfolding diseases.