Biomedical subjects

Murray D Mitchell

Publications and source records attributed to Murray D Mitchell.

29 records · Page 2Linked to original sources

PubMed2004

Characterization of the endocannabinoid system in early human pregnancy.

In recent years, it has been demonstrated that high circulating levels of the endogenous cannabinoid anandamide, resulting from low expression of its metabolizing enzyme fatty acid amide hydrolase (FAAH), may contribute to spontaneous miscarriage and poor outcome in women undergoing in vitro fertilization. The site of action of this compound, however, has not been determined. In this study, we examined the distribution of the cannabinoid receptors, CB1 and CB2, and the endocannabinoid-metabolizing enzyme FAAH in first trimester human placenta. Here, we show that FAAH is expressed throughout the human first trimester placenta, in extravillous trophoblast columns, villous cytotrophoblasts, syncytiotrophoblasts, and macrophages. Furthermore, FAAH mRNA levels appear to be regulated during gestation, with levels peaking at 11 wk before declining again. The immune system-associated cannabinoid CB2 receptors were localized only to placental macrophages. Interestingly, the cannabinoid receptor CB1 was not identified in first trimester placenta despite having previously been shown to be present in placental tissues at term. These findings suggest that the placenta may form a barrier preventing maternal-fetal transfer of anandamide and/or modulate local levels of anandamide by regulation of FAAH expression with gestation.

Cannabinoid Receptor Modulators↗

PubMed2003

3'-azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta.

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, beta-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, beta-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier.

Anti-HIV Agents↗

PubMed2003

Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production.

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 microg/ml) induced a rapid increase in TNF-alpha production, peaking at 4 h. In contrast, IL-10, IL-1beta, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-alpha was a primary regulator of IL-1beta, IL-10, and PGE2 production, while IL-1beta stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-alpha and PGE2 production. IL-10 treatment markedly decreased TNF-alpha and IL-1beta production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-alpha may be a potential target for treatment intervention; IL-10 could be one such therapeutic.

Adjuvants, Immunologic↗

PubMed2003

Macrophage inhibitory cytokine 1 in fetal membranes and amniotic fluid from pregnancies with and without preterm labour and premature rupture of membranes.

The placenta and fetal membranes are the site of expression of macrophage inhibitory cytokine (MIC-1), a member of the transforming growth factor (TGF)-beta superfamily. We hypothesized that MIC-1 may act as an immune regulator in pregnancy complications associated with intrauterine inflammation. Decidual cells, chorionic trophoblasts and amnion epithelial cells were identified by immunohistochemistry as the predominant MIC-1-containing cell type in term membranes. Amnion and choriodecidual explants all produced MIC-1 in culture, the latter having the greatest production rate (206 +/- 74.5 pg/mg tissue/24 h, n=6; mean +/- SEM). Production was not responsive to stimulation by pro-inflammatory cytokines. MIC-1 was detectable in 217 transabdominal amniotic fluid (AF) samples taken from 15 to 41 weeks gestation, concentrations ranging from 0.9-51.1 ng/ml. AF MIC-1 concentrations in pregnancies with premature rupture of membranes (PROM) or preterm labour, either with or without microbial invasion of the amniotic cavity, were not significantly different from those delivered at term either with or without labour. Treatment with MIC-1 (0.25-25 ng/ml) did not alter production of interleukin-6 or -8 by amnion or choriodecidual cells in vitro. We conclude that AF MIC-1 is derived from the fetal membranes and decidua, but that MIC-1 is unlikely to be involved in the pathophysiology of preterm birth or PROM.

Amniotic Fluid↗

PubMed2003

Peroxisome proliferator-activated receptor isoform expression changes in human gestational tissues with labor at term.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that are involved in lipid metabolism, differentiation, proliferation, cell death, and inflammation. Three subtypes have been identified: PPAR-alpha, -delta, and -gamma. We have previously shown presence of PPAR-gamma mRNA in the amnion, choriodecidua, and placenta, and its level of expression was unchanged with labor. To evaluate whether PPAR-alpha and -delta subtypes are present in intrauterine tissues, placentae were obtained from women at term after spontaneous vaginal delivery (TSL; n = 15) and elective caesarean section before labor (TNL; n = 15). Northern blot analyses were used to evaluate the mRNA for PPARs. Activities of PPARs were assessed using JEG3 choriocarcinoma cells transfected with a PPAR-response element reporter construct (pTK-PPREx3-luc) and treated with PPAR ligands. The PPAR-gamma-specific ligand rosiglitazone induced PPAR response element (PPRE)-mediated activity in a concentration-dependent manner, whereas the PPAR-gamma-specific irreversible inhibitor GW9662 fully inhibited this induction. However, GW9662 only partially inhibited 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2)-induced luciferase activity, suggesting that 15d-PGJ2 may also activate either of the other isoforms. PPAR-alpha and -delta are expressed in the amnion, choriodecidua, and placental villous tissues. In the amnion, although for PPAR-alpha no significant difference in expression was observed with labor, PPAR-delta expression increased significantly (p < 0.001). In the choriodecidua, expression of PPAR-alpha declined with labor (p < 0.01), whereas, as in the amnion, PPAR-delta expression increased (p < 0.05). In the placenta, both PPAR-alpha and -delta expression increased with labor (p < 0.005). The changes observed with labor suggest that regulation of PPAR expression and function may have roles to the mechanisms that maintain pregnancy or initiate labor.

Amnion↗

PubMed2002

UDP-glucuronosyltransferase activity, expression and cellular localization in human placenta at term.

The activity, expression and localization of the UDP-glucuronosyltransferases (UGTs) were investigated in human placenta at term. UGT activity (measured with the substrate 4-methylumbelliferone (4-MU)) was observed in all 25 placentas sampled and maximum velocity (V(max)) ranged 13-fold from 5.1+/-0.9 to 66.9+/-17.5 nmol/min/mg protein (mean+/-SD). Substrate affinity (K(m)) ranged 5-fold from 246+/-24 to 1124+/-422 microM. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of the isoforms UGT2B4, 2B7, 2B10, 2B11 and 2B15 was observed in all (12/12) placentas sampled and expression of UGT2B17 was noted in 8/12 placentas. Northern analysis of the UGT2B7 isoform in 12 placentas revealed a 10-fold difference in expression with RT-PCR variability and the 13-fold variation observed in UGT activity. The presence of UGT2B4 and 2B7 proteins (52 and 56kDa, respectively) was demonstrated by Western blotting. The sites of placental UGT2B transcription (in situ hybridization) and protein expression (immunohistochemistry) were located in the syncytium of the placental trophoblasts bordering the placental villi. UGT1A proteins could not be observed with immunohistochemistry or Western blotting and expression could not be observed with RT-PCR. Our discovery of UGT expression and activity at the site of maternal-fetal exchange is consistent with a role for UGTs in detoxification of exogenous and endogenous ligands and the maintenance of placental function through clearance and regulation of steroid hormones.

Female↗

PubMed2002

Regulation of prostaglandin production in an immortalized human myometrial cell line by cytokines and non-steroidal anti-inflammatory drugs.

OBJECTIVE: To test the effect of selective and non-selective inhibitors of prostaglandin H synthase-2 (PGHS-II) on basal and cytokine-stimulated prostaglandin (PG) production by the immortalized human myometrial cell line, UTLRp16. STUDY DESIGN: UTLRp16 cells were treated with interleukin (IL)-1beta (0.1, 1, 10ng/ml) and tumor necrosis factor (TNF)-alpha (1, 3, 10ng/ml) in the presence or absence of indomethacin, etodolac, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2 (5H)-furanone (DFU) or nimesulide for 16h (1.6-1000nM). PG production was then measured by radioimmunoassay. RESULTS: IL-1beta and TNF-alpha both stimulated production of PGE(2) and 6-keto-PGF(1alpha) in a concentration-dependent manner. DFU showed the most PGHS-II selectivity with IL-beta-stimulated PG production, with a IC(50)(basal)/IC(50)(stimulated) ratio of 177.8, followed by nimesulide (122.5), etodolac (23.5) and indomethacin (2.7). DFU was not as selective in TNF-alpha stimulated cells (ratio 99.5). CONCLUSION: PGHS-II-selective inhibitors may be effective in the prevention of cytokine-driven myometrial PG production associated with preterm labor.

6-Ketoprostaglandin F1 alpha↗

PubMed2002

Serum activin A, inhibin A, and follistatin concentrations in preeclampsia or small for gestational age pregnancies.

OBJECTIVE: To determine whether maternal serum activin A, inhibin A, and follistatin concentrations in idiopathic small for gestational age (SGA) pregnancies are similar to those in normal pregnancies or elevated as in preeclampsia. METHODS: Maternal serum activin A, inhibin A, and follistatin concentrations were determined in 1) nulliparous women with idiopathic SGA (birth weight <10th percentile; n = 18), preeclampsia (systolic blood pressure > or =140 mmHg or diastolic blood pressure > or =90 mmHg plus proteinuria > or =2+ or >0.3 g/24h; n = 22), and normotensive controls, matched for gestational age at sampling (n = 22), and 2) a longitudinal series of samples collected at five intervals throughout pregnancy from nulliparous women with idiopathic SGA (n = 19), preeclampsia (n = 22), preeclampsia plus SGA (n = 15), or who had uncomplicated pregnancies (n = 20). RESULTS: Serum concentrations of activin A and inhibin A were similar in idiopathic SGA pregnancies to controls. In preeclampsia, activin A and inhibin A levels were markedly increased compared with controls or women with idiopathic SGA (P <.001), particularly in those with early-onset disease. Follistatin concentrations were only modestly (<twofold) elevated in preeclampsia (P <.001). In the longitudinal study, serum activin A or inhibin A concentrations were increased in women who later developed preeclampsia, whereas in women with idiopathic SGA pregnancy, a small overall increase in activin A levels was observed. CONCLUSIONS: In contrast to women with preeclampsia, normotensive women with SGA pregnancies do not have markedly elevated circulating levels of activin A and inhibin A. These data support the hypothesis that increased serum activin A concentrations in preeclampsia may be a manifestation of maternal disease rather than just a marker of abnormal placentation.

Activins↗

PubMed2002

Metabolizing enzyme localization and activities in the first trimester human placenta: the effect of maternal and gestational age, smoking and alcohol consumption.

BACKGROUND: The rationale for this study was to assess the expression, activity and localization of the enzymes uridine diphosphate glucuronosyltransferase (UGT), beta-glucuronidase, cytochrome P450 1A (CYP1A) and cytochrome P450 2E1 (CYP2E1) in first trimester human placenta and to gauge the effects of maternal variables on placental metabolism. METHODS: CYP1A, CYP2E1, UGT and beta-glucuronidase activities were assessed in 25 placentas using ethoxyresorufin, chlorzoxazone, 4-methylumbelliferone and 4-methylumbelliferone glucuronide respectively. Protein expression and localization were detected by immunoblot and immunohistochemistry. All statistics were non-parametric. RESULTS: UGT, beta-glucuronidase and CYP1A activities were detected in all placentas sampled; CYP2E1 was undetectable. CYP1A, UGT1A UGT2B proteins were detected in all placentas (n = 6) tested and CYP2E1 in 4/6 placentas sampled and were localized to the syncytium. UGT and CYP1A activities were significantly elevated in the placentas of mothers who smoked (P < 0.05 and P < 0.001 respectively) and were greatest in women who both smoked and drank alcohol (P < 0.05 and P < 0.01 respectively). Enzyme activities were significantly negatively correlated with gestational age (P < 0.05, r = 0.54, UGT) and maternal age respectively (P < 0.001, r = 0.63, CYP1A). beta-Glucuronidase activity did not differ with patient variables. CONCLUSIONS: Metabolism of compounds by the human placenta in the first trimester may be affected by maternal and environmental factors altering the activity of constitutive metabolizing enzymes.

Adolescent↗

PubMed2002

Identification of suppressors of cytokine signaling (SOCS) proteins in human gestational tissues: differential regulation is associated with the onset of labor.

Inflammatory cytokines secreted by the placenta and fetal membranes are believed to play an important role in the initiation of parturition. The suppressor of cytokine signaling (SOCS) proteins regulate signal transduction by several cytokines that have been reported to affect gestational tissues. The presence, distribution and roles of SOCS proteins, however, have not been described in human gestational tissues. Using reverse transcriptase (RT)-PCR and Western blot analysis we investigated the expression of SOCS1, SOCS2, and SOCS3 mRNA and protein, respectively, by human villous placenta, amnion and choriodecidua (n = 3-4). Tissues were obtained from uncomplicated pregnancies at term after either spontaneous labor and vaginal delivery or caesarean section (before labor). Messenger RNAs for SOCS1, SOCS2, and SOCS3 were expressed in all tissue types, irrespective of labor status. SOCS proteins were, however, only detectable in villous placenta and in one case in the choriodecidua. Labor was associated with abrogated expression of SOCS1 and SOCS3 proteins in villous placenta and the choriodecidua sample. Following labor the band for SOCS2 protein increased slightly in size which may indicate post-translational modification of SOCS2. Reduced expression of SOCS proteins in gestational tissues may provide a mechanism by which inflammatory cytokines enter into a positive feedback loop of inflammatory changes leading to delivery.

Blotting, Western↗

PubMed2002

Expression of angiogenic and neurotrophic factors in the human amnion and choriodecidua.

OBJECTIVE: Our objective was to identify the novel or differential expression of growth or development associated genes in the human gestational membranes that might play roles in pregnancy or in term or preterm parturition. STUDY DESIGN: Complementary DNA arrays were probed with [alpha(33)P]dCTP-labeled-complementary DNA that was prepared from the RNA of reflected amnion and choriodecidua that represent term not-in-labor, term spontaneous labor, and preterm labor with and without chorioamnionitis (n = 4 per group). Differential expression (term not-in-labor vs term spontaneous labor or preterm labor with chorioamnionitis vs preterm labor without chorioamnionitis) was evaluated by Wilcoxon tests. RESULTS: All 16 amnion samples expressed angiogenic factors (endothelin-2 and -3, vascular endothelial growth factor, and vascular endothelial growth factor-B) and neurotrophic factors (ephrin-A2, ephrin receptors-A2, -B1, -B3, -B4, and -B5, neuropilin-2, p75/nerve growth factor receptor and semaphorin-F). In both amnion and choriodecidua, the expression of vascular endothelial growth factor and the angiopoietin receptor, Tie-2, were greater with term spontaneous labor than with term not-in-labor (P <.05); increased VEGF receptor-2 (flk-1) expression was observed in term spontaneous labor choriodecidua (P <.05) but not amnion. Ephrin-A1 expression increased with term spontaneous labor in both tissues (P <.05). Semaphorin-F expression decreased with preterm labor with chorioamnionitis in choriodecidua (P <.05), although the trend was not significant in amnion (P =.1). CONCLUSION: Neurotrophic and angiogenic factor genes are expressed in amnion and choriodecidual membranes. Several of the genes exhibit differential expression with labor at term or in association with infection preterm, which suggests roles in or associated with these processes.

Amnion↗