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E.E. Kozlowska, R.D. Dix; Measurement of Human Perforin mRNA Levels within Whole Blood: A Pilot Feasibility Study to Quantify Cellular Immunity for Possible Use in Patients with AIDS-Related HCMV Retinitis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4615.
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Purpose: Cytotoxic CD8+ T cells and NK cells kill virus-infected cells by distinct pathways that include perforin-mediated cytotoxicity and receptor-mediated (Fas/FasL) cytotoxicity. We have shown previously that PKO mice deficient in the perforin cytotoxic pathway are susceptible to experimental MCMV retinitis, whereas gld mice deficient in the Fas/FasL cytotoxic pathway exhibit a resistance to retinitis that is observed in immunologically normal mice. Moreover, mice with retrovirus-induced immunodeficiency syndrome (MAIDS) that are susceptible to MCMV retinitis exhibit a significant decrease in splenic and intraocular perforin mRNA levels. We therefore hypothesized that measurement of perforin mRNA levels within whole blood might be a better quantification of cellular immunity (and susceptibility to HCMV retinitis during HIV-1 disease) than measurement of absolute CD4+ and CD8+ T-cell numbers. Methods: Total RNA from whole blood of immunologically normal persons was reversed transcribed using primers specific for human perforin mRNA and subjected to real-time RT-PCR assay using the SmartCycler apparatus and technology for detection of SYBER Green I dye amplification. Standard curves for direct quantification of human perforin mRNA were constructed using human perforin cDNA template copies. PCR amplification of rRNA was performed as an internal control. Results: Human perforin mRNA was detected within whole blood of immunologically normal persons at a level of ~169 copies per 0.1 ml of whole blood. Conclusions: We have developed a real-time RT-PCR assay to detect and quantify human perforin mRNA within samples of whole blood from immunologically normal persons. Use of purified lymphocytes from whole blood should increase the ability of the assay to detect a measurable and meaningful decrease in human perforin mRNA levels during the progression of HIV-1-induced immunosuppression. This assay might be useful as a predictor in the clinical setting for those patients with HIV-1 disease who are destined to develop HCMV retinitis.
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