Milan Zdravkovic

Role of the Fas/Fas ligand pathway in apoptotic cell death induced by the human T cell lymphotropic virus type I Tax transactivator

Two distinct human diseases have been described in association with human T cell lymphotropic virus type I (HTLV-I) infection: adult T cell leukaemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Although comprehensive understanding of specific mechanisms underlying pathogenesis of either disease has not yet been achieved, the viral regulatory protein Tax is believed to play a significant role. Previous studies demonstrated the potential of Tax to transform host cells. Here, it is shown that the Tax transactivator has in addition the potential to induce T cell death by apoptosis. Using an inducible system (Jurkat cell line JPX-9), significant apoptotic cell death upon Tax expression was observed. In an attempt to detect the cellular genes mediating this effect, it was found that induction of Tax was associated with marked upregulation of the Fas ligand (FasL) gene. Tax-induced apoptosis was inhibited when the Fas/FasL pathway was interrupted by YVAD-cmk, the inhibitor of ICE-like proteases. Transient expression experiments provided additional support for the putative role of endogenous FasL in Tax-induced apoptosis. Upon cotransfection with Tax-expressing plasmid, the transcriptional activity of the FasL promoter was found to be significantly upregulated in Jurkat cells and several other cell lines, as measured by reporter gene expression. Furthermore, cotransfection using different Tax mutants demonstrated that both CREB and NF-kappaB activation domains of Tax protein were required for the transactivation to take effect.

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.

Functional characteristics of human trophoblast interferons

Human trophoblast populations from first‐ and third‐trimester placentas produce interferons (IFNs) in the presence of growth factors (CSF and PDGF) or when infected with virus. The highly invasive extravillous trophoblast population produced a higher level of IFNs (three‐ to eightfold, P < 0.05) than the noninvasive villous trophoblast population when stimulated with growth factors and/or virus. The level of IFN produced was dependent on the type of trophoblast population, the type of inducer and the stage of differentiation of the trophoblasts. Tandem immunoaffinity chromatography of the virus‐induced trophoblast IFNs resulted in the isolation of trophoblast IFN‐α and ‐β types. The purified trophoblast IFNs have antiviral, antiproliferative and immunoregulatory properties. Furthermore, the trophoblast IFNs inhibited the expression of proto‐oncogenes such as EGF‐R, c‐erbB2 and c‐fms reported to be involved in normal trophoblast growth and differentiation. These data suggest essential roles of interferons in normal human development during pregnancy.

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.

Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture

The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-alpha and -beta. One hundred IU tro-IFN-alpha and -beta/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-alpha and -beta/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

Immunosuppressive effects of human placental trophoblast interferon-β 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.

Vertical transmission of HIV: Possible mechanisms and placental responses

Summary Most studies to date indicate that the most frequent mode of vertical infection of HIV is transplacentally after hematogenous infection. Since virus is not reaching the fetus in about 70 per cent of the cases a strong defense system may exist. Based upon macroscopic and light microscopic examinations of the human placenta, responses to HIV infection are minimal however, the presence of the virus in most cases can be demonstrated in fetal placental monocytes (Hofbauer cells) and oftern in the trophoblastic cells. Virus presence in other cell types is reported less frequently. Infection of the trophoblastic cells appears possible both with free virus attaching to CD4 receptors, via virus-antibody complexes, and via maternal leukocytes attaching to trophoblastic cells. In vitro evidence further suggests that trophoblastic cells may both secrete free virus and deliver to CD4 positive lymphocytes. Trophoblast infection with HIV seems to be non-lytic but productive and the penetration further into the fetal tissue likely occurs via infection of Hofbauer cells. The trophoblastic cells are capable of producing both alpha, beta, and gamma interferon, in particular, during the first trimester. Their role(s) in placental response to HIV is under study. Maternal antibodies to HIV may enhance trophoblastic cell uptake of HIV. Furthermore, the maternal cellular cytotoxic immunune response to infected trophoblastic cells seems muted by unknown factor(s). Cytokines may play a central role in placental responses to HIV and external modification of placental defenses should include attempts to modify transmission to the fetus during pregnancy.

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.

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.

Basal and interferon-induced 2′,5′-oligoadepylate synthetase activity in human placental trophoblast and trophoblast-derived malignant cell lines

Human placental trophoblasts produce interferon (tro-IFNs) when stimulated with viral inducers. Since the antiviral and cellular functions of IFNs are partly mediated by the 2,5-oligoadenylate synthetase (2-5A synthetase) pathway, the aim of the present study was to determine the basal and IFN-induced levels of 2-5A synthetase in villous trophoblast cultures. A considerable basal level of 2-5A synthetase was observed in syncytiotrophoblast cultures from both first and third trimester. In contrast no basal activity was detectable in placental fibroblast- and trophoblast-derived malignant cell lines (Far, FEG-3, and BeWo). Stimulation with tro-IFN-beta, -alpha and leucocyte-IFN (leu-IFN)-alpha increased the enzyme activity in first and third trimester human syncytiotrophoblast cultures. Treatment with recombinant-IFN (rec-IFN)-gamma significantly enhanced 2-5A synthetase activity in first trimester syncytiotrophoblast, but had no effect on third trimester syncytiotrophoblast. Tro-IFN-beta, -alpha and leu-IFN-alpha induced high levels of 2-5A synthetase activity in placental fibroblast, BeWo and FEG-3 cell-lines, whereas rec-IFN-gamma treatment did not induce 2-5A synthetase activity in any of these cells. No detectable 2-5A synthetase activity was found in the Far cell line. The capability of cells deriving from the fetoplacental unit to raise an antiviral response by the induction of 2-5A synthetase may be important in defending the fetus against viral infection from the mother. Furthermore 2-5A synthetase in cells of the fetoplacental unit may play a role in their normal growth and development.