IMMUNOLOGY CORE SERVICES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目的：免疫学服务核心旨在提供1）猕猴免疫应答的标准化测量，2）向外部研究人员提供有关灵长类动物免疫学研究实验设计的建议，3）开发用于评估该动物模型免疫应答的新技术，4）有关灵长类动物免疫学有用的技术和试剂的信息，和5）培训人员使用免疫学测定法进行猕猴工作。 所提供的大部分服务支持艾滋病研究。 此外，CNPRC的其他感染性和非感染性研究也可使用标称试验抗原的检测方法。 该中心于2000年8月开始运行，有四个服务或工作区，分别对应于体液免疫（仅在初始样品处理时需要BSL-2）、细胞免疫（在整个检测期间需要BSL-2密封）、过敏单元和分子核心单元。 体液免疫测定包括通过ELISA检测针对病毒抗原的抗体。 此外，通过ELISA测量标称抗原破伤风类毒素、霍乱毒素和匙孔血蓝蛋白的抗体水平。 细胞分析包括检测抗病毒细胞毒性T淋巴细胞（CTL）或自然杀伤细胞（NK）以及淋巴细胞对病毒和标称抗原的增殖。 混合淋巴细胞反应可用于遗传学和移植。 细胞因子/趋化因子分泌细胞通过ELISPOT测定。 分子核心单元通过实时PCR分析和定量细胞因子/趋化因子mRNA转录水平。 另外的靶基因扩增可用于某些凋亡基因和一些转录因子。已将SIV DNA和RNA检测/定量以及提供等分试样的SIVmac攻毒储备液（滴定用于粘膜接种）添加到核心中。 过敏科负责提供过敏和肺部相关服务，包括用于气雾剂输送和全身致敏程序的过敏原的表征和制备。 此外，还开发了检测组胺和类胰蛋白酶水平的免疫测定法，以及检测过敏原特异性IgE/IgG的免疫测定法。 还可为采集的血液和支气管肺泡灌洗样本的制备、分析和存档提供支持。 此外，核心已经开发和验证了新的淋巴细胞表型面板和细胞因子流量测定，并尽快由UCD委员会批准率将提供这些。 我们目前有几个外部调查人员与冷冻细胞正在寻求这项服务。 我们还提交了免疫组织化学测定的补充率，如上次基本赠款更新所建议的。如果获得批准，这项服务可以在不久的将来添加到免疫学核心。最后，一些额外的基因靶点已被添加到核心的基因表达服务，我们正在协助外部UCD调查人员开发激光捕获显微镜和RNA提取，并根据时间和材料收回我们的成本。 核心还分发了2种无细胞SIV感染性储备，即SIVmac 251和SIVmac 239。 将储备液分成0.5和1 ml等份，并在液氮中冷冻。 储备液滴度为10^5 TCID 50，含有约10^9 vRNA拷贝/ml。 对于两种贮备液，10 TCID 50的IV接种感染了2/2只动物，这些动物具有持续的高血浆vRNA和下降的CD 4 + T细胞。 我们还发现，1 TCID 50的两个股票感染1/2的猴静脉接种，虽然血浆vRNA水平较低，在这些感染的动物。 免疫学核心在本预算年度提供的服务包括血液处理服务;屋尘螨特异性IgG ELISA;过敏原制备和分发;细胞增殖分析; PBMC和组织的实时PCR; SIV病毒储备生产和分发。

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. Objective: The Immunology Service Core is designed to provide 1) standardized measures of immune response in macaques, 2) advice to outside investigators on experimental design of primate immunology studies, 3) development of new technology for assessing immune responses in this animal model, 4) information on techniques and reagents that are useful for primate immunology, and 5) training of personnel in the use of immunology assays for work with macaques. Much of the services provided support AIDS research. In addition, assays to nominal test antigens were available for other infectious and non-infectious research at the CNPRC. The Core, which began operation in August 2000, has four services or work areas corresponding to humoral immunity (requires BSL-2 only for initial sample processing), cellular immunity (requires BSL-2 containment throughout the time of assay), an allergy unit, and a molecular core unit. Humoral immune assays include the detection of antibodies to viral antigens by ELISA. In addition, antibody levels to the nominal antigens tetanus toxoid, cholera toxin and keyhole limpet hemocyanin are measured by ELISA. Cellular assays include the detection of antiviral cytotoxic T lymphocytes (CTL) or natural killer cells (NK) and lymphocyte proliferation to viral and nominal antigens. A mixed lymphocyte reaction is available for genetics and transplantation. Cytokine/chemokine-secreting cells are assayed by ELISPOT. The molecular core unit analyzes and quantitates cytokine/chemokine mRNA transcript levels by real-time PCR. Additional target gene amplification is available for certain apoptosis genes and some transcription factors. SIV DNA and RNA detection/quantitation and the provision of aliquots of SIVmac challenge stocks, titered for mucosal inoculation, have been added to the Core. The allergy unit is responsible for providing allergy and pulmonary related services, including characterization and preparation of allergen for aerosol delivery and systemic sensitization procedures. In addition, immunoassays to detect histamine and tryptase levels, as well as immunoassays to detect allergen-specific IgE/IgG have been developed. Support for preparation, analysis and archiving of collected blood and bronchoalveolar lavage samples is also available. In addition the Core has developed and validated new lymphocyte phenotyping panels and a multicolor cytokine flow assay and as soon as rates are approved by UCD committees these will be offered. We currently have several outside investigators with frozen cells that are seeking this service. We have also submitted a recharge rate for immunohistochemistry assays as proposed in the last base grant renewal. If approved, this service can be added to the Immunology Core in the near future. Finally, a number of additional gene targets have been added to the gene expression service of the Core and we are assisting outside UCD investigators develop laser capture microscopy and RNA extraction and recovering our costs based on time and materials. The Core also distributes 2 infectious stocks of cell-free SIV, namely SIVmac251 and SIVmac239. The stocks are divided into 0.5 and 1 ml aliquots and frozen in LN2. The stocks have titers of 10^5 TCID50 and contain approx. 10^9 vRNA copies/ml. For both stocks, IV inoculation of 10 TCID50 infects 2 of 2 animals and the animals have sustained high plasma vRNA and declining CD4+ T cells. We also found that 1 TCID50 of both stocks infects 1 of 2 monkeys inoculated IV, although the plasma vRNA levels were lower in these infected animals. Services provided by the Immunology Core in the current budget year include blood processing services; house dust-mite-specific IgG ELISAs; allergen preparation and distribution; cellular proliferation assays; real-time PCR for both PBMC and tissue; and SIV viral stock production and distribution.