![Fig. 1. (A) Increase of HHV-8 DNA load in PBMC from 2 AIDS-KS patient after culture (days 6 to 7) in the presence of TCM or RTCM or in its absence (RPMI). Shown are the autoradigrams of serial-dilution PCR experiments performed with primers specific for HHV-8 ORF 26 (VP23) and for the human β-globin gene used as a control of the amount of the genomic DNA analyzed. PCR products were hybridized with specific oligonucleoutide probes. Numbers above lanes represent dilution factors (for VP23) or aliquots of cell extracts corresponding to the indicated number of cells (for β-globin), respectively. Cell extracts were diluted in salmon sperm DNA as described in Materials and Methods. (C) Amplification of HHV-8 DNA from PBMC (day 0) and long-term cultures (21 or 28 days) of PBMC from 4 patients with KS (2 AIDS-KS, 2 CKS) and a PT patient. Floating (F) and adherent (A) cells were separately harvested at day 21 or 28 from PBMC cultured in the presence (RTCM) or absence (RPMI) of IC and the same number of cells (105) were analyzed with primer set 3. Negative controls (NC) are PCR reactions performed without DNA template or aliquots of salmon sperm DNA processed with PBMC. Positive controls were made with the indicated numbers of molecules (Mol) of plasmid p557-19. PCR products were transferred to nylon membranes and hybridized to a 32[P]-labeled oligonucleotide probe internal to the amplified sequences. Ethidium bromide stainings showed amplification of β-globin gene sequences from the same specimens. PBMC from the 2 CKS patients and the PT patient were analyzed also at day 7 and 14 with negative results. (D) PCR analysis of HHV-8 DNA with PBMC cultured with or without RTCM, γIFN, TNF, or IL-6. The same cell number was analyzed with primer set 3. NC is the negative control, consisting of either salmon sperm DNA processed with the specimens or PCR reactions lacking DNA template. (a) NKS-AIDS patient; (b through e) AIDS-KS patients; (f) 50 and 5 molecules of a positive control plasmid. PBMC from the patient shown in (a) were cultured for 11 days; PBMC from the other patients were cultured for 3 to 5 days. Fresh RTCM was added at days 0 and 3 of culture; single cytokines were added at days 0 and 2 for the patients shown in (a) and (b) and at days 0 and 4 for the other patients, respectively. γlFN was used at a concentration of 10, 50, or 100 IU/mL, as indicated in parenthesis. TNF was used at 30 ng/mL and IL-6 was used at 100 IU/mL, respectively. Experiments repeated with TNF or IL-6 (at 100 or 1,000 IU/mL) on 2 other patients that responded to RTCM gave similar results. All samples were positive for β-globin amplification, as shown by ethidium bromide staining of the PCR products. (B) Detection of HHV-8 DNA in PBMC (day 0) and in floating (F) or adherent (A) cells from 4 AIDS-KS patients (AIDS-KS), an asymptomatic homosexual man (HIV+), and a PT patient (PT) cultured (6 to 7 days) in the presence of TCM or RTCM or in its absence (RPMI). The same cell number (105) was analyzed with primer set 3. PC are positive controls made with the indicated numbers of molecules of plasmid p557-19. NC is negative control made without adding template DNA. PCR products were transferred to nylon membranes and hybridized to a32[P]-labeled oligonucleotide probe internal to the amplified sequences. Ethidium bromide staining shows amplification of β-globin gene sequences from the same specimens.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/93/12/10.1182_blood.v93.12.4044/4/blod41239001bw.jpeg?Expires=1735536905&Signature=h9HD6fQTl0NW0he3oMT6IH6r1CYNekZrlTM2Cddss6XU4grO4~swIEhYcmJvJJE07wvKGF0viJheLTB2baEgl0GpXG4AebMVSxlxxWPvaQANSj3IuE~H-zK1yy4UBeSVkOz8JrumCyVPP6fhOkqho8GAqUAaYv3grbzuHfwFmL0VSRDs3RA7HNWkc5627iTIbZmhaUS1o6aKXuZlafHiDoNHgQODNDdoMRQVprRN7zr6EDMe1mX0aSJ-ijYUgzyQTWYWP-Qxq5abEBZibviLm77mqXUjo4pJ~t2TRv9~o4y85oaKMobEtdD7RvKjsVgAnxzQcH52Htf2sUyGdF1xbw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
(A) Increase of HHV-8 DNA load in PBMC from 2 AIDS-KS patient after culture (days 6 to 7) in the presence of TCM or RTCM or in its absence (RPMI). Shown are the autoradigrams of serial-dilution PCR experiments performed with primers specific for HHV-8 ORF 26 (VP23) and for the human β-globin gene used as a control of the amount of the genomic DNA analyzed. PCR products were hybridized with specific oligonucleoutide probes. Numbers above lanes represent dilution factors (for VP23) or aliquots of cell extracts corresponding to the indicated number of cells (for β-globin), respectively. Cell extracts were diluted in salmon sperm DNA as described in Materials and Methods. (C) Amplification of HHV-8 DNA from PBMC (day 0) and long-term cultures (21 or 28 days) of PBMC from 4 patients with KS (2 AIDS-KS, 2 CKS) and a PT patient. Floating (F) and adherent (A) cells were separately harvested at day 21 or 28 from PBMC cultured in the presence (RTCM) or absence (RPMI) of IC and the same number of cells (105) were analyzed with primer set 3. Negative controls (NC) are PCR reactions performed without DNA template or aliquots of salmon sperm DNA processed with PBMC. Positive controls were made with the indicated numbers of molecules (Mol) of plasmid p557-19. PCR products were transferred to nylon membranes and hybridized to a 32[P]-labeled oligonucleotide probe internal to the amplified sequences. Ethidium bromide stainings showed amplification of β-globin gene sequences from the same specimens. PBMC from the 2 CKS patients and the PT patient were analyzed also at day 7 and 14 with negative results. (D) PCR analysis of HHV-8 DNA with PBMC cultured with or without RTCM, γIFN, TNF, or IL-6. The same cell number was analyzed with primer set 3. NC is the negative control, consisting of either salmon sperm DNA processed with the specimens or PCR reactions lacking DNA template. (a) NKS-AIDS patient; (b through e) AIDS-KS patients; (f) 50 and 5 molecules of a positive control plasmid. PBMC from the patient shown in (a) were cultured for 11 days; PBMC from the other patients were cultured for 3 to 5 days. Fresh RTCM was added at days 0 and 3 of culture; single cytokines were added at days 0 and 2 for the patients shown in (a) and (b) and at days 0 and 4 for the other patients, respectively. γlFN was used at a concentration of 10, 50, or 100 IU/mL, as indicated in parenthesis. TNF was used at 30 ng/mL and IL-6 was used at 100 IU/mL, respectively. Experiments repeated with TNF or IL-6 (at 100 or 1,000 IU/mL) on 2 other patients that responded to RTCM gave similar results. All samples were positive for β-globin amplification, as shown by ethidium bromide staining of the PCR products. (B) Detection of HHV-8 DNA in PBMC (day 0) and in floating (F) or adherent (A) cells from 4 AIDS-KS patients (AIDS-KS), an asymptomatic homosexual man (HIV+), and a PT patient (PT) cultured (6 to 7 days) in the presence of TCM or RTCM or in its absence (RPMI). The same cell number (105) was analyzed with primer set 3. PC are positive controls made with the indicated numbers of molecules of plasmid p557-19. NC is negative control made without adding template DNA. PCR products were transferred to nylon membranes and hybridized to a32[P]-labeled oligonucleotide probe internal to the amplified sequences. Ethidium bromide staining shows amplification of β-globin gene sequences from the same specimens.
