Continuous in vitro culture of Babesia microti

 描述(申请人提供)：体外培养寄生虫的能力导致了对宿主-寄生虫关系、寄生虫生物学和病理生理学的理解的快速进步。相反，缺乏体外寄生虫培养系统会严重阻碍对该特定病原体的研究，如微小巴贝斯虫引起的巴贝斯虫病。这是真的，尽管越来越多的证据表明微小杆菌在美国的出现和传播，以及它作为对输血医学的威胁的主导地位。我们建议的总体目标是在人红细胞中建立一个连续的微小杆菌体外培养系统，以便为未来的实验研究提供一个合适的平台。众所周知，作为巴贝斯虫细胞载体的人红细胞并不是循环中统一、均一的细胞群体，在其120天的生命周期中经历了许多与年龄相关的变化。我们的假设是，这些异质红细胞不是微小杆菌入侵和生长的平等宿主。我们提案中的两个特定目标将检验这一假设，并评估当呈现一个具有最佳年龄和特征的统一红细胞种群时，寄生虫是否会入侵和增殖人类红细胞。特定目的1解决了支持微小杆菌体外最佳入侵和复制的特定人红细胞亚群的鉴定，而特定目的2将定义支持微小杆菌体外入侵和复制所需的培养条件(气体、介质、摇动和静态)。通过将我们在寄生虫学方面的丰富经验(Lobo和Narla、恶性疟原虫和B.disgens)与NYBC提供的红细胞专业知识(Narla)和试剂(微小杆菌的人类来源、脐带细胞资源、抗体和抗原)相结合，我们将深入了解微小杆菌所青睐的特定红细胞谱系。这项研究是对寄生虫繁殖所需培养条件的第一次系统分析，也是寻找诊断、流行病学、治疗和预防人类巴贝斯虫病的试剂以及协助开发筛选血液制品的新方法的必要的第一步。

 DESCRIPTION (provided by applicant): The ability to cultivate parasites in vitro has resulted in rapid progress in the understanding of host-parasite relationships, parasite biology and pathophysiology. Conversely, the lack of a parasite in vitro culture system can severely hamper studies on that specific pathogen as seen in Babesia microti-caused babesiosis. This is true despite the mounting evidence for the emergence and spread of B. microti in the US and its dominating status as a threat to transfusion medicine. The overall goal of our proposal is to establish a continuous in vitro culture system of B. microti in human RBCs to enable a platform amenable for future experimental investigation. It is well known that human RBCs which serve as the cellular vehicle host for Babesia are not a uniform, homogenous population of cells in circulation and undergo many age related transformations in their 120 day life span. Our hypothesis is that these heterogeneous RBCs are not equal hosts for B. microti invasion and growth. The two specific aims in our proposal will test this hypothesis and assess if parasite invasion and proliferation in human RBCs would occur when presented with a uniform RBC population of the optimum age and features. Specific Aim 1 addresses the identification of specific human RBC sub-populations that would support optimum invasion and replication of B. microti in vitro and Specific Aim 2 will define culture conditions (gas, media, shaking vs static) needed to support this invasion and replication of B. microti in vitro. By combining our extensive experience in parasitology (Lobo and Narla, P. falciparum and B. divergens) together with the red cell expertise (Narla) and reagents (Human sources of B. microti, umbilical cord cell resources, antibodies, and antigens) available at NYBC, we will obtain insights into the specific RBC lineage favored by B. microti. This study is the first systematic analysis of culture conditions required for parasite propagation and is a necessary first step in the search for reagents for diagnosis, epidemiology, treatment and prevention of human babesiosis, as well as to assist in developing novel methods to screen blood products.