This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Lyme neuroborreliosis is a disease of the nervous system caused by the spirochete B. burgdorferi, resulting in cognitive deficits that may be due to impaired neuronal function. Various cytokines and chemokines have been detected in the cerebrospinal fluid of Lyme neuroborreliosis patients and we and others have assumed that inflammation plays a role in pathogenesis. We hypothesized that B. burgdorferi spirochetes induce cytokines, chemokines and immune mediators in the central nervous system that contribute to inflammation and neuroglial damage. To test our hypothesis we set up an ex vivo model consisting of fresh brain slices from the cortex of normal rhesus monkeys, and allowed live spirochetes to penetrate the tissue. In order to obtain a global overview of the immune response mounted by brain cells against spirochetes, tissues stimulated with spirochetes or medium alone were subjected to Real Time PCR (human) array analysis for transcripts of common inflammatory mediators. We then identified the actual cells that were producing immune mediators in situ, by blocking protein export from the cells with brefeldin A, followed by immunofluorescence staining with appropriate antibodies and confocal microscopy. Apoptosis was quantified by the TUNEL assay. A transcriptional fold increase of 2.31 (p=0.016) for the chemokine IL-8, and 7.43 (p=0.005) for the cytokine TNF-alpha was quantified by PCR array. We detected IL-6 in astrocytes, IL-1b in microglia and endothelial cells, IL-8 in astrocytes, microglia and endothelial cells and MCP-1 in endothelial cells. The immune mediator COX-2 was detected in astrocytes, microglia and endothelial cells. Concomitant apoptosis of oligodendrocytes and neurons was also observed in spirochete-stimulated tissues. These results provide proof of concept for our proposed hypothesis for Lyme neuroborreliosis pathogenesis, that B. bugdorferi is able to induce in brain tissues an inflammatory environment that leads to neuroglial damage.
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