This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Techniques for identifying infectious agents in tissues and characterizing the host immune response following HIV infection in blood are well developed. However, these techniques are of limited value for in situ examination either of the host immune response or when multiple markers are needed to identify cells producing cytokines at the blood brain barrier (BBB). Most of these problems are due to limitations of traditional microscopy in resolving more than two labels simultaneously, particularly if they are in close proximity to one another. This problem is further compounded if the tissues have a thickness greater than 5 or 10 micrometer. To address these limitations, we have developed confocal microscopy techniques, which allow for simultaneous visualization of three or more labels at a much higher resolution than is possible by epifluorescence microscopy, and have examined tissue sections up to 100 micrometer in thickness. We have also developed a protocol for identifying cells producing cytokines in tissues. We begin by allowing the accumulation of intracellular cytokines by inhibiting protein secretion, after which we detect these cytokines by immunofluorescence. Using these techniques, we have begun to examine the immunophenotype and location of cytokine-producing cells in situ at the BBB. We can now identify cells expressing various groupings of cytokines in combination with different macrophage markers in the BBB of animals infected with simian immunodeficiency virus (SIV). Antibodies to specific cytokines [interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)] have been used in conjunction with cell type specific markers (macrophages CD14, CD68, CD163, HAM 56 and Ln5; and T cells CD3). Initial studies show that many macrophages are producing IL-6, and that all above-mentioned cytokines are expressed to some degree in areas of inflammation in proximity to the BBB of SIV-infected rhesus macaques. By determining the cell types producing specific cytokines in situ, insights into the immune system at work in the brain have been revealed.
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