In Situ Detection of Host-Spirochete Interaction by Iso*

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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.