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Sample records for secreted human placental

  1. Effect of leptin on the regulation of placental hormone secretion in cultured human placental cells.

    PubMed

    Coya, Raquel; Martul, Pedro; Algorta, Jaime; Aniel-Quiroga, Ma Angeles; Busturia, Ma Angeles; Señarís, Rosa

    2006-11-01

    Placenta is an important source of leptin during pregnancy that contributes to the high plasma leptin levels in pregnant women. Leptin and its functional receptors are synthesized in trophoblast cells that, in turn, secrete gestational hormones supporting a paracrine or autocrine role for leptin in the endocrine activity of the placenta. In the present study we examined the effect of leptin on in vitro release of gestational hormones (human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone, estrogens and testosterone) by human term placental cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Progesterone, hCG, hPL, estradiol, estrone, estriol and testosterone levels were measured by different assays in culture media of cells maintained in monolayer culture after incubation for 12, 24, 48 or 72 h with leptin or placebo. Incubation with leptin did not modify hCG, hPL, progesterone, estriol and estrone secretion for any of the doses and times assayed. However, leptin led to a dose-dependent decrease in estradiol release. This effect was observed when treatment with recombinant human leptin spanned from 12 to 72 h. At this time an increase in testosterone levels was observed in leptin-treated cells versus placebo. These results indicate that leptin can be considered a gestational hormone implied in the endocrine function of the placenta, with an important role in control of the production of steroid reproductive hormones in placental cells in vitro. PMID:17145648

  2. EFFECT OF BROMODICHLOROMETHANE ON CHORIONIC GONADOTROPHIN SECRETION BY HUMAN PLACENTAL TROPHOBLAST CULTURES

    EPA Science Inventory

    EFFECT OF BROMODICHLOROMETHANE ON CHORIONIC GONADOTROPHIN SECRETION BY HUMAN PLACENTAL TROPHOBLAST CULTURES

    Jiangang Chen1, Gordon C. Douglas1?,Twanda L. Thirkill1?, Peter N. Lohstroh1, Susan R. Bielmeier2, Michael G. Narotsky3, Deborah S. Best3, Randy A. Harrison3, Kala ...

  3. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

    PubMed

    Díaz, Paula; Sibley, Colin P; Greenwood, Susan L

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3-5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10-1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  4. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    PubMed Central

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  5. Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

    PubMed

    Coya, Raquel; Martul, Pedro; Algorta, Jaime; Aniel-Quiroga, Ma Angeles; Busturia, Ma Angeles; Señarís, Rosa

    2005-07-01

    The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro. PMID:16048798

  6. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    SciTech Connect

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  7. Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor

    PubMed Central

    Flores-Pliego, Arturo; Espejel-Nuñez, Aurora; Castillo-Castrejon, Marisol; Meraz-Cruz, Noemi; Beltran-Montoya, Jorge; Zaga-Clavellina, Veronica; Nava-Salazar, Sonia; Sanchez-Martinez, Maribel; Vadillo-Ortega, Felipe; Estrada-Gutierrez, Guadalupe

    2015-01-01

    Background The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. Methods Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. Results Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. Conclusions In this work we confirm that

  8. Effect of bromodichloromethane on chorionic gonadotrophin secretion by human placental trophoblast cultures.

    PubMed

    Chen, Jiangang; Douglas, Gordon C; Thirkill, Twanda L; Lohstroh, Peter N; Bielmeier, Susan R; Narotsky, Michael G; Best, Deborah S; Harrison, Randy A; Natarajan, Kala; Pegram, Rex A; Overstreet, James W; Lasley, Bill L

    2003-11-01

    Bromodichloromethane (BDCM) is a trihalomethane found in drinking water as a by-product of disinfection processes. BDCM is hepatotoxic and nephrotoxic in rodents and has been reported to cause strain-specific full-litter resorption in F344 rats during the luteinizing hormone-dependent phase of pregnancy. In humans, epidemiological studies suggest an association between exposure to BDCM in drinking water and increased risk of spontaneous abortion. To begin to address the mechanism(s) of BDCM-induced spontaneous abortion, we hypothesized that BDCM targets the placenta. Primary cultures of human term trophoblast cells were used as an in vitro model to test this hypothesis. Trophoblasts were allowed to differentiate into multinucleated syncytiotrophoblast-like colonies, after which they were incubated for 24 h with different concentrations of BDCM (20 nM to 2 mM). Culture media were collected and assayed for immunoreactive and bioactive chorionic gonadotropin (CG). Cultures exposed to BDCM showed a dose-dependent decrease in the secretion of immunoreactive CG as well as bioactive CG. The lowest effective BDCM concentration was 20 nM, approximately 35-times higher than the maximum concentration reported in human blood (0.57 nM). Trophoblast morphology and viability were similar in controls and cultures exposed to BDCM. We conclude that BDCM perturbs CG secretion by differentiated trophoblasts in vitro. This suggests that the placenta is a likely target of BDCM toxicity in the human and that this could be related to the adverse pregnancy outcomes associated with BDCM. PMID:12970577

  9. The Effects of Culture Conditions on the Glycosylation of Secreted Human Placental Alkaline Phosphatase Produced in Chinese Hamster Ovary Cells

    PubMed Central

    Nam, Jong Hyun; Zhang, Fuming; Ermonval, Myriam; Linhardt, Robert J.; Sharfstein, Susan T.

    2009-01-01

    The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33°C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions. PMID:18553404

  10. Palmitic acid induces interleukin-1β secretion via NLRP3 inflammasomes and inflammatory responses through ROS production in human placental cells.

    PubMed

    Shirasuna, Koumei; Takano, Hiroki; Seno, Kotomi; Ohtsu, Ayaka; Karasawa, Tadayoshi; Takahashi, Masafumi; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito

    2016-08-01

    Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1β secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1β release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1β secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1β, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1β, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications. PMID:27300134

  11. Zika Virus Infects Human Placental Macrophages.

    PubMed

    Quicke, Kendra M; Bowen, James R; Johnson, Erica L; McDonald, Circe E; Ma, Huailiang; O'Neal, Justin T; Rajakumar, Augustine; Wrammert, Jens; Rimawi, Bassam H; Pulendran, Bali; Schinazi, Raymond F; Chakraborty, Rana; Suthar, Mehul S

    2016-07-13

    The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier. PMID:27247001

  12. BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL TROPHOBLAST DIFFERENTIATION

    EPA Science Inventory

    BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL
    TROPHOBLAST DIFFERENTIATION
    Jiangang Chen, Twanda L. Thirkill, Peter N. Lohstroh, Susan R. Bielmeier, Michael
    G. Narotsky, Deborah S. Best, Randy A. Harrison, Kala Natarajan, Rex A. Pegram,
    Bill L. Lasley, and Gordon C. Do...

  13. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    PubMed

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition

  14. Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

    PubMed Central

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition

  15. Finite Element Modeling of Human Placental Tissue

    PubMed Central

    Yu, Mao; Manoogian, Sarah; Duma, Stefan M.; Stitzel, Joel D.

    2009-01-01

    Motor vehicle crashes account for a large portion of placental abruption and fetal losses. To better understand the material properties of the human placenta, a Finite Element (FE) model of human placenta tissue was created and verified using data from uniaxial tension tests. Sixty-four tensile tests at three different strain rates of 7% strain/s, 70% strain/s, and 700% strain/s from six whole human placentas were used for model development. Nominal stresses were calculated by dividing forces at the grips by the original cross-sectional area. Nominal strains were calculated by dividing cross-head displacement by the original gauge length. A detailed methodology for interpreting experimental data for application to material model development is presented. A model of the tension coupon was created in LS-DYNA and stretched in the same manner as the uniaxial tension tests. The behavior of the material was optimized to the uniaxial tension test using a multi-island genetic algorithm. The results demonstrate good correlation between experiments and the model, with an average difference of 2% between the optimized FE and experimental first principal stress at the termination state. The material parameters found in this study can be utilized in FE models of placental tissues for behavior under dynamic loading. PMID:20184849

  16. Human placental trophoblasts confer viral resistance to recipient cells

    PubMed Central

    Delorme-Axford, Elizabeth; Donker, Rogier B.; Mouillet, Jean-Francois; Chu, Tianjiao; Bayer, Avraham; Ouyang, Yingshi; Wang, Tianyi; Stolz, Donna B.; Sarkar, Saumendra N.; Morelli, Adrian E.; Sadovsky, Yoel; Coyne, Carolyn B.

    2013-01-01

    Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal–fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections. PMID:23818581

  17. Bromodichloromethane inhibits human placental trophoblast differentiation.

    PubMed

    Chen, Jiangang; Thirkill, Twanda L; Lohstroh, Peter N; Bielmeier, Susan R; Narotsky, Michael G; Best, Deborah S; Harrison, Randy A; Natarajan, Kala; Pegram, Rex A; Overstreet, James W; Lasley, Bill L; Douglas, Gordon C

    2004-03-01

    Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans. PMID:14691210

  18. Animal models of human placentation--a review.

    PubMed

    Carter, A M

    2007-04-01

    This review examines the strengths and weaknesses of animal models of human placentation and pays particular attention to the mouse and non-human primates. Analogies can be drawn between mouse and human in placental cell types and genes controlling placental development. There are, however, substantive differences, including a different mode of implantation, a prominent yolk sac placenta, and fewer placental hormones in the mouse. Crucially, trophoblast invasion is very limited in the mouse and transformation of uterine arteries depends on maternal factors. The mouse also has a short gestation and delivers poorly developed young. Guinea pig is a good alternative rodent model and among the few species known to develop pregnancy toxaemia. The sheep is well established as a model in fetal physiology but is of limited value for placental research. The ovine placenta is epitheliochorial, there is no trophoblast invasion of uterine vessels, and the immunology of pregnancy may be quite different. We conclude that continued research on non-human primates is needed to clarify embryonic-endometrial interactions. The interstitial implantation of human is unusual, but the initial interaction between trophoblast and endometrium is similar in macaques and baboons, as is the subsequent lacunar stage. The absence of interstitial trophoblast cells in the monkey is an important difference from human placentation. However, there is a strong resemblance in the way spiral arteries are invaded and transformed in the macaque, baboon and human. Non-human primates are therefore important models for understanding the dysfunction that has been linked to pre-eclampsia and fetal growth restriction. Models that are likely to be established in the wake of comparative genomics include the marmoset, tree shrew, hedgehog tenrec and nine-banded armadillo. PMID:17196252

  19. Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion.

    PubMed

    Tong, Stephen; Kaitu'u-Lino, Tu'uhevaha J; Onda, Kenji; Beard, Sally; Hastie, Roxanne; Binder, Natalie K; Cluver, Cathy; Tuohey, Laura; Whitehead, Clare; Brownfoot, Fiona; De Silva, Manarangi; Hannan, Natalie J

    2015-11-01

    Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells. PMID:26324507

  20. Human placental coated vesicles contain receptor-bound transferrin.

    PubMed Central

    Booth, A G; Wilson, M J

    1981-01-01

    Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation. Images PLATE 2 PLATE 1 Fig. 1. Fig. 2. Fig. 3. PMID:6272755

  1. Bisphenol A disrupts gene expression in human placental trophoblast cells.

    PubMed

    Rajakumar, Chandrew; Guan, Haiyan; Langlois, David; Cernea, Maria; Yang, Kaiping

    2015-06-01

    This study examined the effect of bisphenol A (BPA) on human placental gene expression using primary trophoblast cells as an in vitro model system. Trophoblast cells were isolated from human placentas at term, cultured and then exposed to environmentally relevant concentrations of BPA (0.1-2 μg/ml) for up to 24h, after which levels of 11β-HSD2 mRNA, protein and activity were determined by standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. The mRNA levels of several other prominent placental hormones/factors were also assessed by qRT-PCR. BPA dramatically increased levels of 11β-HSD2 activity, protein and mRNA in a time- and concentration-dependent manner (> 4-fold). BPA also augmented aromatase, glucose transporter-1, CRH, and hCG mRNA levels while reducing the level of leptin mRNA. These findings demonstrate that BPA severely disrupts human placental gene expression in vitro, which suggests that exposure to BPA may contribute to altered placental function and consequent pregnancy complications. PMID:25784278

  2. Angiogenin expression during early human placental development; association with blood vessel formation.

    PubMed

    Pavlov, Nadine; Frendo, Jean-Louis; Guibourdenche, Jean; Degrelle, Séverine A; Evain-Brion, Danièle; Badet, Josette

    2014-01-01

    The placenta is a transient organ essential for fetal development. During human placental development, chorionic villi grow in coordination with a large capillary network resulting from both vasculogenesis and angiogenesis. Angiogenin is one of the most potent inducers of neovascularisation in experimental models in vivo. We and others have previously mapped angiogenin expression in the human term placenta. Here, we explored angiogenin involvement in early human placental development. We studied, angiogenin expression by in situ hybridisation and/or by RT-PCR in tissues and primary cultured trophoblastic cells and angiogenin cellular distribution by coimmunolabelling with cell markers: CD31 (PECAM-1), vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), Tie-2, von Willebrand factor, CD34, erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Extravillous and villous cytotrophoblasts, isolated and differentiated in vitro, expressed and secreted angiogenin. Angiogenin was detected in villous trophoblastic layers, and structured and nascent fetal vessels. In decidua, it was expressed by glandular epithelial cells, vascular cells and macrophages. The observed pattern of angiogenin expression is compatible with a role in blood vessel formation and in cross-talk between trophoblasts and endothelial cells. In view of angiogenin properties, we suggest that angiogenin may participate in placental vasculogenesis and organogenesis. PMID:25093183

  3. The endocannabinoid system: A novel player in human placentation.

    PubMed

    Costa, M A

    2016-06-01

    Cannabis sativa is the most consumed illegal drug around the world. Its consumption during pregnancy is associated with gestational complications, particularly with fetal growth restriction. Endocannabinoids (eCBs) are lipid molecules that act by activating the G-protein coupled cannabinoid receptors, which are also target of the phytocannabinoid Δ(9)-tetrahydrocannabinol (THC). The endocannabinoid system (ECS) participates in distinct biological processes, including pain, inflammation, neuroprotection, and several reproductive events. In addition, an abnormal expression of ECS is associated with infertility and miscarriages. This manuscript will review and discuss the expression of ECS in normal and pathological human placentas, and the role of eCBs and THC in trophoblast proliferation, apoptosis, differentiation, and function. The current evidence points towards a role of ECS in human placentation, shedding light on the contribution of the eCBs in the coordination of human placentation, and in the cellular mechanisms underlying the deleterious effects of cannabis consumption during pregnancy. PMID:26965993

  4. A microphysiological model of the human placental barrier.

    PubMed

    Blundell, Cassidy; Tess, Emily R; Schanzer, Ariana S R; Coutifaris, Christos; Su, Emily J; Parry, Samuel; Huh, Dongeun

    2016-08-01

    During human pregnancy, the fetal circulation is separated from maternal blood in the placenta by two cell layers - the fetal capillary endothelium and placental trophoblast. This placental barrier plays an essential role in fetal development and health by tightly regulating the exchange of endogenous and exogenous materials between the mother and the fetus. Here we present a microengineered device that provides a novel platform to mimic the structural and functional complexity of this specialized tissue in vitro. Our model is created in a multilayered microfluidic system that enables co-culture of human trophoblast cells and human fetal endothelial cells in a physiologically relevant spatial arrangement to replicate the characteristic architecture of the human placental barrier. We have engineered this co-culture model to induce progressive fusion of trophoblast cells and to form a syncytialized epithelium that resembles the syncytiotrophoblast in vivo. Our system also allows the cultured trophoblasts to form dense microvilli under dynamic flow conditions and to reconstitute expression and physiological localization of membrane transport proteins, such as glucose transporters (GLUTs), critical to the barrier function of the placenta. To provide a proof-of-principle for using this microdevice to recapitulate native function of the placental barrier, we demonstrated physiological transport of glucose across the microengineered maternal-fetal interface. Importantly, the rate of maternal-to-fetal glucose transfer in this system closely approximated that measured in ex vivo perfused human placentas. Our "placenta-on-a-chip" platform represents an important advance in the development of new technologies to model and study the physiological complexity of the human placenta for a wide variety of applications. PMID:27229450

  5. Triazole fungicide tebuconazole disrupts human placental trophoblast cell functions.

    PubMed

    Zhou, Jinghua; Zhang, Jianyun; Li, Feixue; Liu, Jing

    2016-05-01

    Triazole fungicides are one of the top ten classes of current-use pesticides. Although exposure to triazole fungicides is associated with reproductive toxicity in mammals, limited information is available regarding the effects of triazole fungicides on human placental trophoblast function. Tebuconazole (TEB) is a common triazole fungicide that has been extensively used for fungi control. In this work, we showed that TEB could reduce cell viability, disturb normal cell cycle distribution and induce apoptosis of human placental trophoblast cell line HTR-8/SVneo (HTR-8). Bcl-2 protein expression decreased and the level of Bax protein increased after TEB treatment in HTR-8 cells. The results demonstrated that this fungicide induced apoptosis of trophoblast cells via mitochondrial pathway. Importantly, we found that the invasive and migratory capacities of HTR-8 cells decreased significantly after TEB administration. TEB altered the expression of key regulatory genes involved in the modulation of trophoblast functions. Taken together, TEB suppressed human trophoblast invasion and migration through affecting the expression of protease, hormones, angiogenic factors, growth factors and cytokines. As the invasive and migratory abilities of trophoblast are essential for successful placentation and fetus development, our findings suggest a potential risk of triazole fungicides to human pregnancy. PMID:26852204

  6. Hemodynamic aspects of normal human feto-placental (umbilical) circulation.

    PubMed

    Acharya, Ganesh; Sonesson, Sven-Erik; Flo, Kari; Räsänen, Juha; Odibo, Anthony

    2016-06-01

    Understanding the changes in normal circulatory dynamics that occur during the course of pregnancy is essential for improving our knowledge of pathophysiological mechanisms associated with feto-placental diseases. The umbilical circulation is the lifeline of the fetus, and it is accessible for noninvasive assessment. However, not all hemodynamic parameters can be reliably measured in utero using currently available technology. Experimental animal studies have been crucial in validating major concepts related to feto-placental circulatory physiology, but caution is required in directly translating the findings of such studies into humans due to species differences. Furthermore, it is important to establish normal reference ranges and take into account gestational age associated changes while interpreting the results of clinical investigation. Therefore, it is necessary to critically evaluate, synthesize and summarize the knowledge available from the studies performed on human pregnancies to be able to appropriately apply them in clinical practice. This narrative review is an attempt to present contemporary concepts on hemodynamics of feto-placental circulation based on human studies. PMID:27130575

  7. Characterization of urokinase receptor expression by human placental trophoblasts.

    PubMed

    Zini, J M; Murray, S C; Graham, C H; Lala, P K; Karikó, K; Barnathan, E S; Mazar, A; Henkin, J; Cines, D B; McCrae, K R

    1992-06-01

    The processes of implantation and placentation are both dependent on the invasion and remodeling of the uterine endometrium and vasculature by trophoblasts. Because the secretion and autocrine binding of urokinase (uPA) appears to be a common mechanism used by cells to facilitate plasmin-dependent tissue invasion, we measured the production of uPA and expression of uPA receptors by trophoblasts. Prourokinase bound specifically, reversibly, and with high affinity to cultured trophoblasts, via the uPA epidermal growth factor-like domain. Trophoblasts derived from two first-trimester placentae bound more prourokinase than cells isolated from term placentae. Furthermore, in vitro differentiation of cultured cytotrophoblasts into syncytiotrophoblasts was associated with diminished expression of urokinase receptors and a parallel decrease in the cellular content of uPA receptor mRNA. Trophoblasts also secreted prourokinase and plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). Although prourokinase was secreted in amounts sufficient to endogenously saturate trophoblast uPA receptors, trophoblasts secreted greater amounts of PAI-1 and PAI-2 than uPA, and no net plasminogen activator activity was detected in trophoblast conditioned medium. In contrast, plasminogen added directly to cultured trophoblasts was readily converted to plasmin. Although the invasion and remodeling of uterine tissues by trophoblasts is a complex process dependent on several proteases of varying specificity, our findings suggest that the expression and modulation of urokinase receptors on the trophoblast cell surface may play an important role in this process. PMID:1316787

  8. Human placental trophoblast invasion and differentiation: a particular focus on Wnt signaling

    PubMed Central

    Knöfler, Martin; Pollheimer, Jürgen

    2013-01-01

    Wingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. Wnt signaling components also play crucial roles in murine placental development controlling trophoblast lineage determination, chorioallantoic fusion and placental branching morphogenesis. However, the role of the pathway in human placentation, trophoblast development and differentiation is only partly understood. Here, we summarize our present knowledge about Wnt signaling in the human placenta and discuss its potential role in physiological and aberrant trophoblast invasion, gestational diseases and choriocarcinoma formation. Differentiation of proliferative first trimester cytotrophoblasts into invasive extravillous trophoblasts is associated with nuclear recruitment of β -catenin and induction of Wnt-dependent T-cell factor 4 suggesting that canonical Wnt signaling could be important for the formation and function of extravillous trophoblasts. Indeed, activation of the pathway was shown to promote trophoblast invasion in different in vitro trophoblast model systems as well as trophoblast cell fusion. Methylation-mediated silencing of inhibitors of Wnt signaling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β -catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signaling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast invasion and incomplete spiral artery remodeling. Moreover, changes in Wnt signaling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, the current literature suggests a critical role of Wnt signaling in physiological and abnormal trophoblast function. PMID

  9. The Comparison of Adipose Stem Cell and Placental Stem Cell in Secretion Characteristics and in Facial Antiaging.

    PubMed

    Xu, Yan; Guo, Shilei; Wei, Cui; Li, Honglan; Chen, Lei; Yin, Chang; Zhang, Chuansen

    2016-01-01

    Background. Mesenchymal stem cells are the most commonly used seed cells in biomedical research and tissue engineering. Their secretory proteins have also been proven to play an important role in tissue healing. Methods. We isolated adipose stem cells and placental stem cells and performed analysis examining characteristics. The secretory proteins were extracted from conditioned medium and analyzed by MALDI-TOF/TOF. The antiaging effect of conditioned mediums was evaluated by the results of facial skin application. Results. Adipose stem cells and placental stem cells were found to be very similar in their surface markers and multipotency. The specific proteins secreted from adipose stem cells were more adept at cell adhesion, migration, wound healing, and tissue remodeling, while the proteins secreted by placental stem cells were more adept at angiogenesis, cell proliferation, differentiation, cell survival, immunomodulation, and collagen degradation. While these two types of conditioned medium could improve the facial index, the improvement of Melanin index after injection of the adipose stem cell conditioned medium was much more significant. Conclusion. The results suggest that the secreted proteins are ideal cell-free substances for regeneration medicine, especially in the antiaging field. PMID:27057176

  10. The Comparison of Adipose Stem Cell and Placental Stem Cell in Secretion Characteristics and in Facial Antiaging

    PubMed Central

    Xu, Yan; Guo, Shilei; Wei, Cui; Li, Honglan; Chen, Lei; Yin, Chang; Zhang, Chuansen

    2016-01-01

    Background. Mesenchymal stem cells are the most commonly used seed cells in biomedical research and tissue engineering. Their secretory proteins have also been proven to play an important role in tissue healing. Methods. We isolated adipose stem cells and placental stem cells and performed analysis examining characteristics. The secretory proteins were extracted from conditioned medium and analyzed by MALDI-TOF/TOF. The antiaging effect of conditioned mediums was evaluated by the results of facial skin application. Results. Adipose stem cells and placental stem cells were found to be very similar in their surface markers and multipotency. The specific proteins secreted from adipose stem cells were more adept at cell adhesion, migration, wound healing, and tissue remodeling, while the proteins secreted by placental stem cells were more adept at angiogenesis, cell proliferation, differentiation, cell survival, immunomodulation, and collagen degradation. While these two types of conditioned medium could improve the facial index, the improvement of Melanin index after injection of the adipose stem cell conditioned medium was much more significant. Conclusion. The results suggest that the secreted proteins are ideal cell-free substances for regeneration medicine, especially in the antiaging field. PMID:27057176

  11. Human placental cathepsin B1. Isolation and some physical properties

    PubMed Central

    Swanson, Arnold A.; Martin, Bill J.; Spicer, Sam S.

    1974-01-01

    A reproducible procedure for the isolation, from human placenta, of a cathepsin B1 in a homogeneous state, demonstrated by electrophoretic, ultracentrifugal and enzymic criteria, was carried out. The pH optimum was near pH5.5. The placental enzyme catalysed the release of acid-soluble u.v.-dense products from haemoglobin and myoglobin. It was inhibited by heavy metals and several compounds which react with the thiol groups. The optimum temperature was between 37° and 42°C. The molecular weight of the enzyme was calculated to be 24250. ImagesPLATE 1Fig. 5. PMID:4824207

  12. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... placental lactogen are used in the diagnosis and clinical management of high-risk pregnancies involving fetal distress associated with placental insufficiency. Measurements of HPL are also used in...

  13. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... placental lactogen are used in the diagnosis and clinical management of high-risk pregnancies involving fetal distress associated with placental insufficiency. Measurements of HPL are also used in...

  14. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... placental lactogen are used in the diagnosis and clinical management of high-risk pregnancies involving fetal distress associated with placental insufficiency. Measurements of HPL are also used in...

  15. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... placental lactogen are used in the diagnosis and clinical management of high-risk pregnancies involving fetal distress associated with placental insufficiency. Measurements of HPL are also used in...

  16. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... placental lactogen are used in the diagnosis and clinical management of high-risk pregnancies involving fetal distress associated with placental insufficiency. Measurements of HPL are also used in...

  17. Effect of microcystin-LR on human placental villous trophoblast differentiation in vitro.

    PubMed

    Douglas, Gordon C; Thirkill, Twanda L; Kumar, Priyadarsini; Loi, Minerva; Hilborn, Elizabeth D

    2016-04-01

    Microcystin-LR is a cyanobacterial toxin found in surface and recreational waters that inhibits protein phosphatases and may disrupt the cytoskeleton. Microcystins induce apoptosis in hepatocytes at ≤2.0 µM. Nothing is known about the effects of microcystins on human placental trophoblast differentiation and function. The differentiation of villous trophoblasts to form syncytiotrophoblast occurs throughout pregnancy and is essential for normal placental and fetal development. To investigate the effects of microcystin, villous cytotrophoblasts were isolated from term placentas using an established method and exposed to microcystin-LR. Microcystin-LR below the cytotoxic dose of 25 µM did not cause cell rounding or detachment, had no effect on apoptosis, and no effect on the morphological differentiation of mononucleated cytotrophoblasts to multinucleated syncytiotrophoblast. However, secretion of human chorionic gonadotropin (hCG) increased in a microcystin-LR dose-dependent manner. When incubated with l-buthionine sulphoximine (BSO) to deplete glutathione levels, trophoblast morphological differentiation proceeded normally in the presence of microcystin-LR. Microcystin-LR did not disrupt the trophoblast microtubule cytoskeleton, which is known to play a role in trophoblast differentiation. Immunofluorescence studies showed that trophoblasts express organic anion transport protein 1B3 (OATP1B3), a known microcystin transport protein. In comparison to hepatocytes, trophoblasts appear to be more resistant to the toxic effects of microcystin-LR. The physiological implications of increased hCG secretion in response to microcystin-LR exposure remain to be determined. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 427-439, 2016. PMID:25346179

  18. Virus-Free Human Placental Cell Lines To Study Genetic Functions | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.

  19. Up-regulation of placental leptin by human chorionic gonadotropin.

    PubMed

    Maymó, Julieta L; Pérez Pérez, Antonio; Sánchez-Margalet, Víctor; Dueñas, José L; Calvo, Juan Carlos; Varone, Cecilia L

    2009-01-01

    Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction

  20. Human placental perfusion method in the assessment of transplacental passage of antiepileptic drugs

    SciTech Connect

    Myllynen, Paeivi . E-mail: paivi.k.myllynen@oulu.fi; Pienimaeki, Paeivi; Vaehaekangas, Kirsi

    2005-09-01

    Epilepsy is one of the most common neurological diseases, affecting about 0.5 to 1% of pregnant women. It is commonly accepted that older antiepileptic drugs bear teratogenic potential. So far, no agreement has been reached about the safest antiepileptic drug during pregnancy. It is known that nearly all drugs cross the placenta at least to some extent. Nowadays, there is very little information available of the pharmacokinetics of drugs in the feto-placental unit. Detailed information about drug transport across the placenta would be valuable for the development of safe and effective treatments. For reasons of safety, human studies on placental transfer are restricted to a limited number of drugs. Interspecies differences limit the extrapolation of animal data to humans. Several in vitro methods for the study of placental transfer have been developed over the past decades. The placental perfusion method is the only experimental method that has been used to study human placental transfer of substances in organized placental tissue. The aim of this article is to review human placental perfusion data on antiepileptic drugs. According to perfusion data, it seems that most of the antiepileptic drugs are transferred across the placenta meaning significant fetal exposure.

  1. A prospective study to compare serum human placental lactogen and menstrual dates for determining gestational age.

    PubMed

    Whittaker, P G; Lind, T; Lawson, J Y

    1987-01-01

    In a group of 575 healthy pregnant women with certain menstrual dates the estimation of the length of gestation from maternal serum human placental lactogen concentrations has been compared with gestational age calculated from the last menstrual period and ultrasonic measurements of the fetal biparietal diameter. In 412 of these patients labor started spontaneously, and the estimated dates of delivery determined by these three methods were also compared. In the range of 9 to 17 weeks of pregnancy, gestational age can be determined by human placental lactogen measurement to within 7 days (+/- 1 SD) which compares favorably with other methods. Regarding the prediction of the expected date of delivery, 88% were delivered within 2 weeks of the date predicted by last menstrual period, 82% within 2 weeks of the sonar date, and 80% by the date determined by human placental lactogen assessment. Prediction of delivery in a further group of 139 women with uncertain dates gave 73% within 2 weeks by sonar date and 69% within 2 weeks by human placental lactogen determination. We suggest human placental lactogen measurements should become part of routine antenatal care complementing rather than replacing the role of ultrasonic scanning. For those doctors and patients who wish to avoid more exposure to ultrasonic scanning than absolutely necessary, human placental lactogen estimates offer an alternative method for assessing the length of gestation. PMID:3541617

  2. Dickkopf-1 induced apoptosis in human placental choriocarcinoma is independent of canonical Wnt signaling

    SciTech Connect

    Peng Sha; Miao Chenglin; Li Jing; Fan Xiujun; Cao Yujing; Duan Enkui . E-mail: duane@ioz.ac.cn

    2006-11-24

    Placental choriocarcinoma, a reproductive system carcinoma in women, has about 0.81% occurrence frequency in China, which leads to over 90% lethality due to indistinct pathogenesis and the absence of efficient therapeutic treatment. In the present study, using immunostaining and reverse transcription PCR, we reported that Dickkopf-1 (Dkk-1) is prominently expressed in human cytotrophoblast (CTB) cell, but absent in the human placental choriocarcinoma cell line JAR and JEG3, implicating an unknown correlation between Dkk-1 and carcinogenesis of placental choriocarcinoma. Further, through exogenous introduction of Dkk-1, we found repressed proliferation in JAR and JEG3, induced apoptosis in JAR, and discovered significant tumor suppression effects of Dkk-1 in placental choriocarcinoma. Moreover we found that this function of Dkk-1 is achieved through c-Jun N-terminal kinase (JNK), whereas the canonical Wnt pathway may not have a great role. This discovery is not symphonic to previous functional understanding of Dkk-1, a canonical Wnt signaling antagonist. Together, our data indicate the possible correlation between Dkk-1 and human placental choriocarcinoma and suggest potential applications of Dkk-1 in treatment of human placental choriocarcinomas.

  3. Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi.

    PubMed Central

    Aboagye-Mathiesen, G; Laugesen, J; Zdravkovic, M; Ebbesen, P

    1996-01-01

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

  4. Validation of murine and human placental explant cultures for use in sex steroid and phase II conjugation toxicology studies.

    PubMed

    Sato, Brittany L; Ward, Monika A; Astern, Joshua M; Kendal-Wright, Claire E; Collier, Abby C

    2015-02-01

    Human primary placental explant culture is well established for cytokine signaling and toxicity, but has not been validated for steroidogenic or metabolic toxicology. The technique has never been investigated in the mouse. We characterized human and mouse placental explants for up to 96 h in culture. Explant viability (Lactate dehydrogenase) and sex steroid levels were measured in media using spectrophotometry and ELISA, respectively. Expression and activities of the steroidogenic (3β-hydroxysteroid dehydrogenase, Cytochrome P45017A1, Cytochrome P45019), conjugation (UDP-glucuronosyltransferase, sulfotransferase (SULT)), and regeneration (β-glucuronidase, arylsulfatase C (ASC)) enzymes were determined biochemically in tissues with fluorimetric and spectrophotometric assays, and western blot. Explants were viable up to 96 h, but progesterone, estrone, and 17β-estradiol secretion decreased. Steroidogenic enzyme expression and activities were stable in mouse explants and similar to levels in freshly isolated tissues, but were lower in human explants than in fresh tissue (P<0.01). Human and mouse explants exhibited significantly less conjugation after 96 h, SULT was not detected in the mouse, and neither explants had active ASC, although proteins were expressed. Mouse explants may be useful for steroid biochemistry and endocrine disruption studies, but not metabolic conjugation. In contrast, human explants may be useful for studying conjugation for <48 h, but not for steroid/endocrine studies. PMID:25283089

  5. Validation of murine and human placental explant cultures for use in sex steroid and phase II conjugation toxicology studies

    PubMed Central

    Sato, Brittany L.; Ward, Monika A.; Astern, Joshua M.; Kendal-Wright, Claire E.; Collier, Abby C.

    2014-01-01

    Human primary placental explant culture is well established for cytokine signaling and toxicity, but has not been validated for steroidogenic or metabolic toxicology. The technique has never been investigated in the mouse. We characterized human and mouse placental explants for up to 96hr in culture. Explant viability (Lactate dehydrogenase) and sex steroid levels were measured in media using spectrophotometry and ELISA, respectively. Expression and activities of the steroidogenic (3β-hydroxysteroid dehydrogenase, Cytochrome P45017A1, Cytochrome P45019), conjugation (UDP-glucuronosyltransferase, sulfotransferase (SULT)), and regeneration (β-glucuronidase, arylsulfatase C (ASC)) enzymes were determined biochemically in tissues with fluorimetric and spectrophotometric assays, and western blot. Explants were viable up to 96hr, but progesterone, estrone, and 17β-estradiol secretion decreased. Steroidogenic enzyme expression and activities were stable in mouse explants and similar to levels in freshly isolated tissues, but were lower in human explants than in fresh tissue (P<0.01). Human and mouse explants exhibited significantly less conjugation after 96hr, SULT was not detected in the mouse, and neither explants had active ASC, although proteins were expressed. Mouse explants may be useful for steroid biochemistry and endocrine disruption studies, but not metabolic conjugation. In contrast, human explants may be useful for studying conjugation for <48hr, but not for steroid/endocrine studies. PMID:25283089

  6. A role for GPR55 in human placental venous endothelial cells

    PubMed Central

    Kremshofer, Julia; Siwetz, Monika; Berghold, Veronika M.; Lang, Ingrid; Huppertz, Berthold; Gauster, Martin

    2015-01-01

    Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labour. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placenta. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 μM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function. PMID:25869640

  7. A role for GPR55 in human placental venous endothelial cells.

    PubMed

    Kremshofer, Julia; Siwetz, Monika; Berghold, Veronika M; Lang, Ingrid; Huppertz, Berthold; Gauster, Martin

    2015-07-01

    Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labor. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placentas. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 µM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI-enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function. PMID:25869640

  8. Placental endoplasmic reticulum stress negatively regulates transcription of placental growth factor via ATF4 and ATF6β: implications for the pathophysiology of human pregnancy complications

    PubMed Central

    Mizuuchi, Masahito; Cindrova‐Davies, Tereza; Olovsson, Matts; Charnock‐Jones, D Stephen; Burton, Graham J

    2016-01-01

    Abstract Low maternal circulating concentrations of placental growth factor (PlGF) are one of the hallmarks of human pregnancy complications, including fetal growth restriction (FGR) and early‐onset pre‐eclampsia (PE). Currently, PlGF is used clinically with other biomarkers to screen for high‐risk cases, although the mechanisms underlying its regulation are largely unknown. Placental endoplasmic reticulum (ER) stress has recently been found to be elevated in cases of FGR, and to an even greater extent in early‐onset PE complicated with FGR. ER stress activates the unfolded protein response (UPR); attenuation of protein translation and a reduction in cell growth and proliferation play crucial roles in the pathophysiology of these complications of pregnancy. In this study, we further identified that ER stress regulates release of PlGF. We first observed that down‐regulation of PlGF protein was associated with nuclear localization of ATF4, ATF6α and ATF6β in the syncytiotrophoblast of placentae from PE patients. Transcript analysis showed a decrease of PlGF mRNA, and an increase from genes encoding those UPR transcription factors in placentae from cases of early‐onset PE, but not of late‐onset (>34 weeks) PE, compared to term controls. Further investigations indicated a strong correlation between ATF4 and PlGF mRNA levels only (r = − 0.73, p < 0.05). These results could be recapitulated in trophoblast‐like cells exposed to chemical inducers of ER stress or hypoxia–reoxygenation. The stability of PlGF transcripts was unchanged. The use of small interfering RNA specific for transcription factors in the UPR pathways revealed that ATF4 and ATF6β, but not ATF6α, modulate PlGF transcription. To conclude, ATF4 and ATF6β act synergistically in the negative regulation of PlGF mRNA expression, resulting in reduced PlGF secretion by the trophoblast in response to stress. Therefore, these results further support the targeting of placental ER stress as a

  9. Animal Models to Study Placental Development and Function throughout Normal and Dysfunctional Human Pregnancy

    PubMed Central

    Grigsby, Peta L.

    2016-01-01

    Abnormalities of placental development and function are known to underlie many pathologies of pregnancy, including spontaneous preterm birth, fetal growth restriction and preeclampsia. A growing body of evidence also underscores the importance of placental dysfunction in the lifelong health of both mother and offspring. However, our knowledge regarding placental structure and function throughout pregnancy remains limited. Understanding the temporal growth and functionality of the human placenta throughout the entirety of gestation is important if we are to gain a better understanding of placental dysfunction. The utilization of new technologies and imaging techniques that could enable safe monitoring of placental growth and function in vivo has become a major focus area for the National Institutes of Child Health & Human Development, as evident by the establishment of the “Human Placenta Project”. Many of the objectives of the Human Placenta Project will necessitate pre-clinical studies and testing in appropriately designed animal models that can be readily translated to the clinical setting. This review will describe the advantages and limitations of relevant animals such as the guinea pig, sheep and non-human primate models that have been used to study the role of the placenta in fetal growth disorders, preeclampsia or other maternal diseases during pregnancy. PMID:26752715

  10. Random Secretion of Growth Hormone in Humans

    NASA Astrophysics Data System (ADS)

    Prank, Klaus; Kloppstech, Mirko; Nowlan, Steven J.; Sejnowski, Terrence J.; Brabant, Georg

    1996-08-01

    In normal humans, growth hormone (GH) is secreted from a gland located adjacent to the brain (pituitary) into the blood in distinct pulses, but in patients bearing a tumor within the pituitary (acromegaly) GH is excessively secreted in an irregular manner. It has been hypothesized that GH secretion in the diseased state becomes random. This hypothesis is supported by demonstrating that GH secretion in patients with acromegaly cannot be distinguished from a variety of linear stochastic processes based on the predictability of the fluctuations of GH concentration in the bloodstream.

  11. Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion

    PubMed Central

    Astern, J.M.; Collier, A.C.; Kendal-Wright, C.E.

    2012-01-01

    Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation. PMID:23151382

  12. Placental membrane aging and HMGB1 signaling associated with human parturition

    PubMed Central

    Menon, Ramkumar; Behnia, Faranak; Polettini, Jossimara; Saade, George R; Campisi, Judith; Velarde, Michael

    2016-01-01

    Aging is associated with the onset of several diseases in various organ systems; however, different tissues may age differently, rendering some of them dysfunctional sooner than others. Placental membranes (fetal amniochorionic membranes) protect the fetus throughout pregnancy, but their longevity is limited to the duration of pregnancy. The age-associated dysfunction of these membranes is postulated to trigger parturition. Here, we investigated whether cellular senescence—the loss of cell division potential as a consequence of stress—is involved in placental membrane function at term. We show telomere reduction, p38 MAPK activation, increase in p21 expression, loss of lamin B1 loss, increase in SA-β-galactosidase, and senescence-associated secretory phenotype (SASP) gene expression in placental membranes after labor and delivery (term labor [TL]) compared to membranes prior to labor at term (term, not-in-labor [TNIL]). Exposing TNIL placental membranes to cigarette smoke extract, an oxidative stress inducer, also induced markers of cellular senescence similar to those in TL placental membranes. Bioinformatics analysis of differentially expressed SASP genes revealed HMGB1 signaling among the top pathways involved in labor. Further, we show that recombinant HMGB1 upregulates the expression of genes associated with parturition in myometrial cells. These data suggest that the natural physiologic aging of placental tissues is associated with cellular senescence and human parturition. PMID:26851389

  13. Animal Models to Study Placental Development and Function throughout Normal and Dysfunctional Human Pregnancy.

    PubMed

    Grigsby, Peta L

    2016-01-01

    Abnormalities of placental development and function are known to underlie many pathologies of pregnancy, including spontaneous preterm birth, fetal growth restriction, and preeclampsia. A growing body of evidence also underscores the importance of placental dysfunction in the lifelong health of both mother and offspring. However, our knowledge regarding placental structure and function throughout pregnancy remains limited. Understanding the temporal growth and functionality of the human placenta throughout the entirety of gestation is important if we are to gain a better understanding of placental dysfunction. The utilization of new technologies and imaging techniques that could enable safe monitoring of placental growth and function in vivo has become a major focus area for the National Institutes of Child Health and Human Development, as evident by the establishment of the "Human Placenta Project." Many of the objectives of the Human Placenta Project will necessitate preclinical studies and testing in appropriately designed animal models that can be readily translated to the clinical setting. This review will describe the advantages and limitations of relevant animals such as the guinea pig, sheep, and nonhuman primate models that have been used to study the role of the placenta in fetal growth disorders, preeclampsia, or other maternal diseases during pregnancy. PMID:26752715

  14. Human placental cell and tissue uptake of doxorubicin and its liposomal formulations.

    PubMed

    Soininen, Suvi K; Repo, Jenni K; Karttunen, Vesa; Auriola, Seppo; Vähäkangas, Kirsi H; Ruponen, Marika

    2015-12-01

    The anticancer drug doxorubicin and its liposomal formulations are in clinical use, doxorubicin also during pregnancy. However, little is known about how doxorubicin and its liposomal formulations are taken up by placental cells and whether they can cross human placenta. We therefore investigated quantitative cellular uptake and toxicity of doxorubicin and its two liposomal formulations, pH-sensitive liposomal doxorubicin (L-DOX) and commercially available pegylated liposomal doxorubicin (PL-DOX), in human placental choriocarcinoma (BeWo) cells. PL-DOX showed significantly lower cellular uptake and toxicity compared with doxorubicin and L-DOX. In preliminary studies with human placental perfusion, PL-DOX did not cross the placenta at all in 4h, whereas doxorubicin and L-DOX crossed the placenta at low levels (max 12% of the dose). Furthermore, PL-DOX did not accumulate in placental tissue while doxorubicin did (up to 70% of the dose). Surface pegylation probably explains the low placental cell and tissue uptake of PL-DOX. Formulation of doxorubicin thus seems to enable a decrease of fetal exposure. PMID:26383631

  15. Cathepsin B, cathepsin L, and cystatin C in the porcine uterus and placenta: potential roles in endometrial/placental remodeling and in fluid-phase transport of proteins secreted by uterine epithelia across placental areolae.

    PubMed

    Song, Gwonhwa; Bailey, Daniel W; Dunlap, Kathrin A; Burghardt, Robert C; Spencer, Thomas E; Bazer, Fuller W; Johnson, Greg A

    2010-05-01

    Cathepsins (CTSB and CTSL1) and their inhibitor, cystatin C (CST3), remodel uterine endometrium and placenta for transport of gases, micronutrients, and macromolecules essential for development and growth of the conceptus (embryo/fetus and placental membranes). We examined the temporal/spatial control of expression for CTSB, CTSL1, and CST3 mRNAs in endometria and placentae of pigs using three developmental models: 1) pigs were hysterectomized during the estrous cycle or pregnancy; 2) cyclic pigs were injected with estrogen to induce pseudopregnancy and were hysterectomized; and 3) pigs were ovariectomized, injected with progesterone, and hysterectomized. The abundance of CTSB, CTSL1, and CST3 mRNAs increased in endometrial epithelia during pregnancy and in response to exogenous progesterone but not estrogen. CST3 was also expressed in cells scattered within the stratum compactum stroma. Progesterone decreased epithelial but increased stromal compartment expression of CST3. CTSB increased in all chorionic epithelia, but CTSL1 was limited to chorionic epithelia that form areolae to absorb secretions from uterine glands. Based on the placental and endometrial distribution of CTSL1, we examined expression in the neonatal enterocytes known to transport immunoglobulins from colostrum. CTSL1 was also expressed in enterocytes of intestine from neonatal piglets. Therefore, CTSL1 is expressed by endometrial epithelia, placental areolae, and neonatal intestine, and it may function in the transport of macromolecules across these epithelia. Our results support the idea that reciprocal interactions between CSTL1, CTSB, and CST3 may be required to remodel endometrial and placental tissues for close apposition between maternal and fetal vasculatures and to facilitate transplacental transport of gases, micronutrients (amino acids, glucose), and macromolecules (proteins). Cysteine proteases and their inhibitors may also specifically modify proteins for successful utilization and

  16. Different metabolic activity in placental and reflected regions of the human amniotic membrane.

    PubMed

    Banerjee, Asmita; Weidinger, Adelheid; Hofer, Martin; Steinborn, Ralf; Lindenmair, Andrea; Hennerbichler-Lugscheider, Simone; Eibl, Johann; Redl, Heinz; Kozlov, Andrey V; Wolbank, Susanne

    2015-11-01

    Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications. PMID:26386652

  17. Effect of Microcystin-LR on human placental villous trophoblast differentiation in vitro

    EPA Science Inventory

    Microcystin-LR is a cyanobacterial toxin found in surface and recreational waters that inhibits protein phosphatases and may disrupt the cytoskeleton. Microcystins induce apoptosis in hepatocytes at ≤2.0 μM. Nothing is known about the effects of microcystins on human placental tr...

  18. Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

    PubMed

    Cindrova-Davies, T; van Patot, M Tissot; Gardner, L; Jauniaux, E; Burton, G J; Charnock-Jones, D S

    2015-03-01

    Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions. PMID:25391298

  19. Effect of leptin on progesterone, human chorionic gonadotropin, and interleukin-6 secretion by human term trophoblast cells in culture.

    PubMed

    Cameo, Paula; Bischof, Paul; Calvo, Juan Carlos

    2003-02-01

    Leptin, the 16-kDa protein product of the obese gene, was originally seen as an adipocyte-derived signaling molecule. Recently, it has been suggested to be involved in some functions during pregnancy, particularly in the placenta. In the present study, we investigated the role of leptin in the secretion of hCG, progesterone, and interleukin-6 (IL-6) by human term trophoblast cells in culture. Placentae were obtained from cesarean sections following uncomplicated pregnancies and used immediately after delivery. Leptin, hCG, progesterone, and IL-6 were measured by ELISA, RIA, and immunoradiometric assay in the cultured media of trophoblast cells cultured for 48 and 96 h. Leptin mRNA expression in these cultures was determined by reverse transcription-polymerase chain reaction. Recombinant human leptin added to primary cultures of human term placental trophoblast cells showed a stimulatory effect on hCG and IL-6 secretion and an inhibitory effect on progesterone secretion. Primary cultures of term trophoblast cells expressed leptin mRNA. All these findings suggest a role for leptin in human placental endocrine function. PMID:12533410

  20. Recent progress towards understanding the role of DNA methylation in human placental development.

    PubMed

    Bianco-Miotto, Tina; Mayne, Benjamin T; Buckberry, Sam; Breen, James; Rodriguez Lopez, Carlos M; Roberts, Claire T

    2016-07-01

    Epigenetic modifications, and particularly DNA methylation, have been studied in many tissues, both healthy and diseased, and across numerous developmental stages. The placenta is the only organ that has a transient life of 9 months and undergoes rapid growth and dynamic structural and functional changes across gestation. Additionally, the placenta is unique because although developing within the mother, its genome is identical to that of the foetus. Given these distinctive characteristics, it is not surprising that the epigenetic landscape affecting placental gene expression may be different to that in other healthy tissues. However, the role of epigenetic modifications, and particularly DNA methylation, in placental development remains largely unknown. Of particular interest is the fact that the placenta is the most hypomethylated human tissue and is characterized by the presence of large partially methylated domains (PMDs) containing silenced genes. Moreover, how and why the placenta is hypomethylated and what role DNA methylation plays in regulating placental gene expression across gestation are poorly understood. We review genome-wide DNA methylation studies in the human placenta and highlight that the different cell types that make up the placenta have very different DNA methylation profiles. Summarizing studies on DNA methylation in the placenta and its relationship with pregnancy complications are difficult due to the limited number of studies available for comparison. To understand the key steps in placental development and hence what may be perturbed in pregnancy complications requires large-scale genome-wide DNA methylation studies coupled with transcriptome analyses. PMID:27026712

  1. Framing Postpartum Hemorrhage as a Consequence of Human Placental Biology: An Evolutionary and Comparative Perspective

    PubMed Central

    Abrams, Elizabeth; Rutherford, Julienne

    2011-01-01

    Postpartum hemorrhage (PPH), the leading cause of maternal mortality worldwide, is responsible for 35 percent of maternal deaths. Proximately, PPH results from the failure of the placenta to separate from the uterine wall properly, most often because of impairment of uterine muscle contraction. Despite its prevalence and its well-described clinical manifestations, the ultimate causes of PPH are not known and have not been investigated through an evolutionary lens. We argue that vulnerability to PPH stems from the intensely invasive nature of human placentation. The human placenta causes uterine vessels to undergo transformation to provide the developing fetus with a high plane of maternal resources; the degree of this transformation in humans is extensive. We argue that the particularly invasive nature of the human placenta increases the possibility of increased blood loss at parturition. We review evidence suggesting PPH and other placental disorders represent an evolutionarily novel condition in hominins. PMID:21909154

  2. Genetic recapitulation of human pre-eclampsia risk during convergent evolution of reduced placental invasiveness in eutherian mammals

    PubMed Central

    Elliot, Michael G.; Crespi, Bernard J.

    2015-01-01

    The relationship between phenotypic variation arising through individual development and phenotypic variation arising through diversification of species has long been a central question in evolutionary biology. Among humans, reduced placental invasion into endometrial tissues is associated with diseases of pregnancy, especially pre-eclampsia, and reduced placental invasiveness has also evolved, convergently, in at least 10 lineages of eutherian mammals. We tested the hypothesis that a common genetic basis underlies both reduced placental invasion arising through a developmental process in human placental disease and reduced placental invasion found as a derived trait in the diversification of Euarchontoglires (rodents, lagomorphs, tree shrews, colugos and primates). Based on whole-genome analyses across 18 taxa, we identified 1254 genes as having evolved adaptively across all three lineages exhibiting independent evolutionary transitions towards reduced placental invasion. These genes showed strong evidence of enrichment for associations with pre-eclampsia, based on genetic-association studies, gene-expression analyses and gene ontology. We further used in silico prediction to identify a subset of 199 genes that are likely targets of natural selection during transitions in placental invasiveness and which are predicted to also underlie human placental disorders. Our results indicate that abnormal ontogenies can recapitulate major phylogenetic shifts in mammalian evolution, identify new candidate genes for involvement in pre-eclampsia, imply that study of species with less-invasive placentation will provide useful insights into the regulation of placental invasion and pre-eclampsia, and recommend a novel comparative functional-evolutionary approach to the study of genetically based human disease and mammalian diversification. PMID:25602073

  3. IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

    PubMed

    Abumaree, M H; Brownbill, P; Burton, G; Castillo, C; Chamley, L; Croy, B A; Drewlo, S; Dunk, C; Girard, S; Hansson, S; Jones, S; Jurisicova, A; Lewis, R; Letarte, M; Parast, M; Pehrson, C; Rappolee, D; Schneider, H; Tannetta, D; Varmuza, S; Wadsack, C; Wallace, A E; Zenerino, C; Lash, G E

    2014-02-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue. PMID:24321780

  4. Mono-2-Ethylhexyl Phthalate Induces Oxidative Stress Responses in Human Placental Cells In Vitro

    PubMed Central

    Tetz, Lauren M; Cheng, Adrienne A.; Korte, Cassandra S.; Giese, Roger W.; Wang, Poguang; Harris, Craig; Meeker, John D; Loch-Caruso, Rita

    2013-01-01

    Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180 μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180 μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes. PMID:23360888

  5. Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro

    SciTech Connect

    Tetz, Lauren M.; Cheng, Adrienne A.; Korte, Cassandra S.; Giese, Roger W.; Wang, Poguang; Harris, Craig; Meeker, John D.; Loch-Caruso, Rita

    2013-04-01

    Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180 μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180 μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes. - Highlights: ► MEHP increased reactive oxygen species, oxidative DNA damage, and caspase activity. ► MEHP induced expression of PTGS2, a gene

  6. An international network (PlaNet) to evaluate a human placental testing platform for chemicals safety testing in pregnancy.

    PubMed

    Brownbill, Paul; Chernyavsky, Igor; Bottalico, Barbara; Desoye, Gernot; Hansson, Stefan; Kenna, Gerry; Knudsen, Lisbeth E; Markert, Udo R; Powles-Glover, Nicola; Schneider, Henning; Leach, Lopa

    2016-09-01

    The human placenta is a critical life-support system that nourishes and protects a rapidly growing fetus; a unique organ, species specific in structure and function. We consider the pressing challenge of providing additional advice on the safety of prescription medicines and environmental exposures in pregnancy and how ex vivo and in vitro human placental models might be advanced to reproducible human placental test systems (HPTSs), refining a weight of evidence to the guidance given around compound risk assessment during pregnancy. The placental pharmacokinetics of xenobiotic transfer, dysregulated placental function in pregnancy-related pathologies and influx/efflux transporter polymorphisms are a few caveats that could be addressed by HPTSs, not the specific focus of current mammalian reproductive toxicology systems. An international consortium, "PlaNet", will bridge academia, industry and regulators to consider screen ability and standardisation issues surrounding these models, with proven reproducibility for introduction into industrial and clinical practice. PMID:27327413

  7. Epigenetic regulation of human placental function and pregnancy outcome: considerations for causal inference.

    PubMed

    Januar, Vania; Desoye, Gernot; Novakovic, Boris; Cvitic, Silvija; Saffery, Richard

    2015-10-01

    Epigenetic mechanisms, often defined as regulating gene activity independently of underlying DNA sequence, are crucial for healthy development. The sum total of epigenetic marks within a cell or tissue (the epigenome) is sensitive to environmental influence, and disruption of the epigenome in utero has been associated with adverse pregnancy outcomes. Not surprisingly, given its multifaceted functions and important role in regulating pregnancy outcome, the placenta shows unique epigenetic features. Interestingly however, many of these are only otherwise seen in human malignancy (the pseudomalignant placental epigenome). Epigenetic variation in the placenta is now emerging as a candidate mediator of environmental influence on placental functioning and a key regulator of pregnancy outcome. However, replication of findings is generally lacking, most likely due to small sample sizes and a lack of standardization of analytical approaches. Defining DNA methylation "signatures" in the placenta associated with maternal and fetal outcomes offers tremendous potential to improve pregnancy outcomes, but care must be taken in interpretation of findings. Future placental epigenetic research would do well to address the issues present in epigenetic epidemiology more generally, including careful consideration of sample size, potentially confounding factors, issues of tissue heterogeneity, reverse causation, and the role of genetics in modulating epigenetic profile. The importance of animal or in vitro models in establishing a functional role of epigenetic variation identified in human beings, which is key to establishing causation, should not be underestimated. PMID:26428498

  8. Cross talk between cAMP and p38 MAPK pathways in the induction of leptin by hCG in human placental syncytiotrophoblasts.

    PubMed

    Ge, Y C; Li, J N; Ni, X T; Guo, C M; Wang, W S; Duan, T; Sun, K

    2011-08-01

    Leptin produced by the placental syncytiotrophoblasts participates in a number of processes in pregnancy including implantation, proliferation of the cytotrophoblasts, and nutrient transfer across the placenta. Despite the functional significance of leptin in pregnancy, the regulation of leptin synthesis is poorly understood in human placental syncytiotrophoblasts. In this study, we investigated the role of endogenous human chorionic gonadotropin (hCG) in the regulation of leptin production as well as the underlying mechanism involving the cross talk between cAMP and p38 mitogen-activated protein kinase (MAPK) pathways. We found that neutralization of endogenous hCG with its antibody dose dependently decreased leptin mRNA level and secretion, whereas exogenous hCG increased leptin mRNA level and secretion. Activation of the cAMP pathway with dibutyryl cAMP (db cAMP) or forskolin recapitulated the stimulatory effect of hCG on leptin expression. Inhibition of protein kinase A with H89 not only reduced the basal leptin expression but also attenuated the induced leptin expression by hCG. Treatment of the syncytiotrophoblasts with db cAMP and hCG phosphorylated p38 MAPK. Inhibition of p38 MAPK with SB203580 not only reduced the basal leptin production but also attenuated the leptin-induced production by both hCG and db cAMP. These data suggest that endogenous hCG plays a significant role in maintaining leptin production in human placental syncytiotrophoblasts, and this effect involves a cross talk between cAMP and p38 MAPK pathways. PMID:21562093

  9. Vitamin C Supplementation Ameliorates the Adverse Effects of Nicotine on Placental Hemodynamics and Histology in Non-Human Primates

    PubMed Central

    Lo, Jamie O.; Schabel, Matthias C.; Roberts, Victoria H.J.; Morgan, Terry K.; Rasanen, Juha P.; Kroenke, Christopher D.; Shoemaker, Ms. Sophie R.; Spindel, Eliot R.; Frias, Antonio E.

    2015-01-01

    Objective We previously demonstrated that prenatal nicotine exposure decreases neonatal pulmonary function in non-human primates (NHP) and maternal Vitamin C supplementation attenuates these deleterious effects. However, nicotine’s effect on placental perfusion and development is not fully understood. This study utilizes non-invasive imaging techniques and histological analysis in a NHP model to test the hypothesis that prenatal nicotine exposure adversely effects placental hemodynamics and development, but is ameliorated by Vitamin C. Study Design Time-mated macaques (n=27) in 4 treatment groups: control (n=5), nicotine only (n=4), Vitamin C only (n=9), and nicotine plus Vitamin C (n=9). Nicotine animals received 2mg/kg/day of nicotine bitartrate (~0.7mg/kg/day free nicotine levels in pregnant human smokers) from days 26–160 (term, 168 days). Vitamin C groups received ascorbic acid at 50, 100 or 250mg/kg/day with or without nicotine. All underwent placental Dynamic Contrast-Enhanced MRI (DCE-MRI) at 135–140 days and Doppler ultrasound at 155 days to measure uterine artery and umbilical vein velocimetry and diameter to calculate uterine artery volume blood flow (cQuta) and placental volume blood flow (cQuv). Animals were delivered by cesarean section at 160 days. A novel DCE-MRI protocol was utilized to calculate placental perfusion from maternal spiral arteries. Placental tissue was processed for histopathology. Results Placental volume blood flow (cQuv) was significantly reduced in nicotine only animals compared with controls and nicotine plus Vitamin C groups (p=0.03). Maternal placental blood flow was not different between experimental groups by DCE-MRI ranging from 0.75–1.94 ml/ml/min (p=0.93). Placental histology showed increased numbers of villous cytotrophoblast cell islands (p<0.05) and increased syncytiotrophoblast sprouting (p<0.001) in nicotine only animals, which was mitigated by Vitamin C. Conclusion Prenatal nicotine exposure significantly

  10. Zika Virus Targets Different Primary Human Placental Cells, Suggesting Two Routes for Vertical Transmission.

    PubMed

    Tabata, Takako; Petitt, Matthew; Puerta-Guardo, Henry; Michlmayr, Daniela; Wang, Chunling; Fang-Hoover, June; Harris, Eva; Pereira, Lenore

    2016-08-10

    Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but mother-to-fetus transmission routes are unknown. We infected different primary cell types from mid- and late-gestation placentas and explants from first-trimester chorionic villi with the prototype Ugandan and a recently isolated Nicaraguan ZIKV strain. ZIKV infects primary human placental cells and explants-cytotrophoblasts, endothelial cells, fibroblasts, and Hofbauer cells in chorionic villi and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes-that express Axl, Tyro3, and/or TIM1 viral entry cofactors. ZIKV produced NS3 and E proteins and generated higher viral titers in amniotic epithelial cells from mid-gestation compared to late-gestation placentas. Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants. Our results suggest that ZIKV spreads from basal and parietal decidua to chorionic villi and amniochorionic membranes and that targeting TIM1 could suppress infection at the uterine-placental interface. PMID:27443522

  11. Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

    PubMed

    Sarkar, Prasenjit; Mischler, Adam; Randall, Shan M; Collier, Timothy S; Dorman, Karen F; Boggess, Kim A; Muddiman, David C; Rao, Balaji M

    2016-08-01

    Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development. PMID:27378238

  12. Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion.

    PubMed

    Astern, J M; Collier, A C; Kendal-Wright, C E

    2013-01-01

    Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2',7'-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation. PMID:23151382

  13. Placental Transfer of Rilpivirine in an Ex Vivo Human Cotyledon Perfusion Model

    PubMed Central

    Duro, Dominique; Belissa, Emilie; Peytavin, Gilles

    2015-01-01

    Placental transfers of the HIV nonnucleoside reverse transcriptase inhibitor rilpivirine were investigated in 8 term human cotyledons perfused with rilpivirine (400 ng/ml) in the maternal-to-fetal direction. The mean fetal transfer rate (FTR) (fetal/maternal concentration at steady state from 15 to 90 min) was 26% ± 8% (mean ± standard deviation), and the clearance index (rilpivirine FTR/antipyrine FTR) was 61% ± 20%. This shows that rilpivirine crosses the placenta at a relatively high rate, suggesting that the fetus is exposed to the compound during treatment of the mother. PMID:25691637

  14. TNF-α alters the inflammatory secretion profile of human first trimester placenta.

    PubMed

    Siwetz, Monika; Blaschitz, Astrid; El-Heliebi, Amin; Hiden, Ursula; Desoye, Gernot; Huppertz, Berthold; Gauster, Martin

    2016-04-01

    Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal

  15. Equine placentation.

    PubMed

    Allen, W R; Stewart, F

    2001-01-01

    A tough, elastic glycoprotein capsule envelops the equine blastocyst between Days 6 and 23 after ovulation. It maintains the spherical configuration of, and provides physical support for, the embryo as it traverses the entire uterine lumen during Days 6-17, propelled by myometrial contractions that are stimulated by pulsatile release of prostaglandin F2alpha and prostaglandin E2. The capsule also accumulates constituents of the exocrine secretions of the endometrial glands ('uterine milk') as nutrients for the mobile embryo as it releases its antiluteolytic maternal recognition-of-pregnancy signal to the whole of the surface of the endometrium. Mobility ceases abruptly on Day 17 with a sudden increase in uterine tonicity that 'fixes' the conceptus at the base of one of the uterine horns. At Day 35, the trophoblast of the spherical conceptus has separated into its invasive and non-invasive components. The former, distinguished as the thickened, annulate chorionic girdle, invades the maternal endometrium to form the unique endometrial cups. These secrete a chorionic gonadotrophin that synergizes with pituitary follicle-stimulating hormone to induce secondary luteal development in the maternal ovaries. The cup cells express foreign fetal antigens that stimulate strong maternal humoral and cell-mediated immune responses, which curtail their lifespan. The non-invasive trophoblast of the allantochorion establishes a stable microvillous contact with the endometrial epithelium around Day 40 and, over the next 100 days, develops a complex multibranched interdigitation with the endometrium to form the microcotyledonary haemotrophic exchange units that cover the entire surface of the diffuse epitheliochorial placenta. Reduction in the effective total area of fetomaternal contact at this placental interface, by competition between twin conceptuses for the limited area of available endometrium, by attachment of the allantochorion to an imperfect endometrium in a mare with

  16. NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokołowska, Ewa; Hallmann, Anna; Kaletha, Krystian; Klimek, Jerzy

    2008-06-01

    During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions. PMID:18499441

  17. Unsupervised Placental Gene Expression Profiling Identifies Clinically Relevant Subclasses of Human Preeclampsia.

    PubMed

    Leavey, Katherine; Benton, Samantha J; Grynspan, David; Kingdom, John C; Bainbridge, Shannon A; Cox, Brian J

    2016-07-01

    Preeclampsia (PE) is a complex, hypertensive disorder of pregnancy, demonstrating considerable variability in maternal symptoms and fetal outcomes. Unfortunately, prior research has not accounted for this variability, resulting in a lack of robust biomarkers and effective treatments for PE. Here, we created a large (N=330) clinically relevant human placental microarray data set, consisting of 7 previously published studies and 157 highly annotated new samples from a single BioBank. Applying unsupervised clustering to this combined data set identified 3 clinically significant probable etiologies of PE: "maternal", with healthy placentas and term deliveries; "canonical", exhibiting expected clinical, ontological, and histopathologic features of PE; and "immunologic" with severe fetal growth restriction and evidence of maternal antifetal rejection. Moreover, these groups could be distinguished using a small quantitative polymerase chain reaction panel and demonstrated varying influence of maternal factors on PE development. An additional subclass of PE placentas was also revealed to form because of chromosomal abnormalities in these samples, supported by array-based comparative genomic hybridization analysis. Overall, our findings represent a new paradigm in our understanding of the origins and maternal-placental contributions to the pathology of PE. The study of PE represents a unique opportunity to access human tissue associated with a complex hypertensive disorder, and our novel approach could be applied to other hypertensive and heterogeneous human diseases. PMID:27160201

  18. Effect of placental factors on growth and function of the human fetal adrenal in vitro

    SciTech Connect

    Riopel, L.; Branchaud, C.L.; Goodyer, C.G.; Zweig, M.; Lipowski, L.; Adkar, V.; Lefebvre, Y. )

    1989-11-01

    Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: (1) maximal response to PM was 2-5 times greater; (2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; (3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.

  19. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects. PMID:26709042

  20. Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection.

    PubMed

    Bayer, Avraham; Lennemann, Nicholas J; Ouyang, Yingshi; Bramley, John C; Morosky, Stefanie; Marques, Ernesto Torres De Azeved; Cherry, Sara; Sadovsky, Yoel; Coyne, Carolyn B

    2016-05-11

    During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier. PMID:27066743

  1. Toxic effects of low doses of Bisphenol-A on human placental cells

    SciTech Connect

    Benachour, Nora; Aris, Aziz

    2009-12-15

    Humans are exposed daily to a great number of xenobiotics and their metabolites present as pollutants. Bisphenol-A (BPA) is extensively used in a broad range of products including baby bottles, food-storage containers, medical equipment, and consumer electronics. Thus, BPA is the most common monomer for polycarbonates intended for food contact. Levels of this industrial product are found in maternal blood, amniotic fluid, follicular fluid, placental tissue, umbilical cord blood, and maternal urine. In this study, we investigated toxic effects of BPA concentrations close to levels found in serum of pregnant women on human cytotrophoblasts (CTB). These cells were isolated from fresh placentas and exposed to BPA for 24 h. Our results showed that very low doses of BPA induce apoptosis (2 to 3 times) as assessed using M30 antibody immunofluorescent detection, and necrosis (1.3 to 1.7 times) as assessed through the cytosolic Adenylate Kinase (AK) activity after cell membrane damage. We also showed that BPA increased significantly the tumor-necrosis factor alpha (TNF-alpha) gene expression and protein excretion as measured by real-time RT-PCR and ELISA luminescent test, respectively. Moreover, we observed that induction of AK activation and TNF-alpha gene expression require lower levels of BPA than apoptosis or TNF-alpha protein excretion. Our findings suggest that exposure of placental cells to low doses of BPA may cause detrimental effects, leading in vivo to adverse pregnancy outcomes such as preeclampsia, intrauterine growth restriction, prematurity and pregnancy loss.

  2. Effect of Prostaglandin E2 on Multidrug Resistance Transporters In Human Placental Cells

    PubMed Central

    Lee, Gene T.; Dong, Yafeng; Zhou, Helen; He, Lily; Weiner, Carl P.

    2014-01-01

    Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades. PMID:25261564

  3. Effect of prostaglandin E2 on multidrug resistance transporters in human placental cells.

    PubMed

    Mason, Clifford W; Lee, Gene T; Dong, Yafeng; Zhou, Helen; He, Lily; Weiner, Carl P

    2014-12-01

    Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades. PMID:25261564

  4. A human monoclonal antibody specific to placental alkaline phosphatase, a marker of ovarian cancer

    PubMed Central

    Ravenni, Niccolò; Weber, Marcel; Neri, Dario

    2014-01-01

    Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads. PMID:24247025

  5. CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues

    SciTech Connect

    Matouskova, Magda; Blazkova, Jana; Pajer, Petr; Pavlicek, Adam; Hejnar, Jiri . E-mail: hejnar@img.cas.cz

    2006-04-15

    Syncytin-1 is a captive envelope glycoprotein encoded by one of human endogenous retroviruses W. It is expressed exclusively in the placental trophoblast where it participates in cell-to-cell fusion during differentiation of syncytiotrophobast. In other tissues, however, syncytin-1 expression must be kept in check because inadvertent cell fusion might be dangerous for tissue organization and integrity. We describe here an inverse correlation between CpG methylation of syncytin-1 5' long terminal repeat and its expression. Hypomethylation of the syncytin-1 5' long terminal repeat in the placenta and in the choriocarcinoma-derived cell line BeWo was detected. However, other analyzed primary cells and cell lines non-expressing syncytin-1 contain proviruses heavily methylated in this sequence. CpG methylation of syncytin-1 is resistant to the effect of the demethylating agent 5-azacytidine. The inhibitory role of CpG methylation is further confirmed by transient transfection of in-vitro-methylated syncytin-1 promoter-driven reporter construct. Altogether, we conclude that CpG methylation plays a principal role in the transcriptional suppression of syncytin-1 in non-placental tissues, and, in contrast, demethylation of the syncytin-1 promoter in trophoblast is a prerequisite for its expression and differentiation of multinucleated syncytiotrophoblast.

  6. Functional evidence for oxygen-sensitive voltage-gated potassium channels in human placental vasculature.

    PubMed

    Kiernan, M F; Barrie, A; Szkolar, J; Mills, T A; Wareing, M

    2010-06-01

    Hypoxic fetoplacental vasoconstriction (HFPV), involving voltage-gated potassium (K(V)) channels, has been suggested in human placenta; the identity of these channels remains unclear. Using wire myography, chorionic plate blood vessels were exposed to isoform-specific K(V) channel blockers. Dose-response curves (thromboxane mimetic U46619; 0.1-2000 nM) pre- and post-addition of K(V) channel modulator were analysed. Arterial U46619-induced contraction increased with margatoxin and stromatoxin-1, whilst only correolide increased U46619-induced contraction in veins (P < 0.05 two-way ANOVA). Basal tone was unaffected in arteries or veins. These data implicate K(V)1.2 and/or K(V)2.1 and K(V)1.5 in the control of agonist-induced contraction of human placental arteries and veins respectively. PMID:20451247

  7. The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

    SciTech Connect

    Desforges, M.; Greenwood, S.L.; Glazier, J.D.; Westwood, M.; Sibley, C.P.

    2010-07-16

    Research highlights: {yields} mRNA levels for SNAT1 are higher than other system A subtype mRNAs in primary human cytotrophoblast. {yields} SNAT1 knockdown in cytotrophoblast cells significantly reduces system A activity. {yields} SNAT1 is a key contributor to system A-mediated amino acid transport in human placenta. -- Abstract: System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, {sup 14}C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of {sup 14}C-MeAIB uptake revealed two distinct transport systems; system 1: K{sub m} = 0.38 {+-} 0.12 mM, V{sub max} = 27.8 {+-} 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K{sub m} = 45.4 {+-} 25.0 mM, V{sub max} = 1190 {+-} 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific si

  8. Interaction between human placental microvascular endothelial cells and a model of human trophoblasts: effects on growth cycle and angiogenic profile.

    PubMed

    Troja, Weston; Kil, Kicheol; Klanke, Charles; Jones, Helen N

    2014-01-01

    Abstract Intrauterine growth restriction (IUGR) is a leading cause of perinatal complications, and is commonly associated with reduced placental vasculature. Recent studies demonstrated over-expression of IGF-1 in IUGR animal models maintains placental vasculature. However, the cellular environment of the placental chorionic villous is unknown. The close proximity of trophoblasts and microvascular endothelial cells in vivo alludes to autocrine/paracrine regulation following Ad-HuIGF-1 treatment. We investigated the co-culturing of BeWo Choriocarcinoma and Human Placental Microvascular Endothelial Cells (HPMVECs) on the endothelial angiogenic profile and the effect Ad-HuIGF-1 treatment of one cell has on the other. HPMVECs were isolated from human term placentas and cultured in EGM-2 at 37°C with 5% CO2. BeWo cells were maintained in Ham's F12 nutrient mix with 10% FBS and 1% pen/strep. Co-cultured HPMVECS+BeWo cells were incubated in serum-free control media, Ad-HuIGF-1, or Ad-LacZ at MOI 0 and MOI 100:1 for 48 h. Non-treated cells and mono-cultured cells were compared to co-cultured cells. Angiogenic gene expression and proliferative and apoptotic protein expression were analysed by RT-qPCR and immunocytochemistry, respectively. Statistical analyses was performed using student's t-test with P < 0.05 considered significant. Direct Ad-HuIGF-1 treatment increased HPMVEC proliferation (n = 4) and reduced apoptosis (n = 3). Co-culturing HPMVECs+BeWo cells significantly altered RNA expression of the angiogenic profile compared to mono-cultured HPMVECs (n = 8). Direct Ad-HuIGF-1 treatment significantly increased Ang-1 (n = 4) in BeWo cells. Ad-HuIGF-1 treatment of HPMVECs did not alter the RNA expression of angiogenic factors. Trophoblastic factors may play a key role in placental vascular development and IGF-1 may have an important role in HPMVEC growth. PMID:24760505

  9. Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

    PubMed

    Chatterjee, Piyali; Chiasson, Valorie L; Pinzur, Lena; Raveh, Shani; Abraham, Eytan; Jones, Kathleen A; Bounds, Kelsey R; Ofir, Racheli; Flaishon, Liat; Chajut, Ayelet; Mitchell, Brett M

    2016-04-01

    Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during

  10. Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

    PubMed Central

    Frochaux, Violette; Hildebrand, Diana; Talke, Anja; Linscheid, Michael W.; Schlüter, Hartmut

    2014-01-01

    The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM382↓383SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473. PMID:25329061

  11. Comparative placental transfer, localization, and effects of radionuclides in experimental animal and human pregnancies

    SciTech Connect

    Sikov, M.R.; Meznarich, H.K.; Traub, R.J.

    1991-11-01

    Estimating radiation doses to the human embryo/fetus from radionuclides and predicting effects requires extrapolation of data from studies of laboratory species, with scaling for species-specific developmental stage and gestational time relationships and maturities at birth. Combinations of fetal-to-maternal ratios of concentrations, patterns of deposition, transfer kinetics, and compartmental and physiologic models are used to predict radioactivity levels and radiation doses to the conceptus. There is agreement between values expressing fractional transfer across the placenta ({theta}) with tabulated values for fractional absorption (f{sub 1}) from gastrointestinal (GI) tract or lung for most substances commonly involved in metabolic processes. A tendency toward disagreement for some other materials is thought to involve explanations based on their physicochemistry, toxicity, or the influence of target tissue development on placental transfer kinetics.

  12. Review: Exploration of placentation from human beings to ocean-living species.

    PubMed

    Soma, H; Murai, N; Tanaka, K; Oguro, T; Kokuba, H; Yoshihama, I; Fujita, K; Mineo, S; Toda, M; Uchida, S; Mogoe, T

    2013-03-01

    This review covers four topics. 1) Placental pathology in Himalayan mountain people. To determine morphological changes of the placenta at high altitude, pathological examination was made of 1000 Himalayan placentas obtained in Nepal and Tibet and the results compared with Japanese placentas delivered at sea level. Characteristic findings in the placental villi of the Himalayan group included high incidences of villous chorangiosis and chorangioma. These processes were clarified by ultrastructural observation. 2) Placentation in Sirenians. The giant Takikawa sea cow, which lived 5 million years ago, was discovered on Hokkaido, Japan. It was an ancestor of the dugong as well as the manatees. Sirenia, the sea cow group, shares a common ancestor with Proboscidea, the elephants, even though they now inhabit quite different environments. A comparison was made of their zonary endothelial type of placentation. 3) Placentation in sharks and rays. The remarkable placentation of hammerhead sharks and manta rays is described. 4) Placentation in the Antarctic minke whale. Placental tissue samples of this whale were obtained from the Japan Institute of Cetacean Research. In an ultrastructural study of the utero-placental junction, microfilamental processes of the allantochorionic zone and crypt formation were visualized. PMID:23332416

  13. Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

    PubMed

    Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

    2015-12-01

    Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. PMID:26374858

  14. The role of placental indoleamine 2,3-dioxygenase in human pregnancy

    PubMed Central

    2013-01-01

    Munn et al. made a scientific observation of major biological importance. For the first time they showed that in the mammal the fetus does survive an immune attack mounted by the mother, and that the mechanism responsible for the survival depends on the fetus and placenta 'actively' defending itself from attack by maternal T cells by means of an enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42) dependent localised depletion of L-tryptophan. These findings raise critical questions for disease and its prevention during human pregnancy. Specifically, the role of this mechanism (discovered in mouse) in the human, and the extent to which defective activation of this process is responsible for major clinical diseases are unknown. Therefore some key facts about this enzyme expressed in the human placenta have been studied in order to test whether Munn et al.'s findings in mouse are met for human pregnancy. This short review attempts to describe our experimental work on human placental indoleamine 2,3-dioxygenase. PMID:24328005

  15. Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue.

    PubMed

    Ugele, B; Lange, F

    2001-01-01

    Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary. PMID:11573814

  16. Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

    SciTech Connect

    Guyda, H.J. )

    1991-03-01

    The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and (3H)thymidine incorporation into cell protein. Since benzo(alpha)pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo(alpha)pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured human placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of (3H)thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period.

  17. In vitro toxicological effects of estrogenic mycotoxins on human placental cells: Structure activity relationships

    SciTech Connect

    Prouillac, Caroline; Lecoeur, Sylvaine

    2012-03-15

    Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to α- and β-zearalenol (αZEL and βZEL). These reduced metabolites possess estrogenic properties, αZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of αZEL and βΖΕL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPARγ). Our results demonstrated that in spite of structure similarities between ZEN, αZEL and βZEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules. -- Highlights: ► ZEN and metabolites have differential effect on trophoblast differentiation. ► ZEN and metabolites have differential effect on ABC transporter expression. ► ZEN and metabolites effects involved nuclear receptors interaction.

  18. Parent bisphenol A accumulation in the human maternal-fetal-placental unit.

    PubMed Central

    Schönfelder, Gilbert; Wittfoht, Werner; Hopp, Hartmut; Talsness, Chris E; Paul, Martin; Chahoud, Ibrahim

    2002-01-01

    Bisphenol A (BPA), an endocrine disruptor, is employed in the manufacture of a wide range of consumer products. The suggestion that BPA, at amounts to which we are exposed, alters the reproductive organs of developing rodents has caused concern. At present, no information exists concerning the exposure of human pregnant women and their fetuses to BPA. We therefore investigated blood samples from mothers (n = 37) between weeks 32 and 41 of gestation. Afer the births, we also analyzed placental tissue and umbilical cord blood from the same subjects. We developed a novel chemical derivatization-gas chromatography/mass spectrometry method to analyze parent BPA at concentrations < 1 micro g/mL in plasma and tissues. Concentrations of BPA ranged from 0.3 to 18.9 ng/mL (median = 3.1 ng/mL) in maternal plasma, from 0.2 to 9.2 ng/mL (median = 2.3 ng/mL) in fetal plasma, and from 1.0 to 104.9 ng/g (median = 12.7 ng/g) in placental tissue. BPA blood concentrations were higher in male than in female fetuses. Here we demonstrate parent BPA in pregnant women and their fetuses. Exposure levels of parent BPA were found within a range typical of those used in recent animal studies and were shown to be toxic to reproductive organs of male and female offspring. We suggest that the range of BPA concentrations we measured may be related to sex differences in metabolization of parent BPA or variable maternal use of consumer products leaching BPA. PMID:12417499

  19. Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage

    PubMed Central

    Dilwali, Sonam; Landegger, Lukas D.; Soares, Vitor Y. R.; Deschler, Daniel G.; Stankovic, Konstantina M.

    2015-01-01

    Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects’ degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs. PMID:26690506

  20. Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells

    PubMed Central

    Cavet, M E; West, M; Simmons, N L

    1997-01-01

    Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM). Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl− or K+ in the bathing media. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia. PMID:9283689

  1. Inhibition of human placental aromatase activity by hydroxylated polybrominated diphenyl ethers (OH-PBDEs)

    SciTech Connect

    Canton, Rocio F. Scholten, Deborah E.A.; Marsh, Goeran; Jong, Paul C. de; Berg, Martin van den

    2008-02-15

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in many different polymers, resins and substrates. Due to their widespread production and use, their high binding affinity to particles, and their lipophilic properties, several PBDE congeners can bioaccumulate in the environment. As a result, PBDEs and their hydroxylated metabolites (OH-PBDEs) have been detected in humans and various wildlife samples, such as birds, seals, and whales. Furthermore, certain OH-PBDEs and their methoxylated derivatives (MeO-PBDEs) are natural products in the marine environment. Recently, our laboratory focused on the possible effects on steroidogenesis of PBDEs and OH-PBDEs, e.g. in the human adrenocortical carcinoma (H295R) cell line indicating that some OH-PBDEs can significantly influence steroidogenic enzymes like CYP19 (aromatase) and CYP17. In the present study, human placental microsomes have been used to study the possible interaction of twenty two OH-PBDEs and MeO-PBDEs with aromatase, the enzyme that mediates the conversion of androgens into estrogens. All OH-PBDE derivates showed significant inhibition of placental aromatase activity with IC{sub 50} values in the low micromolar range, while the MeO-PBDEs did not have any effect on this enzyme activity. Enzyme kinetics studies indicated that two OH-PBDEs, 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE47) and 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE47), had a mixed-type inhibition of aromatase activity with apparent K{sub i}/K{sub i}' of 7.68/0,02 {mu}M and 5.01/0.04 {mu}M respectively. For comparison, some structurally related compounds, a dihydroxylated polybrominated biphenyl, which is a natural product (2,2'-dihyroxy-3,3',5,5'-tetrabromobiphenyl (2,2'-diOH-BB80)) and its non-bromo derivative were also included in the study. Again inhibition of aromatase activity could be measured, but their potency was significantly less than those observed for the OH-PBDEs. These results show

  2. Cyclooxygenase-1 and -2 in human placenta and placental bed after normal and pre-eclamptic pregnancies.

    PubMed

    Wetzka, B; Nüsing, R; Charnock-Jones, D S; Schäfer, W; Zahradnik, H P; Smith, S K

    1997-10-01

    In pre-eclampsia, the ratio of prostacyclin:thromboxane production rate is decreased favouring the vasoconstrictive thromboxane. One of the rate-limiting steps in prostaglandin synthesis is cyclooxygenase (COX) activity. Therefore, we investigated the expression of COX-1 and COX-2 in human placenta and placental bed. Tissue specimens from the 29th to 40th week of pregnancy were obtained from Caesarean sections after uncomplicated and pre-eclamptic pregnancies before the onset of labour. COX-1 and COX-2 were localized immunohistochemically with the identification of positive cells by double immunofluorescence staining. The protein and mRNA levels were analysed by immunoblotting and quantitative reverse transcriptase-polymerase chain reaction. Expression of both COX-1 and COX-2 could be observed in placenta and placental bed. COX-1-like immunoreactivity was observed in most cell types with strongest staining in macrophages. Only macrophages, endothelium, vascular leiomyocytes and fibroblasts stained positively for COX-2. In placenta, COX-1 and -2 expression was unchanged after pre-eclampsia. In placental bed, protein and mRNA levels of COX-1 were increased in the pre-eclamptic group (P < 0.05), whereas COX-2 expression did not differ significantly from normal pregnancies. An increased expression of COX-1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed. A therapy with drugs inhibiting COX-1 might be beneficial in this condition. PMID:9402302

  3. Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro

    PubMed Central

    Jones, H.; Crombleholme, T.; Habli, M.

    2014-01-01

    Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor-11 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined amino acid transporter expression and localization in both a mouse model of placental Insufficiency (PI) and a model of human trophoblast, the BeWo Choriocarcinoma cell line. For in vitro human studies, BeWo Choriocarcinoma cells were maintained in F12 complete medium + 10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 h. MOIs of 10:1 and 100:1 were utilized. In BeWo, transfection efficiency was 100% at an MOI of 100:1 and Ad-IGF-1 significantly increased IGF-1 secretion, proliferation and invasion but reduced apoptosis compared to controls. In vitro, amino acid uptake was increased following Ad-IGF-1 treatment and associated with significantly increased RNA expression of SNAT1, 2, LAT1 and 4F2hc. Only SNAT2 protein expression was increased but LAT1 showed relocalization from a perinuclear location to the cytoplasm and cell membrane. For in vivo studies, timed-pregnant animals were divided into four groups on day 18; sham-operated controls, uterine artery branch ligation (UABL), UABL + Ad-hIGF-1 (108 PFU), UABL + Ad-LacZ (108 PFU). At gestational day 20, pups and placentas were harvested by C-section. Only LAT1 mRNA expression changed, showing that a reduced expression of the transporter levels in the PI model could be partially rectified with Ad-hIGF1 treatment. At the protein level, System L was reduced in PI but remained at control levels following Ad-hIGF1. The System A isoforms were differentially regulated with SNAT2 expression diminished but SNAT1 increased in PI and Ad-hIGF1 groups. Enhanced

  4. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts--a comparative study.

    PubMed

    van Meeuwen, J A; Nijmeijer, S; Mutarapat, T; Ruchirawat, S; de Jong, P C; Piersma, A H; van den Berg, M

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts. PMID:18201740

  5. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts - A comparative study

    SciTech Connect

    Meeuwen, J.A. van Nijmeijer, S.; Mutarapat, T.; Ruchirawat, S.; Jong, P.C. de; Piersma, A.H.; Berg, M. van den

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts.

  6. Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

    SciTech Connect

    Lee, M.Y.W.T.; Toomey, N.L.

    1987-02-24

    DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and n-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptides component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase ..cap alpha.. inhibited placental polymerase ..cap alpha.. but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not ..cap alpha... These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.

  7. Opioid control of gonadotrophin secretion in humans.

    PubMed

    Genazzani, A R; Genazzani, A D; Volpogni, C; Pianazzi, F; Li, G A; Surico, N; Petraglia, F

    1993-11-01

    Hypothalamus-pituitary-axis (HPA) is constantly under the modulatory effect of many substances, such as neurotransmitters, neuromodulators and steroid hormones. Recently, the involvement of endogenous opioid peptides (EOP) in the control of the neuroendocrine mechanism modulating gonadotrophin secretion has been supported by several authors. It has been demonstrated that acute morphine administration decreases luteinizing hormone (LH) plasma levels and this is due to an inhibitory modulation on gonadotrophin releasing hormone discharge from the hypothalamic neurons. EOP are usually increased by stressful situations. In stress-induced amenorrhoea, the presence of low LH plasma levels and an abnormal LH pulsatile secretion has been related to an increased opioid activity, thus supporting the integrative role of opioids between hormonal and neuronal afferences of brain. PMID:8276950

  8. Chromosomal Mosaicism in Human Feto-Placental Development: Implications for Prenatal Diagnosis

    PubMed Central

    Grati, Francesca Romana

    2014-01-01

    Chromosomal mosaicism is one of the primary interpretative issues in prenatal diagnosis. In this review, the mechanisms underlying feto-placental chromosomal mosaicism are presented. Based on the substantial retrospective diagnostic experience with chorionic villi samples (CVS) of a prenatal diagnosis laboratory the following items are discussed: (i) The frequency of the different types of mosaicism (confined placental, CPM, and true fetal mosaicisms, TFM); (ii) The risk of fetal confirmation after the detection of a mosaic in CVS stratified by chromosome abnormality and placental tissue involvement; (iii) The frequency of uniparental disomy for imprinted chromosomes associated with CPM; (iv) The incidence of false-positive and false-negative results in CVS samples analyzed by only (semi-)direct preparation or long term culture; and (v) The implications of the presence of a feto-placental mosaicism for microarray analysis of CVS and non-invasive prenatal screening (NIPS). PMID:26237479

  9. Mechanisms of alphafetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy.

    PubMed Central

    Brownbill, P; Edwards, D; Jones, C; Mahendran, D; Owen, D; Sibley, C; Johnson, R; Swanson, P; Nelson, D M

    1995-01-01

    We investigated the mechanisms of alphafetoprotein (AFP) transfer across the human placenta by correlating measurements of AFP transfer with cytochemical localization of AFP. Placental cotyledons were dually perfused in vitro with either the fetal or maternal perfusate containing umbilical cord plasma as a source of AFP. Steady state AFP clearance, corrected for release of endogenous AFP, was 0.973 +/- 0.292 microliter/min per gram in the fetal to maternal direction (n = 10), significantly higher (P < 0.02) than that in the maternal to fetal direction (n = 5; 0.022 +/- 0.013 microliter/min per gram). Clearance of a similarly sized protein, horseradish peroxidase was also asymmetric but clearance of the small tracer creatinine was not. Using a monoclonal antibody, we localized AFP to fibrinoid deposits in regions of villi with discontinuities of the syncytiotrophoblast, to cytotrophoblast cells in these deposits, to syncytiotrophoblast on some villi, and to trophoblast cells in the decidua. We conclude that AFP transfer in the placenta is asymmetric and that there are two available pathways for AFP transfer: (a) from the fetal circulation into the villous core and across fibrinoid deposits at discontinuities in the villous syncytiotrophoblast to enter the maternal circulation; and (b) AFP present in the decidua could enter vessels that traverse the basal plate. Images PMID:7593608

  10. Elemental maps in human allantochorial placental vessels cells: 1. High K + and acetylcholine effects

    NASA Astrophysics Data System (ADS)

    Michelet-Habchi, C.; Barberet, Ph.; Dutta, R. K.; Guiet-Bara, A.; Bara, M.; Moretto, Ph.

    2003-09-01

    Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K + and Ca 2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks' solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K + channels, K Ca) and of high concentration of K + (which blocks the voltage-sensitive K + channels, K df). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K + medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K + medium, a significant increase in Cl and K concentration. These results indicated the importance of K df, K Ca and K ATP channels in the regulation of K + intracellular distribution in VSMC and VEC and the possible intervention of a Na-K-2Cl cotransport and corroborated the previous electrophysiological data.

  11. Differential effects of glyphosate and roundup on human placental cells and aromatase.

    PubMed

    Richard, Sophie; Moslemi, Safa; Sipahutar, Herbert; Benachour, Nora; Seralini, Gilles-Eric

    2005-06-01

    Roundup is a glyphosate-based herbicide used worldwide, including on most genetically modified plants that have been designed to tolerate it. Its residues may thus enter the food chain, and glyphosate is found as a contaminant in rivers. Some agricultural workers using glyphosate have pregnancy problems, but its mechanism of action in mammals is questioned. Here we show that glyphosate is toxic to human placental JEG3 cells within 18 hr with concentrations lower than those found with agricultural use, and this effect increases with concentration and time or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower nontoxic concentrations on aromatase, the enzyme responsible for estrogen synthesis. The glyphosate-based herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation. PMID:15929894

  12. Differential Effects of Glyphosate and Roundup on Human Placental Cells and Aromatase

    PubMed Central

    Richard, Sophie; Moslemi, Safa; Sipahutar, Herbert; Benachour, Nora; Seralini, Gilles-Eric

    2005-01-01

    Roundup is a glyphosate-based herbicide used worldwide, including on most genetically modified plants that have been designed to tolerate it. Its residues may thus enter the food chain, and glyphosate is found as a contaminant in rivers. Some agricultural workers using glyphosate have pregnancy problems, but its mechanism of action in mammals is questioned. Here we show that glyphosate is toxic to human placental JEG3 cells within 18 hr with concentrations lower than those found with agricultural use, and this effect increases with concentration and time or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower nontoxic concentrations on aromatase, the enzyme responsible for estrogen synthesis. The glyphosate-based herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation. PMID:15929894

  13. Cross-tolerance of human placental plasma membranes of smokers to fluidizing effects of alcohol

    SciTech Connect

    Sastry, B.V.R.; Horst, M.A.; Naukam, R.J. )

    1991-03-11

    There is cross-tolerance between ethanol and several centrally acting drugs at the membrane level. In order to evaluate cross-tolerance between maternal smoking during pregnancy and alcohol, the authors have prepared plasma membranes of human term placentas from nonsmokers (NS, n=5) and smokers (S, 24 {plus minus} 8 cigarettes/day, n=5) and studied their microviscosities by steady state fluorescence polarization using trans-1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe. These experiments gave the following results: (a) microviscosity was increased by maternal smoking; (b) alcohol decreased microviscosity of the membranes of smokers; (c) exogenous nicotine did not exert any significant effect on the membranes of smokers and nonsmokers. Therefore, the increase in the rigidity of placental plasma membranes is due to chronic smoking, and these membranes are tolerant to the fluidizing effects of alcohol. Cross-tolerance between smoking and ethanol suggests a common hydrophobic locus of the apparent adaptation at the membrane level.

  14. PPAR Action in Human Placental Development and Pregnancy and Its Complications

    PubMed Central

    Wieser, Fritz; Waite, Leslie; Depoix, Christophe; Taylor, Robert N.

    2008-01-01

    During pregnancy crucial anatomic, physiologic, and metabolic changes challenge the mother and the fetus. The placenta is a remarkable organ that allows the mother and the fetus to adapt to the new metabolic, immunologic, and angiogenic environment imposed by gestation. One of the physiologic systems that appears to have evolved to sustain this metabolic regulation is mediated by peroxisome proliferator-activated receptors (PPARs). In clinical pregnancy-specific disorders, including preeclampsia, gestational diabetes, and intrauterine growth restriction, aberrant regulation of components of the PPAR system parallels dysregulation of metabolism, inflammation and angiogenesis. This review summarizes current knowledge on the role of PPARs in regulating human trophoblast invasion, early placental development, and also in the physiology of clinical pregnancy and its complications. As increasingly indicated in the literature, pregnancy disorders, such as preeclampsia and gestational diabetes, represent potential targets for treatment with PPAR ligands. With the advent of more specific PPAR agonists that exhibit efficacy in ameliorating metabolic, inflammatory, and angiogenic disturbances, further studies of their application in pregnancy-related diseases are warranted. PMID:18288290

  15. Effect of Human Placental Extract on Health Status in Elderly Koreans

    PubMed Central

    Kong, Mihee; Park, Sat Byul

    2012-01-01

    Objectives. Human placental extract (HPE) has begun to be used in Korea in various ways to improve health, even though evidence-based data is insufficient. This study investigated the effects of HPE on health status in elderly Koreans. Design. Randomized, single-blind, and case-control study design. Setting and Participants. Thirty-nine community-dwelling healthy Koreans ≥65 years of age. Intervention. The participants were randomly categorized into a placebo group (n = 17) and HPE group (n = 22). The HPE group received abdominal subcutaneous injections of HPE for 8 weeks. The placebo group was injected with normal saline. Measurements. The degree of health status was surveyed by the Korean health status measure for the elderly (KoHSME V1.0) at baseline and the end of the study. Results. In the HPE group, the scores of physical function, sexual life, and general heath perception at the end of the study period were significantly improved from baseline (P = .007, .020, and .005, resp.), while the health status of the placebo group remained unchanged during the study period. There was a significant difference over the study period between the two groups in the mean change of the physical function score (P = .036). Conclusion. A HPE injection regimen can improve the health status in elderly Koreans. PMID:22454680

  16. The NADPH- and iron-dependent lipid peroxidation in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokolowska, Ewa; Hallmann, Anna; Klimek, Jerzy

    2007-01-01

    In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) - a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and alpha-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM. PMID:16896536

  17. Selected Persistent Organic Pollutants in Human Placental Tissue from the United States

    PubMed Central

    Jones, Rachael M.; Li, An; Stodgell, Christopher J.; Walker, Cheryl; Szabo, Sara; Leuthner, Steve; Durkin, Maureen S.; Moye, Jack; Miller, Richard K.

    2014-01-01

    Emerging and legacy environmental pollutants such as polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (DDE) are found in human placenta, indicating prenatal exposure, but data from the United States are sparse. We sought to determine concentrations of these compounds in human placentae as part of a formative research project conducted by the National Children’s Study Placenta Consortium. A total of 169 tissue specimens were collected at different time points post delivery from 42 human placentae at three U.S. locations, and analyzed by gas chromatography coupled with mass spectrometry following extraction using matrix solid phase dispersion. PBDEs, PCBs, and DDE were detected in all specimens. The concentrations of 10 PBDEs (∑10PBDEs), 32 PCBs (∑32PCBs) and p,p’-DDE were 43–1,723, 76–856 and 10–1,968 pg/g wet weight, respectively, in specimens collected shortly after delivery. Significant geographic differences in PBDEs were observed, with higher concentrations in placentae collected in Davis, CA than in those from Rochester, NY or Milwaukee, WI. We combined these with other published data and noted first-order declining trends for placental PCB and DDE concentrations over the past decades, with half-lives of about 5 and 8 years, respectively. The effect of time to tissue collection from refrigerated placentae on measured concentrations of these three classes of persistent organic pollutants was additionally examined, with no significant effect observed up to 120 hours. The results of this work indicate that widespread prenatal exposure to persistent organic pollutants in the United States continues. PMID:24485817

  18. Endothelin A and B receptors change their expression levels during development of human placental villi.

    PubMed

    Cervar, M; Huppertz, B; Barth, S; Hahn, T; Weiss, U; Kaufmann, P; Desoye, G

    2000-01-01

    Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development. PMID:10940204

  19. Characterization of tryptophan transport in human placental brush-border membrane vesicles.

    PubMed Central

    Ganapathy, M E; Leibach, F H; Mahesh, V B; Howard, J C; Devoe, L D; Ganapathy, V

    1986-01-01

    The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity. PMID:3800932

  20. Differentiation of adipocytes and osteocytes from human adipose and placental mesenchymal stem cells

    PubMed Central

    Mohammadi, Zahra; Afshari, Jalil Tavakkol; Keramati, Mohammad Reza; Alamdari, Daryoush Hamidi; Ganjibakhsh, Meysam; Zarmehri, Azam Moradi; Jangjoo, Ali; Sadeghian, Mohammad Hadi; Ameri, Masoumeh Arab; Moinzadeh, Leila

    2015-01-01

    Objective(s): Mesenchymal stem cells (MSC) can be isolated from adult tissues such as adipose tissue and other sources. Among these sources, adipose tissue (because of easy access) and placenta (due to its immunomodulatory properties, in addition to other useful properties), have attracted more attention in terms of research. The isolation and comparison of MSC from these two sources provides a proper source for clinical experimentation. The aim of this study was to compare the characteristics of MSC isolated from human adipose tissue and placenta. Materials and Methods: Adipose and placental MSC were isolated from the subcutaneous adipose tissues of 10 healthy women (25 to 40 years) and from a fresh term placenta (n= 1), respectively. Stem cells were characterized and compared by flow cytometry using CD29, CD31, CD34, CD44, CD45, CD105, CD166 and HLA-DR markers. Osteocytes and adipocytes were differentiated from isolated human mesenchymal stem cells (HMSC). Results: Adipose and placenta-derived MSC exhibited the same morphological features. ADSC differentiated faster than placenta; however, both were differentiated, taking up to 21 days for osteocyte and 14 days for adipocyte differentiation. About 90% of PLC-MSC and ADSC were positive for CD29, CD44, CD105, and CD166; and negative for CD31, CD34, CD45, and HLA-DR. Conclusion: The two sources of stem cells showed similar surface markers, morphology and differentiation potential and because of their multipotency for differentiating to adipocytes and osteocytes, they can be applied as attractive sources of MSC for regenerative medicine. PMID:25945239

  1. Human Insulinomas Show Distinct Patterns of Insulin Secretion In Vitro.

    PubMed

    Henquin, Jean-Claude; Nenquin, Myriam; Guiot, Yves; Rahier, Jacques; Sempoux, Christine

    2015-10-01

    Insulinomas are β-cell tumors that cause hypoglycemia through inappropriate secretion of insulin. Characterization of the in vitro dynamics of insulin secretion by perifused fragments of 10 human insulinomas permitted their subdivision into three functional groups with similar insulin content. Group A (four patients with fasting and/or postprandial hypoglycemic episodes) showed qualitatively normal responses to glucose, leucine, diazoxide, tolbutamide, and extracellular CaCl2 omission or excess. The effect of glucose was concentration dependent, but, compared with normal islets, insulin secretion was excessive in both low- and high-glucose conditions. Group B (three patients with fasting hypoglycemic episodes) was mainly characterized by large insulin responses to 1 mmol/L glucose, resulting in very high basal secretion rates that were inhibited by diazoxide and restored by tolbutamide but were not further augmented by other agents except for high levels of CaCl2. Group C (three patients with fasting hypoglycemic episodes) displayed very low rates of insulin secretion and virtually no response to stimuli (including high CaCl2 concentration) and inhibitors (CaCl2 omission being paradoxically stimulatory). In group B, the presence of low-Km hexokinase-I in insulinoma β-cells (not in adjacent islets) was revealed by immunohistochemistry. Human insulinomas thus show distinct, though not completely heterogeneous, defects in insulin secretion that are attributed to the undue expression of hexokinase-I in 3 of 10 patients. PMID:26116696

  2. Placental Transfer of Maraviroc in an Ex Vivo Human Cotyledon Perfusion Model and Influence of ABC Transporter Expression

    PubMed Central

    Vinot, C.; Gavard, L.; Tréluyer, J. M.; Manceau, S.; Courbon, E.; Scherrmann, J. M.; Declèves, X.; Duro, D.; Peytavin, G.; Mandelbrot, L.

    2013-01-01

    Nowadays, antiretroviral therapy is recommended during pregnancy to prevent mother-to-child transmission of HIV. However, for many antiretroviral drugs, including maraviroc, a CCR5 antagonist, very little data exist regarding placental transfer. Besides, various factors may modulate this transfer, including efflux transporters belonging to the ATP-binding cassette (ABC) transporter superfamily. We investigated maraviroc placental transfer and the influence of ABC transporter expression on this transfer using the human cotyledon perfusion model. Term placentas were perfused ex vivo for 90 min with maraviroc (600 ng/ml) either in the maternal-to-fetal (n = 10 placentas) or fetal-to-maternal (n = 6 placentas) direction. Plasma concentrations were determined by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Fetal transfer rates (FTR) and clearance indexes (CLI) were calculated as ratios of fetal to maternal concentrations at steady state (mean values between 30 and 90 min) and ratios of FTR of maraviroc to that of antipyrine, respectively. ABC transporter gene expression levels were determined by quantitative reverse transcription (RT)-PCR and ABCB1 protein expression by Western blotting. For the maternal-to-fetal direction, the mean FTR and CLI were 8.0% ± 3.0 and 0.26 ± 0.07, respectively, whereas the mean CLI was 0.52 ± 0.23 for the fetal-to-maternal direction. We showed a significant inverse correlation between maraviroc CLI and ABCC2, ABCC10, and ABCC11 placental gene expression levels (P < 0.05). To conclude, we report a low maraviroc placental transfer probably involving ABC efflux transporters and thus in all likelihood associated with a limited fetal exposition. Nevertheless, these results would need to be supported by in vivo data obtained from paired maternal and cord blood samples. PMID:23295922

  3. miR-34a expression, epigenetic regulation, and function in human placental diseases

    PubMed Central

    Doridot, Ludivine; Houry, Dorothée; Gaillard, Harald; Chelbi, Sonia T; Barbaux, Sandrine; Vaiman, Daniel

    2014-01-01

    Preeclampsia (PE) is the major pregnancy-induced hypertensive disorder responsible for maternal and fetal morbidity and mortality that can be associated with intrauterine growth restriction (IUGR). PE and IUGR are thought to be due to a placental defect, occurring early during pregnancy. Several placental microRNAs (miRNAs) have been shown to be deregulated in the context of placental diseases and could thus play a role in the pathophysiology of PE. Here, we show that pri-miR-34a is overexpressed in preeclamptic placentas and that its placental expression is much higher during the first trimester of pregnancy than at term, suggesting a possible developmental role. We explored pri-miR-34a regulation and showed that P53, a known activator of miR-34a, is reduced in all pathological placentas and that hypoxia can induce pri-miR-34a expression in JEG-3 cells. We also studied the methylation status of the miR-34a promoter and revealed hypomethylation in all preeclamptic placentas (associated or not with IUGR), whereas hypoxia induced a hypermethylation in JEG-3 cells at 72 h. Despite the overexpression of pri-miR-34a in preeclampsia, there was a striking decrease of the mature miR-34a in this condition, suggesting preeclampsia-driven alteration of pri-miR-34a maturation. SERPINA3, a protease inhibitor involved in placental diseases, is elevated in IUGR and PE. We show here that miR-34a overexpression in JEG-3 downregulates SERPINA3. The low level of mature miR-34a could thus be an important mechanism contributing to SERPINA3 upregulation in placental diseases. Overall, our results support a role for miR-34a in the pathophysiology of preeclampsia, through deregulation of the pri-miRNA expression and its altered maturation. PMID:24081307

  4. Differential expression of human placental neurotrophic factors in preterm and term deliveries.

    PubMed

    Dhobale, Madhavi V; Pisal, Hemlata R; Mehendale, Savita S; Joshi, Sadhana R

    2013-12-01

    Neurotrophic factors such as brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in development of the placenta and fetal brain. A series of human and animal studies in our department have shown that micronutrients (folic acid, vitamin B12) and omega 3 fatty acids like DHA are all interlinked in the one carbon cycle. Any alterations in one carbon components will lead to changes in methylation patterns that further affect the gene expression at critical periods of development resulting in complications during pregnancy. This may further contribute to risk for neurodevelopmental disorders in children born preterm. Therefore this study for the first time examines the mRNA levels from preterm and term placentae. A total number of 38 women delivering preterm (<37 weeks gestation) and 37 women delivering at term (=>37 weeks gestation) were recruited. The mRNA levels of BDNF and NGF were analyzed by real time quantitative polymerase chain reaction. Our results indicate that BDNF and NGF mRNA levels were lower in preterm group as compared to term group. There was a positive association of placental BDNF and NGF mRNA levels with cord plasma BDNF and NGF levels. The differential expression of BDNF and NGF gene in preterm placentae may also alter the vascular development in preterm deliveries. Our data suggests that the reduced mRNA levels of BDNF and NGF may possibly be a result of altered epigenetic mechanisms and may have an implication for altered fetal programming in children born preterm. PMID:24076518

  5. Comparative intrauterine development and placental function of ART concepti: implications for human reproductive medicine and animal breeding

    PubMed Central

    Bloise, Enrrico; Feuer, Sky K.; Rinaudo, Paolo F.

    2014-01-01

    BACKGROUND The number of children conceived using assisted reproductive technologies (ART) has reached >5 million worldwide and continues to increase. Although the great majority of ART children are healthy, many reports suggest a forthcoming risk of metabolic complications, which is further supported by the Developmental Origins of Health and Disease hypothesis of suboptimal embryo/fetal conditions predisposing adult cardiometabolic pathologies. Accumulating evidence suggests that fetal and placental growth kinetics are important features predicting post-natal health, but the relationship between ART and intrauterine growth has not been systematically reviewed. METHODS Relevant studies describing fetoplacental intrauterine phenotypes of concepti generated by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) in the mouse, bovine and human were comprehensively researched using PubMed and Google Scholar. Intrauterine growth plots were created from tabular formatted data available in selected reports. RESULTS ART pregnancies display minor but noticeable alterations in fetal and placental growth curves across mammalian species. In all species, there is evidence of fetal growth restriction in the earlier stages of pregnancy, followed by significant increases in placental size and accelerated fetal growth toward the end of gestation. However, there is a species-specific effect of ART on birthweights, that additionally vary in a culture condition-, strain-, and/or stage at transfer-specific manner. We discuss the potential mechanisms that underlie these changes, and how they are affected by specific components of ART procedures. CONCLUSIONS ART may promote measurable alterations to intrauterine growth trajectory and placental function. Key findings include evidence that birthweight is not a reliable marker of fetal stress, and that increases in embryo manipulation result in more deviant fetal growth curves

  6. Constitutive secretion of chemokines by cultured human trabecular meshwork cells.

    PubMed

    Shifera, Amde Selassie; Trivedi, Sheetal; Chau, Phuonglan; Bonnemaison, Lucia H; Iguchi, Rumiko; Alvarado, Jorge A

    2010-07-01

    Trabecular meshwork endothelial (TME) cells secrete a number of factors, such as enzymes and cytokines, which modulate the functions of the cells and the extracellular matrix of the conventional aqueous outflow pathway. TME cells usually secrete these factors in response to stimuli such as mechanical stretching, laser irradiation and pro-inflammatory cytokines. Here, we report that cultured human TME cells isolated from two non-glaucomatous individuals secrete significant quantities of the chemotactic cytokines IL8, CXCL6 and MCP1 in the absence of any stimulation. The secretion of these chemokines was augmented by treatment with the pro-inflammatory cytokines TNFalpha and IL1beta. By way of comparison, there was little or very low production of the three chemokines by human non-pigmented ciliary epithelial cells in the absence of stimulation. Our findings provide support to our recent observations that monocytes, presumably under the influence of chemotactic signals, circulate through the trabecular meshwork in the normal state and also that cytokines regulate the permeability of Schlemm's canal endothelial cells. In addition, the fact that normal TME cells constitutively secrete chemotactic cytokines strengthens the notion that cytokines play a key role in the homeostasis of the outflow of the aqueous humor and, possibly, in the pathogenesis of glaucoma. PMID:20403352

  7. EFFECT OF BROMODICHLOROMETHANE ON HUMAN TROPHOBLAST CHORIONIC GONADOTROPHIN SECRETION

    EPA Science Inventory

    Effect of Bromodichloromethane on Human Trophoblast Chorionic Gonadotrophin Secretion

    Jiangang Chen1, Twanda L. Thirkill1, Peter N. Lohstroh1, Susan R. Bielmeier2, Michael G. Narotsky3, Deborah S. Best3, Randy A. Harrison3, Kala Natarajan1, Rex A. Pegram3, Gordon C. Dougla...

  8. Development and Function of the Human Fetal Adrenal Cortex: A Key Component in the Feto-Placental Unit

    PubMed Central

    Ishimoto, Hitoshi

    2011-01-01

    Continuous efforts have been devoted to unraveling the biophysiology and development of the human fetal adrenal cortex, which is structurally and functionally unique from other species. It plays a pivotal role, mainly through steroidogenesis, in the regulation of intrauterine homeostasis and in fetal development and maturation. The steroidogenic activity is characterized by early transient cortisol biosynthesis, followed by its suppressed synthesis until late gestation, and extensive production of dehydroepiandrosterone and its sulfate, precursors of placental estrogen, during most of gestation. The gland rapidly grows through processes including cell proliferation and angiogenesis at the gland periphery, cellular migration, hypertrophy, and apoptosis. Recent studies employing modern technologies such as gene expression profiling and laser capture microdissection have revealed that development and/or function of the fetal adrenal cortex may be regulated by a panoply of molecules, including transcription factors, extracellular matrix components, locally produced growth factors, and placenta-derived CRH, in addition to the primary regulator, fetal pituitary ACTH. The role of the fetal adrenal cortex in human pregnancy and parturition appears highly complex, probably due to redundant and compensatory mechanisms regulating these events. Mounting evidence indicates that actions of hormones operating in the human feto-placental unit are likely mediated by mechanisms including target tissue responsiveness, local metabolism, and bioavailability, rather than changes only in circulating levels. Comprehensive study of such molecular mechanisms and the newly identified factors implicated in adrenal development should help crystallize our understanding of the development and physiology of the human fetal adrenal cortex. PMID:21051591

  9. Effect of manganese on human placental spin-lattice (T1) and spin-spin (T2) relaxation times

    SciTech Connect

    Angtuaco, T.L.; Mattison, D.R.; Thomford, P.J.; Jordan, J.

    1986-01-01

    Human placentas were obtained immediately following delivery and incubated with manganese chloride (MnCl/sub 2/) in concentrations ranging from 0.002 to 2.0 mM. Proton density, T1 and T2 were measured at times ranging from 5-200 minutes. There was rapid uptake of manganese by the placenta producing a dose-dependent decrease in placental T1 and T2. The major effect of manganese uptake was shortening of T1 suggesting that the contrast between placenta and myometrium will be enhanced predominantly for T1-dependent imaging pulse sequences.

  10. Influence of endurance exercise and diet on human placental development and fetal growth.

    PubMed

    Clapp, J F

    2006-01-01

    The delivery of oxygen and substrate to the maternal-fetal interphase is the major maternal environmental stimulus which either up- or down-regulates feto-placental growth. During pregnancy, sustained exercise sessions cause an intermittent reduction in oxygen and substrate delivery to the interphase that may exceed 50% during the exercise but, it is probable that regular bouts of sustained exercise or exercise training may improve oxygen and substrate delivery at rest. The type of maternal carbohydrate intake (low- versus high-glycemic sources) and food intake frequency also influence substrate availability through their effects on maternal blood glucose levels and insulin sensitivity. As a result, different exercise regimens and/or different types of carbohydrate intake modify feto-placental growth. The magnitude and direction of the effect is determined by their average 24-h effect on oxygen and substrate availability at different time-points in pregnancy. In general, exercise in early and mid pregnancy stimulates placental growth while the relative amount of exercise in late pregnancy determines its effect on late fetal growth. Low-glycemic food sources in the diet decrease growth rate and size at birth while high-glycemic food sources increase it. Thus, it may be possible to improve pregnancy outcomes in both healthy, low-risk women and a variety of high-risk populaces by simply modifying maternal physical activity and dietary carbohydrate intake during pregnancy. PMID:16165206

  11. Zika virus damages the human placental barrier and presents marked fetal neurotropism.

    PubMed

    Noronha, Lucia de; Zanluca, Camila; Azevedo, Marina Luize Viola; Luz, Kleber Giovanni; Santos, Claudia Nunes Duarte Dos

    2016-05-01

    An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism. PMID:27143490

  12. Zika virus damages the human placental barrier and presents marked fetal neurotropism

    PubMed Central

    de Noronha, Lucia; Zanluca, Camila; Azevedo, Marina Luize Viola; Luz, Kleber Giovanni; dos Santos, Claudia Nunes Duarte

    2016-01-01

    An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism. PMID:27143490

  13. Purification and cultivation of human pituitary growth hormone secreting cells

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.

    1979-01-01

    Efforts were directed towards maintenance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro. The production of human growth hormone (hGH) by this means would be of benefit for the treatment of certain human hypopituitary diseases such as dwarfism. One of the primary approaches was the testing of agents which may logically be expected to increase hGH release. The progress towards this goal is summarized. Results from preliminary experiments dealing with electrophoresis of pituitary cell for the purpose of somatotroph separation are described.

  14. Purification and cultivation of human pituitary growth hormone secreting cells

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.

    1978-01-01

    The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

  15. Malignant cancer and invasive placentation

    PubMed Central

    D'Souza, Alaric W.; Wagner, Günter P.

    2014-01-01

    Cancer metastasis is an invasive process that involves the transplantation of cells into new environments. Since human placentation is also invasive, hypotheses about a relationship between invasive placentation in eutherian mammals and metastasis have been proposed. The relationship between metastatic cancer and invasive placentation is usually presented in terms of antagonistic pleiotropy. According to this hypothesis, evolution of invasive placentation also established the mechanisms for cancer metastasis. Here, in contrast, we argue that the secondary evolution of less invasive placentation in some mammalian lineages may have resulted in positive pleiotropic effects on cancer survival by lowering malignancy rates. These positive pleiotropic effects would manifest themselves as resistance to cancer cell invasion. To provide a preliminary test of this proposal, we re-analyze data from Priester and Mantel (Occurrence of tumors in domestic animals. Data from 12 United States and Canadian colleges of veterinary medicine. J Natl Cancer Inst 1971;47:1333-44) about malignancy rates in cows, horses, cats and dogs. From our analysis we found that equines and bovines, animals with less invasive placentation, have lower rates of metastatic cancer than felines and canines in skin and glandular epithelial cancers as well as connective tissue sarcomas. We conclude that a link between type of placentation and species-specific malignancy rates is more likely related to derived mechanisms that suppress invasion rather than different degrees of fetal placental aggressiveness. PMID:25324490

  16. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    SciTech Connect

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet; Casas, Josefina; Lacorte, Sílvia; Porte, Cinta

    2014-06-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  17. Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia

    PubMed Central

    Dobierzewska, Aneta; España-Perrot, Pedro P.; Venegas-Araneda, Pía; Guzmán-Rojas, Alejandra M.; González, María I.; Palominos-Rivera, Macarena; Irarrazabal, Carlos E.; Figueroa-Diesel, Horacio; Varas-Godoy, Manuel; Illanes, Sebastián E.

    2015-01-01

    Introduction Preeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE. Methods Serum samples were collected at 32–36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG–3 cells under 1% and 8% O2 for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured. Results The protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-β-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG–3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be

  18. Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro

    SciTech Connect

    Tesarik, J.; Kopecny, V.; Dvorak, M.

    1984-06-01

    The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of /sup 3/H-methionine and /sup 3/H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

  19. Potential new mechanisms of placental damage in celiac disease: anti-transglutaminase antibodies impair human endometrial angiogenesis.

    PubMed

    Di Simone, Nicoletta; De Spirito, Marco; Di Nicuolo, Fiorella; Tersigni, Chiara; Castellani, Roberta; Silano, Marco; Maulucci, Giuseppe; Papi, Massimiliano; Marana, Riccardo; Scambia, Giovanni; Gasbarrini, Antonio

    2013-10-01

    Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion and characterized by circulating anti-transglutaminase type 2 (anti-TG2) autoantibodies. An epidemiological link between maternal CD and increased risk of pregnancy failure has been established; however, the mechanism underlying this association is still poorly understood. Because proper endometrial angiogenesis and decidualization are prerequisites for placental development, we investigated the effect of anti-TG2 antibodies on the process of endometrial angiogenesis. Binding of anti-TG2 antibodies to human endometrial endothelial cells (HEECs) was evaluated by ELISA. Angiogenesis was studied in vitro on HEECs and in vivo in a murine model. In particular, we investigated the effect of anti-TG2 antibodies on HEEC matrix metalloprotease-2 (MMP-2) activity by gelatin zymography, cytoskeletal organization and membrane properties by confocal microscopy, and activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK) by Western blot analysis. Anti-TG2 antibodies bound to HEECs and decreased newly formed vessels both in vitro and in vivo. Anti-TG2 antibodies impaired angiogenesis by inhibiting the activation of MMP-2, disarranging cytoskeleton fibers, changing the physical and mechanical properties of cell membranes, and inhibiting the intracellular phosphorylation of FAK and ERK. Anti-TG2 antibodies inhibit endometrial angiogenesis affecting the TG2-dependent migration of HEECs and extracellular matrix degradation, which are necessary to form new vessels. Our results identify pathogenic mechanisms of placental damage in CD. PMID:23966323

  20. Effect of Macrophage Migration Inhibitory Factor (MIF) in Human Placental Explants Infected with Toxoplasma gondii Depends on Gestational Age

    PubMed Central

    de Oliveira Gomes, Angelica; de Oliveira Silva, Deise Aparecida; Silva, Neide Maria; de Freitas Barbosa, Bellisa; Franco, Priscila Silva; Angeloni, Mariana Bodini; Fermino, Marise Lopes; Roque-Barreira, Maria Cristina; Bechi, Nicoletta; Paulesu, Luana Ricci; dos Santos, Maria Célia; Mineo, José Roberto; Ferro, Eloisa Amália Vieira

    2011-01-01

    Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age. PMID:21641401

  1. Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles.

    PubMed Central

    Huang, T M; Hung, H C; Chang, T C; Chang, G G

    1998-01-01

    Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2, 4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (pi) for deuterium for the transition state (piT) increased when

  2. Effect of sildenafil on renin secretion in human subjects.

    PubMed

    Chiu, Yeong Jen; Reid, Ian A

    2002-09-01

    Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDE5 is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the

  3. Expression of Placental Members of the Human Growth Hormone Gene Family Is Increased in Response to Sequential Inhibition of DNA Methylation and Histone Deacetylation

    PubMed Central

    Ganguly, Esha; Bock, Margaret E.; Cattini, Peter A.

    2015-01-01

    Abstract The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5′ P and 3′ enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2′-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3′ enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3′ enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of

  4. Human cultured endothelial cells do secrete endothelin-1

    SciTech Connect

    Clozel, M.; Fischli, W. )

    1989-01-01

    Endothelin-1 (ET-1) has been identified in the conditioned medium of porcine endothelial cells. Human endothelin (ET-1) cloned from a placenta cDNA library is similar to porcine, but it is not known whether endothelin itself is secreted by human endothelial cells. To answer this question, a conditioned medium taken every 48 h from confluent cultures of umbilical vein endothelial cells was analyzed by HPLC and all fractions were tested for their ability to inhibit ({sup 125}I)ET-1 binding on human placenta membranes. Only one fraction did inhibit ({sup 125}I)ET-1 binding. When the conditioned medium was spiked with ET-1, the same single fraction inhibited ({sup 125}I)ET-1 binding showing that ET-1, itself, is present in the conditioned medium of human endothelial cells. ET-1 accumulates with time, reaching a plateau at 48 h. ET-1 secretion is not increased by a 24-h incubation of endothelial cells with phorbol myristate acetate, interleukin-1, tumor necrosis factor, thrombin or neuropeptide Y.

  5. [Morphological variability and placental function].

    PubMed

    Malassiné, A

    2001-01-01

    In mammals, the blastocyst defines with the maternal organism, a structure which allows embryonic development during gestation: the placenta. The structure of this organ varies remarkably across species. In this review the different type of placentation have been described in a comparative manner using terms of classification such as: placental materno-fetal interdigitation, matemofetal blood flow interrelationships, layers of the placental interhemal barrier, trophoblast invasiveness and decidual cell reaction, formation of syncytiotrophoblast. The human hemomonochorial placenta is characterized by a strong decidualization of the uterus and a major invasiveness of the extravillous trophoblast. Furthermore, there is a spectrum of placental endocrine activities across species. In some mammals (e.g., mouse and rat) the placenta eclipses the pituitary in the maintenance of ovarian function. In the human and in the sheep, horse, cat and guinea pig, the placenta acquires the ability to substitute for the ovaries in the maintenance of gestation at various time during pregnancy. The human placenta is characterized by a high rate of steroïdogenesis (progesterone and estrogens) and by the production of a primate specific trophoblastic hormone: human chorionic gonadotropin (hCG). Recently, it was demonstrated that mutation of many genes in mice results in embryonic mortality or fetal growth restriction, due to defects in placental development. Furthermore, distinct molecular pathways regulate the differentiation of various trophoblast cell subtype of the mouse placenta. An important question is whether or not placental differentiation in other mammals is regulated by the same molecular mechanisms. Due to the striking diversity in placental structure, endocrine function and gene expression, caution must be exercised in extrapolating findings regarding placental function and development from one species to another. PMID:11575143

  6. Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1998-04-01

    The placenta possesses the ability to metabolize a number of xenobiotics and endogenous compounds by processes similar to those seen in the liver. Animal and in vivo studies have observed that the presence of diabetes alters the expression of hepatic metabolizing enzymes (cytochrome P450 and glutathione S-transferase); however, it is unknown whether similar alterations occur in the human placenta. To evaluate whether diabetes has any effect of placental xenobiotic metabolizing activity, the catalytic activities of 7-ethoxyresorufin O-deethylation (EROD, CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugation with glutathione (glutathione S-transferase, GST) from placentas of diet (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared with matched controls. EROD activity (CYP1A1) ranged from 0.29 to 2.67 pmol/min/mg protein. However, no differences were observed among overt or gestational diabetics and their respective matched controls. CDNB conjugation (GST) ranged from 0.275 to 1.65 units/min/mg protein. In contrast to that observed with CYP1A1, a small but statistically significant reduction in GST activity was noted in overt diabetics as compared with their matched controls and gestational diabetics. CYP2E1, 2D6, and 3A4 enzymatic activities were not detected in human placental tissue. GST protein was detectable in all tissues studied, but no CYP protein could be detected in any of the tissues. Thus, it seems that pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in the exposure of the fetus to harmful electrophiles. However, the full clinical significance of this finding remains to be elucidated. PMID:9531526

  7. Transcriptome analysis of PPARγ target genes reveals the involvement of lysyl oxidase in human placental cytotrophoblast invasion.

    PubMed

    Segond, Nadine; Degrelle, Séverine A; Berndt, Sarah; Clouqueur, Elodie; Rouault, Christine; Saubamea, Bruno; Dessen, Philippe; Fong, Keith S K; Csiszar, Katalin; Badet, Josette; Evain-Brion, Danièle; Fournier, Thierry

    2013-01-01

    Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by β-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion. PMID:24265769

  8. Peptide secreted by human alveolar macrophages releases neutrophil granule contents

    SciTech Connect

    MacArthur, C.K.; Miller, E.J.; Cohen, A.B.

    1987-11-15

    A monoclonal antibody was developed against an 8000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose, and by autoradiography. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-..gamma.., tumor necrosis factor, or interleukin l..cap alpha.. or 1..beta... The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes.

  9. Placental Growth Factor Administration Abolishes Placental Ischemia-Induced Hypertension.

    PubMed

    Spradley, Frank T; Tan, Adelene Y; Joo, Woo S; Daniels, Garrett; Kussie, Paul; Karumanchi, S Ananth; Granger, Joey P

    2016-04-01

    Preeclampsia is a pregnancy-specific disorder of new-onset hypertension. Unfortunately, the most effective treatment is early delivery of the fetus and placenta. Placental ischemia appears central to the pathogenesis of preeclampsia because placental ischemia/hypoxia induced in animals by reduced uterine perfusion pressure (RUPP) or in humans stimulates release of hypertensive placental factors into the maternal circulation. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1), which antagonizes and reduces bioavailable vascular endothelial growth factor and placental growth factor (PlGF), is elevated in RUPP rats and preeclampsia. Although PlGF and vascular endothelial growth factor are both natural ligands for sFlt-1, vascular endothelial growth factor also has high affinity to VEGFR2 (Flk-1) causing side effects like edema. PlGF is specific for sFlt-1. We tested the hypothesis that PlGF treatment reduces placental ischemia-induced hypertension by antagonizing sFlt-1 without adverse consequences to the mother or fetus. On gestational day 14, rats were randomized to 4 groups: normal pregnant or RUPP±infusion of recombinant human PlGF (180 μg/kg per day; AG31, a purified, recombinant human form of PlGF) for 5 days via intraperitoneal osmotic minipumps. On day 19, mean arterial blood pressure and plasma sFlt-1 were higher and glomerular filtration rate lower in RUPP than normal pregnant rats. Infusion of recombinant human PlGF abolished these changes seen with RUPP along with reducing oxidative stress. These data indicate that the increased sFlt-1 and reduced PlGF resulting from placental ischemia contribute to maternal hypertension. Our novel finding that recombinant human PlGF abolishes placental ischemia-induced hypertension, without major adverse consequences, suggests a strong therapeutic potential for this growth factor in preeclampsia. PMID:26831193

  10. IL-6 and its circadian secretion in humans.

    PubMed

    Vgontzas, A N; Bixler, E O; Lin, H-M; Prolo, P; Trakada, G; Chrousos, G P

    2005-01-01

    Interleukin-6 (IL-6) is a pleiotropic cytokine produced by numerous types of immune and nonimmune cells and is involved in many pathophysiologic mechanisms in humans. Many studies suggest that IL-6 is a putative 'sleep factor' and its circadian secretion correlates with sleep/sleepiness. IL-6 is elevated in disorders of excessive daytime sleepiness such as narcolepsy and obstructive sleep apnea. It correlates positively with body mass index and may be a mediator of sleepiness in obesity. Also the secretion of this cytokine is stimulated by total acute or partial short-term sleep loss reflecting the increased sleepiness experienced by sleep-deprived individuals. Studies that evaluated the 24-hour secretory pattern of IL-6 in healthy young adults suggest that IL-6 is secreted in a biphasic circadian pattern with two nadirs at about 08.00 and 21.00, and two zeniths at about 19.00 and 05.00 h. In contrast, following sleep deprivation or in disorders of sleep disturbance, e.g., insomnia, IL-6 peaks during the day and, based on the level of stress system activity, i.e., cortisol secretion, contributes to either sleepiness and deep sleep (low cortisol) or feelings of tiredness and fatigue and poor sleep (high cortisol). In order to address concerns about the potential impact of differences of IL-6 levels between the beginning and the end of the 24-hour blood-drawing experiment, we proceeded with a cosinor analysis of 'detrended' data in young and old healthy individuals. This new analysis did not affect the biphasic circadian pattern of IL-6 secretion in young adults, while it augmented the flattened circadian pattern in old individuals in whom the difference was greater. Finally, IL-6 appears to be somnogenic in rats and exhibits a diurnal rhythm that follows the sleep/wake cycle in these animals. We conclude that IL-6 is a mediator of sleepiness and its circadian pattern reflects the homeostatic drive for sleep. PMID:15905620

  11. Green tea (-)-epigallocatechin gallate induced growth inhibition of human placental choriocarcinoma cells.

    PubMed

    Shih, Li-Jane; Lin, Yu-Ren; Lin, Cheng-Kuo; Liu, Hang-Shen; Kao, Yung-Hsi

    2016-05-01

    This study investigated the pathways involved in the effect of green tea epigallocatechin gallate (EGCG) on mitogenesis in BeWo, JEG-3, and JAR placental choriocarcinoma cells. EGCG inhibited cell proliferation in dose-dependent and time-dependent manners, as indicated by the number of cells and incorporation of bromodeoxyuridine (BrdU). A catechin-specific effect of green tea was evident; EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in suppressing cell growth. When all three of the mitogen-activated protein kinase (MAPK) subfamilies, i.e., ERK, p38, and JNK, were examined, EGCG significantly increased levels of phospho-ERK1/2 (pERK1/2) and phospho-p38 (pp38) and did not alter the total protein levels of ERK1/2, p38 MAPK, JNK, and phospho-JNK. EGCG-induced increases in the levels of pERK1/2 and pp38 proteins were prevented by pre-treatment with specific inhibitors of ERK1/2 MAPK and p38 MAPK, respectively. These inhibitors also suppressed EGCG-induced decreases in both cell number and BrdU incorporation. Moreover, pre-treatment with an AMP-activated protein kinase (AMPK) inhibitor prevented the actions of EGCG on proliferation and AMPK phosphorylation. These data suggest that EGCG mediates choriocarcinoma cell growth via the AMPK, ERK, and p38 pathways, but not JNK pathway. PMID:27208402

  12. The synthesis, secretion and uptake of prorenin in human amnion

    PubMed Central

    Pringle, Kirsty G; Wang, Yu; Lumbers, Eugenie R

    2015-01-01

    Very high concentrations of prorenin protein occur in human amniotic fluid and amnion. The source of amniotic fluid prorenin is likely the decidua, as it has the highest levels of prorenin mRNA (REN). Conversely, REN mRNA levels in amnion and chorion are very low. This study aimed to investigate whether decidual prorenin could cross the amnion and accumulate in amniotic fluid. Late gestation amnion was incubated for 24 h in the presence or absence of recombinant human (rh)prorenin. REN mRNA abundance was determined by qPCR and prorenin protein levels in the supernatant and tissue were measured by an ELISA. Prior to incubation only 3/11 amnion samples had REN mRNA but measurable levels of prorenin protein were found (1.4 ng/mg total protein). After 24 h incubation, REN mRNA was found in all explants and levels were significantly increased (P = 0.03) but prorenin protein levels in amnion were unchanged. Prorenin protein levels in the supernatant were, however, increased (P = 0.048). Incubation with (rh)prorenin significantly increased amnion tissue prorenin levels (2.8 ng/mg total protein, P = 0.001); REN mRNA levels were unchanged. Therefore, amnion explants express small amounts of REN and secrete prorenin protein. Prorenin is also taken up by amnion. We postulate that the amniotic renin angiotensin system (RAS) alters pregnancy outcome through effects on gestation length and amniotic fluid volume. Since human amnion can take up and secrete prorenin protein, the activity of both amnion and amniotic fluid RASs can be amplified by prorenin produced by other intrauterine tissues. PMID:25902786

  13. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo.

    PubMed

    Greening, David W; Ji, Hong; Chen, Maoshan; Robinson, Bruce W S; Dick, Ian M; Creaney, Jenette; Simpson, Richard J

    2016-01-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets. PMID:27605433

  14. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo

    PubMed Central

    Greening, David W.; Ji, Hong; Chen, Maoshan; Robinson, Bruce W. S.; Dick, Ian M.; Creaney, Jenette; Simpson, Richard J.

    2016-01-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets. PMID:27605433

  15. Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation.

    PubMed

    Berger, Markus; Reck, José; Terra, Renata M S; Beys da Silva, Walter O; Santi, Lucélia; Pinto, Antônio F M; Vainstein, Marilene H; Termignoni, Carlos; Guimarães, Jorge A

    2010-10-01

    The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile. PMID:20157842

  16. Activation of human inflammatory cells by secreted phospholipases A2.

    PubMed

    Triggiani, Massimo; Granata, Francescopaolo; Frattini, Annunziata; Marone, Gianni

    2006-11-01

    Secreted phospholipases A(2) (sPLA(2)s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA(2)s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA(2)s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA(2)s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA(2)s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA(2) isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA(2)s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA(2)s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA(2)-enzymatic activity and specific antagonists of sPLA(2) receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases. PMID:16952481

  17. Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations.

    PubMed

    Cong, Xiaojing; Cremer, Christian; Nachreiner, Thomas; Barth, Stefan; Carloni, Paolo

    2016-08-01

    Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity. PMID:27110669

  18. Accurate assessment of early gestational age in normal and diabetic women by serum human placental lactogen concentration.

    PubMed

    Whittaker, P G; Aspillaga, M O; Lind, T

    1983-08-01

    Serum human placental lactogen (hPL) and human chorionic gonadotropin (hCG) were assayed and fetal crown-rump length (CRL) was determined by sonar in three groups of pregnant women--35 with uncomplicated pregnancies, 13 with insulin-dependent diabetes mellitus, and 21 who represented a general pregnancy population. Each patient had a regular cycle and recorded last menstrual period, ovulated spontaneously, and was delivered of a single live baby. Serum hPL concentrations within the range 0.01-0.80 microU/ml in patients in the first group gave estimates of gestation with an SD of 6.3 days which was the same as the SD derived from CRL measurements. When the hPL regression equation was applied to the diabetic mothers the difference between the gestational age estimated from hPL and that estimated from LMP had a mean value of - 0.9 days with an SD of 6.2 days; this difference was not significantly different from zero. The third group of patients had a mean difference between hPL and LMP derived gestational age of 0.7 days (+/- 6.7 SD). Serum hPL offers a method of estimating gestation sufficiently precise to be used as a practical alternative to sonar measurements of CRL. PMID:6135831

  19. Preparation, characterization, and transport of dexamethasone-loaded polymeric nanoparticles across a human placental in vitro model.

    PubMed

    Ali, Hazem; Kalashnikova, Irina; White, Mark Andrew; Sherman, Michael; Rytting, Erik

    2013-09-15

    The purpose of this study was to prepare dexamethasone-loaded polymeric nanoparticles and evaluate their potential for transport across human placenta. Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics: particle size, polydispersity index, zeta potential, and drug encapsulation efficiency. Dexamethasone and nanoparticle transport was subsequently investigated using the BeWo b30 cell line, an in vitro model of human placental trophoblast cells, which represent the rate-limiting barrier for maternal-fetal transfer. Encapsulation efficiency and drug transport were determined using a validated high performance liquid chromatography method. Nanoparticle morphology and drug encapsulation were further characterized by cryo-transmission electron microscopy and X-ray diffraction, respectively. Nanoparticles prepared from poly(lactic-co-glycolic acid) were spherical, with particle sizes ranging from 140 to 298 nm, and encapsulation efficiency ranging from 52 to 89%. Nanoencapsulation enhanced the apparent permeability of dexamethasone from the maternal compartment to the fetal compartment more than 10-fold in this model. Particle size was shown to be inversely correlated with drug and nanoparticle permeability, as confirmed with fluorescently labeled nanoparticles. These results highlight the feasibility of designing nanoparticles capable of delivering medication to the fetus, in particular, potential dexamethasone therapy for the prenatal treatment of congenital adrenal hyperplasia. PMID:23850397

  20. Aberrant tropoelastin secretion in MG-63 human osteosarcoma cells

    SciTech Connect

    Curtiss, S.W.

    1989-01-01

    The secretion of newly synthesized tropoelastin, the soluble precursor of the extracellular matrix protein elastin, is not well understood. MG-63 human osteosarcoma cells were found by immunoblot analysis to synthesize 62 kD and 64 kD tropoelastins. Media from 63 cells labelled for five hours with ({sup 3}H)-valine contain no detectable tropoelastin, unlike media from other tropoelastin-synthesizing cells. Immunoblots of conditioned media and 1Ox-concentrated conditioned media left on the cells for six days also show an absence of tropoelastin from the cell media. No insoluble elastin is associated with the cell layer, as determined by amino acid analysis and electron microscopy of 18-21 day cell cultures. The absence of tropoelastin from the cell medium and elastin from the extracellular matrix indicates that MG63 cells do not secrete tropoelastin as expected, but accumulate it intracellularly. This accumulation is transient: immunoblots and immunofluorescence microscopy show that cells three days after passage have the highest steady-state levels of tropoelastin per cell, that day 8 cells contain lower but still significant amounts of tropoelastin, and that by day 22 tropoelastin is no longer present in the cell cultures. Cell density is a critical factor in the observed pattern of tropoelastin expression. Cells seeded at ten fold their usual initial density have high tropoelastin levels at one day after passage, sooner than cells seeded normally. Tropoelastin also disappears from high density-seeded cells more quickly and is no longer detectable at day 10. Lysosome-like vesicles containing membranous structures appear by immunoelectron microscopy to be the primary site of intracellular tropoelastin localization.

  1. H.p.l.c. separation and study of the charge isomers of human placental glutathione transferase.

    PubMed Central

    Radulovic, L L; Kulkarni, A P

    1986-01-01

    Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo. Images Fig. 2. PMID:3800986

  2. Expression and Functional Activity of the Human Bitter Taste Receptor TAS2R38 in Human Placental Tissues and JEG-3 Cells.

    PubMed

    Wölfle, Ute; Elsholz, Floriana A; Kersten, Astrid; Haarhaus, Birgit; Schumacher, Udo; Schempp, Christoph M

    2016-01-01

    Bitter taste receptors (TAS2Rs) are expressed in mucous epithelial cells of the tongue but also outside the gustatory system in epithelial cells of the colon, stomach and bladder, in the upper respiratory tract, in the cornified squamous epithelium of the skin as well as in airway smooth muscle cells, in the testis and in the brain. In the present work we addressed the question if bitter taste receptors might also be expressed in other epithelial tissues as well. By staining a tissue microarray with 45 tissue spots from healthy human donors with an antibody directed against the best characterized bitter taste receptor TAS2R38, we observed an unexpected strong TAS2R38 expression in the amniotic epithelium, syncytiotrophoblast and decidua cells of the human placenta. To analyze the functionality we first determined the TAS2R38 expression in the placental cell line JEG-3. Stimulation of these cells with diphenidol, a clinically used antiemetic agent that binds TAS2Rs including TAS2R38, demonstrated the functionality of the TAS2Rs by inducing calcium influx. Restriction enzyme based detection of the TAS2R38 gene allele identified JEG-3 cells as PTC (phenylthiocarbamide)-taster cell line. Calcium influx induced by PTC in JEG-3 cells could be inhibited with the recently described TAS2R38 inhibitor probenecid and proved the specificity of the TAS2R38 activation. The expression of TAS2R38 in human placental tissues points to further new functions and hitherto unknown endogenous ligands of TAS2Rs far beyond bitter tasting. PMID:26950109

  3. The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

    PubMed

    Sieppi, E; Vähäkangas, K; Rautio, A; Ietta, F; Paulesu, L; Myllynen, P

    2016-07-01

    Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age. PMID:27036933

  4. Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

    PubMed

    Ilekis, John V; Tsilou, Ekaterini; Fisher, Susan; Abrahams, Vikki M; Soares, Michael J; Cross, James C; Zamudio, Stacy; Illsley, Nicholas P; Myatt, Leslie; Colvis, Christine; Costantine, Maged M; Haas, David M; Sadovsky, Yoel; Weiner, Carl; Rytting, Erik; Bidwell, Gene

    2016-07-01

    Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiology of pregnancy-related disorders, a significant gap exists in the utilization of this information for the development of new drug therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular emphasis was given to the identification of important molecular pathways that could serve as drug targets and the advantages and disadvantages of targeting these particular pathways. This article is a summary of the proceedings of that workshop. A broad number of topics were covered that ranged from basic placental biology to clinical trials. This included research in the basic biology of placentation, such as trophoblast migration and spiral artery remodeling, and trophoblast sensing and response to infectious and noninfectious agents. Research findings in these areas will be critical for the formulation of the development of future treatments and the development of therapies for the prevention of a number of pregnancy disorders of placental origin that include preeclampsia, fetal growth restriction, and uterine inflammation. Research was also presented that summarized ongoing clinical efforts in the United States and in Europe that has tested novel interventions for preeclampsia and fetal growth restriction, including agents such as oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were also proposed to improve fetal growth by the enhancement of nutrient transport to the fetus by modulation of their placental transporters and the targeting of placental

  5. Characterization of Humanized Antibodies Secreted by Aspergillus niger

    PubMed Central

    Ward, Michael; Lin, Cherry; Victoria, Doreen C.; Fox, Bryan P.; Fox, Judith A.; Wong, David L.; Meerman, Hendrik J.; Pucci, Jeff P.; Fong, Robin B.; Heng, Meng H.; Tsurushita, Naoya; Gieswein, Christine; Park, Minha; Wang, Huaming

    2004-01-01

    Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2 to Hex15GlcNAc2. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function. PMID:15128505

  6. Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages*

    PubMed Central

    Karunakaran, Denuja; Kockx, Maaike; Owen, Dylan M.; Burnett, John R.; Jessup, Wendy; Kritharides, Leonard

    2013-01-01

    Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages. PMID:23288845

  7. Programming placental nutrient transport capacity

    PubMed Central

    Fowden, A L; Ward, J W; Wooding, F P B; Forhead, A J; Constancia, M

    2006-01-01

    Many animal studies and human epidemiological findings have shown that impaired growth in utero is associated with physiological abnormalities in later life and have linked this to tissue programming during suboptimal intrauterine conditions at critical periods of development. However, few of these studies have considered the contribution of the placenta to the ensuing adult phenotype. In mammals, the major determinant of intrauterine growth is the placental nutrient supply, which, in turn, depends on the size, morphology, blood supply and transporter abundance of the placenta and on synthesis and metabolism of nutrients and hormones by the uteroplacental tissues. This review examines the regulation of placental nutrient transfer capacity and the potential programming effects of nutrition and glucocorticoid over-exposure on placental phenotype with particular emphasis on the role of the Igf2 gene in these processes. PMID:16439433

  8. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

    PubMed Central

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×106 cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  9. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

    PubMed

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  10. Polybrominated diphenyl ethers (PBDEs) in human samples of mother-newborn pairs in South China and their placental transfer characteristics.

    PubMed

    Chen, Zhuo-Jia; Liu, Han-Yan; Cheng, Zhang; Man, Yu-Bon; Zhang, Kun-Shui; Wei, Wei; Du, Jun; Wong, Ming-Hung; Wang, Hong-Sheng

    2014-12-01

    There are limited data concerning the placenta transfer characteristics and accumulation of polybrominated diphenyl ethers (PBDEs) in infants. However, PBDEs received increasing health concerns due to their endocrine disrupt and neurodevelopment toxicity effects. The present study assessed the accumulation of PBDEs in 30 paired placenta, breast milk, fetal cord blood, and neonatal urine samples collected from five major cities of the South China. The age of mothers ranged from 21 to 39 (mean 27.6±4.56). The ∑PBDE concentrations were 15.8±9.88 ng g(-1) lipid in placenta, 13.2±7.64 ng g(-1) lipid in breast milk, 16.5±19.5 ng g(-1) lipid in fetal cord blood, and 1.80±1.99 ng ml(-1) in neonatal urine. BDE-47 was the predominant congener in all types of human sample. Octa-BDEs such as BDE-196/-197 were detected highly in placenta and cord blood while moderately in breast milk and neonatal urine. Significant (p<0.01) correlations were observed for both total and most individual PBDEs in cord blood-maternal placenta and breast milk-urine paired individual samples. The extent of placental transfer of higher brominated BDEs such as BDE-196/-197 was greater than that of BDE-47. The estimated daily intake (EDI) analysis for breast-fed infants revealed that newborns in these areas were exposed to relatively high levels of PBDEs via breast milk. Our study not only provided systematic fundamental data for PBDE distribution but also revealed the placenta transfer characteristics of PBDE congeners in South China. PMID:25090577

  11. Novel 3D Microscopic Analysis of Human Placental Villous Trees Reveals Unexpected Significance of Branching Angles

    PubMed Central

    Haeussner, Eva; Buehlmeyer, Antonia; Schmitz, Christoph; von Koch, Franz Edler; Frank, Hans-Georg

    2014-01-01

    The villous trees of human placentas delineate the fetomaternal border and are complex three-dimensional (3D) structures. Thus far, they have primarily been analyzed as thin, two-dimensional (2D) histological sections. However, 2D sections cannot provide access to key aspects such as branching nodes and branch order. Using samples taken from 50 normal human placentas at birth, in the present study we show that analysis procedures for 3D reconstruction of neuronal dendritic trees can also be used for analyzing trees of human placentas. Nodes and their branches (e.g., branching hierarchy, branching angles, diameters, and lengths of branches) can be efficiently measured in whole-mount preparations of isolated villous trees using high-end light microscopy. Such data differ qualitatively from the data obtainable from histological sections and go substantially beyond the morphological horizon of such histological data. Unexpectedly, branching angles of terminal branches of villous trees varied inversely with the fetoplacental weight ratio, a widely used clinical parameter. Since branching angles have never before been determined in the human placenta, this result requires further detailed studies in order to fully understand its impact. PMID:25155961

  12. Effect of enteral nutrition on human pancreatic secretions.

    PubMed

    Grant, J P; Davey-McCrae, J; Snyder, P J

    1987-01-01

    The influence on pancreatic secretion of four enteral feeding products was evaluated in a unique patient with an isolated duodenal fistula for whom enteral feeding access was obtained via a gastrostomy with a small Silastic catheter passed through the gastrostomy and through a surgically created gastrojejunostomy. The patient was totally supported by intravenous nutrition during the study. Each enteral feeding solution was administered at full strength at 50 ml/hr for 2 days with a 24-hr collection of pancreatic secretions by the duodenal cutaneous fistula taken on the second day. Infusion of the enteral feeding solutions did not alter volume of fistula drainage. All solutions decreased bicarbonate and amylase secretion but increased lipase and total nitrogen excretion. From this study, it would appear reasonable to administer Vivonex HN and Criticare HN via the jejunum in patients with pancreatic disease, whereas Osmolite would appear less satisfactory, due to its much stronger stimulation of lipase secretion. PMID:3110448

  13. Pigment epithelium-derived factor (PEDF): a novel trophoblast-derived factor limiting feto-placental angiogenesis in late pregnancy.

    PubMed

    Loegl, Jelena; Nussbaumer, Erika; Hiden, Ursula; Majali-Martinez, Alejandro; Ghaffari-Tabrizi-Wizy, Nassim; Cvitic, Silvija; Lang, Ingrid; Desoye, Gernot; Huppertz, Berthold

    2016-07-01

    The rapidly expanding feto-placental vasculature needs tight control by paracrine and endocrine mechanisms. Here, we focused on paracrine influence by trophoblast, the placental epithelium. We aimed to identify differences in regulation of feto-placental angiogenesis in early versus late pregnancy. To this end, the effect of conditioned media (CM) from early and late pregnancy human trophoblast was tested on network formation, migration and proliferation of human feto-placental endothelial cells. Only CM of late pregnancy trophoblast reduced network formation and migration. Screening of trophoblast transcriptome for anti-angiogenic candidates identified pigment epithelium-derived factor (PEDF) with higher expression and protein secretion in late pregnancy trophoblast. Addition of a PEDF-neutralizing antibody restored the anti-angiogenic effect of CM from late pregnancy trophoblast. Notably, human recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two key players in VEGF-induced proliferation and migration, revealed that PEDF altered VEGF signaling, while PEDF alone did not affect phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and targets to modulate the intracellular effect of VEGF. PMID:27278471

  14. Macrophage-derived IL-33 is a critical factor for placental growth.

    PubMed

    Fock, Valerie; Mairhofer, Mario; Otti, Gerlinde R; Hiden, Ursula; Spittler, Andreas; Zeisler, Harald; Fiala, Christian; Knöfler, Martin; Pollheimer, Jürgen

    2013-10-01

    IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy. PMID:23997215

  15. Acetylcholine output and foetal vascular resistance of human perfused placental cotyleda.

    PubMed Central

    Boura, A. L.; Gude, N. M.; King, R. G.; Walters, W. A.

    1986-01-01

    The foetal villous vessels of single cotyleda of human placentae have been perfused with a constant flow of Krebs solution, recording inflow pressure and passing the venous perfusate in cascade over guinea-pig ileum and rat stomach strip preparations in vitro. Each cotyledon released for at least 4 h a substance that was probably acetylcholine. The perfusate caused contractions of both preparations which were inhibited by atropine or hyoscine and potentiated by physostigmine. Contractile activity was destroyed after incubation at 37 degrees C of perfusate with acetylcholinesterase but not with acetylcholinesterase plus physostigmine. When the perfusion temperature was lowered to 34 degrees C or below, acetylcholine output was reduced, the extent depending on the fall in temperature. No change in resistance of the villous vessels occurred during the changes in temperature or in the presence at 37 degrees C of atropine, hyoscine, hexamethonium, (+)-tubocurarine, hemicholinium-3 or bretylium. Submaximal vasoconstrictor responses of the villous vessels to the thromboxane A2-mimetic U46619 were not affected by reduction of the perfusion temperature to 30 degrees C, which lowered acetylcholine-like output by approximately 70%. Responses to U46619, at 37 degrees C, were unchanged during the presence of atropine or hyoscine. Acetylcholine is released into the foetal circulation of the human placenta but no evidence could be obtained that it affects villous vascular smooth muscle tone or vasoconstrictor responses. PMID:3730696

  16. Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization

    SciTech Connect

    Yoshida, Nobutaka; Osawa, Yoshio )

    1991-03-26

    A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B couples with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-{alpha}-phosphatidylcholine with (1{beta}-{sup 3}H,4-{sup 14}C)androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high K{sub m} value for 16{alpha}-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at {minus}80C.

  17. Regulation of follistatin-like protein 1 expression and secretion in primary human skeletal muscle cells.

    PubMed

    Görgens, Sven W; Raschke, Silja; Holven, Kirsten Bjørklund; Jensen, Jørgen; Eckardt, Kristin; Eckel, Jürgen

    2013-05-01

    Follistatin-like protein 1 (Fstl1) is a secreted glycoprotein of the follistatin family. Fstl1 is secreted by C2C12 cells, and Akt1 over-expression in skeletal muscle leads to its induction in muscle and increased circulating levels. So far, secretion of Fstl1 by human myotubes and the effect of exercise on its circulating levels have not been investigated. Here, we examined both the regulation of Fstl1 expression and secretion in primary human skeletal muscle cells and the effect of acute exercise on Fstl1 serum concentrations in humans. We show that human myotubes express and secrete Fstl1 in a differentiation-dependent manner. Furthermore, IFNγ and IL-1β significantly increase Fstl1 secretion. Electrical pulse stimulation (EPS)-induced contractile activity of myotubes did not regulate Fstl1. Interestingly, we observed that 60 min cycling increased serum Fstl1 level by 22%. In conclusion, we demonstrate that Fstl1 is expressed and secreted by human myotubes and plasma Fstl1 levels are increased after exercise. PMID:23419164

  18. A stochastic model for early placental development†

    PubMed Central

    Cotter, Simon L.; Klika, Václav; Kimpton, Laura; Collins, Sally; Heazell, Alexander E. P.

    2014-01-01

    In the human, placental structure is closely related to placental function and consequent pregnancy outcome. Studies have noted abnormal placental shape in small-for-gestational-age infants which extends to increased lifetime risk of cardiovascular disease. The origins and determinants of placental shape are incompletely understood and are difficult to study in vivo. In this paper, we model the early development of the human placenta, based on the hypothesis that this is driven by a chemoattractant effect emanating from proximal spiral arteries in the decidua. We derive and explore a two-dimensional stochastic model, and investigate the effects of loss of spiral arteries in regions near to the cord insertion on the shape of the placenta. This model demonstrates that disruption of spiral arteries can exert profound effects on placental shape, particularly if this is close to the cord insertion. Thus, placental shape reflects the underlying maternal vascular bed. Abnormal placental shape may reflect an abnormal uterine environment, predisposing to pregnancy complications. Through statistical analysis of model placentas, we are able to characterize the probability that a given placenta grew in a disrupted environment, and even able to distinguish between different disruptions. PMID:24850904

  19. Membrane potential difference and intracellular cation concentrations in human placental trophoblast cells in culture.

    PubMed Central

    Greenwood, S L; Clarson, L H; Sides, M K; Sibley, C P

    1996-01-01

    1. The electrochemical gradients for Na+ and K+ were assessed in a cell culture model of trophoblast differentiation. 2. Membrane potential difference (Em), intracellular water and Na+ and K+ contents were measured in choriocarcinoma cells (JAr cell line; 96% of which are undifferentiated trophoblast cells) and in mononucleate and multinucleate (differentiated) cytotrophoblast cells isolated from the human placenta at term. 3. There was a significant fall in Em from -57 mV in JAr cells, to -48 and -40 mV in mono-and multinucleate cytotrophoblast cells, respectively. Treatment with ouabain (1 mM for 15 min) depolarized the JAr cell membrane by 15 mV but did not affect cytotrophoblast cell membrane potential. 4. Intracellular K+ concentration was similar in JAr, mono- and multinucleate cytotrophoblast cells but Na+ concentration was higher in mononucleate cytotrophoblast cells compared with JAr cells. 5. Ouabain treatment (3 mM for 15 min) caused a small increase (4.5%) in cell water in mononucleate cytotrophoblast cells but lowered K+ (approximately 30%) and increased Na+ concentration (approximately 125%) in all the trophoblast cells studied. 6. The K+ equilibrium potential (EK) was more negative than Em in all cells and the difference between EK and Em was smaller in JAr cells (-25 mV) than in mono- and multinucleate cytotrophoblast cells (-33 and -43 mV, respectively). 7. The Na+ equilibrium potential (ENa) was positive in the trophoblast cells and the difference between ENa and Em was 122, 100 and 100 mV in JAr, mono- and multinucleate cytotrophoblast cells, respectively. 8. These results suggest that the electrochemical gradient for K+ is affected by the stage of trophoblast cell differentiation. In contrast, the electrochemical gradient for Na+ is similar in mono- and multinucleate cytotrophoblast cells. Images Figure 1 PMID:8734977

  20. Application of Human Placental Villous Tissue Explants to Study ABC Transporter Mediated Efflux of 2,4-Dinitrophenyl-S-Glutathione

    PubMed Central

    Vaidya, Soniya S.; Walsh, Scott W.; Gerk, Phillip M.

    2011-01-01

    Objective The purpose of this study was to characterize the human term placental villous tissue explant culture model as a tool to study the formation and efflux of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) as a model system for phase II metabolism and ATP-binding cassette (ABC) transporter-mediated cellular efflux. Methods Placental tissue samples were obtained after cesarean section following normal pregnancies (n=9). Cultured villous tissue was monitored up to 48 h to study the effect of time in culture on biochemical parameters, formation and efflux of DNP-SG in the absence or presence of ATPase inhibitor sodium orthovanadate and the protein expression of ABC transporters - multidrug resistance associated protein 2 (MRP2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and enzyme glutathione-S-transferase isoform P1-1 (GSTP1-1). Results Villous tissue structure, tissue viability and expression of BCRP, GSTP1-1 remained unchanged, while expression of MRP2, P-gp and total tissue glutathione decreased with time in culture. Tissue integrity was unchanged up to 24 h but declined at 48 h. However, DNP-SG formation, DNP-SG efflux, and the extent of inhibition of DNP-SG efflux by sodium orthovanadate showed only minor changes through 48 h. Sodium orthovanadate decreased DNP-SG efflux, consistent with inhibition of apical ABC transporters. Conclusion The results support the use of the cultured human term placental villous tissue explants model to study coordinated function of GSTP1-1 and apical ABC transporters in the formation and efflux of the model substrate DNP-SG. PMID:21342117

  1. Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates.

    PubMed

    Wang, Hong; Schuetz, Chris; Arima, Akihiro; Chihaya, Yutaka; Weinbauer, Gerhard F; Habermann, Gunnar; Xiao, Jim; Woods, Cynthia; Grogan, Jane; Gelzleichter, Thomas; Cain, Gary

    2016-08-01

    An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant. PMID:27211603

  2. Human placental extract exerts hair growth-promoting effects through the GSK-3β signaling pathway in human dermal papilla cells.

    PubMed

    Kwon, Tae-Rin; Oh, Chang Taek; Choi, Eun Ja; Park, Hye Min; Han, Hae Jung; Ji, Hyi Jeong; Kim, Beom Joon

    2015-10-01

    Human placental extract (HPE) is widely used in Korea to relieve fatigue. However, its effects on human dermal papilla cells (hDPCs) remain unknown. In the present study, in an effort to develop novel therapies to promote hair growth, we screened HPE. We demonstrate that HPE has hair growth‑promoting activities and induces β‑catenin expression through the inhibition of glycogen synthase kinase‑3β (GSK‑3β) by phosphorylation in hDPCs. Treatment with HPE significantly increased the viability of the hDPCs in a concentration‑dependent manner, as shown by bromodeoxyuridine (BrdU) assay. HPE also significantly increased the alkaline phosphatase (ALP) expression levels. The increased β‑catenin levels and the inhibition of GSK‑3β (Ser9) by phosphorylation suggested that HPE promoted the hair-inductive capacity of hDPCs. We compared the effects of treatment with HPE alone and treatment with HPE in conjunction with minoxidil (MXD). We found that HPE plus MXD effectively inhibited GSK‑3β by phosphorylation (Ser9) in the hDPCs. Moreover, we demonstrated that HPE was effective in inducing root hair elongation in rat vibrissa hair follicles, and that treatment with HPE led to a delay in catagen progression. Overall, our findings suggest that HPE promotes hair growth and may thus provide the basis of a novel therapeutic strategy for the clinical treatment of hair loss. PMID:26311045

  3. Placental Adaptations in Growth Restriction

    PubMed Central

    Zhang, Song; Regnault, Timothy R.H.; Barker, Paige L.; Botting, Kimberley J.; McMillen, Isabella C.; McMillan, Christine M.; Roberts, Claire T.; Morrison, Janna L.

    2015-01-01

    The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions. PMID:25580812

  4. Novel 3D light microscopic analysis of IUGR placentas points to a morphological correlate of compensated ischemic placental disease in humans

    PubMed Central

    Haeussner, Eva; Schmitz, Christoph; Frank, Hans-Georg; Edler von Koch, Franz

    2016-01-01

    The villous tree of the human placenta is a complex three-dimensional (3D) structure with branches and nodes at the feto-maternal border in the key area of gas and nutrient exchange. Recently we introduced a novel, computer-assisted 3D light microscopic method that enables 3D topological analysis of branching patterns of the human placental villous tree. In the present study we applied this novel method to the 3D architecture of peripheral villous trees of placentas from patients with intrauterine growth retardation (IUGR placentas), a severe obstetric syndrome. We found that the mean branching angle of branches in terminal positions of the villous trees was significantly different statistically between IUGR placentas and clinically normal placentas. Furthermore, the mean tortuosity of branches of villous trees in directly preterminal positions was significantly different statistically between IUGR placentas and clinically normal placentas. We show that these differences can be interpreted as consequences of morphological adaptation of villous trees between IUGR placentas and clinically normal placentas, and may have important consequences for the understanding of the morphological correlates of the efficiency of the placental villous tree and their influence on fetal development. PMID:27045698

  5. Novel 3D light microscopic analysis of IUGR placentas points to a morphological correlate of compensated ischemic placental disease in humans.

    PubMed

    Haeussner, Eva; Schmitz, Christoph; Frank, Hans-Georg; Edler von Koch, Franz

    2016-01-01

    The villous tree of the human placenta is a complex three-dimensional (3D) structure with branches and nodes at the feto-maternal border in the key area of gas and nutrient exchange. Recently we introduced a novel, computer-assisted 3D light microscopic method that enables 3D topological analysis of branching patterns of the human placental villous tree. In the present study we applied this novel method to the 3D architecture of peripheral villous trees of placentas from patients with intrauterine growth retardation (IUGR placentas), a severe obstetric syndrome. We found that the mean branching angle of branches in terminal positions of the villous trees was significantly different statistically between IUGR placentas and clinically normal placentas. Furthermore, the mean tortuosity of branches of villous trees in directly preterminal positions was significantly different statistically between IUGR placentas and clinically normal placentas. We show that these differences can be interpreted as consequences of morphological adaptation of villous trees between IUGR placentas and clinically normal placentas, and may have important consequences for the understanding of the morphological correlates of the efficiency of the placental villous tree and their influence on fetal development. PMID:27045698

  6. Human Mammospheres Secrete Hormone-Regulated Active Extracellular Vesicles

    PubMed Central

    Rodriguez-Suarez, Eva; Gil, David; Royo, Felix; Elortza, Felix; Falcon-Perez, Juan M.; Vivanco, Maria dM.

    2014-01-01

    Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression. PMID:24404144

  7. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    PubMed Central

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

  8. A Positive Feedback Loop between Glial Cells Missing 1 and Human Chorionic Gonadotropin (hCG) Regulates Placental hCGβ Expression and Cell Differentiation

    PubMed Central

    Cheong, Mei-Leng; Wang, Liang-Jie; Chuang, Pei-Yun; Chang, Ching-Wen; Lee, Yun-Shien; Lo, Hsiao-Fan; Tsai, Ming-Song

    2015-01-01

    Human chorionic gonadotropin (hCG) is composed of a common α subunit and a placenta-specific β subunit. Importantly, hCG is highly expressed in the differentiated and multinucleated syncytiotrophoblast, which is formed via trophoblast cell fusion and stimulated by cyclic AMP (cAMP). Although the ubiquitous activating protein 2 (AP2) transcription factors TFAP2A and TFAP2C may regulate hCGβ expression, it remains unclear how cAMP stimulates placenta-specific hCGβ gene expression and trophoblastic differentiation. Here we demonstrated that the placental transcription factor glial cells missing 1 (GCM1) binds to a highly conserved promoter region in all six hCGβ paralogues by chromatin immunoprecipitation-on-chip (ChIP-chip) analyses. We further showed that cAMP stimulates GCM1 and the CBP coactivator to activate the hCGβ promoter through a GCM1-binding site (GBS1), which also constitutes a previously identified AP2 site. Given that TFAP2C may compete with GCM1 for GBS1, cAMP enhances the association between the hCGβ promoter and GCM1 but not TFAP2C. Indeed, the hCG-cAMP-protein kinase A (PKA) signaling pathway also stimulates Ser269 and Ser275 phosphorylation of GCM1, which recruits CBP to mediate GCM1 acetylation and stabilization. Consequently, hCG stimulates the expression of GCM1 target genes, including the fusogenic protein syncytin-1, to promote placental cell fusion. Our study reveals a positive feedback loop between GCM1 and hCG regulating placental hCGβ expression and cell differentiation. PMID:26503785

  9. Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

    PubMed

    Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

    2016-09-01

    Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. PMID:27432863

  10. Dopamine-mediated autocrine inhibitory circuit regulating human insulin secretion in vitro.

    PubMed

    Simpson, Norman; Maffei, Antonella; Freeby, Matthew; Burroughs, Steven; Freyberg, Zachary; Javitch, Jonathan; Leibel, Rudolph L; Harris, Paul E

    2012-10-01

    We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-containing secretory granules by vesicular monoamine transporter type 2 in human β-cells, is released in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its action on D2R, which are also expressed on β-cells. We found that antagonism of receptors participating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in vitro observations may represent correlates of the in vivo metabolic changes associated with the use of atypical antipsychotics, such as increased adiposity. PMID:22915827

  11. Dopamine-Mediated Autocrine Inhibitory Circuit Regulating Human Insulin Secretion in Vitro

    PubMed Central

    Simpson, Norman; Maffei, Antonella; Freeby, Matthew; Burroughs, Steven; Freyberg, Zachary; Javitch, Jonathan; Leibel, Rudolph L.

    2012-01-01

    We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-containing secretory granules by vesicular monoamine transporter type 2 in human β-cells, is released in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its action on D2R, which are also expressed on β-cells. We found that antagonism of receptors participating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in vitro observations may represent correlates of the in vivo metabolic changes associated with the use of atypical antipsychotics, such as increased adiposity. PMID:22915827

  12. Primary human chorionic gonadotropin secreting germinoma of the corpus callosum

    PubMed Central

    Chuan Aaron, Foo Song; Dawn, Chong Q. Q.; Kenneth, Chang T. E.; Hoe, Ng Wai; Yen, Soh Shui; Chee Kian, Tham

    2013-01-01

    Background: Primary intracranial germinomas are a rare subset of intracranial tumors derived from mis-incorporated germ cells within the folding neural plate during embryogenesis. Though known to arise from midline structures in the central nervous system (CNS), occurrence within the corpus callosum is exceedingly rare. Case Description: We present a rare case of secreting primary intracranial germinoma with extensive intraventricular metastasis presenting as a multi-cystic butterfly lesion in the genu of the corpus callosum in a young boy. Conclusion: Intracranial germ cell tumors must be considered for any multi-cystic lesion arising from midline structures in the CNS in the preadult population. PMID:24233184

  13. Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion.

    PubMed Central

    Fantini, J; Yahi, N; Baghdiguian, S; Chermann, J C

    1992-01-01

    Selected strains of the human immunodeficiency virus (HIV) types 1 and 2 are able to infect human colon epithelial cells in vitro, suggesting a mechanism for the anal route of HIV transmission. In some cases, HIV is not produced by infected colon cells but can be rescued after coculture with T-lymphoid cells. One of the HIV strains (HIV1-NDK) replicated well in colonic cells. A transmission electron microscope study demonstrated two major structural perturbations in producer colon cells: an unusual number of secretion bodies and the appearance of intracellular lumina with disorganized microvilli, indicating a defect in brush border assembly and differentiation. Either abnormality could account for HIV-induced enteropathy consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens. Moreover, HT29 cells infected with HIV provide a unique model for selection of enterotropic HIV strains. Images PMID:1727501

  14. Long-chain polyunsaturated fatty acids stimulate cellular fatty acid uptake in human placental choriocarcinoma (BeWo) cells.

    PubMed

    Johnsen, G M; Weedon-Fekjaer, M S; Tobin, K A R; Staff, A C; Duttaroy, A K

    2009-12-01

    Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 microM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [(14)C] fatty acids was increased by 20-50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ASCL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development. PMID:19880178

  15. Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells.

    PubMed

    Chang, Seok-Woo; Kim, Ji-Youn; Kim, Mi-Joo; Kim, Ga-Hyun; Yi, Jin-Kyu; Lee, Deok-Won; Kum, Kee-Yeon; Kim, Eun-Cheol

    2016-05-01

    Objective The aim of this study was to evaluate the combined effects of mineral trioxide aggregate (MTA) and human placental extract (HPE) on cell growth, differentiation and in vitro angiogenesis of human dental pulp cells (HDPCs) and to identify underlying signal transduction mechanisms. In vivo dental pulp responses in rats for a pulp-capping agent were examined. Materials and methods MTS assay. ALP activity test, alizarin red S staining and RT-PCR for marker genes were carried out to evaluate cell growth and differentiation. HUVEC migration, mRNA expression and capillary tube formation were measured to evaluate angiogenesis. Signal transduction was analysed using Western blotting and confocal microscopy. The pulps of rat maxillary first molars were exposed and capped with either MTA or MTA plus HPE. Histologic observation and scoring were performed. Results Compared to treatment of HDPCs with either HPE or MTA alone, the combination of HPE and MTA increased cell growth, ALP activity, mineralized nodules and expression of marker mRNAs. Combination HPE and MTA increased migration, capillary tube formation and angiogenic gene expression compared with MTA alone. Activation of Akt, mammalian target of rapamycin (mTOR), p38, JNK and ERK MAPK, Akt, and NF-κB were significantly increased by combining HPE and MTA compared with MTA alone. Pulp capping with MTA plus HPE in rats showed superior dentin bridge formation, odontoblastic layers and dentinal tubules and lower inflammatory cell response, compared to the MTA alone group. Conclusions This study demonstrates for the first time that the use of MTA with HPE promotes cell growth, differentiation and angiogenesis in HDPCs, which were associated with mTOR, MAPK and NF-κB pathways. Direct pulp capping with HPE plus MTA showed superior results when compared with MTA alone. Thus, the combination of MTA and HPE may be useful for regenerative endodontics. PMID:26807656

  16. A single-islet microplate assay to measure mouse and human islet insulin secretion.

    PubMed

    Truchan, Nathan A; Brar, Harpreet K; Gallagher, Shannon J; Neuman, Joshua C; Kimple, Michelle E

    2015-01-01

    One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest. PMID:26452321

  17. PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development.

    PubMed

    Garnier, Vanessa; Traboulsi, Wael; Salomon, Aude; Brouillet, Sophie; Fournier, Thierry; Winkler, Carine; Desvergne, Beatrice; Hoffmann, Pascale; Zhou, Qun-Yong; Congiu, Cenzo; Onnis, Valentina; Benharouga, Mohamed; Feige, Jean-Jacques; Alfaidy, Nadia

    2015-08-15

    PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion. PMID:26081281

  18. Regulation of in vitro PWM-induced IgG secretion in humans

    SciTech Connect

    O'Gorman, M.R.; Oger, J.J.

    1989-02-01

    PWM-induced differentiation of human PBMC into immunoglobulin (Ig) secreting cells is a widely used model of in vivo IgG secretion. We examined the relationship between the amount of IgG secreted in PBMC cultures obtained from individuals who consistently secrete either very high (HR) or very low amounts (LR) of IgG and various in vitro immune function assays. The PBMC obtained from LR contained a higher ratio of cells expressing the T-suppressor/inducer to T-helper/inducer phenotype. The autologous mixed lymphocyte response was lower and the amount of in vivo radiation sensitive suppression was higher in the LR than in the HR. LR E- cells secreted less IgG than the HR E- cells when both were mixed with heterologous HR E+ cells. Monocyte depletion reduced IgG secretion in both LR and HR. These results suggest that each of the subsets Ts, Th (Thi, Tsi), and B lymphocytes are involved in the regulation of PWM-induced Ig secretion and that the functions of each of these subsets differ in HR and LR individuals.

  19. Trophoblast viability in perfused term placental tissue and explant cultures limited to 7-24 hours.

    PubMed

    Di Santo, S; Malek, A; Sager, R; Andres, A-C; Schneider, H

    2003-01-01

    Human term-placental culture techniques such as villous explant or dual perfusion are commonly used to study trophoblast function under control and experimentally manipulated conditions. We have compared trophoblast viability during perfusion and in explants cultured under various conditions by monitoring glucose consumption, protein synthesis and secretion, expression of differentiation-specific genes, induction of stress proteins and apoptotic cell death. The tissue was obtained from term-placentae of uncomplicated pregnancies after elective Caesarean delivery. We observed a severe loss of trophoblast viability in explants irrespective of the culture conditions used. Over 7 h of culture the amount of the differentiation specific placental hormones hCG, hPL and leptin accumulated in the medium dropped significantly. Analysis of their expression by semi-quantitative and real-time RT-PCR revealed that the down-regulation of expression occurred at the transcriptional level. This transcriptional repression was accompanied by induction of the stress-proteins RTP and BiP/GRP78. Analysis of apoptotic cell death by TUNEL assay and immunohistochemical detection of the caspase-3-specific degradation product of cytokeratin 18 revealed prominent cell death after 7 h of culture. These results are in contrast to the findings obtained in perfused placental tissue where, after 7 h of culture, hormone secretion, expression of stress proteins and cell death were similar as in native tissue. This difference between villous explant incubation and dual perfusion is also reflected by a significantly higher consumption of glucose in perfused tissue. PMID:13129686

  20. High-resolution elemental mapping of human placental chorionic villi using synchrotron X-ray fluorescence spectroscopy

    DOE PAGESBeta

    Punshon, Tracy; Chen, Si; Finney, Lydia; Howard, Louisa; Jackson, Brian P.; Karagas, Margaret R.; Ornvold, Kim

    2015-07-03

    The placenta is the organ that mediates transport of nutrients and waste materials between mother and fetus. Synchrotron X-ray fluorescence (SXRF) microanalysis is a tool for imaging the distribution and quantity of elements in biological tissue, which can be used to study metal transport across biological membranes. Our aims were to pilot placental biopsy specimen preparation techniques that could be integrated into an ongoing epidemiology birth cohort study without harming rates of sample acquisition. We studied the effects of fixative (formalin or glutaraldehyde) and storage duration (30 days or immediate processing) on metal distribution and abundance and investigated a thaw-fixationmore » protocol for archived specimens stored at -80° C. We measured fixative elemental composition with and without a placental biopsy via inductively coupled plasma mass spectrometry (ICP-MS) to quantify fixative-induced elemental changes. Formalin-fixed specimens showed hemolysis of erythrocytes. The glutaraldehyde-paraformaldehyde solution in HEPES buffer (GTA-HEPES) had superior anatomical preservation, avoided hemolysis, and minimized elemental loss, although some cross-linking of exogenous Zn was evident. Elemental loss from tissue stored in fixative for 1 month showed variable losses (≈ 40 % with GTA-HEPES), suggesting storage duration be controlled for. Lastly, thawing of tissue held at -80 °C in a GTA-HEPES solution provided high-quality visual images and elemental images« less

  1. High-resolution elemental mapping of human placental chorionic villi using synchrotron X-ray fluorescence spectroscopy.

    PubMed

    Punshon, Tracy; Chen, Si; Finney, Lydia; Howard, Louisa; Jackson, Brian P; Karagas, Margaret R; Ornvold, Kim

    2015-09-01

    The placenta is the organ that mediates transport of nutrients and waste materials between mother and fetus. Synchrotron X-ray fluorescence (SXRF) microanalysis is a tool for imaging the distribution and quantity of elements in biological tissue, which can be used to study metal transport across biological membranes. Our aims were to pilot placental biopsy specimen preparation techniques that could be integrated into an ongoing epidemiology birth cohort study without harming rates of sample acquisition. We studied the effects of fixative (formalin or glutaraldehyde) and storage duration (30 days or immediate processing) on metal distribution and abundance and investigated a thaw-fixation protocol for archived specimens stored at -80 °C. We measured fixative elemental composition with and without a placental biopsy via inductively coupled plasma mass spectrometry (ICP-MS) to quantify fixative-induced elemental changes. Formalin-fixed specimens showed hemolysis of erythrocytes. The glutaraldehyde-paraformaldehyde solution in HEPES buffer (GTA-HEPES) had superior anatomical preservation, avoided hemolysis, and minimized elemental loss, although some cross-linking of exogenous Zn was evident. Elemental loss from tissue stored in fixative for 1 month showed variable losses (≈40 % with GTA-HEPES), suggesting storage duration be controlled for. Thawing of tissue held at -80 °C in a GTA-HEPES solution provided high-quality visual images and elemental images. PMID:26138895

  2. High-resolution Elemental Mapping of Human Placental Chorionic Villi Using Synchrotron X-ray Fluorescence Spectroscopy

    SciTech Connect

    Punshon, Tracy; Chen, Si; Finney, Lydia; Howard, Louisa; Jackson, Brian P.; Karagas, Margaret R.; Ornvold, Kim

    2015-09-01

    The placenta is the organ that mediates transport of nutrients and waste materials between mother and fetus. Synchrotron X-ray fluorescence (SXRF) microanalysis is a tool for imaging the distribution and quantity of elements in biological tissue, which can be used to study metal transport across biological membranes. Our aims were to pilot placental biopsy specimen preparation techniques that could be integrated into an ongoing epidemiology birth cohort study without harming rates of sample acquisition. We studied the effects of fixative (formalin or glutaraldehyde) and storage duration (30 days or immediate processing) on metal distribution and abundance and investigated a thaw-fixation protocol for archived specimens stored at -80 A degrees C. We measured fixative elemental composition with and without a placental biopsy via inductively coupled plasma mass spectrometry (ICP-MS) to quantify fixative-induced elemental changes. Formalin-fixed specimens showed hemolysis of erythrocytes. The glutaraldehyde-paraformaldehyde solution in HEPES buffer (GTA-HEPES) had superior anatomical preservation, avoided hemolysis, and minimized elemental loss, although some cross-linking of exogenous Zn was evident. Elemental loss from tissue stored in fixative for 1 month showed variable losses (a parts per thousand 40 % with GTA-HEPES), suggesting storage duration be controlled for. Thawing of tissue held at -80 A degrees C in a GTA-HEPES solution provided high-quality visual images and elemental images

  3. High-resolution elemental mapping of human placental chorionic villi using synchrotron X-ray fluorescence spectroscopy

    PubMed Central

    Punshon, Tracy; Chen, Si; Finney, Lydia; Howard, Louisa; Jackson, Brian P.; Karagas, Margaret R.; Ornvold, Kim

    2015-01-01

    The placenta is the organ that mediates transport of nutrients and waste materials between mother and fetus. Synchrotron X-ray fluorescence microanalysis (SXRF) is a tool for imaging the distribution and quantity of elements in biological tissue, which can be used to study metal transport across biological membranes. Our aims were to pilot placental biopsy specimen preparation techniques that could be integrated into an ongoing epidemiology birth bohort study without harming rates of sample acquisition. We studied the effects of fixative (formalin or glutaraldehyde) and storage duration (30 days or immediate processing) on metal distribution and abundance and investigated a thaw-fixation protocol for archived specimens stored at −80°C. We measured fixative elemental composition with and without a placental biopsy via ICP-MS to quantify fixative-induced elemental changes. Formalin fixed specimens showed hemolysis of erythrocytes. The glutaraldehyde-paraformaldehyde solution in HEPES buffer (GTA-HEPES) had superior anatomical preservation, avoided hemolysis and minimized elemental loss, although some cross-linking of exogenous Zn was evident. Elemental loss from tissue stored in fixative for 1 month showed variable losses (≈ 40% with GTA-HEPES), suggesting storage duration be controlled for. Thawing of tissue held at −80°C in GTA-HEPES solution provided high quality visual images and elemental images. PMID:26138895

  4. High-resolution elemental mapping of human placental chorionic villi using synchrotron X-ray fluorescence spectroscopy

    SciTech Connect

    Punshon, Tracy; Chen, Si; Finney, Lydia; Howard, Louisa; Jackson, Brian P.; Karagas, Margaret R.; Ornvold, Kim

    2015-07-03

    The placenta is the organ that mediates transport of nutrients and waste materials between mother and fetus. Synchrotron X-ray fluorescence (SXRF) microanalysis is a tool for imaging the distribution and quantity of elements in biological tissue, which can be used to study metal transport across biological membranes. Our aims were to pilot placental biopsy specimen preparation techniques that could be integrated into an ongoing epidemiology birth cohort study without harming rates of sample acquisition. We studied the effects of fixative (formalin or glutaraldehyde) and storage duration (30 days or immediate processing) on metal distribution and abundance and investigated a thaw-fixation protocol for archived specimens stored at -80° C. We measured fixative elemental composition with and without a placental biopsy via inductively coupled plasma mass spectrometry (ICP-MS) to quantify fixative-induced elemental changes. Formalin-fixed specimens showed hemolysis of erythrocytes. The glutaraldehyde-paraformaldehyde solution in HEPES buffer (GTA-HEPES) had superior anatomical preservation, avoided hemolysis, and minimized elemental loss, although some cross-linking of exogenous Zn was evident. Elemental loss from tissue stored in fixative for 1 month showed variable losses (≈ 40 % with GTA-HEPES), suggesting storage duration be controlled for. Lastly, thawing of tissue held at -80 °C in a GTA-HEPES solution provided high-quality visual images and elemental images

  5. Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion

    PubMed Central

    Perrin, Laurent; Loizides-Mangold, Ursula; Skarupelova, Svetlana; Pulimeno, Pamela; Chanon, Stephanie; Robert, Maud; Bouzakri, Karim; Modoux, Christine; Roux-Lombard, Pascale; Vidal, Hubert; Lefai, Etienne; Dibner, Charna

    2015-01-01

    Objective Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. Methods We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. Results Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. Conclusions Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes. PMID:26629407

  6. Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines

    PubMed Central

    Adalsteinsson, Viktor; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B.; Huang, Cindy; Bowman, Brittany; Williamson, Christina; Kwon, Douglas S.; Wittrup, K. Dane; Love, J. Christopher

    2014-01-01

    Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via either or both of the CXCR1 and CXCR2 receptors, exerting profound impacts on tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors. PMID:23995780

  7. Tributyltin alters secretion of interleukin 1 beta from human immune cells.

    PubMed

    Brown, Shyretha; Whalen, Margaret

    2015-08-01

    Tributyltin (TBT) has been used as a biocide in industrial applications such as wood preservation, antifouling paint and antifungal agents. Owing to its many uses, it contaminates the environment and has been found in human blood samples. Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine that promotes cell growth, tissue repair and immune response regulation. Produced predominately by both monocytes and macrophages, IL-1β appears to increase the invasiveness of certain tumors. This study shows that TBT modifies the secretion of IL-1β from increasingly reconstituted preparations of human immune cells. IL-1β secretion was examined after 24-, 48-h or 6-day exposures to TBT in highly enriched human natural killer (NK) cells, monocyte-depleted peripheral blood mononuclear cells (MD-PBMCs), PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes (PBMCs+granulocytes). TBT altered IL-1β secretion from all of the cell preparations. The 200 nM concentration of TBT normally blocked the secretion of IL-1β, whereas lower concentrations (usually 5-50 nM) elevated secretion of IL-1β. Examination of the signaling pathway(s) responsible for the elevated secretion of IL-1β was carried out in MD-PBMCs. Pathways examined were IL-1β processing (Caspase-1), mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB). Results indicated that MAPK pathways (p44/42 and p38) appear to be the targets of TBT that lead to increased IL-1β secretion from immune cells. These results from human immune cells show IL-1β dysregulation by TBT is occurring ex vivo. Thus, the potential for in vivo effects on pro-inflammatory cytokine levels may possibly be a consequence of TBT exposures. PMID:25382723

  8. Simplified matrix solid phase dispersion procedure for the determination of parabens and benzophenone-ultraviolet filters in human placental tissue samples.

    PubMed

    Vela-Soria, F; Rodríguez, I; Ballesteros, O; Zafra-Gómez, A; Ballesteros, L; Cela, R; Navalón, A

    2014-12-01

    In recent decades, the industrial development has resulted in the appearance of a large amount of new chemicals that are able to produce disorders in the human endocrine system. These substances, so-called endocrine disrupting chemicals (EDCs), include many families of compounds, such as parabens and benzophenone-UV filters. Taking into account the demonstrated biological activity of these compounds, it is necessary to develop new analytical procedures to assess the exposure in order to establish, in an accurate way, relationships between EDCs and harmful health effects in population. In the present work, a new method based on a simplified sample treatment by matrix solid phase dispersion (MSPD) followed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, is validated for the determination of four parabens (methyl-, ethyl-, propyl- and butylparaben) and six benzophenone-UV filters (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8 and 4-hydroxybenzophenone) in human placental tissue samples. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-13C6 and benzophenone-d10 were used as surrogates. The found limits of quantification ranged from 0.2 to 0.4 ng g(-1) and inter-day variability (evaluated as relative standard deviation) ranged from 5.4% to 12.8%. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 96% to 104%. The method was satisfactorily applied for the determination of compounds in human placental tissue samples collected at the moment of delivery from 10 randomly selected women. PMID:25456585

  9. The effect of gamma interferon on IL-1 secretion of in vitro differentiated human macrophages.

    PubMed

    Haq, A U; Rinehart, J J; Maca, R D

    1985-12-01

    After being cultured overnight, human monocytes lose their ability to secrete interleukin-1 (IL-1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon-gamma (IFN-gamma), these cells were found to retain their ability to secrete appreciable amounts of IL-1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN-gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN-gamma. In addition to inducing IL-1 secretion, IFN-gamma also appeared to increase the overall production of IL-1, since reinduction of IL-1 secretion was not associated with a decrease in intracellular IL-1 content. When these macrophages were initially cultured with IFN-gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL-1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN-gamma, these macrophages were found to regain their capacity to secrete IL-1. However, the amount of reinduced IL-1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL-1 secretion occurring if monocytes were allowed to mature for 6 days before IFN-gamma pretreatment. In summary, these studies suggest that IFN-gamma may be important in enhancing IL-1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo. PMID:3934302

  10. The Effect of Ovine Secreted Soluble Factors on Human Dermal Papilla Cell Aggregation

    PubMed Central

    Sari, Agnes Rosarina Prita; Rufaut, Nicholas Wolfgang; Jones, Leslie Norman; Sinclair, Rodney Daniel

    2016-01-01

    Context: In androgenetic alopecia, follicular miniaturization and dynamic changes to the hair cycle produce patterned baldness. The most effective treatment for baldness is hair transplantation surgery. The major limitation to hair transplantation is the availability of donor hair from the relatively unaffected occipital scalp. Hair induction with in vitro expansion of donor follicle populations has the potential to overcome this. The major obstacle to this is that in vitro expansion of human dermal papilla cell (DPC) colonies is associated with irreversible loss of aggregative behavior and hair follicle-inductive potential. In contrast, cultured ovine DPCs maintain these properties after extensive proliferation. Aims: To determine whether aggregating ovine DPC secrete factors that enhance the aggregative behavior or inductive potential of human DPC. Subjects and Methods: Fluorescently-labelled ovine DPC were mixed in culture with human DPC at passage number seven-nine, which had lost their aggregative behavior. The effects of different culture substrates and medium compositions on aggregative behavior were determined. Ovine and human papilla cells were co-cultured, separated by a permeable membrane to determine whether the ovine cells secrete soluble factors that affect human papilla cells. Results: In direct co-culture experiments, well-formed aggregates were produced by 90:10 human:ovine and 50:50 human:ovine DPC mixtures. In contrast, unmixed human DPC remained in a monolayer state after 18 days. Both human and ovine DPC had a higher tendency to aggregate in medium containing 20% (v/v) lamb serum (LS) compared to 10% (v/v) fetal calf serum (FCS). In co-culture experiments separated with permeable membrane, the human DPC aggregates were bigger and more rapidly formed with the addition of ovine secreted soluble factors. Conclusions: Soluble factors secreted by ovine DPC and present in LS increase the aggregative behavior of human DPC. These molecules might

  11. On the role of placental major histocompatibility complex and decidual leukocytes in implantation and pregnancy success using non-human primate models

    PubMed Central

    Golos, Thaddeus G.; Bondarenko, Gennadiy I.; Dambaeva, Svetlana V.; Breburda, Edith E.; Durning, Maureen

    2011-01-01

    While there is broad agreement that interactions of the human maternal immune system with the tissues and cells of the implanting embryo are likely to be critical contributors to pregnancy success, there remains a dearth of information which directly confirms this expectation. Although animal models of reproductive function often provide opportunities for confirming such hypotheses, progress in this area has been sporadic due to limitations of traditional laboratory or agricultural animal models, such as rodents, sheep, pigs and cattle. Many of these limitations derive from divergent modes of implantation and placentation across mammalian species. Over the past decade there has been progress in the development of the nonhuman primate as a model in which to address questions of pregnancy success in the area of immunology. The purpose of this review is to compare available model species, summarize current knowledge and recent progress with an emphasis on experimental in vivo manipulations, and suggest areas available for additional study and growth. PMID:19876826

  12. Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells.

    PubMed

    Pedersen, Morten Gram; Ahlstedt, Ingela; El Hachmane, Mickaël F; Göpel, Sven O

    2016-01-01

    Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na(+)/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications. PMID:27535321

  13. Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells

    PubMed Central

    Pedersen, Morten Gram; Ahlstedt, Ingela; El Hachmane, Mickaël F.; Göpel, Sven O.

    2016-01-01

    Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na+/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications. PMID:27535321

  14. Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages.

    PubMed Central

    Muschol, Nicole; Matzner, Ulrich; Tiede, Stephan; Gieselmann, Volkmar; Ullrich, Kurt; Braulke, Thomas

    2002-01-01

    The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of (125)I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [(35)S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [(33)P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed (33)P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [(35)S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain. PMID:12296771

  15. Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages.

    PubMed

    Muschol, Nicole; Matzner, Ulrich; Tiede, Stephan; Gieselmann, Volkmar; Ullrich, Kurt; Braulke, Thomas

    2002-12-15

    The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of (125)I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [(35)S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [(33)P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed (33)P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [(35)S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain. PMID:12296771

  16. Secretion of glucose in human parotid saliva after carbohydrate intake.

    PubMed

    Borg, A; Birkhed, D

    1988-12-01

    The aims of the present investigation were, first, to follow the secretion of free glucose in parotid saliva in various subjects after a single oral intake of different carbohydrates, and second, to compare the salivary glucose concentration with the concentration in blood. Twenty healthy subjects, three women and 17 men, 20-35 yr of age, participated. They were asked not to eat or drink anything from 10 p.m. the night before the examination. 75 g of carbohydrate (glucose, fructose, or sucrose) dissolved in 300 ml water was ingested the next morning at 8 a.m. One experimental series with glucose was performed in triplicate in 10 of the subjects. Approximately 1.5 ml of citric acid-stimulated parotid saliva was collected before (0 min) and 15, 30, 45, 60, and 120 min after the intake. Salivary concentration of glucose was analyzed enzymatically. Most of the 0-min samples showed a variation in glucose concentration from 5 to 25 mumol/l. After the glucose, fructose, and sucrose intakes, the salivary glucose level increased about 2-4 times, especially in the 30-min samples. A large inter- as well as intra-individual variation was found both in the 0-min samples and in the samples collected after the different intakes. The correlation between the glucose concentration in saliva and blood was higher after than before the carbohydrate intakes. PMID:3206201

  17. Calpain secreted by activated human lymphoid cells degrades myelin.

    PubMed

    Deshpande, R V; Goust, J M; Hogan, E L; Banik, N L

    1995-10-01

    Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination. PMID:8568927

  18. Stress-impaired transcription factor expression and insulin secretion in transplanted human islets

    PubMed Central

    Dai, Chunhua; Kayton, Nora S.; Shostak, Alena; Poffenberger, Greg; Cyphert, Holly A.; Aramandla, Radhika; Thompson, Courtney; Papagiannis, Ioannis G.; Shiota, Masakazu; Stafford, John M.; Greiner, Dale L.; Herrera, Pedro L.; Shultz, Leonard D.; Stein, Roland; Powers, Alvin C.

    2016-01-01

    Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity. PMID:27064285

  19. Localization and regulation of the human very low density lipoprotein/apolipoprotein-E receptor: trophoblast expression predicts a role for the receptor in placental lipid transport.

    PubMed

    Wittmaack, F M; Gåfvels, M E; Bronner, M; Matsuo, H; McCrae, K R; Tomaszewski, J E; Robinson, S L; Strickland, D K; Strauss, J F

    1995-01-01

    The very low density lipoprotein/apolipoprotein-E receptor (VLDLR) is the newest member of the low density lipoprotein receptor (LDLR) family. Very little is known about VLDLR localization and regulation. Immunohistochemical analysis of human placenta with a specific polyclonal antibody detected VLDLR in syncytiotrophoblast and intermediate trophoblast cells. VLDLR transcripts were also localized in these cells by in situ hybridization histochemistry. In addition, VLDLR messenger RNA (mRNA) was detected in villous core endothelial cells and cells appearing to be Hofbauer cells. Northern blot analysis of placenta revealed a 2.6-fold increase in VLDLR mRNA at term compared to that in the first trimester. The regulation of VLDLR expression was studied in JEG-3 and BeWo choriocarcinoma cells, two trophoblast-derived cell lines. Treatment of these cells with 8-bromo-cAMP caused a profound suppression of VLDLR message, whereas LDLR transcripts were increased. Incubation of JEG-3 cells with 25-hydroxycholesterol did not lead to sterol negative feedback on VLDLR gene expression, unlike LDLR mRNA, which declined markedly. Insulin (200 mg/L) up-regulated VLDLR message in JEG-3 cells 2-fold, as did the fibrate hypolipidemic drug, clofibric acid. We conclude that 1) VLDLR is expressed in human placental trophoblast cells in a pattern consistent with a role in placental lipid transport; 2) VLDLR expression is high at term relative to that in the first trimester; and 3) the trophoblast VLDLR is subject to down-regulation by cAMP and up-regulation by insulin and fibrate hypolipidemic drugs. PMID:7828550

  20. Prevention of Defective Placentation and Pregnancy Loss by Blocking Innate Immune Pathways in a Syngeneic Model of Placental Insufficiency.

    PubMed

    Gelber, Shari E; Brent, Elyssa; Redecha, Patricia; Perino, Giorgio; Tomlinson, Stephen; Davisson, Robin L; Salmon, Jane E

    2015-08-01

    Defective placentation and subsequent placental insufficiency lead to maternal and fetal adverse pregnancy outcome, but their pathologic mechanisms are unclear, and treatment remains elusive. The mildly hypertensive BPH/5 mouse recapitulates many features of human adverse pregnancy outcome, with pregnancies characterized by fetal loss, growth restriction, abnormal placental development, and defects in maternal decidual arteries. Using this model, we show that recruitment of neutrophils triggered by complement activation at the maternal/fetal interface leads to elevation in local TNF-α levels, reduction of the essential angiogenic factor vascular endothelial growth factor, and, ultimately, abnormal placentation and fetal death. Blockade of complement with inhibitors specifically targeted to sites of complement activation, depletion of neutrophils, or blockade of TNF-α improves spiral artery remodeling and rescues pregnancies. These data underscore the importance of innate immune system activation in the pathogenesis of placental insufficiency and identify novel methods for treatment of pregnancy loss mediated by abnormal placentation. PMID:26071558

  1. Immunoglobulin isotype isolated from human placental extract does not interfere in complement-mediated bacterial opsonization within the wound milieu

    PubMed Central

    Sharma, Kanika; Bhattacharyya, Debasish

    2015-01-01

    The wound healing potency of an aqueous extract of placenta can be evaluated through the presence of numerous regulatory components. The presence of glycans was detected by thin layer chromatography and fluorophore-assisted carbohydrate electrophoresis. Mass spectrometric analysis revealed the existence of multiple fragments of immunoglobulin G (IgG). IgG was present in the extract at a concentration of 25.2 ± 3.97 μg/ml. IgG possesses anti-complementary activity by diverting the complement activation from target surface. Thus, effect of placental IgG on complement–bacteria interaction was investigated through classical and alternative pathway and the preparation was ascertained to be safe with respect to their interference in the process of bacterial opsonization. PMID:25984442

  2. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

    PubMed

    Di Marco, E; Marchisio, P C; Bondanza, S; Franzi, A T; Cancedda, R; De Luca, M

    1991-11-15

    Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing. PMID:1718982

  3. Cortisol-secreting adrenocortical tumours in dogs and their relevance for human medicine.

    PubMed

    Galac, Sara

    2016-02-01

    Spontaneous cortisol-secreting adrenocortical tumours in pet dogs are an attractive animal model for their human counterparts. Adrenal morphology and function are similar in dogs and humans, and adrenocortical tumours have comparable clinical and pathological characteristics. Their relatively high incidence in pet dogs represents a potential source of adrenocortical tumour tissue to facilitate research. The molecular characteristics of canine cortisol-secreting adrenocortical tumours suggest that they will be useful for the study of angiogenesis, the cAMP/protein kinase A pathway, and the role of Steroidogenic Factor-1 in adrenal tumourigenesis. Pet dogs with spontaneous cortisol-secreting adrenocortical tumours may also be useful in clinical testing of new drugs and in investigating the molecular background of adrenocortical tumours. PMID:26123587

  4. Effect of repeated US stimulation on adiponectin secretion by adipocytes of obese human subjects

    NASA Astrophysics Data System (ADS)

    Fujii, Yasutomo; Taniguchi, Nobuyuki; Satoh, Masaaki; Irie, Takasuke; Itoh, Kouichi

    2006-05-01

    To clarify the effect of the repeated sonication on the adiponectin secretion by adipocytes obtained from obese subjects. Using 1-MHz continuous-wave ultrasound at an intensity of 0.50 or 2.1 W/cm2, we sonicated culture flasks of subcutaneous adipocytes obtained from obese human subjects, in a series of 3 sessions of US stimulation applied for a daily total of 15 min. For the measurement of adiponectin secretion, 50 μl of the culture medium was collected from each flask every 24 h after the 1st stimulation. Quantification of adiponectin protein levels in cell culture supernatants was performed with a commercially available ELISA kit recommended by the manufacturer. The adiponectin concentrations in the culture medium of the US stimulation groups rose significantly (p<0.05). Repeated US stimulation may accelerate adiponectin secretion in obese human adipocytes.

  5. In-Vitro Study of the Effect of Anti-Hypertensive Drugs on Placental Hormones and Angiogenic Proteins Synthesis in Pre-Eclampsia

    PubMed Central

    Gangooly, Subrata; Jauniaux, Eric

    2014-01-01

    Introduction Antihypertensive drugs lower the maternal blood pressure in pre-eclampsia (PE) by direct or central vasodilatory mechanisms but little is known about the direct effects of these drugs on placental functions. Objective The aim of our study is to evaluate the effect of labetolol, hydralazine, α-methyldopa and pravastatin on the synthesis of placental hormonal and angiogenic proteins know to be altered in PE. Design Placental villous explants from late onset PE (n = 3) and normotensive controls (n = 6) were cultured for 3 days at 10 and 20% oxygen (O2) with variable doses anti-hypertensive drugs. The levels of activin A, inhibin A, human Chorionic Gonadotrophin (hCG), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) were measured in explant culture media on day 1, 2 and 3 using standard immunoassays. Data at day 1 and day 3 were compared. Results Spontaneous secretion of sEndoglin and sFlt-1 were higher (p<0.05) in villous explants from PE pregnancies compared to controls. There was a significant time dependant decrease in the secretion of sFlt-1 and sEndoglin in PE cases, which was seen only for sFlt-1 in controls. In both PE cases and controls the placental protein secretions were not affected by varying doses of anti-hypertensive drugs or the different O2 concentration cultures, except for Activin, A which was significantly (p<0.05) higher in controls at 10% O2. Interpretation Our findings suggest that the changes previously observed in maternal serum hormones and angiogenic proteins level after anti-hypertensive treatment in PE could be due to a systemic effect of the drugs on maternal blood pressure and circulation rather than a direct effect of these drugs on placental biosynthesis and/or secretion. PMID:25251016

  6. Effect of substance P on immunoglobulin and interferon-gamma secretion by cultured human duodenal mucosa.

    PubMed

    Hart, R; Dancygier, H; Wagner, F; Lersch, C; Classen, M

    1990-01-01

    Recently, we have demonstrated a substance P (SP)-dependent modulation of in vitro IgM and interferon-gamma (IFN-gamma) secretion by human peripheral blood mononuclear cells, as well as lymphokine activities in supernatants of cultured duodenal mucosa. Therefore we investigated other local immunoregulatory effects of SP. Duodenal biopsies of 7 healthy subjects were cultured with Pokeweed mitogen (PWM, 1 microgram/ml) for 4 days at 37 degrees C in 1 ml medium each. SP was added in concentrations ranging from 10(-12)M to 10(-6)M on day 1. Fresh media with fresh PWM were added every day. IgG, IgM, IgA (ELISA) and IFN-gamma (RIA) were determined in the culture supernatants. Values were referred to 5 mg biopsy weight and expressed as % change in basal PWM pulsed secretion, or as units/ml. 10(-6) M and 10(-12) M SP increased secretion of all immunoglobulin isotypes. Compared to controls, 10(-6) M and 10(-12) M SP led to an increase in IgM secretion of up to 73 +/- 23% and 41 +/- 32% and to an increase in IgA secretion up to 96 +/- 35% and 25 +/- 33%, respectively (alpha = 0.02 for both isotypes at 10(-6) M). 10(-12) M to 10(-6) M SP led to a significant dose-dependent increase in IFN-gamma secretion from 7.08 +/- 1.65 up to 21.8 +/- 12.6 units/ml/5 mg. The maximum effect could be seen on culture days 3 and 4. We were able to demonstrate for the first time that SP stimulates PWM pulsed immunoglobulin and IFN-gamma secretion by human duodenal immunocompetent cells. These results support the hypothesis of local neuropeptidergic-immune interactions. PMID:1689696

  7. The effect of cloprostenol on human luteal steroid and prostaglandin secretion in vitro.

    PubMed Central

    McDougall, A N; Walker, F M; Watson, J

    1977-01-01

    1 Human luteal tissue slices from days 18, 21 and 25 of the menstrual cycle were superfused in vitro with Medium 199 alone or containing cloprostenol (1 microgram/ml). Concentrations of progesterone, oestradiol-17beta and prostaglandins F2alpha and E2 were determined in the superfusate samples. 2 Secretion of steroids and prostaglandins was maintained at an approximately constant level throughout the experiments (21 h in one case) when the tissue was perfused with M199 alone. 3 Superfusion with cloprostenol (1 microgram/ml) resulted in an initial depression of progesterone and oestradiol-17beta but this was not maintained, levels returning to control values or showing an increase, while superfusion with cloprostenol continued. Cloprostenol is not therefore considered to be luteolytic at this dose and under these conditions for human luteal tissue in vitro. 4 Superfusion with cloprostenol (1 microgram/ml) also resulted in a large stimulation of secretion of endogenous prostaglandin F2 alpha following a short lag phase. This stimulation was possibly due to the initial depression of progesterone secretion. A short-lived stimulation of prostaglandin E2 secretion was also observed. 5 The significance of the increase in prostaglandin E2 secretion and the interrelationships between the various changes observed with cloprostenol are difficult to interpret. PMID:890210

  8. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

    PubMed

    Devor, D C; Singh, A K; Lambert, L C; DeLuca, A; Frizzell, R A; Bridges, R J

    1999-05-01

    Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance

  9. 1,25(OH)2D3 Induces Placental Vascular Smooth Muscle Cell Relaxation by Phosphorylation of Myosin Phosphatase Target Subunit 1Ser507: Potential Beneficial Effects of Vitamin D on Placental Vasculature in Humans.

    PubMed

    Jia, Xiuyue; Gu, Yang; Groome, Lynn J; Al-Kofahi, Mahmoud; Alexander, J Steven; Li, Weimin; Wang, Yuping

    2016-05-01

    Placental vascular dysfunction has been linked to insufficiency/deficiency of maternal vitamin D levels during pregnancy. In contrast, sufficient maternal vitamin D levels have shown beneficial effects on pregnancy outcomes. To study the role of vitamin D in pregnancy, we tested our hypothesis that vitamin D exerts beneficial effects on placental vasculature. We examined expression of CYP2R1, CYP27B1, vitamin D receptor (VDR), and CYP24A1 in placental vascular smooth muscle cells (VSMCs) in response to 1,25(OH)2D3 We found that VDR expression was inducible, CYP27B1 expression was dose-dependently down-regulated, and CYP24A1 expression was dose-dependently up-regulated in cells treated with 1,25(OH)2D3 These data suggest a feedback autoregulatory system of vitamin D existing in placental VSMCs. Using a VSMC/collagen-gel contraction assay, we evaluated the effect of 1,25(OH)2D3 on placental VSMC contractility. We found that, similar to losartan, 1,25(OH)2D3 could diminish angiotensin II-induced cell contractility. The mechanism of 1,25(OH)2D3-mediated VSMC relaxation was further explored by examination of Rho-associated protein kinase 1 (ROCK1)/phosphorylation of myosin phosphatase target subunit 1 (MYPT1) pathway molecules. Our results showed that p-MYPT1(Thr853) and p-MYPT1(Thr696) were undetectable. However, p-MYPT1(Ser507), but not p-MYPT1(Ser668), was significantly up-regulated in cells treated with losartan plus angiotensin II. Similar effects were also seen in cells treated with 1,25(OH)2D3 plus angiotensin II or 1,25(OH)2D3 plus losartan plus angiotensin II. Because MYPT1 serine phosphorylation could activate myosin light chain phosphatase (MLCP), and MLCP activation is an important regulatory machinery of smooth muscle cell relaxation, up-regulation of MYPT1(Ser507) phosphorylation could be a mechanism of vitamin D and/or losartan mediated placental VSMC relaxation. PMID:27075619

  10. Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal-Placental Messaging.

    PubMed

    Holder, Beth; Jones, Tessa; Sancho Shimizu, Vanessa; Rice, Thomas F; Donaldson, Beverly; Bouqueau, Marielle; Forbes, Karen; Kampmann, Beate

    2016-02-01

    During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regulation of trans-placental trafficking is therefore essential for the healthy development of the growing fetus. Extracellular vesicle-mediated transfer of protein and nucleic acids from the human placenta into the maternal circulation is well documented; the possibility that this trafficking is bi-directional has not yet been explored but could affect placental function and impact on the fetus.We hypothesized that the ability of the placenta to respond to maternal inflammatory signals is mediated by the interaction of maternal immune cell exosomes with placental trophoblast. Utilizing the BeWo cell line and whole placental explants, we demonstrated that the human placenta internalizes macrophage-derived exosomes in a time- and dose-dependent manner. This uptake was via clathrin-dependent endocytosis. Furthermore, macrophage exosomes induced release of proinflammatory cytokines by the placenta. Taken together, our data demonstrates that exosomes are actively transported into the human placenta and that exosomes from activated immune cells modulate placental cytokine production. This represents a novel mechanism by which immune cells can signal to the placental unit, potentially facilitating responses to maternal inflammation and infection, and thereby preventing harm to the fetus. PMID:26602702

  11. Human luteinized granulosa cells secrete apoB100-containing lipoproteins.

    PubMed

    Gautier, Thomas; Becker, Steffi; Drouineaud, Véronique; Ménétrier, Franck; Sagot, Paul; Nofer, Jerzy-Roch; von Otte, Sören; Lagrost, Laurent; Masson, David; Tietge, Uwe J F

    2010-08-01

    Thus far, liver, intestine, heart, and placenta have been shown to secrete apolipoprotein (apo)B-containing lipoproteins. In the present study, we first investigated lipoproteins in human follicular fluid (FF), surrounding developing oocytes within the ovary, as well as in corresponding plasma samples (n = 12). HDL cholesterol within FF correlated well with plasma HDL cholesterol (r = 0.80, P < 0.01), whereas VLDL cholesterol did not, indicating that VLDL in FF might originate directly from the granulosa cells producing FF. Primary human granulosa cells expressed apoB, microsomal triglyceride transfer protein, and apoE, but not the apoB-editing enzyme apobec-1. Using (3)H-leucine, we show that granulosa cells secrete apoB100-containing lipoproteins and that secretion can be stimulated by adding oleate to the medium (+83%). With electron microscopy, apoB-containing lipoproteins within the secretory pathway of human granulosa cells were directly visualized. Finally, we found a positive relationship between apoB levels in FF and improved fertility parameters in a population of 27 women undergoing in vitro fertilization. This study demonstrates that human granulosa cells assemble and secrete apoB100-containing lipoproteins, thereby identifying a novel cell type equipped with these properties. These results might have important implications for female infertility phenotypes as well as for the development of drugs targeting the VLDL production pathway. PMID:20407020

  12. Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2).

    PubMed

    Haghpassand, M; Wilder, D; Moberly, J B

    1996-07-01

    Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride

  13. Placentation in mammals: Definitive placenta, yolk sac, and paraplacenta.

    PubMed

    Carter, A M; Enders, A C

    2016-07-01

    An overview is given of variations in placentation with particular focus on yolk sac, paraplacenta, and other structures important to histotrophic nutrition. The placenta proper varies in general shape, internal structure, and the number of tissues in the interhemal barrier. Yolk sac membranes persist to term in insectivores, colugos, rodents, and lagomorphs. In the latter two orders, they are of known importance for maternal-fetal transfer of antibodies, vitamins, lipids, and proteins. The detached yolk sac of bats is also active throughout gestation. A vascular paraplacenta, or smooth chorioallantois, has known functions in ruminants and carnivores and is found in several other orders of mammal where its function has yet to be explored. In human gestation, the chorion (avascular chorioallantois) is important for hormone synthesis. The true chorion of squirrels and hedgehogs is avascular but may nevertheless allow transfer from mother to fetus through the exocelom. Hemophagous areas with columnar trophoblast are paraplacental structures in carnivores and elephants but occur also within the placenta as in hyenas and moles. In shrews, it is the yolk sac that ingests and processes red cells. Areolas and chorionic vesicles are other structures important for absorption of uterine secretions and ingestion of cellular debris. In conclusion, we find that paraplacental structures, while showing less variation than the placenta proper, contribute not just to the integrity of overall placentation, but in various ways to maternal-fetal interrelationships. PMID:27155730

  14. Vectorial bicarbonate transport by Capan-1 cells: a model for human pancreatic ductal secretion.

    PubMed

    Szucs, Akos; Demeter, Irma; Burghardt, Beáta; Ovári, Gabriella; Case, R Maynard; Steward, Martin C; Varga, Gábor

    2006-01-01

    Human pancreatic ducts secrete a bicarbonate-rich fluid but our knowledge of the secretory process is based mainly on studies of animal models. Our aim was to determine whether the HCO(3)(-) transport mechanisms in a human ductal cell line are similar to those previously identified in guinea-pig pancreatic ducts. Intracellular pH was measured by microfluorometry in Capan-1 cell monolayers grown on permeable filters and loaded with BCECF. Epithelial polarization was assessed by immunolocalization of occludin. Expression of mRNA for key electrolyte transporters and receptors was evaluated by RT-PCR. Capan-1 cells grown on permeable supports formed confluent, polarized monolayers with well developed tight junctions. The recovery of pH(i) from an acid load, induced by a short NH(4)(+) pulse, was mediated by Na(+)-dependent transporters located exclusively at the basolateral membrane. One was independent of HCO(3)(-) and blocked by EIPA (probably NHE1) while the other was HCO(3)(-)-dependent and blocked by H(2)DIDS (probably pNBC1). Changes in pH(i) following blockade of basolateral HCO(3)(-) accumulation confirmed that the cells achieve vectorial HCO(3)(-) secretion. Dose-dependent increases in HCO(3)(-) secretion were observed in response to stimulation of both secretin and VPAC receptors. ATP and UTP applied to the apical membrane stimulated HCO(3)(-) secretion but were inhibitory when applied to the basolateral membrane. HCO(3)(-) secretion in guinea-pig ducts and Capan-1 cell monolayers share many common features, suggesting that the latter is an excellent model for studies of human pancreatic HCO(3)(-) secretion. PMID:17167230

  15. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    PubMed

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells. PMID:26716748

  16. Characteristics and Quantities of HIV Host Cells in Human Genital Tract Secretions

    PubMed Central

    Politch, Joseph A.; Marathe, Jai; Anderson, Deborah J.

    2014-01-01

    Human immunodeficiency virus (HIV)–infected leukocytes have been detected in genital secretions from HIV-infected men and women and may play an important role in the sexual transmission of HIV. However, they have been largely overlooked in studies on mechanisms of HIV transmission and in the design and testing of HIV vaccine and microbicide candidates. This article describes the characteristics and quantities of leukocytes in male and female genital secretions under various conditions and also reviews evidence for the involvement of HIV-infected cells in both horizontal and vertical cell-associated HIV transmission. Additional research is needed in this area to better target HIV prevention strategies. PMID:25414414

  17. IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

    PubMed

    Barbaux, S; Erwich, J J H M; Favaron, P O; Gil, S; Gallot, D; Golos, T G; Gonzalez-Bulnes, A; Guibourdenche, J; Heazell, A E P; Jansson, T; Laprévote, O; Lewis, R M; Miller, R K; Monk, D; Novakovic, B; Oudejans, C; Parast, M; Peugnet, P; Pfarrer, C; Pinar, H; Roberts, C T; Robinson, W; Saffery, R; Salomon, C; Sexton, A; Staff, A C; Suter, M; Tarrade, A; Wallace, J; Vaillancourt, C; Vaiman, D; Worton, S A; Lash, G E

    2015-04-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction. PMID:25703592

  18. Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

    PubMed

    Pérez-Pérez, Antonio; Toro, Ayelén R; Vilarino-Garcia, Teresa; Guadix, Pilar; Maymó, Julieta L; Dueñas, José L; Varone, Cecilia L; Sánchez-Margalet, Víctor

    2016-06-01

    Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling. PMID:27238720

  19. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  20. Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.

    PubMed

    McNamara, B; Winter, D C; Cuffe, J E; O'Sullivan, G C; Harvey, B J

    1999-08-15

    1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. PMID:10432355

  1. A Role for SPARC in the Moderation of Human Insulin Secretion

    PubMed Central

    Harries, Lorna W.; McCulloch, Laura J.; Holley, Janet E.; Rawling, Thomas J.; Welters, Hannah J.; Kos, Katarina

    2013-01-01

    Aims/Hypothesis We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine)/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes. Methods We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS) in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines. Results SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05). SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01). Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01). Conclusions Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC’s modulation of obesity-induced insulin resistance in adipose tissue. PMID:23840838

  2. Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3

    PubMed Central

    Cowley, Elizabeth A; Linsdell, Paul

    2002-01-01

    Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K+ channels involved in basal, cAMP- and Ca2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (Isc). The non-specific K+ channel inhibitor Ba2+ inhibited the basal Isc by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K+ conductance is required for maintenance of basal anion secretion. Using the K+ channel blockers clofilium and clotrimazole, we found basal Isc to be sensitive to clofilium, with a small clotrimazole-sensitive component. By stimulating the cAMP and Ca2+ pathways, we determined that cAMP-stimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K+ channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K+ conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated Isc. Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K+ channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K+ channel types, possibly involving one or more class of KCNQ1-containing channel complexes. PMID:11826162

  3. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    PubMed

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  4. Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

    PubMed

    Loera-Arias, María J; Villatoro-Hernández, Julio; Parga-Castillo, Miguel A; Salcido-Montenegro, Alejandro; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Montes-de-Oca-Luna, Roberto; Saucedo-Cárdenas, Odila

    2014-12-01

    Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications. PMID:25214209

  5. INDUCTION BY EPIDERMOPHYTON FLOCCOSUM OF HUMAN FIBROBLAST MATRIX METALLOPROTEINASE-9 SECRETION IN VITRO.

    PubMed

    Kitisin, Thitinan; Luplertlop, Natthanej

    2015-03-01

    Skin infection from pathogenic dermatophyte, Epidermophytonfloccosum, can cause serious health complications, especially in immuno-compromised patients. Proteolytic enzymes secreted from E. floccosum are required for host tissue degradation, facilitating fungal invasion. However, little is known regarding host matrix metalloproteinase (MMP) expression during E. floccosum infection. In this study human foreskin fibroblast (HFF) cell line was used to determine MMP-9 protease activity by gelatin zymography and amount by ELISA. E. floccosum-induced HFF secretion of MMP-9 in a time dependent manner, but HFF cell viability decreased. Treatment with an MMP inhibitor (SB-3CT) caused reduction in E. floccosum-induced secreted MMP-9 and improvement in HFF cell viability. These findings indicate a possible control measure for protecting skin from E. floccosum infection. PMID:26513930

  6. Purification and cultivation of human pituitary growth hormone secreting cells

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.

    1984-01-01

    A multiphase study was conducted to examine the properties of growth hormone cells. Topics investigated included: (1) to determine if growth hormone (GH) cells contained within the rat pituitary gland can be separated from the other hormone producing cell types by continuous flow electrophoresis (CFE); (2) to determine what role, if any, gravity plays in the electrophoretic separation of GH cells; (3) to compare in vitro GH release from rat pituitary cells previously exposed to microgravity conditions vs release from cells not exposed to microgravity; (4) to determine if the frequency of different hormone producing pituitary cell types contained in cell suspensions can be quantitated by flow cytometry; and (5) to determine if GH contained within the human post mortem pituitary gland can be purified by CFE. Specific experimental procedures and results are included.

  7. Angiotensin II-stimulated secretion of arginine vasopressin is inhibited by atrial natriuretic peptide in humans.

    PubMed

    Matsukawa, Toshiyoshi; Miyamoto, Takenori

    2011-03-01

    We investigated the effect of the intravenous infusion of atrial natriuretic peptide (ANP) on the response of plasma arginine vasopressin (AVP) levels to intravenous infusion of angiotensin II (ANG II) in healthy individuals. Intravenous infusion of ANP (10 ng·kg(-1)·min(-1)) slightly but significantly decreased plasma AVP levels, while intravenous infusion of ANG II (10 ng·kg(-1)·min(-1)) resulted in slightly increased plasma AVP levels. ANG II infused significant elevations in arterial blood pressure and central venous pressure (CVP). Because the elevation in blood pressure could have potentially inhibited AVP secretion via baroreceptor reflexes, the effect of ANG II on blood pressure was attenuated by the simultaneous infusion of nitroprusside. ANG II alone produced a remarkable increase in plasma AVP levels when infused with nitroprusside, whereas the simultaneous ANP intravenous infusion (10 ng·kg(-1)·min(-1)) abolished the increase in plasma AVP levels induced by ANG II when blood pressure elevation was attenuated by nitroprusside. Thus, ANG II increased AVP secretion and ANP inhibited not only basal AVP secretion but also ANG II-stimulated AVP secretion in humans. These findings support the hypothesis that circulating ANP modulates AVP secretion, in part, by antagonizing the action of circulating ANG II. PMID:21123762

  8. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

    PubMed Central

    Verhoeven, A J; Neve, B P; Jansen, H

    1999-01-01

    Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked. PMID:9854035

  9. Placental Features of Late-Onset Adverse Pregnancy Outcome

    PubMed Central

    Higgins, Lucy E.; Wareing, Mark; Greenwood, Susan L.; Jones, Rebecca L.; Sibley, Colin P.; Johnstone, Edward D.; Heazell, Alexander E. P.

    2015-01-01

    Objective Currently, no investigations reliably identify placental dysfunction in late pregnancy. To facilitate the development of such investigations we aimed to identify placental features that differ between normal and adverse outcome in late pregnancy in a group of pregnancies with reduced fetal movement. Methods Following third trimester presentation with reduced fetal movement (N = 100), placental structure ex vivo was measured. Placental function was then assessed in terms of (i) chorionic plate artery agonist responses and length-tension characteristics using wire myography and (ii) production and release of placentally derived hormones (by quantitative polymerase chain reaction and enzyme linked immunosorbant assay of villous tissue and explant conditioned culture medium). Results Placentas from pregnancies ending in adverse outcome (N = 23) were ~25% smaller in weight, volume, length, width and disc area (all p<0.0001) compared with those from normal outcome pregnancies. Villous and trophoblast areas were unchanged, but villous vascularity was reduced (median (interquartile range): adverse outcome 10 (10–12) vessels/mm2 vs. normal outcome 13 (12–15), p = 0.002). Adverse outcome pregnancy placental arteries were relatively insensitive to nitric oxide donated by sodium nitroprusside compared to normal outcome pregnancy placental arteries (50% Effective Concentration 30 (19–50) nM vs. 12 (6–24), p = 0.02). Adverse outcome pregnancy placental tissue contained less human chorionic gonadotrophin (20 (11–50) vs. 55 (24–102) mIU/mg, p = 0.007) and human placental lactogen (11 (6–14) vs. 27 (9–50) mg/mg, p = 0.006) and released more soluble fms-like tyrosine kinase-1 (21 (13–29) vs. 5 (2–15) ng/mg, p = 0.01) compared with normal outcome pregnancy placental tissue. Conclusion These data provide a description of the placental phenotype of adverse outcome in late pregnancy. Antenatal tests that accurately reflect elements of this phenotype may

  10. Ion channels and regulation of insulin secretion in human β-cells

    PubMed Central

    Fridlyand, Leonid E.; Jacobson, David A.; Philipson, L.H.

    2013-01-01

    In mammals an increase in glucose leads to block of ATP dependent potassium channels in pancreatic β cells leading to membrane depolarization. This leads to the repetitive firing of action potentials that increases calcium influx and triggers insulin granule exocytosis. Several important differences between species in this process suggest that a dedicated human-oriented approach is advantageous as extrapolating from rodent data may be misleading in several respects. We examined depolarization-induced spike activity in pancreatic human islet-attached β-cells employing whole-cell patch-clamp methods. We also reviewed the literature concerning regulation of insulin secretion by channel activity and constructed a data-based computer model of human β cell function. The model couples the Hodgkin-Huxley-type ionic equations to the equations describing intracellular Ca2+ homeostasis and insulin release. On the basis of this model we employed computational simulations to better understand the behavior of action potentials, calcium handling and insulin secretion in human β cells under a wide range of experimental conditions. This computational system approach provides a framework to analyze the mechanisms of human β cell insulin secretion. PMID:23624892