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Sample records for secreted human placental

  1. 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 ...

  2. 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.

  3. Altered folate metabolism modifies cell proliferation and progesterone secretion in human placental choriocarcinoma JEG-3 cells.

    PubMed

    Moussa, Carolyne; Ross, Nikia; Jolette, Philippe; MacFarlane, Amanda J

    2015-09-28

    Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nM) demonstrated 13% (P<0.001) and 26% (P<0.001) lower proliferation, 5.5% (P=0.025) and 7.5% (P=0.004) lower invasion capacity, and 5 to 7.5% (P=0.004-0.025) lower viability compared with control (20 nM) or supplemented (100 nM) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17.7% (P<0.0001) lower proliferation and 61% (P=0.014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10.3% (P=0.001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

  4. 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.

  5. 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

  6. 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.

  7. Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans.

    PubMed Central

    Sasaki, A; Shinkawa, O; Yoshinaga, K

    1989-01-01

    To clarify the physiological role of placental corticotropin-releasing hormone (CRH), we measured plasma CRH, ACTH, and cortisol throughout pregnancy. Cerebrospinal fluid (CSF) CRH levels and ACTH responsiveness to synthetic CRH were also quantified in pregnant and nonpregnant women. Maternal plasma CRH levels, which increased progressively during pregnancy, correlated well with both ACTH and cortisol in early labor, delivery, and postpartum samples, and also with cortisol levels in samples before labor. CSF CRH levels in term pregnant women did not differ from those of nonpregnant women. CRH infusion that attained similar plasma CRH levels to those found in late pregnancy elicited significant ACTH release in vivo and regular CRH test provoked normal ACTH response during early pregnancy but no response during late pregnancy. We concluded that: (a) maternal pituitary-adrenal axis correlates well with plasma CRH levels, which are high enough to provoke ACTH release from maternal pituitary; (b) hypothalamic CRH secretion in term pregnant women is not exaggerated; and (c) maternal pituitary is responsive to synthetic CRH in early but not late pregnancy, suggesting that maternal pituitary-adrenal axis is already activated by high circulating CRH. Placental CRH may be an important stimulator of the maternal pituitary-adrenal axis during pregnancy. Images PMID:2556451

  8. Retinal Angiogenesis Effects of TGF-β1 and Paracrine Factors Secreted From Human Placental Stem Cells in Response to a Pathological Environment.

    PubMed

    Kim, Kyung-Sul; Park, Ji-Min; Kong, TaeHo; Kim, Chul; Bae, Sang-Hun; Kim, Han Wool; Moon, Jisook

    2016-01-01

    Abnormal angiogenesis is a primary cause of many eye diseases, including diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Mesenchymal stem cells (MSCs) are currently being investigated as a treatment for several such retinal diseases based on their neuroprotective and angiogenic potentials. In this study, we evaluated the role of systemically injected human placental amniotic membrane-derived MSCs (AMSCs) on pathological neovascularization of proliferative retinopathy. We determined that AMSCs secrete higher levels of transforming growth factor-β (TGF-β1) than other MSCs, and the secreted TGF-β1 directly suppresses the proliferation of endothelial cells under pathological conditions in vitro. Moreover, in a mouse model of oxygen-induced retinopathy, intraperitoneally injected AMSCs migrated into the retina and suppressed excessive neovascularization of the vasculature via expression of TGF-β1, and the antineovascular effect of AMSCs was blocked by treatment with TGF-β1 siRNA. These findings are the first to demonstrate that TGF-β1 secreted from AMSCs is one of the key factors to suppress retinal neovascularization in proliferative retinopathy and further elucidate the therapeutic function of AMSCs for the treatment of retinal neovascular diseases.

  9. Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue.

    PubMed

    Gregoraszczuk, E L; Ptak, A; Karpeta, A; Fiedor, E; Wróbel, A; Milewicz, T; Falandysz, J

    2014-01-01

    Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2 ng/ml HCBz or PeCBz for 6-72 h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

  10. Excess LIGHT contributes to placental impairment, increased secretion of vasoactive factors, hypertension, and proteinuria in preeclampsia.

    PubMed

    Wang, Wei; Parchim, Nicholas F; Iriyama, Takayuki; Luo, Renna; Zhao, Cheng; Liu, Chen; Irani, Roxanna A; Zhang, Weiru; Ning, Chen; Zhang, Yujin; Blackwell, Sean C; Chen, Lieping; Tao, Lijian; Hicks, M John; Kellems, Rodney E; Xia, Yang

    2014-03-01

    Preeclampsia, a prevalent hypertensive disorder of pregnancy, is believed to be secondary to uteroplacental ischemia. Accumulating evidence indicates that hypoxia-independent mediators, including inflammatory cytokines and growth factors, are associated with preeclampsia, but it is unclear whether these signals directly contribute to placental damage and disease development in vivo. We report that LIGHT, a novel tumor necrosis factor superfamily member, is significantly elevated in the circulation and placentas of preeclamptic women compared with normotensive pregnant women. Injection of LIGHT into pregnant mice induced placental apoptosis, small fetuses, and key features of preeclampsia, hypertension and proteinuria. Mechanistically, using neutralizing antibodies specific for LIGHT receptors, we found that LIGHT receptors herpes virus entry mediator and lymphotoxin β receptor are required for LIGHT-induced placental impairment, small fetuses, and preeclampsia features in pregnant mice. Accordingly, we further revealed that LIGHT functions through these 2 receptors to induce secretion of soluble fms-like tyrosine kinase-1 and endothelin-1, 2 well-accepted pathogenic factors in preeclampsia, and thereby plays an important role in hypertension and proteinuria in pregnant mice. Lastly, we extended our animal findings to human studies and demonstrated that activation of LIGHT receptors resulted in increased apoptosis and elevation of soluble fms-like tyrosine kinase-1 secretion in human placental villous explants. Overall, our human and mouse studies show that LIGHT signaling is a previously unrecognized pathway responsible for placental apoptosis, elevated secretion of vasoactive factors, and subsequent maternal features of preeclampsia, and reveal new therapeutic opportunities for the management of the disease.

  11. 21 CFR 862.1585 - Human placental lactogen test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Human placental lactogen test system. 862.1585 Section 862.1585 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1585 Human placental lactogen test system. (a) Identification. A human placental...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Human placental lactogen test system. 862.1585 Section 862.1585 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1585 Human placental lactogen test system. (a) Identification. A human placental...

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Human placental lactogen test system. 862.1585 Section 862.1585 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1585 Human placental lactogen test system. (a) Identification. A human placental...

  14. Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

    PubMed

    Lahiani, Adi; Zahavi, Efrat; Netzer, Nir; Ofir, Racheli; Pinzur, Lena; Raveh, Shani; Arien-Zakay, Hadar; Yavin, Ephraim; Lazarovici, Philip

    2015-02-01

    Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 10⁵ PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

  15. 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...

  16. 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

  17. Epidermal growth factor stimulates mouse placental lactogen I but inhibits mouse placental lactogen II secretion in vitro.

    PubMed Central

    Yamaguchi, M; Ogren, L; Endo, H; Thordarson, G; Kensinger, R; Talamantes, F

    1992-01-01

    This study was undertaken to determine whether epidermal growth factor (EGF) regulates the secretion of mouse placental lactogen (mPL)-I and mPL-II. Primary cell cultures were prepared from placentas from days 7, 9, and 11 of pregnancy and cultured for up to 5 days. Addition of EGF (20 ng/ml) to the medium resulted in significant stimulation of mPL-I secretion by the second day of culture in cells from days 7 and 9 of pregnancy and significant inhibition of mPL-II secretion by the third or fourth day of culture in cells from days 7, 9, and 11. Dose-response studies carried out with cells from day 7 of pregnancy demonstrated that the minimum concentration of EGF that stimulated mPL-I secretion and inhibited mPL-II secretion was 1.0 ng/ml. EGF did not affect the DNA content of the cells or cell viability, assessed by trypan blue exclusion, nor did it have a general effect on protein synthesis. There are three types of PL-containing giant cells in mouse placental cell cultures: cells that contain either mPL-I or mPL-II and cells that contain both hormones. Immunocytochemical analysis and the reverse hemolytic plaque assay indicated that EGF treatment was accompanied by a significant increase in the number of cells that produce mPL-I, but among the PL cells that contained mPL-I, there was no change in the fraction of cells that contained only mPL-I or the fraction that contained both mPL-I and mPL-II. In contrast, EGF treatment did affect the distribution of mPL-II among PL cells. In control cultures, about 75% of the cells that contained mPL-II also contained mPL-I, but in EGF-treated cultures, all of the cells that contained mPL-II also contained mPL-I. These data suggest that EGF regulates mPL-I and mPL-II secretion at least partly by regulating PL cell differentiation. PMID:1454826

  18. Human placental lactogen decreases regional blood flow in anesthetized pigs.

    PubMed

    Grossini, E; Molinari, C; Battaglia, A; Mary, D A S G; Ribichini, F; Surico, N; Vacca, G

    2006-01-01

    In 22 pigs anesthetized with sodium pentobarbitone, changes in blood flow caused by infusion of human placental lactogen into the left renal, external iliac, and anterior descending coronary arteries were assessed using electromagnetic flowmeters. In 17 pigs, infusion of human placental lactogen whilst keeping the heart rate and arterial pressure constant decreased coronary, renal and iliac flow. In 5 additional pigs, increasing the dose of human placental lactogen produced a dose-related decrease in regional blood flow. The mechanisms of the above response were studied in 15 of the 17 pigs by repeating the experiment of infusion. The human placental lactogen-induced decrease in regional blood flow was not affected by blockade of cholinergic receptors (5 pigs) or of alpha-adrenergic receptors (5 pigs), but it was abolished by blockade of beta2-adrenergic receptors (5 pigs). The present study showed that intra-arterial infusion of human placental lactogen primarily decreased coronary, renal and iliac blood flow. The mechanism of this response was shown to be due to the inhibition of a vasodilatory beta2-adrenergic receptor-mediated effect.

  19. 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.

  20. Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

    PubMed

    Williams, Carmen J; Chu, Alison; Jefferson, Wendy N; Casero, David; Sudhakar, Deepthi; Khurana, Nevil; Hogue, Claire P; Aryasomayajula, Chinmayi; Patel, Priya; Sullivan, Peggy; Padilla-Banks, Elizabeth; Mohandessi, Shabnam; Janzen, Carla; Wadehra, Madhuri

    2017-03-14

    Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2(-/-) ) were generated. Emp2(-/-) females are fertile but have reduced litter sizes when carrying Emp2(-/-) but not Emp2(+/-) fetuses. Placentas of Emp2(-/-) fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2(-/-) fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans and suggest it may be a new biomarker for placental insufficiency.

  1. 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

  2. Placental lactogen secretion during prolonged-pregnancy in the rat: the ovary plays a pivotal role in the control of placental function.

    PubMed

    Shiota, K; Furuyama, N; Takahashi, M

    1991-10-01

    The serum of rats at mid-pregnancy contains at least 2 distinct placental lactogen (PL)-like substances tentatively termed placental lactogen-alpha (PL-alpha) and placental lactogen-beta (PL-beta) (Endocrinol Japon 38: 533-540, 1991). We have investigated the secretory patterns of three placental lactogens (PL-alpha, PL-beta and placental lactogen-II) during normal pregnancy and in two prolonged-pregnancy models. Pregnancy was prolonged by the introduction of new corpora lutea by inducing ovulation on day 15 of pregnancy by successive treatments with PMSG (30 IU/rat, sc on day 12) and hCG (10 IU/rat, iv on day 14), and in the second model by progesterone implants on day 15 of pregnancy. During normal pregnancy, each of the 3 PLs exhibited only one secretory peak in the serum; PL-alpha and PL-beta on day 12 and placental lactogen II (PL-II) on day 20. Interestingly, in the rats with new sets of corpora lutea, serum PL-alpha and PL-beta levels began to increase again on day 18 and showed peaks on day 20 for PL-alpha and on day 22 for PL-beta. In this model, the initiation of PL-II secretion was not affected, but high levels were maintained until day 26, when parturition occurred. In rats receiving either PMSG or hCG, the secretory patterns of the PLs were similar to as those during normal pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. 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

    2016-08-02

    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.

  4. 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.

  5. Bidirectional Transfer Study of Polystyrene Nanoparticles across the Placental Barrier in an ex Vivo Human Placental Perfusion Model

    PubMed Central

    Grafmueller, Stefanie; Manser, Pius; Diener, Liliane; Diener, Pierre-André; Maeder-Althaus, Xenia; Maurizi, Lionel; Jochum, Wolfram; Krug, Harald F.; Buerki-Thurnherr, Tina; von Mandach, Ursula

    2015-01-01

    Background Nanoparticle exposure in utero might not be a major concern yet, but it could become more important with the increasing application of nanomaterials in consumer and medical products. Several epidemiologic and in vitro studies have shown that nanoparticles can have potential toxic effects. However, nanoparticles also offer the opportunity to develop new therapeutic strategies to treat specifically either the pregnant mother or the fetus. Previous studies mainly addressed whether nanoparticles are able to cross the placental barrier. However, the transport mechanisms underlying nanoparticle translocation across the placenta are still unknown. Objectives In this study we examined which transport mechanisms underlie the placental transfer of nanoparticles. Methods We used the ex vivo human placental perfusion model to analyze the bidirectional transfer of plain and carboxylate modified polystyrene particles in a size range between 50 and 300 nm. Results We observed that the transport of polystyrene particles in the fetal to maternal direction was significantly higher than for the maternal to fetal direction. Regardless of their ability to cross the placental barrier and the direction of perfusion, all polystyrene particles accumulated in the syncytiotrophoblast of the placental tissue. Conclusions Our results indicate that the syncytiotrophoblast is the key player in regulating nanoparticle transport across the human placenta. The main mechanism underlying this translocation is not based on passive diffusion, but is likely to involve an active, energy-dependent transport pathway. These findings will be important for reproductive toxicology as well as for pharmaceutical engineering of new drug carriers. Citation Grafmueller S, Manser P, Diener L, Diener PA, Maeder-Althaus X, Maurizi L, Jochum W, Krug HF, Buerki-Thurnherr T, von Mandach U, Wick P. 2015. Bidirectional transfer study of polystyrene nanoparticles across the placental barrier in an ex vivo human

  6. 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.

  7. Higher order organization of human placental aromatase.

    PubMed

    Ghosh, Debashis; Jiang, Wenhua; Lo, Jessica; Egbuta, Chinaza

    2011-07-01

    Aromatase (CYP19A1) is an integral membrane enzyme that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens. All human estrogens are synthesized from their androgenic precursors by this unique cytochrome P450. The crystal structure of active aromatase purified from human placenta has recently been determined in complex with its natural substrate androstenedione in the high-spin ferric state of heme. Hydrogen bond forming interactions and tight packing hydrophobic side chains closely complement puckering of the steroid backbone, thereby providing the molecular basis for the androgenic specificity of aromatase. In the crystal, aromatase molecules are linked by a head-to-tail intermolecular interaction via a surface loop between helix D and helix E of one aromatase molecule that penetrates the heme-proximal cavity of the neighboring, crystallographically related molecule, thus forming in tandem a polymeric aromatase chain. This intermolecular interaction is similar to the aromatase-cytochrome P450 reductase coupling and is driven by electrostatics between the negative potential surface of the D-E loop region and the positively charged heme-proximal cavity. This loop-to-proximal site link in aromatase is rather unique--there are only a few of examples of somewhat similar intermolecular interactions in the entire P450 structure database. Furthermore, the amino acids involved in the intermolecular contact appear to be specific for aromatase. Higher order organization of aromatase monomers may have implications in lipid integration and catalysis.

  8. 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...

  9. 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...

  10. Molecular Regulation of Human Placental Growth Factor (PlGF) Gene Expression in Placental Villi and Trophoblast Cells is Mediated via the Protein Kinase A Pathway

    PubMed Central

    Depoix, Christophe; Tee, Meng Kian; Taylor, Robert N.

    2011-01-01

    Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated. We verified this result in two choriocarcinoma cell lines, BeWo and JEG-3, and also in first trimester placental villous explants by quantifying PlGF mRNA (real time PCR) and PlGF protein secreted into the supernatant (ELISA). Similar effects were noted for vascular endothelial growth factor (VEGF) mRNA and protein expression. Treatment with cholera toxin and the use of a specific inhibitor of protein kinase A (PKA) blocked these effects, indicating that the cAMP/PKA pathway is responsible for the cAMP-induced upregulation of PlGF and that one or more G protein coupled receptor(s) was involved. We identified two functional cAMP responsive elements (CRE) in the PlGF promoter and demonstrated that the CRE binding protein, CREB, contributes to the regulation of PlGF gene expression. We speculate that defects in this signaling pathway may lead to abnormal secretion of PlGF protein as observed in the pregnancy-related diseases preeclampsia and intrauterine growth restriction. PMID:21135203

  11. The roles of placental growth hormone and placental lactogen in the regulation of human fetal growth and development.

    PubMed

    Handwerger, S; Freemark, M

    2000-04-01

    The human growth hormone (hGH)/human placental lactogen (hPL) gene family, which consists of two GH and three PL genes, is important in the regulation of maternal and fetal metabolism and the growth and development of the fetus. During pregnancy, pituitary GH (hGH-N) expression in the mother is suppressed; and hGH-V, a GH variant expressed by the placenta, becomes the predominant GH in the mother. hPL, which is the product of the hPL-A and hPL-B genes, is secreted into both the maternal and fetal circulations after the sixth week of pregnancy. hGH-V and hPL act in concert in the mother to stimulate insulin-like growth factor (IGF) production and modulate intermediary metabolism, resulting in an increase in the availability of glucose and amino acids to the fetus. In the fetus, hPL acts via lactogenic receptors and possibly a unique PL receptor to modulate embryonic development, regulate intermediary metabolism and stimulate the production of IGFs, insulin, adrenocortical hormones and pulmonary surfactant. hGH-N, which is expressed by the fetal pituitary, has little or no physiological actions in the fetus until late in pregnancy due to the lack of functional GH receptors on fetal tissues. hGH-V, which is also a potent somatogenic hormone, is not released into the fetus. Taken together, studies of the hGH/hPL gene family during pregnancy reveal a complex interaction of the hormones with one another and with other growth factors. Additional investigations are necessary to clarify the relative roles of the family members in the regulation of fetal growth and development and the factors that modulate the expression of the genes.

  12. IFPA Meeting 2012 Workshop Report I: comparative placentation and animal models, advanced techniques in placental histopathology, human pluripotent stem cells as a model for trophoblast differentiation.

    PubMed

    Ackerman, W E; Carter, A M; De Mestre, A M; Golos, T G; Jeschke, U; Kusakabe, K; Laurent, L C; Parast, M M; Roberts, R M; Robinson, J M; Rutherford, J; Soma, H; Takizawa, T; Ui-Tei, K; Lash, G E

    2013-03-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 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 models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.

  13. Human Term Placental Cells: Phenotype, Properties and New Avenues in Regenerative Medicine

    PubMed Central

    Caruso, Maddalena; Evangelista, Marco; Parolini, Ornella

    2012-01-01

    The human placenta has long been the subject of scientific interest due to the important roles which it performs during pregnancy in sustaining the fetus and maintaining fetomaternal tolerance. More recently, however, researchers have begun to investigate the possibility that the placenta’s utility may extend beyond fetal development to act as a source of cells with clinically relevant properties. Indeed, several groups have reported the isolation of cells from different placental regions which display both multilineage differentiation potential and immunomodulatory properties in vitro. Furthermore, these cells have also been shown to secrete soluble factors involved in pathophysiological processes that may aid tissue repair. Cells with such features will clearly find application in the field of regenerative medicine for the repair/regeneration of damaged or diseased tissues or organs. In line with these promising findings, several preclinical and clinical studies conducted to date argue in strong favor of the therapeutic utility of placenta-derived cells for the treatment of several diseases. Although much work remains to be conducted in order to fully understand the properties of placental cells and the mechanisms which underlie their beneficial effects in vivo, data reported to date nonetheless provide compelling evidence in support of the placenta as a cell source for use in regenerative medicine. PMID:24551761

  14. Vulnerability of primitive human placental trophoblast to Zika virus.

    PubMed

    Sheridan, Megan A; Yunusov, Dinar; Balaraman, Velmurugan; Alexenko, Andrei P; Yabe, Shinichiro; Verjovski-Almeida, Sergio; Schust, Danny J; Franz, Alexander W; Sadovsky, Yoel; Ezashi, Toshihiko; Roberts, R Michael

    2017-02-28

    Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKV(U)) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKV(C)). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.

  15. Vulnerability of primitive human placental trophoblast to Zika virus

    PubMed Central

    Sheridan, Megan A.; Yunusov, Dinar; Balaraman, Velmurugan; Alexenko, Andrei P.; Yabe, Shinichiro; Verjovski-Almeida, Sergio; Schust, Danny J.; Franz, Alexander W.; Ezashi, Toshihiko; Roberts, R. Michael

    2017-01-01

    Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction. PMID:28193876

  16. 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.

  17. The Human Placenta Project: placental structure, development, and function in real time.

    PubMed

    Guttmacher, A E; Maddox, Y T; Spong, C Y

    2014-05-01

    Despite its crucial role in the health of both the fetus and the pregnant woman, the placenta is the least understood human organ. Since a growing body of evidence also underscores the importance of placental development in the lifelong health of both mother and offspring, this lack of knowledge about placental structure and function is particularly concerning. Given modern approaches and technologies and the ability to develop new methods, we propose a coordinated "Human Placenta Project", with the ultimate goal of understanding human placental structure, development, and function in real time.

  18. 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.

  19. Characteristics of thyroxine 5'-deiodination in cultured human placental cells. Regulation by iodothyronines.

    PubMed Central

    Hidal, J T; Kaplan, M M

    1985-01-01

    Human and rat placental homogenates convert L-thyroxine (T4) to 3,5,3'-L-triiodothyronine (T3) via a pathway termed type II iodothyronine deiodination. To study regulation of this pathway, cell dispersions were prepared from human placental chorionic-decidual membrane. Dispersed cells deiodinated T4 and 3,3',5'-triiodothyronine (rT3), but not T3, at the 5' position. The reaction was only slightly inhibited by 1 mM 6-n-propylthiouracil, enhanced by dithiothreitol, and substantially inhibited by 50 nM iopanoic acid. Incubation of the cells in thyroid hormone-depleted medium induced a near doubling of T4 5'-deiodination in 36-48 h, with a significant rise seen as early as 12 h. Addition of T4, rT3, or T3 to hormone-depleted medium impaired the rise in type II deiodination in a dose-dependent fashion. T4 and rT3 were equipotent in this regard, and T3 was 2-3 times less potent. T4 was effective in physiological concentrations, 6.5-13 nM in medium containing 10% calf serum, and the effect of T4 was not due to its conversion to either T3 or rT3. In cells with deiodinase activity raised by 48 h incubation in thyroid hormone-depleted medium, addition of T4, T3, or rT3 reversed the increase in 8-24 h. Secretion of prolactin and beta hCG by the dispersed cells was not substantially affected by thyroid hormone deprivation. The increase in type II deiodination during thyroid hormone deprivation appears to depend on a signal from the thyroxine molecule, per se, and could potentially defend intracellular, and/or circulating, T3 pools in pathological states of mild-to-moderate hypothyroxinemia. PMID:2413075

  20. 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.

  1. Proteome of human minor salivary gland secretion.

    PubMed

    Siqueira, W L; Salih, E; Wan, D L; Helmerhorst, E J; Oppenheim, F G

    2008-05-01

    Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

  2. Lipid Raft- and Src Family Kinase-Dependent Entry of Coxsackievirus B into Human Placental Trophoblasts

    PubMed Central

    Delorme-Axford, Elizabeth; Sadovsky, Yoel

    2013-01-01

    Maternal-fetal transmission of group B coxsackieviruses (CVB) during pregnancy has been associated with a number of diverse pathological outcomes, including hydrops fetalis, fetal myocarditis, meningoencephalitis, neurodevelopmental delays, congenital skin lesions, miscarriage, and/or stillbirth. Throughout pregnancy, the placenta forms a critical antimicrobial protective barrier at the maternal-fetal interface. Despite the severity of diseases accompanying fetal CVB infections, little is known regarding the strategies used by CVB to gain entry into placental trophoblasts. Here we used both a trophoblast cell line and primary human trophoblasts to demonstrate the mechanism by which CVB gains entry into polarized placental trophoblasts. Our studies revealed that the kinetics of CVB entry into placental trophoblasts are similar to those previously described for polarized intestinal epithelial cells. Likewise, CVB entry into placental trophoblasts requires decay-accelerating factor (DAF) binding and involves relocalization of the virus from the apical surface to intercellular tight junctions. In contrast, we have identified a divergent mechanism for CVB entry into polarized trophoblasts that is clathrin, caveolin-1, and dynamin II independent but requires intact lipid rafts. In addition, we found that members of the Src family of tyrosine kinases were required for CVB entry. Our studies highlight the complexity of viral entry into human placental trophoblasts and may serve as a model for mechanisms used by diverse pathogens to penetrate the placental barrier. PMID:23720726

  3. 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.

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

    PubMed

    Mizuuchi, Masahito; Cindrova-Davies, Tereza; Olovsson, Matts; Charnock-Jones, D Stephen; Burton, Graham J; Yung, Hong Wa

    2016-03-01

    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 potential new therapeutic

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

    PubMed

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

    2016-02-01

    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.

  6. 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.

  7. 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.

  8. 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

    2015-12-03

    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.

  9. Infectious Achilles Tendinitis After Local Injection of Human Placental Extracts: A Case Report.

    PubMed

    Kim, Yoon-Chung; Ahn, Jae Hoon; Kim, Man-Soo

    2015-01-01

    Local injections of corticosteroids or human placental extracts are sometimes used for the treatment of resistant tendinitis or fasciitis. We report a case of infectious Achilles tendinitis complicated by calcaneal osteomyelitis after injection of human placental extracts for the Achilles tendinitis. She was treated with excision of the infected bone and tendon, followed by V-Y lengthening of the proximal portion of the Achilles tendon in a single stage. At 2 years postoperative, she remained symptom free without any signs of recurrence, and the follow-up magnetic resonance imaging scan demonstrated a well-maintained Achilles tendon with normal signal intensity.

  10. Transcriptional enhancer within the human placental lactogen and growth hormone multigene cluster.

    PubMed Central

    Rogers, B L; Sobnosky, M G; Saunders, G F

    1986-01-01

    Human placental lactogen (hPL) and human growth hormone (hGH) are members of a multigene family that share amino acid sequence homology and similarity in gene structure and nucleotide sequence, but differ in both function and expression. To determine the sequence requirements for tissue specific expression recombinant plasmids containing the members of the hPL-hGH multigene family and flanking regions were analyzed by both transient and stable transfection assays. We have identified a transcriptional enhancer in a 1.0 kb region located 2.0 kb downstream of the hPL3 structural gene. This enhancer sequence is not strictly cell-type specific since it functions in cell lines of both placental (JEG-3) and pituitary (18-54,SF) origin. However, its efficiency is several fold higher in placental cells than in pituitary cells. Images PMID:3774541

  11. Maternal growth factor regulation of human placental development and fetal growth.

    PubMed

    Forbes, Karen; Westwood, Melissa

    2010-10-01

    Normal development and function of the placenta is critical to achieving a successful pregnancy, as normal fetal growth depends directly on the transfer of nutrients from mother to fetus via this organ. Recently, it has become apparent from both animal and human studies that growth factors within the maternal circulation, for example the IGFs, are important regulators of placental development and function. Although these factors act via distinct receptors to exert their effects, the downstream molecules activated upon ligand/receptor interaction are common to many growth factors. The expression of numerous signaling molecules is altered in the placentas from pregnancies affected by the fetal growth complications, fetal growth restriction, and macrosomia. Thus, targeting these molecules may lead to more effective treatments for complications of pregnancy associated with altered placental development. Here, we review the maternal growth factors required for placental development and discuss their mechanism of action.

  12. 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...

  13. Placental-related diseases of pregnancy: involvement of oxidative stress and implications in human evolution

    PubMed Central

    Jauniaux, Eric; Poston, Lucilla; Burton, Graham J.

    2007-01-01

    Miscarriage and pre-eclampsia are the most common disorders of human pregnancy. Both are placental-related and exceptional in other mammalian species. Ultrasound imaging has enabled events during early pregnancy to be visualized in vivo for the first time. As a result, a new understanding of the early materno–fetal relationship has emerged and, with it, new insight into the pathogenesis of these disorders. Unifying the two is the concept of placental oxidative stress, with associated necrosis and apoptosis of the trophoblastic epithelium of the placental villous tree. In normal pregnancies, the earliest stages of development take place in a low oxygen (O2) environment. This physiological hypoxia of the early gestational sac protects the developing fetus against the deleterious and teratogenic effects of O2 free radicals (OFRs). In miscarriage, development of the placento–decidual interface is severely impaired leading to early and widespread onset of maternal blood flow and major oxidative degeneration. This mechanism is common to all miscarriages, with the time at which it occurs in the first trimester depending on the aetiology. In contrast, in pre-eclampsia the trophoblastic invasion is sufficient to allow early pregnancy phases of placentation but too shallow for complete transformation of the arterial utero–placental circulation, predisposing to a repetitive ischaemia–reperfusion (I/R) phenomenon. We suggest that pre-eclampsia is a three-stage disorder with the primary pathology being an excessive or atypical maternal immune response. This would impair the placentation process leading to chronic oxidative stress in the placenta and finally to diffuse maternal endothelial cell dysfunction. PMID:16682385

  14. Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia.

    PubMed

    Liao, Wu-Xiang; Laurent, Louise C; Agent, Sally; Hodges, Jennifer; Chen, Dong-Bao

    2012-04-01

    Preeclampsia is characterized by dysfunctional endothelium and impaired angiogenesis. Recent studies suggest that the neuronal guidance SLIT/ROBO system regulates tumor angiogenesis. This study investigated if SLIT and ROBO are differentially expressed in healthy term and preeclamptic placentas and if hypoxia regulates SLIT and ROBO expression in placental trophoblast and endothelial cells. Total RNA and protein were extracted from placental tissues of healthy term (n = 5) and preeclamptic (n = 6) pregnancies and used for SLIT/ROBO expression analyses with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative-PCR, and immunoblotting. Paraffin-embedded tissues were processed to localize SLIT/ROBO proteins in placental villi by immunohistochemistry. BeWo choriocarcinoma cells and human umbilical vein endothelial cells (HUVEC) were treated with 2% or 10% oxygen or the hypoxia mimetic deferoxamine mesylate (100 μM) to test if hypoxia regulates SLIT/ROBO expression. SLIT2, SLIT3, ROBO1, and ROBO4 mRNA and proteins were detected in the placenta. SLIT2 and ROBO1 proteins localized in the syncytiotrophoblast, and SLIT3, ROBO1, and ROBO4 in capillary endothelium of the placental villi. Levels of ROBO1 and ROBO4 as well as sFLT1 (soluble fms-like tyrosine kinase-1) proteins were significantly greater in preeclamptic placentas compared to normal controls. Hypoxia significantly increased both mRNA and protein levels of SLIT2 in BeWo cells and of SLIT3, ROBO1, and ROBB4 in HUVEC. Thus, trophoblast and endothelial coexpression of SLIT/ROBO suggests an autocrine/paracrine regulatory system for regulating placental function. Differential expression of SLITs and ROBOs in healthy term and preeclamptic placentas and hypoxia regulation of their expressions in placental cells implicate a potential pathophysiological role for this system in preeclampsia.

  15. Placental-related diseases of pregnancy: Involvement of oxidative stress and implications in human evolution.

    PubMed

    Jauniaux, Eric; Poston, Lucilla; Burton, Graham J

    2006-01-01

    Miscarriage and pre-eclampsia are the most common disorders of human pregnancy. Both are placental-related and exceptional in other mammalian species. Ultrasound imaging has enabled events during early pregnancy to be visualized in vivo for the first time. As a result, a new understanding of the early materno-fetal relationship has emerged and, with it, new insight into the pathogenesis of these disorders. Unifying the two is the concept of placental oxidative stress, with associated necrosis and apoptosis of the trophoblastic epithelium of the placental villous tree. In normal pregnancies, the earliest stages of development take place in a low oxygen (O2) environment. This physiological hypoxia of the early gestational sac protects the developing fetus against the deleterious and teratogenic effects of O2 free radicals (OFRs). In miscarriage, development of the placento-decidual interface is severely impaired leading to early and widespread onset of maternal blood flow and major oxidative degeneration. This mechanism is common to all miscarriages, with the time at which it occurs in the first trimester depending on the aetiology. In contrast, in pre-eclampsia the trophoblastic invasion is sufficient to allow early pregnancy phases of placentation but too shallow for complete transformation of the arterial utero-placental circulation, predisposing to a repetitive ischaemia-reperfusion (I/R) phenomenon. We suggest that pre-eclampsia is a three-stage disorder with the primary pathology being an excessive or atypical maternal immune response. This would impair the placentation process leading to chronic oxidative stress in the placenta and finally to diffuse maternal endothelial cell dysfunction.

  16. 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

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

    PubMed

    Elliot, Michael G; Crespi, Bernard J

    2015-03-05

    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.

  18. 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

  19. Establishment of hybridomas secreting monoclonal antibodies to placental alkaline phosphatase and development of an enzyme immunoassay for its determination.

    PubMed

    Kinoshita, Y; Okamoto, T; Mano, H; Furuhashi, Y; Goto, S; Tomoda, Y

    1990-06-01

    We established seven hybridomas secreting murine IgG monoclonal antibodies (MoAbs) to placental alkaline phosphatase (PLAP). The seven hybridomas were designated (1) 7C6, (2) 6G10, (3) 5B9, (4) 6D5, (5) 6B5, (6) 11G6 and (7) 3E10, respectively. The characteristics of these hybridomas were evaluated by radioimmunoassay (RIA) with 125I-PLAP. Their reactivity with the intestinal alkaline phosphatase, one of the alkaline phosphatase isozymes, was (1) 0.04, (2) 0.2, (3) 1.4, (4) 1.8, (5) 0, (6) 4.0 and (7) 6.2(%), respectively. None of them showed signs of cross-reactivity with the liver-type alkaline phosphatase, also one of the alkaline phosphatase isozymes, within a PLAP concentration of 2,000 IU/l. The subtype of 5B9 was IgG1, and that of the others was IgG2a. We then used 7C6, to develop a sensitive, specific and convenient enzyme immunoassay (EIA) for the determination of PLAP, and assayed sera from patients with various gynecologic diseases. The incidence of increased PLAP was 6.4% in patients with benign diseases, 21.5% in cervical cancer, 36.4% in endometrial carcinoma, and 39.5% in malignant ovarian tumors. The specificity for malignant diseases seemed to be higher than that of CA125. Among endometrial carcinomas, well-differentiated adenocarcinoma had the highest incidence of an increased concentration. Among malignant ovarian tumors, serous cystadenocarcinoma, endometrioid carcinoma, dysgerminoma and Krukenberg's tumor showed a higher incidence than the other types.

  20. Choline inadequacy impairs trophoblast function and vascularization in cultured human placental trophoblasts.

    PubMed

    Jiang, Xinyin; Jones, Sara; Andrew, Benjamin Y; Ganti, Anita; Malysheva, Olga V; Giallourou, Natasa; Brannon, Patsy M; Roberson, Mark S; Caudill, Marie A

    2014-08-01

    Maternal choline intake during gestation may influence placental function and fetal health outcomes. Specifically, we previously showed that supplemental choline reduced placental and maternal circulating concentrations of the anti-angiogenic factor, fms-like tyrosine kinase-1 (sFLT1), in pregnant women as well as sFLT1 production in cultured human trophoblasts. The current study aimed to quantify the effect of choline on a wider array of biomarkers related to trophoblast function and to elucidate possible mechanisms. Immortalized HTR-8/SVneo trophoblasts were cultured in different choline concentrations (8, 13, and 28 µM [control]) for 96-h and markers of angiogenesis, inflammation, apoptosis, and blood vessel formation were examined. Choline insufficiency altered the angiogenic profile, impaired in vitro angiogenesis, increased inflammation, induced apoptosis, increased oxidative stress, and yielded greater levels of protein kinase C (PKC) isoforms δ and ϵ possibly through increases in the PKC activators 1-stearoyl-2-arachidonoyl-sn-glycerol and 1-stearoyl-2-docosahexaenoyl-sn-glycerol. Notably, the addition of a PKC inhibitor normalized angiogenesis and apoptosis, and partially rescued the aberrant gene expression profile. Together these results suggest that choline inadequacy may contribute to placental dysfunction and the development of disorders related to placental insufficiency by activating PKC.

  1. Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells

    PubMed Central

    ISHIKAWA, TOMOKO; TAKIZAWA, TAKAMI; IWAKI, JUN; MISHIMA, TAKUYA; UI-TEI, KUMIKO; TAKESHITA, TOSHIYUKI; MATSUBARA, SHIGEKI; TAKIZAWA, TOSHIHIRO

    2015-01-01

    The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments. PMID:25778799

  2. 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.

  3. Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion.

    PubMed

    Schneider, H

    2009-03-01

    In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the

  4. High activity of alpha-glycerophosphate oxidation by human placental mitochondria.

    PubMed

    Swierczyński, J; Scislowski, P; Aleksandrowicz, Z

    1976-03-11

    Human term placental mitochondria oxidize alpha-glycerophosphate at an unusually high rate as compared to other substrates. The apparent Km both for oxidation and alpha-glycerophosphate dehydrogenase (EC 1.1.99.5) activity of DL-alpha glycerophosphate determined in a medium containing 2mM EDTA and 5 mM MgSO4 was approx. 0.7 mM. EDTA inhibited the alpha-glycerophosphate oxidation if the later was used at low concentrations. A subsequent addition of MgSO4 or CaCl2 restored the original activity. EDTA had no effect on mitochondrial respiration at high concentration of alpha-glycerophosphate. Possible physiological role of relatively high activity of human placental mitochondrial alpha-glycerophosphate dehydrogenase is discussed.

  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. 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

  7. Concentrations of Polybrominated Diphenyl Ethers (PBDEs) and 2,4,6-Tribromophenol in Human Placental Tissues

    PubMed Central

    Leonetti, Christopher; Butt, Craig M.; Hoffman, Kate; Miranda, Marie Lynn; Stapleton, Heather M.

    2015-01-01

    Legacy environmental contaminants such as polybrominated diphenyl ethers (PBDEs) are widely detected in human tissues. However, few studies have measured PBDEs in placental tissues, and there are no reported measurements of 2,4,6-tribromophenol (2,4,6-TBP) in placental tissues. Measurements of these contaminants are important for understanding potential fetal exposures, as these compounds have been shown to alter thyroid hormone regulation in vitro and in vivo. In this study, we measured a suite of PBDEs and 2,4,6-TBP in 102 human placental tissues collected between 2010–2011 in Durham County, North Carolina, USA. The most abundant PBDE congener detected was BDE-47, with a mean concentration of 5.09 ng/g lipid (range: 0.12–141 ng/g lipid; detection frequency 91%); however, 2,4,6-TBP was ubiquitously detected and present at higher concentrations with a mean concentration of 15.4 ng/g lipid (range:1.31–316 ng/g lipid; detection frequency 100%). BDE-209 was also detected in more than 50% of the samples, and was significantly associated with 2,4,6-TBP in placental tissues, suggesting they may have a similar source, or that 2,4,6-TBP may be a degradation product of BDE-209. Interestingly, BDE-209 and 2,4,6-TBP were negatively associated with age (rs=−0.16; p=0.10 and rs=−0.17; p=0.08, respectively). The results of this work indicate that PBDEs and 2,4,6-TBP bioaccumulate in human placenta tissue and likely contribute to prenatal exposures to these environmental contaminants. Future studies are needed to determine if these joint exposures are associated with any adverse health measures in infants and children. PMID:26700418

  8. Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice

    PubMed Central

    Pernicone, Elizabeth; Korkes, Henri A.; Burke, Suzanne D.; Rajakumar, Augustine; Thadhani, Ravi I.; Roberts, Drucilla J.; Bhasin, Manoj; Karumanchi, S. Ananth

    2016-01-01

    Decidual NK (dNK) cells, a distinct type of NK cell, are thought to regulate uterine spiral artery remodeling, a process that allows for increased blood delivery to the fetal-placental unit. Impairment of uterine spiral artery remodeling is associated with decreased placental perfusion, increased uterine artery resistance, and obstetric complications such as preeclampsia and intrauterine growth restriction. Ex vivo manipulation of human peripheral blood NK (pNK) cells by a combination of hypoxia, TGFß-1 and 5-aza-2’-deoxycytidine yields cells with phenotypic and in vitro functional similarities to dNK cells, called idNK cells. Here, gene expression profiling shows that CD56Bright idNK cells derived ex vivo from human pNK cells, and to a lesser extent CD56Dim idNK cells, are enriched in the gene expression signature that distinguishes dNK cells from pNK cells. When injected into immunocompromised pregnant mice with elevated uterine artery resistance, idNK cells homed to the uterus and reduced the uterine artery resistance index, suggesting improved placental perfusion. PMID:27736914

  9. Exposure to ethanol and nicotine induces stress responses in human placental BeWo cells.

    PubMed

    Repo, Jenni K; Pesonen, Maija; Mannelli, Chiara; Vähäkangas, Kirsi; Loikkanen, Jarkko

    2014-01-13

    Human placental trophoblastic cancer BeWo cells can be used as a model of placental trophoblasts. We found that combined exposure to relevant exposure concentrations of ethanol (2‰) and nicotine (15 μM) induces an increase in the amount of reactive oxygen species (ROS). Neither ethanol or nicotine alone, nor their combination affected cell viability. However, nicotine decreased cell proliferation, both alone and combined with ethanol. Nicotine increased the expression of the endoplasmic reticulum (ER)-stress related protein GRP78/BiP, but not another marker of ER-stress, IRE1α. We also studied the effects of nicotine and/or ethanol on phosphorylation and expression of three mitogen-activated protein kinases (MAPKs), i.e. JNK, p38 and ERK1/2. Nicotine decreased the phosphorylation of JNK and also had similar effect on total amount of this protein. Phosphorylation and expression of p38 were increased 1.7- and 1.6-fold, respectively, by nicotine alone, and 1.9- and 2.1-fold by the combined treatment. Some increase (1.8-fold) was also seen in the phosphorylation of ERK2 at 48 h, in cells exposed to both ethanol and nicotine. This study shows that ethanol and nicotine, which harm the development of fetus may induce both oxidative and ER stress responses in human placental trophoblastic cells, implicating these mechanisms in their fetotoxic effects.

  10. TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

    PubMed Central

    Girard, Thomas J.; Tuley, Elodee

    2012-01-01

    Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ. PMID:22144186

  11. Expression and function of potassium channels in the human placental vasculature.

    PubMed

    Wareing, Mark; Bai, Xilian; Seghier, Fella; Turner, Claire M; Greenwood, Susan L; Baker, Philip N; Taggart, Michael J; Fyfe, Gregor K

    2006-08-01

    In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.

  12. Triamcinolone up-regulates GLUT 1 and GLUT 3 expression in cultured human placental endothelial cells.

    PubMed

    Kipmen-Korgun, Dijle; Ozmen, Asli; Unek, Gozde; Simsek, Mehmet; Demir, Ramazan; Korgun, Emin Turkay

    2012-01-01

    The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown. In the present study, HPECs were cultured 24 h in the presence or absence of 0.5, 5 and 50 µmol · l(-1) of synthetic GC triamcinolone (TA). The glucose carrier proteins GLUT 1, GLUT 3 and GC receptor (GR) were detected in the HPECs. We showed increased expression of GLUT 1 and GLUT 3 proteins and messenger RNA (mRNA) levels (p < 0.05) after 24-h cell culture in the presence of 0.5, 5 and 50 µmol · l(-1) of TA. In contrast, GR protein and mRNA expressions were down-regulated (p < 0.05) with 0.5, 5 and 50 µmol · l(-1) of TA 24-h cell culture. The results demonstrate that GCs are potent regulators of placental GLUT 1 and GLUT 3 expression through GR. Excessive exposure to GCs causes maternal and fetal hypoglycemia and diminished fetal growth. We speculate that to compensate for fetal hypoglycemia and diminished fetal growth, the expression of placental endothelial glucose transporters might be increased.

  13. Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling.

    PubMed

    Chen, Cheng-Yi; Tsai, Chin-Han; Chen, Chia-Yu; Wu, Yi-Hsin; Chen, Chie-Pein

    2016-03-03

    The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.

  14. A proposed study on the transplacental transport of parabens in the human placental perfusion model.

    PubMed

    Mathiesen, Line; Zuri, Giuseppina; Andersen, Maria H; Knudsen, Lisbeth E

    2013-12-01

    Human exposure to parabens as a preservative used in personal care products is of increasing concern, as there is evidence from in vivo and in vitro studies of hormone disruption in association with exposure to parabens. Transport across the placenta could be critical for risk assessment, but the available data are sparse. The aim is to develop a method for estimating fetal exposure, via the placenta, to the most commonly-used parabens, by using a human placental perfusion model. The use of human tissue is vital for determining human fetal exposure, because animal studies are of little relevance, since the placenta exhibits significant interspecies variation. An HPLC model is currently being established to simultaneously quantify four different parabens, namely, methylparaben, ethylparaben, propylparaben and butylparaben, and their main metabolite, p-hydroxybenzoic acid. With this model, we aim to determine the transport kinetics of these parabens across the human placenta, and to investigate placental metabolism, including differences in transport due to molecular characteristics. This will facilitate assessment of the risks associated with the use of paraben-containing products during pregnancy.

  15. Bidirectional placental transfer of Bisphenol A and its main metabolite, Bisphenol A-Glucuronide, in the isolated perfused human placenta.

    PubMed

    Corbel, T; Gayrard, V; Puel, S; Lacroix, M Z; Berrebi, A; Gil, S; Viguié, C; Toutain, P-L; Picard-Hagen, N

    2014-08-01

    The widespread human exposure to Bisphenol A (BPA), an endocrine disruptor interfering with developmental processes, raises the question of the risk for human health of BPA fetal exposure. In humans, highly variable BPA concentrations have been reported in the feto-placental compartment. However the human fetal exposure to BPA still remains unclear. The aim of the study was to characterize placental exchanges of BPA and its main metabolite, Bisphenol A-Glucuronide (BPA-G) using the non-recirculating dual human placental perfusion. This high placental bidirectional permeability to the lipid soluble BPA strongly suggests a transport by passive diffusion in both materno-to-fetal and feto-to-maternal direction, leading to a calculated ratio between fetal and maternal free BPA concentrations of about 1. In contrast, BPA-G has limited placental permeability, particularly in the materno-to-fetal direction. Thus the fetal exposure to BPA conjugates could be explained mainly by its limited capacity to extrude BPA-G.

  16. Structural comparisons of two allelic variants of human placental alkaline phosphatase.

    PubMed

    Millán, J L; Stigbrand, T; Jörnvall, H

    1985-01-01

    A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

  17. The placental transfer of IgG subclasses in human pregnancy.

    PubMed Central

    Pitcher-Wilmott, R W; Hindocha, P; Wood, C B

    1980-01-01

    Total IgG concentrations and the concentrations of the four subclasses of IgG were estimated in thirty-four pairs of maternal and foetal sera from pregnancies of various gestations ranging from 28 to 42 weeks using the method of radial immunodiffusion. It was found that: (1) all subclasses of IgG cross the human placenta freely, (2) foetal levels of IgG and each subclass of IgG exceed maternal levels in full-term pregnancies and (3) there was a close linear relationship between gestational age and the placental transfer of IgG and each of its subclasses. PMID:7438556

  18. Placental Transfer of Darunavir in an Ex Vivo Human Cotyledon Perfusion Model

    PubMed Central

    Duro, Dominique; Belissa, Emilie; Peytavin, Gilles

    2014-01-01

    Placental transfer of the HIV protease inhibitor darunavir was investigated in 5 term human cotyledons perfused with darunavir (1,000 ng/ml) in the maternal to fetal direction. The mean (± the standard deviation [SD]) fetal transfer rate (FTR) (fetal/maternal concentration at steady state from 30 to 90 min) was 15.0% ± 2.1%, and the mean (±SD) clearance index (darunavir FTR/antipyrine FTR) was 40.3% ± 5.8%. This shows that darunavir crosses the placenta at a relatively low rate, resulting in fetal exposure. PMID:24982090

  19. Barium, TEA and sodium sensitive potassium channels are present in the human placental syncytiotrophoblast apical membrane.

    PubMed

    Díaz, P; Vallejos, C; Guerrero, I; Riquelme, G

    2008-10-01

    The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno-fetal exchange. There is ample evidence suggesting the presence of potassium (K(+)) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K(+) currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K(+) channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba(+2)). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba(+2) respectively. The more frequent conductance was approximately 73pS, however several levels of current were detected suggesting the presence of more than one type of K(+) channel. In addition, sodium (Na(+)) sensitivity was detected in the patch current thus, over 10 mmol/L Na(+) reduced the seal current to 38%. These results were corroborated by the total current experiments where the K(+) current elicited in injected oocytes with apical purified membrane was blocked by Ba(+2) and TEA. The total current was also affected by Na(+), becoming larger when a Na(+)-free solution was used. Our results show the existence of at least two types of Ba(+2)-sensitive K(+) channels including a TEA sensitive sub-population, and some of them Na(+) sensitive K(+) channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K(+) channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

  20. 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.

  1. 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.

  2. 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

  3. The role of nitric oxide in the peripheral vasoconstriction caused by human placental lactogen in anaesthetized pigs.

    PubMed

    Molinari, C; Grossini, E; Mary, D A S G; Ribichini, F; Surico, N; Vacca, G

    2006-05-01

    Regional intra-arterial infusion of human placental lactogen in anaesthetized pigs has been shown to cause coronary, renal and iliac vasoconstriction by antagonizing the vasodilatory effects of beta2-adrenergic receptors. Since nitric oxide is known to modulate or mediate beta2-adrenergic effects, the present study was planned in the same experimental model to determine the role of nitric oxide in the above vascular responses to human placental lactogen. In eight pigs anaesthetized with sodium pentobarbitone, changes in anterior descending coronary, left renal and left internal iliac blood flow caused by intra-arterial infusion of human placental lactogen at constant heart rate and arterial blood pressure were assessed using electromagnetic flowmeters. Intra-arterial infusion of the human placental lactogen caused decreases in coronary, renal and iliac blood flow which, respectively, averaged 16.7, 8.1 and 12.2% of the baseline values. The role of nitric oxide in this response was studied in the same pigs by repeating the experiments, after measured blood flows had returned to baseline values, following intra-arterial administration of N(omega)-nitro-L-arginine methyl ester (L-NAME). The subsequent intra-arterial infusion of human placental lactogen did not cause any significant changes in measured blood flows, even when performed after reversing the increase in arterial blood pressure and coronary, renal and iliac resistance caused by L-NAME with continuous intravenous infusion of papaverine. These results indicate that the coronary, renal and iliac vasoconstriction caused by human placental lactogen, known to involve antagonism of beta2-adrenergic vasodilatory effects, was mediated by inhibition of nitric oxide release.

  4. 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.

  5. Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier.

    PubMed

    Noguchi, Saki; Nishimura, Tomohiro; Fujibayashi, Ayasa; Maruyama, Tetsuo; Tomi, Masatoshi; Nakashima, Emi

    2015-09-01

    Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [(3) H]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a Km of 20 μM, and was increased in the absence of chloride. [(3) H]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS.

  6. Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

    PubMed Central

    Aagaard, Kjersti M.; Lahon, Anismrita; Suter, Melissa A.; Arya, Ravi P.; Seferovic, Maxim D.; Vogt, Megan B.; Hu, Min; Stossi, Fabio; Mancini, Michael A.; Harris, R. Alan; Kahr, Maike; Eppes, Catherine; Rac, Martha; Belfort, Michael A.; Park, Chun Shik; Lacorazza, Daniel; Rico-Hesse, Rebecca

    2017-01-01

    Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations. PMID:28128342

  7. Comparative toxicity, oxidative stress and endocrine disruption potential of plasticizers in JEG-3 human placental cells.

    PubMed

    Pérez-Albaladejo, Elisabet; Fernandes, Denise; Lacorte, Silvia; Porte, Cinta

    2017-02-01

    Plasticizers are suspected to be toxic and/or to modulate or disrupt the endocrine system of humans and to cross the placental barrier, being embryonic and fetal development a particularly vulnerable period. This work investigates the comparative toxicity and ability to interfere with the synthesis of steroids and to generate reactive oxygen species (ROS) of a selected number of plasticizers, including bisphenol A (BPA), nonyl- (NP) and octylphenol (OP), benzyl butyl phthalate (BBP), dibutyl phthalate (DBP), di(2-ethylhexyl)phthalate (DEHP) and dimethyl phthalate (DMP), in the human placenta JEG-3 cells. Moreover, the bioavailability of chemicals in culture medium has been investigated. After 24h exposure, OP and NP showed the highest cytotoxicity (EC50: 36-40μM) followed by BPA (138-219μM), whereas no significant toxicity was observed for phthalates. Notwithstanding, BBP and DBP significantly decreased P450 aromatase activity (experimental IC50: 14-15μM), while NP and OP (20μM) increased the activity. Overall, this study evidences the differential toxicity and ability to modulate placental aromatase activity of some of the compounds nowadays used as plasticizers, and highlights the need of an accurate determination of the bioavailability of chemicals to improve the sensitivity of in-vitro tests.

  8. 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

  9. The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria.

    PubMed

    Klimek, J

    1988-01-19

    Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.

  10. 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.

  11. Fc gamma-receptor activity of isolated human placental syncytiotrophoblast plasma membrane.

    PubMed Central

    Brown, P J; Johnson, P M

    1981-01-01

    Fc gamma-receptor activity of isolated human placental syncytiotrophoblast microvillous plasma membrane (StMPM) vesicle preparations has been determined in an immunoradiometric assay using Sepharose-immobilized protein A to separate free 125I-labelled human IgG from membrane-bound 125I-IgG. This receptor assay has been optimalized in terms of buffer pH and molarity, and used to demonstrate that prior 60 min washing of isolated membranes in 3 M KCl to remove extrinsic membrane-bound protein substantially increases the membrane-binding capacity for IgG. Inhibition studies have determined the syncytiotrophoblast Fc gamma-receptor equilibrium constant for association (Ka) as 4.0 x 10(7) M-1 at 37 degrees and the number of available Fc gamma-receptor sites as 1.5 x 10(14) per mg membrane protein. PMID:7461733

  12. Comprehensive genome-wide proteomic analysis of human placental tissue for the Chromosome-Centric Human Proteome Project.

    PubMed

    Lee, Hyoung-Joo; Jeong, Seul-Ki; Na, Keun; Lee, Min Jung; Lee, Sun Hee; Lim, Jong-Sun; Cha, Hyun-Jeong; Cho, Jin-Young; Kwon, Ja-Young; Kim, Hoguen; Song, Si Young; Yoo, Jong Shin; Park, Young Mok; Kim, Hail; Hancock, William S; Paik, Young-Ki

    2013-06-07

    As a starting point of the Chromosome-Centric Human Proteome Project (C-HPP), we established strategies of genome-wide proteomic analysis, including protein identification, quantitation of disease-specific proteins, and assessment of post-translational modifications, using paired human placental tissues from healthy and preeclampsia patients. This analysis resulted in identification of 4239 unique proteins with high confidence (two or more unique peptides with a false discovery rate less than 1%), covering 21% of approximately 20, 059 (Ensembl v69, Oct 2012) human proteins, among which 28 proteins exhibited differentially expressed preeclampsia-specific proteins. When these proteins are assigned to all human chromosomes, the pattern of the newly identified placental protein population is proportional to that of the gene count distribution of each chromosome. We also identified 219 unique N-linked glycopeptides, 592 unique phosphopeptides, and 66 chromosome 13-specific proteins. In particular, protein evidence of 14 genes previously known to be specifically up-regulated in human placenta was verified by mass spectrometry. With respect to the functional implication of these proteins, 38 proteins were found to be involved in regulatory factor biosynthesis or the immune system in the placenta, but the molecular mechanism of these proteins during pregnancy warrants further investigation. As far as we know, this work produced the highest number of proteins identified in the placenta and will be useful for annotating and mapping all proteins encoded in the human genome.

  13. 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

  14. Time- and dose-dependent effects of roundup on human embryonic and placental cells.

    PubMed

    Benachour, N; Sipahutar, H; Moslemi, S; Gasnier, C; Travert, C; Séralini, G E

    2007-07-01

    Roundup is the major herbicide used worldwide, in particular on genetically modified plants that have been designed to tolerate it. We have tested the toxicity and endocrine disruption potential of Roundup (Bioforce on human embryonic 293 and placental-derived JEG3 cells, but also on normal human placenta and equine testis. The cell lines have proven to be suitable to estimate hormonal activity and toxicity of pollutants. The median lethal dose (LD(50)) of Roundup with embryonic cells is 0.3% within 1 h in serum-free medium, and it decreases to reach 0.06% (containing among other compounds 1.27 mM glyphosate) after 72 h in the presence of serum. In these conditions, the embryonic cells appear to be 2-4 times more sensitive than the placental ones. In all instances, Roundup (generally used in agriculture at 1-2%, i.e., with 21-42 mM glyphosate) is more efficient than its active ingredient, glyphosate, suggesting a synergistic effect provoked by the adjuvants present in Roundup. We demonstrated that serum-free cultures, even on a short-term basis (1 h), reveal the xenobiotic impacts that are visible 1-2 days later in serum. We also document at lower non-overtly toxic doses, from 0.01% (with 210 microM glyphosate) in 24 h, that Roundup is an aromatase disruptor. The direct inhibition is temperature-dependent and is confirmed in different tissues and species (cell lines from placenta or embryonic kidney, equine testicular, or human fresh placental extracts). Furthermore, glyphosate acts directly as a partial inactivator on microsomal aromatase, independently of its acidity, and in a dose-dependent manner. The cytotoxic, and potentially endocrine-disrupting effects of Roundup are thus amplified with time. Taken together, these data suggest that Roundup exposure may affect human reproduction and fetal development in case of contamination. Chemical mixtures in formulations appear to be underestimated regarding their toxic or hormonal impact.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

    PubMed Central

    Grafmüller, Stefanie; Manser, Pius; Krug, Harald F.; Wick, Peter; von Mandach, Ursula

    2013-01-01

    Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles. Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results. The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data. PMID:23851364

  20. Determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model.

    PubMed

    Grafmüller, Stefanie; Manser, Pius; Krug, Harald F; Wick, Peter; von Mandach, Ursula

    2013-06-18

    Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ (1). Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 (2) and continuously modified by Schneider et al. in 1972 (3), can serve as an excellent model to study the transfer of xenobiotics or particles. Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results. The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

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

    PubMed Central

    Rosario, Fredrick J.; Shehab, Majida Abu; Powell, Theresa L.; Gupta, Madhulika B.; Jansson, Thomas

    2015-01-01

    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

  2. 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.

  3. Cadmium reduces 11 beta-hydroxysteroid dehydrogenase type 2 activity and expression in human placental trophoblast cells.

    PubMed

    Yang, Kaiping; Julan, Laura; Rubio, Fran; Sharma, Anju; Guan, Haiyan

    2006-01-01

    Cadmium, a common environmental pollutant and a major constituent of tobacco smoke, has been identified as a new class of endocrine disruptors with a wide range of detrimental effects on mammalian reproduction. During human pregnancy, maternal cadmium exposure, via the environment and/or cigarette smoking, leads to fetal growth restriction (FGR), but the underlying mechanisms are unknown. Although a substantial amount of evidence suggests that cadmium may affect fetal growth indirectly via the placenta, the molecular targets remain to be identified. Given that reduced placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2, encoded by HSD11B2 gene) is causally linked to FGR, the present study was undertaken to examine the hypothesis that cadmium induces FGR in part by targeting placental HSD11B2. Using cultured human trophoblast cells as a model system, we showed that cadmium exposure resulted in a time- and concentration-dependent decrease in 11 beta-HSD2 activity, such that an 80% reduction was observed after 24-h treatment at 1 microM. It also led to a similar decrease in levels of 11 beta-HSD2 protein and mRNA, suggesting that cadmium reduced 11 beta-HSD2 expression. Furthermore, cadmium diminished HSD11B2 promoter activity, indicative of repression of HSD11B2 gene transcription. In addition, the effect of cadmium was highly specific, in that other divalent metals (Zn(2+), Mg(2+), and Mn(2+)) as well as nicotine and cotinine (a major metabolite of nicotine) did not alter 11 beta-HSD2 activity. Taken together, these findings demonstrate that cadmium reduces human placental 11 beta-HSD2 expression and activity by suppressing HSD11B2 gene transcription. Thus the present study identifies placental 11 beta-HSD2 as a novel molecular target of cadmium. It also reveals a molecular mechanism by which this endocrine disruptor may affect human placental function and, consequently, fetal growth and development.

  4. The effect of opiates on the activity of human placental aromatase/CYP19.

    PubMed

    Zharikova, Olga L; Deshmukh, Sujal V; Kumar, Meena; Vargas, Ricardo; Nanovskaya, Tatiana N; Hankins, Gary D V; Ahmed, Mahmoud S

    2007-01-15

    Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiates L-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E(2) and E(3) formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E(3) formation was more pronounced than that on E(2) due to the lower affinity of 16-OHT than testosterone to aromatase. The K(i) values for the opiates that inhibited E(3) formation were sufentanil, 7 +/- 1 microM; LAAM, 13 +/- 8 microM; fentanyl, 25 +/- 5 microM; oxycodone, 92 +/- 22 microM; codeine, 218 +/- 69 microM; (+)-pentazocine, 225 +/- 73 microM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (-)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E(3) formation by 124-160% of control but had no effect on E(2) formation. Moreover, oxycodone and codeine did not inhibit E(2) formation and the IC(50) values for fentanyl, sufentanil, and (+)-pentazocine were >1000 microM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

  5. 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.

  6. 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

  7. 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

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

    PubMed

    Cantón, Rocío F; Scholten, Deborah E A; Marsh, Göran; de Jong, Paul C; van den Berg, Martin

    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(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(i)/K(i)' of 7.68/0,02 microM and 5.01/0.04 microM 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 that a

  9. 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

  10. 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.

  11. 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.

  12. Human placental transfer of perfluoroalkyl acid precursors: Levels and profiles in paired maternal and cord serum.

    PubMed

    Yang, Lin; Wang, Zhen; Shi, Yu; Li, Jingguang; Wang, Yuxin; Zhao, Yunfeng; Wu, Yongning; Cai, Zongwei

    2016-02-01

    Perfluoroalkyl acids (PFAAs) precursors, the indirect source of PFAA exposure, have been observed in environmental and human samples. However, the maternal-fetal transfer of these chemicals has not been well examined. In this study, 50 paired maternal and cord serum samples collected in Jiangsu province of China were analyzed for fifteen PFAA precursors. Among the detected PFAAs, 6:2 fluorotelomer sulfonate (6:2 FTS), N-methyl- and N-ethyl-perfluorooctanesulfonamidoacetates had comparable detection rate in both maternal and cord sera, while the mean concentrations and detection rates of 8:2 FTS and perfluorooctane sulfonamide (PFOSA) were higher in maternal sera compared to cord sera (Mann-Whitney U test, P < 0.05). Analysis of variance and least significant difference tests showed that the youngest maternal age group (21-24 years old) had the highest concentration of 6:2 FTS in cord sera. Maternal serum PFOSA was found significantly correlated with the cord serum perfluorooctanesulfonate (PFOS) (Spearman test, r = 0.361, P = 0.010), indicating that maternal serum PFOSA might be an indirect source of PFOS in fetuses. The obtained results suggested the potential prenatal exposure and human placental transfer of perfluoroalkyl acid precursors.

  13. Phylogenetic Origin of Human Chromosomes 7, 16, and 19 and their Homologs in Placental Mammals

    PubMed Central

    Richard, Florence; Lombard, Martine; Dutrillaux, Bernard

    2000-01-01

    The origin of human chromosomes (HSA) 7, 16, and 19 was studied by comparing data obtained from chromosome banding, chromosome painting, and gene mapping in species belonging to 11 orders of placental mammals (Eutherians). This allowed us to propose the reconstruction of their presumed ancestral forms. The HSA7 homologs were composed of two parts, the largest forming an acrocentric. The smallest formed one arm of a small submetacentric; the other arm was composed of sequences homologous to the short arm of HSA16 (HSA16p). The sequences homologous to the long arm of HSA16 (HSA16q) were associated with sequences homologous to the long arm of HSA19 (HSA19q) and formed another submetacentric. From their origin, these chromosomes underwent the following rearrangements to give rise to current human chromosomes: centromeric fission of the two submetacentrics in ancestors of all primates (∼80 million years ago); fusion of the HSA19p and HSA19q sequences, originating the current HSA19, in ancestors of all simians (∼55 million years ago); fusions of the HSA16p and HSA16q sequences, originating the current HSA16 and the two components of HSA7 before the separation of Cercopithecoids and Hominoids (∼35 million years ago); and finally, pericentric and paracentric inversions of the homologs to HSA7 after the divergence of orangutan and gorilla, respectively. Thus, compared with HSA16 and HSA19, HSA7 is a fairly recent chromosome shared by man and chimpanzee only. PMID:10810086

  14. Gas chromatography-mass spectrometric study of 19-oxygenation of the aromatase inhibitor 19-methylandrostenedione with human placental microsomes.

    PubMed

    Numazawa, Mitsuteru; Nagaoka, Masao; Handa, Wakako; Yamada, Akane

    2006-06-01

    To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.

  15. 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

  16. Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas.

    PubMed

    Bassetti, M; Brina, M; Spada, A; Giannattasio, G

    1989-11-01

    A morphological study has been carried out on 20 GH-secreting adenomas removed from acromegalic normoprolactinemic patients, on 29 PRL-secreting adenomas removed from hyperprolactinemic patients without signs of acromegaly and on one normal human anterior pituitary gland collected at autopsy. The protein A-gold immunoelectron microscopic technique has been utilized in order to verify the presence of mixed cells producing both GH and PRL (somatomammotrophs) in these pituitary tissues. In the normal pituitary a considerable number of somatomammotrophs (15-20%) was found, thus supporting the idea that these cells are normal components of the human anterior pituitary gland. In 10 GH-secreting adenomas and in 10 PRL-secreting adenomas somatomammotrophs were present in a variable number (from 4 to 20% of the whole cell population in GH adenomas and from 1 to 47% in PRL tumors). It can be concluded therefore that these cells, largely present in all GH/PRL-secreting adenomas, can also be found in GH-secreting and PRL-secreting tumors without clinical evidence of a mixed secretion. Adenomatous somatomammotrophs displayed ultrastructural features of adenomatous somatotrophs and mammotrophs (prominent Golgi complexes, abundant rough endoplasmic reticulum, irregular nuclei). The size and the number of granules were variable. In some cells GH and PRL were stored in distinct secretory granules, in others in mixed granules or both in mixed and distinct granules, thus suggesting that in adenomatous somatomammotrophs the efficiency of the mechanisms of sorting of the two hormones varies from one cell to another.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. In vitro transport mechanism of psoralen in a human placental cell line (BeWo cells).

    PubMed

    Guo, Jie; Song, Dianrong; Han, Fang; Zhang, Wei; Wang, Yanan; Wang, Yuhua; Du, Wenxin

    2015-01-01

    The mechanism by which psoralen is transported across the placenta was investigated in the BeWo human placental cell line derived from choriocarcinoma in a transwell assay system using liquid chromatography-mass spectrometry/mass spectrometry detection. Psoralen uptake by BeWo cells increased linearly over the concentration range of 0.01 µM to 100 µM (r (2) = 0.997) and was not saturable. Psoralen uptake by BeWo cells was not affected by temperature (4 °C, room temperature, and 37 °C; p > 0.05). Psoralen transport increased linearly over 180 min (r (2) = 0.988) with 3.08 ± 0.26 %, 5.47 ± 0.21 %, 7.54 ± 0.06 %, 9.40 ± 0.37 %, 11.49 ± 0.31 %, and 12.46 ± 0.61 % transferred from the apical chamber to the basolateral chamber in the transwell assays at 30, 60, 90, 120, 150, and 180 min, respectively. The rate of transport showed the same tendency, increasing linearly from 0.13 ± 0.01 pmol/s to 0.58 ± 0.03 pmol/s over the concentration range of 25 µM to 100 µM (r (2) = 0.989). The apparent permeability coefficient for psoralen (100 µM) was 5.62 ± 0.24 × 10(-6) cm/s and 5.53 ± 0.47 × 10(-6) cm/s before and after treatment with verapamil (100 µM), respectively (p > 0.05). The efflux value for psoralen was approximately 1. These data show that psoralen is well absorbed and crosses the placental barrier via passive diffusion in the BeWo cell line.

  18. The Placental Variant of Human Growth Hormone Reduces Maternal Insulin Sensitivity in a Dose-Dependent Manner in C57BL/6J Mice.

    PubMed

    Liao, Shutan; Vickers, Mark H; Stanley, Joanna L; Ponnampalam, Anna P; Baker, Philip N; Perry, Jo K

    2016-03-01

    The human placental GH variant (GH-V) is secreted continuously from the syncytiotrophoblast layer of the placenta during pregnancy and is thought to play a key role in the maternal adaptation to pregnancy. Maternal GH-V concentrations are closely related to fetal growth in humans. GH-V has also been proposed as a potential candidate to mediate insulin resistance observed later in pregnancy. To determine the effect of maternal GH-V administration on maternal and fetal growth and metabolic outcomes during pregnancy, we examined the dose-response relationship for GH-V administration in a mouse model of normal pregnancy. Pregnant C57BL/6J mice were randomized to receive vehicle or GH-V (0.25, 1, 2, or 5 mg/kg · d) by osmotic pump from gestational days 12.5 to 18.5. Fetal linear growth was slightly reduced in the 5 mg/kg dose compared with vehicle and the 0.25 mg/kg groups, respectively, whereas placental weight was not affected. GH-V treatment did not affect maternal body weights or food intake. However, treatment with 5 mg/kg · d significantly increased maternal fasting plasma insulin concentrations with impaired insulin sensitivity observed at day 18.5 as assessed by homeostasis model assessment. At 5 mg/kg · d, there was also an increase in maternal hepatic GH receptor/binding protein (Ghr/Ghbp) and IGF binding protein 3 (Igfbp3) mRNA levels, but GH-V did not alter maternal plasma IGF-1 concentrations or hepatic Igf-1 mRNA expression. Our findings suggest that at higher doses, GH-V treatment can cause hyperinsulinemia and is a likely mediator of the insulin resistance associated with late pregnancy.

  19. Purification and properties of molecular-weight variants of human placental alkaline phosphatase

    PubMed Central

    Ghosh, Nimai K.; Fishman, William H.

    1968-01-01

    1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8·6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4·2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined. ImagesFig. 3.Fig. 5.Fig. 7.Fig. 8.Fig. 9. PMID:4970595

  20. Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.

    PubMed Central

    Lee, F S; Vallee, B L

    1990-01-01

    Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures. Images PMID:2408043

  1. 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.

  2. 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.

  3. Membrane-active antimicrobial peptides and human placental lysosomal extracts are highly active against mycobacteria.

    PubMed

    Jena, Prajna; Mishra, Bibhuti; Leippe, Matthias; Hasilik, Andrej; Griffiths, Gareth; Sonawane, Avinash

    2011-05-01

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manifests discreet strategies to subvert host immune responses, which enable the pathogen to survive and multiply inside the macrophages. This problem is further worsened by the emergence of multidrug resistant mycobacterial strains, which make most of the anti-tuberculous drugs ineffective. It is thus imperative to search for and design better therapeutic strategies, including employment of new antibiotics. Recently, naturally produced antimicrobial molecules such as enzymes, peptides and their synthetic analogs have emerged as compounds with potentially significant therapeutical applications. Although, many antimicrobial peptides have been identified only very few of them have been tested against mycobacteria. A major limitation in using peptides as therapeutics is their sensitivity to enzymatic degradation or inactivity under certain physiological conditions such as relatively high salt concentration. Here, we show that NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, and Ci-MAM-A24, a synthetic salt-tolerant peptide derived from immune cells of Ciona intestinalis, efficiently kill Mycobacterium smegmatis and Mycobacterium bovis-BCG. In addition, NK-2 and Ci-MAM-A24 showed a synergistic killing effect against M. smegmatis, no cytotoxic effect on mouse macrophages at bactericidal concentrations, and were even found to kill mycobacteria residing inside the macrophages. We also show that human placental lysosomal contents exert potent killing effect against mycobacteria under acidic and reducing growth conditions. Electron microscopic studies demonstrate that the lysosomal extract disintegrate bacterial cell membrane resulting in killing of mycobacteria.

  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.

  5. 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

  6. 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.

  7. Monocarboxylate transporter 8 modulates the viability and invasive capacity of human placental cells and fetoplacental growth in mice.

    PubMed

    Vasilopoulou, Elisavet; Loubière, Laurence S; Heuer, Heike; Trajkovic-Arsic, Marija; Darras, Veerle M; Visser, Theo J; Lash, Gendie E; Whitley, Guy S; McCabe, Christopher J; Franklyn, Jayne A; Kilby, Mark D; Chan, Shiao Y

    2013-01-01

    Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.

  8. Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

    PubMed Central

    Vasilopoulou, Elisavet; Loubière, Laurence S.; Heuer, Heike; Trajkovic-Arsic, Marija; Darras, Veerle M.; Visser, Theo J.; Lash, Gendie E.; Whitley, Guy S.; McCabe, Christopher J.; Franklyn, Jayne A.; Kilby, Mark D.; Chan, Shiao Y.

    2013-01-01

    Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo. PMID

  9. Production of interferons in human placental trophoblast subpopulations and their possible roles in pregnancy.

    PubMed Central

    Aboagye-Mathiesen, G; Tóth, F D; Zdravkovic, M; Ebbesen, P

    1994-01-01

    The human cytotrophoblasts are the first fetal cells to arise during embryogenesis and are the progenitor cells to villous (noninvasive), syncytiotrophoblast (noninvasive), "intermediate" extravillous (invasive), and "anchoring" extravillous (invasive) trophoblast subpopulations. These trophoblast subpopulations were isolated from first- and third-trimester placentae and were stimulated with Sendai virus, granulocyte-macrophage colony-stimulating factors (GM-CSF), and platelet-derived growth factor (PDGF) to produce interferons (IFNs). GM-CSF and PDGF induced very low levels of IFN in first-trimester extravillous and villous trophoblast subpopulations. Highly proliferating and invasive intermediate extravillous trophoblast cultures produced five- to eightfold more IFNs than villous trophoblast cultures and two- to fivefold more IFN than the syncytiotrophoblast cultures when stimulated with Sendai virus. Syncytiotrophoblast cultures produced higher levels of IFNs (up to twofold) than villous trophoblast cultures when stimulated with the same virus. Pretreatment of first-trimester extravillous and villous trophoblast cultures with GM-CSF and PDGF followed by infection with Sendai virus resulted in greater IFN production than when the cultures were stimulated with virus alone. The levels of IFN produced were dependent on the type of trophoblast, the type of inducer, and the stage of differentiation of the trophoblasts. The purified trophoblast IFNs have potent antiviral activities when assayed on human amniotic WISH cells, and they inhibited proliferation of normal trophoblasts and trophoblast-derived malignant cells in vitro without any toxicity. Furthermore, the trophoblast IFNs activated NK cell activity and suppressed mitogen-stimulated lymphocyte proliferation at concentrations of between 10 and 1,000 IU/ml. The possible functions of the trophoblast IFNs during pregnancy are discussed with respect to human placental and fetal protection and development. Images

  10. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

  11. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus.

    PubMed

    Stenqvist, Ann-Christin; Nagaeva, Olga; Baranov, Vladimir; Mincheva-Nilsson, Lucia

    2013-12-01

    Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability.

  12. Comparative N-Glycoproteomic and Phosphoproteomic Profiling of Human Placental Plasma Membrane between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

    PubMed Central

    Wang, Fuqiang; Wang, Ling; Shi, Zhonghua; Liang, Gaolin

    2013-01-01

    Preeclampsia is a serious complication of pregnancy, which affects 2–8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane. PMID:24260401

  13. In vitro studies of ferric carboxymaltose on placental permeability using the dual perfusion model of human placenta.

    PubMed

    Malek, Antoine

    2010-01-01

    An in vitro perfusion model of human placenta was used to study the transplacental passage of iron applied in the form of the drug compound ferric carboxymaltose (FCM) which had been radio-labelled with 59Fe. In four placental perfusion experiments, two simulated circuits for the maternal and fetal sides of the placenta were set up with two experimental phases each lasting 3 h. FCM was added to the maternal circuit at the beginning of each phase to a final iron concentration of 11 mM, which is at least 10 times higher than the maximal predicted level in blood after an administration of 200 mg iron as FCM. The effects of adding transferrin at a physiological concentration of 1.67 mg/ ml were also tested. The concentration profiles of 59Fe showed a 10% decrease within the first 30 min of perfusion on the maternal side. Thereafter the radioactivity levels remained unchanged. The addition of transferrin had no effect on the tissue uptake of 59Fe-FCM. No transferred iron radioactivity could be detected in the fetal circuit. Despite a loss of approximately 10% of the radio-labelled iron observed on the maternal side, only 0.5-2% of the radioactivity was detected in the placental tissue after perfusion. No free iron could be detected at the end of perfusion on the maternal side using ultrafiltration or acid precipitation methods. In addition, the production of transferrin receptor remained unchanged, with similar concentrations in placental tissue before and after perfusion. No effects of FCM on placental viability were observed in terms of energy metabolism (glucose consumption and lactate production), hormone release or placental permeability (assessed by the transfer rates of creatinine and antipyrine). However, two additional observations were made: firstly, a significant reduction in the rate of cell death compared to control conditions was observed in the presence of FCM; secondly, the integrity of the fetal capillary system was improved on the fetal side of the

  14. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells.

    PubMed

    Benachour, Nora; Séralini, Gilles-Eric

    2009-01-01

    We have evaluated the toxicity of four glyphosate (G)-based herbicides in Roundup formulations, from 10(5) times dilutions, on three different human cell types. This dilution level is far below agricultural recommendations and corresponds to low levels of residues in food or feed. The formulations have been compared to G alone and with its main metabolite AMPA or with one known adjuvant of R formulations, POEA. HUVEC primary neonate umbilical cord vein cells have been tested with 293 embryonic kidney and JEG3 placental cell lines. All R formulations cause total cell death within 24 h, through an inhibition of the mitochondrial succinate dehydrogenase activity, and necrosis, by release of cytosolic adenylate kinase measuring membrane damage. They also induce apoptosis via activation of enzymatic caspases 3/7 activity. This is confirmed by characteristic DNA fragmentation, nuclear shrinkage (pyknosis), and nuclear fragmentation (karyorrhexis), which is demonstrated by DAPI in apoptotic round cells. G provokes only apoptosis, and HUVEC are 100 times more sensitive overall at this level. The deleterious effects are not proportional to G concentrations but rather depend on the nature of the adjuvants. AMPA and POEA separately and synergistically damage cell membranes like R but at different concentrations. Their mixtures are generally even more harmful with G. In conclusion, the R adjuvants like POEA change human cell permeability and amplify toxicity induced already by G, through apoptosis and necrosis. The real threshold of G toxicity must take into account the presence of adjuvants but also G metabolism and time-amplified effects or bioaccumulation. This should be discussed when analyzing the in vivo toxic actions of R. This work clearly confirms that the adjuvants in Roundup formulations are not inert. Moreover, the proprietary mixtures available on the market could cause cell damage and even death around residual levels to be expected, especially in food and feed

  15. Clinical use of placental hormones in pregnancy management.

    PubMed

    De Bonis, M; Vellucci, F L; Di Tommaso, M; Voltolini, C; Torricelli, M; Petraglia, F

    2012-09-01

    Across human pregnancy, placenta represents a transit of oxygen and nutrients from the mother to the fetus and actively produces a large number of hormones that serve to regulate and balance maternal and fetal physiology. An abnormal secretion of placental hormones may be part of the pathogenesis of the main obstetric syndrome, from early to late pregnancy, in particular chromosomopathies, miscarriage, gestational trophoblastic diseases, preeclampsia, gestational diabetes, and pre-term delivery. The possibility to measure placental hormones represents an important tool not only for the diagnosis and management of gestational disorders, but it is also fundamental in the early identification of women at risk for these pregnancy complications. In the last decades, the use of ultrasound examination has provided additional biophysical markers, improving the early diagnosis of gestational diseases. In conclusion, while few placental hormones have sufficient sensitivity for clinical application, there are promising new biochemical and biophysical markers that, if used in combination, may provide a valid screening tool.

  16. Placental hypoxia during placental malaria

    PubMed Central

    Boeuf, Philippe; Tan, Aimee; Romagosa, Cleofe; Radford, Jane; Mwapasa, Victor; Molyneux, Malcolm E.; Meshnick, Steven R.; Hunt, Nicholas H.; Rogerson, Stephen J.

    2009-01-01

    Background Placental malaria causes fetal growth retardation (FGR), which has been linked epidemiologically to placental monocyte infiltrates. We investigated whether parasite or monocyte infiltrates were associated with placental hypoxia, as a potential mechanism underlying malarial FGR. Methods We studied the hypoxia markers hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), placental growth factor, VEGF receptor 1 and its soluble form and VEGF receptor 2. We used real time PCR (in 59 women) to examine gene transcription, immunohistochemistry (in 30 women) to describe protein expression and laser capture microdissection (in 23 women) to examine syncytiotrophoblast-specific changes in gene expression. We compared gene and protein expression in relation to malaria infection, monocytes infiltrates and birth weight. Results we could not associate any hallmark of placental malaria with a transcription, expression or tissue distribution profile characteristic of a response to hypoxia but found higher HIF-1α (P=.0005) and lower VEGF levels (P=.0026) in the syncytiotrophoblast of malaria cases versus asymptomatic controls. Conclusion our data are inconsistent with a role for placental hypoxia in the pathogenesis of malaria-associated FGR. The laser capture microdissection study was small, but suggests that malaria affects syncytiotrophoblast gene transcription, and proposes novel potential mechanisms for placental malaria-associated FGR. PMID:18279052

  17. Human Placental Alkaline Phosphatase as a Tracking Marker for Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Balmayor, Elizabeth Rosado; Flicker, Magdalena; Käser, Tobias; Saalmüller, Armin

    2013-01-01

    Abstract Currently, adult mesenchymal stem cells (MSCs) are being evaluated for a wide variety of therapeutic approaches. It has been suggested that MSCs possess regenerative properties when implanted or injected into damaged tissue. However, the efficacy of MSCs in several of the proposed treatments is still controversial. To further explore the therapeutic potential of these cells, it is necessary to trace the fate of individual donor or manipulated cells in the host organism. Recent studies from our lab showed that human placental alkaline phosphatase (hPLAP) is a marker with great potential for cell tracking. However, a potential concern related to this marker is its enzymatic activity, which might alter cell behavior and differentiation by hydrolyzing substrates in the extracellular space and thereby changing the cellular microenvironment. Therefore, the aim of this study was to characterize bone marrow MSCs (BMSCs) derived from hPLAP-transgenic inbred F344 rats (hPLAP-tg) in comparison to wild type (wt) BMSCs. Here, we show that BMSCs from wt and hPLAP-tg donors are indistinguishable in terms of cell morphology, viability, adhesion, immune phenotype, and proliferation as well as in their differentiation capacity over six passages. The expression of the hPLAP marker enzyme was not impaired by extensive in vitro cultivation, osteogenic, adipogenic, or chondrogenic differentiation, or seeding onto two- or three-dimensional biomaterials. Thus, our study underscores the utility of genetically labeled BMSCs isolated from hPLAP-tg donors for long-term tracking of the fate of transplanted MSCs in regenerative therapies. PMID:24083090

  18. Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

    PubMed Central

    Hung, H C; Chang, G G

    2001-01-01

    Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally

  19. Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

    PubMed

    Hung, H C; Chang, G G

    2001-12-01

    Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally

  20. Effect of sulglycotide on gastric bicarbonate secretion in humans.

    PubMed

    Guslandi, M; Nannini, D; Tittobello, A

    1985-01-01

    The effect of sulglycotide, a cytoprotective agent with a healing effect on ulcers, on gastric bicarbonate secretion in humans was evaluated. Fifteen healthy volunteers were treated with sulglycotide 400 mg t.i.d. for 10 days. Before and after treatment the bicarbonate content of basal gastric juice was determined by Feldman and Barnett's method. Sulglycotide was found to increase significantly (p less than 0.0001) basal HCO3- production from the human stomach, thus strengthening the gastric mucosal defences. It was concluded that the cytoprotective and therapeutic properties of the drug are partially related to stimulation of gastric alkaline secretion.

  1. 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

  2. Cobalt ions induce chemokine secretion in primary human osteoblasts.

    PubMed

    Queally, J M; Devitt, B M; Butler, J S; Malizia, A P; Murray, D; Doran, P P; O'Byrne, J M

    2009-07-01

    Chemokines are major regulators of the inflammatory response and have been shown to play an important role in periprosthetic osteolysis. Titanium particles have previously been shown to induce IL-8 and MCP-1 secretion in osteoblasts. These chemokines result in the chemotaxis and activation of neutrophils and macrophages, respectively. Despite a resurgence in the use of cobalt-chromium-molybdenum alloys in metal-on-metal arthroplasty, cobalt and chromium ion toxicity in the periprosthetic area has been insufficiently studied. In this study we investigate the in vitro effect of cobalt ions on primary human osteoblast activity. We demonstrate that cobalt ions rapidly induce the protein secretion of IL-8 and MCP-1 in primary human osteoblasts. This elevated chemokine secretion is preceded by an increase in the transcription of the corresponding chemokine gene. Using a Transwell migration chemotaxis assay we also demonstrate that the chemokines secreted are capable of inducing neutrophil and macrophage migration. Furthermore, cobalt ions significantly inhibit osteoblast function as demonstrated by reduced alkaline phosphatase activity and calcium deposition. In aggregate these data demonstrate that cobalt ions can activate transcription of the chemokine genes IL-8 and MCP-1 in primary human osteoblasts. Cobalt ions are not benign and may play an important role in the pathogenesis of osteolysis by suppressing osteoblast function and stimulating the production and secretion of chemokines that attract inflammatory and osteoclastic cells to the periprosthetic area.

  3. Placental Vitamin D-Binding Protein Expression in Human Idiopathic Fetal Growth Restriction

    PubMed Central

    Wookey, Alice F.; Chollangi, Tejasvy; Yong, Hannah E. J.

    2017-01-01

    Vitamin D-binding protein is a multifunctional serum protein with multiple actions related to normal health. Vitamin D-binding protein transports vitamin D and influences the metabolism of this key hormone but it also has additional immunomodulatory and actin-clearing properties. We investigated whether vitamin D-binding protein expression is altered in fetal growth restriction-associated placental dysfunction. Protein was extracted from 35 placentae derived from 17 healthy control subjects and 18 gestation-matched subjects with fetal growth restriction (FGR). FGR subjects were further subdivided as idiopathic (n = 9) and nonidiopathic (n = 9). Vitamin D-binding protein and 25(OH) vitamin D were measured by ELISA and normalized to protein concentration. The results showed significantly reduced levels of placental vitamin D-binding protein (control versus FGR, p < 0.05, Student's t-test) that were strongly associated with idiopathic fetal growth restriction (p < 0.01, Kruskal-Wallis), whereas levels of vitamin D-binding protein were not associated with placental 25(OH) vitamin D stores (p = 0.295, Pearson's correlation). As such, vitamin D-binding protein may be a factor in unexplained placental dysfunction associated with idiopathic fetal growth restriction and may potentially serve as a biomarker of this disease. PMID:28293436

  4. 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

  5. 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...

  6. 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.

  7. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  8. Ontological Differences in First Compared to Third Trimester Human Fetal Placental Chorionic Stem Cells

    PubMed Central

    Jones, Gemma N.; Moschidou, Dafni; Puga-Iglesias, Tamara-Isabel; Kuleszewicz, Katarzyna; Vanleene, Maximilien; Shefelbine, Sandra J.; Bou-Gharios, George; Fisk, Nicholas M.; David, Anna L.; De Coppi, Paolo; Guillot, Pascale V.

    2012-01-01

    Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous. PMID:22962584

  9. Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro.

    PubMed

    LaMorte, A; Kumar, N; Bardin, C W; Sundaram, K

    1994-02-01

    Part of the biological effects of testosterone (T) are mediated by its enzymatic reduction to 5 alpha-dihydrotestosterone (DHT) or aromatization to estradiol (E2). 7 alpha-Methyl-19-nortestosterone (MENT) is a synthetic androgen that is considerably more potent than T. Previous studies have shown that MENT is not 5 alpha-reduced. The studies reported here were undertaken to determine whether MENT undergoes enzymatic aromatization in vitro. Human placental microsomes were used as the source of the aromatase. Radioactive or nonradioactive T or MENT was incubated with the microsomes in the presence of NADPH and the metabolites extracted out with ethyl ether. Following evaporation of ether, the residue was dissolved in benzene-petroleum ether and extracted with 0.4 N NaOH which selectively removes phenolic metabolites of the androgens. When either radioactive T or MENT was incubated with the aromatase in the presence of NADPH, there was a 20-fold increase in the amount of radioactivity extracted with NaOH. In contrast, if the incubation was carried out in the absence of NADPH or in the presence of R76713, an aromatase inhibitor, most of the radioactivity remained in the benzene-petroleum ether phase. To further identify the enzymatic reaction products, thin layer chromatography (TLC) was performed. The Rf value for MENT was 0.22 while that of the major reaction product was 0.34, which corresponded with the RF value of the estrogen, 7 alpha-methyl-estradiol (MeE2). This was further verified by using a second solvent system for the chromatographic separation. In an effort to ascertain whether the metabolites bind to estrogen receptors (ER), rat uterine cytosol was used. NaOH extracts of medium following incubation of nonradioactive MENT with microsomes showed competitive inhibition of [3H]E2 binding to rat uterine ER. Furthermore, after [3H]MENT was incubated with microsomes, the radioactive metabolite extracted in NaOH showed specific binding to the ER which could

  10. Secretion of human interferon alpha 2b by Streptomyces lividans.

    PubMed

    Pimienta, E; Fando, R; Sánchez, J C; Vallin, C

    2002-02-01

    Biologically active human interferon alpha 2b (HuIFNalpha-2b) was secreted into the culture medium by Streptomyces lividans transformed with recombinant plasmids coding for HuIFNalpha-2b fused to the Streptomyces exfoliatus M11 lipase A signal sequence. Levels were low, 15 or 100 ng/ml, depending on the plasmid used. Neither processed nor unprocessed HuIFNalpha-2b was detected in cell lysates of the transformants secreting the recombinant product. However, the secreted recombinant product was found to partially degrade when cultures reached the stationary phase by the action of an, as yet, unidentified mycelium-associated factor. Experimental evidence suggests that the degrading factor is related to mycelium-associated proteolytic activity.

  11. Human placental insulin binding in normal and well-controlled diabetic patients.

    PubMed

    Nelson, D M; Ortman-Nabi, J; Curran, E M

    1990-01-01

    Previous studies of insulin binding to placentas of both insulin-dependent and untreated gestational diabetic patients have described placentas from diabetics to contain fewer insulin receptors than placentas from nondiabetic gravidas. However, these studies were done using membrane fractions prepared from the placentas and at a time when adequacy of antepartum glycemic control in the diabetic patients was not routinely evaluated by self blood sugar measurement or hemoglobin A1 assay. The current study compares specific 125I-insulin binding in vitro to intact placental villi from 15 normal patients with insulin binding to intact villi obtained from 15 insulin-dependent diabetic mothers whose fasting and postprandial blood sugars and hemoglobin A1 levels were maintained in a range normal for term pregnancy. We demonstrate that insulin binding to intact placental villi is the same in this group of diabetic patients as in the nondiabetic patients.

  12. Role of human placental apical membrane transporters in the efflux of glyburide, rosiglitazone, and metformin

    PubMed Central

    HEMAUER, Sarah J.; PATRIKEEVA, Svetlana L.; NANOVSKAYA, Tatiana N.; HANKINS, Gary D.V.; AHMED, Mahmoud S.

    2010-01-01

    Objective Substrates of placental efflux transporters could compete for a single transporter, which could result in an increase in the transfer of each substrate to the fetal circulation. Our aim was to determine the role of placental transporters in the biodisposition of oral hypoglycemic drugs that could be used as monotherapy or in combination therapy for gestational diabetes. Study design Inside-out brush border membrane vesicles from term placentas were used to determine the efflux of glyburide, rosiglitazone, and metformin by P-gp, Breast Cancer Resistance Protein (BCRP), and Multidrug Resistance Protein (MRP1). Results Glyburide was transported by MRP1 (43 ± 4%); BCRP (25 ± 5%); and P-gp (9 ± 5%). Rosiglitazone was transported predominantly by P-gp (71 ± 26%). Metformin was transported by P-gp (58 ± 20%) and BCRP (25 ± 14%). Conclusion Multiple placental transporters contribute to efflux of glyburide, rosiglitazone, and metformin. Administration of drug combinations could lead to their competition for efflux transporters. PMID:20350646

  13. 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

  14. A new liquid chromatography-tandem mass spectrometry method for determination of parabens in human placental tissue samples.

    PubMed

    Jiménez-Díaz, I; Vela-Soria, F; Zafra-Gómez, A; Navalón, A; Ballesteros, O; Navea, N; Fernández, M F; Olea, N; Vílchez, J L

    2011-05-15

    Endocrine disruptors are a group of organic compounds widely used, which are ubiquitous in the environment and in biological samples. The main effect of these compounds is associated with their ability to mimic or block the action of natural hormones in living organisms, including humans. Parabens (esters of p-hydroxybenzoic acid) belong to this group of compounds. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to asses the presence of parabens most commonly used in industrial applications (methyl-, ethyl-, propyl- and butyl-paraben) in samples of human placental tissue. The method involves the extraction of the analytes from the samples using ethyl acetate, followed by a clean-up step using centrifugation prior to their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative mode. Deuterated bisphenol A (BPA-d(16)) was used as surrogate. Found detection limits (LOD) ranged from 0.03 to 0.06 ng g(-1) and quantification limits (LOQ) from 0.1 to 0.2 ng g(-1), while inter- and intra-day variability was under 13.8%. The method was validated using standard addition calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 82% to 108%. This method was satisfactorily applied for the determination of parabens in 50 placental tissue samples collected from women who live in the province of Granada (Spain).

  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.

    PubMed

    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.

  17. 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

  18. 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.

  19. 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.

  20. Placental insufficiency

    MedlinePlus

    ... other drugs Certain medicines can also increase the risk of placental insufficiency. In some cases, the placenta: May have an abnormal shape May not grow big enough (more likely if you are carrying twins or other multiples) Does not attach correctly to ...

  1. Placental Transporter Localization and Expression in the Human: the importance of species, sex and gestational age differences1.

    PubMed

    Walker, Natasha; Filis, Panagiotis; Soffientini, Ugo; Bellingham, Michelle; O'Shaughnessy, Peter J; Fowler, Paul A

    2017-03-07

    The placenta is a critical organ during pregnancy, essential for the provision of an optimal intrauterine environment, with fetal survival, growth and development relying on correct placental function. It must allow nutritional compounds and relevant hormones to pass into the fetal bloodstream and metabolic waste products to be cleared. It also acts as a semi-permeable barrier to potentially harmful chemicals both endogenous and exogenous. Transporter proteins allow for bidirectional transport and are found in the syncytiotrophoblast of the placenta and endothelium of fetal capillaries. The major transporter families in the human placenta are ABC and SLC and insufficiency of these transporters may lead to deleterious effects on the fetus. Transporter expression levels are gestation-dependent and this is of considerable clinical interest as levels of drug resistance may be altered from one trimester to the next. This highlights the importance of these transporters in mediating correct and timely transplacental passage of essential compounds but also for efflux of potentially toxic drugs and xenobiotics. We review the current literature on placental molecular transporters with respect to their localization and ontogeny, the influence of fetal sex and the relevance of animal models. We conclude that a paucity of information exists and further studies are required to unlock the enigma of this dynamic organ.

  2. 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

  3. 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.

  4. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

    SciTech Connect

    Fisher, G.J.; Kelley, L.K.; Smith, C.H.

    1987-01-01

    As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na/sup +/ or K/sup +/ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

  5. Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

    PubMed

    Kulkarni, A P; Sajan, M

    1999-11-15

    In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.

  6. The control of steroidogenesis by human fetal adrenal cells in tissue culture. IV. The effect of exposure to placental steroids.

    PubMed

    Fujieda, K; Faiman, C; Feyes, F I; Winter, J S

    1982-01-01

    The effect upon steroidogenesis of adding various steroids produced by the placenta was studied in short term cultures of human fetal adrenal cells. The addition of high concentrations (10(3) ng/ml) of estrone or estriol inhibited the production of cortisol, but only the former elicited a parallel increase in dehydroepiandrosterone (DHA) production. Estradiol was effective in inhibiting delta-4-3-ketosteroid production at concentrations of 10-100 ng/ml, levels which approach those found in the fetal circulation, while DHA production was increased at concentrations of 1 microgram/ml. The addition of progesterone (4 microgram/ml) to the medium caused increased production of cortisol and corticosterone, but had no effect on DHA production. Pregnenolone (4 microgram/ml) increased the basal production of DHA and slightly impaired both basal and ACTH-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect of estrogen is not influenced by other peptide hormones such as hCG, human prl, beta-lipotropin, corticotropin-like intermediate lobe peptide, or beta-endorphin. A revised model of

  7. 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.

  8. Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells.

    PubMed

    Weedon-Fekjaer, M Susanne; Dalen, Knut Tomas; Solaas, Karianne; Staff, Anne Cathrine; Duttaroy, Asim K; Nebb, Hilde Irene

    2010-07-01

    Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

  9. Placental Permeability of Lead

    PubMed Central

    Carpenter, Stanley J.

    1974-01-01

    The detection of lead in fetal tissues by chemical analysis has long been accepted as prima facie evidence for the permeability of the placenta to this nonessential trace metal. However, only a few investigations, all on lower mammalian species, have contributed any direct experimental data bearing on this physiological process. Recent radioactive tracer and radioautographic studies on rodents have shown that lead crosses the placental membranes rapidly and in significant amounts even at relatively low maternal blood levels. While it is not possible to extrapolate directly the results of these experiments to humans because of differences in placental structure and other factors, the results do serve as a warning of the possible hazard to the human embryo and fetus of even low levels of lead in the maternal system. PMID:4857497

  10. 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.

  11. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  12. 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.

  13. Pathogens and the Placental Fortress

    PubMed Central

    Robbins, Jennifer R.

    2011-01-01

    Summary Placental infections are major causes of maternal and fetal disease. This review introduces a new paradigm for placental infections based on current knowledge of placental defenses and how this barrier can be breached. Transmission of pathogens from mother to fetus can occur at two sites of direct contact between maternal cells and specialized fetal cells (trophoblasts) in the human placenta: (i) maternal immune and endothelial cells juxtaposed to extravillous trophoblasts in the uterine implantation site and (ii) maternal blood surrounding the syncytiotrophoblast. Recent findings suggest that the primary vulnerability is in the implantation site. We explore evidence that the placental syncytiotrophoblast evolved as a defense against pathogens, and that inflammation-mediated spontaneous abortion may benefit mother and pathogen. PMID:22169833

  14. S100P is a potential molecular target of cadmium-induced inhibition of human placental trophoblast cell proliferation.

    PubMed

    Zhou, Taimei; Wang, Haiying; Zhang, Shen; Jiang, Xinglin; Wei, Xiaolong

    2016-11-01

    Cadmium, a common and highly toxic pollutant, has been known to accumulate high concentrations in placenta with deleterious effects on placental structure and function. Cadmium inhibits cell proliferation in placenta via targeting metal binding proteins. S100P, a Ca(2+)-binding protein, plays an important role in promoting cell proliferation and our previous study found its downregulation was linked to cadmium exposure in Guiyu, a famous e-waste recycling town in China. So, the present study was aimed to define whether cadmium inhibited cell proliferation through interfering with S100P. Using human trophoblast-derived HTR-8/SVneo cells as a model in vitro, we showed that cadmium exposure led to decreases in both cell proliferation and S100P expression. Knockdown of S100P in HTR-8/SVneo cells led to an obvious decrease of cell proliferation, and upregulation of S100P resulted in a significant increase of cell proliferation. Furthermore, after 24h of exposure to cadmium (20μM), cells transfected with pcDNA3.1-S100P showed a 1.3-fold higher S100P protein level, 38% higher proliferation evaluated with MTT assay than cells with no transfection, indicating that S100P expression attenuated cadmium-induced inhibition of cell proliferation. Taken together, we demonstrate that cadmium inhibits S100P expression and cell proliferation in placenta, meanwhile, S100P expression affects cell proliferation. Thus, our study is the first to indicate that cadmium may induce inhibition of placental trophoblast cell proliferation through targeting S100P.

  15. Acute effects of ethanol on the transfer of nicotine and two dietary carcinogens in human placental perfusion.

    PubMed

    Veid, Jenni; Karttunen, Vesa; Myöhänen, Kirsi; Myllynen, Päivi; Auriola, Seppo; Halonen, Toivo; Vähäkangas, Kirsi

    2011-09-10

    Many mothers use, against instructions, alcohol during pregnancy. Simultaneously mothers are exposed to a wide range of other environmental chemicals. These chemicals may also harm the developing fetus, because almost all toxic compounds can go through human placenta. Toxicokinetic effects of ethanol on the transfer of other environmental compounds through human placenta have not been studied before. It is known that ethanol has lytic properties and increases the permeability and fluidity of cell membranes. We studied the effects of ethanol on the transfer of three different environmental toxins: nicotine, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and NDMA (N-nitrosodimethylamine) in placental perfusion. We tested in human breast cancer adenocarcinoma cell line MCF-7 whether ethanol affects ABCG2/BCRP, which is also the major transporter in human placenta. We found that the transfer of ethanol is comparable to that of antipyrine, which points to passive diffusion as the transfer mechanism. Unexpectedly, ethanol had no statistically significant effect on the transfer of the other studied compounds. Neither did ethanol inhibit the function of ABCG2/BCRP. These experiments represent only the effects of acute exposure to ethanol and chronic exposure remains to be studied.

  16. Aromatase activity modulation by lindane and bisphenol-A in human placental JEG-3 and transfected kidney E293 cells.

    PubMed

    Nativelle-Serpentini, C; Richard, S; Séralini, G-E; Sourdaine, P

    2003-08-01

    Aromatase is the cytochrome P-450 involved in converting androgens to estrogens. The cytochrome P-450 family plays a central role in the oxidative metabolism of compounds including environmental pollutants. Since lindane and bisphenol-A (BPA) are two well-characterized endocrine disruptors that have been detected in animals and humans, it was important to learn whether they could affect aromatase activity and consequently estrogen biosynthesis. The present study investigates the effects of BPA and lindane on cytotoxicity, aromatase activity and mRNA levels in human placental JEG-3 cells and transfected human embryonal kidney 293 cells. Both cell lines were exposed to increasing concentrations of lindane (25, 50 and 75 microM) and bisphenol-A (25, 50 and 100 microM) over different time periods (10 min-18 h). As a result, none of these concentrations showed cytotoxicity. After short pre-incubation times (10 min-6 h), aromatase activity was enhanced by both compounds. Longer time incubation (18 h), however, produced dose-related inhibition. Lindane and BPA had no significant effects on CYP19 mRNA levels. Therefore, lindane and BPA modulate aromatase activity suggesting an interaction with the cytochrome P-450 aromatase. This study highlights the endocrine-modulating properties of lindane and bisphenol-A.

  17. Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-1N).

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Kono, A

    1991-07-01

    Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

  18. 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

  19. Ovarian control of pituitary hormone secretion in early human pregnancy.

    PubMed

    Emmi, A M; Skurnick, J; Goldsmith, L T; Gagliardi, C L; Schmidt, C L; Kleinberg, D; Weiss, G

    1991-06-01

    To determine the influence of ovarian relaxin on the secretion of pituitary GH and PRL in vivo, we evaluated circulating serum hormone levels in 17 pregnant patients with functional corpora lutea (group I) and compared them to levels in 10 patients with premature ovarian failure (POF; group II) who became pregnant with egg donation and did not have corpora lutea. Group II patients had exogenous hormonal support. Serum relaxin (RLX), GH, PRL, estradiol (E2), and progesterone levels were measured weekly by RIA from weeks 4-8 of pregnancy. Analysis of variance and covariance were used to determine hormonal relationships. Serum RLX was present in the natural pregnancy group, with a mean of 1.94 micrograms/L over the study period. Serum RLX was undetectable in the POF patients (less than 0.16 micrograms/L). No significant difference in PRL or progesterone levels between the two groups was noted. E2 levels showed an upward trend in both groups with time and were significantly higher in patients of the POF group than in group I women (P = 0.001). GH levels were significantly higher in the natural cycle patients (P = 0.02) despite lower E2 levels. These data provide additional support for the concept that RLX production in early pregnancy originates from the corpus luteum. They suggest that a luteal product, probably RLX, stimulates GH secretion in early pregnancy. This is a previously undescribed role for RLX in pituitary physiology during human pregnancy.

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

    PubMed Central

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

    2013-01-01

    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 140–298 nm, and encapsulation efficiency ranging from 52–89%. 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

  1. PLACENTAL GROWTH FACTOR ADMINISTRATION ABOLISHES PLACENTAL ISCHEMIA-INDUCED HYPERTENSION

    PubMed Central

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

    2016-01-01

    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 as 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 (VEGF) and placental growth factor (PlGF), is elevated in RUPP rats and preeclampsia. Although PlGF and VEGF are both natural ligands for sFlt-1, VEGF 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 four groups: normal pregnant (NP) or RUPP ± infusion of rhPlGF (180 μg/kg/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 NP rats. Infusion of rhPlGF 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 rhPlGF abolishes placental ischemia-induced hypertension, without major adverse consequences, suggests a strong therapeutic potential for this growth factor in preeclampsia. PMID:26831193

  2. Placental growth factor reduces blood pressure in a uteroplacental ischemia model of preeclampsia in non-human primates

    PubMed Central

    Makris, Angela; Yeung, Kristen R; Lim, Shirlene M; Sunderland, Neroli; Heffernan, Scott; Thompson, John F; Iliopoulos, Jim; Killingsworth, Murray C; Yong, Jim; Xu, Bei; Ogle, Robert F; Thadhani, Ravi; Karumanchi, S. Ananth; Hennessy, Annemarie

    2016-01-01

    An imbalance in the angiogenesis axis during pregnancy manifests as clinical preeclampsia due to endothelial dysfunction. Circulating sFLT-1 (soluble fms-like tyrosine kinase 1) increases and PlGF (placental growth factor) reduces prior to and during disease. We investigated the clinical and biochemical effects of replenishing the reduced circulating PlGF with recombinant human PlGF (rhPlGF) and thus restoring the angiogenic balance. Hypertensive proteinuria was induced in a non-human primate (Papio hamadryas) by uterine artery ligation at 136 days gestation (of an 182 day pregnancy). Two weeks after uteroplacental ischemia (UPI), rhPlGF (rhPlGF, n=3) or normal saline (control, n=4) was administered by subcutaneous injection (100μg/kg/day) for 5 days. Blood pressure (BP) was monitored by intra-arterial radiotelemetry, sFLT-1 and PlGF by ELISA. UPI resulted in experimental preeclampsia evidenced by increased BP, proteinuria and endotheliosis on renal biopsy and elevated sFLT-1. PlGF significantly reduced after UPI. rhPlGF reduced SBP in the treated group (-5.2mmHg+0.8mmHg;from 132.6+6.6mmHg to 124.1+7.6mmHg) compared to an increase in SBP in controls (6.5mmHg+3mmHg; from 131.3+1.5mmHg to 138.6+1.5mmHg). Proteinuria reduced in the treated group (-72.7±55.7mg/mmol) but increased in the control group. Circulating sFLT-1 was not affected by the administration of PlGF, however a reduction in placental sFLT-1 mRNA expression was demonstrated. There was no significant difference in the weights or lengths of the neonates in the rhPlGF or control group, however, this study was not designed to assess fetal safety or outcomes. Increasing circulating PlGF by the administration of rhPlGF improves clinical parameters in a primate animal model of experimental preeclampsia. PMID:27091894

  3. Leptin secretion and leptin receptor in the human stomach

    PubMed Central

    Sobhani, I; Bado, A; Vissuzaine, C; Buyse, M; Kermorgant, S; Laigneau, J; Attoub, S; Lehy, T; Henin, D; Mignon, M; Lewin, M

    2000-01-01

    BACKGROUND AND AIM—The circulating peptide leptin produced by fat cells acts on central receptors to control food intake and body weight homeostasis. Contrary to initial reports, leptin expression has also been detected in the human placenta, muscles, and recently, in rat gastric chief cells. Here we investigate the possible presence of leptin and leptin receptor in the human stomach.
METHODS—Leptin and leptin receptor expression were assessed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot analysis on biopsy samples from 24 normal individuals. Fourteen (10 healthy volunteers and four patients with non-ulcer dyspepsia and normal gastric mucosa histology) were analysed for gastric secretions. Plasma and fundic mucosa leptin content was determined by radioimmunoassay.
RESULTS—In fundic biopsies from normal individuals, immunoreactive leptin cells were found in the lower half of the fundic glands. mRNA encoding ob protein was detected in the corpus of the human stomach. The amount of fundic leptin was 10.4 (3.7) ng leptin/g mucosa, as determined by radioimmunoassay. Intravenous infusions of pentagastrin or secretin caused an increase in circulating leptin levels and leptin release into the gastric juice. The leptin receptor was present in the basolateral membranes of fundic and antral gastric cells. mRNA encoding Ob-RL was detected in both the corpus and antrum, consistent with a protein of ~120 kDa detected by immunoblotting.
CONCLUSION—These data provide the first evidence of the presence of leptin and leptin receptor proteins in the human stomach and suggest that gastric epithelial cells may be direct targets for leptin. Therefore, we conclude that leptin may have a physiological role in the human stomach, although much work is required to establish this.


Keywords: leptin; leptin receptor; human stomach; gastrin; secretin PMID:10896907

  4. Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture.

    PubMed Central

    Kerneis, S; Bernet, M F; Coconnier, M H; Servin, A L

    1994-01-01

    Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations. These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively. Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed. All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity. Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation. The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity. Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border. Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7959203

  5. Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase.

    PubMed Central

    Hoylaerts, M F; Manes, T; Millán, J L

    1992-01-01

    Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its

  6. Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants.

    PubMed

    Rojo, Gemma; Castillo, Christian; Duaso, Juan; Liempi, Ana; Droguett, Daniel; Galanti, Norbel; Maya, Juan Diego; López-Muñoz, Rodrigo; Kemmerling, Ulrike

    2014-04-01

    Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs.

  7. Human Pregnancy Specific Beta-1-Glycoprotein 1 (PSG1) Has a Potential Role in Placental Vascular Morphogenesis1

    PubMed Central

    Ha, Cam T.; Wu, Julie A.; Irmak, Ster; Lisboa, Felipe A.; Dizon, Anne M.; Warren, James W.; Ergun, Suleyman; Dveksler, Gabriela S.

    2010-01-01

    Previous studies suggest that human pregnancy specific beta-1-glycoproteins (PSGs) play immunomodulatory roles during pregnancy; however, other possible functions of PSGs have yet to be explored. We have observed that PSGs induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties. The present study investigates a potential role for PSG1, the most abundant PSG in maternal serum, as a possible inducer of proangiogenic growth factors known to play an important role in establishment of the vasculature at the maternal-fetal interface. To this end, we measured TGFB1, vascular endothelial growth factors (VEGFs) A and C, and placental growth factor (PGF) protein levels in several cell types after PSG1 treatment. In addition, tube formation and wound healing assays were performed to investigate a possible direct interaction between PSG1 and endothelial cells. PSG1 induced up-regulation of both TGFB1 and VEGFA in human monocytes, macrophages, and two human extravillous trophoblast cell lines. We did not observe induction of VEGFC or PGF by PSG1 in any of the cells tested. PSG1 treatment resulted in endothelial tube formation in the presence and absence of VEGFA. Site-directed mutagenesis was performed to map the essential regions within the N-domain of PSG1 required for functional activity. We found that the aspartic acid at position 95, previously believed to be required for binding of PSGs to cells, is not required for PSG1 activity but that the amino acids implicated in the formation of a salt bridge within the N-domain are essential for PSG1 function. PMID:20335639

  8. 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

    2016-03-03

    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.

  9. Volume-activated amino acid efflux from term human placental tissue: stimulation of efflux via a pathway sensitive to anion transport inhibitors.

    PubMed

    Shennan, D B; McNeillie, S A

    1995-04-01

    The effect of a hyposmotic challenge and hence cell-swelling upon the efflux of a variety of solutes from isolated human placental tissue has been examined. A hyposmotic shock increased the fractional release of taurine, the most abundant free amino acid in placental tissue, via a pathway sensitive to niflumic acid, DIDS (4,4'-Diisothiocyanatostilbene-2',2'-disulphonic acid,) NPPB (5-Nitro-2(3-phenylpropylamino)benzoic acid) and DIOA (R(+)[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden -5-y) oxy] acetic acid). In contrast, tamoxifen was without effect. The cell-swelling induced efflux of taurine was attenuated (40 per cent) by replacing external Cl- with NO3-. The efflux of glutamic acid was also markedly increased by a hyposmotic challenge. Niflumic acid inhibited both basal and volume-activated glutamic acid efflux. A hyposmotic shock also increased alpha-aminoisobutyric acid efflux but not that of 3-O-methylglucose and SO4(2)-. The results suggest that the human placenta can respond to cell-swelling by releasing organic osmolytes such as amino acids via a pathway which is sensitive to anion transport inhibitors. However, it appears that the volume-activated amino acid transport system is independent from the placental anion-exchange pathways. The efflux of these compounds may act with K+ and Cl- efflux to effect a regulatory volume decrease in placental tissue. In addition, volume-activated transport may play a role in transplacental amino acid transfer.

  10. Human Parotid Gland Alpha-Amylase Secretion as a Function of Chronic Hyperbaric Exposure

    DTIC Science & Technology

    1979-01-01

    parotid ...Pullman, WA 99163 Gilman, S. C, G. J. Fischer, R. J. Biersner, R. D. Thornton, and D. A. Miller. 1979. Human parotid gland alpha-amylase secretion...as a function of chronic hyperbaric exposure. Undersea Biomed. Res. 6(3):303-307.—Secretion of a-amylase by the human parotid gland increased

  11. 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

  12. 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

  13. Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian in segregation and localization of mutation site in the gene

    SciTech Connect

    Tsavaler, L.; Penhallow, R.C.; Sussman, H.H. )

    1988-10-01

    The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site.

  14. Combination Effects of (Tri)Azole Fungicides on Hormone Production and Xenobiotic Metabolism in a Human Placental Cell Line

    PubMed Central

    Rieke, Svenja; Koehn, Sophie; Hirsch-Ernst, Karen; Pfeil, Rudolf; Kneuer, Carsten; Marx-Stoelting, Philip

    2014-01-01

    Consumers are exposed to multiple residues of different pesticides via the diet. Therefore, EU legislation for pesticides requires the evaluation of single active substances as well as the consideration of combination effects. Hence the analysis of combined effects of substances in a broad dose range represents a key challenge to current experimental and regulatory toxicology. Here we report evidence for additive effects for (tri)azole fungicides, a widely used group of antifungal agents, in the human placental cell line Jeg-3. In addition to the triazoles cyproconazole, epoxiconazole, flusilazole and tebuconazole and the azole fungicide prochloraz also pesticides from other chemical classes assumed to act via different modes of action (i.e., the organophosphate chlorpyrifos and the triazinylsulfonylurea herbicide triflusulfuron-methyl) were investigated. Endpoints analysed include synthesis of steroid hormone production (progesterone and estradiol) and gene expression of steroidogenic and non-steroidogenic cytochrome-P-450 (CYP) enzymes. For the triazoles and prochloraz, a dose dependent inhibition of progesterone production was observed and additive effects could be confirmed for several combinations of these substances in vitro. The non-triazoles chlorpyrifos and triflusulfuron-methyl did not affect this endpoint and, in line with this finding, no additivity was observed when these substances were applied in mixtures with prochloraz. While prochloraz slightly increased aromatase expression and estradiol production and triflusulfuron-methyl decreased estradiol production, none of the other substances had effects on the expression levels of steroidogenic CYP-enzymes in Jeg-3 cells. For some triazoles, prochloraz and chlorpyrifos a significant induction of CYP1A1 mRNA expression and potential combination effects for this endpoint were observed. Inhibition of CYP1A1 mRNA induction by the AhR inhibitor CH223191 indicated AhR receptor dependence of this effect. PMID

  15. 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.

  16. 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.

  17. 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

  18. Association between altered placental human chorionic gonadotrophin (hCG) production and the occurrence of cryptorchidism: a retrospective study

    PubMed Central

    2014-01-01

    Background An increase in cryptorchidism has been reported in many countries. One mechanism could be low fetal testosterone production possibly secondary to altered placental human chorionic gonadotrophin (hCG) release. Our Objective was to compare hCG values from maternal blood between boys with cryptorchidism and normal boys. Methods Total hCG and α-fetoprotein (AFP) values [12–16 weeks of gestation; from the double test for Down syndrome screening) were compared between cases of cryptorchidism and normal control boys who were matched for maternal age, maternal smoking, gestational age at time of hCG measurement (±1 day), birth weight and birth term. Measurements were performed in a single laboratory; values were expressed as absolute values (KU/L) and multiples of the median (MoM). Boys whose mothers had had a complicated pregnancy were excluded. Groups were compared using the Student’s t test. Log transformation was used to normalize hCG, MoM hCG, AFP and MoM AFP distribution, and values were expressed as geometric means (-1, + 1 tolerance factor). Results Total hCG and MoM hCG levels were significantly lower in the 51 boys with cryptorchidism compared to 306 controls (21.4 (12.3; 37) KU/L vs 27.7 (15.9; 47.9) KU/L and 0.8 (0.5; 1.2) MoM vs 1.0 (0.6; 1.6) MoM, respectively, p < 0.01). By contrast, AFP and MoM AFP levels were similar between groups. Conclusion This study showed a link between low maternal serum hCG levels and cryptorchidism in boys from uncomplicated pregnancy, while normal AFP levels indicated a normal fetoplacental unit. Whether these abnormalities were due to endogenous or exogenous factors remains to be determined. PMID:25064170

  19. Combination effects of (tri)azole fungicides on hormone production and xenobiotic metabolism in a human placental cell line.

    PubMed

    Rieke, Svenja; Koehn, Sophie; Hirsch-Ernst, Karen; Pfeil, Rudolf; Kneuer, Carsten; Marx-Stoelting, Philip

    2014-09-17

    Consumers are exposed to multiple residues of different pesticides via the diet. Therefore, EU legislation for pesticides requires the evaluation of single active substances as well as the consideration of combination effects. Hence the analysis of combined effects of substances in a broad dose range represents a key challenge to current experimental and regulatory toxicology. Here we report evidence for additive effects for (tri)azole fungicides, a widely used group of antifungal agents, in the human placental cell line Jeg-3. In addition to the triazoles cyproconazole, epoxiconazole, flusilazole and tebuconazole and the azole fungicide prochloraz also pesticides from other chemical classes assumed to act via different modes of action (i.e., the organophosphate chlorpyrifos and the triazinylsulfonylurea herbicide triflusulfuron-methyl) were investigated. Endpoints analysed include synthesis of steroid hormone production (progesterone and estradiol) and gene expression of steroidogenic and non-steroidogenic cytochrome-P-450 (CYP) enzymes. For the triazoles and prochloraz, a dose dependent inhibition of progesterone production was observed and additive effects could be confirmed for several combinations of these substances in vitro. The non-triazoles chlorpyrifos and triflusulfuron-methyl did not affect this endpoint and, in line with this finding, no additivity was observed when these substances were applied in mixtures with prochloraz. While prochloraz slightly increased aromatase expression and estradiol production and triflusulfuron-methyl decreased estradiol production, none of the other substances had effects on the expression levels of steroidogenic CYP-enzymes in Jeg-3 cells. For some triazoles, prochloraz and chlorpyrifos a significant induction of CYP1A1 mRNA expression and potential combination effects for this endpoint were observed. Inhibition of CYP1A1 mRNA induction by the AhR inhibitor CH223191 indicated AhR receptor dependence this effect.

  20. ROCKing cytokine secretion balance in human T cells.

    PubMed

    Zanin-Zhorov, Alexandra; Waksal, Samuel D

    2015-04-01

    Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.

  1. Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans.

    PubMed

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, K; Rask-Madsen, J

    1996-01-01

    The proton pump inhibitor, omeprazole, surprisingly resulted in higher rates of proximal duodenal mucosal bicarbonate secretion than previously reported using an H2 receptor antagonist for gastric acid inhibition. Gastroduodenal perfusions were performed in healthy volunteers to evaluate whether this incidental finding is explained by more potent gastric acid inhibition by omeprazole or might be caused by the different mode of drug action. Basal and stimulated gastric and duodenal bicarbonate secretion rates were measured in the same subjects in control experiments (n = 17) and after pretreatment with high dose omeprazole (n = 17) and ranitidine (n = 9), respectively, by use of a technique permitting simultaneous measurements. Concentrations of bicarbonate were measured in the respective effluents by the method of back titration. Both omeprazole and ranitidine completely inhibited gastric acid secretion (pH 6.9 v 6.8; p > 0.05). Omeprazole caused higher rates of basal (mean (SEM)) (597 (48) v 351 (39) mumol/h; p < 0.02) and vagally stimulated (834 (72) v 474 (66) mumol/h; p < 0.02), but not acid stimulated (3351 (678) v 2550 (456) mumol/h; p > 0.05) duodenal bicarbonate secretion compared with control experiments. Also the combination of omeprazole and ranitidine increased (p = 0.05) duodenal bicarbonate secretion, while ranitidine alone caused no change in either basal or stimulated secretion. In the stomach basal as well as vagally stimulated bicarbonate secretion was independent of the means of acid inhibition. These results show that the proton pump inhibitor, omeprazole, promotes proximal duodenal mucosal bicarbonate secretion apparently independent of its gastric acid inhibitory effect. The mechanism of action remains speculative.

  2. Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans.

    PubMed Central

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, K; Rask-Madsen, J

    1996-01-01

    The proton pump inhibitor, omeprazole, surprisingly resulted in higher rates of proximal duodenal mucosal bicarbonate secretion than previously reported using an H2 receptor antagonist for gastric acid inhibition. Gastroduodenal perfusions were performed in healthy volunteers to evaluate whether this incidental finding is explained by more potent gastric acid inhibition by omeprazole or might be caused by the different mode of drug action. Basal and stimulated gastric and duodenal bicarbonate secretion rates were measured in the same subjects in control experiments (n = 17) and after pretreatment with high dose omeprazole (n = 17) and ranitidine (n = 9), respectively, by use of a technique permitting simultaneous measurements. Concentrations of bicarbonate were measured in the respective effluents by the method of back titration. Both omeprazole and ranitidine completely inhibited gastric acid secretion (pH 6.9 v 6.8; p > 0.05). Omeprazole caused higher rates of basal (mean (SEM)) (597 (48) v 351 (39) mumol/h; p < 0.02) and vagally stimulated (834 (72) v 474 (66) mumol/h; p < 0.02), but not acid stimulated (3351 (678) v 2550 (456) mumol/h; p > 0.05) duodenal bicarbonate secretion compared with control experiments. Also the combination of omeprazole and ranitidine increased (p = 0.05) duodenal bicarbonate secretion, while ranitidine alone caused no change in either basal or stimulated secretion. In the stomach basal as well as vagally stimulated bicarbonate secretion was independent of the means of acid inhibition. These results show that the proton pump inhibitor, omeprazole, promotes proximal duodenal mucosal bicarbonate secretion apparently independent of its gastric acid inhibitory effect. The mechanism of action remains speculative. PMID:8566861

  3. 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.

  4. Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

    PubMed Central

    Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K.

    2016-01-01

    ABSTRACT Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/− Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus. PMID:27923925

  5. S-Nitrosylation of secreted recombinant human glypican-1.

    PubMed

    Svensson, Gabriel; Mani, Katrin

    2009-12-01

    Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.

  6. Matrix metalloproteinase 9 is a distal-less 3 target-gene in placental trophoblast cells

    PubMed Central

    Clark, Patricia A.; Xie, Jianjun; Li, Sha; Zhang, Xuesen; Coonrod, Scott

    2013-01-01

    Matrix metalloproteinases (MMPs) are enzymes that regulate extracellular matrix composition and contribute to cell migration. Microarray studies in mouse placenta suggested that MMP-9 transcript abundance was dependent on distal-less 3 (Dlx3), a placental-specific transcriptional regulator; however, it was not clear if this was a direct or indirect effect. Here we investigate mechanism(s) for Dlx3-dependent MMP-9 gene transcription and gelatinase activity in placental trophoblasts. Initial studies confirmed that MMP-9 activity was reduced in placental explants from Dlx3−/− mice and that murine MMP-9 promoter activity was induced by Dlx3 overexpression. Two binding sites within a murine MMP-9 promoter fragment bound Dlx3, and mutations in both elements reduced basal MMP-9-luciferase reporter activity and abolished regulation by Dlx3. Chromatin immunoprecipitation studies in JEG3 cells confirmed Dlx3 binding to the endogenous human MMP-9 promoter at three distinct sites and knockdown of human Dlx3 resulted in reduced endogenous MMP-9 transcripts and secreted activity. These studies provide novel evidence that Dlx3 is involved directly in the transcriptional regulation of mouse and human MMP-9 gene expression in placental trophoblasts. PMID:23657566

  7. Human corpus luteum secretion of relaxin, oxytocin, and progesterone.

    PubMed

    Khan-Dawood, F S; Goldsmith, L T; Weiss, G; Dawood, M Y

    1989-03-01

    To determine whether the human corpus luteum is a source of relaxin and oxytocin, we measured the concentrations of these peptides in plasma obtained from the ovarian veins of ovaries with and without a corpus luteum and compared these to peripheral plasma levels. Peripheral and ovarian venous blood samples were obtained from 34 nonpregnant women, 13 during the luteal phase and 21 during the follicular phase of their cycles, and from a 6-week pregnant woman. Plasma relaxin, oxytocin, and progesterone concentrations were determined by sensitive and specific RIAs. Plasma relaxin levels were not detectable (less than 0.16 microgram/L) in peripheral or ovarian venous plasma not draining a corpus luteum. The mean relaxin concentration in plasma draining an ovary with a corpus luteum was 0.41 +/- 0.09 (+/- SE) microgram/L. Oxytocin levels also were significantly higher in plasma draining an ovary with a corpus luteum (6.70 +/- 1.86 pmol/L) than in that draining the ovary with no corpus luteum (1.58 +/- 0.09 pmol/L; P less than 0.01) or in peripheral plasma (1.58 +/- 0.09 pmol/L; P less than 0.025). The mean progesterone concentration also was highest in plasma draining an ovary with a corpus luteum (210.2 +/- 50.5 nmol/L) compared with those in plasma from the contralateral ovarian vein (40.3 +/- 16.5 nmol/L P less than 0.005) and peripheral plasma (30.2 +/- 5.7 nmol/L; P less than 0.005) during the luteal phase. In a woman who was 6 weeks pregnant, plasma draining the ovary with a corpus luteum had 1.9 micrograms relaxin/L, but only 0.49 pmol/L oxytocin; the latter was similar to concentrations in noncorpus luteum-bearing ovarian venous plasma. These findings indicate that the human corpus luteum secretes relaxin, oxytocin, and progesterone. Both ovarian oxytocin and relaxin may function as paracrine or autocrine modulators of luteal function.

  8. Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells.

    PubMed

    Egawa, M; Kamata, H; Kushiyama, A; Sakoda, H; Fujishiro, M; Horike, N; Yoneda, M; Nakatsu, Y; Ying, Guo; Jun, Zhang; Tsuchiya, Y; Takata, K; Kurihara, H; Asano, T

    2008-12-01

    BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

  9. The phenotype of human placental macrophages and its variation with gestational age.

    PubMed Central

    Goldstein, J.; Braverman, M.; Salafia, C.; Buckley, P.

    1988-01-01

    The antigenic phenotype of human villous stromal macrophages (M phi s) from first and third trimester placentas was analyzed using a large number of monoclonal antibodies (MAbs) to monocyte (Mo)/M phi-associated cell membrane determinants. The purpose of this study was to investigate M phi phenotypic heterogeneity to create a database for the correlation of M phi phenotype with specific immunologic functions. The results showed that villous stromal mononuclear cells express many cell surface antigens found on Mo and M phi s and that they are morphologically diverse, ranging in appearance from classic Hofbauer cells to spindle-shaped cells with long cytoplasmic processes. Villous stromal M phi s were the numerically dominant cell type in this structure and exhibited some major phenotypic differences from M phi s in other tissues. Comparison of first- and third-trimester placentas revealed variation in antigen expression with increasing gestational age, in particular of class II major histocompatibility complex (MHC) determinants: HLA-DR and HLA-DP antigen density was low on first-trimester villous M phi s and much higher on third-trimester M phi s while HLA-DQ was undetectable in the first trimester but present on cells in third trimester placentas. The CD1 (T6) antigen, found on Langerhans (LH) cells and cortical thymocytes, was detected on villous M phi s by two thirds of the MAbs directed against different epitopes on this determinant. Furthermore, comparison with similar studies of lymphoid tissues showed that villous M phi s and dendritic cells share the expression of a number of other cell surface antigens. Finally, it was shown that M phi s in first- and third-trimester villi exhibit strong reactivity with MAbs (Leu 3a,b) to the CD4 antigen that serves as the receptor for the human immunodeficiency virus (HIV), suggesting that these cells may be a portal of entry or reservoir for this virus in the fetuses of pregnant, HIV+ women. Images Figure 1 Figure 1 PMID

  10. Placenta Maps: In Utero Placental Health Assessment of the Human Fetus.

    PubMed

    Miao, Haichao; Mistelbauer, Gabriel; Karimov, Alexey; Alansary, Amir; Davidson, Alice; Lloyd, David; Damodaram, Mellisa; Story, Lisa; Hutter, Jana; Hajnal, Joseph; Rutherford, Mary; Preim, Bernhard; Kainz, Bernhard; Groller, M Eduard

    2017-02-24

    The human placenta is essential for the supply of the fetus. To monitor the fetal development, imaging data is acquired using ultrasound (US). Although it is currently the gold-standard in fetal imaging, it might not capture certain abnormalities of the placenta. Magnetic resonance imaging (MRI) is a safe alternative for the in utero examination while acquiring the fetus data in higher detail. Nevertheless, there is currently no established procedure for assessing the condition of the placenta and consequently the fetal health. Due to maternal respiration and inherent movements of the fetus during examination, a quantitative assessment of the placenta requires fetal motion compensation, precise placenta segmentation and a standardized visualization, which are challenging tasks. Utilizing advanced motion compensation and automatic segmentation methods to extract the highly versatile shape of the placenta, we introduce a novel visualization technique that presents the fetal and maternal side of the placenta in a standardized way. Our approach enables physicians to explore the placenta even in utero. This establishes the basis for a comparative assessment of multiple placentas to analyze possible pathologic arrangements and to support the research and understanding of this vital organ. Additionally, we propose a three-dimensional structure-aware surface slicing technique in order to explore relevant regions inside the placenta. Finally, to survey the applicability of our approach, we consulted clinical experts in prenatal diagnostics and imaging. We received mainly positive feedback, especially the applicability of our technique for research purposes was appreciated.

  11. Effects of 5-hydroxytryptamine on human isolated placental chorionic arteries and veins.

    PubMed Central

    Reviriego, J.; Marín, J.

    1989-01-01

    1. Effects of 5-hydroxytrypamine (5-HT) on cylindrical segments of human chorionic arteries and veins were investigated. Concentrations of 5-HT (up to 3 x 10(-6) M) produced concentration-dependent contractions; higher concentrations induced a reduction of the maximal response. These responses were antagonized by methysergide and ketanserin in a non-competitive manner. The contractions elicited by low 5-HT concentrations were more affected by methysergide (10(-7) M) than by ketanserin (10(-7) M). Ketanserin apparently increased the responses to high 5-HT concentrations in veins. Arteries appeared to be more sensitive to both drugs than veins. Single concentrations of 5-HT elicited transient contractions in both kinds of vessel. 2. Marked tachyphylaxis was seen in segments exposed to high concentrations of 5-HT or in which a concentration-response curve was determined. 3. Contractions induced by 5-HT were reduced in a Ca2+-free medium. Veins were more affected by the Ca2+ antagonists, nifedipine (10(-7) M), nicardipine (10(-5) M) and diltiazem (10(-5) M) than arteries. 4. 5-HT (10(-6) M) enhanced 45Ca2+ uptake in those vessels in which a concentration-response curve had not been previously determined. In veins, this increase was reduced by the three Ca2+ antagonists. 5. The results indicate that 5-HT responses in these vessels were greatly dependent on extracellular Ca2+. A type of 5-HT1-receptor may mediate responses to low 5-HT concentrations, while higher concentrations may activate 5-HT2-receptors. 5-HT may desensitize the latter by interconversion between a high affinity and low affinity state, as suggested by others, a change prevented in part by ketanserin. PMID:2743086

  12. A time-course regulatory and kinetic expression study of steroid metabolizing enzymes by calcitriol in primary cultured human placental cells.

    PubMed

    Noyola-Martínez, Nancy; Halhali, Ali; Zaga-Clavellina, Verónica; Olmos-Ortiz, Andrea; Larrea, Fernando; Barrera, David

    2017-03-01

    1,25-dihydroxivitamin D3 (calcitriol), is a secoesteroid involved in several placental functions. In particular, we and others showed that calcitriol regulates peptides, proteins, cytokines and hormones production in human trophoblastic cells. On the other hand, calcitriol modifies the activity and expression of some steroidogenic enzymes, a process that is considered tissue-specific. However, the effects of calcitriol on the expression of enzymes involved in the synthesis of sex steroids in placental tissue have not yet been entirely studied. The aim of the present study was to investigate the effects of calcitriol upon gene expression of several steroid enzymes such as cytochrome P450scc (CYP11A1), type 1 3β-hydroxysteroid dehydrogenase(3β-HSDI), 17β-HSD3, 17α-hydroxylase/17,20 lyase (CYP17A1) and aromatase (CYP19A1) in primary cultures of human placental cells. Cell cultures were performed using placentas obtained immediately after delivery by caesarean section from normotensive healthy women and calcitriol effects were evaluated, at level of transcription, by qPCR. The results showed that: 1) from basal expression values of the five genes studied, 3β-HSDI was the most expressed gene (P<0.05); 2) basal expression of all enzymes was significantly higher in cultured syncytiotrophoblast than in cytotrophoblasts (P<0.05); 3) the presence of calcitriol in cultured trophoblast cells generally resulted in a stimulatory effect of CYP11A1, CYP19A1 and 17β-HSD3 gene expression at 3h of treatment whereas 3β-HSDI was induced at 6h (P<0.05). However, a time-dependent variable was also observed; 4) protein expression of CYP11A1 and 3β-HSDI were not modified significantly by calcitriol, however that of CYP19A1 was regulated in similar fashion as gene expression. In conclusion, calcitriol affected in a time-dependent manner the expression of steroids metabolizing enzymes in human placental cell cultures.

  13. Human placental estradiol 17. beta. -dehydrogenase: evidence for inverted substrate orientation (wrong-way binding) at the active site

    SciTech Connect

    Murdock, G.L.; Warren, J.C.; Sweet, F.

    1988-06-14

    Human placental estradiol 17..beta..-dehydrogenase was affinity labeled with 17lambda-estradiol 17-(bromo(2-/sup 14/C)acetate) (10 ..mu..M) or 17..beta..-estradiol 17-(bromo(2-/sup 14/C)acetate) (10 ..mu..M). The steroid bromoacetates competitively inhibit the enzyme (against 17..beta..-estradiol) with K/sub i/ values of 90 ..mu..M (17..cap alpha.. bromoacetate) and 134 ..mu..M(17..beta.. bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with t/sub 1/2/ = 110 min (17..cap alpha.. bromoacetate) and t/sub 1/2/ = 220 min (17..beta.. bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N/sup ..pi../-(carboxy(/sup 14/C)methyl histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N/sup ..pi../-(carboxy(/sup 14/C)methyl)histidine. Both the 17..cap alpha.. bromoacetate and also the 17..beta.. bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(..pi..CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involve in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17..cap alpha.. bromoacetate and 17..beta.. bromoacetate D-ring analogue of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone. Moreover, as each of the estradiol 17-(bromoacetates) undergoes the reversible binding step at the enzyme active site, its D-ring is in a reversed binding position relative to that of the natural substrate 17..beta..-estradiol as it undergoes catalytic hydrogen transfer at the same active site.

  14. Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

    PubMed

    Bellazi, L; Mornet, E; Meurice, G; Pata-Merci, N; De Mazancourt, P; Dieudonné, M-N

    2011-10-01

    During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

  15. Parallel secretion of pancreastatin and somatostatin from human pancreastatin producing cell line (QGP-1N).

    PubMed

    Funakoshi, A; Tateishi, K; Kitayama, N; Jimi, A; Matsuoka, Y; Kono, A

    1993-05-01

    In this investigation we studied pancreastatin (PST) secretion from a human PST producing cell line (QGP-1N) in response to various secretagogues. Immunocytochemical study revealed the immunoreactivity of PST and somatostatin (SMT) in the same cells of a monolayer culture. Ki-ras DNA point mutation on codon 12 was found. Carbachol stimulated secretion of PST and SMT and intracellular Ca2+ mobilization in the range of 10(-6)-10(-4) M. The secretion and Ca2+ mobilization were inhibited by atropine, a muscarinic receptor antagonist. Phorbol ester and calcium ionophore (A23187) stimulated secretion of PST and SMT. The removal of extracellular calcium suppressed both secretions throughout stimulation with 10(-5) M carbachol. Fluoride, a well-known activator of guanine nucleotide binding (G) protein, stimulated intracellular Ca2+ mobilization and secretion of PST and SMT in a dose-dependent manner in the range of 5-40 mM. Also, 10(-5) M carbachol and 20 mM fluoride stimulated inositol 1,4,5-triphosphate production. However, cholecystokinin and gastrin-releasing peptide did not stimulate Ca2+ mobilization or secretion of the two peptides. These results suggest that secretion of PST and SMT from QGP-1N cells is regulated mainly by acetylcholine in a parallel fashion through muscarinic receptors coupled to the activation of polyphosphoinositide breakdown by a G-protein and that increases in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  16. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    PubMed

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

  17. Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

    PubMed

    Yamada, Takahiro; Takemura, Yoshizumi; Niisato, Naomi; Mitsuyama, Etsuko; Iwasaki, Yoshinobu; Marunaka, Yoshinori

    2009-03-13

    We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

  18. L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2.

    PubMed

    Lahjouji, Karim; Elimrani, Ihsan; Lafond, Julie; Leduc, Line; Qureshi, Ijaz A; Mitchell, Grant A

    2004-08-01

    Maternofetal transport of l-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that l-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K(m) = 11.09 +/- 1.32 microM; V(max) = 41.75 +/- 0.94 pmol.mg protein(-1).min(-1)), and was unchanged over the pH range from 5.5 to 8.5. l-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of l-carnitine, including d-carnitine, acetyl-d,l-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-l-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na(+)-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for l-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans.

  19. The evolution of epitheliochorial placentation.

    PubMed

    Carter, Anthony M; Enders, Allen C

    2013-01-01

    Epitheliochorial placentation is a derived condition and has evolved separately in strepsirrhine primates and laurasiatherians (pangolins, whales, and hoofed mammals). Usually it is associated with a long gestation period, small litters, and precocial young. Oxygen transfer is facilitated by indenting of the uterine and trophoblast epithelia by maternal and fetal capillaries, respectively. Histotrophic nutrition is important, and adaptations include areolas and hemophagous regions. In pigs and horses, for example, iron is transported as uteroferrin secreted from the uterine glands and taken up by areolas. In the horse, invasive trophoblast cells form cups within the endometrium that are the source of equine chorionic gonadotropin. In ruminants, binucleate trophoblast cells fuse with uterine epithelial cells to form trinucleate cells or plaques that secrete pregnancy hormones. There is evidence of immunosuppression in connection with these more invasive types of trophoblasts. The epitheliochorial condition may be advantageous for long pregnancies in large animals.

  20. Immature human chorionic gonadotropin (hCG) in first trimester placental cells is bound to an ATP-binding protein forming high-molecular-weight hCG.

    PubMed

    Shimojo, M; Sakakibara, R; Ishiguro, M

    1993-07-01

    Human chorionic gonadotropin (hCG) in first trimester placental cells is made up of immature alpha- and beta-subunits containing only N-linked high-mannose sugar chains, which are of 21 kDa for the alpha-subunit and 23 and 19 kDa for the beta-subunit. However, the apparent molecular weight of immature hCG from placental cell extracts has been estimated from gel filtration to be much higher (100-200 kDa; high molecular weight-hCG, HMW-hCG) based on gel filtration than the theoretical value (approximately 44 kDa) of the alpha beta dimer (alpha beta-hCG). We prepared a gel-filtered fraction containing HMW-hCG and investigated treatments for converting it to alpha beta-hCG. We found that the molecular weight of HMW-hCG was decreased to close to that of alpha beta-hCG by treatment with acetone, proteases, or chelating agents. These treatments also shifted the isoelectric point of HMW-hCG from the acidic region (pI = 4-6) to the alkaline (pI = 9-11), approximating to that of alpha beta-hCG. We also found that HMW-hCG, but not acetone-treated HMW-hCG, bound to ATP-agarose resin. These results suggested that the immature alpha beta-hCG molecule in placental cells may be bound to an acidic ATP-binding protein to form HMW-hCG.

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

    PubMed

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

    2016-01-01

    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.

  2. Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response

    PubMed Central

    Park, Sang Gyu; Kim, Hye Jin; Min, You Hong; Choi, Eung-Chil; Shin, Young Kee; Park, Bum-Joon; Lee, Sang Won; Kim, Sunghoon

    2005-01-01

    Although aminoacyl-tRNA synthetases (ARSs) are essential for protein synthesis, they also function as regulators and signaling molecules in diverse biological processes. Here, we screened 11 different human ARSs to identify the enzyme that is secreted as a signaling molecule. Among them, we found that lysyl-tRNA synthetase (KRS) was secreted from intact human cells, and its secretion was induced by TNF-α. The secreted KRS bound to macrophages and peripheral blood mononuclear cells to enhance the TNF-α production and their migration. The mitogen-activated protein kinases, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, and Gαi were determined to be involved in the signal transduction triggered by KRS. All of these activities demonstrate that human KRS may work as a previously uncharacterized signaling molecule, inducing immune response through the activation of monocyte/macrophages. PMID:15851690

  3. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

    PubMed

    Sleep, D; Belfield, G P; Goodey, A R

    1990-01-01

    We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.

  4. Dibutyltin-induced alterations of interleukin 1beta secretion from human immune cells.

    PubMed

    Brown, Shyretha; Tehrani, Shahin; Whalen, Margaret M

    2017-02-01

    Dibutyltin (DBT) is used to stabilize polyvinyl chloride plastics (including pipes that distribute drinking water) and as a de-worming agent in poultry. DBT is found in human blood, and DBT exposures alter the secretion of tumor necrosis factor alpha and interferon gamma from lymphocytes. Interleukin (IL)-1β is a proinflammatory cytokine that regulates cellular growth, tissue restoration and immune response regulation. IL-1β plays a role in increasing invasiveness of certain tumors. This study reveals that exposures to DBT (24 h, 48 h and 6 days) modify the secretion of IL-1β from increasingly reconstituted preparations of human immune cells (highly enriched human natural killer cells, monocyte-depleted [MD] peripheral blood mononuclear cells [PBMCs], PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes). DBT altered IL-1β secretion from all cell preparations. Higher concentrations of DBT (5 and 2.5 μm) decreased the secretion of IL-1β, while lower concentrations of DBT (0.1 and 0.05 μm) increased the secretion of IL-1β. Selected signaling pathways were examined in MD-PBMCs to determine if they play a role in DBT-induced elevations of IL-1β secretion. Pathways examined were IL-1β converting enzyme (caspase 1), mitogen-activated protein kinases and nuclear factor kappa B. Caspase 1 and mitogen-activated protein kinase pathways appear to be utilized by DBT in increasing IL-1β secretion. These results indicate that DBT alters IL-1β secretion from human immune cells in an ex. vivo system utilizing several IL-1β regulating signaling pathways. Thus, DBT may have the potential to alter IL-1β secretion in an in vivo system. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Immunoreactive relaxin in human cervico-vaginal secretion.

    PubMed

    Fuchs, U; Seeger, H; Voelter, W; Lippert, T H

    1988-01-01

    Relaxin was measured in cervico-vaginal secretion of non-pregnant women of reproductive age using the heterologous radioimmuno-assay for porcine relaxin. It was detected in about three-quarters of the samples collected. The mean value of the 529 samplers tested was 599 pg/ml. There was a slight trend to higher relaxin values during the second week of the menstrual cycle.

  6. Differential placental expression profile of human Growth Hormone/Chorionic Somatomammotropin genes in pregnancies with pre-eclampsia and gestational diabetes mellitus

    PubMed Central

    Männik, Jaana; Vaas, Pille; Rull, Kristiina; Teesalu, Pille; Laan, Maris

    2012-01-01

    The human GH/CSH cluster consisting of one pituitary-expressed (GH1) and four placenta-expressed loci has been implicated in maternal metabolic adaptation to pregnancy, regulation of intrauterine and postnatal growth. We investigated how the mRNA expression profile of placental GH2, CSH1 and CSH2 genes and their alternative transcripts correlates with maternal pre-eclampsia (PE) and/or gestational diabetes mellitus (GD). The expression of studied genes in PE placentas (n = 17) compared to controls (n = 17) exhibited a trend for reduced transcript levels. The alternative transcripts retaining intron 4, GH2-2 and CSH1-2 showed significantly reduced expression in PE cases without growth restriction (P = 0.007, P = 0.008, respectively). In maternal GD (n = 23), a tendency of differential expression was detected only for the GH2 gene and in pregnancies with large-for-gestational-age newborns. Our results, together with those reported by others, are consistent with a pleiotropic effect of placental hGH/CSH genes at the maternal-fetal interface relating to the regulation of fetal growth and the risk of affected maternal metabolism. PMID:22387044

  7. TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

    PubMed Central

    Mitrovic, Sandra; Nogueira, Cristina; Cantero-Recasens, Gerard; Kiefer, Kerstin; Fernández-Fernández, José M; Popoff, Jean-François; Casano, Laetitia; Bard, Frederic A; Gomez, Raul; Valverde, Miguel A; Malhotra, Vivek

    2013-01-01

    Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca2+-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca2+ signal. ATP-induced MUC5AC secretion depended strongly on Ca2+ influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca2+ entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca2+ and by inhibition of the Na+/Ca2+ exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na+ entry to promote Ca2+ uptake via an NCX to trigger MUC5AC secretion. DOI: http://dx.doi.org/10.7554/eLife.00658.001 PMID:23741618

  8. The effect of oxytocin on oestradiol-17 beta and testosterone secretion by cultured human granulosa cells.

    PubMed

    Clamagirand, C; Plevrakis, I; Bussenot, I; Parinaud, J; Vieitez, G; Grandjean, H

    1991-07-01

    The effect of oxytocin at different concentrations was tested on the secretion of oestradiol-17 beta and testosterone by cultured human granulosa cells obtained by follicular punctures during in-vitro fertilization (IVF) attempts. Oxytocin had no effect on testosterone secretion, either in the absence or the presence of follicle stimulating hormone (FSH). It had no effect on oestradiol-17 beta in the absence of FSH. However, it decreased the FSH-stimulated secretion of oestradiol-17 beta in a certain number of cases. This inhibitory effect appears to be associated with cells more responsive to FSH and was identified in women found to be successful in achieving pregnancy during IVF attempts.

  9. Detrimental effects of ethanol and its metabolite acetaldehyde, on first trimester human placental cell turnover and function.

    PubMed

    Lui, Sylvia; Jones, Rebecca L; Robinson, Nathalie J; Greenwood, Susan L; Aplin, John D; Tower, Clare L

    2014-01-01

    Fetal alcohol spectrum disorder (FASD) describes developmental issues from high maternal alcohol intake, which commonly results in fetal growth restriction and long term morbidity. We aimed to investigate the effect of alcohol and acetaldehyde, on the first trimester placenta, the period essential for normal fetal organogenesis. Normal invasion and establishment of the placenta during this time are essential for sustaining fetal viability to term. We hypothesise that alcohol (ethanol) and acetaldehyde have detrimental effects on cytotrophoblast invasion, turnover and placental function. Taurine is an important amino acid for neuronal and physiological development, and so, its uptake was assayed in cells and placental explants exposed to alcohol or acetaldehyde. First trimester villous explants and BeWo cells were treated with 0, 10, 20, 40 mM ethanol or 0, 10, 20, 40 µM acetaldehyde. The invasive capacity of SGHPL4, a first trimester extravillous cytotrophoblast cell line, was unaffected by ethanol or acetaldehyde (p>0.05; N = 6). The cells in-cycle were estimated using immunostaining for Ki67. Proliferating trophoblast cells treated with ethanol were decreased in both experiments (explants: 40% at 20 mM and 40 mM, p<0.05, N = 8-9) (cell line: 5% at 20 mM and 40 mM, p<0.05, N = 6). Acetaldehyde also reduced Ki67-positive cells in both experiments (explants at 40 µM p<0.05; N = 6) (cell line at 10 µM and 40 µM; p<0.05; N = 7). Only in the cell line at 20 µM acetaldehyde demonstrated increased apoptosis (p<0.05; N = 6). Alcohol inhibited taurine transport in BeWo cells at 10 mM and 40 mM (p<0.05; N = 6), and in placenta at 40 mM (p<0.05; N = 7). Acetaldehyde did not affect taurine transport in either model (P<0.05; N = 6). Interestingly, system A amino acid transport in placental explants was increased at 10 µM and 40 µM acetaldehyde exposure (p<0.05; N = 6). Our results demonstrate that exposure to both genotoxins may contribute to the pathogenesis of FASD by

  10. Cidea control of lipid storage and secretion in mouse and human sebaceous glands.

    PubMed

    Zhang, Shasha; Shui, Guanghou; Wang, Guanqun; Wang, Chao; Sun, Shuhong; Zouboulis, Christos C; Xiao, Ran; Ye, Jing; Li, Wei; Li, Peng

    2014-05-01

    Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that Cidea is expressed at high levels in lipid-laden mature sebocytes and that Cidea deficiency led to dry hair and hair loss in aged mice. In addition, Cidea-deficient mice had markedly reduced levels of skin surface lipids, including triacylglycerides (TAGs) and wax diesters (WDEs), and these mice were defective in water repulsion and thermoregulation. Furthermore, we observed that Cidea-deficient sebocytes accumulated a large number of smaller-sized lipid droplets (LDs), whereas overexpression of Cidea in human SZ95 sebocytes resulted in increased lipid storage and the accumulation of large LDs. Importantly, Cidea was highly expressed in human sebaceous glands, and its expression levels were positively correlated with human sebum secretion. Our data revealed that Cidea is a crucial regulator of sebaceous gland lipid storage and sebum lipid secretion in mammals and humans.

  11. Affinity of bronchial secretion glycoproteins and cells of human bronchial mucosa for Ricinus communis lectins.

    PubMed

    Lhermitte, M; Lamblin, G; Degand, P; Roussel, P; Mazzuca, M

    1977-01-01

    The coupling of Ricinus communis lectins to Sephadex G 25 was used in order to study mucins and other glycoproteins from human bronchial secretion. The major part of human bronchial mucins and other glycoproteins such as immunoglobulins A, bronchotransferrin and alpha1-antichymotrypsin were isolated by this procedure. A parallel study of human bronchial mucosa was achieved with peroxidase labeled Ricinus communis lectins; this study characterized goblet cells and mucous cells which contain mucins, and serous cells which are involved in the synthesis or the secretion of the other glycoproteins.

  12. 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

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

    DOE PAGES

    Punshon, Tracy; Chen, Si; Finney, Lydia; ...

    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

  14. 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

  15. Correlation between human maternal-fetal placental transfer and molecular weight of PCB and dioxin congeners/isomers.

    PubMed

    Mori, Chisato; Nakamura, Noriko; Todaka, Emiko; Fujisaki, Takeyoshi; Matsuno, Yoshiharu; Nakaoka, Hiroko; Hanazato, Masamichi

    2014-11-01

    Establishing methods for the assessment of fetal exposure to chemicals is important for the prevention or prediction of the child's future disease risk. In the present study, we aimed to determine the influence of molecular weight on the likelihood of chemical transfer from mother to fetus via the placenta. The correlation between molecular weight and placental transfer rates of congeners/isomers of polychlorinated biphenyls (PCBs) and dioxins was examined. Twenty-nine sample sets of maternal blood, umbilical cord, and umbilical cord blood were used to measure PCB concentration, and 41 sample sets were used to analyze dioxins. Placental transfer rates were calculated using the concentrations of PCBs, dioxins, and their congeners/isomers within these sample sets. Transfer rate correlated negatively with molecular weight for PCB congeners, normalized using wet and lipid weights. The transfer rates of PCB or dioxin congeners differed from those of total PCBs or dioxins. The transfer rate for dioxin congeners did not always correlate significantly with molecular weight, perhaps because of the small sample size or other factors. Further improvement of the analytical methods for dioxin congeners is required. The findings of the present study suggested that PCBs, dioxins, or their congeners with lower molecular weights are more likely to be transferred from mother to fetus via the placenta. Consideration of chemical molecular weight and transfer rate could therefore contribute to the assessment of fetal exposure.

  16. 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.

  17. 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

    2014-12-05

    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.

  18. 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.

  19. Placental phenotype and the insulin-like growth factors: resource allocation to fetal growth.

    PubMed

    Sferruzzi-Perri, Amanda N; Sandovici, Ionel; Constancia, Miguel; Fowden, Abigail L

    2017-03-24

    The placenta is the main determinant of fetal growth and development in utero. It supplies all the nutrients and oxygen required for fetal growth and secretes hormones that facilitate maternal allocation of nutrients to the fetus. Furthermore, the placenta responds to nutritional and metabolic signals in the mother by altering its structural and functional phenotype which can lead to changes in maternal resource allocation to the fetus. The molecular mechanisms by which the placenta senses and responds to environmental cues are poorly understood. This review discusses the role of the insulin-like growth factors (IGFs) in controlling placental resource allocation to fetal growth, particularly in response to adverse gestational environments. In particular, it assesses the impact of the IGFs and their signalling machinery on placental morphogenesis, substrate transport and hormone secretion, primarily in the laboratory species, although it draws on data from human and other species where relevant. It also considers the role of the IGFs as environmental signals in linking resource availability, to fetal growth through changes in the morphological and functional phenotype of the placenta. As altered fetal growth is associated with increased perinatal morbidity and mortality and a greater risk of developing adult-onset diseases in later life, understanding the role of IGFs during pregnancy in regulating placental resource allocation to fetal growth is important for identifying the mechanisms underlying the developmental programming of offspring phenotype by suboptimal intrauterine growth. This article is protected by copyright. All rights reserved.

  20. Review: Placental programming of postnatal diabetes and impaired insulin action after IUGR.

    PubMed

    Gatford, K L; Simmons, R A; De Blasio, M J; Robinson, J S; Owens, J A

    2010-03-01

    Being born small due to poor growth before birth increases the risk of developing metabolic disease, including type 2 diabetes, in later life. Inadequate insulin secretion and decreasing insulin sensitivity contribute to this increased diabetes risk. Impaired placental growth, development and function are major causes of impaired fetal growth and development and therefore of IUGR. Restricted placental growth (PR) and function in non-human animals induces similar changes in insulin secretion and sensitivity as in human IUGR, making these valuable tools to investigate the underlying mechanisms and to test interventions to prevent or ameliorate the risk of disease after IUGR. Epigenetic changes induced by an adverse fetal environment are strongly implicated as causes of later impaired insulin action. These have been well-characterised in the PR rat, where impaired insulin secretion is linked to epigenetic changes at the Pdx-1 promotor and reduced expression of this transcription factor. Present research is particularly focussed on developing intervention strategies to prevent or reverse epigenetic changes, and normalise gene expression and insulin action after PR, in order to translate this to treatments to improve outcomes in human IUGR.

  1. 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.

  2. 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

  3. Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon.

    PubMed

    Woodward, B J; Lenton, E A; Turner, K

    1993-09-01

    Of 48 spare human pre-embryos achieving the expanded blastocyst stage, 22 (45.6%) secreted significant amounts of human chorionic gonadotrophin (HCG) (> 5 IU/l/day). Of these, nine remained intrazonal, seven partially hatched and six fully hatched. Embryonic production of HCG in vitro appeared to be time-dependent, starting after a certain minimum time (approximately 160 h post-insemination) and rising exponentially, with maximal HCG production around day 10. Hatching was not a prerequisite for HCG secretion, since similar amounts were produced by intrazonal blastocysts. Blastocysts derived from abnormally fertilized oocytes also began secreting HCG exponentially but secretion was delayed and the upper limit of maximum HCG secretion rate was comparatively low. The actual amount of HCG is thought to reflect the number of viable trophectoderm cells producing the hormone. HCG doubling times for blastocysts in vitro were rapid when compared to implanting blastocysts of a similar age in vivo, with 19/22 (86.4%) blastocysts having a doubling time of < 10 h. Provided a pre-embryo can secrete HCG and maintain an adequate doubling time, sufficient HCG should be produced for initial stages of embryonic recognition in vivo. Since intrazonal blastocysts are capable of fulfilling both of these criteria, the limiting factor in realizing their full potential may be escaping from the zona pellucida.

  4. Lubiprostone stimulates secretion from tracheal submucosal glands of sheep, pigs, and humans.

    PubMed

    Joo, N S; Wine, J J; Cuthbert, A W

    2009-05-01

    Lubiprostone, a putative ClC-2 chloride channel opener, has been investigated for its effects on airway epithelia (tracheas). Lubiprostone is shown to increase submucosal gland secretion in pigs, sheep, and humans and to increase short-circuit current (SCC) in the surface epithelium of pigs and sheep. Use of appropriate blocking agents and ion-substitution experiments shows anion secretion is the driving force for fluid formation in both glands and surface epithelium. From SCC concentration-response relations, it is shown that for apical lubiprostone K(d) = 10.5 nM with a Hill slope of 1.08, suggesting a single type of binding site and, from the speed of the response, close to the apical surface, confirmed the rapid blockade by Cd ions. Responses to lubiprostone were reversible and repeatable, responses being significantly larger with ventral compared with dorsal epithelium. Submucosal gland secretion rates following basolateral lubiprostone were, respectively, 0.2, 0.5, and 0.8 nl gl(-1) min(-1) in humans, sheep, and pigs. These rates dwarf any contribution surface secretion adds to the accumulation of surface liquid under the influence of lubiprostone. Lubiprostone stimulated gland secretion in two out of four human cystic fibrosis (CF) tissues and in two of three disease controls, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (COPD/IPF), but in neither type of tissue was the increase significant. Lubiprostone was able to increase gland secretion rates in normal human tissue in the continuing presence of a high forskolin concentration. Lubiprostone had no spasmogenic activity on trachealis muscle, making it a potential agent for increasing airway secretion that may have therapeutic utility.

  5. GPR54 peptide agonists stimulate insulin secretion from murine, porcine and human islets.

    PubMed

    Bowe, James E; Foot, Victoria L; Amiel, Stephanie A; Huang, Gao Cai; Lamb, Morgan W; Lakey, Jonathan; Jones, Peter M; Persaud, Shanta J

    2012-01-01

    This study was designed to determine the effects of 10 and 13 amino acid forms of kisspeptin on dynamic insulin secretion from mammalian islets since it is not clear from published data whether the shorter peptide is stimulatory while the longer peptide inhibits insulin release. Insulin secretion was measured by radioimmunoassay following perifusion of human, pig, rat and mouse isolated islets with kisspeptin-10 or kisspeptin-13 in the presence of 20 mM glucose. Both peptides stimulated rapid, reversible potentiation of glucose-stimulated insulin secretion from islets of all species tested. These data indicate that both kisspeptin-10 and kisspeptin-13, which is an extension of kisspeptin-10 by three amino acids, act directly at islet β-cells of various species to potentiate insulin secretion, and suggest that inhibitory effects reported in earlier studies may reflect differences in experimental protocols.

  6. Circadian disruption, Per3, and human cytokine secretion.

    PubMed

    Guess, Jaclyn; Burch, James B; Ogoussan, Kisito; Armstead, Cheryl A; Zhang, Hongmei; Wagner, Sara; Hebert, James R; Wood, Patricia; Youngstedt, Shawn D; Hofseth, Lorne J; Singh, Udai P; Xie, Dawen; Hrushesky, William J M

    2009-12-01

    Circadian disruption has been linked with inflammation, an established cancer risk factor. Per3 clock gene polymorphisms have also been associated with circadian disruption and with increased cancer risk. Patients completed a questionnaire and provided a blood sample prior to undergoing a colonoscopy (n = 70). Adjusted mean serum cytokine concentrations (IL-6, TNF-alpha, gamma-INF, IL-1ra, IL-1-beta, VEGF) were compared among patients with high and low scores for fatigue (Multidimensional Fatigue Inventory), depressive symptoms (Beck Depression Inventory II), or sleep disruption (Pittsburgh Sleep Quality Index), or among patients with different Per3 clock gene variants. Poor sleep was associated with elevated VEGF, and fatigue-related reduced activity was associated with elevated TNF-alpha concentrations. Participants with the 4/5 or 5/5 Per3 variable tandem repeat sequence had elevated IL-6 concentrations compared to those with the 4/4 genotype. Biological processes linking circadian disruption with cancer remain to be elucidated. Increased inflammatory cytokine secretion may play a role.

  7. 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.

  8. 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

  9. 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

    2016-08-18

    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.

  10. 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

  11. Effects of glucosamine infusion on insulin secretion and insulin action in humans.

    PubMed

    Monauni, T; Zenti, M G; Cretti, A; Daniels, M C; Targher, G; Caruso, B; Caputo, M; McClain, D; Del Prato, S; Giaccari, A; Muggeo, M; Bonora, E; Bonadonna, R C

    2000-06-01

    Glucose toxicity (i.e., glucose-induced reduction in insulin secretion and action) may be mediated by an increased flux through the hexosamine-phosphate pathway. Glucosamine (GlcN) is widely used to accelerate the hexosamine pathway flux, independently of glucose. We tested the hypothesis that GlcN can affect insulin secretion and/or action in humans. In 10 healthy subjects, we sequentially performed an intravenous glucose (plus [2-3H]glucose) tolerance test (IVGTT) and a euglycemic insulin clamp during either a saline infusion or a low (1.6 micromol x min(-1) x kg(-1)) or high (5 micromol x min(-1) x kg(-1) [n = 5]) GlcN infusion. Beta-cell secretion, insulin (SI*-IVGTT), and glucose (SG*) action on glucose utilization during the IVGTT were measured according to minimal models of insulin secretion and action. Infusion of GlcN did not affect readily releasable insulin levels, glucose-stimulated insulin secretion (GSIS), or the time constant of secretion, but it increased both the glucose threshold of GSIS (delta approximately 0.5-0.8 mmol/l, P < 0.03-0.01) and plasma fasting glucose levels (delta approximately 0.3-0.5 mmol/l, P < 0.05-0.02). GlcN did not change glucose utilization or intracellular metabolism (glucose oxidation and glucose storage were measured by indirect calorimetry) during the clamp. However, high levels of GlcN caused a decrease in SI*-IVGTT (delta approximately 30%, P < 0.02) and in SG* (delta approximately 40%, P < 0.05). Thus, in humans, acute GlcN infusion recapitulates some metabolic features of human diabetes. It remains to be determined whether acceleration of the hexosamine pathway can cause insulin resistance at euglycemia in humans.

  12. 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Differentially Suppresses Angiogenic Responses in Human Placental Vein and Artery Endothelial Cells

    PubMed Central

    Li, Yan; Wang, Kai; Zou, Qing-Yun; Magness, Ronald R.; Zheng, Jing

    2015-01-01

    Placental angiogenesis is dramatically increased during pregnancy in association with the elevated placental blood flows to support the rapidly growing fetus. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant and a ligand of aryl hydrocarbon receptor (AhR). Herein, we investigated the effects of TCDD on proliferation, migration, and viability of fetoplacental endothelial cells in response to a complete growth medium which contained serum and growth supplement using human umbilical cord vein (HUVECs) and artery (HUAECs) cells as models. We found that TCDD dose- and time-dependently inhibited (p < 0.05) proliferation of HUVECs and HUAECs. Treatment with TCDD at 10 nM for 6 days inhibited (p < 0.05) migration (by ~30%) of HUAECs, but not HUVECs. TCDD at 10 nM also attenuated (p < 0.05) viability of HUVECs and HUAECs. Interestingly, specific AhR siRNA blocked (p < 0.05) the TCDD-inhibited cellular responses in HUAECs, but not HUVECs. Nonetheless, TCDD at 10 nM neither affected the cell cycle process, nor did it induce cell apoptosis in HUVECs and HUAECs. In addition, TCDD at 10 nM also did not alter activation of ERK1/2 and AKT1 in HUVECs and HUAECs. Collectively, TCDD suppresses proliferation and/or migration (two key steps of angiogenesis) of HUVECs and HUAECs independent and dependent of AhR, respectively. These data suggest that TCDD inhibited growth of HUVECs and HUAECs via decreasing cell viability. Thus, TCDD may inhibit fetoplacental angiogenesis, leading to negative pregnancy outcomes. PMID:26275813

  13. Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function.

    PubMed

    Gentilin, Erica; Tagliati, Federico; Terzolo, Massimo; Zoli, Matteo; Lapparelli, Marcello; Minoia, Mariella; Ambrosio, Maria Rosaria; Degli Uberti, Ettore C; Zatelli, Maria Chiara

    2013-09-01

    Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

  14. [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.

  15. Hofbauer cells of M2a, M2b and M2c polarization may regulate feto-placental angiogenesis.

    PubMed

    Loegl, J; Hiden, U; Nussbaumer, E; Schliefsteiner, C; Cvitic, S; Lang, I; Wadsack, C; Huppertz, B; Desoye, G

    2016-11-01

    The human placenta comprises a special type of tissue macrophages, the Hofbauer cells (HBC), which exhibit M2 macrophage phenotype. Several subtypes of M2-polarized macrophages (M2a, M2b and M2c) exist in almost all tissues. Macrophage polarization depends on the way of macrophage activation and leads to the expression of specific cell surface markers and the acquisition of specific functions, including tissue remodeling and the promotion of angiogenesis. The placenta is a highly vascularized and rapidly growing organ, suggesting a role of HBC in feto-placental angiogenesis. We here aimed to characterize the specific polarization and phenotype of HBC and investigated the role of HBC in feto-placental angiogenesis. Therefore, HBC were isolated from third trimester placentas and their phenotype was determined by the presence of cell surface markers (FACS analysis) and secretion of cytokines (ELISA). HBC conditioned medium (CM) was analyzed for pro-angiogenic factors, and the effect of HBC CM on angiogenesis, proliferation and chemoattraction of isolated primary feto-placental endothelial cells (fpEC) was determined in vitro Our results revealed that isolated HBC possess an M2 polarization, with M2a, M2b and M2c characteristics. HBC secreted the pro-angiogenic molecules VEGF and FGF2. Furthermore, HBC CM stimulated the in vitro angiogenesis of fpEC. However, compared with control medium, chemoattraction of fpEC toward HBC CM was reduced. Proliferation of fpEC was not affected by HBC CM. These findings demonstrate a paracrine regulation of feto-placental angiogenesis by HBC in vitro Based on our collective results, we propose that the changes in HBC number or phenotype may affect feto-placental angiogenesis.

  16. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42.

    PubMed Central

    Shinjo, K; Koland, J G; Hart, M J; Narasimhan, V; Johnson, D I; Evans, T; Cerione, R A

    1990-01-01

    We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein. Images PMID:2124704

  17. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42.

    PubMed

    Shinjo, K; Koland, J G; Hart, M J; Narasimhan, V; Johnson, D I; Evans, T; Cerione, R A

    1990-12-01

    We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.

  18. Molecular cloning of the gene for the human placental GTP-binding protein G sub p (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    SciTech Connect

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A. ); Johnson, D.I. ); Evans, T. )

    1990-12-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G{sub p} (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G{sub p} protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.

  19. 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.

  20. Thimerosal induces TH2 responses via influencing cytokine secretion by human dendritic cells.

    PubMed

    Agrawal, Anshu; Kaushal, Poonam; Agrawal, Sudhanshu; Gollapudi, Sastry; Gupta, Sudhir

    2007-02-01

    Thimerosal is an organic mercury compound that is used as a preservative in vaccines and pharmaceutical products. Recent studies have shown a TH2-skewing effect of mercury, although the underlying mechanisms have not been identified. In this study, we investigated whether thimerosal can exercise a TH2-promoting effect through modulation of functions of dendritic cells (DC). Thimerosal, in a concentration-dependent manner, inhibited the secretion of LPS-induced proinflammatory cytokines TNF-alpha, IL-6, and IL-12p70 from human monocyte-derived DC. However, the secretion of IL-10 from DC was not affected. These thimerosal-exposed DC induced increased TH2 (IL-5 and IL-13) and decreased TH1 (IFN-gamma) cytokine secretion from the T cells in the absence of additional thimerosal added to the coculture. Thimerosal exposure of DC led to the depletion of intracellular glutathione (GSH), and addition of exogenous GSH to DC abolished the TH2-promoting effect of thimerosal-treated DC, restoring secretion of TNF-alpha, IL-6, and IL-12p70 by DC and IFN-gamma secretion by T cells. These data suggest that modulation of TH2 responses by mercury and thimerosal, in particular, is through depletion of GSH in DC.

  1. Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

    1985-01-01

    The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

  2. A quality system for placental blood banking.

    PubMed

    Sirchia, G; Rebulla, P; Mozzi, F; Lecchi, L; Lazzari, L; Ratti, I

    1998-06-01

    A Quality System for Placental Blood Banking aimed at the transplantation of haematopoietic stem cells to related and unrelated allogeneic recipients is described. It includes the organizational structure, procedures, processes and resources needed to implement quality management. The Quality System described in this article is based on ISO 9002, a model for quality assurance in production, installation and servicing developed in 1987 and revised in 1994 by the International Organization for Standardization. ISO 9002 includes 20 clauses that provide guidance for the implementation of the Quality System. The development of the Quality System is started by the Placental Blood Bank Medical Director with the definition of a General Quality Plan including: (1) the written description of the Mission, Objectives, Technical and Organizational Policies, and Staff Organization Chart; (2) the definition and acquisition of adequate financial, human and structural resources; (3) the appointment of a Quality System Head, who must identify the Placental Blood Banking process together with the Placental Blood Bank personnel; implement a documentation plan; identify quality indicators; start regular internal audit; report audit results to the Medical Director for review. Following staff training and qualification, the Quality System is launched. The Placental Blood Bank can then undergo audit by an external inspector and be finally certified for compliance to ISO 9002. The Quality System must be maintained and subjected to external audit at regular intervals so that certification is confirmed.

  3. Comparison of pulsatile vs. continuous administration of human placental growth hormone in female C57BL/6J mice.

    PubMed

    Liao, Shutan; Vickers, Mark H; Evans, Angharad; Stanley, Joanna L; Baker, Philip N; Perry, Jo K

    2016-10-01

    Exogenous growth hormone has different actions depending on the method of administration. However, the effects of different modes of administration of the placental variant of growth hormone on growth, body composition and glucose metabolism have not been investigated. In this study, we examined the effect of pulsatile vs. continuous administration of recombinant variant of growth hormone in a normal mouse model. Female C57BL/6J mice were randomized to receive vehicle or variant of growth hormone (2 or 5 mg/kg per day) by daily subcutaneous injection (pulsatile) or osmotic pump for 6 days. Pulsatile treatment with 2 and 5 mg/kg per day significantly increased body weight. There was also an increase in liver, kidney and spleen weight via pulsatile treatment, whereas continuous treatment did not affect body weight or organ size. Pulsatile treatment with 5 mg/kg per day significantly increased fasting plasma insulin concentration, whereas with continuous treatment, fasting insulin concentration was not significantly different from the vehicle-treated control. However, a dose-dependent increase in fasting insulin concentration and decrease in insulin sensitivity, as assessed by HOMA, was observed with both modes of treatment. At 5 mg/kg per day, hepatic growth hormone receptor expression was increased compared to vehicle-treated animals, by both modes of administration. Pulsatile variant of growth hormone did not alter the plasma insulin-like growth factor-1 concentration, whereas a slight decrease was observed with continuous variant of growth hormone treatment. Neither pulsatile nor continuous treatment affected hepatic insulin-like growth factor-1 mRNA expression. Our findings suggest that pulsatile variant of growth hormone treatment was more effective in stimulating growth but caused marked hyperinsulinemia in mice.

  4. The secretion of hormones during the culture of human preimplantation embryos with corona cells.

    PubMed

    Shutt, D A; Lopata, A

    1981-04-01

    Following in vitro fertilization of human preovulatory oocytes from spontaneously ovulating women, determinations were made of the secretion into the culture medium of progesterone, estradiol, human chorionic gonadotropin (hCG), and the prostaglandins (PG) E2 and F2 alpha, over a 3- to 4-day period of embryo development. It was found that the corona cells associated with the egg could account, between days 2 and 3, for a mean daily secretion of 50 ng of progesterone and approximately 100 pg of estradiol, PGE2, and PGF2 alpha, respectively. Mechanical removal of the corona cells after about 48 hours for the examination of the egg for cleavage reduced the mean amount of progesterone produced on day 3 to 2 ng, and a concomitant decrease in estradiol. PGE2, and PGF2 alpha was observed. Steroid secretion could be restored on day 3 to more than 50% of that secreted on day 2 by returning some detached corona cells to the culture medium containing the embryo. hCG was not detected (less than 2 mIU/ml) in the culture medium at any stage of embryo culture over the 3- to 4-day period.

  5. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

    PubMed Central

    Rufo, P A; Jiang, L; Moe, S J; Brugnara, C; Alper, S L; Lencer, W I

    1996-01-01

    Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals. PMID:8903326

  6. Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion

    PubMed Central

    Cuman, Carly; Van Sinderen, Michelle; Gantier, Michael P.; Rainczuk, Kate; Sorby, Kelli; Rombauts, Luk; Osianlis, Tiki; Dimitriadis, Evdokia

    2015-01-01

    Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure. PMID:26629549

  7. Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

    PubMed

    Schwenke, Maren; Knöfler, Martin; Velicky, Philipp; Weimar, Charlotte H E; Kruse, Michelle; Samalecos, Annemarie; Wolf, Anja; Macklon, Nick S; Bamberger, Ana-Maria; Gellersen, Birgit

    2013-01-01

    Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of

  8. Non-steroidal anti-inflammatory drugs and prostaglandin effects on pepsinogen secretion by dispersed human peptic cells.

    PubMed Central

    Lanas, A I; Nerín, J; Esteva, F; Sáinz, R

    1995-01-01

    The effects of aspirin and ibuprofen on pepsinogen secretion were studied in isolated human peptic cells prepared from endoscopically obtained biopsy specimens after collagenase digestion, mechanical disruption, and percoll gradient centrifugation. Pharmacological concentrations of aspirin and ibuprofen (10(-8)-10(-4) M), potentiated histamine (10(-6)-10(-4)M) and forskolin (10(-5)M) stimulated pepsinogen secretion without affecting basal secretion, acetylcholine (10(-6)M) stimulated pepsinogen secretion or cell vitality. Augmentation of secretagogue stimulated pepsinogen secretion was dependent on extracellular calcium because potentiation was abolished by calcium depletion of the medium. Cimetidine inhibited the potentiation effect on histamine but not on forskolin stimulated pepsinogen secretion, thus suggesting that this augmentation was independent of histamine H2 receptors. Of interest, potentiation was also independent of endogenous prostaglandin inhibition because exogenous addition of prostaglandin E2 and D2 increased both basal and acetylcholine stimulated pepsinogen secretion in a dose dependent way, but they did not modify histamine or histamine plus aspirin or ibuprofen stimulated pepsinogen secretion. In conclusion, aspirin and ibuprofen potentiate secretagogue stimulated pepsinogen secretion by dispersed human peptic cells and this might be an additional mechanism of non-steroidal anti-inflammatory drug (NSAID) induced gastric injury. This potentiation effect is regulated by calcium, independent of endogenous prostaglandin inhibition and seems to act on pepsinogen secretion at a post-receptor site. PMID:7797113

  9. Simian virus 5 is a poor inducer of chemokine secretion from human lung epithelial cells: identification of viral mutants that activate interleukin-8 secretion by distinct mechanisms.

    PubMed

    Young, Virginia A; Parks, Griffith D

    2003-06-01

    We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI-) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.

  10. Effects of Methoxychlor and Its Metabolite Hydroxychlor on Human Placental 3β-Hydroxysteroid Dehydrogenase 1 and Aromatase in JEG-3 Cells.

    PubMed

    Liu, Shiwen; Mao, Baiping; Bai, Yanfang; Liu, Jianpeng; Li, Huitao; Li, Xiaoheng; Lian, Qingquan; Ge, Ren-Shan

    2016-01-01

    Progesterone and estradiol produced by the human placenta are critical for maintenance of pregnancy and fetal development. In the human placenta, 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) is responsible for the formation of progesterone from pregnenolone and aromatase (CYP19A1) for the production of estradiol from androgen. Insecticide methoxychlor (MXC) and its metabolite hydroxychlor (HPTE) may disrupt the activities of these 2 enzymes. In this study, we investigated the effects of MXC and HPTE on steroid production in human placental JEG-3 cells and on HSD3B1 and CYP19A1 activities. MXC and HPTE inhibited progesterone and estradiol production in JEG-3 cells. MXC and HPTE were potent HSD3B1 inhibitors with the half maximal inhibitory concentration (IC50) values of 2.339 ± 0.096 and 1.918 ± 0.078 μmol/l, respectively. MXC had no inhibition on CYP19A1 at 100 μmol/l, while HPTE was a weak inhibitor with IC50 of 97.16 ± 0.10 μmol/l. When pregnenolone was used to determine the inhibitory mode, MXC and HPTE were found to be competitive inhibitors of HSD3B1. When cofactor NAD+ was used, MXC and HPTE were the noncompetitive inhibitors of HSD3B1. When testosterone was used, HPTE was a mixed inhibitor of CYP19A1. In conclusion, MXC and HPTE are potent inhibitors of human HSD3B1, and HPTE is a weak CYP19A1 inhibitor.

  11. Human iPSC-derived Immature Astroglia Promote Oligodendrogenesis by increased TIMP-1 Secretion

    PubMed Central

    Jiang, Peng; Chen, Chen; Liu, Xiao-Bo; Pleasure, David E.; Liu, Ying; Deng, Wenbin

    2016-01-01

    SUMMARY Astrocytes, once considered passive support cells, are increasingly appreciated as dynamic regulators of neuronal development and function, in part via secreted factors. The extent to which they similarly regulate oligodendrocytes, or proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) is less well understood. Here, we generated astrocytes from human pluripotent stem cells (hiPSC-Astros) and demonstrate that immature astrocytes - as opposed to mature - promoted oligodendrogenesis in vitro. In the PVL mouse model of neonatal hypoxic/ischemic encephalopathy, associated with cerebral palsy in humans, transplanted immature hiPSC-Astros promote myelinogenesis and behavioral outcome. We further identified TIMP-1 as a selectively upregulated component secreted from immature hiPSC-Astros. Accordingly, in the rat PVL model, intranasal administration of conditioned medium from immature hiPSC-Astros promoted oligodendrocyte maturation in a TIMP-1 dependent manner. Our findings suggest stage-specific developmental interactions between astroglia and oligodendroglia, with important therapeutic implications for promoting myelinogenesis. PMID:27134175

  12. Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

    PubMed

    Wawrzyniak, Ivan; Texier, Catherine; Poirier, Philippe; Viscogliosi, Eric; Tan, Kevin S W; Delbac, Frédéric; El Alaoui, Hicham

    2012-09-01

    Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

  13. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment.

    PubMed

    Almeida, Catarina R; Caires, Hugo R; Vasconcelos, Daniela P; Barbosa, Mário A

    2016-04-12

    Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

  14. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment

    PubMed Central

    Almeida, Catarina R.; Caires, Hugo R.; Vasconcelos, Daniela P.; Barbosa, Mário A.

    2016-01-01

    Summary Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. PMID:27052313

  15. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    PubMed Central

    Bachnoff, Niv; Cohen-Kutner, Moshe; Atlas, Daphne

    2011-01-01

    A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α11.2, the pore forming subunit of CaV1.2. PMID:22216029

  16. Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells.

    PubMed

    Tang, Ying-Sheng; Khan, Rehana A; Xiao, Suhong; Hansen, Deborah K; Stabler, Sally P; Kusumanchi, Praveen; Jayaram, Hiremagalur N; Antony, Aśok C

    2017-04-01

    and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1.

  17. Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

    PubMed Central

    Vasilopoulou, E.; Loubière, L.S.; Lash, G.E.; Ohizua, O.; McCabe, C.J.; Franklyn, J.A.; Kilby, M.D.; Chan, S.Y.

    2014-01-01

    normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory ‘switch’ in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S) This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research – Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose. PMID:24626803

  18. Full-Length Human Placental sFlt-1-e15a Isoform Induces Distinct Maternal Phenotypes of Preeclampsia in Mice

    PubMed Central

    Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L.; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S.; Hernandez-Andrade, Edgar; Than, Nandor Gabor

    2015-01-01

    Objective Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Methods Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Results Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the

  19. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  20. Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

    PubMed

    Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V; Walton, Ross; Johnston, Sebastian L; Solari, Roberto

    2014-10-01

    The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be

  1. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  2. Interaction of differentiated human adipocytes with macrophages leads to trogocytosis and selective IL-6 secretion

    PubMed Central

    Sárvári, A K; Doan-Xuan, Q-M; Bacsó, Z; Csomós, I; Balajthy, Z; Fésüs, L

    2015-01-01

    Obesity leads to adipose tissue inflammation that is characterized by increased release of proinflammatory molecules and the recruitment of activated immune cells. Although macrophages are present in the highest number among the immune cells in obese adipose tissue, not much is known about their direct interaction with adipocytes. We have introduced an ex vivo experimental system to characterize the cellular interactions and the profile of secreted cytokines in cocultures of macrophages and human adipocytes differentiated from either mesenchymal stem cells or a preadipocyte cell line. As observed by time-lapse microscopy, flow, and laser-scanning cytometry, macrophages phagocytosed bites of adipocytes (trogocytosis), which led to their de novo, phagocytosis and NF-κB-dependent synthesis, then release of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1. IL-6 secretion was not accompanied by secretion of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-8, except MCP-1. LPS-induced release of TNF-α, IL-8 and MCP-1 was decreased in the presence of the differentiated adipocytes but the IL-6 level did not subside suggesting that phagocytosis-dependent IL-6 secretion may have significant regulatory function in the inflamed adipose tissue. PMID:25611388

  3. Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.

    PubMed

    Barile, Lucio; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Moccetti, Tiziano; Vassalli, Giuseppe

    2012-01-01

    The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34⁺ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  4. Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion.

    PubMed

    Mizgier, Maria L; Cataldo, Luis R; Gutierrez, Juan; Santos, José L; Casas, Mariana; Llanos, Paola; Contreras-Ferrat, Ariel E; Moro, Cedric; Bouzakri, Karim; Galgani, Jose E

    2017-01-01

    Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100 nmol/L) for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.

  5. Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion

    PubMed Central

    Cataldo, Luis R.; Gutierrez, Juan; Santos, José L.; Casas, Mariana; Contreras-Ferrat, Ariel E.; Moro, Cedric; Bouzakri, Karim

    2017-01-01

    Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100 nmol/L) for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets. PMID:28286777

  6. Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes

    PubMed Central

    2014-01-01

    Background Chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers contribute to a considerable amount of mortality in the U.S. annually. The inability of these wounds to heal has now been associated with the presence of microbial biofilms. The aim of this study was to determine if products secreted by S. aureus biofilms play an active role in chronic wounds by promoting inflammation, which is a hallmark of chronic wounds. Methods In vitro experiments were conducted to examine changes in gene expression profiles and inflammatory response of human epithelial keratinocytes (HEKa) exposed to products secreted by S. aureus grown in biofilms or products secreted by S. aureus grown planktonically. Results After only two hours of exposure, gene expression microarray data showed marked differences in inflammatory, apoptotic, and nitric oxide responses between HEKa cells exposed to S. aureus biofilm conditioned media (BCM) and HEKa cells exposed to S. aureus planktonic conditioned media (PCM). As early as 4 hours post exposure, ELISA results showed significant increases in IL-6, IL-8, TNFα, and CXCL2 production by HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or controls. Nitric oxide assay data also showed significant increases in nitric oxide production by HEKa cells treated with BCM compared to HEKa cells treated with PCM, or controls. Conclusions Taken together, these results support and extend previous findings that indicate products secreted by S. aureus biofilms directly contribute to the chronic inflammation associated with chronic wounds. PMID:24936153

  7. Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.

    PubMed

    da Silva, Bianca Alcântara; Sodré, Cátia Lacerda; Souza-Gonçalves, Ana Luiza; Aor, Ana Carolina; Kneipp, Lucimar Ferreira; Fonseca, Beatriz Bastos; Rozental, Sonia; Romanos, Maria Teresa Villela; Sola-Penna, Mauro; Perales, Jonas; Kalume, Dário Eluan; dos Santos, André Luis Souza

    2012-01-01

    Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P

  8. Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes

    SciTech Connect

    Imamura, K.; Spriggs, D.; Ohno, T.; Kufe, D.

    1989-05-01

    Botulinum toxins are potent neurotoxins which block the release of neurotransmitters. The effects of these toxins on hematopoietic cells, however, are unknown. Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF). In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined. The results demonstrate that biotulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate. Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process. This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-(/gamma/-thio)t-riphosphate into these cells. The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholiphase C and another which acts as a substrate for botulinum toxin type D. ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with M/sub r/s of 45,000, 21,000, and 17,000. While the role of these substrates in exocytosis is unknown, the results suggest that the M/sub r/ 21,000 substrate is involved in a process other than TNF secretion.

  9. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

  10. Intracellular Organisms as Placental Invaders

    PubMed Central

    Vigliani, Marguerite B.; Bakardjiev, Anna I.

    2015-01-01

    In this article we present a novel model for how the human placenta might get infected via the hematogenous route. We present a list of diverse placental pathogens, like Listeria monocytogenes or Cytomegalovirus, which are familiar to most obstetricians, but others, like Salmonella typhi, have only been reported in case studies or small case series. Remarkably, all of these organisms on this list are either obligate or facultative intracellular organisms. These pathogens are able to enter and survive inside host immune cells for at least a portion of their life cycle. We suggest that many blood-borne pathogens might arrive at the placenta via transportation inside of maternal leukocytes that enter the decidua in early pregnancy. We discuss mechanisms by which extravillous trophoblasts could get infected in the decidua and spread infection to other layers in the placenta. We hope to raise awareness among OB/GYN clinicians that organisms not typically associated with the TORCH list might cause placental infections and pregnancy complications. PMID:27695204

  11. The production and secretion of complement component C1q by human mast cells.

    PubMed

    van Schaarenburg, Rosanne A; Suurmond, Jolien; Habets, Kim L L; Brouwer, Mieke C; Wouters, Diana; Kurreeman, Fina A S; Huizinga, Tom W J; Toes, René E M; Trouw, Leendert A

    2016-10-01

    C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN- γ or via FcεRI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue.

  12. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells

    PubMed Central

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O’Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  13. Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability.

    PubMed

    Fitzsimmons, C; Proudfoot, D; Bowyer, D E

    1999-02-01

    Extracellular matrix remodelling occurs during atherosclerosis dictating the structure of the plaque and thus the resistance to rupture. Monocytes and macrophages are believed to play a role in this remodelling. In the present study, filter-separated co-culture has been used to study the effect of monocytes on procollagen turnover by human vascular smooth muscle cells (VSMC). In this system, freshly isolated human peripheral blood monocytes inhibited procollagen secretion from VSMC without affecting either degradation of procollagen, or DNA synthesis by the VSMC. Insertion of a 12 kDa dialysis membrane between the two cell types and treatment with indomethacin showed that the inhibitory factor was of low molecular weight and was cyclooxygenase-dependent. Pre-incubation of each cell type with indomethacin demonstrated that monocyte, but not VSMC cyclooxygenase was required. Thus, the inhibitory effect on procollagen secretion was due, most likely, to monocyte prostaglandins. Neither inhibition of thromboxane synthetase, nor blocking IL-1 activity, reduced the inhibitory activity. Addition of prostaglandins PGE1, PGE2 and PGF2alpha to VSMC cultures caused a reduction in procollagen secretion which was equivalent to, but was not additive with, the maximal effect achieved by monocytes. Monocytes and macrophages are a major source of prostaglandins and these molecules are likely to play an important role in collagen turnover within lesions.

  14. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    SciTech Connect

    Giblin, P.; Kavathas, P. ); Ledbetter, J.A. )

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  15. Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer.

    PubMed

    Porter, Charlene; Armstrong-Fisher, Sylvia; Kopotsha, Tim; Smith, Bryan; Baker, Terry; Kevorkian, Lara; Nesbitt, Andrew

    2016-08-01

    Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus.

  16. Placental corticotrophin-releasing hormone, local effects and fetomaternal endocrinology.

    PubMed

    King, B R; Nicholson, R C; Smith, R

    2001-12-01

    The human placenta produces corticotrophin-releasing hormone (CRH) in exponentially increasing amounts during pregnancy with peak levels during labour. CRH in human pregnancy appears to be involved in many aspects of pregnancy including placental bloodflow, placental prostaglandin production, myornetrial function, fetal pituitary and adrenal function and the maternal stress axis. Since fetal cortisol levels are associated with pulmonary development and maturity, placental CRH may have an indirect role in fetal development.Although the precise role of placental CRH in the regulation of gestational length and timing of parturition is unclear it appears to be involved in a placental clock. While glucocorticoids inhibit hypothalamic CRH production they stimulate CRH gene expression in the placenta.This difference may allow the fetal and maternal stress axes to influence this placental clock.Maternal CRH levels are elevated in many pathological conditions of pregnancy where fetal well-being is compromised, and in these situations it may act to maintain a stable intrauterine environment. Therefore, CRH appears to link placental function, maternal well-being, fetal well-being and fetal development to the duration of gestation and the timing of parturition.

  17. The composition f human saliva secreted in response to a gustatory stimulus and to pilocaprine.

    PubMed

    Dawes, C

    1966-03-01

    1. The composition of human saliva secreted in response to sour lemon drops (S.L.D.), and pilocarpine, was studied.2. At a given flow rate, pilocarpine-stimulated submandibular and parotid saliva contained less sodium and potassium and an equivalent amount of inorganic phosphate, and parotid saliva also contained more calcium and protein than did the corresponding types of S.L.D.-stimulated saliva.3. Prolonged S.L.D. stimulation did not cause a depletion in the protein concentration of either parotid or submandibular saliva and neither this procedure nor pilocarpine stimulation altered the proportions of the different proteins secreted.4. Pilocarpine was judged to be an inadequate substitute for more physiological, gustatory stimuli.

  18. Secretion of the human T cell leukemia virus type I transactivator protein tax.

    PubMed

    Alefantis, Timothy; Mostoller, Kate; Jain, Pooja; Harhaj, Edward; Grant, Christian; Wigdahl, Brian

    2005-04-29

    Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

  19. Interleukin 2 modulates ion secretion and cell proliferation in cultured human small intestinal enterocytes

    PubMed Central

    O'Loughlin, E; Pang, G; Noltorp, R; Koina, C; Batey, R; Clancy, R

    2001-01-01

    AIMS—To determine if interleukin 2 (IL-2) alters epithelial transport and barrier function in cultured human small intestinal enterocytes.
METHODS—Confluent monolayers of small intestinal cells derived from duodenal biopsies were treated with IL-2 0.2-50 U/ml for 24 hours prior to study. Transport measurements were performed under short circuited conditions in Ussing chambers, with and without the secretagogues forskolin and 3-isobutyl-1-methyl xanthine (IBMX). Serosal to mucosal flux of 3[H] mannitol (permeability) and 3[H] thymidine uptake (proliferation) were measured. IL-2 receptor and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA were identified using reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS—IL-2 did not alter baseline electrical parameters but caused a significant increase in cAMP dependent chloride secretion. The effect was mediated by the IL-2 receptor and paralleled a rapid increase in tyrosine phosphorylation, janus kinase 1, and signal transducers and activators of transcription (STATs) 1, 3, and 5. IL-2 significantly increased proliferation but at a lower dose than observed for enhanced secretion but did not alter permeability. IL-2 receptor β and γc chains and CFTR mRNA were identified by RT-PCR.
CONCLUSIONS—IL-2 treatment enhances cAMP stimulated chloride secretion and cellular proliferation in a human small intestinal cell line expressing a functional IL-2 receptor.


Keywords: interleukin 2; ion secretion; cell proliferation; enterocytes; small intestine PMID:11600465

  20. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

    PubMed

    Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

    2016-08-09

    Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

  1. Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats

    PubMed Central

    Jin, Luyuan; Qin, Lizheng; Xia, Dengsheng; Liu, Xibao; Fan, Zhipeng; Zhang, Chunmei; Gu, Liankun; He, Junqi; Ambudkar, Indu S.; Deng, Dajun; Wang, Songlin

    2014-01-01

    Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1 h later (p < 0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1 h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastricmucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p < 0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was

  2. Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats.

    PubMed

    Jin, Luyuan; Qin, Lizheng; Xia, Dengsheng; Liu, Xibao; Fan, Zhipeng; Zhang, Chunmei; Gu, Liankun; He, Junqi; Ambudkar, Indu S; Deng, Dajun; Wang, Songlin

    2013-04-01

    Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was

  3. Potential roles of placental human beta-defensin-3 and apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G in prevention of intrauterine transmission of hepatitis B virus.

    PubMed

    Bai, Xiaoxia; Tian, Ting; Wang, Peng; Yang, Xiaofu; Wang, Zhengping; Dong, Minyue

    2015-03-01

    Approximately 5% of newborns were infected by hepatitis B virus (HBV) via intrauterine transmission and this is the main reason for high prevalence of HBV in endemic regions. However, the mechanisms by which intrauterine transmission is avoided in most cases remain elusive and placental natural anti-microbial factors may play a role in the prevention of HBV intrauterine transmission. The expression levels of human β-defensin-3 (HBD-3), apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G (A3G) and mannose binding lectin (MBL) were determined in the placenta of 30 HBV-seronegative pregnant women (controls), 7 HBV-seropositive pregnant women with infants infected via intrauterine transmission (infected group) and 30 HBV-seropositive pregnant women with non-infected infants (non-infected group). The expression of HBD-3, A3G, and MBL of placental trophoblast cell line Swan71 was determined after exposed to HBV. There were significant differences in placental HBD-3 and A3G levels among three groups, but the expression of MBL did not significantly differ. The expressions of HBD-3 and A3G were higher in non-infected group than controls and infected group, but not significantly different between infected group and controls. The exposure to HBV increased significantly the expression of HBD-3, A3G, and MBL by Swan 71. It may be concluded HBV up-regulates HBD-3 and A3G expression in vivo and in vitro in placental trophoblast and lack of this up-regulation is possibly associated with intrauterine transmission of HBV.

  4. Placental Nutrient Transport and Intrauterine Growth Restriction

    PubMed Central

    Gaccioli, Francesca; Lager, Susanne

    2016-01-01

    Intrauterine growth restriction refers to the inability of the fetus to reach its genetically determined potential size. Fetal growth restriction affects approximately 5–15% of all pregnancies in the United States and Europe. In developing countries the occurrence varies widely between 10 and 55%, impacting about 30 million newborns per year. Besides having high perinatal mortality rates these infants are at greater risk for severe adverse outcomes, such as hypoxic ischemic encephalopathy and cerebral palsy. Moreover, reduced fetal growth has lifelong health consequences, including higher risks of developing metabolic and cardiovascular diseases in adulthood. Numerous reports indicate placental insufficiency as one of the underlying causes leading to altered fetal growth and impaired placental capacity of delivering nutrients to the fetus has been shown to contribute to the etiology of intrauterine growth restriction. Indeed, reduced expression and/or activity of placental nutrient transporters have been demonstrated in several conditions associated with an increased risk of delivering a small or growth restricted infant. This review focuses on human pregnancies and summarizes the changes in placental amino acid, fatty acid, and glucose transport reported in conditions associated with intrauterine growth restriction, such as maternal undernutrition, pre-eclampsia, young maternal age, high altitude and infection. PMID:26909042

  5. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein.

    PubMed

    Xu, Juan; Zhang, Chunhua

    2016-05-01

    Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.

  6. Nonlinear dynamics in pulsatile secretion of parathyroid hormone in normal human subjects

    NASA Astrophysics Data System (ADS)

    Prank, Klaus; Harms, Heio; Brabant, Georg; Hesch, Rolf-Dieter; Dämmig, Matthias; Mitschke, Fedor

    1995-03-01

    In many biological systems, information is transferred by hormonal ligands, and it is assumed that these hormonal signals encode developmental and regulatory programs in mammalian organisms. In contrast to the dogma of endocrine homeostasis, it could be shown that the biological information in hormonal networks is not only present as a constant hormone concentration in the circulation pool. Recently, it has become apparent that hormone pulses contribute to this hormonal pool, which modulates the responsiveness of receptors within the cell membrane by regulation of the receptor synthesis, movement within the membrane layer, coupling to signal transduction proteins and internalization. Phase space analysis of dynamic parathyroid hormone (PTH) secretion allowed the definition of a (in comparison to normal subjects) relatively quiet ``low dynamic'' secretory pattern in osteoporosis, and a ``high dynamic'' state in hyperparathyroidism. We now investigate whether this pulsatile secretion of PTH in healthy men exhibits characteristics of nonlinear determinism. Our findings suggest that this is conceivable, although on the basis of presently available data and techniques, no proof can be established. Nevertheless, pulsatile secretion of PTH might be a first example of nonlinear deterministic dynamics in an apparently irregular hormonal rhythm in human physiology.

  7. Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts

    PubMed Central

    Villa, Oscar; Brookes, Steven J; Thiede, Bernd; Heijl, Lars; Lyngstadaas, Staale P

    2015-01-01

    Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–electrospray ionization–tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography–electrospray ionization–tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis. PMID:26090085

  8. Cadherin engagement improves insulin secretion of single human β-cells.

    PubMed

    Parnaud, Geraldine; Lavallard, Vanessa; Bedat, Benoît; Matthey-Doret, David; Morel, Philippe; Berney, Thierry; Bosco, Domenico

    2015-03-01

    The aim of this study was to assess whether cadherin-mediated adhesion of human islet cells was affected by insulin secretagogues and explore the role of cadherins in the secretory activity of β-cells. Experiments were carried out with single islet cells adherent to chimeric proteins made of functional E-, N-, or P-cadherin ectodomains fused to the Fc fragment of immunoglobulin (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on an inert substrate. We observed that cadherin expression in islet cells was not affected by insulin secretagogues. Adhesion tests showed that islet cells attached to N-cad/Fc and E-cad/Fc acquired, in a time- and secretagogue-dependent manner, a spreading form that was inhibited by blocking cadherin antibodies. By reverse hemolytic plaque assay, we showed that glucose-stimulated insulin secretion of single β-cells was increased by N-cad/Fc and E-cad/Fc adhesion compared with control. In the presence of E-cad/Fc and after glucose stimulation, we showed that total insulin secretion was six times higher in spreading β-cells compared with round β-cells. Furthermore, cadherin-mediated adhesion induced an asymmetric distribution of cortical actin in β-cells. Our results demonstrate that adhesion of β-cells to E- and N-cadherins is regulated by insulin secretagogues and that E- and N-cadherin engagement promotes stimulated insulin secretion.

  9. Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts.

    PubMed

    Villa, Oscar; Brookes, Steven J; Thiede, Bernd; Heijl, Lars; Lyngstadaas, Staale P; Reseland, Janne E

    2015-01-01

    Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography-electrospray ionization-tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

  10. Cigarette smoke extract induces placental growth factor release from human bronchial epithelial cells via ROS/MAPK (ERK-1/2)/Egr-1 axis

    PubMed Central

    Wu, Dong; Yuan, Yalian; Lin, Zhixiu; Lai, Tianwen; Chen, Min; Li, Wen; Lv, Quanchao; Yuan, Binfan; Li, Dongmin; Wu, Bin

    2016-01-01

    Etiological evidence demonstrates that there is a significant association between cigarette smoking and chronic airway inflammatory disease. Abnormal expression of placental growth factor (PlGF) has been reported in COPD, and its downstream signaling molecules have been reported to contribute to the pathogenesis of airway epithelial cell apoptosis and emphysema. However, the signaling mechanisms underlying cigarette smoke extract (CSE)-induced PlGF expression in airway microenvironment remain unclear. Herein, we investigated the effects of reactive oxygen species (ROS)-dependent activation of the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase1/2 [ERK-1/2])/early growth response-1 (Egr-1) pathway on CSE-induced PlGF upregulation in human bronchial epithelium (HBE). The data obtained with quantitative reverse transcription polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining analyses showed that CSE-induced Egr-1 activation was mainly mediated through production of ROS and activation of the MAPK (ERK-1/2) cascade. The binding of Egr-1 to the PlGF promoter was corroborated by an ELISA-based DNA binding activity assay. These results demonstrate that ROS activation of the MAPK (ERK-1/2)/Egr-1 pathway is a main player in the regulatory mechanism for CSE-induced PlGF production and that the use of an antioxidant could partly abolish these effects. Understanding the mechanisms of PlGF upregulation by CSE in the airway microenvironment may provide rational therapeutic interventions for cigarette smoking-related airway inflammatory diseases. PMID:27980400

  11. A study of the distribution of aluminum in human placental tissues based on alkaline solubilization with determination by electrothermal atomic absorption spectrometry

    PubMed Central

    Kruger, Pamela C.; Schell, Lawrence M.; Stark, Alice D.; Parsons, Patrick J.

    2010-01-01

    Summary Aluminum (Al) is a nonessential element known to induce neurotoxic effects, such as dialysis dementia, in patients on hemodialysis, with compromised kidney function. The role of Al in the progression of some neurodegenerative diseases, such as Alzheimer’s disease (AD), is controversial, and remains unclear. The effects of Al on other vulnerable populations, such as fetuses and infants, have been infrequently studied. In the present study, Al has been measured in human placenta samples, comprising ~160 each of placenta bodies, placenta membranes, and umbilical cords, using electrothermal atomic absorption spectrometry (ETAAS) after atmospheric pressure digestion with tetramethylammonium hydroxide (TMAH) and ethylenediaminetetraacidic acid (EDTA). The sensitivity, or characteristic mass (m0), for Al at the 309.3-nm line was found to be 30 ± 4 pg. The instrumental detection limit (IDL) (3s) for Al in solution was calculated as 0.72 μg L-1, while the method detection limit (MDL) (3s) was 0.25 μg g-1. Accuracy was assessed through analysis of quality control (QC) materials, including certified reference materials (CRMs), in-house reference materials (RMs), and spike recovery experiments, of varying matrices. Placental tissue analyses revealed geometric mean concentrations of approximately 0.5 μg g-1 Al in placenta bodies (n=165) and membranes (n=155), while Al concentrations in the umbilical cord (n=154) were about 0.3 μg g-1. Al was detected in 95% of placenta bodies, and 81% of placenta membranes, but only in 46% of umbilical cords. PMID:21072353

  12. Influence of cetirizine and levocetirizine on two cytokines secretion in human airway epithelial cells.

    PubMed

    Shih, Mei-Yin; Hsu, Jeng-Yuan; Weng, Yueh-Shan; Fu, Lin-Shien

    2008-01-01

    Recent studies suggest that several second-generation antihistamines can modulate various inflammatory reactions besides their H(1)-receptor antagonism. The antihistamine cetirizine is a racemic mixture of levocetirizine and dextrocetirizine. The aim of this study was to investigate the effects of these two antihistamines (cetirizine and levocetirizine) on granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 secretion in A549 human airway epithelial cells. A549 cells were preincubated with cetirizine (0.1, 1, 2.5, 5, and 10 microM) or levocetirizine (0.1, 1, 2.5, 5, and 10 microM) individually for 16 hours and were then stimulated with IL-1beta for 8 hours. The levels of GM-CSF and IL-8 in cultured supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that cetirizine (5 and 10 microM) and levocetirizine (2.5, 5, and 10 microM) significantly suppressed GM-CSF secretion from A549 cells stimulated with IL-1beta (p<0.05). Cetirizine (10 microM) and levocetirizine (5 and 10 microM) significantly suppressed IL-8 secretion after A549 was stimulated. The suppressive effect was comparable between levocetirizine, 2.5 microM, and cetirizine, 5 microM, as well as levocetirizine, 5 microM, and cetirizine, 10 microM. Moreover, levocetirizine, 5 microM, was better than cetirizine, 5 microM, on suppressing IL-8 secretion, but such a difference did not appear in other conditions. Our results suggest that cetirizine and levocetirizine at higher concentrations can reduce the release of GM-CSF and IL-8 from A549 cells stimulated with IL-1beta. These observations indicate that the two second-generation antihistamines may exert anti-inflammatory effects beyond histamine H(1)-receptor antagonist, and levocetirizine plays a major role in terms of this activity.

  13. Proopiomelanocortin, glucocorticoid, and CRH receptor expression in human ACTH-secreting pituitary adenomas.

    PubMed

    Cassarino, Maria Francesca; Sesta, Antonella; Pagliardini, Luca; Losa, Marco; Lasio, Giovanni; Cavagnini, Francesco; Pecori Giraldi, Francesca

    2017-03-01

    ACTH-secreting pituitary tumors are by definition partially autonomous, i.e., secrete ACTH independent of physiological control. However, only few, small-sized studies on proopiomelanocortin (POMC) and its regulation by corticotropin-releasing hormone (CRH) or glucocorticoids are available. Objective of the present study was to report on constitutive and CRH- and dexamethasone-regulated POMC, CRH (CRH-R1), and glucocorticoid receptor (NR3C1) gene expression in a large series of human corticotrope adenomas. Fifty-three ACTH-secreting adenomas were incubated with 10 nM CRH or 10 nM dexamethasone for 24 h. POMC, CRH-R1, NR3C1, and its alpha and beta isoforms were quantified and medium ACTH measured. Constitutive POMC expression proved extremely variable, with macroadenomas exhibiting higher levels than microadenomas. POMC increased during CRH in most specimens; conversely, changes induced by dexamethasone were varied, ranging from decrease to paradoxical increase. No correlation between POMC and ACTH was detected in any experimental condition. CRH-R1 expression was not linked to the response to CRH while NR3C1 was expressed at greater levels in specimens who failed to inhibit during dexamethasone; glucocorticoid receptor α was the more abundant isoform and subject to down-regulation by dexamethasone. Our results demonstrate a considerable variability in POMC expression among tumors and no correlation between POMC and ACTH, suggesting that POMC peptide processing/transport plays a major role in modulating ACTH secretion. Further, CRH-R1 and NR3C1 expression were not linked to the expected ligand-induced outcome, indicating that receptor signaling rather than abundance determines corticotrope responses. Our findings pave the way to new avenues of research into Cushing's disease pathophysiology.

  14. A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

    PubMed

    Al-Romaiyan, A; Liu, B; Asare-Anane, H; Maity, C R; Chatterjee, S K; Koley, N; Biswas, T; Chatterji, A K; Huang, G-C; Amiel, S A; Persaud, S J; Jones, P M

    2010-09-01

    Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

  15. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  16. Expression and function of thyroid hormone transporters in the microvillous plasma membrane of human term placental syncytiotrophoblast.

    PubMed

    Loubière, L S; Vasilopoulou, E; Glazier, J D; Taylor, P M; Franklyn, J A; Kilby, M D; Chan, Shiao Y

    2012-12-01

    The transplacental passage of thyroid hormones (THs) from mother to fetus in humans has been deduced from observational clinical studies and is important for normal fetoplacental development. To investigate the transporters that regulate TH uptake by syncytiotrophoblast (the primary barrier to maternal-fetal exchange, which lies in direct contact with maternal blood), we isolated the microvillous plasma membrane (MVM) of human term syncytiotrophoblasts. We have demonstrated that MVM vesicles express plasma membrane TH transporter proteins, including system-L (L-type amino acid transporter 1 and CD98), monocarboxylate transporters (MCTs) 8 and 10, organic anion-transporting polypeptides 1A2 and 4A1. We provide the first definitive evidence that the human syncytiotrophoblast MVM is capable of rapid, saturable T(4) and T(3) uptake at similar rates and in a Na(+)-independent manner. These two major forms of THs could not significantly inhibit each others' uptake, suggesting that each is mediated by largely different transporters. No single transporter was noted to play a dominant role in either T(4) or T(3) uptake. Using combinations of transporter inhibitors that had an additive effect on TH uptake, we provide evidence that 67% of saturable T(4) uptake is facilitated by system-L and MCT10 with a minor role played by organic anion-transporting polypeptides, whereas 87% of saturable T(3) uptake is mediated by MCT8 and MCT10. Our data demonstrate that syncytiotrophoblast may control the quantity and forms of THs taken up by the human placenta. Thus, syncytiotrophoblast could be critical in regulating transplacental TH supply from the mother to the fetus.

  17. Human placental glucose dehydrogenase: IEF polymorphism in two Italian populations and enzyme activity in the six common phenotypes.

    PubMed

    Scacchi, R; Corbo, R M; Calzolari, E; Laconi, G; Palmarino, R; Lucarelli, P

    1985-01-01

    Glucose dehydrogenase (hexose-6-phosphate dehydrogenase) has been assayed qualitatively and quantitatively in more than 600 human placentae collected in two Italian populations. The gene frequencies for GDH1, GDH2 and GDH3 were, respectively, 0.66, 0.21 and 0.12 in Continental Italy and 0.65, 0.23 and 0.12 in Sardinia. Among the six common phenotypes there was no difference in catalytic activity.

  18. Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from human lung mast cells.

    PubMed Central

    Leung, K B; Flint, K C; Brostoff, J; Hudspith, B N; Johnson, N M; Lau, H Y; Liu, W L; Pearce, F L

    1988-01-01

    Sodium cromoglycate and nedocromil sodium produced a dose dependent inhibition of histamine secretion from human pulmonary mast cells obtained by bronchoalveolar lavage and by enzymatic dissociation of lung parenchyma. Both compounds were significantly more active against the lavage cells than against the dispersed lung cells, and nedocromil sodium was an order of magnitude more effective than sodium cromoglycate against both cell types. Tachyphylaxis was observed with the parenchymal cells but not with the lavage cells. Nedocromil sodium and sodium cromoglycate also inhibited histamine release from the lavage cells of patients with sarcoidosis and extrinsic asthma. PMID:2462755

  19. Adhering maternal platelets can contribute to the cytokine and chemokine cocktail released by human first trimester villous placenta.

    PubMed

    Blaschitz, A; Siwetz, M; Schlenke, P; Gauster, M

    2015-11-01

    Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analyzing the inflammatory secretion profile of human placenta.

  20. The expression and post-transcriptional regulation of FSTL1 transcripts in placental trophoblasts

    PubMed Central

    Mouillet, Jean-Francois; Mishima, Takuya; Paffaro, Andrea Mollica do Amarante; Parks, Tony W.; Ziegler, Judy A.; Chu, Tianjiao; Sadovsky, Yoel

    2015-01-01

    Introduction Follistatin-like-1 (FSTL1) is a widely expressed secreted protein with diverse but poorly understood functions. Originally described as a pro-inflammatory molecule, it has recently been reported to play a role in signaling pathways that regulate development and homeostasis. Distinctively, FSTL1 harbors within its 3′-UTR the sequence encoding microRNA-198 (miR-198), shown to be inversely regulated relative to FSTL1 expression and to exhibit opposite actions on cellular processes such as cell migration. We sought to investigate the expression of FSTL1 and to assess its interplay with miR-198 in human trophoblasts. Methods We used a combination of northern blot analyses, quantitative PCR, small RNA sequencing, western blot and immunohistochemistry to characterize FSTL1 and miR-198 expression in placental trophoblasts. We also used reporter assays to examine the post-transcriptional regulation of FSTL1 and assess its putative regulation by miR-198. Results We detected the expression of FSTL1 transcript in both the human extravillous trophoblast line HTR-8/SVneo and in primary term human villous trophoblasts. We also found that the expression of FSTL1 was largely restricted to extravillous trophoblasts. Hypoxia enhanced the expression of FSTL1 protein in cultured primary villous trophoblasts. Interestingly, we did not detect any evidence for expression or function of mature miR-198 in human trophoblasts. Discussion Our data indicate that placental FSTL1 is expressed particularly in extravillous trophoblasts. We also found no evidence for placental expression of miR-198, or for its regulation of FSTL1, implying that the post-transcriptional regulation of FSTL1 by miR-198 is tissue specific. PMID:26386648

  1. Human antibodies against dengue enhance dengue viral infectivity without suppressing type I interferon secretion in primary human monocytes.

    PubMed

    Kou, Zhihua; Lim, Joanne Y H; Beltramello, Martina; Quinn, Matthew; Chen, Huiyuan; Liu, Shengyong; Liu, Shengyo-ng; Martinez-Sobrido, Luis; Martnez-Sobrido, Luis; Diamond, Michael S; Schlesinger, Jacob J; de Silva, Aravinda; Sallusto, Federica; Jin, Xia

    2011-02-05

    It remains unclear whether antibody-dependent-enhancement (ADE) of dengue infection merely augments viral attachment and entry through Fcγ receptors or immune complex binding to Fcγ receptors triggers an intrinsic signaling cascade that changes the viral permissiveness of the cell. Using human dengue-immune sera and novel human monoclonal antibodies against dengue in combination with virologic and immunologic techniques, we found that ADE infection increased the proportion of infected primary human monocytes modestly from 0.2% ± 0.1% (no Ab) to 1.7% ± 1.6% (with Ab) but the total virus output markedly from 2 ± 2 (× 10(3)) FFU to 120 ± 153 (× 10(3))FFU. However, this increased virus production was not associated with a reduced secretion of type I interferon or an elevated secretion of anti-inflammatory cytokine, IL-10. These results demonstrate that the regulation of virus production in ADE infection of primary human monocytes is more complex than previously appreciated.

  2. Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells.

    PubMed

    Green, Alastair D; Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

    2015-10-01

    We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity.

  3. Reevaluation of Fatty Acid Receptor 1 as a Drug Target for the Stimulation of Insulin Secretion in Humans

    PubMed Central

    Wagner, Robert; Kaiser, Gabriele; Gerst, Felicia; Christiansen, Elisabeth; Due-Hansen, Maria E.; Grundmann, Manuel; Machicao, Fausto; Peter, Andreas; Kostenis, Evi; Ulven, Trond; Fritsche, Andreas; Häring, Hans-Ulrich; Ullrich, Susanne

    2013-01-01

    The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists. PMID:23378609

  4. Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

    PubMed Central

    Chang, C N; Matteucci, M; Perry, L J; Wulf, J J; Chen, C Y; Hitzeman, R A

    1986-01-01

    Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion. Images PMID:3023906

  5. Leukotriene B4 potentiates CpG signaling for enhanced cytokine secretion by human leukocytes.

    PubMed

    Gaudreault, Eric; Gosselin, Jean

    2009-08-15

    TLRs are known to be important in innate host defense against a variety of microbial infections. In particular, TLR9 has been associated with immune defense against different foreign organisms by recognition of unmethylated DNA sequences. In this report, we provide evidence that leukotriene B(4) (LTB(4)) has the capacity to modulate TLR9 expression on human neutrophils. The effect of LTB(4) was found to be specific, because related leukotrienes such as LTC(4) and LTD(4) or neutrophil agonists IL-8 and C5a failed to modulate TLR9 expression in neutrophils. Using fluorochrome-tagged CpG DNA, we observed that LTB(4) treatment also increased TLR9 ligand binding in neutrophils. Moreover, LTB(4) stimulation potentiates CpG-mediated signaling via an endosome-independent mechanism in human neutrophils, leading to enhanced secretion of proinflammatory cytokines. The increase in cytokine secretion by LTB(4) following CpG stimulation of neutrophils was associated with the activation of TGF-beta-activated kinase (TAK-1) as well as p38 and c-Jun (JNK) kinases. In contrast, in PBMC LTB(4) leads to an increase in cytokine secretion following CpG stimulation but via a MyD88- and endosome-dependent mechanism. As observed in neutrophils, PBMC stimulation with LTB(4) in the presence of CpG also results in enhanced TAK-1, p38, and JNK phosphorylation/activation. These data provide new evidence underlying the immunomodulatory properties of LTB(4) leading to antimicrobial defense.

  6. Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways

    PubMed Central

    Cabrera, Lissette; Saavedra, Andrea; Rojas, Susana; Cid, Marcela; Valenzuela, Cristina; Gallegos, David; Careaga, Pamela; Basualto, Emerita; Haensgen, Astrid; Peña, Eduardo; Rivas, Coralia; Vera, Juan Carlos; Gallardo, Victoria; Zúñiga, Leandro; Escudero, Carlos; Sobrevia, Luis; Wareing, Mark; González, Marcelo

    2016-01-01

    HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs.Short-term incubation with insulin increases the NO synthesis in HUVECs.Insulin induces relaxation in human placental vascular bed.Insulin attenuates the constriction induced by hydrogen peroxide in human placenta.The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This

  7. Netrins and Their Roles in Placental Angiogenesis

    PubMed Central

    Dakouane-Giudicelli, Mbarka; Alfaidy, Nadia; de Mazancourt, Philippe

    2014-01-01

    Netrins, a family of laminin-related proteins, were originally identified as axonal guidance molecules. Subsequently, netrins were found to modulate various biological processes including morphogenesis, tumorogenesis, adhesion, and, recently, angiogenesis. In human placenta, the most vascularized organ, the presence of netrins has also been reported. Recent studies demonstrated the involvement of netrins in the regulation of placental angiogenesis. In this review we focused on the role of netrins in human placental angiogenesis. Among all netrins examined, netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. These opposite effects appear to be related to the endothelial cell phenotype studied and seem also to depend on the receptor type to which netrin binds, that is, the canonical receptor member of the DCC family, the members of the UNC5 family, or the noncanonical receptor members of the integrin family or DSCAM. PMID:25143950

  8. Neurotrophins: Role in Placental Growth and Development.

    PubMed

    Sahay, A S; Sundrani, D P; Joshi, S R

    2017-01-01

    Neurotrophins, a family of closely related proteins, were originally identified as growth factors for survival, development, and function of neurons in both the central and peripheral nervous systems. Subsequently, neurotrophins have been shown to have functions in immune and reproductive systems. Neurotrophins like nerve growth factor and brain-derived neurotrophic factor (BDNF) are known to play an important role during pregnancy in the process of placental angiogenesis and maturation. Several studies have demonstrated the presence of neurotrophins in the human placenta. The current chapter reviews studies demonstrating the role of neurotrophins during pregnancy particularly in placental development. This chapter also focuses on the regional changes in neurotrophins in the human placenta and its interactions with other growth factors. Future research is needed to understand the mechanisms through which neurotrophins influence the growth and development of the placenta and pregnancy outcome.

  9. Transport of digoxin-loaded polymeric nanoparticles across BeWo cells, an in vitro model of human placental trophoblast

    PubMed Central

    Albekairi, Norah A; Al-Enazy, Sanaalarab; Ali, Shariq; Rytting, Erik

    2015-01-01

    Background: Fetal arrhythmias can lead to fetal congestive heart failure and hydrops fetalis. Digoxin (the first-line treatment) has low transplacental permeability and high risk of maternal side effects. Biodegradable digoxin-loaded PEGylated poly(lactic-co-glycolic acid) nanoparticles may increase digoxin transport across BeWo b30 cell monolayers (an in vitro model of trophoblast in human placenta) by reducing the drug's interaction with P-gp. Results/methodology: The nanoparticles showed high encapsulation efficiency and sustained release over 48 h. Transport studies revealed significantly increased permeability across BeWo cell layers of digoxin-loaded nanoparticles when compared with free digoxin. P-gp inhibition also increased the permeability of digoxin, but not digoxin-loaded nanoparticles. Conclusion: This represents a novel treatment strategy for fetal cardiovascular disease which may improve maternal and fetal outcomes. PMID:26652279

  10. Zinc uptake by human placental microvillous membrane vesicles: effects of gestational age and maternal serum zinc levels.

    PubMed

    Vargas Zapata, C L; Trugo, N M; Donangelo, C M

    2000-02-01

    Zinc uptake by syncytiotrophoblast microvillous membrane vesicles (SMMV) from human placentas was characterized and the effects of maternal serum zinc levels at term and of gestational age on kinetic parameters were evaluated. Zinc uptake at pH 7.2 was rapid for the first 2 min, followed by a slower increase, approaching equilibrium after 30 min. Uptake was saturable at a zinc concentration of 30 micromol/L, higher than the upper range of the physiological serum zinc level. Kinetic analysis of uptake at 1 min in SMMV from term placenta showed similar Km values (mean: 6.9+/-0.6 micromol/L) for different levels of maternal serum zinc. However, Vmax was higher (p < 0.05) in SMMV from mothers with serum zinc lower than 7.6 micromol/L compared to those with higher serum zinc levels (35.8+/-1.6 and 26.6+/-1.6 nmol 65Zn/mg protein/min, respectively). Km values were similar in term (>37 wk of gestation) and preterm (20-25 wk of gestation) placentas, whereas Vmax was higher (p < 0.05) in the preterm (34.3+/-1.6 nmol Zn/mg protein/min) compared to term placentas from mothers with serum zinc levels above 7.6 micromol/L. These results suggest that whereas afffinity for zinc was not altered with gestational age or maternal serum zinc levels, zinc-uptake capacity in human placenta is influenced both by gestational age and by low levels of maternal serum zinc in order to ensure an adequate maternal-fetal zinc transfer.

  11. The spectrophotometric sulfo-phospho-vanillin assessment of total lipids in human meibomian gland secretions.

    PubMed

    McMahon, Anne; Lu, Hua; Butovich, Igor A

    2013-05-01

    Human meibomian gland secretions (meibum) are the major lipid component of the human preocular tear film. The predominant lipid classes found in meibum include waxes (WE), cholesteryl esters (CE), and varying amounts of cholesterol (Chl). The classical sulfo-phospho-vanillin assay (SPVA), adapted for a microplate reader, was used to quantitate lipids in meibum. To account for varying reactivities of different lipids in SPVA, a model meibomian lipid mixture (MMx) that approximated the WE/CE/Chl composition of meibum was developed and used to quantitate meibomian lipids. The overall SPV responses of MMx and meibum were found to be close, with similar intermediate and final reaction products for both. Saturated WE that had not been expected to be reactive were found to be SPV-positive. A reaction mechanism for these compounds in SPVA which involves the formation of alkenyl ethers is proposed and discussed. Tested proteins were non-reactive in SPVA. Thus, by comparing the results of gravimetric analyses of meibum samples with the results of a properly calibrated SPVA, it was estimated that the SPV-reactive lipid content of dry meibum in tested samples was about 78 % (w/w). The SPV method can also be adopted for analyzing other types of complex lipids secretions, such as sebum, as well as whole lipid extracts from other lipid-enriched organs and tissues, if proper standards are chosen.

  12. Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro.

    PubMed

    Nicoll, Jim; Gorbunov, Evgeniy A; Tarasov, Sergey A; Epstein, Oleg I

    2013-01-01

    Purpose. To investigate the mechanism of action in peripheral tissues of novel complex drug containing release-active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase (Subetta), which has shown efficacy in animal models of diabetes. Methods. Human mature adipocytes were incubated either with Subetta, with one of negative controls (placebo or vehicle), with one of nonspecific controls (release-active dilutions of antibodies to cannabinoid receptor type I or release-active dilutions of rabbit nonimmune serum), or with dimethyl sulfoxide (DMSO) at 37°C in a humidified incubator at 5% CO2 for three days. Rosiglitazone was used as reference drug. Secretion of adiponectin was measured by quantitative enzyme-linked immunosorbent assay (ELISA). Results. Only Subetta significantly stimulates adiponectin production by mature human adipocytes. Nonspecific controls did not significantly affect adiponectin secretion, resulting in adiponectin levels comparable to background values of the negative controls and DMSO. Conclusion. Increasing adiponectin production in absence of insulin by Subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug. These in vitro results give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

  13. Human umbilical cord-derived mesenchymal stem cells can secrete insulin in vitro and in vivo.

    PubMed

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2014-01-01

    Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decreased insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells offers novel ways of diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord-derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. hUDSCs were transduced with nonintegrated lentivirus harboring PDX1 (nonintegrated LV-PDX1) and was cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), and somatostatin were detected by quantitative RT-PCR (P < 0.05). PDX1 and insulin proteins were shown by immunocytochemistry analysis. Insulin secretion of hUDSCs(PDX1+) in the high-glucose medium was 1.8 μU/mL. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to a normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin-producing cells by transduction with nonintegrated LV-PDX1. These hUDSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

  14. Secretion of thioredoxin after in vitro activation of human B cells.

    PubMed

    Ericson, M L; Hörling, J; Wendel-Hansen, V; Holmgren, A; Rosén, A

    1992-10-01

    The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.

  15. Abolished InsP3R2 function inhibits sweat secretion in both humans and mice.

    PubMed

    Klar, Joakim; Hisatsune, Chihiro; Baig, Shahid M; Tariq, Muhammad; Johansson, Anna C V; Rasool, Mahmood; Malik, Naveed Altaf; Ameur, Adam; Sugiura, Kotomi; Feuk, Lars; Mikoshiba, Katsuhiko; Dahl, Niklas

    2014-11-01

    There are 3 major sweat-producing glands present in skin; eccrine, apocrine, and apoeccrine glands. Due to the high rate of secretion, eccrine sweating is a vital regulator of body temperature in response to thermal stress in humans; therefore, an inability to sweat (anhidrosis) results in heat intolerance that may cause impaired consciousness and death. Here, we have reported 5 members of a consanguineous family with generalized, isolated anhidrosis, but morphologically normal eccrine sweat glands. Whole-genome analysis identified the presence of a homozygous missense mutation in ITPR2, which encodes the type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2), that was present in all affected family members. We determined that the mutation is localized within the pore forming region of InsP3R2 and abrogates Ca2+ release from the endoplasmic reticulum, which suggests that intracellular Ca2+ release by InsP3R2 in clear cells of the sweat glands is important for eccrine sweat production. Itpr2-/- mice exhibited a marked reduction in sweat secretion, and evaluation of sweat glands from Itpr2-/- animals revealed a decrease in Ca2+ response compared with controls. Together, our data indicate that loss of InsP3R2-mediated Ca2+ release causes isolated anhidrosis in humans and suggest that specific InsP3R inhibitors have the potential to reduce sweat production in hyperhidrosis.

  16. Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

    PubMed

    Dirix, Violette; Mielcarek, Nathalie; Debrie, Anne-Sophie; Willery, Eve; Alonso, Sylvie; Versheure, Virginie; Mascart, Françoise; Locht, Camille

    2014-07-01

    In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

  17. Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

    SciTech Connect

    Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.

    2003-12-01

    We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.

  18. Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts.

    PubMed

    Seo, Young-Kwon; Song, Kye-Yong; Kim, Young-Jin; Park, Jung-Keug

    2007-07-01

    In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.

  19. P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation

    PubMed Central

    Buvinic, Sonja; Poblete, M Inés; Donoso, M Verónica; Delpiano, Ana María; Briones, René; Miranda, Ramiro; Huidobro-Toro, J Pablo

    2006-01-01

    The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y1R and P2Y2R revealed a 6- to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y2R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y1R and P2Y2R were detected by RT-PCR and the mRNA coding for the P2Y4R, P2Y6R and P2Y11R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y1R and P2Y2R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration-dependent manner by 10–100 nm indomethacin or 10 nm GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y1R antagonist, reduced the 2-MeSADP- but not the UTP-evoked contractions. Perfusion of cotyledons with 2-MeSADP or UTP evoked concentration-dependent reductions in perfusion pressure mediated by the NO–cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2-MeSADP but not by UTP. In summary, P2Y1R and P2Y2R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses. PMID:16543271

  20. A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion.

    PubMed

    Ravassard, Philippe; Hazhouz, Yasmine; Pechberty, Séverine; Bricout-Neveu, Emilie; Armanet, Mathieu; Czernichow, Paul; Scharfmann, Raphael

    2011-09-01

    Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.

  1. Placental C4d deposition is a feature of defective placentation: observations in cases of preeclampsia and miscarriage.

    PubMed

    Kim, Eun Na; Yoon, Bo Hyun; Lee, Joong Yeup; Hwang, Doyeong; Kim, Ki Chul; Lee, JoonHo; Shim, Jae-Yoon; Kim, Chong Jai

    2015-06-01

    Placental C4d deposition is frequent in preeclampsia, and shallow placentation is a characteristic of both preeclampsia and miscarriage. This study was conducted to determine the relationship among placental C4d, maternal human leukocyte antigen (HLA) antibodies, and placental pathology in preeclampsia and miscarriage cases. The patient population (N = 104) included those with (1) preterm preeclampsia with fetal growth restriction (PE-FGR; n = 21), (2) preterm preeclampsia (PE; n = 20), (3) spontaneous preterm delivery (sPTD; n = 39), and (4) miscarriage (n = 24). C4d immunohistochemistry was performed, and the presence of maternal plasma HLA antibodies was examined. C4d staining of the syncytiotrophoblast was more frequent in PE-FGR patients (76.2 %) than in PE (10.0 %; p < 0.001) and sPTD (2.6 %; p < 0.001) patients. Maternal HLA antibody-positive rate was not different among the study groups. There was a significant correlation between C4d immunoreactivity and placental pathology consistent with maternal vascular underperfusion (p < 0.001) but not with maternal HLA antibody status. In miscarriages, the positive rates of C4d, HLA class I, and HLA class II antibodies were 58.3, 25.0, and 12.5 %, respectively. There was no correlation between the presence of maternal HLA class I or II antibodies and placental C4d immunoreactivity. This study confirms frequent placental C4d deposition in preeclampsia with fetal growth restriction and miscarriage. The association between placental C4d deposition and pathological findings of maternal vascular underperfusion suggests that C4d staining of the syncytiotrophoblast is a consequence of defective placentation rather than of a specific maternal immune response against fetal HLA. The study also demonstrates the usefulness of C4d as a biomarker of placentas at risk.

  2. Placental Protein 13 (PP13) – A Placental Immunoregulatory Galectin Protecting Pregnancy

    PubMed Central

    Than, Nándor Gábor; Balogh, Andrea; Romero, Roberto; Kárpáti, Éva; Erez, Offer; Szilágyi, András; Kovalszky, Ilona; Sammar, Marei; Gizurarson, Sveinbjorn; Matkó, János; Závodszky, Péter; Papp, Zoltán; Meiri, Hamutal

    2014-01-01

    Galectins are glycan-binding proteins that regulate innate and adaptive immune responses, and some confer maternal-fetal immune tolerance in eutherian mammals. A chromosome 19 cluster of galectins has emerged in anthropoid primates, species with deep placentation and long gestation. Three of the five human cluster galectins are solely expressed in the placenta, where they may confer additional immunoregulatory functions to enable deep placentation. One of these is galectin-13, also known as Placental Protein 13 (PP13). It has a “jelly-roll” fold, carbohydrate-recognition domain and sugar-binding preference resembling other mammalian galectins. PP13 is predominantly expressed by the syncytiotrophoblast and released from the placenta into the maternal circulation. Its ability to induce apoptosis of activated T cells in vitro, and to divert and kill T cells as well as macrophages in the maternal decidua in situ, suggests important immune functions. Indeed, mutations in the promoter and an exon of LGALS13 presumably leading to altered or non-functional protein expression are associated with a higher frequency of preeclampsia and other obstetrical syndromes, which involve immune dysregulation. Moreover, decreased placental expression of PP13 and its low concentrations in first trimester maternal sera are associated with elevated risk of preeclampsia. Indeed, PP13 turned to be a good early biomarker to assess maternal risk for the subsequent development of pregnancy complications caused by impaired placentation. Due to the ischemic placental stress in preterm preeclampsia, there is increased trophoblastic shedding of PP13 immunopositive microvesicles starting in the second trimester, which leads to high maternal blood PP13 concentrations. Our meta-analysis suggests that this phenomenon may enable the potential use of PP13 in directing patient management near to or at the time of delivery. Recent findings on the beneficial effects of PP13 on decreasing blood pressure

  3. The new framework for understanding placental mammal evolution.

    PubMed

    Asher, Robert J; Bennett, Nigel; Lehmann, Thomas

    2009-08-01

    An unprecedented level of confidence has recently crystallized around a new hypothesis of how living placental mammals share a pattern of common descent. The major groups are afrotheres (e.g., aardvarks, elephants), xenarthrans (e.g., anteaters, sloths), laurasiatheres (e.g., horses, shrews), and euarchontoglires (e.g., humans, rodents). Compared with previous hypotheses this tree is remarkably stable; however, some uncertainty persists about the location of the placental root, and (for example) the position of bats within laurasiatheres, of sea cows and aardvarks within afrotheres, and of dermopterans within euarchontoglires. A variety of names for sub-clades within the new placental mammal tree have been proposed, not all of which follow conventions regarding priority and stability. More importantly, the new phylogenetic framework enables the formulation of new hypotheses and testing thereof, for example regarding the possible developmental dichotomy that seems to distinguish members of the newly identified southern and northern radiations of living placental mammals.

  4. Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

    PubMed

    Loganath, A; Peh, K L; Wong, P C

    2002-03-01

    With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.

  5. A Combined Synthetic-Fibrin Scaffold Supports Growth and Cardiomyogenic Commitment of Human Placental Derived Stem Cells

    PubMed Central

    Lisi, Antonella; Grimaldi, Settimio; Marchese, Rodolfo; Soldani, Giorgio

    2012-01-01

    Aims A potential therapy for myocardial infarction is to deliver isolated stem cells to the infarcted site. A key issue with this therapy is to have at one's disposal a suitable cell delivery system which, besides being able to support cell proliferation and differentiation, may also provide handling and elastic properties which do not affect cardiac contractile function. In this study an elastic scaffold, obtained combining a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (s-IPN) with fibrin, was used as a substrate for in vitro studies of human amniotic mesenchymal stromal cells (hAMSC) growth and differentiation. Methodology/Principal Findings After hAMSC seeding on the fibrin side of the scaffold, cell metabolic activity and proliferation were evaluated by WST-1 and bromodeoxyuridine assays. Morphological changes and mRNAs expression for cardiac differentiation markers in the hAMSCs were examined using immunofluorescence and RT-PCR analysis. The beginning of cardiomyogenic commitment of hAMSCs grown on the scaffold was induced, for the first time in this cell population, by a nitric oxide (NO) treatment. Following NO treatment hAMSCs show morphological changes, an increase of the messenger cardiac differentiation markers [troponin I (TnI) and NK2 transcription factor related locus 5 (Nkx2.5)] and a modulation of the endothelial markers [vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR)]. Conclusions/Significance The results of this study suggest that the s-IPN PEtU-PDMS/fibrin combined scaffold allows a better proliferation and metabolic activity of hAMSCs cultured up to 14 days, compared to the ones grown on plastic dishes. In addition, the combined scaffold sustains the beginning of hAMSCs differentiation process towards a cardiomyogenic lineage. PMID:22509287

  6. S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

    PubMed

    Guizzardi, F; Rodighiero, S; Binelli, A; Saino, S; Bononi, E; Dossena, S; Garavaglia, M L; Bazzini, C; Bottà, G; Conese, M; Daffonchio, L; Novellini, R; Paulmichl, M; Meyer, G

    2006-01-01

    Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This

  7. Polyaromatic compounds alter placental protein synthesis in pregnant rats

    SciTech Connect

    Shiverick, K.T.; Ogilvie, S.; Medrano, T. )

    1991-03-15

    The administration of the polyaromatic compounds {beta}-naphthoflavone ({beta}NF) and 3-methylcholanthrene (3MC) to pregnant rats during mid-gestation has been shown to produce marked feto-placental growth retardation. This study examined secretory protein synthesis in placental tissue from rats following administration of {beta}NF on gestation days (gd) 11-14 or 3MC on gd 12-14. Explants of placental basal zone tissue were cultured for 24 hours in serum-free medium in the presence of ({sup 3}H)leucine. Secreted proteins were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis followed by either fluorography or immunostaining. Total incorporation of ({sup 3}H)leucine into secreted proteins was not altered in BZ explants from {beta}NF or 3MC-treated animals. However a selective decrease was observed in ({sup 3}H)leucine incorporation into a major complex of proteins with apparent molecular weight of 25-30,000 and isoelectric point between 5.3 to 5.7. This group of proteins has been further identified as being related to rat pituitary growth hormone (GH) using N-terminal amino acid microsequencing of individual spots from 2-D SDS-PA gels. This is the first report that synthesis of GH-related proteins by rat placenta is decreased following {beta}NF and 3MC administration, a change which may underlie the feto-placental growth retardation associated with these polyaromatic compounds.

  8. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.

    PubMed

    Fang, Shijing; Crews, Anne L; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong; Adler, Kenneth B

    2013-04-15

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

  9. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

    PubMed Central

    Fang, Shijing; Crews, Anne L.; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong

    2013-01-01

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation. PMID:23377348

  10. Heparan Sulfate Proteoglycans and Their Binding Proteins in Embryo Implantation and Placentation

    PubMed Central

    Kirn-Safran, Catherine; D’Souza, Sonia S.; Carson, Daniel D.

    2008-01-01

    Complex interactions occur among embryonic, placental and maternal tissues during embryo implantation. Many of these interactions are controlled by growth factors, extracellular matrix and cell surface components that share the ability to bind heparan sulfate (HS) polysaccharides. HS is carried by several classes of cell surface and secreted proteins called HS proteoglycan that are expressed in restricted patterns during implantation and placentation. This review will discuss the expression of HS proteoglycans and various HS binding growth factors as well as extracellular matrix components and HS-modifying enzymes that can release HS-bound proteins in the context of implantation and placentation. PMID:17766150

  11. Xanthohumol impairs glucose uptake by a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo cells) and impacts the process of placentation.

    PubMed

    Correia-Branco, Ana; Azevedo, Cláudia F; Araújo, João R; Guimarães, João T; Faria, Ana; Keating, Elisa; Martel, Fátima

    2015-10-01

    In this study, we aimed to investigate modulation of glucose uptake by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line by a series of compounds and to study its consequences upon cell proliferation, viability and migration. We observed that uptake of (3)H-deoxy-d-glucose ((3)H-DG; 10 nM) was time-dependent, saturable, inhibited by cytochalasin B (50 and 100 µM), phloretin (0.5 mM) and phloridzin (1 mM), insulin-insensitive and sodium-independent. In the short term (30 min), neither 5-HT (100-1000 µM), melatonin (10 nM) nor the drugs of abuse ethanol (100 mM), nicotine (100 µM), cocaine (25 µM), amphetamine (10-25 µM) and 3,4-methylenedioxy-N-methamphetamine (10 µM) affected (3)H-DG uptake, while dexamethasone (100-1000 µM), fluoxetine (100-300 µM), quercetin, epigallocatechin-3-gallate (30-1000 µM), xanthohumol (XH) and resveratrol (1-500 µM) decreased it. XH was the most potent inhibitor [IC50 = 3.55 (1.37-9.20) µM] of (3)H-DG uptake, behaving as a non-competitive inhibitor of (3)H-DG uptake, both after short- and long-term (24 h) treatment. The effect of XH (5 µM; 24 h) upon (3)H-DG uptake involved mammalian target of rapamycin, tyrosine kinases and c-Jun N-terminal kinases intracellular pathways. Moreover, XH appeared to decrease cellular uptake of lactate due to inhibition of the monocarboxylate transporter 1. Additionally, XH (24 h; 5 µM) decreased cell viability, proliferation, culture growth and migration. The effects of XH upon cell viability and culture growth, but not the antimigratory effect, were mimicked by low extracellular glucose conditions and reversed by high extracellular glucose conditions. We thus suggest that XH, by inhibiting glucose cellular uptake and impairing HTR-8/SVneo cell viability and proliferation, may have a deleterious impact in the process of placentation.

  12. Nicotinic acid induces secretion of prostaglandin D2 in human macrophages: an in vitro model of the niacin flush.

    PubMed

    Meyers, C Daniel; Liu, Paul; Kamanna, Vaijinath S; Kashyap, Moti L

    2007-06-01

    Nicotinic acid is a safe, broad-spectrum lipid agent shown to prevent cardiovascular disease, yet its widespread use is limited by the prostaglandin D2 (PGD2) mediated niacin flush. Previous research suggests that nicotinic acid-induced PGD2 secretion is mediated by the skin, but the exact cell type remains unclear. We hypothesized that macrophages are a source of nicotinic acid-induced PGD2 secretion and performed a series of experiments to confirm this. Nicotinic acid (0.1-3 mM) induced PGD2 secretion in cultured human macrophages, but not monocytes or endothelial cells. The PGD2 secretion was dependent on the concentration of nicotinic acid and the time of exposure. Nicotinuric acid, but not nicotinamide, also induced PGD2 secretion. Pre-incubation of the cells with aspirin (100 microM) entirely prevented the nicotinic acid effects on PGD2 secretion. The PGD2 secreting effects of nicotinic acid were additive to the effects of the calcium ionophore A23187 (6 microM), but were independent of extra cellular calcium. These findings, combined with recent in vivo work, provide evidence that macrophages play a significant role in mediating the niacin flush and may lead to better strategies to eliminate this limiting side effect.

  13. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    PubMed

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

  14. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    PubMed Central

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  15. Angiotensin II Type 1 Receptor-Dependent GLP-1 and PYY Secretion in Mice and Humans

    PubMed Central

    Pais, Ramona; Rievaj, Juraj; Larraufie, Pierre

    2016-01-01

    Angiotensin II (Ang II) is the key hormone mediator of the renin angiotensin system, which regulates blood pressure and fluid and electrolyte balance in the body. Here we report that in the colonic epithelium, the Ang II type 1 receptor is highly and exclusively expressed in enteroendocrine L cells, which produce the gut hormones glucagon-like peptide-1 and peptide YY (PYY). Ang II stimulated glucagon-like peptide-1 and PYY release from primary cultures of mouse and human colon, which was antagonized by the specific Ang II type 1 receptor blocker candesartan. Ang II raised intracellular calcium levels in L cells in primary cultures, recorded by live-cell imaging of L cells specifically expressing the fluorescent calcium sensor GCaMP3. In Ussing chamber recordings, Ang II reduced short circuit currents in mouse distal colon preparations, which was antagonized by candesartan or a specific neuropeptide Y1 receptor inhibitor but insensitive to amiloride. We conclude that Ang II stimulates PYY secretion, in turn inhibiting epithelial anion fluxes, thereby reducing net fluid secretion into the colonic lumen. Our findings highlight an important role of colonic L cells in whole-body fluid homeostasis by controlling water loss through the intestine. PMID:27447725

  16. Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes

    PubMed Central

    Liao, Mei-Chen; Muratore, Christina R.; Gierahn, Todd M.; Sullivan, Sarah E.; Srikanth, Priya; De Jager, Philip L.; Love, J. Christopher

    2016-01-01

    Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. SIGNIFICANCE STATEMENT We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single

  17. CD4+ T cells are important mediators of oxidative stress that cause hypertension in response to placental ischemia.

    PubMed

    Wallace, Kedra; Cornelius, Denise C; Scott, Jeremy; Heath, Judith; Moseley, Janae; Chatman, Krystal; LaMarca, Babbette

    2014-11-01

    Preeclampsia is associated with oxidative stress, which is suspected to play a role in hypertension, placental ischemia, and fetal demise associated with the disease. Various cellular sources of oxidative stress, such as neutrophils, monocytes, and CD4(+) T cells have been suggested as culprits in the pathophysiology of preeclampsia. The objective of this study was to examine a role of circulating and placental CD4(+) T cells in oxidative stress in response to placental ischemia during pregnancy. CD4(+) T cells and oxidative stress were measured in preeclamptic and normal pregnant women, placental ischemic and normal pregnant rats, and normal pregnant recipient rats of placental ischemic CD4(+) T cells. Women with preeclampsia had significantly increased circulating (P=0.02) and placental CD4(+) T cells (P=0.0001); lymphocyte secretion of myeloperoxidase (P=0.004); and placental reactive oxygen species (P=0.0004) when compared with normal pregnant women. CD4(+) T cells from placental ischemic rats cause many facets of preeclampsia when injected into normal pregnant recipient rats on gestational day 13. On gestational day 19, blood pressure increased in normal pregnant recipients of placental ischemic CD4(+) T cells (P=0.002) compared with that in normal pregnant rats. Similar to preeclamptic patients, CD4(+) T cells from placental ischemic rats secreted significantly more myeloperoxidase (P=0.003) and induced oxidative stress in cultured vascular cells (P=0.003) than normal pregnant rat CD4(+)Tcells. Apocynin, a nicotinamide adenine dinucleotide phosphate inhibitor, attenuated hypertension and all oxidative stress markers in placental ischemic and normal pregnant recipient rats of placental ischemic CD4(+)Tcells (P=0.05). These data demonstrate an important role for CD4(+) T cells in mediating another factor, oxidative stress, to cause hypertension during preeclampsia.

  18. Secreted aspartic peptidases of Candida albicans liberate bactericidal hemocidins from human hemoglobin.

    PubMed

    Bocheńska, Oliwia; Rąpała-Kozik, Maria; Wolak, Natalia; Braś, Grażyna; Kozik, Andrzej; Dubin, Adam; Aoki, Wataru; Ueda, Mitsuyoshi; Mak, Paweł

    2013-10-01

    Secreted aspartic peptidases (Saps) are a group of ten acidic hydrolases considered as key virulence factors of Candida albicans. These enzymes supply the fungus with nutrient amino acids as well as are able to degrade the selected host's proteins involved in the immune defense. Our previous studies showed that the human menstrual discharge is exceptionally rich in bactericidal hemoglobin (Hb) fragments - hemocidins. However, to date, the genesis of such peptides is unclear. The presented study demonstrates that the action of C. albicans isozymes Sap1-Sap6, Sap8 and Sap9, but not Sap7 and Sap10, toward human hemoglobin leads to limited proteolysis of this protein and generates a variety of antimicrobial hemocidins. We have identified these peptides and checked their activity against selected microorganisms representative for human vagina. We have also demonstrated that the process of Hb hydrolysis is most effective at pH 4.0, characteristic for vagina, and the liberated peptides showed pronounced killing activity toward Lactobacillus acidophilus, and to a lower degree, Escherichia coli. However, only a very weak activity toward Staphylococcus aureus and C. albicans was noticed. These findings provide interesting new insights into pathophysiology of human vaginal candidiasis and suggest that C. albicans may be able to compete with the other microorganisms of the same physiological niche using the microbicidal peptides generated from the host protein.

  19. Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor.

    PubMed Central

    Fu, Y K; Arkins, S; Fuh, G; Cunningham, B C; Wells, J A; Fong, S; Cronin, M J; Dantzer, R; Kelley, K W

    1992-01-01

    Recombinant human growth hormone (HuGH) and human prolactin (HuPRL), but not GH of bovine or porcine origin, prime human neutrophils for enhanced superoxide anion (O2-) secretion. Since HuGH, but not GH of other species, effectively binds to the HuPRL receptor (HuPRL-R), we used a group of HuGH variants created by site-directed mutagenesis to identify the receptor on human neutrophils responsible for HuGH priming. A monoclonal antibody (MAb) directed against the HuPRL-R completely abrogated O2- secretion by neutrophils incubated with either HuGH or HuPRL, whereas a MAb to the HuGH-R had no effect. The HuGH variant K172A/F176A, which has reduced affinity for both the HuGH-binding protein (BP) and the HuPRL-BP, was unable to prime human neutrophils. This indicates that priming is initiated by a ligand-receptor interaction, the affinity of which is near that defined for receptors for PRL and GH. Another HuGH variant, K168A/E174A, which has relatively low affinity for the HuPRL-BP but slightly increased affinity for the HuGH-BP, had much reduced ability to prime neutrophils. In contrast, HuGH variant E56D/R64M, which has a similar affinity as wild-type HuGH for the HuPRL-BP but a lower affinity for the HuGH-BP, primed neutrophils as effectively as the wild-type HuGH. Finally, binding of HuGH to the HuPRL-BP but not to the HuGH-BP has been shown to be zinc dependent, and priming of neutrophils by HuGH was also responsive to zinc. Collectively, these data directly couple the binding of HuGH to the HuPRL-R with one aspect of functional activation of human target cells. Images PMID:1310696

  20. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-01-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl− and HCO3− secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3− net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl− and HCO3− net fluxes. Ieq and HCO3− fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3− is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3−. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3− secretion, suggesting that HCO3− transport under these conditions requires catalysed synthesis of carbonic acid. Cl− was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50–70% of Cl− and fluid transport was bumetanide-insensitive, suggesting basolateral Cl− loading by a sodium–potassium–chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO3− gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3− secretion was increased by bilateral Cl− removal and therefore did not require apical Cl−/HCO3− exchange. The results suggest a model in which most HCO3− is recycled basolaterally by exchange with Cl−, and the resulting HCO3−-dependent Cl− transport provides an osmotic driving force for

  1. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    PubMed

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  2. Azurocidin, a natural antibiotic from human neutrophils: expression, antimicrobial activity, and secretion.

    PubMed

    Almeida, R P; Vanet, A; Witko-Sarsat, V; Melchior, M; McCabe, D; Gabay, J E

    1996-06-01

    The azurophil granules of human PMN contain four antibiotic proteins, the serprocidins, which have extensive homology to one another and to serine proteases. Azurocidin, a member of this family, is a 29-kDa glycoprotein with broad spectrum antimicrobial activity and chemotactic activity toward monocytes. Insect cells transfected with a baculovirus vector carrying azurocidin cDNA produced a recombinant azurocidin protein. We purified the recombinant azurocidin protein from the culture medium of the infected cells and showed that it retained the antimicrobial activity of the native neutrophil-derived molecule. In addition, we present evidence that a 49-amino-acid region of the recombinant azurocidin protein is required for its secretion from insect cells.

  3. Placental mTOR links maternal nutrient availability to fetal growth.

    PubMed

    Roos, Sara; Powell, Theresa L; Jansson, Thomas

    2009-02-01

    The mTOR (mammalian target of rapamycin) signalling pathway functions as a nutrient sensor, both in individual cells and, more globally, in organs such as the fat body in Drosophila and the hypothalamus in the rat. The activity of placental amino acid transporters is decreased in IUGR (intrauterine growth restriction), and recent experimental evidence suggests that these changes contribute directly to the restricted fetal growth. We have shown that mTOR regulates the activity of the placental L-type amino acid transporter system and that placental mTOR activity is decreased in IUGR. The present review summarizes the emerging evidence implicating placental mTOR signalling as a key mechanism linking maternal nutrient and growth factor concentrations to amino acid transport in the human placenta. Since fetal growth is critically dependent on placental nutrient transport, placental mTOR signalling plays an important role in the regulation of fetal growth.

  4. Combinatorial human progenitor cell transplantation optimizes islet regeneration through secretion of paracrine factors.

    PubMed

    Bell, Gillian I; Meschino, Michael T; Hughes-Large, Jennifer M; Broughton, Heather C; Xenocostas, Anargyros; Hess, David A

    2012-07-20

    Transplanted human bone marrow (BM) and umbilical cord blood (UCB) progenitor cells activate islet-regenerative or revascularization programs depending on the progenitor subtypes administered. Using purification of multiple progenitor subtypes based on a conserved stem cell function, high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), we have recently shown that transplantation of BM-derived ALDH(hi) progenitors improved systemic hyperglycemia and augmented insulin secretion by increasing islet-associated proliferation and vascularization, without increasing islet number. Conversely, transplantation of culture-expanded multipotent-stromal cells (MSCs) derived from BM ALDH(hi) cells augmented total beta cell mass via formation of beta cell clusters associated with the ductal epithelium, without sustained islet vascularization. To identify paracrine effectors produced by islet-regenerative MSCs, culture-expanded BM ALDH(hi) MSCs were transplanted into streptozotocin-treated nonobese diabetic/severe combine immune deficient (SCID) mice and segregated into islet-regenerative versus nonregenerative cohorts based on hyperglycemia reduction, and subsequently compared for differential production of mRNA and secreted proteins. Regenerative MSCs showed increased expression of matrix metalloproteases, epidermal growth factor receptor (EGFR)-activating ligands, and downstream effectors of Wnt signaling. Regenerative MSC supernatant also contained increased levels of pro-angiogenic versus pro-inflammatory cytokines, and augmented the expansion of ductal epithelial but not beta cells in vitro. Conversely, co-culture with UCB ALDH(hi) cells induced beta cell but not ductal epithelial cell proliferation. Sequential transplantation of MSCs followed by UCB ALDH(hi) cells improved hyperglycemia and glucose tolerance by increasing beta cell mass associated with the ductal epithelium and by augmenting intra-islet capillary densities. Thus, combinatorial human progenitor cell

  5. Safety of repeated transplantations of neurotrophic factors-secreting human mesenchymal stromal stem cells

    PubMed Central

    2014-01-01

    Background Therapies based on mesenchymal stem cells (MSC) have been shown to have potential benefit in several clinical studies. We have shown that, using a medium-based approach, MSC can be induced to secrete elevated levels of neurotropic factors, which have been shown to have protective effects in animal models of neurodegenerative diseases. These cells, designated MSC-NTF cells (Neurotrophic factor-secreting MSC, also known as NurOwn™) derived from the patient's own bone marrow, have been recently used for Phase I/II and Phase IIa clinical studies in patients with Amyotrophic Lateral Sclerosis (ALS). In these studies, ALS patients were subjected to a single administration of autologous MSC-NTF cells. The data from these studies indicate that the single administration of MSC-NTF cells is safe and well tolerated. In a recently published case report, it was shown that repeated MSC-NTF injections in an ALS patient treated on a compassionate basis were safe and well tolerated [Muscle Nerve 49:455-457, 2014]. Methods In the current study we studied the toxicity and tolerability of three consecutive intramuscular injections (IM) of cryopreserved human MSC-NTF cells in C57BL/B6 mice to investigate the effect of repeated administration of these cells. Results Monitoring of clinical signs and immune reactions showed that repeated injections of the cells did not lead to any serious adverse events. Pathology, histology and blood biochemistry parameters tested were found to be within normal ranges with no sign of tumor formation. Conclusions Based on these results we conclude that repeated injections of human MSC-NTF are well tolerated in mice. The results of this study suggest that if the outcomes of additional clinical studies point to the need for repeated treatments, such option can be considered safe. PMID:25097724

  6. Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

    PubMed Central

    1992-01-01

    Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity- purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of

  7. Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

    PubMed Central

    Smith, Ida M.; Christensen, Jeffrey E.; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  8. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    PubMed

    Smith, Ida M; Christensen, Jeffrey E; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  9. Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

    PubMed

    Ao, Mei; Domingue, Jada C; Khan, Nabihah; Javed, Fatima; Osmani, Kashif; Sarathy, Jayashree; Rao, Mrinalini C

    2016-06-01

    Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of

  10. Organic cation transporter-mediated renal secretion of ipratropium and tiotropium in rats and humans.

    PubMed

    Nakanishi, Takeo; Haruta, Tsunemitsu; Shirasaka, Yoshiyuki; Tamai, Ikumi

    2011-01-01

    Ipratropium bromide (ipratropium) and tiotropium bromide (tiotropium), anticholinergic agents with bronchodilating properties, are used to treat patients with chronic obstructive pulmonary disease. Because they are actively secreted into urine, the interaction of these agents with organic cation transporters (OCTs/Octs) was examined in rat kidney slices and in cultured cells expressing rat Oct (rOct) or human OCT (hOCT). Uptake of radiolabeled ipratropium in rat kidney slices was significantly inhibited by OCT/Oct substrates including cimetidine, imipramine, and quinidine, but not by organic anion transporter substrates (e.g., p-aminohippuric acid and estrone-3-sulfate). [(3)H]Tiotropium uptake showed similar characteristics. Reverse transcription-polymerase chain reaction showed that, in rat kidney, mRNA expression of rOct2 was the highest, followed by rOct1, but little rOct3 was detected. In vitro, rOct1 and rOct2 transported both anticholinergics, but rOct3 accepted only ipratropium. Ipratropium uptake by rat kidney slices consisted of two components with K(m) values of 0.114 ± 0.06 and 24.5 ± 2.21 μM. The K(m) value of rOct2-mediated ipratropium uptake (0.143 ± 0.03 μM) was consistent with that of the high-affinity component. The OCT/Oct inhibitor corticosterone, at a concentration of 1 μM (IC(50), 1.11 ± 0.20 μM for rOct2-mediated ipratropium transport), inhibited ipratropium by 18.4%, suggesting that rOct2 is involved in renal secretion of ipratropium. In a similar manner, ipratropium and tiotropium were taken up by cultured cells expressing hOCT1 and hOCT2 but not hOCT3. We conclude that OCT2/Oct2 plays a role in renal secretion of both anticholinergics in these species. Coadministration of these anticholinergics with cationic drugs recognized by OCT2/Oct2 may decrease renal clearance, resulting in increased systemic exposure.

  11. Effects of mebendazole on protein biosynthesis and secretion in human-derived fibroblast cultures.

    PubMed

    Soto, H; Massó, F; Cano, S; Díaz de León, L

    1996-07-26

    Previous results of our group revealed that mebendazole, a broad spectrum anthelmintic drug with antimicrotubular properties, used for the treatment of liver cirrhosis, decreased total collagen content and biosynthesis in liver upon treatment. In the present study, we have evaluated the effects of mebendazole (5-50 micrograms/mL) on protein synthesis, secretion, and deposition in human-derived fibroblast cultures. The results showed a decrease in cell viability (18.5 +/- 0.9%) at 50 micrograms/mL. [3H]Thymidine incorporation diminished gradually with increasing mebendazole concentrations, reaching a plateau (53.67%) between 30 and 50 micrograms/mL. In late logarithmic phase cultures, the drug caused a decrease of [3H]proline incorporation (43.10%) and collagen biosynthesis (58.61%) in the extracellular matrix. This correlated with an increase in radioactivity in total proteins (51.28%) of the intracellular fraction. Similar results were obtained when mebendazole was assayed in post-confluent fibroblast cultures. The electrophoretic patterns of the extracellular matrix showed a decrease of radioactive collagenous components (alpha chains and beta dimers). By contrast, in the intracellular fraction an increase of radioactive collagen precursors (pro alpha chains) was observed. Immunofluorescence studies and immunotransfer analysis, using polyclonal anti-type I collagen antibodies, revealed an accumulation of intracellular collagen which included: collagen pro alpha chains, alpha chains, and low molecular weight peptides. The results obtained suggest that mebendazole interferes with the transcellular mobilization of proteins, resulting in a decrease of secretion and deposition of extracellular matrix proteins, and an accumulation of intracellular collagenous components. The intracellular accumulation of newly synthesized proteins could cause a feedback regulation in fibroblast cultures.

  12. Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

    SciTech Connect

    Bronzert, D.A.; Pantazis, P.; Antoniades, H.N.; Kasid, A.; Davidson, N.; Dickson, R.B.; Lippman, M.E.

    1987-08-01

    The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of (/sup 3/H) thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95/sup 0/C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approx. =30 kDa on NaDodSO/sub 4//polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO/sub 4//polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth.

  13. Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N).

    PubMed

    Squires, P E; Amiranoff, B; Dunne, M J

    1994-10-01

    Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 microM) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 microM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 microM acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 microM), verapamil (100 microM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.

  14. Combined Use of Etomidate and Dexmedetomidine Produces an Additive Effect in Inhibiting the Secretion of Human Adrenocortical Hormones.

    PubMed

    Gu, Hongbin; Zhang, Mazhong; Cai, Meihua; Liu, Jinfen

    2015-11-16

    BACKGROUND The direct effects of etomidate were investigated on the secretion of cortisol and its precursors by dispersed cells from the adrenal cortex of human of animals. Dexmedetomidine (DEX) is an anesthetic agent that may interfere with cortisol secretion via an unknown mechanism, such as involving inhibition of 11b-hydroxylase and the cholesterol side-chain cleavage enzyme system. The aim of this study was to determine whether dexmedetomidine (DEX) has a similar inhibitory effect on adrenocortical function, and whether combined use of etomidate (ETO) and DEX could produce a synergistic action in inhibiting the secretion of human adrenocortical hormones. MATERIAL AND METHODS Human adrenocortical cells were exposed to different concentrations of ETO and DEX. The dose-effect model between the ETO concentration and the mean secretion of cortisone (CORT) and aldosterone (ALDO) per hour was estimated. RESULTS Hill's equation well-described the dose-effect correlation between the ETO concentration and the amount of ALDO and CORT secretion. When the DEX concentration was introduced into the model by using E0 (basal secretion) as the covariate, the goodness of fit of the ETO-CORT dose-effect model was improved significantly and the objective function value was reduced by 4.55 points (P<0.05). The parameters of the final ETO-ALDO pharmacodynamics model were EC50=9.74, Emax=1.20, E0=1.33, and γ=18.5; the parameters of the final ETO-CORT pharmacodynamics model were EC50=9.49, Emax=8.16, E0=8.57, and γ=37.0. In the presence of DEX, E0 was 8.57-0.0247×(CDEX-4.6), and the other parameters remained unchanged. All parameters but γ were natural logarithm conversion values. CONCLUSIONS Combined use of DEX and ETO reduced ETO's inhibitory E0 (basal secretion) of CORT from human adrenocortical cells in a dose-dependent manner, suggesting that combined use of ETO and DEX produced an additive effect in inhibiting the secretion of human adrenocortical hormones.

  15. Source of peritoneal proteoglycans. Human peritoneal mesothelial cells synthesize and secrete mainly small dermatan sulfate proteoglycans.

    PubMed Central

    Yung, S.; Thomas, G. J.; Stylianou, E.; Williams, J. D.; Coles, G. A.; Davies, M.

    1995-01-01

    This study describes experiments that compare the proteoglycans (PGs) extracted from the dialysate from patients receiving continuous peritoneal ambulatory dialysis (CAPD) with those secreted by metabolically labeled human peritoneal mesothelial cells in vitro. The PGs isolated from both sources were predominantly small chondroitin sulfate/dermatan sulfate PGs. Western blot of the core proteins obtained after chondroitin ABC lyase treatment with specific antibodies identified decorin and biglycan. With [35S]sulfate and [35S]methionine as labeling precursors it was shown that dermatan sulfate rather than chondroitin sulfate were the major glycosaminoglycan chains and that decorin was the predominant species. These data provide the first evidence that human peritoneal mesothelial cells may be the principal source of PGs in the peritoneum. Given the proposed functions of decorin and biglycan, the results suggest that these PGs may be involved in the control of transforming growth factor-beta activity and collagen fibril formation in the peritoneum. Images Figure 2 Figure 7 Figure 8 PMID:7856761

  16. Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

    PubMed

    Conley, A J; Corbin, C J; Browne, P; Mapes, S M; Place, N J; Hughes, A L; Glickman, S E

    2007-07-01

    At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to

  17. Placental Glucose transporter 3 (GLUT3) is Up-regulated in Human Pregnancies Complicated by Late-onset Intrauterine Growth Restriction

    PubMed Central

    Janzen, Carla; Lei, Margarida Y.Y.; Cho, John; Sullivan, Peggy; Shin, Bo-Chul; Devaskar, Sherin U.

    2013-01-01

    Introduction Transport of glucose from maternal blood across the placental trophoblastic tissue barrier is critical to sustain fetal growth. The mechanism by which GLUTs are regulated in trophoblasts in response to ischemic hypoxia encountered with intra-uterine fetal growth restriction (IUGR) has not been suitably investigated. Objective To investigate placental expression of GLUT1, GLUT3 and GLUT4 and possible mechanisms of GLUT regulation in idiopathic IUGR. Methods We analyzed clinical, biochemical and histological data from placentas collected from women affected by idiopathic full-term IUGR (n=10) and gestational age-matched healthy controls (n=10). Results We found increased GLUT3 protein expression in the trophoblast (cytotrophoblast greater than syncytiotrophoblast) on the maternal aspect of the placenta in IUGR compared to normal placenta, but no differences in GLUT1 or GLUT4 were found. No differential methylation of the GLUT3 promoter between normal and IUGR placentas was observed. Increased GLUT3 expression was associated with an increased nuclear concentration of HIF-1α, suggesting hypoxia may play a role in the up-regulation of GLUT3. Discussion Further studies are needed to elucidate whether increased GLUT3 expression in IUGR is a marker for defective villous maturation or an adaptive response of the trophoblast in response to chronic hypoxia. Conclusions Patients with IUGR have increased trophoblast expression of GLUT3, as found under the low-oxygen conditions of the first trimester. PMID:24011442

  18. Mono-(2-ethylhexyl) phthalate induces apoptosis through miR-16 in human first trimester placental cell line HTR-8/SVneo.

    PubMed

    Meruvu, Sunitha; Zhang, Jian; Bedi, Yudhishtar Singh; Choudhury, Mahua

    2016-03-01

    Phthalates have been linked to adverse pregnancy complications. Mono-(2-ethylhexyl) phthalate, an active metabolite of di-(2-ethylhexyl) phthalate and an endocrine disruptor, has been shown to induce apoptosis in various cell types including placental cells. However, the mechanism of action of MEHP induced apoptosis is still unknown. We hypothesized that apoptosis may be mediated in part through altered microRNA(s) in placenta under MEHP exposure. In the present study, we report that MEHP increases miR-16 expression in a time- and dose-dependent manner (p<0.05), while inducing apoptosis in HTR-8/SVneo. Cells treated with MEHP showed a dose-dependent increase in cytotoxicity and reactive oxygen species along with decreased cell viability. Consistent with significant increase in apoptosis analyzed by flow cytometry, we detected decreased anti-apoptotic BCL-2 at transcriptional and translational levels with MEHP (p<0.05). Knockdown of miR-16 did not decrease the BCL-2/BAX protein expression ratio in the presence of MEHP when compared to negative control demonstrating that MEHP induces apoptosis directly through miR-16. In conclusion, our study demonstrates for the first time that MEHP induces miR-16, which in turn, alters BCL-2/BAX ratio leading to increased apoptosis. This study provides a novel insight into MEHP induced epigenetic regulation in placental apoptosis which may lead to pregnancy complications.

  19. PGE2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells

    PubMed Central

    Fujii, Satoru; Suzuki, Kohei; Kawamoto, Ami; Ishibashi, Fumiaki; Nakata, Toru; Murano, Tatsuro; Ito, Go; Shimizu, Hiromichi; Mizutani, Tomohiro; Oshima, Shigeru; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Araki, Akihiro; Ohtsuka, Kazuo; Okamoto, Ryuichi; Watanabe, Mamoru

    2016-01-01

    Intestinal epithelial cells (IECs) play an indispensable role in maintaining body fluid balance partly through their ability to regulate anion/fluid secretion. Yet in various inflammatory gastrointestinal diseases, over-secretion of anions results in symptoms such as severe diarrhoea. Endogenous mediators, such as vasoactive intestinal peptide or prostaglandin E2 (PGE2), regulate intestinal anion/fluid secretion, but their direct effect on purified human IECs has never been described in detail. Based on a previously described intestinal organoid swelling model, we established a 3D-scanner-assisted quantification method to evaluate the anion/fluid secretory response of cultured human IECs. Among various endogenous secretagogues, we found that PGE2 had the lowest EC50 value with regard to the induction of swelling of the jejunal and colonic organoids. This PGE2-mediated swelling response was dependent on environmental Cl− concentrations as well as on several channels and transporters as shown by a series of chemical inhibitor studies. The concomitant presence of various inflammatory cytokines with PGE2 failed to modulate the PGE2-mediated organoid swelling response. Therefore, the present study features PGE2 as a direct and robust mediator of anion/fluid secretion by IECs in the human intestine. PMID:27827428

  20. Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon.

    PubMed

    Kaji, Izumi; Yasuoka, Yukiko; Karaki, Shin-Ichiro; Kuwahara, Atsukazu

    2012-04-01

    In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10(-6)-10(-3) M) induced Cl(-) and HCO(3)(-) secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E(2); however, this secretion was not affected by TTX, atropine, or extracellular Ca(2+) depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP(4) receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP(4) receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

  1. Sex-Specific Placental Responses in Fetal Development

    PubMed Central

    2015-01-01

    The placenta is an ephemeral but critical organ for the survival of all eutherian mammals and marsupials. It is the primary messenger system between the mother and fetus, where communicational signals, nutrients, waste, gases, and extrinsic factors are exchanged. Although the placenta may buffer the fetus from various environmental insults, placental dysfunction might also contribute to detrimental developmental origins of adult health and disease effects. The placenta of one sex over the other might possess greater ability to respond and buffer against environmental insults. Given the potential role of the placenta in effecting the lifetime health of the offspring, it is not surprising that there has been a resurging interest in this organ, including the Human Placental Project launched by the National Institutes of Child Health and Human Development. In this review, we will compare embryological development of the laboratory mouse and human chorioallantoic placentae. Next, evidence that various species, including humans, exhibit normal sex-dependent structural and functional placental differences will be examined followed by how in utero environmental changes (nutritional state, stress, and exposure to environmental chemicals) might interact with fetal sex to affect this organ. Recent data also suggest that paternal state impacts placental function in a sex-dependent manner. The research to date linking placental maladaptive responses and later developmental origins of adult health and disease effects will be explored. Finally, we will focus on how sex chromosomes and epimutations may contribute to sex-dependent differences in placental function, the unanswered questions, and future directions that warrant further consideration. PMID:26241064

  2. Secretion of neurotensin from a human pancreatic islet cell carcinoma cell line (QGP-1N).

    PubMed

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1993-12-10

    Effects of various secretagogues on secretion of neurotensin from a pancreatic islet cell carcinoma cell line (QGP-1N) were examined. Carbachol stimulated secretion of neurotensin concentration-dependently in the range of 10(-6) - 10(-4) M. The neurotensin secretion stimulated with 10(-5) M carbachol was completely inhibited by atropine at 10(-5) M. Phorbol ester and calcium ionophore (A23187) stimulated secretion of neurotensin. The removal of extracellular Ca2+ suppressed the secretion through the stimulation with 10(-5) M carbachol. Fluoride, an activator of guanine nucleotide-binding (G) protein, stimulated secretion of neurotensin. Neurotensin released into culture medium through stimulation with carbachol coeluted with neurotensin 1-13 on a gel-chromatography. Our results suggest that secretion of neurotensin from QGP-1N cells is mainly regulated by acetylcholine through muscarinic receptors coupled to G protein and that an increase in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  3. Peroxisome proliferator-activated receptor gamma (PPARG) modulates free fatty acid receptor 1 (FFAR1) dependent insulin secretion in humans

    PubMed Central

    Wagner, Robert; Hieronimus, Anja; Lamprinou, Apostolia; Heni, Martin; Hatziagelaki, Erifili; Ullrich, Susanne; Stefan, Norbert; Staiger, Harald; Häring, Hans-Ulrich; Fritsche, Andreas

    2014-01-01

    Genetic variation in FFAR1 modulates insulin secretion dependent on non-esterified fatty acid (NEFA) concentrations. We previously demonstrated lower insulin secretion in minor allele carriers of PPARG Pro12Ala in high-NEFA environment, but the mode of action could not been revealed. We tested if this effect is mediated by FFAR1 in humans. Subjects with increased risk of diabetes who underwent oral glucose tolerance tests were genotyped for 7 tagging SNPs in FFAR1 and PPARG Pro12Ala. The FFAR1 SNPs rs12462800 and rs10422744 demonstrated interactions with PPARG on insulin secretion. FFAR1 rs12462800 (p = 0.0006) and rs10422744 (p = 0.001) were associated with reduced insulin secretion in participants concomitantly carrying the PPARG minor allele and having high fasting FFA. These results suggest that the minor allele of the PPARG SNP exposes its carriers to modulatory effects of FFAR1 on insulin secretion. This subphenotype may define altered responsiveness to FFAR1-agonists, and should be investigated in further studies. PMID:25161890

  4. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

    SciTech Connect

    Matzuk, M.M.; Krieger, M.; Corless, C.L.; Boime, I.

    1987-09-01

    Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results reveal that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.

  5. A somatostatin-secreting cell line established from a human pancreatic islet cell carcinoma (somatostatinoma): release experiment and immunohistochemical study.

    PubMed

    Iguchi, H; Hayashi, I; Kono, A

    1990-06-15

    Production and secretion of somatostatin (SRIF) were studied using a carcinoembryonic antigen (CEA)-producing cell line (QGP-1) established from a human pancreatic islet cell carcinoma. High concentrations of SRIF (274 +/- 51 ng/mg of protein, mean +/- SD, n = 5) and CEA (3083 +/- 347 ng/mg of protein, mean +/- SD, n = 5) were present in QGP-1 cells, and the basal secretion rates of SRIF and CEA by the cells (n = 5) were 46.4 +/- 4.8 and 1690 +/- 78 pg/10(5) cells/h, respectively. Immunohistochemical studies revealed the presence of SRIF in xenografts of QGP-1 cells and colocalization of SRIF and CEA. Secretion of SRIF by QGP-1 cells was stimulated in the presence of high K+ (50 mmol) and theophylline (10 mmol), but arginine (10 mmol) and glucose (300 mg/dl) had no effect on the SRIF secretion. The QGP-1 cell line may be useful for studying the regulation mechanism of SRIF secretion.

  6. Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF.

    PubMed

    Weiser, W Y; Remold, H G; David, J R

    1985-01-01

    Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

  7. Selective novel inverse agonists for human GPR43 augment GLP-1 secretion.

    PubMed

    Park, Bi-Oh; Kim, Seong Heon; Kong, Gye Yeong; Kim, Da Hui; Kwon, Mi So; Lee, Su Ui; Kim, Mun-Ock; Cho, Sungchan; Lee, Sangku; Lee, Hyun-Jun; Han, Sang-Bae; Kwak, Young Shin; Lee, Sung Bae; Kim, Sunhong

    2016-01-15

    GPR43/Free Fatty Acid Receptor 2 (FFAR2) is known to be activated by short-chain fatty acids and be coupled to Gi and Gq family of heterotrimeric G proteins. GPR43 is mainly expressed in neutrophils, adipocytes and enteroendocrine cells, implicated to be involved in inflammation, obesity and type 2 diabetes. However, several groups have reported the contradictory data about the physiological functions of GPR43, so that its roles in vivo remain unclear. Here, we demonstrate that a novel compound of pyrimidinecarboxamide class named as BTI-A-404 is a selective and potent competitive inverse agonist of human GPR43, but not the murine ortholog. Through structure-activity relationship (SAR), we also found active compound named as BTI-A-292. These regulators increased the cyclic AMP level and reduced acetate-induced cytoplasmic Ca(2+) level. Furthermore, we show that they modulated the downstream signaling pathways of GPR43, such as ERK, p38 MAPK, and NF-κB. It was surprising that two compounds augmented the secretion of glucagon-like peptide 1 (GLP-1) in NCI-H716 cell line. Collectively, these novel and specific competitive inhibitors regulate all aspects of GPR43 signaling and the results underscore the therapeutic potential of them.

  8. Mechanism and function of type IV secretion during infection of the human host

    PubMed Central

    Gonzalez-Rivera, Christian; Bhatty, Minny; Christie, Peter J.

    2015-01-01

    Bacterial pathogens employ type IV secretion systems (T4SSs) for various purposes to aid in survival and proliferation in eukaryotic host. One large T4SS subfamily, the conjugation systems, confers a selective advantage to the invading pathogen in clinical settings through dissemination of antibiotic resistance genes and virulence traits. Besides their intrinsic importance as principle contributors to the emergence of multiply drug-resistant ‘superbugs’, detailed studies of these highly tractable systems have generated important new insights into the mode of action and architectures of paradigmatic T4SSs as a foundation for future efforts aimed at suppressing T4SS machine function. Over the past decade, extensive work on the second large T4SS subfamily, the effector translocators, has identified a myriad of mechanisms employed by pathogens to subvert, subdue, or bypass cellular processes and signaling pathways of the host cell. An overarching theme in the evolution of many effectors is that of molecular mimicry. These effectors carry domains similar to those of eukaryotic proteins and exert their effects through stealthy interdigitation of cellular pathways, often with the outcome not of inducing irreversible cell damage but rather of reversibly modulating cellular functions. This chapter summarizes the major developments for the actively studied pathogens with an emphasis on the structural and functional diversity of the T4SSs and the emerging common themes surrounding effector function in the human host. PMID:27337453

  9. CD8+ Lymphocytes Suppress Human Immunodeficiency Virus 1 Replication by Secreting Type I Interferons

    PubMed Central

    Teque, Fernando; Walker, Robert L.; Meltzer, Paul S.; Killian, J. Keith

    2013-01-01

    CD8+ cells can suppress human immunodeficiency virus 1 (HIV-1) replication by releasing soluble factors. In 26 years of intensive research efforts, the identity of the major CD8+ cell antiviral factor has remained elusive. To investigate the mechanism for this antiviral immune response, we performed gene expression analyses on primary CD4+ cells that were exposed to HIV-suppressing CD8+ cells or CD8+ cell-conditioned medium having HIV-suppressing activity. These experiments revealed increased levels of multiple genes stimulated by type I interferons (IFN; eg, IFN-α and IFN-β). Further evaluation revealed that primary CD8+ cells, particularly those from elite controllers and other asymptomatic HIV-1-infected individuals, secrete IFN, and this response directly contributes to the in vitro suppression of HIV replication in CD4+ cells. This novel immune response, likely mediated by memory CD8+ T cells, may play an important role in a wide variety of viral infections, cancers, and autoimmune diseases. PMID:23402527

  10. Regulation of vascular endothelial growth factor secretion in human meningioma cells.

    PubMed

    Tsai, J C; Hsiao, Y Y; Teng, L J; Shun, C T; Chen, C T; Goldman, C K; Kao, M C

    1999-02-01

    Previously, we induced vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) secretion in glioma cell lines by using physiologic concentrations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). We hypothesized that VEGF/VPF might enhance the blood supply required for the unregulated growth of tumors, and that it acts as the central mediator of tumor angiogenesis. The objective of this study was to determine whether the expression of VEGF/VPF by meningiomas is regulated by growth factors or sex hormones. By means of an enzyme-linked immunosorbent assay of CH-157MN meningioma cell supernatants, we demonstrated that EGF and bFGF similarly induce VEGF secretion by CH-157MN meningioma cells. At the maximum concentrations of EGF (50 ng/mL) and bFGF (50 ng/mL) used in this study, VEGF secretion was induced to 140% to 160% above baseline constitutive secretion. PDGF-BB homodimer did not enhance VEGF secretion significantly. Estradiol (up to 10(-7) mol/L), progesterone (up to 10(-5) mol/L), or testosterone (up to 10(-5) mol/L) did not stimulate or inhibit VEGF secretion in CH-157MN meningioma cells (p > 0.05). Furthermore, we demonstrated that dexamethasone decreased VEGF secretion to 32% of baseline constitutive secretion. This might explain the effect of corticosteroids in alleviating peritumoral brain edema in meningiomas. These results suggest that VEGF secretion in CH-157MN meningioma cells is mainly regulated by growth factors and corticosteroids, but not by sex hormones. Understanding the regulation of VEGF/VPF secretion in meningiomas might contribute to the development of a new therapeutic strategy.

  11. Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

    SciTech Connect

    Ogura, M.; Tanabe, N.; Nishioka, J.; Suzuki, K.; Saito, H.

    1987-07-01

    A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with (/sup 35/S)-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

  12. Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta

    SciTech Connect

    Robinson, B.G.; Emanuel, R.L.; Frim, D.M.; Majzoub, J.A. )

    1988-07-01

    Primary cultures of purified human cytotrophoblasts have been used to examine the expression of the corticotropin-releasing hormone (CRH) gene in placenta. The authors report here that glucocorticoids stimulate placental CRH synthesis and secretion in primary cultures of human placenta. This stimulation is in contrast to the glucocorticoid suppression of CRH expression in hypothalamus. The positive regulation of CRH by glucocorticoids suggests that the rise in CRH preceding parturition could result from the previously described rise in fetal glucocorticoids. Furthermore, this increase in placental CRH could stimulate, via adrenocorticotropic hormone, a further rise in fetal glucocorticoids, completing a positive feedback loop that would be terminated by delivery.

  13. Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta.

    PubMed Central

    Robinson, B G; Emanuel, R L; Frim, D M; Majzoub, J A

    1988-01-01

    Primary cultures of purified human cytotrophoblasts have been used to examine the expression of the corticotropin-releasing hormone (CRH) gene in placenta. We report here that glucocorticoids stimulate placental CRH synthesis and secretion in primary cultures of human placenta. This stimulation is in contrast to the glucocorticoid suppression of CRH expression in hypothalamus. The positive regulation of CRH by glucocorticoids suggests that the rise in CRH preceding parturition could result from the previously described rise in fetal glucocorticoids. Furthermore, this increase in placental CRH could stimulate, via adrenocorticotropic hormone, a further rise in fetal glucocorticoids, completing a positive feedback loop that would be terminated by delivery. Images PMID:2839838

  14. Nitric oxide and oxidative stress in placental explant cultures.

    PubMed

    Goncalves, Juvic M; Casart, Ysabel C; Camejo, María I

    2016-01-01

    Placental explant culture, and cellular cytolysis and cellular differentiation have been previously studied. However, oxidative stress and nitric oxide profiles have not been evaluated in these systems. The aim of this study was to determine the release of lipid peroxidation and nitric oxide from placental explants cultured over a seven day period. Placental explants were maintained for seven days in culture and the medium was changed every 24 hours. The response was assessed in terms of syncytiotrophoblast differentiation (human chorionic gonadotropin, hCG), cellular cytolysis (lactate dehydrogenase, LDH), oxidative stress (thiobarbituric acid reactive substances, TBARS), and nitric oxide (NO). Levels of hCG increased progressively from day two to attain its highest level on days four and five after which it decreased gradually. In contrast, the levels of LDH, TBARS, and NO were elevated in the early days of placental culture when new syncytiotrophoblast from cytotrophoblast were forming and also in the last days of culture when tissue was declining. In conclusion, the levels of NO and lipid peroxidation follow a pattern similar to LDH and contrary to hCG. Future placental explant studies to evaluate oxidative stress and NO should consider the physiological changes inherent during the time of culture.

  15. Exercise-mimicking treatment fails to increase Fndc5 mRNA & irisin secretion in primary human myotubes.

    PubMed

    Kurdiova, Timea; Balaz, Miroslav; Mayer, Alexander; Maderova, Denisa; Belan, Vitazoslav; Wolfrum, Christian; Ukropec, Jozef; Ukropcova, Barbara

    2014-06-01

    Irisin, myokine secreted by skeletal muscle, was suggested to mediate some of exercise health benefits via "browning" of white adipose tissue. However, mounting evidence contradicts the regulatory role of exercise for muscle irisin production/secretion in humans. Thus, we explored the direct effect of exercise-mimicking treatment on irisin in human primary muscle cells in vitro. Human primary muscle cell cultures were established from lean, obese prediabetic and type-2-diabetic individuals. Complex metabolic phenotyping included assessment of insulin sensitivity (euglycemic hyperinsulinemic clamp) and adiposity content&distribution (MRI&MRS). In vitro exercise-mimicking treatment (forskolin+ionomycin) was delivered in 1-h pulse/day during differentiation. Fndc5 mRNA (qRT-PCR) and secreted irisin (ELISA) were determined in cells and media. Exercise-mimicking treatment more than doubled Pgc1α mRNA in differentiated muscle cells. Nevertheless, Fndc5 mRNA was reduced by 18% and irisin in media by 20%. Moreover, Fncd5 mRNA was increased in myotubes derived from individuals with type-2-diabetes, independent on exercise-mimicking treatment. Fndc5 mRNA in cells was positively related to fasting glycemia (p=0.0001) and negatively to whole-body insulin sensitivity (p<0.05). Collectively, our data do not support the role of exercise-related signaling pathways in irisin regulation in human skeletal muscle and confirm our previous observations on increased Fndc5 expression in muscle cells from individuals with type-2-diabetes.

  16. Beta2-Adrenoceptor Stimulation Suppresses TLR9-Dependent IFNA1 Secretion in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Weisheit, Christina; Knüfermann, Pascal; Baumgarten, Georg; Hoeft, Andreas; Poth, Jens M.

    2013-01-01

    Introduction IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs. Methods PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay. Results TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells. Conclusion We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice

  17. Dental Calculus Stimulates Interleukin-1β Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes

    PubMed Central

    Montenegro Raudales, Jorge Luis; Yoshimura, Atsutoshi; SM, Ziauddin; Kaneko, Takashi; Ozaki, Yukio; Ukai, Takashi; Miyazaki, Toshihiro; Latz, Eicke; Hara, Yoshitaka

    2016-01-01

    Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in

  18. Temperature Oscillations Drive Cycles in the Activity of MMP-2,9 Secreted by a Human Trabecular Meshwork Cell Line

    PubMed Central

    Li, Stanley Ka-lok; Banerjee, Juni; Jang, Christopher; Sehgal, Amita; Stone, Richard A.; Civan, Mortimer M.

    2015-01-01

    Purpose. Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. Methods. Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. Results. Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. Conclusions. Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels. PMID:25655795

  19. In vivo Cigarette Smoke Exposure Decreases CCL20, SLPI, and BD-1 Secretion by Human Primary Nasal Epithelial Cells

    PubMed Central

    Jukosky, James; Gosselin, Benoit J.; Foley, Leah; Dechen, Tenzin; Fiering, Steven; Crane-Godreau, Mardi A.

    2016-01-01

    Smokers and individuals exposed to second-hand cigarette smoke have a higher risk of developing chronic sinus and bronchial infections. This suggests that cigarette smoke (CS) has adverse effects on immune defenses against pathogens. Epithelial cells are important in airway innate immunity and are the first line of defense against infection. Airway epithelial cells not only form a physical barrier but also respond to the presence of microbes by secreting antimicrobials, cytokines, and chemokines. These molecules can lyse infectious microorganisms and/or provide signals critical to the initiation of adaptive immune responses. We examined the effects of CS on antimicrobial secretions of primary human nasal epithelial cells (PHNECs). Compared to non-CS-exposed individuals, PHNEC from in vivo CS-exposed individuals secreted less chemokine ligand (C-C motif) 20 (CCL20), Beta-defensin 1 (BD-1), and SLPI apically, less BD-1 and SLPI basolaterally, and more CCL20 basolaterally. Cigarette smoke extract (CSE) exposure in vitro decreased the apical secretion of CCL20 and beta-defensin 1 by PHNEC from non-CS-exposed individuals. Exposing PHNEC from non-CS exposed to CSE also significantly decreased the levels of many mRNA transcripts that are involved in immune signaling. Our results show that in vivo or in vitro exposure to CS alters the secretion of key antimicrobial peptides from PHNEC, but that in vivo CS exposure is a much more important modifier of antimicrobial peptide secretion. Based on the gene expression data, it appears that CSE disrupts multiple immune signaling pathways in PHNEC. Our results provide mechanistic insight into how CS exposure alters the innate immune response and increases an individual’s susceptibility to pathogen infection. PMID:26793127

  20. Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells.

    PubMed

    Zhou, Yan; Tao, Jing; Yu, Hao; Ni, Jinjing; Zeng, Lingbing; Teng, Qihui; Kim, Kwang Sik; Zhao, Guo-Ping; Guo, Xiaokui; Yao, Yufeng

    2012-03-01

    Type VI secretion systems (T6SSs) are involved in the pathogenicity of several gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

  1. Lanreotide inhibits human jejunal secretion induced by prostaglandin E1 in healthy volunteers.

    PubMed

    Sobhani, I; René, E; Ramdani, A; Bayod, F; Sabbagh, L C; Thomas, F; Mignon, M

    1996-02-01

    1. Somatostatin inhibits hormonal secretions in the gastrointestinal tract. Somatostatin analogues are used in the treatment of VIPome-related watery diarrhoea. In addition, more than 10% of patients with AIDS suffer from diarrhoea likely due to the increased intestinal secretion of water and ions. However, the direct effect of somatostatin on the flux of water and ions in the intestine has not been, so far, analyzed in vivo. The aim of the present study was to evaluate the effect of lanreotide, a somatostatin analogue, on the movements of water and ions in the jejunum in man. 2. Accordingly, 10 healthy volunteers (age 18-35 years, mean 27) and two patients with AIDS (26 and 33 years) suffering from water diarrhoea (> 800 ml day-1) underwent intestinal perfusion using a four lumen tube with proximal occluding balloon. The segment tested was 25 cm long. The jejunum was infused by an isotonic control saline solution containing polyethylene glycol (PEG) as nonabsorbable marker. Basal jejunal secretions were measured in all subjects. Prostaglandin E1 (PGE1) was administered intraluminally to stimulate jejunal secretion in healthy volunteers. The effect of intravenous lanreotide on the jejunal PGE1-induced secretions of water and electrolytes was analysed in healthy subjects and on the basal secretions in AIDS patients. Each period was analyzed on the basis of three (10 min) successive intestinal juice collections after 20-30 min equilibration time. The antisecretory effect of lanreotide was evaluated in each subject as the difference between fluxes compared to the control period. 3. In healthy volunteers, PGE1 induced secretion of H2O, Na+, K+ and Cl- in the jejunum and lanreotide reduced significantly PGE1-induced response. In both AIDS patients basal fluxes of water and ions were reduced by lanreotide in a dose-dependent manner. 4. Somatostatin can reduce stimulated-jejunal secretion of ions and water in normal subjects and may improve water diarrhoea in AIDS

  2. Interleukin-11 alters placentation and causes preeclampsia features in mice

    PubMed Central

    Winship, Amy L.; Koga, Kaori; Menkhorst, Ellen; Van Sinderen, Michelle; Rainczuk, Katarzyna; Nagai, Miwako; Cuman, Carly; Yap, Joanne; Zhang, Jian-Guo; Simmons, David; Young, Morag J.; Dimitriadis, Evdokia

    2015-01-01

    Preeclampsia (PE) is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 wk gestation. Abnormal extravillous trophoblast (EVT) invasion and remodeling of uterine spiral arterioles is thought to contribute to PE development. Interleukin-11 (IL11) impedes human EVT invasion in vitro and is elevated in PE decidua in women. We demonstrate that IL11 administered to mice causes development of PE features. Immunohistochemistry shows IL11 compromises trophoblast invasion, spiral artery remodeling, and placentation, leading to increased systolic blood pressure (SBP), proteinuria, and intrauterine growth restriction, although nonpregnant mice were unaffected. Real-time PCR array analysis identified pregnancy-associated plasma protein A2 (PAPPA2), associated with PE in women, as an IL11 regulated target. IL11 increased PAPPA2 serum and placental tissue levels in mice. In vitro, IL11 compromised primary human EVT invasion, whereas siRNA knockdown of PAPPA2 alleviated the effect. Genes regulating uterine natural killer (uNK) recruitment and differentiation were down-regulated and uNK cells were reduced after IL11 treatment in mice. IL11 withdrawal in mice at onset of PE features reduced SBP and proteinuria to control levels and alleviated placental labyrinth defects. In women, placental IL11 immunostaining levels increased in PE pregnancies and in serum collected from women before development of early-onset PE, shown by ELISA. These results indicate that elevated IL11 levels result in physiological changes at the maternal–fetal interface, contribute to abnormal placentation, and lead to the development of PE. Targeting placental IL11 may provide a new treatment option for PE. PMID:26655736

  3. IGF-1 drives chromogranin A secretion via activation of Arf1 in human neuroendocrine tumour cells

    PubMed Central

    Münzberg, Christin; Höhn, Katharina; Krndija, Denis; Maaß, Ulrike; Bartsch, Detlef K; Slater, Emily P; Oswald, Franz; Walther, Paul; Seufferlein, Thomas; von Wichert, Götz

    2015-01-01

    Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells. PMID:25754106

  4. IGF-1 drives chromogranin A secretion via activation of Arf1 in human neuroendocrine tumour cells.

    PubMed

    Münzberg, Christin; Höhn, Katharina; Krndija, Denis; Maaß, Ulrike; Bartsch, Detlef K; Slater, Emily P; Oswald, Franz; Walther, Paul; Seufferlein, Thomas; von Wichert, Götz

    2015-05-01

    Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells.

  5. Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

    PubMed Central

    Sloop, Kyle W.; Willard, Francis S.; Brenner, Martin B.; Ficorilli, James; Valasek, Kathleen; Showalter, Aaron D.; Farb, Thomas B.; Cao, Julia X.C.; Cox, Amy L.; Michael, M. Dodson; Gutierrez Sanfeliciano, Sonia Maria; Tebbe, Mark J.; Coghlan, Michael J.

    2010-01-01

    OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization. PMID:20823098

  6. Gene therapy of multiple sclerosis using interferon β-secreting human bone marrow mesenchymal stem cells.

    PubMed

    Ryu, Chung Heon; Park, Kwang Ywel; Hou, Yun; Jeong, Chang Hyun; Kim, Seong Muk; Jeun, Sin-Soo

    2013-01-01

    Interferon-beta (IFN- β ), a well-established standard treatment for multiple sclerosis (MS), has proved to exhibit clinical efficacy. In this study, we first evaluated the therapeutic effects for MS using human bone marrow-derived mesenchymal stem cells (hBM-MSCs) as delivery vehicles with lesion-targeting capability and IFN- β as therapeutic gene. We also engineered hBM-MSCs to secret IFN- β (MSCs-IFN β ) via adenoviral transduction and confirmed the secretory capacity of MSCs-IFN β by an ELISA assay. MSCs-IFN β -treated mice showed inhibition of experimental autoimmune encephalomyelitis (EAE) onset, and the maximum and average score for all animals in each group was significantly lower in the MSCs-IFN β -treated EAE mice when compared with the MSCs-GFP-treated EAE mice. Inflammatory infiltration and demyelination in the lumbar spinal cord also significantly decreased in the MSCs-IFN β -treated EAE mice compared to PBS- or MSCs-GFP-treated EAE mice. Moreover, MSCs-IFN β treatment enhanced the immunomodulatory effects, which suppressed proinflammatory cytokines (IFN-γ and TNF-α) and conversely increased anti-inflammatory cytokines (IL-4 and IL-10). Importantly, injected MSCs-IFN β migrated into inflamed CNS and significantly reduced further injury of blood-brain barrier (BBB) permeability in EAE mice. Thus, our results provide the rationale for designing novel experimental protocols to enhance the therapeutic effects for MS using hBM-MSCs as an effective gene vehicle to deliver the therapeutic cytokines.

  7. Gene Therapy of Multiple Sclerosis Using Interferon β-Secreting Human Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Ryu, Chung Heon; Park, Kwang Ywel; Hou, Yun; Jeong, Chang Hyun; Kim, Seong Muk; Jeun, Sin-Soo

    2013-01-01

    Interferon-beta (IFN-β), a well-established standard treatment for multiple sclerosis (MS), has proved to exhibit clinical efficacy. In this study, we first evaluated the therapeutic effects for MS using human bone marrow-derived mesenchymal stem cells (hBM-MSCs) as delivery vehicles with lesion-targeting capability and IFN-β as therapeutic gene. We also engineered hBM-MSCs to secret IFN-β (MSCs-IFNβ) via adenoviral transduction and confirmed the secretory capacity of MSCs-IFNβ by an ELISA assay. MSCs-IFNβ-treated mice showed inhibition of experimental autoimmune encephalomyelitis (EAE) onset, and the maximum and average score for all animals in each group was significantly lower in the MSCs-IFNβ-treated EAE mice when compared with the MSCs-GFP-treated EAE mice. Inflammatory infiltration and demyelination in the lumbar spinal cord also significantly decreased in the MSCs-IFNβ-treated EAE mice compared to PBS- or MSCs-GFP-treated EAE mice. Moreover, MSCs-IFNβ treatment enhanced the immunomodulatory effects, which suppressed proinflammatory cytokines (IFN-γ and TNF-α) and conversely increased anti-inflammatory cytokines (IL-4 and IL-10). Importantly, injected MSCs-IFNβ migrated into inflamed CNS and significantly reduced further injury of blood-brain barrier (BBB) permeability in EAE mice. Thus, our results provide the rationale for designing novel experimental protocols to enhance the therapeutic effects for MS using hBM-MSCs as an effective gene vehicle to deliver the therapeutic cytokines. PMID:23710456

  8. Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation.

    PubMed

    Díaz-Delfín, Julieta; Domingo, Pere; Giralt, Marta; Villarroya, Francesc

    2013-03-01

    Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patients with CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequently appear in HIV-infected patients under HAART, there is no information available on the effects of MVC on adipose tissue. Here we studied the effects of MVC during and after the differentiation of human adipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured the acquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity, adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally, we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed no alterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistent with this, there were no changes in the mRNA levels of PPARγ or SREBP-1c, two master regulators of adipogenesis. In addition, MVC caused a significant decrease in the gene expression and release of pro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflammation-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes in differentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rather shows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatory cytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissue development, metabolism, and inflammation, and thus could be a potentially beneficial component of antiretroviral therapy.

  9. A nanoscaffold impregnated with human wharton's jelly stem cells or its secretions improves healing of wounds.

    PubMed

    Tam, Kimberley; Cheyyatraviendran, Suganya; Venugopal, Jayarama; Biswas, Arijit; Choolani, Mahesh; Ramakrishna, Seeram; Bongso, Ariff; Fong, Chui-Yee

    2014-04-01

    Wound healing is a major problem in diabetic patients and current methods of treatment have met with limited success. Since skin cell renewal is under the control of mesenchymal stem cells (MSCs) treatment of wounds has been attempted with the application of exogenous bone marrow MSCs (hBMMSCs). However, hBMMSCs have the limitations of painful harvest, low cell numbers and short-lived stemness properties unlike MSCs from the Wharton's jelly of human umbilical cords (hWJSCs). Since nanoscaffolds provide three dimensional architectural patterns that mimic in vivo stem cell niches and aloe vera has antibacterial properties we evaluated the use of an aloe vera-polycaprolactone (AV/PCL) nanoscaffold impregnated with green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs + AV/PCL) or its conditioned medium (hWJSC-CM + AV/PCL) for healing of excisional and diabetic wounds. In skin fibroblast scratch-wound assays exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL, fibroblasts migrated significantly faster from edges of scratches into vacant areas together with increased secretion of collagen I and III, elastin, fibronectin, superoxide dismutase, and metalloproteinase-1 (MMP-1) compared to controls. After one application of GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL excisional and diabetic wounds in mice showed rapid wound closure, reepithelialization, and increased numbers of sebaceous glands and hair follicles compared to controls. The same wounds exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL also showed positive keratinocyte markers (cytokeratin, involucrin, filaggrin) and increased expression of ICAM-1, TIMP-1, and VEGF-A compared to controls. AV/PCL nanoscaffolds in combination with hWJSCs appear to have synergistic benefits for wound healing.

  10. Insulin secretion and interleukin-1β dependent mechanisms in human diabetes remission after metabolic surgery.

    PubMed

    Chen, Chih-Yen; Lee, Wei-Jei; Asakawa, A; Fujitsuka, N; Chong, Keong; Chen, Shu-Chun; Lee, Shou-Dong; Inui, A

    2013-01-01

    To compare endocrine, metabolic, and inflammatory changes induced by gastric bypass (GB) and sleeve gastrectomy (SG) in patients with type 2 diabetes mellitus (T2DM), and to investigate the mechanisms of success after metabolic surgery. Sixteen GB and 16 SG patients were followed up before and at 1 year after surgery. The 75-g oral glucose tolerance test (OGTT) was performed before and after surgery. Glucose homeostasis, serum interleukin-1β, plasma gut hormones and adipokines, and the United Kingdom Prospective Diabetes Study (UKPDS) ten-year cardiovascular risks were evaluated. The diabetes remission rate was significantly higher in GB than SG. Changes in the area under the curve (AUC) for glucose were greater in those with complete and partial remission after GB and remitters after SG than non-remitters after SG, whereas changes in AUC for C-peptide were higher in complete and partial remitters after GB than non-remitters after SG. Insulinogenic index was enhanced and serum interleukin-1β was reduced in complete remitters after GB and remitters after SG. Logistic regression analysis confirmed that insulinogenic index and interleukin-1β, not insulin resistance, were the factors determining the success of diabetes remission after metabolic surgeries. GB and SG significantly reduced the ten-year risk of coronary heart disease and fatal coronary heart disease in T2DM patients after surgery, while GB had the additional benefit of reduced stroke risk. Human diabetes remission after metabolic surgery is through insulin secretion and interleukin-1β dependent mechanisms. GB is superior to SG in cardiocerebral risk reduction in Asian non-morbidly obese, not well-controlled T2DM patients.

  11. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

    PubMed Central

    Bacos, Karl; Gillberg, Linn; Volkov, Petr; Olsson, Anders H; Hansen, Torben; Pedersen, Oluf; Gjesing, Anette Prior; Eiberg, Hans; Tuomi, Tiinamaija; Almgren, Peter; Groop, Leif; Eliasson, Lena; Vaag, Allan; Dayeh, Tasnim; Ling, Charlotte

    2016-01-01

    Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA methylation genome-wide in islets from 87 non-diabetic donors, aged 26–74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D. PMID:27029739

  12. Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion.

    PubMed Central

    Akita, S; Webster, J; Ren, S G; Takino, H; Said, J; Zand, O; Melmed, S

    1995-01-01

    Leukemia inhibitory factor (LIF) gene expression was detected in human fetal pituitary tissue by expression of LIF mRNA transcripts, protein immunocytochemistry, and immunoelectron microscopy. Fetal LIF immunoreactivity colocalized with 30% of ACTH-expressing cells, approximately 20% of somatotrophs, and approximately 15% of non-hormone-expressing cells. LIF was also strongly expressed in normal adult pituitary and in four growth hormone-producing and two ACTH-producing adenomas, but not in eight nonfunctioning pituitary tumors. Culture of fetal cells expressing surface LIF-binding sites demonstrated predominance of in vitro ACTH secretion as compared with other pituitary hormones. In AtT-20 murine cells, LIF (ED50 10 pM) stimulated basal proopiomelanocortin mRNA levels by 40% and corticotropin-releasing hormone-induced ACTH secretion (two- to threefold), as did oncostatin M (ED50 30 pM), a related peptide. ACTH responses were not further enhanced by both cytokines together, which is consistent with their shared receptor. Anti-LIF antiserum neutralized basal and LIF-induced ACTH secretion, suggesting autocrine regulation of ACTH by LIF. The results show that human pituitary cells express the LIF gene and LIF-binding sites, predominantly in corticotrophs. Pituitary LIF expression and LIF regulation of proopiomelanocortin and ACTH reflect an intrapituitary role for LIF in modulating early embryonic determination of specific human pituitary cells and as a paracrine or autocrine regulator of mature ACTH. Images PMID:7883977

  13. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes.

    PubMed

    Bacos, Karl; Gillberg, Linn; Volkov, Petr; Olsson, Anders H; Hansen, Torben; Pedersen, Oluf; Gjesing, Anette Prior; Eiberg, Hans; Tuomi, Tiinamaija; Almgren, Peter; Groop, Leif; Eliasson, Lena; Vaag, Allan; Dayeh, Tasnim; Ling, Charlotte

    2016-03-31

    Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA methylation genome-wide in islets from 87 non-diabetic donors, aged 26-74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D.

  14. Epithelial Cell Secretions from the Human Female Reproductive Tract Inhibit Sexually Transmitted Pathogens and Candida albicans but not Lactobacillus

    PubMed Central

    Wira, CR; Ghosh, M; Smith, JM; Shen, L; Connor, RI; Sundstrom, P; Frechette, Gregory M.; Hill, EM; Fahey, JV

    2011-01-01

    Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens which threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), HIV-1, and Lactobacillus crispatus, prior to being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. Cytokines and chemokines analysis in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans. PMID:21048705

  15. Transplantation of insulin-secreting cells differentiated from human adipose tissue-derived stem cells into type 2 diabetes mice.

    PubMed

    Nam, Ji Sun; Kang, Hyun Mi; Kim, Jiyoung; Park, Seah; Kim, Haekwon; Ahn, Chul Woo; Park, Jin Oh; Kim, Kyung Rae

    2014-01-10

    Currently, there are limited ways to preserve or recover insulin secretory capacity in human pancreas. We evaluated the efficacy of cell therapy using insulin-secreting cells differentiated from human eyelid adipose tissue-derived stem cells (hEAs) into type 2 diabetes mice. After differentiating hEAs into insulin-secreting cells (hEA-ISCs) in vitro, cells were transplanted into a type 2 diabetes mouse model. Serum levels of glucose, insulin and c-peptide were measured, and changes of metabolism and inflammation were assessed in mice that received undifferentiated hEAs (UDC group), differentiated hEA-ISCs (DC group), or sham operation (sham group). Human gene expression and immunohistochemical analysis were done. DC group mice showed improved glucose level, and survival up to 60 days compared to those of UDC and sham group. Significantly increased levels of human insulin and c-peptide were detected in sera of DC mice. RT-PCR and immunohistochemical analysis showed human gene expression and the presence of human cells in kidneys of DC mice. When compared to sham mice, DC mice exhibited lower levels of IL-6, triglyceride and free fatty acids as the control mice. Transplantation of hEA-ISCs lowered blood glucose level in type 2 diabetes mice by increasing circulating insulin level, and ameliorating metabolic parameters including IL-6.

  16. Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

    PubMed Central

    Williams, Rachel C.; Skelton, Andrew J.; Todryk, Stephen M.; Rowan, Andrew D.; Preshaw, Philip M.; Taylor, John J.

    2016-01-01

    Introduction Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. Methods and Results We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. Conclusions We conclude that leptin selectively enhances the expression and secretion of certain matrix

  17. A low dose euglycemic infusion of recombinant human insulin-like growth factor I rapidly suppresses fasting-enhanced pulsatile growth hormone secretion in humans.

    PubMed Central

    Hartman, M L; Clayton, P E; Johnson, M L; Celniker, A; Perlman, A J; Alberti, K G; Thorner, M O

    1993-01-01

    To determine if insulin-like growth factor I (IGF-I) inhibits pulsatile growth hormone (GH) secretion in man, recombinant human IGF-I (rhIGF-I) was infused for 6 h at 10 micrograms.kg-1.h-1 during a euglycemic clamp in 10 normal men who were fasted for 32 h to enhance GH secretion. Saline alone was infused during an otherwise identical second admission as a control. As a result of rhIGF-I infusion, total and free IGF-I concentrations increased three- and fourfold, respectively. Mean GH concentrations fell from 6.3 +/- 1.6 to 0.59 +/- 0.07 micrograms/liter after 120 min. GH secretion rates, calculated by a deconvolution algorithm, decreased with a t 1/2 of 16.6 min and remained suppressed thereafter. Suppression of GH secretion rates occurred within 60 min when total and free IGF-I concentrations were 1.6-fold and 2-fold above baseline levels, respectively, and while glucose infusion rates were < 1 mumol.kg-1.min-1. During saline infusion, GH secretion rates remained elevated. Infusion of rhIGF-I decreased the mass of GH secreted per pulse by 84% (P < 0.01) and the number of detectable GH secretory pulses by 32% (P < 0.05). Plasma insulin and glucagon decreased to nearly undetectable levels after 60 min of rhIGF-I. Serum free fatty acids, beta-hydroxybutyrate, and acetoacetate were unaffected during the first 3 h of rhIGF-I but decreased thereafter to 52, 32, and 50% of levels observed during saline. We conclude that fasting-enhanced GH secretion is rapidly suppressed by a low-dose euglycemic infusion of rhIGF-I. This effect of rhIGF-I is likely mediated through IGF-I receptors independently of its insulin-like metabolic actions. PMID:8514857

  18. [Endocrino-pharmacological study of reproduction: Role and biosynthesis of steroid hormones in the feto-placental unit].

    PubMed

    Hirai, M; Masubuchi, Y; Komoriyama, K

    1981-03-01

    Although considerable information is available concerning steroidogenesis in the human fetus, the function of the different steroids formed during pregnancy and the factors regulating this delicate hormones balance are poorly understood. During human pregnancy, the placenta synthesizes large quantities of progesterone, estradiol, estrone and estriol and secretes these hormones into both the maternal and fetal circulations; progesterone from maternal lipoprotein-cholesterol, estradiol and estrone from maternal and fetal dehydroepiandrosterone sulfate (DHAS), and estriol largely from fetal 16 alpha-OH-DHAS. It has been demonstrated that preimplantation blastocysts of several animal species have the capacity to accumulate steroids to pregnenolone to progesterone, and to interconvert estrone and estradiol. Estetrol (E4), 15 alpha-hydroxy derivative of estriol is an interesting compound, since its formation is relatively unique to fetal liver function. Of special interest is that placental sulfatase deficiencies result in an extension of the gestation, and Cesarean section has to be done. This raises the question of the role of estrogens in determining the onset of labor, much as in the case of anencephaly. In general, progesterone may decline prior to an abortion, but there has not been a direct application to clinical practice. Estrogen levels during pregnancy are influenced by factors other than fetal well-being and include fetal weight, placental enzyme function, fetal adrenal function, maternal intestinal flora, maternal renal excretion and maternal liver function. Although not yet extensively utilized, such a dynamic test as the infusion of DHAS may yield useful information within a short period in otherwise complicated cases related to fetal and placental function.

  19. Does hypercalcaemia or calcium antagonism affect human melatonin secretion or renal excretion?

    PubMed

    Wikner, J; Wetterberg, L; Röjdmark, S

    1997-05-01

    Patients with primary hyperparathyroidism have higher serum melatonin concentrations during active disease than after surgical cure. Whether this is caused by hypercalcaemia per se, increased parathyroid hormone secretion or other mechanisms is unknown. We decided to elucidate whether exogenous hypercalcaemia influences melatonin secretion. For this purpose, eight healthy volunteers were infused with calcium and saline on separate days and in random order (experiment A). Hypercalcaemia inhibited nocturnal melatonin secretion by 20% but left urinary melatonin excretion unaffected. If exogenous hypercalcaemia inhibits melatonin secretion, it is reasonable to assume that calcium channel blockers such as verapamil might have the opposite effect. This was investigated in experiment B, in which eight healthy subjects were treated on separate occasions with oral verapamil and placebo. Verapamil did not affect nocturnal melatonin secretion but increased melatonin excretion by 145%. As 6-sulphatoxy-melatonin is the main melatonin metabolite excreted by the kidneys, it was considered important to find out whether verapamil would also influence the excretion of 6-sulphatoxy-melatonin. This was investigated in experiment C, in which eight healthy volunteers were treated, on separate occasions, with oral verapamil and placebo. In this experiment also, verapamil increased urinary melatonin excretion significantly (by 67%), but left excretion of 6-sulphatoxy-melatonin unaffected. These findings imply that verapamil influences the renal and/or hepatic handling of melatonin.

  20. HIV-1 Nef breaches placental barrier in rat model.

    PubMed

    Singh, Poonam; Agnihotri, Saurabh Kumar; Tewari, Mahesh Chandra; Kumar, Sadan; Sachdev, Monika; Tripathi, Raj Kamal

    2012-01-01

    The vertical transmission of HIV-1 from the mother to fetus is known, but the molecular mechanism regulating this transmission is not fully characterized. The fetus is highly protected by the placenta, which does not permit microbial pathogens to cross the placental barrier. In the present study, a rat model was established to observe the effect of HIV-1 protein Nef on placental barrier. Evans blue dye was used to assay permeability of placental barrier and fourteen day pregnant Sprague Dawley rats were injected intravenously with 2% Evans blue dye along with various concentrations of recombinant Nef. After an hour, animals were sacrificed and dye migration was observed through the assimilation of peripheral blood into fetus. Interestingly, traces of recombinant Nef protein were detected in the embryo as well as amniotic fluid and amniotic membrane along with placenta and uterus. Our study indicates that recombinant HIV-1-Nef protein breaches the placental barrier and allows the migration of Evans blue dye to the growing fetus. Further the concentration of Nef protein in blood is directly proportional to the intensity of dye migration and to the amount of Nef protein detected in uterus, placenta, amniotic membrane, amniotic fluid and embryo. Based on this study, it can be concluded that the HIV-1 Nef protein has a direct effect on breaching of the placental barrier in the model we have established in this study. Our observations will be helpful to understand the molecular mechanisms related to this breach of placental barrier by Nef in humans and may be helpful to identify specific Nef inhibitors.

  1. Induction of synthesis and secretion of interleukin 1 beta in the human monocytic THP-1 cells by human serum albumins modified with methylglyoxal and advanced glycation endproducts.

    PubMed

    Westwood, M E; Thornalley, P J

    1996-04-01

    Human serum albumin modified with 1-2 methylglyoxal residues per molecule of protein (MGmin-HSA) stimulated the synthesis and secretion of interleukin 1 beta (IL-1 beta) from human monocytic THP-1 cells in vitro. It was a more potent inducer of IL-1 beta synthesis than human serum albumin highly-modified with glucose-derived advanced glycation endproducts (AGE-HSA). With 20 microM ligand. IL-1 beta synthesis was (pg/10(6) cells): MGmin-HSA 484.5 +/- 50.3; AGE-HSA 30.6 +/- 2.0 (n = 3). IL-1 beta synthesis increased markedly with MGmin-HSA concentrations > 5 microM. IL-1 beta synthesis and secretion from monocytes in response to methylglyoxal-modified proteins in vivo may contribute to the development of macro- and micro-angiopathy, particularly in diabetes mellitus.

  2. Hyperpolarization of the Membrane Potential Caused by Somatostatin in Dissociated Human Pituitary Adenoma Cells that Secrete Growth Hormone

    NASA Astrophysics Data System (ADS)

    Yamashita, Naohide; Shibuya, Naohiko; Ogata, Etsuro

    1986-08-01

    Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 ± 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 ± 4.3 mV in 6 mM K+ medium and -97.2 ± 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 ± 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.

  3. Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

    PubMed Central

    Veit, Guido; Bossard, Florian; Goepp, Julie; Verkman, A. S.; Galietta, Luis J. V.; Hanrahan, John W.; Lukacs, Gergely L.

    2012-01-01

    Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr∆F508/∆F508 immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia. PMID:22973054

  4. PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma.

    PubMed

    Siggins, Sarah; Jauhiainen, Matti; Olkkonen, Vesa M; Tenhunen, Jukka; Ehnholm, Christian

    2003-09-01

    Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.

  5. Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion.

    PubMed

    Ferrero, Mariana C; Fossati, Carlos A; Rumbo, Martín; Baldi, Pablo C

    2012-10-01

    In spite of the frequent acquisition of Brucella infection by the oral route in humans, the interaction of the bacterium with cells of the intestinal mucosa has been poorly studied. Here, we show that different Brucella species can invade human colonic epithelial cell lines (Caco-2 and HT-29), in which only smooth species can replicate efficiently. Infection with smooth strains did not produce a significant cytotoxicity, while the rough strain RB51 was more cytotoxic. Infection of Caco-2 cells or HT-29 cells with either smooth or rough strains of Brucella did not result in an increased secretion of TNF-α, IL-1β, MCP-1, IL-10 or TGF-β as compared with uninfected controls, whereas all the infections induced the secretion of IL-8 and CCL20 by both cell types. The MCP-1 response to flagellin from Salmonella typhimurium was similar in Brucella-infected or uninfected cells, ruling out a bacterial inhibitory mechanism as a reason for the weak proinflammatory response. Infection did not modify ICAM-1 expression levels in Caco-2 cells, but increased them in HT-29 cells. These results suggest that Brucella induces only a weak proinflammatory response in gut epithelial cells, but produces a significant CCL20 secretion. The latter may be important for bacterial dissemination given the known ability of Brucella to survive in dendritic cells.

  6. Placental growth factor mediates aldosterone-dependent vascular injury in mice.

    PubMed

    Jaffe, Iris Z; Newfell, Brenna G; Aronovitz, Mark; Mohammad, Najwa N; McGraw, Adam P; Perreault, Roger E; Carmeliet, Peter; Ehsan, Afshin; Mendelsohn, Michael E

    2010-11-01

    In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

  7. Placental Hypoxia During Early Pregnancy Causes Maternal Hypertension and Placental Insufficiency in the Hypoxic Guinea Pig Model.

    PubMed

    Thompson, Loren P; Pence, Laramie; Pinkas, Gerald; Song, Hong; Telugu, Bhanu P

    2016-12-01

    Chronic placental hypoxia is one of the root causes of placental insufficiencies that result in pre-eclampsia and maternal hypertension. Chronic hypoxia causes disruption of trophoblast (TB) development, invasion into maternal decidua, and remodeling of maternal spiral arteries. The pregnant guinea pig shares several characteristics with humans such as hemomonochorial placenta, villous subplacenta, deep TB invasion, and remodeling of maternal arteries, and is an ideal animal model to study placental development. We hypothesized that chronic placental hypoxia of the pregnant guinea pig inhibits TB invasion and alters spiral artery remodeling. Time-mated pregnant guinea pigs were exposed to either normoxia (NMX) or three levels of hypoxia (HPX: 16%, 12%, or 10.5% O2) from 20 day gestation until midterm (39-40 days) or term (60-65 days). At term, HPX (10.5% O2) increased maternal arterial blood pressure (HPX 57.9 ± 2.3 vs. NMX 40.4 ± 2.3, P < 0.001), decreased fetal weight by 16.1% (P < 0.05), and increased both absolute and relative placenta weights by 10.1% and 31.8%, respectively (P < 0.05). At midterm, there was a significant increase in TB proliferation in HPX placentas as confirmed by increased PCNA and KRT7 staining and elevated ESX1 (TB marker) gene expression (P < 0.05). Additionally, quantitative image analysis revealed decreased invasion of maternal blood vessels by TB cells. In summary, this animal model of placental HPX identifies several aspects of abnormal placental development, including increased TB proliferation and decreased migration and invasion of TBs into the spiral arteries, the consequences of which are associated with maternal hypertension and fetal growth restriction.

  8. Notch signalling in placental development and gestational diseases.

    PubMed

    Haider, S; Pollheimer, J; Knöfler, M

    2017-01-16

    Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.

  9. Maternal fructose drives placental uric acid production leading to adverse fetal outcomes

    PubMed Central

    Asghar, Zeenat A.; Thompson, Alysha; Chi, Maggie; Cusumano, Andrew; Scheaffer, Suzanne; Al-Hammadi, Noor; Saben, Jessica L.; Moley, Kelle H.

    2016-01-01

    Maternal metabolic diseases increase offspring risk for low birth weight and cardiometabolic diseases in adulthood. Excess fructose consumption may confer metabolic risks for both women and their offspring. However, the direct consequences of fructose intake per se are unknown. We assessed the impact of a maternal high-fructose diet on the fetal-placental unit in mice in the absence of metabolic syndrome and determined the association between maternal serum fructose and placental uric acid levels in humans. In mice, maternal fructose consumption led to placental inefficiency, fetal growth restriction, elevated fetal serum glucose and triglyceride levels. In the placenta, fructose induced de novo uric acid synthesis by activating the activities of the enzymes AMP deaminase and xanthine oxidase. Moreover, the placentas had increased lipids and altered expression of genes that control oxidative stress. Treatment of mothers with the xanthine oxidase inhibitor allopurinol reduced placental uric acid levels, prevented placental inefficiency, and improved fetal weights and serum triglycerides. Finally, in 18 women delivering at term, maternal serum fructose levels significantly correlated with placental uric acid levels. These findings suggest that in mice, excess maternal fructose consumption impairs placental function via a xanthine oxidase/uric acid-dependent mechanism, and similar effects may occur in humans. PMID:27125896

  10. Maternal fructose drives placental uric acid production leading to adverse fetal outcomes.

    PubMed

    Asghar, Zeenat A; Thompson, Alysha; Chi, Maggie; Cusumano, Andrew; Scheaffer, Suzanne; Al-Hammadi, Noor; Saben, Jessica L; Moley, Kelle H

    2016-04-29

    Maternal metabolic diseases increase offspring risk for low birth weight and cardiometabolic diseases in adulthood. Excess fructose consumption may confer metabolic risks for both women and their offspring. However, the direct consequences of fructose intake per se are unknown. We assessed the impact of a maternal high-fructose diet on the fetal-placental unit in mice in the absence of metabolic syndrome and determined the association between maternal serum fructose and placental uric acid levels in humans. In mice, maternal fructose consumption led to placental inefficiency, fetal growth restriction, elevated fetal serum glucose and triglyceride levels. In the placenta, fructose induced de novo uric acid synthesis by activating the activities of the enzymes AMP deaminase and xanthine oxidase. Moreover, the placentas had increased lipids and altered expression of genes that control oxidative stress. Treatment of mothers with the xanthine oxidase inhibitor allopurinol reduced placental uric acid levels, prevented placental inefficiency, and improved fetal weights and serum triglycerides. Finally, in 18 women delivering at term, maternal serum fructose levels significantly correlated with placental uric acid levels. These findings suggest that in mice, excess maternal fructose consumption impairs placental function via a xanthine oxidase/uric acid-dependent mechanism, and similar effects may occur in humans.

  11. Insulin-secreting cells from human eyelid-derived stem cells alleviate type I diabetes in immunocompetent mice.

    PubMed

    Kang, Hyun Mi; Kim, Jiyoung; Park, Seah; Kim, Jinyoung; Kim, Haekwon; Kim, Kyung Sik; Lee, Eun Jig; Seo, Sung Ig; Kang, Sung Goo; Lee, Jong-Eun; Lim, Hyunjung

    2009-08-01

    Various attempts have been made to develop stem cell-based therapy to alleviate type I diabetes using animal models. However, it has been a question whether human insulin produced from explanted cells is solely responsible for the normoglycemia of diabetic animals. In this study, we isolated neural crest-like stem cells from the human eyelid fat and examined their therapeutic potentials for diabetes. The human eyelid adipose-derived stem cells (HEACs) displayed characteristics of neural crest cells. Using a two-step culture condition combined with nicotinamide, activin, and/or GLP-1, we differentiated HEACs into insulin-secreting cells and examined in vivo effects of differentiated cells by transplantation experiments. Following differentiation in vitro, HEACs released insulin and c-peptide in a glucose-dependent manner. Upon their transplantation under kidney capsules of streptozotocin-treated immunocompetent mice, we observed normalization of hyperglycemia in 10 of 20 recipient mice until sacrifice after 2 months. Only the human, but not the mouse, insulin and c-peptide were detected in the blood of recipient mice. Removal of the kidneys transplanted with HEACs resulted in a sharp increase of blood glucose level. Removed kidney tissues showed distinct expression of various human genes including insulin, and colocalization of the human insulin and the human nuclear protein in many cells. However, they showed diminished or null expression of some immune-related genes. In conclusion, human insulin alone produced from eyelid-derived stem cells following differentiation into insulin-secreting cells and transplantation could normalize type I diabetes in mice.

  12. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types incl