A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a ''crazy pavement'' and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule Ecadherin. The isolated villous trophoblasts in culture expressed integrins ␣6 and ␤4 and reduced levels of ␤1 subunits, whereas the proliferating extravillous trophoblast cultures expressed ␣1, ␣3, and ␣5 and high levels of ␤1 integrin subunits, vitronectin receptor (␣V␤3/␤5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72-and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.

Interferons (IFN) are produced by the placenta during pregnancy, and they can be detected in the maternal and fetal blood. Although the antiviral 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-alpha on fetal acquisition of herpes simplex virus (HSV). Nine mothers with a known history of genital HSV infection were studied. In five cases IFN-alpha was detected in the placenta, maternal, and fetal blood, whereas in three cases IFN-alpha could not be detected. in the remaining case, IFN-alpha 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-alpha 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.

SUMMARY We examined if Fc receptor‐mediated antibody‐dependent enhancement (FcR‐ADE) or complement‐mediated antibody‐dependent enhancement (C′‐ADE) of virus infection can contribute to increasing replication of HIV‐1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR‐ADE and C′‐ADE may result in enhanced virus release from HIV‐1‐infected ST cells. We show that FcR‐ADF of HIV‐1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'‐ADE uses both CD4 and CR2‐like receptors. FcR‐ADE: seems to be more efficient in enhancing HIV‐I replication than C′‐ADE. While FcR‐ADE leads to increased internalization of HIV‐1. C′‐ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR‐ADE arc reactive with the gp120 viral envelope antigen, whereas antibodies involved in C′‐ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR‐ADH and C′‐ADE may contribute lo the spread of HIV‐1 from mother to the fetus.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.

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