Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia.

During normal human pregnancy a subpopulation of fetal cytotrophoblast stem cells differentiate and invade the uterus and its arterioles. In the pregnancy disease preeclampsia, cytotrophoblast differentiation is abnormal and invasion is shallow. Thus, the placenta is relatively hypoxic. We investigated whether lowering oxygen tension affects cytotrophoblast differentiation and invasion. Previously we showed that when early gestation cytotrophoblast stem cells are cultured under standard conditions (20% O2) they differentiate/invade, replicating many aspects of the in vivo process. Specifically, the cells proliferate at a low rate and rapidly invade extracellular matrix (ECM) substrates, a phenomenon that requires switching their repertoire of integrin cell-ECM receptors, which are stage-specific antigens that mark specific transitions in the differentiation process. In this study we found that lowering oxygen tension to 2% did not change many of the cells' basic processes. However, there was a marked increase in their incorporation of [3H]thymidine and 5-bromo-2'-deoxyuridine (BrdU). Moreover, they failed to invade ECM substrates, due at least in part to their inability to completely switch their integrin repertoire. These changes mimic many of the alterations in cytotrophoblast differentiation/invasion that occur in preeclampsia, suggesting that oxygen tension plays an important role in regulating these processes in vivo.

Ca2+ entry through L-type voltage-sensitive Ca2+ channels stimulates the release of human chorionic gonadotrophin and placental lactogen by placental explants.

The release of human chorionic gonadotrophin (hCG) and placental lactogen (hPL) by human placental explants can be stimulated by Ca2+ entry. The aim of the present study was to characterize the modality of Ca2+ entry in the presence of high extracellular K+ concentration ([K+]o). A rise in [K+]o from 5 to > or = 50 mM induced a rapid and marked increase in the release of hCG and hPL from human term placental explants. The stimulatory effects of an excess [K+]o on the release of hCG and hPL were blocked in the absence of extracellular Ca2+ or in the presence of 0.5 mM Co2+. The presence of 50 microM methoxyverapamil, 20 microM nifedipine or 40 microM Cd2+ in the medium inhibited the stimulatory effects of [K+]o addition. Lastly, 40 microM Ni2+ failed to affect the increases in hCG and hPL releases elicited by [K+]o addition. Our data clearly show that a rise in [K+]o stimulates the release of hCG and hPL from placental explants. These secretory effects can be viewed as resulting from a Ca2+ entry through voltage-sensitive Ca2+ channels of the L-type.

A 2-year follow-up of an anti-HIV immune reaction in HIV-1 gp160-immunized healthy seronegative humans: evidence for persistent cell-mediated immunity.

The first trial of an anti-HIV immunization, using a recombinant vaccinia virus expressing gp160 (rV) for priming and paraformaldehyde-fixed rV-infected PBLs and soluble gp 160 for boosting, clearly showed an in vitro HIV-protective immune reaction. This result led us to carry out an additional 2 year Phase I clinical trial in 25 HIV-seronegative volunteers, using HIV gp 160 antigens for immunization in four different protocols. The 2 year trial showed (a) the safety of the preparations, (b) a transient humoral immunity following each boost, and (c) a long-lasting memory T-cell response. Memory cytotoxic T-lymphocytes (CTLs) induced by gp 160 antigen with or without vaccinia vector lysed HLA class I restricted target cells expressing HIV-1 env antigens. These results are consistent with CTLs being an effective component of an AIDS vaccine to control cell-to-cell viral replication, dissemination in the organism, and subsequent evolution toward AIDS.

The release of human chorionic gonadotrophin and placental lactogen by placental explants can be stimulated by Ca2+ entry through a Na(+)-Ca2+ exchange process.

Isosmotical replacement of extracellular Na+ ([Na+]o) by K+, choline and to a lesser extent by saccharose stimulated the release of chorionic gonadotrophin and placental lactogen from human term placental explants. The effect of [Na+]o removal on the release of both hormones was concentration-dependent and was inhibited in the absence of extracellular Ca2+ or in the presence of 0.5 mM Co2+, a Ca2+ entry blocker. Blockers of the voltage-sensitive Ca2+ channels (20 microM nifedipine and 50 microM methoxyverapamil) or Na+ channels (5 microM tetrodotoxin) did not affect the stimulatory effects of [Na+]o omission. By contrast, Mg2+ and Sr2+ (10 mM) as well as amiloride (2 mM) and its analogue 2',4'-dimethylbenzamil (50 microns), all known to affect the Na(+)-Ca2+ exchange, markedly reduced the increase in hormone release elicited by [Na+]o removal. Lastly, the secretory responses to [Na+]o deprivation were increased in the presence of 2 mM ouabain, an inhibitor of the Na(+)-K+ ATPase. These results indicate for the first time that [Na+]o omission provokes a Ca(2+)-dependent stimulation of human chorionic gonadotrophin and placental lactogen releases. The pharmacological dissection of the secretory effects of [Na+]o removal supports the existence of a process of Na(+)-Ca2+ exchange in placental cells.

Stimulatory effects of extracellular calcium on chorionic gonadotrophin and placental lactogen release by human placental explants.

The effects of sudden modifications of extracellular calcium ion concentration on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) release were investigated using term placental explants incubated in Krebs Ringer solution. The hCG and hPL releases were both stimulated when the extracellular Ca2+ concentration was increased. The hCG and hPL secretions elicited by the addition of extracellular Ca2+ were larger when placental explants were preincubated in alpha-calcium (no added calcium + EGTA) than in normo-calcium (1.5 mM). Removal of extracellular Ca2+ from the medium also elicited an increase in hCG and hPL release. However, this stimulatory effect of Ca2+ omission was partly suppressed by washing the explants prior to incubation in the alpha-calcium medium, and was completely abolished when alpha-calcium medium was supplemented with 1 mM cobalt. Our results indicate that changes in Ca2+ modify hCG and hPL release from term placental explants in a manner concordant with the 'stimulus-secretion coupling' concept.

Inhibitory effects of human chorionic gonadotropin (hCG) preparations on HIV infection of human placenta in vitro.

Human chorionic gonadotropin (hCG) has been implicated in modifying Kaposi sarcoma lesions in HIV positive patients and in reducing HIV infection in human lymphocytes and human choriocarcinoma cells. These anti-HIV effects of hCG may contribute to the limited maternal to fetal transmission of HIV infection (25-35 per cent without treatment). However, it is unknown whether such high dosages of hCG have any effect on vertical transmission of HIV or on the infection of the human placenta with cell-free HIV. We have investigated in a dose dependent manner the effects of hCG on HIV-1 infection of human term placentae. Using commercially available hCG preparations, the ability to modify the infection of placental explants in vitro was examined. Sigma hCG and ICN beta-hCG (0.1, 1, 10 IU/ml) and APL hCG and Sigma and Serono recombinant hCG (0.1, 1, 10, 100 IU/ml) were added during 6 h of pre-incubation and the 4 days of culture (3 days following the 24 h exposure to HIV-1 Ba-L strain). Cell-free HIV infection of the placental explants was documented using DNA-PCR detection of Gag and LTR regions of HIV. Each experimental condition was repeated in different placentae (n=5) and each PCR amplification was performed in duplicate with each primer set (total=20). Our results demonstrate that there is a dose dependent inhibition of HIV-1 infection in the human placenta above the physiologic levels (0.2 IU/ml) of hCG produced during incubation. At the highest concentration used (100 IU/ml), 80 per cent inhibition of HIV infection was achieved with urinary extract hCG and about 50 per cent with recombinant hCG. beta-hCG alone appears to possess an efficacy equivalent to the complete hCG molecule. In this in vitro study, hCG demonstrates specific anti-HIV inhibitory properties that cannot be solely attributed to urinary contamination of the commercial preparations. Such inhibitory action of hCG may be present at varying levels throughout gestation based upon the circulating levels of hCG and its production by the placenta. Knowledge of the specific mechanisms underlying this inhibition is necessary before clinical applications can be considered.

Marginal transfer of ReoPro (Abciximab) compared with immunoglobulin G (F105), inulin and water in the perfused human placenta in vitro.

ReoPro (Abciximab), a Fab fragment of a human-murine chimeric monoclonal antibody, binds to glycoprotein IIb/IIIa receptors on human platelets and inhibits platelet aggregation. Can ReoPro transit the human placenta since it does not have an Fc (domain) as does IgG? This question was addressed using an in vitro term human placental lobular dual perfusion model. ReoPro, along with 3H(2)O, inulin or 125I-F105 human IgG(1), were added to the maternal reservoir for 6 or >12 h, ReoPro was equivalent to, or exceeded, clinically relevant plasma concentrations (0.3-3 microg/ml). 3H(2)O rapidly appeared in the fetal circuit, while fetal 14C-inulin never equilibrated with the maternal inulin. After 6 h, 125I-F105 was present with fetal/maternal percentages-0.55 per cent. ReoPro was not detectable (<3.9 ng/ml) in the fetal circuit during or at the end of any perfusion. Using immunohistochemistry, ReoPro was only detected attached to maternal and fetal platelets, and to the trophoblastic surface of the placental villi. Only pharmaceutically insignificant amounts of ReoPro were detected in the fetal circuit, which demonstrates a barrier capacity of the human term placenta for this Fab fragment compared with IgG.

Production of granulocyte colony-stimulating factor by the human placenta at various stages of development.

The human placenta is capable of producing a variety of haematopoietic growth factors in vitro. It is not clear, however, whether the placenta produces such factors in vivo and if so, whether placental production of haematopoietic growth factors has a physiological role in fetal haematopoietic development. As a step toward making this determination, we assessed whether the onset of placental production of granulocyte colony-stimulating factor (G-CSF), in vivo, coincides with the onset of granulocytopoiesis in the developing fetus. To make this assessment, we obtained human placentae between 10 weeks of gestation and term and studied production of G-CSF in several ways. First, we sought to determine whether the onset of production of G-CSF mRNA in the placenta immediately precedes the appearance of neutrophil development in the fetus. Second, we assessed the effect of gestational age on the capacity of the placenta to generate G-CSF in vitro, by incubating cubes of placenta, with or without including interleukin-1 alpha (IL-1 alpha) in the culture media, and quantifying G-CSF in the cell culture supernatants 24 h later. Third, we assessed the rate of G-CSF production by the placenta, by perfusing two normal, term placentae using a membrane-oxygenator system, and quantifying G-CSF, at intervals, in the perfusates. We found: (1) no evidence that placental production of G-CSF is involved in regulating granulocytopoiesis in the fetus, (2) that the healthy placenta contains little or no G-CSF mRNA in vivo, (3) the placenta at term has a far greater capacity to produce G-CSF, when stimulated, than does the placenta before term, and (4) that although the placenta does not normally produce G-CSF in vivo, it has the capacity of generating very large quantities of G-CSF continuously over at least several days.

Long-term dual perfusion of isolated human placental lobules with improved oxygenation for infectious diseases research.

An improved method for long-term perfusion of the isolated human term placental lobule has been developed to investigate the maternofetal transfer of infectious agents, in particular the human immunodeficiency virus (HIV). The purpose of this paper is to describe those modifications that allow for substantially prolonged perfusions in in a biohazard environment. The method described has been adapted from previous models. The perfusion apparatus has been modified for use within a biohazard hood, and, intravenous bags contain the medium for circulation of perfusates in closed circuits. A Mera Silox-S 0.3 membrane oxygenator delivers more oxygen to the tissue, and, Electromedic Cardioplegia heat exchangers warm the perfusate prior to oxygenation. Viability criteria (glucose consumption, lactate production, de novo production of human placental lactogen (hPL), volume loss, flow, temperature, pressure, oxygen transfer, carbon dioxide production, absence of IgM transfer and light and electron microscopy) demonstrate that the placental tissue remains in a functional state throughout the perfusion. Oxygen and glucose consumption are both stable over time; lactate levels remain constant; and hPL continues to be produced. These significant modifications of the perfusion system have permitted the investigators to increase the duration of perfusion to 48 h while preserving normal metabolic function of ultrastructurally intact tissue as demonstrated by ultra structural observations. This perfusion model device provides biohazard precautions and may be applied to other studies of placental physiology.

Granulocyte colony-stimulating factor in preterm and term pregnancy, parturition, and intra-amniotic infection.

OBJECTIVE: [corrected] To determine the sources of granulocyte colony-stimulating factor (G-CSF) in amniotic fluid and to examine its relation to labor and clinically diagnosed intra-amniotic infection. METHODS: We assessed G-CSF and G-CSF receptor expression in placentas (n = 50) from 5-40 weeks' gestation, and G-CSF concentrations were measured in amniotic fluid (n = 146), bronchoalveolar lavage fluid (n = 8), and paired maternal serum, cord blood, neonatal serum, and neonatal urine samples (n = 16). RESULTS: Immunohistochemical staining and messenger RNA analysis showed placental expression of G-CSF and G-CSF receptor throughout gestation. The number of decidual stromal cells expressing G-CSF receptor was significantly higher in women with intra-amniotic infection compared with women without infection (27 +/- 2 versus 18 +/- 3 cells per high power field, P =.02). Amniotic fluid concentrations of G-CSF were not significantly different in noninfected preterm compared with term samples (1708 +/- 1673 versus 1612 +/- 2100 pg/mL, P =.9). Labor was not associated with a significant increase in amniotic fluid G-CSF concentrations (1864 +/- 3151 versus 1612 +/- 2100 pg/mL, P =.77, term labor versus no labor; 3335 +/- 5364 versus 1708 +/- 1673 pg/mL, P =.09, preterm). Concentrations of G-CSF in maternal serum, amniotic fluid, bronchoalveolar lavage fluid, and neonatal urine were increased during intra-amniotic infection (all P <.05). CONCLUSION: Amniotic fluid G-CSF concentrations were similar in preterm and term pregnancies and were not significantly influenced by labor. Intra-amniotic infection was associated with an increased number of placental cells expressing the G-CSF receptor and higher concentrations of G-CSF in amniotic fluid, maternal serum, neonatal urine, and neonatal bronchoalveolar lavage samples.

Immunogenicity of combined anti-HIV and anti-suppressive vaccine preparations.

HIV-1 antigens generate in man both a humoral and cellular immune reaction. However, in ARC/AIDS patients, the cellular response is inhibited by HIV-1 which induces an antiproliferative (suppressive) effect on activated T cells. To overcome this inhibition and up-regulate the cellular response, we designed a new vaccine strategy directed both against HIV-1 and immunosuppression and we used an immunizing preparation composed of HIV-1 antigens combined with immunoregulatory peptides prepared in a biologically inactivated but immunogenic form. In mice, this preparation induced anti-HIV-1 antibodies and a cell-mediated cytotoxicity directed against H2 restricted cells carrying HIV-1 antigens.

Free vitamin B12 and transcobalamin II-vitamin B12 complex uptake by the visceral yolk sac of the Sprague-Dawley rat: effect of inhibitors.

Exogenous free vitamin B12 or B12 bound to human transcobalamin II (TCII) accumulated in the near-term rat visceral yolk sac. The rates of their uptakes in vitro and in vivo increased rapidly with time then reached a plateau, which supports a saturable transport/binding process as the rate-limiting step for the uptake of free and TCII complexed B12. Both uptakes were significantly decreased by trypan blue, colchicine, and low temperature but not by ouabain. Such inhibition suggests that the absorption of free and bound B12 is via an endocytosis process dependent upon energy but not the magnesium-dependent sodium/potassium-activated ATPase. Thus, the role of the visceral yolk sac in vitamin transfer to the conceptus and the alterations in yolk sac function associated with birth defects and diminished growth can be integrally related.

[Malaria: the end of a multimillenia reign?]. / Malaria: la fin d'un règne multimillénaire?

Malaria is one of the oldest and more pervasive diseases of mankind with half of the world's population still meeting with Plasmodium. The resistance of the parasite and its carrier, anopheles, at the current levels of antimalaria programs results in recurrence of this illness among the less developed tropical zones. The high risk groups for malaria are pregnant women and children, resulting in a delay of intra-uterine growth, neonatal/infantile morbidity and mortality. Recent progress in molecular biology provide new perspectives for preventive and curative solutions, more specifically in the relationship between host and parasite.

Human immunodeficiency virus infection: in situ polymerase chain reaction localization in human placentas after in utero and in vitro infection.

OBJECTIVE: We compared localization of human immunodeficiency virus type 1 within human placentas infected in utero with localization within human placental explants infected in vitro. STUDY DESIGN: Placental tissues from 3 cases of vertical transmission of human immunodeficiency virus type 1 were studied. Human placental explants from 6 term pregnancies not complicated by human immunodeficiency virus type 1 infection were infected in vitro with human immunodeficiency virus type 1(Ba-L). Sections from each placental explant and each placenta infected in utero were analyzed for human immunodeficiency virus type 1 localization by means of in situ polymerase chain reaction. RESULTS: Human immunodeficiency virus type 1 was primarily localized within syncytiotrophoblast, Hofbauer cells, and extravillous mononuclear cells in placental tissue sections from cases of in utero infection. Within placental explants human immunodeficiency virus type 1 deoxyribonucleic acid was found in syncytiotrophoblast and Hofbauer cells. The distributions of viral localization were similar in placentas infected in utero and placental explants infected in vitro. CONCLUSION: Human immunodeficiency virus type 1 can be localized to specific human placental cells (eg, syncytiotrophoblast) after either in utero or in vitro infection, which demonstrates the specificity and selectivity of human immunodeficiency virus infection in the human placenta.

Subpopulations of T and B cells in perinatally HIV-infected and noninfected age-matched children compared with those in adults.

Peripheral blood mononuclear cells were quantified for the subsets of CD4, CD8, and CD19 lymphocytes by using CD45RA (2H4), CD29(4B4), CD57, CD5, CD10, Leu8, HLA-DR, and TCR gamma delta-1 monoclonal antibodies and dual color immunofluorescence. A comparative analysis of lymphocyte subpopulations was made among 52 HIV-infected and 50 age-matched control children and 30 HIV-seropositive and 27 negative control adults. A significant decrease in the CD4+CD45RA+ "naive" cells was much more marked in HIV-infected children than in HIV-infected adults. A significant percentage increase in the CD4+CD29+ "memory" cells was observed in HIV-infected children but not in infected adults; however, the absolute numbers were usually decreased in all age groups. The mean percentage and absolute numbers of CD4+CD7+ and CD4+Leu8+ cells were decreased in HIV-infected children, although usually not significantly. The CD3+TCR gamma delta-1+ did not show any change in the infected children tested. The mean percentage and absolute number of the CD8+HLA-DR+ cells increased significantly in HIV-infected persons of all ages. The CD8+CD57+ cells were increased in percentage and absolute number in HIV-infected children ages 1-4 and 4-8 years. In the adults, no change was noted in either the percentage or absolute number of CD19+CD5+ B cells, a finding similar to that noted in HIV-infected children above 1 year of age. Although adults showed a significant decrease in both percentage and numbers of CD5- B cells, an increase was noted in the 7- to 12-month-old HIV-infected children. The CD19+CD10+ cells showed a slight but significant decrease in the youngest age group and a significant increase in the older age groups of HIV-infected children. These findings indicate that several lymphocyte subpopulations are altered differentially during HIV infection in children of varying ages and in adults.

Fast scan magnetic resonance imaging and Doppler ultrasonography of uteroplacental hemodynamics in the rhesus monkey (Macaca mulatta).

Fast scan magnetic resonance imaging, color Doppler ultrasonography, and magnetic resonance angiography were used to demonstrate the uteroplacental hemodynamics in rhesus monkeys. "Jets" of maternal blood spurting into the placenta were observed with, or without, the use of a contrast medium from 114 days to the term of pregnancy. Cumulative intravenous doses of cocaine hydrochloride brought about changes in the sequences of imaging of the maternal blood inflow to the placenta. These methods appear suitable for non-invasive monitoring of the effects of drugs on placental function.

Culture of first-trimester and full-term human chorionic villus explants: role of human chorionic gonadotropin and human placental lactogen as a viability index.

In long-term cultures of human chorionic villus explants, the viability of the tissue must be controlled to ensure the reliability of functional studies. Ionic levels (pH), gas concentrations (pO2, pCO2) and metabolic markers (glucose, lactate) in the culture medium are often utilized. Analyses of hormone, enzyme and protein levels are also frequently used to estimate viability. The purpose of this study was to evaluate whether in vitro release and immunoreactivity of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were correlated with the viability of first-trimester and full-term chorionic villus explants as determined by histopathology. Villus explants of first-trimester and full-term pregnancies were incubated in 6-well plates of RPMI medium which was supplemented with 10% fetal calf serum. Incubations were performed for 10 days, and the plates were kept at 37 degrees C under a water-saturated atmosphere containing 5% CO2 and 95% O2. The medium was replaced every day and samples of supernatant were frozen for later testing of hCG (first trimester) or hPL (full term), glucose consumption and lactate production. The tissue was also fixed and embedded for light-microscopic examination and immunocytochemistry. The hCG release remained stable during 6-7 days at a high level before decreasing, whereas hPL release decreased during the first 5-6 days then stabilized at a relatively low level. Only hCG kinetics were significantly different between tissue incubated with and without cycloheximide or iodoacetic acid. Both hCG and hPL immunoreactivity were not significantly different between tissue cultures with, and without, addition of cycloheximide or iodoacetic acid and even with morphological evidence of trophoblast and endothelial necrosis. The immunoreactivity for both hormones remains highly positive when the significant release has stopped, and does not reflect the tissue viability.