In Situ Detection of Host-Spirochete Interaction by Iso*

描述（由申请人提供）：我们建议研究宿主的各个方面 莱姆病螺旋体，伯氏疏螺旋体的反应，使用最近 所描述的技术，等温滚环扩增（RCA）。RCA是一种 一种新的极其灵敏的信号放大方法， 单个抗原-抗体复合物或点突变的检测水平 在核苷酸序列中。我们将利用这一点来研究 蛋白抗原和核酸信号。 在目标1中，我们提出检测多株伯氏疏螺旋体， 患者滑液使用RCA放大来自每个 螺旋体在目标2中，我们将在初步实验结果的基础上， 用原位免疫放射分析法检测B组巨噬细胞的功能状态。 感染伯氏菌的小鼠。我们研究了巨噬细胞与 B。burgdorferi在一些细节，并已表明，在体外， 摄入并杀死螺旋体，但在体内，螺旋体不能从体内清除。 宿主，导致莱姆病的后期并发症。的功能状态 小鼠皮肤中的组织巨噬细胞， 哺乳动物宿主，将通过原位等温RCA分析确定 他们的细胞因子谱在一个时间过程之前，期间， 传播B。burgdorferi。总之，该提案采用了一种新颖的， 强大的技术来解决螺旋体的几种可能的机制， 免疫系统的逃避。

DESCRIPTION (provided by applicant): We propose to study aspects of host response to the Lyme spirochete, Borrelia burgdorferi, using the recently described technique, isothermal rolling circle amplification (RCA). RCA is a new and extremely sensitive method of signal amplification that extends our level of detection to a single antigen- antibody complex or to a point mutation in a nucleotide sequence. We will take advantage of this to examine both protein antigens and nucleic acid signals in biological fluids and in tissues. In Aim 1, we propose to detect multiple strains of Borrelia burgdorferi in patient synovial fluids using RCA to amplify the minute signal from each spirochete. In Aim 2, we will build on the results of initial experiments, to use in situ immunoRCA to examine the functional state of macrophages in B. burgdorferi-infected mice. We have examined the interaction of macrophages with B. burgdorferi in some detail and have shown that in vitro, the cells easily ingest and kill spirochetes, yet in vivo, spirochetes are not cleared from the host, leading to later complications of Lyme disease. The functional state of tissue macrophages in murine skin, the main reservoir for spirochetes in the mammalian host, will be determined by in situ isothermal RCA through analysis of their cytokine profiles over a time course before, during, and after dissemination of B. burgdorferi. In summary, this proposal employs a novel and powerful technique to address several possible mechanisms of spirochetal evasion of the immune system.