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Biomedical subjects

V Zachar

Publications and source records attributed to V Zachar.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1989

Nucleotide sequence of HIV1-NDK: a highly cytopathic strain of the human immunodeficiency virus.

A highly cytopathic strain of HIV1, named HIV1-NDK, has been isolated from a Zaïrian patient affected with AIDS. This isolate is 10(4) times more cytopathic and infectious than the prototype. To correlate the high cytopathic properties of this strain with genetic variations, we have cloned and sequenced the genome of this isolate. The principal feature which could be drawn from the fine analysis of the HIV1-NDK sequence is that the variability is not clustered in one particular region but rather spread out all along the genome. Only minor differences seem to be responsible for the acute biological effect of HIV1-NDK.

Amino Acid Sequence↗
PubMed1989

Increased migration inhibition factor production by leukocytes of patients with herpes zoster given the leukocyte ultrafiltrate.

Using the direct leukocyte migration inhibition assay, the cell-mediated immune response was followed in together 47 patients suffering from herpes zoster. Of these, 19 persons were treated with partially purified and concentrated lysed leukocyte ultrafiltrate. Enhancement and/or earlier production of the leukocyte migration inhibition factor--in the presence of varicella-zoster virus antigen--was observed by leukocytes from patients given the ultrafiltrate on days 2-3 since the onset of the vesicular stage of the disease. Highly significant (alpha = 1%, P less than 0.01) differences in the migration inhibition values were observed between non-treated patients and patients given one dose of the ultrafiltrate during the days 3-12 after appearance of the first herpes zoster vesicles; on days 13-30 these values were not significant.

Adolescent↗
PubMed1988

A biometrical view on normal values of CD4 and CD8 lymphocyte counts in peripheral blood.

Statistical methods have been used to determine an optimal approach to the definition of the reference range for CD4 ("helper") and CD8 ("suppressor/cytotoxic") T cell numbers and the CD4/CD8 ratio in the peripheral blood. A graphical presentation of the absolute values for CD4 and CD8 for 85 healthy blood donors showed that a reference ellipse defined by fitting a gaussian distribution to logarithmically transformed data for absolute counts of CD4 and CD8 cells was superior to the fitting of an ellipse to untransformed data. Further analysis for another 147 subjects showed that the 95% tolerance prediction for the CD4/CD8 ratio in health could be stated with 95% confidence as 0.6 to 5.0. This approach allows clear definition of reference ranges for T cell tests in health and would also be applicable to results for patients with a disease such as HIV 1 infection in which a reference range for "well" patients exists and a change in the T cell ratio is of prognostic significance.

Biometry↗
PubMed1988

Varicella-zoster virus IgG antibodies during primoinfection in competent and transfer factor modulated immunocompromised host: comparison of three indirect assays.

Ten patients with acute leukaemia and next three with Hodgkin's or non-Hodgkin's lymphoma, suffering from varicella-zoster-virus (VZV) primoinfection, were given 1 to 2 doses of ultrafiltrate of the human leukocytes lysate (LLU) containing transfer factor (TF) activity (1 dose being equivalent to the product of 10(8) leukocytes). Only LLU administered to patients with acute lymphocytic leukaemia (ALL) at early phases of the illness (days 1 and 2) displayed a notable benefit on the clinical course of varicella. No influence upon the infection, on the other hand, was observed following LLU administration to subjects with lymphoma. The convalescent levels of IgG antibodies to VZV, as detected by indirect immunoperoxidase assay to membrane antigen (IPAMA), demonstrated no significant difference between infected competent and immunocompromised untreated and LLU treated individuals. The performance characteristics of IPAMA are compared with indirect immunofluorescence method (IFA) and non-competitive enzyme-linked immunosorbent assay (ELISA) on the same panel of specimens.

Antibodies, Viral↗
PubMed1987

Varicella-zoster virus IgM antibody fraction separated by minicolumn gel filtration in serodiagnosis of acute infection.

Gel filtration through Sephadex G-200 or Ultrogel AcA 34 minicolumns was applied to isolate IgM antibodies from sera of patients with varicella and herpes zoster (HZ). Serum samples were stained with Sudan III and Evan's Blue in order to monitor the separation process visually when running several columns at the same time. The presence of specific IgM antibodies was demonstrated by enzyme linked immunosorbent assay (ELISA). Rapid separation of stained sera by diffusion chromatography on minicolumns with successive determination of specific IgM antibodies in ELISA proved suitable for serological diagnosis of varicella, whereas the significance of IgM-specific antibody response in HZ seems uncertain.

Acute Disease↗