Pleiotropic actions of forskolin result in phosphatidylserine exposure in primary trophoblasts.

Forskolin is an extract of the Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels. In the field of trophoblast biology, forskolin is one of the primary treatments used to induce trophoblastic cellular fusion. The syncytiotrophoblast (ST) is a continuous multinucleated cell in the human placenta that separates maternal from fetal circulations and can only expand by fusion with its stem cell, the cytotrophoblast (CT). Functional investigation of any aspect of ST physiology requires in vitro differentiation of CT and de novo ST formation, thus selecting the most appropriate differentiation agent for the hypothesis being investigated is necessary as well as addressing potential off-target effects. Previous studies, using forskolin to induce fusion in trophoblastic cell lines, identified phosphatidylserine (PS) externalization to be essential for trophoblast fusion and showed that widespread PS externalization is present even after fusion has been achieved. PS is a membrane phospholipid that is primarily localized to the inner-membrane leaflet. Externalization of PS is a hallmark of early apoptosis and is involved in cellular fusion of myocytes and macrophages. We were interested to examine whether PS externalization was also involved in primary trophoblast fusion. We show widespread PS externalization occurs after 72 hours when fusion was stimulated with forskolin, but not when stimulated with the cell permeant cAMP analog Br-cAMP. Using a forskolin analog, 1,9-dideoxyforskolin, which stimulates membrane transporters but not adenylate cyclase, we found that widespread PS externalization required both increased intracellular cAMP levels and stimulation of membrane transporters. Treatment of primary trophoblasts with Br-cAMP alone did not result in widespread PS externalization despite high levels of cellular fusion. Thus, we concluded that widespread PS externalization is independent of trophoblast fusion and, importantly, provide evidence that the common differentiation agent forskolin has previously unappreciated pleiotropic effects on trophoblastic cells.

Fibrocyte-like cells from intrauterine growth restriction placentas have a reduced ability to stimulate angiogenesis.

Intrauterine growth restriction (IUGR) is a common complication of pregnancy whereby the fetus fails to achieve its genetic growth potential. Malformation of the placental vasculature is observed in IUGR and may be due to the development of the placenta in a chronically hypoxic environment. Recently, we identified that the predominant stromal cells in the angiogenic zones of the placenta are fibrocyte-like cells. The conditioned medium from fibrocyte-like cells (FcCM) has been shown to stimulate angiogenesis in vitro. Thus, we hypothesized that FcCM from IUGR cells would have a reduced ability to stimulate angiogenesis and that chronic hypoxia would decrease the ability of both normal and IUGR fibrocyte-like cells to stimulate angiogenesis. IUGR FcCM had a reduced ability to stimulate endothelial tubule-like structure formation and an increased ability to stimulate endothelial migration compared with normal FcCM. However, normal and IUGR FcCM produced in chronic hypoxia did not alter endothelial proliferation, migration, or tubule-like structure formation. IUGR FcCM was found to have reduced levels of the pro-angiogenic cytokine IL-8 and increased levels of the anti-angiogenic factors activin-A and pigment epithelium-derived growth factor. Thus, alterations in the ability of IUGR fibrocyte-like cells to stimulate angiogenesis may contribute to the development of vascular malformation in IUGR, but in vitro these changes cannot be attributed to a chronically hypoxic environment.

Differential proteomic analysis of highly purified placental cytotrophoblasts in pre-eclampsia demonstrates a state of increased oxidative stress and reduced cytotrophoblast antioxidant defense.

Proteomics were performed using highly (99.99%) purified cytotrophoblasts from six normal and six pre-eclamptic placentas. Eleven proteins were found which decreased in pre-eclampsia (actin, glutathione S-transferase, peroxiredoxin 6, aldose reductase, heat shock protein 60 (Hsp60), two molecular forms of heat shock protein 70 (Hsp70) ß-tubulin, subunit proteasome, ezrin, protein disulfide isomerase, and phosphoglycerate mutase 1). Only one protein, α-2-HS-glycoprotein (fetuin), was found to increase its expression. Western blots of actin, Hsp70, ezrin, and glutatione S-transferase confirmed decrease in protein expression. Many of the proteins that decreased are consistent with a state of oxidative stress in the pre-eclamptic placenta and a decreased cytotrophoblast defense against and response to oxidative stress.

Invasion of the leukocytes into the fetal-maternal interface during pregnancy.

No other organ in the body undergoes such an invasion of selective cells (leukocytes) and release of homing molecules, CAMs, proinflammatory cytokines, and mediators or undergoes similar extensive remodeling of tissues over such a short period of time as the pregnant uterus. This is especially interesting, as an infectious process involving microorganisms does not exist in a healthy pregnancy and delivery. Furthermore, after delivery of the baby and placenta, the uterus involutes and returns to its normal monthly cycling, and most of the leukocytes are swept away or leave. In this review, we discuss leukocyte infiltration and recruitment and the potential roles of each subpopulation of leukocytes in relation to pregnancy and the problems of preterm birth, pre-eclampsia, and intrauterine growth restriction.

Isolation of non-activated monocytes from human umbilical cord blood.

PROBLEM: Methods for monocyte purification are common but few work with umbilical cord monocytes that do not activate the cell for subsequent culture analysis. METHODS OF STUDY: The collection procedure avoids use of needles and procedures that variably activate blood clotting and uses a purification procedure that involves diluted Ficoll, autologous serum to remove platelets and 42% and 51% Percoll step gradients for the final purification. The resulting monocytes were stimulated with bacterial lipopolysaccharide and formalin-treated bacteria Escherichia coli and group B streptococci (GBS) to secrete TNF-alpha and IL-1beta, measured by ELISA. RESULTS: The purification procedure results in non-active but stimulation-competent monocytes with high yields (2.3-9 x 10(7) cells) and purity (from 70% to 98%). CONCLUSION: We describe a procedure that is easy, uses common reagents and provides a uniformly high yield and purity of non-activated fetal monocytes for studies of innate defense responses.

Inflammatory processes in preterm and term parturition.

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Keratinocyte conditioned medium abrogates the modulatory effects of IGF-1 and TGF-beta1 on collagenase expression in dermal fibroblasts.

Overexpression of wound healing-promoting factors such as transforming growth factor-1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) during the healing process has been implicated in the development of dermal fibrosis in patients following thermal injury, surgical incision, and deep trauma. However, the mechanism through which the expression of these two fibrogenic factors is slowed down and/or abrogated in the late stages of the healing process is not known. Here, we hypothesize that keratinocyte-releasable factors counteract the fibrogenic role of both IGF-1 and TGF-beta1 in fibroblasts. To test this hypothesis, the levels of collagenase (MMP-1), as an index for extracellular matrix degradation, in dermal fibroblasts in response to either keratinocyte-conditioned medium (KCM) or our recently identified keratinocyte-releasable stratifin in the presence and absence of either IGF-1, TGF-beta1, or both were evaluated. The results of Northern analysis showed a significant increase in collagenase mRNA expression in cells treated with KCM in the presence of both IGF-1 and TGF-beta1. The effect was, at least in part, due to keratinocyte-derived stratifin that was present in KCM. This was ascertained as the levels of MMP-1 mRNA were markedly reduced when cells were treated with stratifin-immuno-depleted KCM. The results of Western blot analysis showed an increase in the level of MMP-1 protein in stratifin-treated fibroblasts and this was consistent with the level of MMP-1 mRNA expression detected by Northern analysis. However, in contrast to KCM, whose efficacy on MMP-1 expression was modestly reduced by either IGF-1 and TGF-beta1, or a combination of both, these factors abrogated the MMP-1 stimulatory effect of stratifin in fibroblasts. In summary, the results of this study revealed that both stratifin and KCM stimulate the expression of MMP-1-in fibroblasts and this effect can be abrogated by either IGF-1, TGF-beta1, or a combination of both.

Proinflammatory cytokines inhibit human placental 11beta-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways.

Excessive fetal exposure to glucocorticoids has been implicated in the etiology of adult metabolic and cardiovascular disease. Placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) may protect the fetus from excessive glucocorticoid exposure. Maternal stress may be accompanied by elevated levels of cortisol and increased proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha)]. We hypothesize that proinflammatory cytokines inhibit human placental 11beta-HSD activity. We incubated explant cultures of term human placental villi in the presence or absence of 10 ng/ml IL-1beta, IL-6, or TNF-alpha, with or without agonists or antagonists of intracellular Ca2+ and adenylyl cyclase. Activity for 11beta-HSD2 was estimated using a radioisotope assay, and mRNA was measured using quantitative RT-PCR. All cytokines significantly (P < or = 0.05) reduced 11beta-HSD2 activity (>75% suppression); maximal inhibition occurred within 2 h and was maintained for at least 24 h. The IL-1beta-induced inhibitory activity was attenuated using a Ca2+ channel blocker (nifedipine), an intracellular Ca2+ antagonist [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate], or the adenylyl cyclase stimulant forskolin. Conversely, 11beta-HSD2 activity was diminished in the presence of the Ca2+ ionophore A-23187 or the adenylyl cyclase inhibitor SQ-22536. mRNA levels for 11beta-HSD2 were not changed by any of the treatments. Proinflammatory cytokines inhibit human placental 11beta-HSD2 activity through a mechanism that involves increased intracellular Ca2+ and inhibition of adenylyl cyclase. This could result in excessive fetal exposure to maternal cortisol. This mechanism might mediate part of the increased risk of metabolic and cardiovascular disease in adult offspring.

Enhanced monocyte binding to human cytomegalovirus-infected syncytiotrophoblast results in increased apoptosis via the release of tumour necrosis factor alpha.

We have shown that monocytes bound to intercellular adhesion molecules (ICAM-1) on syncytialized placental trophoblasts (ST) induce trophoblast apoptosis, and that ST infection by human cytomegalovirus (HCMV) up-regulates ICAM-1. We hypothesize that the focal loss of trophoblast seen in HCMV-infected placenta is mediated by increased adherence of monocytes at sites of infection. We find that ST cultures (differentiated from primary cytotrophoblasts) increase monocyte binding when infected with HCMV. Monocyte adhesion was inhibited by antibodies to ICAM-1 and its ligand leukocyte function-associated molecule (LFA-1) on monocytes. When co-cultured with adhering monocytes, infected ST cultures had higher levels of apoptosis than infected cultures alone. Although trophoblast apoptosis clustered around adhering monocytes, it occurred only in non-infected cells. Blocking monocyte binding with ICAM-1 and LFA-1 antibodies reduced the rate of apoptosis to that of the infected culture. Co-cultures incubated with TNFalpha antibody and EGF inhibited both monocyte- and HCMV-induced apoptosis but did not block binding. We conclude that HCMV stimulates ST culture expression of ICAM-1, which binds to LFA-1 on monocytes that release TNFalpha, thereby inducing apoptosis of neighbouring uninfected trophoblasts. The above data indicates that trophoblast loss associated with HCMV infection can be caused by increased monocyte adhesion to ST.

Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation.

Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadotropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3-7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.

Sphingosine-1-phosphate inhibition of placental trophoblast differentiation through a G(i)-coupled receptor response.

The failure of placental trophoblasts to differentiate properly is thought to play an important role in the cause of pregnancy disorders such as preeclampsia. We looked at the effects of the bioactive lipid sphingosine-1-phosphate (S1P) on the differentiation of primary human cytotrophoblasts (CTs) into syncytiotrophoblasts (STs) in culture. We found that S1P inhibited CT differentiation measured by human chorionic gonadotropin (hCG) secretion and the expression of placental alkaline phosphatase but had no effect on their fusion into multinucleated syncytialized cells. G-protein-linked S1P receptors 1, 2, and 3 were found in CTs by reverse transcriptase-polymerase chain reaction, and receptor 1 was found by Western blot analysis. Disruption of G(i) signaling with pertussis toxin reversed the inhibitory effects of S1P. S1P reduced intracellular cAMP, and the addition of 8-bromo-cAMP reversed S1P inhibition of hCG secretion. Therefore, we suggest that S1P inhibits the differentiation of CTs into STs through G(i)-coupled S1P receptor interaction(s), leading to the inhibition of adenylate cyclase and reduced production of intracellular cAMP. This is the first reported effect of S1P on placental trophoblast function.

PL74, a novel member of the transforming growth factor-beta superfamily, is overexpressed in preeclampsia and causes apoptosis in trophoblast cells.

PL74, a novel member of the TGFbeta superfamily that has highest expression in placenta, is a multifunctional peptide that can induce differentiation, inhibit inflammatory stimulation of TNFalpha, and execute apoptosis after p53 overexpression and cytotoxic injury. To study its expression and function in placenta and preeclampsia, we first determined mRNA expression in nine normal and 10 preeclamptic placentas. PL74 mRNA was overexpressed by 57.3% in preeclampsia. Transfection of PL74 into term cytotrophoblasts resulted in increased apoptosis by terminal uridine deoxynucleotidyl nick end labeling labeling (control, 2.8 +/- 0.5%; PL74, 19.1 +/- 0.2%; P < 0.005). Addition of PL74 protein to HTR8/SVneo extravillous cytotrophoblast cells showed a dose-response (0-100 ng/ml) inhibition of [3H]thymidine uptake and increase in apoptosis shown by terminal uridine deoxynucleotidyl nick end labeling and histone-associated DNA fragment ELISA (control, 0.11 +/- 0.01 absorbance units; PL74, 0.21 +/- 0.01; P < 0.01). PL74 did not alter cytotrophoblast invasion using a Matrigel in vitro invasion assay. Cytokine regulation of PL74 mRNA expression in term cytotrophoblasts showed that epidermal growth factor and IFNgamma increased PL74 expression, but TGFbeta and TNFalpha had no effect. Transfection of antisense PL74 into term cytotrophoblast cells resulted in an inhibition of spontaneous differentiation at 2 and 24 h of culture (control vector, 30.8 +/- 3.1% and 26.4 +/- 1.2%; antisense PL74, 17.6 +/- 1.8%and 12.6 +/- 1.4% syncytial units, at 2 and 24 h respectively; P < 0.01). We conclude that PL74 is overexpressed in preeclampsia and may thus promote apoptosis of cytotrophoblasts at the expense of differentiation. PL74 secretion is induced by IFNgamma and may play a role in abnormal placental responses in preeclampsia.

Transforming growth factor-beta induces differentiation of the labyrinthine trophoblast stem cell line SM10.

The mammalian placenta consists of different trophoblast cell types that assist in the variety of functions required for the maintenance of pregnancy. In rodents, labyrinthine trophoblasts of the placenta are especially important, because they are capable of differentiating into fused labyrinthine cells, which form the feto-maternal exchange surface. Even though the molecular signals triggering labyrinthine trophoblast differentiation are poorly understood, transforming growth factor-beta (TGF-beta) has been shown to be present in the placental environment and alter trophoblast development. In this study, we investigated the effects of TGF-beta on the differentiation of the labyrinthine trophoblast stem cell lines SM10 and HRP-1. RT-PCR analyses demonstrated that while the molecular expression of labyrinthine-specific lineage markers (Esx1, Tfeb, and Tec) was maintained in TGF-beta-treated SM10 and HRP-1 cells, TGF-beta induced the down-regulation of trophoblast stem cell markers Id2 and Cdx2. In contrast, TGF-beta induced the expression of a marker of differentiated labyrinthine trophoblasts, Gcm1, only in the SM10 cell line. Furthermore, we demonstrated an increased glucose uptake in the TGF-beta-treated SM10 cells, indicative of functional differentiation. Finally, cell fusion in TGF-beta-treated SM10 and HRP-1 cells was investigated by western blotting analysis of placental alkaline phosphatase and cadherin-11 and by microscopic analyses of cell morphology using green fluorescent protein (GFP) and rhodamine phalloidin staining. The western blotting and morphological analyses indicate TGF-beta-induced cell fusion and morphological differentiation in the SM10 cell line. The SM10 cell line will provide a new and unique model for detailed analysis of TGF-beta-induced molecular events associated with labyrinthine trophoblast differentiation and function.

The effects of preeclampsia and oxygen environment on endothelial release of matrix metalloproteinase-2.

BACKGROUND: There is evidence of altered vascular endothelial function in women with preeclampsia as well as in the endothelial cells from umbilical vessels of preeclamptic pregnancies. Matrix metalloproteinase (MMP)-2 is elevated in the plasma of preeclamptic women and is a mediator of vascular reactivity; however, whether MMP-2 release is altered in preeclamptic endothelial cells is unknown. We hypothesize that MMP-2 release is enhanced in endothelial cells from preeclamptic compared with uncomplicated pregnancies and that this phenomenon may be mediated by an oxygen-dependent mechanism. Our specific hypothesis is that cells from normal pregnancies will demonstrate enhanced MMP-2 release at low oxygen (< 0.5%, 2%) compared to high oxygen (20%), thus mimicking the behavior of preeclamptic cells. METHODS: Human umbilical vein endothelial cells (HUVECs) from preeclamptic pregnancies (n = 4) and normal pregnancies (n = 4) were incubated for 12 hr in standard culture conditions (20% oxygen). In a separate series of experiments, HUVECs from normal pregnancies (n = 6) were incubated for 12 hr at < 0.5%, 2%, and 20% oxygen. Supernatants were analyzed for MMP-2 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2. RESULTS: The HUVECs from women with preeclampsia demonstrated significantly enhanced release of MMP-2 (p < 0.05), TIMP-1 (p < 0.001), and TIMP-2 (p = 0.01) compared to normal cells. MMP-2 release from HUVECs from uncomplicated pregnancies was significantly elevated at 2% oxygen compared to < 0.5% and 20% oxygen (p < 0.05). TIMP-1 and -2 secretion was not altered with varying oxygen. CONCLUSIONS: Preeclamptic endothelial cells demonstrate significantly enhanced MMP-2, TIMP-1 and TIMP-2 release compared to normal cells. Our data show that there are significant effects of oxygen tension on MMP-2 release from normal cells; however, the magnitude of the enhanced release is small when compared to the differences in MMP-2 release in cells from preeclamptic and normal pregnancies. Furthermore, TIMP-1 and -2 release is not affected by changes in oxygen. It is unlikely that oxygen is a key mediator of the enhanced MMP-2, TIMP-1 and TIMP-2 release observed in preeclamptic cells.

A proprietary extract from North American ginseng (Panax quinquefolium) enhances IL-2 and IFN-gamma productions in murine spleen cells induced by Con-A.

A patented aqueous extract from North American ginseng (Panax quinquefolium), containing mainly oligosaccharides and polysaccharides, is commercially available over the counter as COLD-FX (CVT-E002). This proprietary extract is used for the treatment of upper respiratory tract infections. Its in vitro stimulating effects on the immunoglobulin production by B lymphocytes and on natural immune responses by peritoneal exudates macrophages have been previously reported. Using C57 BL/6 mice, an ex vivo study was conducted to examine Con-A-induced splenocytic productions of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) as markers of acquired immune responses. CVT-E002 (10-500 microg/ml) significantly increased Con-A-induced IL-2 and IFN-gamma productions in spleen cells in a dose-dependent manner. Such response was seen by the ginseng extract originated from three different lots, suggesting consistency between the lots.

Adrenomedullin is decreased in preeclampsia because of failed response to epidermal growth factor and impaired syncytialization.

To explore the mechanisms of adrenomedullin (ADM) regulation in normal and preeclamptic (PE) states, we determined placental production of ADM and ADM regulation by cytokines. Isolated, purified cytotrophoblast cultures from normal (n=8) and PE (n=10) placentas were cultured for 3 days in the absence or presence of 10 ng/mL epidermal growth factor (EGF), 1 ng/mL transforming growth factor (TGF)-beta1, 10 ng/mL tumor necrosis factor (TNF)-alpha, or 100 U/mL interferon (IFN)-gamma. Cells were also cultured for 3 days in 10% fetal bovine serum for determination of syncytial formation by desmoplakin staining. Pieces of normal and PE placentas were snap-frozen for ADM mRNA measurement. Results showed that basal ADM production into culture medium by radioimmunoassay was significantly lower in PE placental cells. EGF significantly stimulated ADM production in normal trophoblasts but did not in PE placentas. None of the factors TNF-alpha, TGF-beta1, or IFN-gamma altered ADM secretion in either normal or PE placentas. ADM expression by Northern blot analysis demonstrated a 34.3+/-8.3% reduction in mRNA expression in PE placentas. Syncytialization, as assessed by desmoplakin-outlined syncytial units, was decreased in PE placentas (day 3: normal, 16.7+/-1.3%; PE, 5.5+/-2.0%; P<0.01, ANOVA). However, there was a normal increment in syncytialization in response to EGF in normal and PE trophoblast preparations (EGF day 3: normal, 43.8+/-5.6%; PE, 46.1+/-12.3%). We conclude that spontaneous placental syncytialization is impaired in PE and that ADM production is markedly reduced in PE, possibly owing to an impaired EGF response. These abnormalities indicate poor placental production of ADM as the likely cause of a failed compensatory increase in maternal serum ADM levels in PE.

Infection of placental trophoblasts by Toxoplasma gondii.

How the intracellular parasite Toxoplasma gondii causes placental inflammation and infects the fetus is unknown. By use of a culture model of primary human trophoblasts, we examined the consequences of infection by a virulent strain of T. gondii. Infection fractions (parasitophorous vacuoles per trophoblast nuclei) < or =0.9 were observed 1 day after challenge at an inoculum ratio of T. gondii to nuclei of 10. The culture content of infectious T. gondii increased 45-fold in 48 h. Two days after infection, almost 30% of trophoblast nuclei became apoptotic, and 30%-35% of nuclei were lost. Almost 90% of apoptotic nuclei were not adjacent to a parasitophorous vacuole, suggesting infection protected against apoptosis. However, there was no T. gondii-dependent accumulation of putative cytotoxic factors, such as tumor necrosis factor-alpha, that could mediate paracrine killing. Both mature and immature trophoblasts can be productively infected, and uninfected, but not infected, cells undergo apoptosis.

Human cytomegalovirus-caused damage to placental trophoblasts mediated by immediate-early gene-induced tumor necrosis factor-alpha.

Infection of the fetal epithelium (trophoblast) lining the villous placenta by human cytomegalovirus (HCMV) accompanies placental inflammations and fetal intrauterine growth restriction. However, the consequences of infection on the villous trophoblast have not been explored. We show that HCMV infection of primary immature (cytotrophoblast-like) or mature (syncytiotrophoblast-like) cultures results in loss of half of the cells within 24 hours of virus challenge. Two-color immunofluorescence of HCMV immediate early (IE) gene expression and apoptosis (terminal dUTP nick-end labeling) revealed apoptosis only in uninfected cells. Antibody to tumor necrosis factor (TNF)-alpha completely inhibited infection-induced trophoblast apoptosis and cell loss, as did co-incubation with epidermal growth factor, known to inhibit trophoblast apoptosis. Transfection with HCMV immediate early- (IE)1-72 and IE2-86, but not IE2-55, expression plasmids induced paracrine trophoblast apoptosis inhibitable by epidermal growth factor or antibody to TNF-alpha. These results show that HCMV infection of villous trophoblasts leads to rapid loss of neighboring cells mediated by viral IE protein-induced TNF-alpha secretion. We propose that HCMV infection damages the placental trophoblast barrier by accelerating trophoblast turnover and decreasing its capacity for renewal.

Trophoblasts are productively infected by CD4-independent isolate of HIV type 1.

Evidence for HIV-1 infection of trophoblasts is discordant. Utilizing highly purified full-term trophoblasts, we demonstrate that full-term trophoblasts express CXCR4 but are negative for CCR5 and CD4 cell surface proteins. Full-term trophoblasts were refractory to infection by HIV-1 IIIB and primary isolates of HIV-1. However, full-term trophoblasts could be infected by a CD4-independent, CXCR4-utilizing HIV-1 strain, as demonstrated by substantial p24 (5.5 ng/ml) levels and HIV-1 gag/pol DNA content (3050 copies/microg) 7 days postinfection. These data illustrate that trophoblasts express the essential host factors for productive HIV-1 infection and that the block to HIV-1 infection may be at the level of entry. In additional, our data suggest that CD4-independent mechanisms of infection may play a role in promoting in utero HIV-1 transmission.