IMMUNOLOGY CORE SERVICES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 目的：免疫学服务核心旨在提供1)猕猴免疫反应的标准化测量，2)向外部研究人员提供关于灵长类免疫学研究的实验设计的建议，3)在该动物模型中评估免疫反应的新技术的发展，4)关于灵长类免疫学有用的技术和试剂的信息，以及5)培训工作人员使用免疫学分析处理猕猴。提供的大部分服务都支持艾滋病研究。 此外，CNPRC还为其他感染性和非感染性研究提供了标称试验抗原的检测。2000年8月开始运行的核心有四个服务或工作领域，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(在整个化验过程中需要BSL-2遏制)、过敏单元和分子核心单元。体液免疫分析包括用酶联免疫吸附试验检测病毒抗原抗体。此外，用酶联免疫吸附试验检测了标称抗原破伤风类毒素、霍乱毒素和锁孔帽血蓝蛋白的抗体水平。细胞检测包括检测抗病毒的细胞毒性T淋巴细胞(CTL)或自然杀伤细胞(NK)以及淋巴细胞对病毒和名义抗原的增殖。混合淋巴细胞反应可用于遗传学和移植。细胞因子/趋化因子分泌细胞用ELISPOT法检测。 分子核心单元通过实时定量聚合酶链式反应分析和定量细胞因子/趋化因子mRNA的转录水平。对于某些凋亡基因和某些转录因子，可以进行额外的靶基因扩增。核心增加了SIV DNA和RNA检测/定量以及SIVmac挑战菌种的等量供应，滴定用于粘膜接种。 过敏股负责提供与过敏和肺部相关的服务，包括用于气雾剂输送和全身致敏程序的过敏原的特征和制备。此外，还开发了检测组胺和类胰蛋白酶水平的免疫检测方法，以及检测过敏原特异性IgE/IgG的免疫检测方法。还可为采集的血液和支气管肺泡灌洗样本的准备、分析和存档提供支持。 此外，CORE还开发和验证了新的淋巴细胞表型表型板和多色细胞因子流动分析，一旦UCD委员会批准了这些比率，就将提供这些。我们目前有几个冷冻细胞的外部调查人员正在寻求这项服务。我们还提交了免疫组织化学分析的补充率，就像上次基础拨款续签中所建议的那样。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中。最后，Core的基因表达服务增加了一些额外的基因靶点，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。 核心还分发两种非细胞SIV感染性库存，即SIVmac251和SIVmac239。将原料分成0.5毫升和1毫升等份，并将其冷冻在液氮中。这些股票的滴度为10^5 TCID50，含有大约。10~9vRNA拷贝/ml。静脉接种10TCID50后，2只动物中2只感染，血浆vRNA升高，CD4+T细胞下降。我们还发现，两个种群中的1个TCID50感染了2只接种IV的猴子中的1只，尽管这些感染动物的血浆vRNA水平较低。 免疫核心在本预算年度提供的服务包括血液处理服务；室内尘螨特异性免疫球蛋白EISA；过敏原的制备和分发；细胞增殖分析；PBMC和组织的实时聚合酶链式反应；以及SIV病毒库存的生产和分发。

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.