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Maternal-Fetal Transmission of Human Immunodeficiency Virus: A Review of Possible Routes and Cellular Mechanisms of Infection Gordon C. Douglas and Barry F. King

From the Department ofCell Biology and Human Anatomy. School ofMedicine, University of California. Davis, California

The great majority of children with AIDS acquire the disease from their mothers during the perinatal period or during the pregnancy [1-6], Thus, the prevalence of human immunodeficiency virus (HIV) infections among children is increasing in a manner that closely follows the spread of the disease among women. It is predicted that 4 million women and 1 million children will be infected worldwide by 1992 [7, 8]. HIV may be transmitted to an infant via the mother's milk, by contact with maternal blood and/or genital secretions during labor, and via the placenta [9-11]. The exact proportion of fetal/infant HIV infections acquired by these different routes is unclear, but in utero transmission via the placenta is thought to playa major role [12, 13]. Prevention of transmission during pregnancy presents a difficult challenge because very little is known of the route or cellular mechanisms by which HIV or indeed any other virus travels from the mother to the embryo or fetus during this period. If we accept that transmission of HIV to the fetus can occur, it then becomes important to clarify when during gestation transmission might take place and by what mechanism. In

Received 27 January 1992; revised 30 April 1992. Financial support: University of California Davis Medical Center Research Fund, Pediatric AIDS Foundation, and National Institutes of Health (grants A132307 and HD 11658). Reprints or correspondence: Dr. G. C. Douglas. Department ofCell Biology and Human Anatomy, School of Medicine, University of California, Davis. California 95616-8643. Clinical Infectious Diseases 1992;15:678-91 © 1992 by The University of Chicago. AIl rights reserved. 1058-4838/92/1504-0017$02.00

theory, infection could occur at any number of times and places during gestation. It could begin with gametic infection or it could occur during development and transport of the embryo in the oviduct and uterus, during the peri-implantation period, or via the placenta during the remainder of gestation, Our purpose in this review is to consider the various routes and mechanisms by which HIV might be transmitted to the embryo and fetus. Since relatively little is known about the routes in early gestation, we will emphasize what is known about the role of the placenta in the maternal-fetal transmission of HIV. The more general reviews by Kalter [14] and Benirschke and Kaufmann [15] should be consulted for information relating to other viral infections of the placenta. Naz and Ellaurie [4] have discussed the implications of HIV infection of sperm. The review by Schwartz and Nahmias [13] examines analogies between HIV infection of the placenta and other perinatal viral infections. An earlier review by Valente and Main [16] also discusses potential routes by which HIV may cross the placenta. Oxtoby [9] and Andiman and Modlin (17] have recently reviewed infection during the perinatal period, so this topic is only briefly covered in the present review.

Mechanisms of HIV Entry into Cells Susceptible cells may become infected with HIV either by interaction with free virus particles and/or by interaction with other HIV-infected cells. While there is considerable information relating to the former mechanism, cell-mediated

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The prevalence of human immunodeficiency virus (HIV) infections among children is increasing in a manner that closely follows the spread of the disease among women. Despite the fact that in utero transmission via the placenta is thought to playa major role in the spread of HIV to the pediatric population, little is known about the timing, route{s), and cellular mechanisms by which maternal-fetal transmission occurs. This review attempts to use a developmental and cellular approach to assess the available clinical and laboratory data pertaining to maternal-fetal HIV transmission. While much of this review focuses on the role of the placenta, particularly the placental trophoblast, on the transmission of HIV, potential routes of infection during early development are also discussed. Clinical studies indicate that the placental trophoblast can be infected with HIV but have shed no light on how the virus gains entry to this tissue. While some laboratory studies confirm that trophoblast cells and placental macrophages can be infected with HIV in vitro, many studies are difficult to interpret because of inadequate characterization of the placental cells used. The role of CD4 in the infection of trophoblast remains controversial and clearly warrants a systematic examination. It is also apparent that viral tropism has not received enough attention and more studies using different strains of HIV are required. Thus, several basic questions remain to be answered before strategies to prevent maternal-fetal transmission of HIV can be developed.

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infection has received comparatively little attention, despite the fact that it may constitute a major route of transmission of infection in vivo [I8].

Entry of Free Virus Particles

Cell-Mediated Transmission of HIV In this scheme, interaction of an infected cell with an uninfected target cell results in transmission of virus to the uninfected cell. This may involve cell-cell fusion or some other process that somehow generates an HIV-permissive state in the uninfected cell. Fusion of uninfected cells with HIV-infected cells has been demonstrated [35-39]. In some cases the phenomenon requires expression of the viral CD4-binding protein, gp 120, on the surface of the infected cell and expression of CD4 antigen on the target cell [35-37]. However, other cell surface components may playa role in permitting cell-cell fusion [40]. These include the leukocyte adhesion receptor LFA-I [41] and the putative HIV fusion protein, gp41 [42, 43]. Most studies on cell-mediated HIV infection have been carried out using lymphocytes or lymphocytic cell lines, and very little is known about cell-mediated infection of epithelia, including trophoblast. Recently, Bourinbaiar and Phillips [44] suggested that monocyte-mediated infection of in-

testinal epithelial cells may proceed by more than one route (cell-cell fusion, endocytosis of virus released from the cells, and virus-cell fusion). Direct interaction of lymphocytes with epithelia may result in cell-cell binding. Information derived from other (non-HIV-related) studies shows that lymphoid cells can bind to certain epithelia, and several cell adhesion systems have been characterized [45-50]. The best characterized are the LFA-l jintercellular adhesion molecule-l and CD2j LFA-3 pathways, which involve members of the immunoglobulin gene superfamily and the integrin family. In these systems, a surface molecule on the homing cell binds to a surface ligand expressed on the target cell; for example, T lymphocytes express CD2, a cell surface glycoprotein that binds to the LFA-3 molecule present on the surface of a variety of cells. In addition to mediating cell-cell binding, interaction of adhesion molecules may result in activation of the cells involved, with important functional and developmental consequences. The expression ofadhesion molecules on some epithelial cells can be induced by mediators such as interferons and interleukins [45, 51, 52]. Some intriguing findings relating to the product ofthe HIV tat gene may have important implications for cell-mediated infection. This protein plays a role in transactivation of HIV gene expression but also appears to be released from infected cells, whereupon it has cytokine-like effects on certain target cells [53]. It is interesting that another report indicates that the tat gene product also demonstrates integrin binding properties [54] and may therefore playa role in cell adhesion reactions.

Infection During Early Development A number of different routes of infection from parent to embryo are possible during early development. These include infection of the gametes or transmission via the fluids immediately surrounding the developing embryo in the oviduct and uterus. Virus could be present in the embryonic environment in the female tract as a result of infection of the oviduct and uterus or from seminal fluid. Infection of Gametes HIV is thought not to infect germ cells; thus, gametes would not be directly infected by the virus [55] and HIV would not be transmitted vertically as occurs with many endogenous retroviruses [56]. There is little doubt that infected semen plays a role in the sexual transmission of HIV [4, 57-60]. Human semen contains small numbers of white blood cells (WBCs), including granulocytes, macrophages, and CD4-positive and CD8-positive lymphocytes. The number ofWBCs present is greatly increased in HIV-seropositive men [60]. HIV has been isolated from mononuclear cells obtained from semen of HIV-seropositive men [61, 62], and virus can be found in cell-free preparations of human semen.

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There is considerable evidence indicating a role for CD4 surface antigen in the infection of certain cells [19, 20], although the actual mechanism of CD4-mediated entry is unresolved. In lymphoblastoid CEM cells, HIV binds to cellsurface CD4 and appears to be internalized by receptor-mediated endocytosis [21]. Parallel ultrastructural studies suggested that fusion of internalized virus with the endosomal membrane occurs, followed by uncoating and replication of new viral particles within the cytoplasm. In the monoblastoid cell line U937, CD4-mediated internalization leading to infection also occurs. However, a CD4-independent, noninfectious mode of HIV entry also seems to occur [21]. Apparent CD4-independent entry of HIV into other cells occurs as well [22-25]. HIV is internalized by lymphoblastoid 1M cells by means ofa 10w-pH-dependent, endocytic event [26]. Some enveloped viruses, such as Sendai virus, enter cells by direct, pH-independent fusion with the plasma membrane rather than with the membrane of acidic endosomes [27]. Some investigators have suggested that this is a mode ofentry for HIV [28, 29]. The phenomenon ofantibody-dependent enhancement of virus infection is another means by which HIV can enter cells [30]. In this scheme, virus is internalized as an immune complex via interaction with cell surface IgG (Fe) receptors or with complement receptors. Antibody-dependent enhancement of HIV binding and infection of certain cells has been demonstrated in vitro [24, 31-34].

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Infection at Fertilization and Post-Fertilization Stages We have mentioned the possibility that the gametes may be infected, but other potential routes of infection of the embryo warrant consideration. Even if the fertilizing sperm itself is not infected, free virus or HIV-infected leukocytes present in the ejaculate may reach the site of fertilization in the upper oviduct. Since there are a variety of barriers to the movement of sperm to the site of fertilization, it is questionable whether virus or leukocytes would be transferred sufficiently far or quickly enough to be factors at the time of fertilization. However, if virus was bound or passively adsorbed to motile sperm, it might reach the site. When the ejaculate is virus-free and the ovulated oocyte is uninfected, the embryo may still be susceptible to infection as a result of the presence of virus or infected leukocytes in the female genital tract. HIV has been cultured from samples of vaginal and cervical fluid [67-71] and isolated from cells (macrophages, endothelial cells, and lymphocytes) of the cervix [3, 70]. There is also evidence of a leukocytic infiltration of the cervix and uterus following insemination [72]. If leukocytes are infected, the likelihood of intrauterine infection could be increased. In the context of transmission of HIV to the embryo, these findings are relevant in several ways. First, uninfected motile sperm may bind virus shed into the vaginal and cervical secretions and then go on to infect the zygote. Infection also could occur at parturition during the infant's passage down the birth canal. Less is known about infections of the fluids of the oviduct, where fertilization and early development occur. However, it is known that the oviduct contains macrophages [73], and it seems likely that virus could be shed into oviductal secretions.

Infection at Preimplantation Stages Following ovulation, the ovum is surrounded by cumulus cells and their associated intercellular matrix. These cells are soon lost, however, and are unlikely to present anything but a temporary barrier to infection of the ovum. After fertilization, cell divisions lead to the formation of a morula. These stages take place in the oviduct, and during this time the developing embryo is surrounded by a thick extracellular layer, the zona pellucida (ZP) (figure I). Thus, for a virus (or any other pathogen) to infect the embryo during these stages of development, it would have to penetrate the ZP. While there does not seem to be information available with respect to the capacity of HIV to penetrate the ZP, data are available on the capacity of other viruses to penetrate it [65, 66, 74]. In general, the ZP appears to provide a barrier to the passage of infectious agents. However, the effectiveness of the zona as a barrier varies according to both the virus and the species challenged. Several viruses are able to penetrate the zona oflaboratory animals [75, 76]. On the other hand, the ZP of bovine, ovine, and porcine embryos are resistant to penetration by many viruses [66]. Many of the studies cited above examined infection of the embryo in vitro before and after removal of the ZP, and in most cases virus was capable of infecting zona-free embryos. Although direct evidence ofthis mechanism is lacking, it is possible that the ZP temporarily protects the embryo from HIV from the time the oocyte is surrounded by the zona (in the ovarian follicle) until the zona is shed (hatching). In the human, the latter event occurs after arrival of the morula in the uterus, about 3.5 days after ovulation (figure 1). A zonafree blastocyst has been recovered from the human uterus about 6 days postovulation [77]. At this point, any protection afforded by the zona would end, and the blastocyst would be susceptible to infection either by free virus or cellassociated virus in the uterine environment. As mentioned in previous sections, virus in the uterus may be derived from shedding from the uterine mucosa or may be derived from residual seminal fluid. This stage of embryo vulnerability would last through the initial stages of implantation.

Infection at Implantation Implantation of the human embryo probably occurs --79 days postovulation [78, 79]. This process is initiated by adherence of the blastocyst to the uterine epithelium and is rapidly followed by trophoblastic penetration of the epithelium and underlying stroma. With invasion of the uterine mucosa, the developing conceptus is exposed to still other potential avenues of infection. As noted above, the mucosa of the uterus (at least in the cervix) may contain infected cells (see "Infection at Fertilization and Post-Fertilization Stages" section). The invading trophoblast (mostly syncytiotrophoblast) would encounter these cells within a few days of

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The susceptibility of sperm to HIV remains controversial. Anderson et al. [60] found that HIV did not bind to motile sperm and failed to detect CD4 on the cells. Ashida and Scofield [63], on the other hand, were able to detect CD4 on a small subpopulation of sperm. Similarly, a molecule that recognized anti-CD4 antibodies was detected on sperm [57, 64]. It has been suggested that these conflicting results are due to differences in sperm isolation techniques and fixation protocols [58, 59]. As pointed out by Naz and Ellaurie [4], further studies are required before the role of sperm in HIV infection can be established. To our knowledge, there is no direct evidence demonstrating the capacity of HIV to infect developing oocytes. However, there is evidence that other viruses can infect developing mammalian oocytes [65]. While this potential pathway of infection needs to be considered, it is generally thought that gametic infection is not a significant factor in transmission of infection to the embryo [65, 66].

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morula stage, still surrounded by the zona. After shedding the zona in the uterine lumen, the blastocyst (upper left) briefly resides free in the lumen before implantation (TROPH = trophoblastic layer; ICM = inner cell mass).

implantation [80]. Indeed, a transitory leukocytic infiltration may accompany implantation. Trophoblast invades maternal endometrial capillaries within a few days ofimplantation in both the human and nonhuman primates [81, 82]. Contact of trophoblast with maternal blood poses additional hazards of HIV infection for the conceptus since, throughout the remainder of gestation, placental trophoblast will be bathed in maternal blood. The significance of this pathway is considered in the next section.

Infection During Later Development Anatomical Considerations: Transmission Across Placental Villi and the Chorioamnion

Placental villi. In humans, most maternal-fetal exchanges are mediated by the placenta. The placental syncytiotrophoblast plays a major role in these interactions since it borders the intervillous space and is therefore in direct con-

tact with maternal blood, probably as early as day 10 of pregnancy (Carnegie stage 5b; [81 D. Syncytiotrophoblast forms a continuous multinucleated epithelium that, for the most part, presents an effective barrier against the transport ofmacromolecules and maternal blood cells. An exception is IgG, which is selectively transported to the fetus [83-85]. Microorganisms such as rubella virus, cytomegalovirus, poliovirus, and gibbon ape leukemia virus (a retrovirus) are also reported to be transported across the placenta, but the mechanism is not known [ 13-15]. Syncytiotrophoblast must be traversed by HIV in order to reach underlying cytotrophoblastic cells, placental macrophages (Hofbauer cells), fibroblasts, and endothelial cells lining the fetal capillaries (figure 2). Exposure to these nontrophoblastic placental cells, and to macrophages in particular, may well be ofimportance in limiting or preventing further dissemination of virus. Paradoxically, some studies in other systems suggest that monocytes/ macrophages and other accessory cells may actually function as reservoirs, slowly releasing potentially infective virus [86].

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Figure 1. Diagrams of early stages of embryonic development. Fertilization and early cleavage occur in the upper oviduct. The ovum shortly after ovulation (upper right) is surrounded by cumulus cells (CC) and the zona pellucida (ZP). The upper center diagram is of a

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A

FS

AMNIOTIC CAVITY

c Figure 2. A, diagram of the general arrangement of the placenta and fetal membranes at term. Areas indicated by the boxes are enlarged in Band C. B, an enlargement of the base of the placenta as seen in the first trimester. The most likely route ofviral transmission is across free villi. Virus would have to cross syncytiotrophoblast (ST), a layer of cytotrophoblast (CT), and the trophoblastic basement membrane to reach fetal cells in the core of the placental villus. Fetal cells in the villus core include macrophages (Hofbauer

Studies of HIV infection in these non-trophoblastic cells are clearly required but would not address the question of how the virus reaches the villous mesenchyme in the first place. It should also be noted that while preparations of placental fibroblasts are relatively easy to obtain [87], current isolation methods yield only an enriched population of placental macrophages [88-90]. This situation limits the kind of experiments that can be performed (see below). The possibility that HIV or HIV-infected blood cells could reach the villous core either by passive diffusion or migration through pathological lesions in the syncytiotrophoblastic layer must also be considered. Results of physiological and morphological studies of a variety of hemochorial placentas have suggested the presence of channels through the trophoblastic layer [91, 92]. The existence of such channels in the human placenta is unclear, and even in animal studies it is difficult to demonstrate continuity of transtrophoblastic channels. Nonetheless, these putative channels need to be considered as a potential route of virus transfer across the syncytiotrophoblast. Discontinuities in the placental barrier could also arise pathologically, through some traumatic event. Leakage of blood across the placental barrier, when it occurs, is usually in a fetal-to-maternal direction because of pressure differentials between the fetal circulation and the intervillous space. Nelson et al. [93] noted the presence, in normal human placentas, of small fibrin-associated lesions where the syncytiotrophoblast layer was incomplete. Placental permeability in such regions is not known, but areas such as this represent potential routes of maternal-fetal transmission that essentially bypass the syncytiotrophoblast. In addition, the available information indicates that the transplacental passage of maternal cells is an exceedingly rare event [I6, 94]. If passive movement of HIV or HIV-infected cells across the placenta does contribute to fetal infection, then it may be difficult to prevent. Anchoring villi and chorioamnion. While it is generally assumed that maternal-fetal transmission of HIV would be across the syncytiotrophoblast of the free (terminal) placental villi described above, two other potential routes of infection warrant consideration. One possible route involves the anchoring villi. As the placenta develops, specialized struc-

cells) (FM), fibroblasts (FF), and endothelium of capillaries (FC). A possible alternative pathway might be via movement of virus or infected maternal cells across the region of cytotrophoblastic cell columns associated with the anchoring villi. The underlying uterine stroma is a complex mixture of extracellular matrix as well as fetal and maternal cells. LGL = large granular lymphocyte; SGC = syncytiotrophoblastic giant cell; UG = uterine gland; UPA = uteroplacental artery. C, an enlargement of the chorioamnion at term shown in A. For maternal-fetal transfer to occur by this pathway, virus or infected cells in the maternal decidua would have to cross a layer of fetal cytotrophoblast (CT), a layer of fetal stroma (FS) containing fibroblasts and macrophages, and the amniotic epithelium (AE) to reach the amniotic fluid.

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AE

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Evidence of HIV Infection of the Placenta

Evidencefrom clinical studies. The first evidence suggesting the occurrence of transplacental transmission of HI V was epidemiological [5, 100, 101]. Some studies reported the isolation of HIV from placental tissue [l00, 102]. U nfortunately, the presence of maternal blood in such samples usually made it impossible to determine whether the virus came from placental cells per se or from contaminating blood cells [103, 104]. Other studies in which HIV was detected in amniotic fluid or in tissues from infants (humans and nonhuman primates) delivered by cesarian section provided better evidence of transplacental transmission [97, 105-109]. Direct evidence of the infection of placental tissue with HIV has recently been described. Using immunohistochemical and in situ hybridization techniques, researchers have detected HIV antigens and genetic material in first-trimester trophoblastic samples obtained from HIV-infected women [110]. Chandwani et al. [99] examined term placental tissue from HIV-seropositive women and found HIV antigens and nucleic acid in what appeared to be trophoblast. Mano and Chermann [I I 1] used the polymerase chain reaction (PCR) to reveal HIV proviral DNA in cells isolated from second-trimester placentas obtained from HIV-infected women. However, the nature of the technique and the fact that mixed-cell preparations were used do not permit the identity of the infected cells to be determined. Peuchmaur et

al. [112] examined placentas from HIV-infected women and failed to detect either HIV antigens or HIV RNA sequences using immunohistochemical techniques. It is not known whether the susceptibility of the placenta to HIV infection varies with gestational stage. The results of Lewis et al. [110] and earlier results in which HIV was isolated from a IS-week fetus [108] indicate that infection and/ or transmission can occur early in gestation. Courgnaud et al. [113] were able to detect HIV DNA sequences in fetal tissues obtained from HIV-infected but asymptomatic women from as early as 16 weeks of gestation, again suggesting that transplacental transmission of HIV could be an early event. These results contrast with a study reported by Ehrnst et al. [6]. They detected no HIV in newborn infants born to HIV-infected (viremic but asymptomatic) mothers; however, 26% of these infants were subsequently found to be HIV-infected after the neonatal period. The authors concluded that there is a low rate of transmission of HIV during the first and second trimester and that, in most cases, vertical transmission occurs close to or at the time of delivery. These conclusions have been criticized on the basis of the sampling methods and statistical analyses used [114]. For the most part, the above clinical studies indicate that placental cells, including trophoblast, can be infected with HIV, and they substantiate a role for the placenta in maternal/fetal transmission of virus. However, they do not reveal how HIV enters syncytiotrophoblast or otherwise gains access to placental tissue. Evidence from placental explant systems. Theoretically, in vitro systems allow greater manipulation of experimental variables and permit basic questions regarding mechanisms to be addressed. Four experimental systems are available. These are placental explants, isolated placental cells, continuous cultures of choriocarcinoma cells, and placental perfusion systems [115]. Each has inherent advantages and disadvantages and, for the unwary, potential pitfalls. To our knowledge, perfusion systems have not been employed to study HIV infection, so the following discussion will focus on the first three aforementioned techniques. Until very recently, all of the in vitro studies of HIV infection of trophoblast had been performed using placental villous explants. For a detailed description of trophoblast explant/organ culture techniques, see the reviews by Stromberg [116] and Loke [117]. It is as well to remember that the villous tissue pieces used in these experiments comprise syncytiotrophoblast, cytotrophoblast, macrophages, fibroblasts, and endothelial cells. The advantage of this system, as seen by some researchers, is that the cells remain in a natural topography. However, syncytiotrophoblast is particularly fragile if optimal culture conditions are not met [117]. Furthermore, damaged cells will inevitably be found at cut surfaces. If such systems are used, the viability should be constantly monitored. Maury et al. [118] reported that when term placental explants were exposed to HIV, viral antigens

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tures known as anchoring villi and cell columns are formed at the junction of fetal and maternal tissue [15, 95] (figure 2). Their unusual organization affords a potential pathway for viral entry into fetal tissues that does not involve passage of virus across syncytiotrophoblast. HIV theoretically could pass from uterine cells and stroma, go between cytotrophoblastic cells of the cell columns, and enter fetal stroma. The cytotrophoblastic cells are joined by desmosomes but lack tight junctions. It is doubtful that any maternal cells gain entry to the fetal stroma by this pathway, but it cannot be ruled out. Macrophages in the fetal connective tissue are thought to be of fetal origin. Another potential route of HIV infection of the fetus is across the chorioamnion (figure 2C). Here, in late gestation, cellular trophoblast abuts maternal decidua. The decidua contains maternal blood vessels, macrophages, and lymphocytes and thus may serve to expose the adjacent trophoblast to HIV. From here, virus or cells may cross the fetal connective tissue and amnion and enter the amniotic fluid. At present it is unclear whether HIV may reach the fetus by this route. However, the chorioamnion is implicated as a pathway for bacterial infection of the fetus [96]. HIV has been isolated from amniotic fluid and cells, but the pathway by which virus enters the fluid is unknown [97]. The correlation between chorioamnionitis and HIV transmission is unclear and warrants further study [98, 99].

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tions are not suitable for studies that use biochemical assays of virus infection-such as reverse transcriptase release, p24 antigen capture, and PCR-since they do not allow discrimination of different cell types. Subsequent improvements in trophoblast isolation procedures have been described that, on the basis of intermediate filament immunocytochemistry, appear to produce 100% cytotrophoblastic cells [122, 123]. Another important point is that most experiments dealing with purification oftrophoblast actually produce villous cytotrophoblastic cells. Attempts to isolate viable syncytiotrophoblast are generally unsuccessful since this tissue is sensitive to physical shock and to trypsin [117]. This does not pose a problem to most investigators since it is generally assumed that cytotrophoblast will differentiate to form multinucleated syncytiotrophoblast when placed in culture. This assumption should not be made without individual confirmation with use of a reliable assay. We have developed a method to assess syncytiotrophoblastic formation rapidly and reliably by staining cultures with antibodies to the desmosomal protein, desmoplakin [124]. When epithelial cells are stained with antibodies to desmosomal proteins, the intercellular borders are revealed as a pavement-like pattern. As cytotrophoblast differentiates to form syncytiotrophoblast, this pavement-like pattern gradually disappears because of reorganization of desmosomes [124]. Staining trophoblastic cultures simultaneously with anti-desmoplakin and antinuclear antibodies provides a means of distinguishing multinucleated cells from mere aggregates ofmononucleated cytotrophoblastic cells-a task that is difficult with the phase-contrast microscope. Mano and Chermann [Ill] have described results of experiments in which they attempted to infect isolated placental trophoblastic cells and placental macrophages with cellfree HIV (LAVBru)' After addition of HIV, they monitored their cultures by immunofluorescence staining for p25 antigen, reverse transcriptase assay, and PCR. The trophoblastic cultures showed no evidence of HIV infection when an immunofluorescence assay was performed and reverse transcriptase was measured. The PCR analyses, however, indicated the presence of proviral DNA sequences. Unfortunately, and as pointed out by the authors, the sensitivity of the technique and the likelihood that the trophoblastic cultures were not pure preclude any meaningful interpretation of this finding. At best it can be said that some unidentified placental cells (trophoblast, macrophage, fibroblast, endothelial) were infected. The enriched macrophage cultures (consisting of 60%-80% phagocytic cells) were found to be susceptible to HIV infection but less so than fetal monocytederived macrophages, as determined by p25 antigen localization, reverse transcriptase assay, and PCR analysis. Zachar et al. [125] studied the interaction of cell-free HIV with human choriocarcinoma cells. They found that the cells supported transient HIV replication during initial stages of infection. Choriocarcinoma cell lines actively replicate,

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could subsequently be detected by immunofluorescence microscopy in cells within villous tissue. The staining pattern of their micrograph, however, does not suggest that syncytiotrophoblast was positive for antigens. Antigens did appear to be localized within mesenchymal cells in the villous core. These studies give no indication as to the route of infection since virus could gain access to the villous mesenchyme via cut surfaces, exposed fetal capillaries, and/or damaged cells. The same researchers stated that first-trimester-tissue explants could also be infected with HIV, but no specific cell type was mentioned and no data were presented. Amirhessami-Aghili and Spector [119] have recently reported that first-trimester human placental explants could be infected with HIV in vitro and claimed that HIV antigen was detected in trophoblast and placental macrophages. However, interpretation of their micrographs is difficult because there appears to be little difference in staining intensity between the section from the experimentally infected explant and that from the uninfected control. Evidencefrom isolated placental cells. The use of purified populations of placental cells offers advantages for studying cellular mechanisms of HIV entry and infection. However, certain points need to be raised concerning the use of such systems lest incorrect assumptions and invalid conclusions be made. Many methods have been described for the purification of cytotrophoblastic cells, and earlier methods have been reviewed by Stromberg [116] and Loke [117]. These earlier protocols suffered from deficiencies in the methods available to assess cell purity. In 1986, Kliman et al. [120] modified a method originally described by Hall et al. [121] to produce highly purified trophoblastic cells. Purity was assessed by staining the cells with newly available antibodies to intermediate filament proteins. Trophoblast, in common with other epithelial cells, stains positively for cytokeratin and negatively for vimentin, whereas macrophages, fibroblasts, lymphocytes, and endothelial cells stain positively for vimentin and are cytokeratin-negative. Kliman et al. [120] reported that their preparation consisted of about 95% cytotrophoblast. This degree of purity is acceptable to many researchers. If the cells are to be analyzed for HIV infection by a morphological technique such as immunofluorescence microscopy or in situ hybridization, then the presence of a few contaminating macrophages or fibroblasts does not present a problem since they can be readily identified with the appropriate staining method. Even here, however, it is important to note that these contaminating cells may secrete molecules capable of modulating the activity of trophoblast. Furthermore, addition offree HIV to such mixed placental-cell preparations may result in infection of placental macrophages, which could then go on to infect the trophoblastic cells by a cell-mediated process (see the section on "Interaction ofMaternal Lymphocytes and Trophoblast"). Thus, experiments using mixed-cell preparations must be interpreted with caution. Enriched trophoblastic prepara-

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(LAVBru) for up to 24 hours, after which the cultures were monitored for up to 30 days by p24 antigen capture assay, reverse transcriptase assay, and electron microscopy for evidence of virus uptake and replication. No evidence of replication was found and no viral particles were observed bound to the trophoblast plasma membrane. In the second series of experiments, syncytiotrophoblastic cells were cocultured for 24 hours with HIV-infected MOLT-4 cells, stained using an anti-HI V antibody, and examined by immunofluorescence microscopy. The lymphocyte-derived MOLT cells were strongly positive, as expected, but most of the trophoblastic colonies also showed a punctate staining pattern. The MOLT cells appeared to be adherent to the trophoblast. Examination of similar cultures with the electron microscope also revealed MOLT cells adherent to trophoblast but no evidence of cell-cell fusion. In contrast to the studies with cell-free virus, virions were observed in coated pits at the syncytiotrophoblastic cell surface and in endosomes or multivesicular bodies in the cytoplasm. These observations are consistent with an endocytosis-mediated mechanism of virus entry. Virions were also observed budding from the trophoblast plasma membrane, activity indicating that these cells can support HIV replication. This observation upholds the idea that placental syncytiotrophoblast could be infected with HIV by interaction with virus-infected maternal lymphocytes. The above results indicate that trophoblast can be infected with HIV in vivo and in vitro. In many in vitro studies, however, it is not clear whether the cells were cytotrophoblastic or syncytiotrophoblastic at the time of virus entry, and cell purity was often not described. The transfection studies of Zachar et al. [125] indicate that the ability of trophoblast to support viral replication is dependent on the particular HIV isolate used. Target-cell tropism of HIV is an issue that has received very little attention with respect to infection oftrophoblast, so these potentially important findings warrant further investigation.

Are There Binding Sites for HIV or HIV/ Antibody Complexes on Placental Cells?

While the above studies indicate that trophoblast can be infected with HIV, what information is available concerning the cellular mechanism by which the virus enters these cells and, in particular, syncytiotrophoblast? As mentioned earlier, the CD4 molecule seems to playa major role in mediating HIV infection in many cells. In an examination of firsttrimester and term placental tissue sections, Goldstein et al. [ 131] found that placental macrophages were positive for CD4 but did not discuss trophoblast; examination of their micrographs suggests no evidence of positive CD4 staining of trophoblast. Maury et al. [118], on the other hand, reported that some trophoblastic cells (Hperhaps cytotrophoblast")

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whereas term cytotrophoblastic cells show little replication in vitro. Since HIV usually only replicates in actively dividing cells [126), this may explain why HIV replication can be more readily detected in choriocarcinoma cells than in isolated trophoblastic cells. Zachar et al. [125) did not state whether their choriocarcinoma cultures had undergone differentiation to form syncytiotrophoblast. If they were differentiating, then the transient nature of the viral replication could perhaps be explained by the transition from actively dividing cytotrophoblast-like cells to post-mitotic differentiated cells. This would be an important point to establish. Zachar et al. also sought to determine whether primary cultures of term trophoblastic cells can support HIV replication [125). In one of their studies, immunomagnetic beadpurified trophoblastic cells were transfected with three different HIV proviral clones and were found to support viral replication. On the basis of examination with the phase-contrast microscope, the authors stated that the cytotrophoblastic cells had formed syncytiotrophoblast. It should be noted that there are reservations concerning the reliability of this method for assessing the formation of multinucleated cells [124, 127, 128]. In another experiment within the same study, trophoblastic cells were incubated with HIV isolates (RF, NDK, and 3B) and monitored for HIV infection by means of immunofluorescence microscopy and PCR analysis. Only low levels of infection were found; by immunofluorescence,

Maternal-fetal transmission of human immunodeficiency virus: a review of possible routes and cellular mechanisms of infection.

The prevalence of human immunodeficiency virus (HIV) infections among children is increasing in a manner that closely follows the spread of the diseas...
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