Transmission of HIV to the placenta, fetus and mother and implications of gametic infection.

Therapeutic interventions and public education are reducing pediatric AIDS cases in developed countries, but the number of HIV-infected women and children is still a major global concern. The finding that human sperm-associated HIV can be transmitted to oocytes following in vitro fertilization provides a novel viewpoint from which to consider not only the problem of HIV transmission to children but also transmission to women. In the present paper we will first discuss some recent findings that offer new perspectives on the role of the placenta, and particularly the trophoblast, in maternal-fetal transmission of HIV. Results will be presented showing that cell-mediated infection of syncytiotrophoblast cells requires direct contact between infected lymphocytes and trophoblast. We will also discuss possible routes of transmission of HIV to both mothers and their offspring in the light of data providing evidence of gametic infection. These hypothetical routes include trophoblast-mediated infection of maternal uterine cells during implantation and trophoblast-mediated infection of maternal blood cells during pregnancy. Clearly, more studies are needed in order to assess the significance and relative contribution of these routes in the transmission of HIV.

Kinetics of HIV infection of human placental syncytiotrophoblast cultures: an ultrastructural and immunocytochemical study.

We previously demonstrated that syncytiotrophoblast (ST) cells from term human placentas could be infected when cocultured with HIV-infected lymphocytic cells. Here, we have used fluorescence microscopy and transmission electron microscopy to examine the kinetics of this infection process. Molt-4 clone 8 cells infected with HIV-1Lai or filtered supernatant from these cultures were incubated with ST cells for different times. In cell-associated infection, immunofluorescence microscopy revealed that some ST colonies were positive for HIV core proteins (p24,p55) after 1 hr. The number of positive colonies and the intensity of the ST-associated fluorescence increased with time. Transmission electron microscopy showed viral particles with HIV morphology associated with the ST cell surface at 1 hr. Immature virions with budding morphology were observed at 2 hr. In cell-free infection, positive p24,p55 staining was first detected in a few ST colonies at 4 hr. The number of positive colonies increased with time. At 24 hr, the fluorescence pattern and intensity resembled that seen with cell-mediated infection at 4 hr. Transmission electron microscopy revealed an increasing number of viral particles associated with the ST cell plasma membrane with respect to time, and budding virions first appeared at 8 hr. These results demonstrate that HIV infection of placental ST cells proceeds very rapidly in culture and that, furthermore, cell-associated infection of ST is much more efficient than the infection with cell-free virus.

Simian immunodeficiency virus infection via amniotic fluid: a model to study fetal immunopathogenesis and prophylaxis.

The rising prevalence of infection with the human immunodeficiency virus type 1 (HIV-1) in young women will increase the number of infected children worldwide. Because HIV-1 seems to be transmitted mostly intrapartum, fetal infection probably occurs mainly via skin or mucous membrane exposure. A model for this route of fetal infection has been established in primates. After injecting the simian immunodeficiency virus (SIV) into amniotic fluid during late gestation, six of seven rhesus monkeys were born infected. All infected neonates were viable and showed signs of disease, such as low birth weights, lymphadenopathy, and rashes. Cytotoxic T-cell responses to SIV were absent in neonates, but present in mothers. The high fetal infection rate allows studies of lentiviral immunopathogenesis during ontogeny and the development of strategies to prevent maternal HIV-1 transmission.

Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells.

Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha-mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia.

N-carboxymethylchitosan-N,O-sulfate as an anti-HIV-1 agent.

N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome.

Postexposure chemoprophylaxis with ZDV or ZDV combined with interferon-alpha: failure after inoculating rhesus monkeys with a high dose of SIV.

Presently, no information is available regarding the efficacy of chemoprophylaxis in controlled human trials following accidental exposure to the human immunodeficiency virus (HIV). Using the closely related simian immunodeficiency virus (SIV) in rhesus monkeys, which develop a disease closely resembling human AIDS, we tested the efficacy of either single-agent 3'-azido-3'-deoxythymidine (ZDV) or the combination of ZDV plus recombinant human interferon-alpha (IFN-alpha). Treatment was started 3 h following inoculation of a high dose of SIV and continued for 21 days. SIV-inoculated control animals remained untreated. Virus was recovered from all monkeys on day 8, and by week 7 all had seroconverted. In contrast to monkeys treated with ZDV alone, animals given combination therapy had lower levels of p27 gag antigen compared to untreated controls on day 8 (p = 0.043). We conclude that neither treatment regimen could prevent infection after high-dose virus exposure; however, combination therapy may have depressed the level of virus replication more effectively than ZDV alone.

Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate esters of 3'-azido-3'-deoxythymidine.

The water-soluble ammonium salt of 3'-azido-5'-(O-ethoxycarbonylphosphinyl)-3'-deoxythymidine (ECP-AZT), the prototype of a novel class of compounds incorporating two active antiretroviral agents, in this case 3'-azido-3'-deoxythymidine (AZT) and phosphonoformic acid (PFA), within the same structure, was synthesized and tested as an inhibitor of the replication of human immunodeficiency virus type 1 (HIV-1) in Jurkat cells, a CD4+ human T-lymphocyte cell line. The corresponding 5'-(O-methoxycarbonylphosphinyl) derivative (MCP-AZT) was also prepared. The rationale for the synthesis of ECP-AZT and MCP-AZT was that they may be cleaved intracellularly to AZT and PFA via hydrolysis of the phosphate ester bond or to AZT 5'-monophosphate by oxidative cleavage of the carbon-phosphorus bond. ECP-AZT was found to block viral replication at a 50% inhibitory concentration (IC50) of ca. 10(-6) M as measured by reverse transcriptase (RT) activity in supernatants from cultures of infected cells. Little or no inhibition of cell growth was observed at this concentration, and there was less than 20% inhibition of cell growth at 10(-4) M. AZT itself was a more potent inhibitor of HIV-1 replication than ECP-AZT, but was also more cytotoxic. The antiviral selectivity of ECP-AZT, defined as the ratio IC50 (virus inhibition)/IC50(cell growth inhibition), was in the range considered to be therapeutic for anti-AIDS nucleosides.

Murine models for evaluating antiretroviral therapy.

The pandemic of the acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus type 1 (HIV-1), requires rapid development of effective therapy and prevention. Analysis of candidate anti-HIV-1 drugs in animals is problematic since no ideal animal model for HIV-1 infection and disease exists. For many reasons, including small size, availability of inbred strains, immunological reagents, and lymphokines, murine systems have been used for in vivo analysis of antiretroviral agents. Here we review currently available murine models involving HIV-1 in transgenic mice and in chimeric mice reconstituted with human cells, as well as murine systems using retroviruses of the subfamily Oncovirinae rather than Lentivirinae. We report our results on various antiretroviral treatment strategies, including chemoprophylaxis after acute retroviral exposure, therapy of chronic viremia, quantitative analysis of combination therapy, and therapy during pregnancy and in the neonatal period aimed at preventing viremia in the offspring. Due to our highly effective postexposure treatment protocols with 3'-azido-3'-deoxythymidine (zidovudine) combined with recombinant human interferon-alpha A/D, retrovirus-inoculated mice developed immunity to the virus to which they were exposed, which will allow us to determine the nature of protective antiretroviral immunity in inbred mice.

Retinoic acid and epidermal growth factor binding in retinoid-mediated invasion suppressed human lung carcinoma cells.

The effect of retinoic acid (RA)-induced suppression of in vitro invasive ability of A549 human lung carcinoma cells on cellular binding of RA and epidermal growth factor (EGF) was investigated. RA inhibition of cell invasive potential was accompanied by a significant increase in specific high affinity cellular retinoic acid binding protein (CRABP) level. An approximately 2.7-fold increase in cytosolic CRABP was found in RA-treated cells (450 fm/mg protein compared to 167 fm/mg control cell protein). In contrast, 125I-EGF ligand binding was similar for control and treated cells.

Reversibility of retinoid effect on sialyltransferase activity, sialic acid content and invasive ability of human lung carcinoma cells.

The effect of retinoid induced suppression of in vitro invasive ability of A549 human lung carcinoma cells on sialyltransferase activity and sialic acid content was investigated. Inhibition by retinol acetate of cell invasive potential was accompanied by a significant decrease in the enzyme activity of intact cells as well as total and cell surface neuraminidase-releasable sialic acid contents. Moreover, reversibility of the invasion-suppressed A549 cell phenotype resulted in a return of invasion potential, sialyltransferase activity and surface sialic acid content to invasive cell levels. These findings suggest that membrane-bound sialic acid plays a role in invasiveness of A549 cells.

Human cell lines stably expressing HIV env and tat gene products.

A DNA fragment containing the tat, rev and env genes of the human immunodeficiency virus type 1 was inserted into the retroviral vector pZIPneoAU3. The resulting plasmid penvAU3 was transfected into HeLa and psi CRIP cells. Resulting recombinant retroviruses were used to infect HeLa and Jurkat cells. Immunoprecipitation analysis of stable transformants showed the expression of HIV env glycoproteins gp160, gp120 and gp41. Transactivation assays with a plasmid containing the gene for chloramphenicol acetyltransferase linked to HIV promoter-enhancer sequences demonstrated the expression of functional tat. These cells constitute virus-free tools for functional and structural studies of native env and tat.

Inhibition by retinoids of in vitro invasive ability of human lung carcinoma cells.

The effect of retinoid treatment of A549 human lung carcinoma cells on in vitro cell invasion using the human amnion basement membrane (BM) was investigated. A 2-day retinoid pretreatment of the cells resulted in a significant reduction in their invasive ability. The most effective retinoid, retinol acetate, inhibited cell migration through the BM and degradation of [3H] proline labeled BM components by 50% at noncytotoxic concentrations of 0.09, and 3 micrograms/ml, respectively. Inhibition by retinol acetate of A549 cell invasive potential was accompanied by a significant decrease in type IV collagenase activity and no change in transglutaminase activity.

Inhibition of invasion activity in vitro by a novel class of antitumor agents: silatrane derivatives.

The effect of several silatranes on in vitro invasion of the human amnion basement membrane (BM) by A549 human lung carcinoma cells was examined. Cells treated for two days with the derivatives were examined for invasive activity in the absence of the compounds. From silatrane dose-invasion response curves, an 80% inhibition of invasiveness compared to untreated cells was obtained with 40 micrograms/mg of 1-vinyl silatrane, 50 micrograms/ml of 1-(p-aminophenyl) silatrane, 80 micrograms/mg of 1-(3-phenylthiocarbamidopropyl) silatrane, 66 micrograms/ml of parent silatrane or 171 micrograms/ml of 1-bromosilatrane. Treatment with these doses had no effect on viability, growth or BM attachment of A549 cells.

Effects of retinoids on invasion of organ cultures of chick chorioallantoic membrane by adenovirus transformed cells.

Invasion of chick chorioallantoic membrane (CAM) organ cultures by rat 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 (3Y1/12-10 cells) was inhibited by several retinoids tested. The anti-invasive activity of the retinoids was dependent on retinoid concentration and continuous (4 d) exposure of the CAM. The 50% retinoid dose (dose effective in achieving a response in half of the organ cultures) that inhibited invasion was 0.85 micrograms/ml of retinol palmitate, 0.39 micrograms/ml of retinoic acid, or 0.16 micrograms/ml of retinol acetate. This dose was of the same order of magnitude as that which induced CAM differentiation, and was three- to fourfold less than the dose that caused cytotoxic damage of CAM. In addition, the retinoids inhibited 3Y1/12-10 cell growth by approximately 40% at levels over 10-fold higher than those needed for anti-invasion activity. The findings suggest that the anti-invasive activity of retinoids was at least partly due to direct induction of cell differentiation of the CAM host tissue.