Biomedical subjects

M H Sullivan

Publications and source records attributed to M H Sullivan.

At least 37 records · Page 2Linked to original sources

PubMed1997

The regulation of progesterone and hCG production from placental cells by interleukin-1beta.

We have investigated the roles of interleukin-1beta as a regulator of progesterone and chorionic gonadotrophin production from human placental cells. In primary placental cells IL-1beta increased hCG synthesis through a cyclic AMP-independent pathway, and was without effect on progesterone or cyclic AMP production. Since dibutyryl cyclic AMP increased progesterone production, this suggests that there is no coupling between the IL-1beta receptor and the adenylate cyclase enzyme in these cells. Immortalised trophoblast cells responded to IL-1beta by increasing progesterone production through a cyclic AMP-dependent mechanism, but hCG production by these cells was unaffected by IL-1beta or dibutyryl cyclic AMP. Further studies are needed to identify the role of IL-1beta as a possible regulator of progesterone production in primary placental cells. While hCG production in first-trimester trophoblast was increased by dibutyryl cyclic AMP and IL-1beta, both these effects may involve other factors such as IL-6, and their second messenger systems.

Cells, Cultured↗

PubMed1997

Second messengers and the control of progesterone production from first trimester trophoblast.

Basal progesterone production from first trimester placental cells in culture was high during the first 24 h of culture and fell to less than 30% of the initial level after 96 h in vitro. 22(R)-Hydroxycholesterol had a similar effect on progesterone production at all incubation times, indicating that the decline in basal steroidogenesis was not due to a loss of mitochondrial or post-mitochondrial enzymes. Continuous stimulation with dibutyryl (db) cyclic AMP maintained progesterone synthesis at a relatively constant high level despite the fall in basal progesterone production, and the optimum concentration of db cyclic AMP was 1.0 mM. The calcium ionophore A23187 had no effect on progesterone incubation during short-term cultures (<4 h), and inhibited steroidogenesis after 24 h. Repeated addition of A23187 during 96 h of culture also inhibited progesterone production. These findings indicate that progesterone production in human trophoblast is supported by a local factor which maintains a high level of steroid production through a cyclic AMP-dependent mechanism. The inhibitory effects of calcium ionophore in trophoblast differ from the stimulatory effects of this compound in other steroidogenic cells, but the reasons for the difference are not known at present.

Adenosine Monophosphate↗

PubMed1997

The regulation of platelet aggregation in vitro by interleukin-1beta and tumor necrosis factor-alpha: changes in pregnancy and in pre-eclampsia.

Platelet activation occurs in early pregnancy in women at risk of developing pre-eclampsia. Cytokines have been implicated in the pathogenesis of pre-eclampsia, so we determined the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the in vitro aggregation of human platelets. IL-1beta increased aggregation of platelets from non-pregnant and pre-eclamptic women, and inhibited the aggregation of platelets from normal pregnant women. This latter effect was linked to a diminished P-selectin expression on ADP-stimulated whole blood platelets in normal pregnant women (p = 0.011). Platelet aggregation in response to ADP was found to be inhibited after preincubation with TNF-alpha in non-pregnant (38%, p = 0.01) and in normal pregnant women (54%, p = 0.001) and not affected in pre-eclamptic women. The inhibitory effects of TNF-alpha were mediated through the P75 receptor for TNF-alpha.

Adenosine Diphosphate↗

PubMed1996

Morphological interactions of human first trimester placental villi co-cultured with decidual explants.

Abnormalities of pregnancy such as pre-eclampsia and intrauterine growth retardation are characterized by shallow trophoblastic invasion of the placental bed, the precise molecular pathophysiology of which remains to be fully elucidated. An in-vitro model involving a co-culture of first trimester placental villi and decidua parietalis explants (of 8-12 weeks gestation) was developed and used to characterize the migration and local invasion of trophoblast cells. Trophoblast proliferation (confirmed by Ki-67 immunostaining), differentiation and loose attachment of placental villi to the underlying decidual epithelium or stroma occurred within the first 24 h of co-culture. This was followed by erosion of the syncytial layer of the placental villi and commencement of a progressive cytotrophoblast invasion after 48 h of co-culture, which continued until 120 h, when the experiments were terminated. E-cadherin was expressed at the interfaces between trophoblast cells within the villi, but expression of this adhesion molecule seemed to be down-regulated in the invasive trophoblast cells. Our results suggest that the model could be useful in investigating the factors that control early human placentation and the feto-maternal interface.

Antibodies, Monoclonal↗

PubMed1996

Preparation of a population of macrophages from human third trimester decidua.

Human decidua in the third trimester of pregnancy contains at least four different cell types (stromal cells, macrophages, T-lymphocytes and granulocytes). The contributions of these cells to the overall function of the tissue are not known, although it is anticipated that both stromal cells and macrophages will produce the prostaglandins involved in labour. In addition, the decidua produces cytokines, but the cellular source has not been identified. As a first stage to addressing these questions, we have developed magnetic separation methods to obtain enriched populations of decidual macrophages which contain at least 5 x 10(6) cells. We found that a preparation of up to 10(7) cells which are up to 80% pure (as assessed by a fluorescence-activated cell sorting procedure) and approximately 90% pure (as assessed by immunocytochemistry) can be obtained reliably with this method. These cells are viable for at least 48 h in culture after the enrichment procedure, and produce prostaglandins after stimulation with interleukin-1 beta.

Cell Separation↗

PubMed1996

A comparison of cytokine and hormone production by decidual cells and tissue explants.

Previous work has shown that enzymatic digestion of human placental tissue can induce the production of the cytokine interleukin-1 beta. Most studies of the feto-maternal interface of human pregnancy have used decidual cells prepared in a similar way, but the effects of tissue dissociation on the production of growth factors, cytokines, prostaglandins or hormones have not been investigated. Our studies show human decidual explants produce substantially lower levels of a range of factors than do human decidual cells cultured under the same conditions, indicating that induction may be a general process during the dissociation of tissues in vitro as the production of interleukins-1 beta, -6 and -8, granulocyte-macrophage colony-stimulating factor, transforming growth factor-beta 2, tissue necrosis factor-alpha, prostaglandins E2 and F2 alpha, and prolactin were all affected. The induction of cytokine production (expressed per mg tissue protein) ranged from 10- to 300-fold, indicating that isolated cells cultured in vitro may not reflect accurately the in vivo situation.

Cell Culture Techniques↗

PubMed1995

Interleukin-1 beta independently stimulates production of prostaglandin E2 and cyclic AMP from human decidual cells.

Interleukin-1 beta (IL-1 beta) increased the production of cyclic AMP and prostaglandin E2 (PGE2) by cultured human decidual cells during 24 h of stimulation, but not over short incubation times (< 6 h). At concentrations of IL-1 beta ranging from 1 to 100 pg/ml, there were parallel changes in cyclic AMP and PGE2 levels, but 1000 pg of IL-1 beta/ml inhibited cyclic AMP production while still stimulating PGE2 synthesis. The possible link between cyclic AMP and PGE2 was therefore studied further. Inhibition of IL-1 beta-stimulated PGE2 synthesis by indomethacin and direct addition of PGE2 had no effect on cyclic AMP levels, indicating that PGE2 did not increase cyclic AMP production by human decidual cells and confirming the independent synthesis of cyclic AMP and PGE2. The increase in cyclic AMP production induced by IL-1 beta is dependent on protein synthesis, but it is not known which component of the adenylate cyclase is increased. A phosphodiesterase inhibitor potentiated the effects of IL-1 beta on cyclic AMP synthesis, indicating that the cytokine may increase cyclic AMP metabolism. We suggest that high concentrations of IL-1 beta activate phosphodiesterase activity more than adenylate cyclase, which gives rise to the low levels of cyclic AMP noted above. IL-1 beta also decreased forskolin-stimulated cyclic AMP production, which again indicates increased cyclic AMP metabolism. Since most concentrations of IL-1 beta alone increased cyclic AMP levels, this stimulation must out-weigh the increase in metabolism apparent in the presence of forskolin, phosphodiesterase inhibitor or high levels of interleukin. It is clear that IL-1 beta increased decidual PGE2 production independently of cyclic AMP, and that other second messenger must mediate the action of this cytokine.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Endothelin-1 regulates human decidual cells through both A- and B-type receptors.

The aims of this study were to investigate the effects of endothelin-1 (ET-1) on first trimester human decidual cells, identify the ET receptor sub-types through which these effects are mediated and assess the role of cyclic AMP in any effects of ET-1. ET-1 increased prostaglandin production by first trimester decidual cells, which was consistent with similar studies in third trimester decidual cells. The endothelin A-type receptor (ETA) was coupled to the increased prostaglandin production from first trimester cells, as shown by the inhibitory effects of BQ610, an ETA receptor antagonist. The time course of this effect was unusual, with a rapid increase peaking after 6 h of stimulation, followed by a longer effect over 12-24 h, which was paralleled by changes in cyclic AMP production. No evidence was obtained for any involvement of cyclic AMP in mediating the effects of ET-1 on prostaglandin production. ET-1 also increased decidual prolactin production with a maximum effect after 6 h of stimulation. This was mediated through the ETB receptor and may be linked to increased cyclic AMP production, indicating that the ETA and ETB receptors were coupled to different second messenger systems and affected decidual cell function in different ways.

Cyclic AMP↗

PubMed1995

Formation and metabolism of 14,15-epoxyeicosatrienoic acid by human reproductive tissues.

Human granulosa-luteal cells cultured in the presence of arachidonic acid produced low levels of the epoxygenase metabolite 14,15-epoxy-5,8,11-(Z,Z,Z)-eicosatrienoic acid (14,15-EpETrE) as determined by HPLC analysis and gas chromatography mass spectrometry. When authentic 14,15-[3H]EpETrE was incubated with these cells in the absence of serum it was metabolised initially to the dihydroxy derivative (14,15-dihydroxy-5,8,11-eicosatrienoic acid, 14,15-DiHETrE) and subsequently to a number of more polar metabolites as determined by HPLC. Fetal calf serum protected 14,15-EpETrE from metabolism for at least 2 h. A similar pattern of metabolism was obtained when 14,15-[3H]EpETrE was incubated with a human choriocarcinoma cell line (BeWo). Microsomes from this cell line converted arachidonic acid to a large number of radioactive metabolites including 14,15-DiHETrE and 11,12-DiHETrE although there was no evidence for the parent epoxides. These results extend earlier findings that human reproductive tissues produce epoxygenase metabolites, and demonstrate the rapid metabolism of these compounds by intact cells in the absence of serum.

8,11,14-Eicosatrienoic Acid↗

PubMed1995

Treatment of pseudomyxoma peritonei in a geriatric patient population.

Pseudomyxoma peritonei of appendiceal origin is a disease characterized by the progressive accumulation of mucus ascites within the abdomen and pelvis. Metastatic disease outside the peritoneal cavity is unusual. Gastrointestinal function is lost from external compression of stomach, small bowel, and large bowel. New surgical and regional chemotherapy treatments were used to provide palliation of two patients aged 88 and 85 years. The selection criteria and methodologies for treatment of geriatric patients with pseudomyxoma peritonei are presented.

Aged↗

PubMed1995

Interleukin-1 beta-stimulated PGE2 production from early first trimester human decidual cells is inhibited by dexamethasone and progesterone.

Cytokines are known to increase the production of prostaglandins by human decidual cells, but negative regulators have not been identified. We have examined the effects of dexamethasone and progesterone on prostaglandin (PG) E2 synthesis by cultured human first trimester decidual cells. The numbers of cyclooxygenase (COX) enzyme positive cells were visualised by immunocytochemistry, using antibodies specific for COX-1 and COX-2. Interleukin-1 beta stimulated the production of prostaglandins E2 and F2 alpha dose-dependently, and this was associated with increased numbers of COX-2 positive cells. Progesterone (10(-7)-10(-6) M) and dexamethasone (10(-7)-10(-6) M) inhibited basal and interleukin-1 beta-stimulated prostaglandin production, and decreased the numbers of COX-2 positive cells. Neither interleukin-1 beta nor the steroids affected numbers of COX-1 positive cells. COX-2 seems to be the main enzyme controlling the synthesis of PGE2 by human decidual cells, and may be negatively regulated by progesterone.

Antibodies↗

PubMed1995

Interleukin-1 beta-stimulated prostaglandin synthesis by human decidual cells is independent of protein kinase C.

Basal prostaglandin E2 (PGE2) synthesis by human decidual cells was stimulated by phorbol myristate acetate (PMA) which activates protein kinase C. Staurosporine, which is an inhibitor of protein kinase C in most systems, also increased basal PGE2 synthesis. Further work is needed to explain this finding, as another inhibitor of protein kinase C, H7, inhibited PGE2 production under similar culture conditions. Interleukin-1 beta (IL-1 beta)-stimulated PGE2 synthesis was potentiated by coincubation with PMA or staurosporine, indicating that IL-1 beta and protein kinase C increase decidual PGE2 synthesis through different mechanisms. Desensitization of the decidual cells for 24 h with PMA did not affect IL-1 beta-stimulated PGE2 synthesis. The complex roles of protein kinase C in regulating decidual prostaglandin synthesis require further investigation, but it is clear that the effects of IL-1 beta are not mediated by protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Decidual adenylate cyclase and prostaglandin production in vitro.

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E2 biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE2 production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE2 synthesis, and was without effect on IL-1 beta-stimulated PGE2 levels. PGE2 synthesis was increased by 24 h culture with IL-1 beta in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE2 levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE2 production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4-7 days prior to stimulation, in contrast with 24 h in our investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.

1. It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre-eclampsia (PE) and intra-uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal-term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG-nitro-L-[2,3,4,5(-3)H]-arginine ([3H]-L-NOARG) binding, quantitative in vitro autoradiography, [3H]-arginine to [3H]-citrulline conversion and Western blotting. 2. Specific, high affinity (KD = 38 nM) [3H]-L-NOARG binding was demonstrated in the villous trophoblast of normal-term placentae. Binding was calcium-independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L-NOARG > or = L-NMMA > or = 7-NI > L-NAME > L-Arg > or = L-NIO > ADMA). 3. [3H]-L-NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal-term placentae. 4. Western blotting, using an endothelial NOS peptide antiserum, demonstrated a approximately 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal-term placentae. 5. Specific [3H]-L-NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]-L-NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal-term controls. 6. The role of trophoblast-derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]-L-NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.

Adult↗

PubMed1994

An endogenous inhibitor of prostaglandin synthesis in human decidua.

Human first trimester decidual tissue has been reported to contain an inhibitor of cyclo-oxygenase enzyme, but a careful reading of the literature indicates that the inhibitor may be human albumin. Our own studies also showed that high levels of albumin were present in samples from homogenised decidual tissues. We therefore digested the tissue to obtain isolated cells and washed them extensively to minimise contamination with blood. Homogenates of such cells did not bind arachidonic acid, and inhibited cyclo-oxygenase enzyme-1 (COX-1) activity. The cellular factor inhibited COX-1 at concentrations of 60-100 micrograms protein/ml, and was therefore more potent than human albumin (0.5-2.0 mg/ml). The inhibitory activity was lost after heat treatment (100 degrees C for 10 mins) and after digestion with trypsin or papain, but was resistant to mercaptoethanol and urea. Preliminary data indicated that the protein had a molecular weight > 30 kD.

Arachidonic Acid↗

PubMed1994

Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface.

Feto-placental vessels lack innervation, hence control of this circulation is dependent on locally produced and circulating vasoactive factors. Functional studies have presented evidence that nitric oxide, a potent vasodilator and platelet anti-aggregating agent, may be generated into the feto-placental circulation, contributing to control of vascular tone. In view of the absence of nerves supplying the placenta the source of NO is likely to be endothelial. We have therefore investigated the localization of endothelial constitutive nitric oxide synthase (ecNOS) in human normal full-term placentae, using immunocytochemistry, with rabbit antiserum to a synthetic peptide, corresponding to amino acid residues 1172-1186 of human and bovine ecNOS. On Western blots of partially purified NO synthase extracted from placenta, the peptide antiserum reacted exclusively with a single protein band of approximately 135kDA. Immunoreactivity in tissue sections was localized to endothelium of umbilical artery and vein, and appeared uniform in sections at different levels along the cord. Staining in chorionic vessels was much more variable; it was present mainly in the larger vessels close to the cord where it had a patchy distribution. Staining was not seen in the endothelium of small feto-placental vessels. Strong immunoreactivity was evident in the syncytiotrophoblast of the placenta, although the intensity of staining was variable, being weaker along stem villi and strongest along terminal villi. The differential distribution and intensity of nitric oxide synthase immunoreactivity in the human placenta might indicate that locally produced, and in particular trophoblast-derived nitric oxide may play a pivotal role both in control of feto-placental vascular tone and as a platelet anti-aggregating agent in the utero-placental circulation.

Amino Acid Oxidoreductases↗

PubMed1994

Regulation by interleukin-1 beta of growth and collagenase production by choriocarcinoma cells.

Impaired trophoblastic invasion and proliferation have been implicated in the pathogenesis of eclampsia, pre-eclampsia, spontaneous abortions and intra-uterine growth retardation (IUGR). First trimester trophoblast cells (which do not grow in culture) and choriocarcinoma (BeWo) (which grow spontaneously, and are used as a model for proliferating trophoblast) were incubated with interleukin-1 beta (IL-1 beta). BeWo cell growth was decreased dose-dependently by exogenous IL-1 beta at concentrations of 100-1000 pg/ml. This effect was first detected after 24 h of incubation with IL-1 beta, and persisted for up to 96 h of culture. In contrast, trophoblast cells isolated from first trimester placental tissue showed no growth response when stimulated with IL-1 beta. The levels of active interstitial collagenase produced by BeWo cells were increased by IL-1 beta (100-1000 pg/ml), which paralleled the decrease in cell growth. First trimester trophoblast cells produced lower levels of collagenase and this was not affected by incubation of the cells by IL-1 beta. These results indicate that IL-1 beta may regulate placental development, but further development of culture systems for first trimester trophoblast will be needed before this result can be confirmed.

Cell Division↗

PubMed1994

Transfer of cytokines through human fetal membranes.

Intact human fetal membranes (amnion, chorion and decidua) were incubated with 125I-labelled cytokines added to the fetal or maternal sides of the membrane. The transfer of 125I-labelled interleukin-6 (IL-6), 125I-labelled tumour necrosis factor alpha (TNF-alpha), 125I-labelled interleukin-1 alpha (IL-1 alpha) and 125I-labelled interleukin-1 beta (IL-1 beta) was determined by measurement of radioactivity in a gamma counter and the integrity of the cytokines was assessed by acid precipitation and by radioimmunoassay. IL-1 alpha and IL-1 beta were transferred through human fetal membranes in both feto-maternal and materno-fetal directions at similar rates. Only 2-4% of the cytokine originally added appeared to be intact on the opposing side of the membrane after 24 h of culture. Transfer of intact TNF-alpha (5-7%) and IL-6 (8-17%) was greater than that of the IL-1 isomers. Low but variable amounts of the four cytokines tested may be transferred through the human fetal membrane. This finding suggested that concentrations of cytokines in amniotic fluid would not reflect those produced by decidua if the fetal membranes are intact.

Biological Transport↗