Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be hard, if not impossible. and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still 1643913-93-2 IC50 expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA activation. These results indicated a much Rabbit Polyclonal to GNA14 more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo. Human herpesvirus 1643913-93-2 IC50 8 (HHV-8) is usually associated with Kaposi’s sarcoma (KS), main effusion lymphoma (PEL), and a subset of multicentric Castleman’s disease (2, 19, 28). Serological examinations have revealed that almost all KS patients have anti-HHV-8 antibodies (3, 12, 22, 30). Immunohistochemical studies have directly exhibited that the proliferating spindle-shaped cells of KS lesions express a latency-associated nuclear antigen (LANA) (6, 15, 16, 21), strongly suggesting that HHV-8 is usually the pathogenic agent of KS. While there are some controversies about the source of the spindle-shaped cells, endothelial cells appear to be the main candidate, as inferred from the manifestation of endothelium-specific molecules (1, 4, 11, 25C27, 29). Thus, attempts to transmit HHV-8 to endothelial cells have been conducted (7, 8, 18, 20, 23). In general, there are two modes of viral transmission: direct contact between target and supplier cells (so-called cell-mediated transmission) and cell-free transmission. In previously reported HHV-8 contamination experiments, cell-free transmission has been investigated; no detailed work on cell-mediated transmission has been reported. In the mean time, cell-free transmission was reported for human W cells in vivo in SCID-hu mice (5) and in vitro assays. However, viral transmission to endothelial cells with a cell-free supernatant was reported to be much more hard (23). Flore 1643913-93-2 IC50 et al. exhibited that main human umbilical vein endothelial cells (HUVECs) can be infected with HHV-8 using purified viral particles (7). They prepared purified and concentrated viral particles from the supernatant of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BC-3 cell collection, an HHV-8-positive- and Epstein-Barr virus-negative PEL cell collection, and added the medium of HUVECs at 5 to 10 genome equivalents per cell. Moreover, they also reported that HHV-8 contamination caused long-term proliferation and survival of these cells (7). Moses et al. also succeeded in transmitting HHV-8 to dermal microvascular endothelial cells transfected with human papillomavirus (HPV) At the6 and At the7 genes by exposing these cells to the nonconcentrated culture supernatant of BCBL-1 cells, another HHV-8-positive PEL cell collection (18, 24). Dermal microvascular endothelial cells thus infected with HHV-8 were transformed, lost contact inhibition, and proliferated in soft agar (18). Another group succeeded in transmitting HHV-8 to human neonatal brain endothelial cells using a highly concentrated suspension of HHV-8 particles produced from the 1643913-93-2 IC50 culture supernatant of BCBL-1 cells (23). However, these conditions were too artificial and do not appear to reflect the conditions occurring in vivo because of the use of highly concentrated viral particles or transformed target cells. To our knowledge, neither data on cell-cell contact transmission of HHV-8 in vitro nor evidence for the presence of cell-mediated transmission in vivo has ever been reported. Thus, in this study, we attempted to transmit HHV-8 to human endothelial cells by cell-cell contact using main cultures of HUVECs and BCBL-1 cells. Transmission of HHV-8 to HUVECs by coculturing with BCBL-1 cells. To determine the possibility of HHV-8 contamination, we cocultured HUVECs and TPA-treated BCBL-1 cells. TPA treatment is usually known to increase the production of HHV-8 particles (24). HUVECs were obtained from healthy donors with their informed consent and were cultured in an RPMI 1640-based conditioned medium made up of 10% fetal calf serum and 30 g of endothelial cell growth product (Upstate Biotechnology, Lake Placid, N.Y.)/ml on chamber glass photo slides coated.