Pseudotypes of vesicular stomatitis virus-bearing envelope antigens of certain HIV-1 strains permissively infect human syncytiotrophoblasts cultured in vitro: implications for in vivo infection of syncytiotrophoblasts by cell-free HIV-1.

Intrauterine infection of the fetus is clearly an important mode of vertical transmission of human immunodeficiency virus type 1 (HIV-1). The syncytiotrophoblast layer of the human placenta must be traversed by HIV-1 in order to reach underlying cells and fetal capillaries. Although HIV-1 has been detected in the syncytiotrophoblast layer in situ, there is conflicting evidence regarding infection of syncytiotrophoblast cells with cell-free virus. The phenotypic mixing between HIV-1 and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) pseudotypes were found to enter syncytiotrophoblast cells. In contrast, VSV pseudotyped with envelope glycoproteins of RF, MN, or Ada-M strains of HIV-1 did not infect syncytiotrophoblasts. Plating efficiency of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) was 10-fold lower on syncytiotrophoblasts than on T-cells and macrophages, respectively. Incubation of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) viruses with appropriate HIV-1 neutralizing sera before infection strongly inhibited entry of pseudotyped VSV into syncytiotrophoblast cells. These findings demonstrated that infection of syncytiotrophoblasts with VSV(HIV-1) pseudotypes was mediated by Env from IIIB and Ba-L strains of HIV-1. Monoclonal antibodies (MAb) to CD4, CXCR4, CCR5, and CCR3 were tested for their ability to block VSV(HIV-1) infection of syncytiotrophoblast cells. Neither the anti-CD4 nor the anti-CXCR4, anti-CCR5, and anti-CCR3 MAb had any inhibitory effect on infection of syncytiotrophoblast cells with VSV(HIV-1) pseudotypes. Results from this study suggest that cell-free HIV-1 can enter syncytiotrophoblasts and the susceptibility of these cells to penetration by the virus is strain dependent. Pseudotype infection merely demonstrates that the first steps in HIV-1 replication are possible in syncytiotrophoblast cells.

Prevalence and age distribution of human herpesvirus-8 specific antibodies in Hungarian blood donors.

Sera of blood donors were investigated by a peptide ELISA and indirect immunofluorescence assay to assess the prevalence of HHV-8 infection in the Hungarian population. A 14 amino acid long synthetic oligopeptide from the carboxyterminus of orf65/small virus capsid antigen was used as antigen in the ELISA. ELISA results were confirmed by recombinant orf65 antigen Western blot. Antibodies to the latent nuclear antigen were detected by the immunofluorescence assay. Nine of 12 sera obtained from patients with classical Kaposi sarcoma were reactive by ELISA whereas all were positive by immunofluorescence. Four of 482 (0.83%) healthy blood donors had anti-orf65 peptide antibodies and 17/1089 (1.56%) had antibodies to the latent nuclear antigen. In a group of children ages 1-14 years, antibodies to the latent nuclear antigen (0/29) were not detected. The prevalence of antibodies to the latent nuclear antigen showed a moderate but significant increase in correlation with senescence. In the Kaposi sarcoma patients, the titre of antibodies to the latent nuclear antigen was significantly higher than in the healthy seropositive donors. The overall HHV-8 seroprevalence by the two assays was 2.28% (11/482) in the Hungarian blood donor group.

Induction of HIV-1 replication in latently infected syncytiotrophoblast cells by contact with placental macrophages: role of interleukin-6 and tumor necrosis factor-alpha.

The syncytiotrophoblast (ST) layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Although certain strains of human immunodeficiency virus type 1 (HIV-1) may enter ST cells, the trophoblast does not exhibit permissiveness for HIV-1. The present study tested the possibility that placental macrophages might induce replication of HIV-1 carried in ST cells and, further, that infected ST cells would be capable of transmitting virus into neighboring macrophages. For this purpose, we investigated HIV-1 replication in ST cells grown alone or cocultured with uninfected placental macrophages. The macrophage-tropic Ba-L strain of HIV-1, capable of entering ST cells, was used throughout our studies. We demonstrated that interactions between ST cells and macrophages activated HIV-1 from latency and induced its replication in ST cells. After having become permissive for viral replication, ST cells delivered HIV-1 to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HIV-1 gene expression in ST cells was mediated by marked tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) release from macrophages, an effect caused by contact between the different placental cells. Results of this study suggest an interactive role for the ST layer and placental macrophages in the dissemination of HIV-1 among placental tissue. Data reported here may also explain why macrophage-tropic HIV-1 strains are transmitted preferentially during pregnancy.

Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

Role of interleukin-8 and transforming growth factor-beta1 in enhancement of human cytomegalovirus replication by human T cell leukemia-lymphoma virus type I in macrophages coinfected with both viruses.

Human cytomegalovirus (HCMV) is one of the most frequent opportunistic agents causing severe illness in chronic human T cell leukemia-lymphoma virus type I (HTLV-I) infection. Our previous studies have shown that coinfection of macrophages with HCMV and HTLV-I significantly enhances HCMV replication, resulting in release of infectious HCMV from dually infected cells. We found that double infection of macrophages with HCMV and HTLV-I induced a rapid production of substantial amounts of interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1). Results of transfection studies demonstrated that the tax gene product of HTLV-I was able to induce secretion of IL-8 and TGF-beta1. In addition to its cytokine-inducing effect, the Tax protein could interact with HCMV synergistically to result in production of much higher levels of IL-8 and TGF-beta1 than expected on the basis of their separate activities. Treatment of dually infected macrophage cultures with neutralizing antibodies to IL-8 and TGF-beta1 led to a nearly 1000-fold decrease in release of infectious HCMV from coinfected cells. Similar results were obtained when anti-IL-8 and anti-TGF-beta1 treatments were combined in macrophage cultures transfected with the tax gene before HCMV infection. Our results suggest that the stimulatory effect of HTLV-I Tax protein on HCMV replication in coinfected macrophages is largely mediated by high levels of IL-8 and TGF-beta1 production.

Changes in oncogene expression implicated in evolution of chronic granulocytic leukemia from its chronic phase to acceleration.

Expression of nine oncogenes was investigated in cell samples from fifteen patients with Philadelphia chromosome (Ph)-positive chronic granulocytic leukemia (CGL) both at diagnosis and at the onset of accelerated phase (AP) of the disease. The bcr-abl fusion gene, the H-ras gene and the c-myb gene were universally expressed. In comparison with the chronic phase (CP) of the disease, an increase in the levels of bcr-abl-, c-myb- and H-ras-related transcripts was found in three, two and three AP samples, respectively. Elevation of the bcr-abl-related message was associated with duplication of the Ph chromosome and amplification of the bcr-abl fusion gene in one AP sample. No CP samples were positive for c-myc or c-sis expression. On the contrary, c-myc and c-sis were expressed in three and four AP samples, respectively. The presence of c-myc-related transcript was associated with trisomy 8 with or without amplification of the c-myc oncogene in leukemia cells of two patients with CGL in AP. No changes of oncogene expression were found in four AP samples. However, we observed deletions of chromosome 13 and 17 or i(17q) in three of them, suggesting that tumor suppressor gene alterations may also be responsible for the development of AP of CGL. Our data indicate that heterogeneous alterations in oncogenes and tumor suppressor genes accompany the evolution of CGL-CP to the AP of the disease.

Reciprocal interactions between human cytomegalovirus and human T cell leukemia-lymphoma virus type I in monocyte-derived macrophages cultured in vitro.

Infection of macrophages with human cytomegalovirus (HCMV) has been shown to be nonlytic and exclusively cell associated. Human T cell leukemia-lymphoma virus type I (HTLV-I) is capable of establishing productive infection in macrophages. We studied the interactions between HCMV and HTLV-I in monocyte-derived macrophages cultured in vitro. We found that coinfection of macrophages with HCMV and HTLV-I significantly enhanced HCMV replication, resulting in release of infectious HCMV from dually infected cells. On the other hand, HCMV inhibited HTLV-I replication in macrophages coinfected with both viruses. Reciprocal interactions between HCMV and HTLV-I were mediated by their trans-acting proteins. Results of transfection studies demonstrated that the tax gene product of HTLV-I alone was capable of upregulating HCMV production. In a transient gene expression assay the immediate-early 2 (IE2) protein of HCMV alone could inhibit HTLV-I replication, whereas the IE1 protein, which had no effect by itself, produced a synergistic inhibitory effect together with the IE2 protein. Results from this study suggest that in vivo double infection of macrophages with HCMV and HTLV-I may contribute to the dissemination of HCMV infection in patients suffering from HTLV-I-associated T cell leukemia-lymphoma.

Epstein-Barr virus permissively infects human syncytiotrophoblasts in vitro and induces replication of human T cell leukemia-lymphoma virus type I in dually infected cells.

Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1), as well as human T-cell leukemia-lymphoma virus type I (HTLV-I), may interact in the pathogenesis of human retroviral infections. The placental syncytiotrophoblast layer represents a barrier protecting the fetal compartment from exposure to retroviruses. We studied the interactions of EBV with HIV-1 and HTLV-I in human term syncytiotrophoblast cells to investigate the significance of double infections in transplacental transmission of human retroviruses. We found that syncytiotrophoblast cells could be productively infected with EBV. Dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. In contrast, the restricted permissiveness of syncytiotrophoblasts for HIV-1 was not influenced by coinfection of the cells with EBV. Infection of syncytiotrophoblast cells with EBV, but not HTLV-I, induced interleukin-2 and interleukin-6 secretion, and augmented secretion occurred on coinfection with both viruses. Coinfection of syncytiotrophoblast cells with EBV and HTLV-I induced tumor necrosis factor-beta and transforming growth factor-beta 1 secretion, but infection with either virus alone did not lead to secretion of these cytokines. Permissive replication cycle of HTLV-I was induced by the EBV immediate-early gene product Zta. Pseudotype formation between EBV and HTLV-I in coinfected syncytiotrophoblast cells was not found. Our data suggest that activation of HTLV-I gene expression by EBV in coinfected syncytiotrophoblast cells may be a mechanism for transplacental transmission of HTLV-I.

Compact growth of Staphylococcus haemolyticus in soft agar is not due to hydrophobic interaction between the cocci.

It was hypothesized that the formation of compact colony in soft-agar both in the presence and absence of serum, characteristic mainly for strains of the species Staphylococcus haemolyticus among coagulase-negative staphylococci [Szúcs et al. Acta Microbiologica Hungarica 40:181-189 (1993)] was due to hydrophobic interaction between cocci. The effect of a number of surface active agents on this phenomenon was examined. Neither 0.1% and 1%. Tween-80 nor 5% and 10% ethylene glycol and polyethylene glycol nor 0.1%-4% trypsin influenced the colony morphology in soft-agar prepared in modified Staphylococcus 110 broth. Bovine lactoferrin and apolactoferrin at concentrations of 0.1%-0.4% made compact colonies transient to diffuse ones. Thus, cocci are not adhered to each other in compact ball-like colonies by hydrophobic interaction or trypsin-sensitive proteins. It is possible that still unknown polysaccharide-binding proteins or other trypsin-resistant proteins are responsible for the formation of compact colonies by Staphylococcus haemolyticus in soft-agar.

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

A comparative study on growth in soft-agar, adherence to glass and haemolysis types of coagulase-negative staphylococci.

Growth properties of coagulase-negative staphylococci in the presence and in the absence of human and rabbit serum in soft-agar prepared in modified Staphylococcus 110 broth were studied. The adherent growth was examined in modified Staphylococcus 110 broth and 1% glucose-meat broth. Of 100 strains examined 69% exhibited diffuse, 18% compact, 7% transient and 6% mixed growth. Compact type colonies were mainly characteristic of Staphylococcus haemolyticus strains. The presence of serum failed to influence the types of colony morphology in any of the strains. Sixty-three percent of the strains showed adherent growth; none of the S. haemolyticus strains produced adherent growth. The glucose-meat broth, unlike modified Staphylococcus 110 broth, was suitable to study adherence. The coincidence of the compact colony morphology in soft-agar and the absence of adherent growth seems to be a taxonomic sign for the species S. haemolyticus and differentiate it from the species Staphylococcus epidermidis.

The effect of methicillin on the fatty acid composition of total polar lipid in methicillin resistant Staphylococcus aureus.

The effect of two concentrations of methicillin on the fatty acid (FA) distribution in intracellular total polar lipid (TPL) of the log-phase cultures of a methicillin resistant Staphylococcus aureus strain No. 5814R was studied during a period of 2 h. Half the MIC of methicillin (= 1000 micrograms/ml) caused 18.6% increase in branched-FAs and a same decrease in straight-FAs, while one MIC (= 2000 micrograms/ml) of the drug induced a moderate change in those of TPL. The ratio of branched-FAs to straight-FAs increased from 1.24 to 1.56 in the presence of 1/2 x MIC of methicillin and reduced from 1.24 to 0.87 in the presence of 1 X MIC of the antibiotic. In TPL of the control cultures it gradually decreased from 1.24 to 0.77. It is concluded that under the effect of methicillin, FA composition of TPL in methicillin resistant cocci does not change as dramatically as in methicillin sensitive ones indicating lipid synthesis in methicillin resistant S. aureus to be less sensitive to the action of methicillin than in methicillin susceptible strains. This may contribute to the resistance against the lytic effect of the drug. Membrane lipid properties seem to be involved in the mechanisms of methicillin resistance.