CYTOKINE PRODUCING CELLS AT THE BLOOD BRAIN BARRIER IN AIDS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在血液中感染艾滋病毒后，识别组织中的感染源和表征宿主免疫反应的技术已经得到很好的发展。然而，这些技术在原位检测宿主免疫反应或需要多个标记物来识别血脑屏障(BBB)产生细胞因子的细胞时的价值有限。这些问题中的大多数是由于传统显微镜在同时分辨两个以上的标记方面的局限性，特别是当它们彼此非常接近的时候。如果组织的厚度大于5或10微米，这个问题就会进一步复杂化。为了解决这些局限性，我们开发了共聚焦显微镜技术，允许以比荧光显微镜更高的分辨率同时显示三个或更多标记，并检查了厚度达100微米的组织切片。我们还开发了一种识别组织中产生细胞因子的细胞的方案。我们首先通过抑制蛋白质分泌来允许细胞内细胞因子的积累，然后我们用免疫荧光法检测这些细胞因子。利用这些技术，我们已经开始在血脑屏障原位检测细胞因子产生细胞的免疫表型和位置。我们现在可以在感染猴免疫缺陷病毒(SIV)的动物的血脑屏障中鉴定出表达各种细胞因子的细胞，并结合不同的巨噬细胞标志物。针对特定细胞因子的抗体[白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(干扰素-γ)]与细胞类型特异性标记物(巨噬细胞CD14、CD68、CD163、Ham 56和LN5；以及T细胞CD3)一起使用。初步研究表明，许多巨噬细胞正在产生IL-6，上述细胞因子在SIV感染猕猴血脑屏障附近的炎症区域都有不同程度的表达。通过在原位确定产生特定细胞因子的细胞类型，已经揭示了对大脑中发挥作用的免疫系统的洞察。

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.