(A) Detection of latent and lytic viral gene expression in total PBMC at day 0 and in floating or adherent cells cultured with or without IC from 2 patients with AIDS-KS. (a) RT-PCR and hybridization for T0.7; (b) RT-PCR and hybridization for VP23; (c) RT-PCR and hybridization for T0.7 in the absence of reverse transcriptase; (d) RT-PCR and ethidium bromide staining for human β-actin. NC are negative controls, including Jurkat cells and PCR reactions without DNA template. RT⁻ indicates β-actin RT-PCR reactions performed from the same samples of RNA in the absence of reverse transcriptase. The same number of RTCM-treated and untreated cells were processed and RNA normalized by spectrophotometric determination. (B) ISH detection of HHV-8 lytic (Vp23) gene expression in PBMC from an AIDS-KS patient after culture with RTCM. ISH of (A) PBMC cultured for 72 hours without RTCM (original magnification × 25) and (B) corresponding dark field; (C) PBMC cultured for 72 hours with RTCM (original magnification × 25), arrows point to ISH-positive cells and (D) corresponding dark field; (E) PBMC from a patient not infected by HHV-8 (original magnification × 25) and (F) corresponding dark field; (G) higher magnification showing positive cells with monocytic morphology; a cell with lymphocyte morphology is negative. Several microscopic fields were analyzed and a similar density of positive cells was present in cells cultured in the presence of RTCM. In contrast, cells cultured in the presence of RPMI were always negative. Hybridization to a latency-associated HHV-8 gene probe could not be performed, because cells were not sufficient.