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1.
Standard proliferation assays used for analysis of T cell function have significant shortcomings, including limited sensitivity, lack of quantitative readouts, and considerable variability. Recently, flow cytometric methods have been developed to allow multiparametric detection of cell surface antigens and intracellular cytokine expression in response to polyclonal stimuli and antigen. We have optimized an intracellular cytokine staining assay in the non-human primate model of AIDS, which allows us to identify antigen-specific T lymphocytes at the single cell level with high sensitivity, while reducing background staining to a minimum. Central to our optimized protocol is the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification that results in up to 3-fold enhancement of the frequency of cytokine-secreting CD4(+) T cells following superantigen or antigen-specific stimulation. Optimization of the antigen concentration and duration of antigenic stimulation resulted in a convenient and highly reproducible assay, which permits delineation of antigen-specific cells at the single cell level, thereby providing new insights into pathogen-specific immune responses and allowing detailed phenotypic analysis of extremely low frequency events.  相似文献   

2.
Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis   总被引:13,自引:0,他引:13  
Dengue virus produces a mild acute febrile illness, dengue fever (DF) and a severe illness, dengue hemorrhagic fever (DHF). The characteristic feature of DHF is increased capillary permeability leading to extensive plasma leakage in serous cavities resulting in shock. The pathogenesis of DHF is not fully understood. This paper presents a cascade of cytokines, that in our view, may lead to DHF. The main feature is the early generation of a unique cytokine, human cytotoxic factor (hCF) that initiates a series of events leading to a shift from Th1-type response in mild illness to a Th2-type response resulting in severe DHF. The shift from Th1 to Th2 is regulated by the relative levels of interferon-gamma and interleukin (IL)-10 and between IL-12 and transforming growth factor-beta, which showed an inverse relationship in patients with DF.  相似文献   

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