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Biomedical subjects

V Zachar

Publications and source records attributed to V Zachar.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cell-mediated transmission of human T cell lymphotropic virus type I to human malignant trophoblastic cells results in long-term persistent infection.

We investigated permissiveness of the malignantly transformed trophoblast (choriocarcinoma) cell lines JAR, BeWo and JEG-3 to the human T cell lymphotropic virus type I (HTLV-I). After co-culture with the productively infected cell line MT-2 the choriocarcinoma cell lines were analysed for infection over a period of three months. The presence of HTLV-I viral DNA was examined by PCR using primers targeting the gag, pol, env and pX specific sequences. All amplified segments were found consistently in the cell cultures throughout the period of study. Further analysis that aimed to characterize the size variation of the integrated proviral DNA by Southern blotting revealed the presence of integrated proviral sequences which consisted, for the most part, of incomplete genomes. The presence of the full-length HTLV-I genome, however, was unequivocally confirmed in clonally expanded cell cultures derived from the originally infected parental cells. In order to analyse virus expression at the transcriptional level, we used reverse transcriptase (RT)-mediated PCR that was targeted at intra-exon regions (gag, pol, env and pX), and the splicing sites of the env and pX-tax/rex mRNAs. When compared with MT-2 cells, substantially lower levels of all transcripts were found in all the cell lines analysed. We were unsuccessful in attempts to detect viral protein expression using polyvalent or Tax- and Gag-specific monoclonal antibodies by Western blot analysis or immunoprecipitation, and we could not detect any RT activity released into the supernatant of the infected cells either. Collectively, these data suggest that the trophoblastic cells may become persistently but essentially non-productively infected with HTLV-I.

Base Sequence↗
PubMed1994

Immunosuppressive effects of human placental trophoblast interferon-beta on lymphocytes in vitro.

Human placental trophoblast cells produce predominantly interferon-beta-type (IFN-beta) when stimulated with viral inducers. The aim of the present study was to determine the in vitro antiproliferative effect of the trophoblast interferon-beta (tro-IFN-beta) on mitogen-stimulated and resting lymphocytes. The antiproliferative effect of the tro-IFN-beta was compared to human recombinant IFN-beta. All activities of tro-IFN-beta and human recombinant IFN-beta ranging between 10-1000 IU/ml showed suppression of proliferative responses on mitogen-stimulated and resting lymphocytes compared to cultures without IFN treatment. The inhibitory level of both tro-IFN-beta and recombinant IFN-beta was significantly higher on the stimulated than on the resting lymphocytes. Although there was a variation in the inhibition of lymphocyte proliferation by both IFNs with respect to time, there was no statistically significant difference in the antiproliferative effect of the IFNs on both resting and mitogen-stimulated lymphocytes. Since IFNs are produced locally by the placenta during pregnancy, our data suggest that in addition to the antiviral activity, the human tro-IFN-beta may participate in the local control of the maternal immune response during pregnancy at the fetomaternal interface.

Cell Division↗
PubMed1994

In vitro cytotoxic activity of cord blood NK cells against herpes simplex virus type-1 infected purified human term villous cytotrophoblast.

Transplacental infection of the fetus with herpes simplex virus (HSV) is associated with high morbidity. The present study was undertaken to shed light on the possible participation of the fetal immune system in the elimination of HSV from placental unit. In a chromium release assay cultured term villous trophoblast cells, regardless of infection with HSV-1, were found resistant to lysis by cord blood natural killer (CBNK) cells. In contrast to this, CBNK cells exhibited a basal level of cytotoxic activity against placental fibroblasts, which was significantly increased by preceding infection of the target cells with HSV-1. Stimulation of CBNK cells with interferon-beta purified from trophoblast (tro-IFN-beta) increased the killing of both HSV-1 infected and uninfected fibroblast, while HSV-1-infected and uninfected term villous trophoblast cells remained resistant to lysis. IL-2-stimulated CBNK cells were able to lyse villous trophoblast cells at a low level, but no significant difference in the susceptibility of the HSV-1-infected and uninfected trophoblast cell was detected.

Cells, Cultured↗
PubMed1994

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication.

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Base Sequence↗
PubMed1993

Human trophoblast interferons.

The human placental trophoblasts which constitute the first fetal cells and form the major cell layer of the feto-maternal interface are potent producers of interferons (IFNs). The IFN production is dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblasts. First trimester trophoblast populations produce higher levels (5-6 times) of IFN than the third trimester trophoblasts when stimulated with viruses. Non-viral inducers, such as poly(rl).poly(rC), induce exclusively IFN-beta whereas viruses such as Sendai and Newcastle Disease Virus (NDV) induce mixtures of IFN-alpha subtypes and IFN-beta. Differentiation of mononuclear cytotrophoblasts into syncytiotrophoblasts in vitro increase the IFN production. High-performance and immunoaffinity chromatography of the virus-induced trophoblast IFN preparations resulted in the isolation of three antigenically distinct IFNs, namely, alpha I, alpha II1 (omega 1), and beta with molecular masses of 16, 22 and 24 kDa, respectively, on SDS-PAGE. The human trophoblast IFNs have physical and antiviral activities characteristic of the Type 1 IFNs. The possible roles of the trophoblast IFNs in human placental and fetal development are also discussed in this review.

Female↗
PubMed1992

Herpes simplex virus infection of cultured human term trophoblast.

Mononuclear trophoblast cells were isolated from term placentas of uncomplicated pregnancies, purified to homogeneity by negative immunomagnetic separation using monoclonal antibodies to the major histocompatibility complex, and challenged with herpes simplex virus (HSV). The cultures were highly susceptible to virus-induced cytopathic effect as evidenced by cytopathic alteration and inhibition of human chorionic gonadotropin (hCG) secretion. Both HSV I and II underwent a full replicative cycle in the trophoblast, although the production of progeny virus was 10-100 times less than that obtained with placental fibroblasts or choriocarcinoma cells. The permissiveness was independent of in vitro syncytial differentiation of the trophoblast. The results suggest that the trophoblast layer may be involved in intrauterine HSV infection.

Antibodies↗
PubMed1992

Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography.

Human placental trophoblasts, fibroblasts and the trophoblast-derived malignant cell JAR are potent producers of interferons (IFNs) when stimulated with Sendai virus. The three cell lines produced different levels and compositions of IFN-alpha subtypes and IFN-beta. Anti-IFN globulins, Cibacron Blue F3GA and Concanavalin A were covalently immobilized on pressure-stable, macroporous polymeric matrices derivatized with vinyl sulphone (HEMA-BIO 1000 VS and HEMA 1000 VS). These supports were packed in biocompatible PEEK columns and were coupled with switching valves, to develop a tandem high-performance affinity chromatographic (HPAC) method for the isolation, purification and biochemical characterization of the IFNs produced in Sendai virus-stimulated human placental trophoblasts, fibroblasts and trophoblast-derived malignant cell, JAR, cultures. Silver-stained SDS-PAGE and gel densitometric analysis revealed the purity of the purified proteins to be between 94 and 98%. Specific activities of the purified IFNs ranged between 0.37-2.76 x 10(8) IU/mg of protein with cumulative recoveries between 90 and 92.2%. The purified IFN components exhibited quantitatively different antiviral activities in human and bovine cell lines. The utility of the tandem method for the purification and characterization of human type 1 IFNs produced from other cell lines are also discussed.

Chromatography, Affinity↗
PubMed1992

Bacterially expressed core and envelope proteins of human immunodeficiency virus type-1 (HIV-1): comparative evaluation in detection of type-specific antibodies.

Recombinant proteins derived from immunodominant conserved domains of HIV-1 env and gag genes were synthesized in E. coli. An immunoblot system using total cell lysates was employed for the analysis of recombinant bacterial clones. Together 427 serum samples obtained from asymptomatic anti-HIV seropositive individuals, AIDS patients, healthy donors and persons suffering from various conditions were comparatively evaluated for the presence of HIV-1 antibodies using recombinant peptides and commercially available western blot (WB) and ELISA assays. The recombinant antigen product of plasmid pEX41 was found to be superior, with respect to sensitivity and specificity, to the viral gp41 which represents a diagnostically important constituent of the WB.

AIDS Serodiagnosis↗
PubMed1991

Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes.

The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.

Adamantane↗
PubMed1991

Susceptibility of cultured human trophoblasts to infection with human immunodeficiency virus type 1.

Primary cultures of essentially pure human term trophoblasts were studied to determine their ability to support the expression of complete proviral clones of human immunodeficiency virus (HIV) and their permissiveness to this virus. Transient expression of molecular clones derived from two biologically distinct strains, BRU and NDK, resulted in the release of comparable amounts of infectious virions, which were rescued by cocultivation with permissive CEM-SS cells. Trophoblasts were inoculated with three HIV-1 isolates, RF, 3B and NDK, which differ in their cytopathogenicity on T lymphoblastoid cells. Infection of cells by all three strains was demonstrated by the presence of virus-specific proteins in the trophoblasts and the detection of virus gag gene-related DNA sequences by the polymerase chain reaction (PCR), but cells were more susceptible to infection with the RF and NDK strains than with the 3B strain. The virus was readily transmitted to the CEM-SS cells with simultaneous formation of syncytia between the two cell types. Flow cytometry and direct radioimmunoassay revealed no trace of the CD4 receptor on the surface of the cultured trophoblasts and CD4 mRNA could not be detected by Northern blot hybridization, although a minimal amount of CD4-associated mRNA was detected by PCR. Our data suggest that infection of trophoblasts occurs independently of the pathway mediated by CD4.

Base Sequence↗
PubMed1991

Characterization of Sendai virus-induced human placental trophoblast interferons.

Human placental trophoblast cultures produce a mixture of interferons (IFNs) when challenged with Sendai virus. High-performance dye-ligand and immunoaffinity chromatography of a trophoblast IFN (tro-IFN) preparation enabled the isolation of three antigenically distinct IFNs, alpha I, alpha II 1 and beta, with Mrs of 16K, 22K and 24K respectively, by reducing and non-reducing SDS-PAGE. The major IFN, responsible for 75% of the total antiviral activity, was tro-IFN-beta, with the remaining activity being due to tro-IFN-alpha I and tro-IFN-alpha II 1, as determined by an antiviral neutralization test using specific anti-human IFN antibodies. The antiviral activities of the tro-IFNs were stable at pH 2.0 for 24 h and tro-IFN-alpha II 1 and -beta were shown to be glycoproteins. The three tro-IFNs showed different antiviral activities when assayed on human and bovine cell species; tro-IFN-alpha I and alpha II 1 protected both human and bovine (MDBK) cells from virus infection, whereas tro-IFN-beta showed a high degree of species specificity, protecting only the human cell types tested.

Animals↗
PubMed1991

Human transformed trophoblast-derived cells lacking CD4 receptor exhibit restricted permissiveness for human immunodeficiency virus type 1.

We investigated the nature of interaction of the malignantly transformed cell lines of trophoblast origin BeWo, JAR, and JEG-3 with three different human immunodeficiency virus type 1 (HIV-1) isolates (RF, 3B, and NDK). After inoculation with cell-free virus, the persistence of infection was determined for 1 month by monitoring the presence of viral DNA in the cells by the polymerase chain reaction (PCR). Furthermore, the infectious virus in the culture supernatant was assayed with CEM-SS cells, and attempts to rescue the virus by cocultivation with CEM-SS cells were made. Appraised on the basis of the relative amount of viral DNA and the frequency of positive cocultivation. JEG-3 was the most permissive and BeWo was the least permissive cell line. However, when the cells were transfected with two biologically active molecular clones of HIV-1, the BRU and NDK isolates, all three cell lines turned out to support the production of mature virus progeny to the same extent. The abundance of viral DNA sequences in the infected cells varied with the isolate, showing an overall decline from RF to NDK. The amount of viral DNA in the cells and its expression decreased during the period of observation; this decrease was mirrored in an erosion of the virus recovery rate at cocultivation from 71% recovery on day 8 to failure of isolation on day 32. None of the cell lines expressed detectable amounts of cell surface CD4 molecules when assayed by flow microfluorometry and direct radioimmunoassay. Northern (RNA) blot hybridization analysis of both the total RNA and the mRNA did not reveal any CD4-specific message: nonetheless, by using the PCR, sequences specifically related to the CD4 gene were uncovered. The data demonstrate that the trophoblast-derived cell lines are susceptible to infection with HIV and that they support transient viral replication in the initial phases of infection. However, the latent form of infection may persist over a period of several weeks.

Acquired Immunodeficiency Syndrome↗
PubMed1991

In vitro productive infection of human malignant trophoblastic cell line JAR with human immunodeficiency virus type 1 (HIV-1).

Human choriocarcinoma cells of the JAR line, with no demonstrable surface CD4 receptor were infected with human immunodeficiency virus type 1 (HIV-1), strain RF. Primer-directed enzymatic DNA amplification (polymerase chain reaction, PCR) detected the presence of viral DNA when the cultures were investigated at day 5 post-infection (p.i.). The absence of cytopathic changes attributable to virus replication suggested silent infection of these malignant trophoblastic cells. Neither reverse transcriptase (RT) activity nor HIV-specific antigens were found in the culture nutrient medium during JAR cell propagation. However, when the HIV-carrier JAR cells were continuously cultured and the cocultivation was initiated with CEM-SS lymphoblastoid cells after two subsequent passages, rescue of infectious virus was observed.

Animals↗