CYTOKINE PRODUCING CELLS AT THE BLOOD BRAIN BARRIER IN AIDS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。用于鉴定组织中的感染因子和表征血液中的HIV感染后的宿主免疫应答的技术已经发展良好。然而，这些技术对于原位检查宿主免疫应答或当需要多种标志物来鉴定在血脑屏障（BBB）处产生细胞因子的细胞时的价值有限。大多数这些问题是由于传统的显微镜在解决两个以上的标签同时，特别是如果他们是在彼此非常接近的限制。如果组织具有大于5或10微米的厚度，则该问题进一步复杂化。为了解决这些局限性，我们已经开发出共聚焦显微镜技术，它允许同时可视化的三个或更多的标签在一个更高的分辨率比可能的落射荧光显微镜，并检查组织切片高达100微米的厚度。我们还开发了一种用于鉴定组织中产生细胞因子的细胞的方案。我们开始通过抑制蛋白质分泌来允许细胞内细胞因子的积累，之后我们通过免疫荧光检测这些细胞因子。使用这些技术，我们已经开始研究的免疫表型和位置的精氨酸生产细胞原位在血脑屏障。我们现在可以识别细胞表达的各种集团的细胞因子结合不同的巨噬细胞标记物在BBB感染猴免疫缺陷病毒（SIV）的动物。特异性细胞因子[白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）]的抗体已与细胞类型特异性标志物（巨噬细胞<$CD 14、CD 68、CD 163、HAM 56和Ln 5;以及T细胞<$CD 3）联合使用。初步研究显示，许多巨噬细胞产生IL-6，并且所有上述细胞因子在SIV感染的恒河猴的BBB附近的炎症区域中在一定程度上表达。通过确定原位产生特定细胞因子的细胞类型，揭示了免疫系统在大脑中的作用。

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.