IMMUNOLOGY CORE SERVICES

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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.