Vladimir Zachar

High interferon alpha levels in placenta, maternal, and cord blood suggest a protective effect against intrauterine herpes simplex virus infection

Interferons (IFN) are produced by the placenta during pregnancy, and they can be detected in the maternal and fetal blood. Although the anti‐viral potential of IFNs is well established, it remains unclear whether the IFNs associated with pregnancy can prevent transplacental spread of viral infection. The present study was undertaken in order to determine the possible protective effect of placentally produced IFN‐α on fetal acquisition of herpes simplex virus (HSV). Nine mothers with a known history of genital HSV infection were studied. In five cases IFN‐α was detected in the placenta, maternal, and fetal blood, whereas in three cases IFN‐α could not be detected. In the remaining case, IFN‐α was found only in the maternal blood. As corroborated by the serological evidence of early HSV infection in the cord blood, the single case of vertical HSV transmission was observed in the group of IFN nonproducers. Furthermore, virus transmission did not occur in cases where IFN‐α was present in the placenta and simultaneously in the maternal and fetal circulations. Thus, the present data indicate that high levels of IFN during pregnancy may protect the fetus from acquiring a possibly fatal intrauterine HSV infection. J. Med. Virol. 51:210–213, 1997.

Differential interferon production in human first and third trimester trophoblast cultures stimulated with viruses

Stimulation of human placental first and third trimester trophoblast and syncytiotrophoblast cultures with viruses [Newcastle Disease Virus (NDV) and Sendai virus] led to a high interferon (IFN) production. The magnitude of the production was dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produced five to sixfold more IFN than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast at term produced twice as much IFN than the mononuclear term trophoblast when stimulated with the viruses. NDV and Sendai virus produced different levels and composition of IFN-alpha and -beta in both first and third trimester trophoblast and syncytiotrophoblast cultures. Purification of the virus-induced trophoblast interferons (tro-IFNs) by tandem high-performance affinity chromatography resulted in specific activities between 0.7 and 2.7 x 10(8) IU/mg of protein when assayed on human amniotic WISH cells. The tro-IFN-alpha protected both human and bovine MDBK cells from virus infection whereas the tro-IFN-beta protected only the human cell lines tested. The possible roles of the tro-IFNs are discussed in light of the observed differences in trophoblast IFN response.

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.

Differential HIV replication and HIV-induced interferon production in mononuclear phagocytes: relationship to cell maturation

We have investigated the replication of human immunodeficiency virus (HIV) and HIV-induced interferon (IFN) production in human mononuclear phagocytes at 2 different stages of in vitro maturation. Blood monocytes and monocyte-derived macrophages from 6 healthy, HIV-seronegative donors were challenged with HIV1IIIB and HIV2ROD. Freshly separated monocytes produced IFN when inoculated with both HIV types. In these cultures, an inverse correlation was observed between the amount of IFN production and the rate of HIV replication. In contrast to the monocytes, 5-day-old monocyte-derived macrophages did not produce IFN when challenged with HIV, but a significant replication of HIV1IIIB and HIV2ROD was found in all cultures.

Physiological oxygen tension is relevant to MHC-1 expression, spontaneous transformation, and interferon response of in vitro aging murine fibroblasts

Working from the hypothesis that modest deviations from physiological oxygen tension will influence cell characteristics important for infections/immunity and tumor development, cells were studied at three oxygen tensions during in vitro aging. Primary mouse embryo fibroblasts were established and subsequently passaged at 3, 6, and 18 kPa oxygen tension (6 representing normal tissue tension and 18 being the conventionally tension at in vitro cultures). The growth rate was slightly higher at 6 than 3 and 18 kPa. All cultures eventually stopped growing and subsequently transformed to nonmalignant cells with unlimited growth capacity. Cells kept at 3 kPa reached the highest number of cell doublings before crisis. Stimulation with PolyI:C resulted in detectable interferon response only at the high oxygen tension, and after transformation none of the cultures responded with interferon production. Expression of the major histocompatibility complex H-2K was elevated above and below physiological oxygen tension, indicating regulatory processes optimizing MHC expression at about physiological oxygen tension.

A BIOMETRICAL VIEW ON NORMAL VALUES OF CD4 AND CD8 LYMPHOCYTE COUNTS IN PERIPHERAL BLOOD

&NA; 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 ellpse 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.

Lack of protection against vertical transmission of HIV-1 by interferons produced during pregnancy in a cohort from East African republic of Malawi.

Interferons (IFNs) associated with pregnancy were studied for their possible role in inhibition of vertical transmission of the human immunodeficiency virus type 1 (HIV‐1). A study group was composed of 43 HIV‐1‐positive mothers, of whom 15 transmitted the virus to the offspring and 28 did not. The control group included 48 HIV‐1‐negative mother‐infant pairs. The IFN‐α was detected only sporadically in the maternal sera from the groups of transmitters (27%), nontransmitters (21%), and controls (19%). The average levels of IFN‐α were low, 16.3 ± 2.5 pg/ml, 21.4 ± 9.9 pg/ml, and 21.3 ± 9.4 pg/ml among the transmitters, nontransmitters, and control subjects, respectively. In the cord blood, IFN‐α was detected only on two occasions among transmitters, and on a single occasion in the control group. IFN‐β was absent from both maternal and cord blood in the study group, and found to be present in one case in the control group simultaneously in the maternal and fetal sera. In the placentas, on the other hand, both type I and II IFNs were expressed universally in the villous trophoblast, and IFN‐α and ‐β in the stromal macrophages as well. In one case among transmitters, no IFNs were detected; nevertheless, no significant difference with respect to nontransmitters could be confirmed. Our data suggest that although the placental IFNs have an antiviral potential, they are not sufficient to suppress transmission of HIV from mother to infant. J. Med. Virol. 61:195–200, 2000.

Vaccinia virus infection of cultured human first trimester trophoblast

Summary Congenital vaccinia is a well known cause of fetal wastage. To investigate a possible mechanism of virus transmission from mother to fetus, human trophoblast isolated from 7–12 weeks placentae was challenged with vaccinia virus. First trimester trophoblast was susceptible to vaccinia virus infection in vitro , as determined by morphologic alterations, cessation of hCG synthesis, and production of progeny virus. A recombinant virus carrying HIV DNA sequences induced trophoblast expression of gp 160 and replicated to the same extent as the control wild-type strain. The results may have a bearing on the general pathogenesis of fetal infections, and serve as a reminder of the small but not negligible risk of vaccination with live virus in pregnancy.

Clonal analysis suggests provirus expression in a subpopulation of human malignant trophoblast cells harbouring the human T cell lymphotropic virus type-I genome

Previous studies have indicated that the villous trophoblast may be involved in intrauterine HTLV-I infection. Although the data furnished by our group (Liu et al., 1995) have demonstrated that the human trophoblast-derived malignant cell lines JAR and JEG-3 are susceptible to HTLV-I, the infection, even after thorough analysis, appeared to be limited to expression at the transcriptional level. In the present report, we sought to explore virus expression at the single cell level using eight clonally selected cell lines which were derived by limiting dilution from the previously infected parental cultures. Of the three cell lines JAR-H2, JAR-H3, and JEG-H3, all of which harboured full-length provirus, only in two (JAR-H2 and JEG-H3) were the virus-specific tax/rex and env transcripts demonstrated using RT-PCR. When compared with MT-2 cells, the detected steady-state levels of HTLV-I mRNA appeared to be lower by three orders of magnitude. Viral Tax protein displaying a typical intranuclear localization was found in 1-2% of JAR-H2 and JEG-H3 cells. Moreover, an altered phenotype characterized by multinucleated syncytia was observed in these cell cultures with the same frequency as Tax transactivator, implying a fusogenic activity of env protein. Infectious virus, however, could not be rescued from JAR-H2 or JEG-H3 clones by coculture with cord blood mononuclear cells. Our data suggest that trophoblast represents a susceptible, albeit a slightly permissive, host system for HTLV-I.

HUMAN NATURAL KILLER CELLS FAIL TO KILL HERPES SIMPLEX VIRUS TYPE 1-INFECTED TERM VILLUS TROPHOBLAST CELLS

Summary The study was undertaken to determine if HSV infection of trophoblast cells would alter the general resistant state of trophoblast cells to MHC non-restricted cell-mediated cytotoxicity. The NK cell activity against HSV-infected human term placental trophoblast cells was investigated. In a 12-hour 51 Cr release assay, PBMC, or immunosorted peripheral blood CD56 + NK cells from healthy donors were found not to lyse HSV-1-infected trophoblast cells, while HSV-1-infected placental fibroblast cells were preferentially lysed when using the same experimental set-up. Furthermore, using cold target competition assay, no inhibition of NK cell activity against the susceptible target cell K562 was noted when using HSV-infected or uninfected trophoblast cells. Inversely, both HSV-infected and uninfected fibroblast cells demonstrated a competitive inhibition of NK cell lysis of K562 cells. The observed differences between the susceptibility of HSV-infected placental trophoblast and fibroblast cells to NK lysis could not be explained by a difference in the surface expression of HSV-antigens or transferrin receptors. Additionally, no significant differences were observed for de novo production of IFN by effector cells during the cocultivation with HSV-infected trophoblast and fibroblast cells that would explain the observed discrepancies in NK susceptibility. Consequently, these in vitro results suggest that the observed resistance of trophoblast cells to maternal cell-mediated lysis is not modified by HSV-1 infection and that the peripheral blood NK cells are not involved in the prevention of a possible vertical HSV spread through infected trophoblast cells.