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M D Mitchell

Publications and source records attributed to M D Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed2001

Gestational age-dependent effects of lipopolysaccharide on prostaglandin production by murine decidual caps.

We sought to determine whether the gestational age of the pregnant mouse had any relationship with its lipopolysaccharide (LPS) responsiveness. Murine decidual caps from days 13, 15 and 17 of gestation (term is day 20) were dissected out, placed in inserts and equilibrated in media overnight. The following day, media were removed, replaced with fresh media (+/-LPS at 10 microg/mL). After LPS stimulation (24 h), prostaglandin (PG)E2 production by decidual caps from days 13 and 15 increased by 80-fold and 5-fold, respectively. PGF2alpha, 6-keto-PGF1a and TxB2 production also increased. Day 17 decidual caps were unaffected by LPS, pregnant mice inoculated i.p. with LPS (50 microg) at day 13 of gestation induced 100% delivery within 24 h. However, mice treated at days 15 and 17 had an equal occurrence of premature delivery or fetal resorption. This change in LPS responsiveness may indicate changes in the fetal-maternal immune system in late pregnancy.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Nuclear lipid signaling: novel role of eicosanoids.

Nuclear lipid signaling is an established, widespread mechanism that operates in multiple cellular processes including proliferative and differentiative responses to a variety of stimuli. In this literature review with key references highlighted, we put forward the hypothesis that differential flow through various intracrine mechanisms can dictate resultant cellular actions such as mitosis, differentiation, or apoptosis.

Animals↗
PubMed2000

Activin A exerts both pro- and anti-inflammatory effects on human term gestational tissues.

There is a growing appreciation of the importance of activin as a modulator of immune function. The aim of the present study was to determine whether activin A exerts any effects on cytokine and prostaglandin (PG) production by the tissues of pregnancy. Explant cultures were established for amnion, choriodecidual and placental tissues derived from pregnancies delivered at term by Caesarean section (n=5 placentae). Explants were treated with activin A (0.5, 5 and 50 ng/ml) in serum-free Ham's F12/DME media for 24 h (n=3-4 replicates). Production rates of interleukin (IL)-1beta, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and PGE(2)were determined using immunoassay. Differences between treatment groups were analysed by ANOVA followed by Dunnett's test;P< 0.05 was considered to be significant. Amnion IL-6 production exhibited biphasic responses to activin A: at 5 ng/ml activin A, IL-6 production was significantly stimulated (to 246+/-74.6 per cent of control (mean+/-sem), while at 50 ng/ml it was significantly inhibited (to 46+/-7.4 per cent of control). IL-8 and PGE(2)production by amnion showed significant responses to activin A that were similar to those of IL-6. No significant effects of activin A were observed on choriodecidual and placental IL-6, IL-8 and PGE(2)production. However, TNF-alpha production was significantly inhibited by 50 ng/ml activin A in both choriodecidual and placental explants (to 43+/-9.7 per cent and 51+/-6.7 per cent of control, respectively). Placental IL-1beta production was not altered by treatment with activin A at any concentration. These findings support the concept of activin as an immune modulator in tissues of pregnancy.

Activins↗
PubMed2000

Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in amnion with term and preterm labour.

To evaluate the association between intercellular adhesion molecule-1 (ICAM-1) in the amnion and preterm labour and delivery, we have assessed ICAM-1 mRNA abundance by Northern analysis and protein levels by enzyme-linked immunosorbent assay (ELISA), in samples of this tissue after term and preterm delivery. The median ICAM-1 mRNA expression following preterm delivery (PTD, n=30) was 24 times greater (P< 0.05) than following elective caesarean section prior to labour at term (CST, n=14). ICAM-1 expression following vaginal delivery after spontaneous labour at term (SLT, n=11) was seven times greater than in the CST group (P< 0.05). The concentration of ICAM-1 protein in the PTD samples (n=31) was four-fold greater than (P< 0.05) in CST (n=14). It was also three-fold greater than in the SLT (n=15) samples (P< 0.05). The results were substantially the same when a preterm spontaneous labour group (PTL) (n=26), exclusive of deliveries complicated by pre-eclampsia (n=1) or intrauterine growth restriction (n=3), was compared to the CST and SLT groups. The ICAM-1 mRNA expression did not differ significantly (P=0.93) between PTL with (n=12) or without (n=14) indicators of intrauterine infection. The results were similar when ICAM-1 protein concentrations were compared (P=0.43) between these two groups. These findings indicate that ICAM-1 is expressed by the human amnion and that this expression is elevated with preterm labour and delivery.

Amnion↗
PubMed2000

The 15-deoxy-delta(12,14)-prostaglandin J(2)receptor, peroxisome proliferator activated receptor-gamma (PPARgamma) is expressed in human gestational tissues and is functionally active in JEG3 choriocarcinoma cells.

RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n = 15) or elective caesarean section (n = 15) at term. The samples were subjected to Northern analysis, using a 2 kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-gamma. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARgamma. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10 microm 15-deoxy-delta(12,14)prostaglandin J(2)(15dPGJ(2)) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARgamma and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues.

Adult↗
PubMed2000

Tumour necrosis factor-alpha regulation of prostaglandin H synthase-2 transcription is not through nuclear factor-kappaB in amnion-derived AV-3 cells.

Tumour necrosis factor (TNF)-alpha-stimulated prostaglandin (PG) E(2)biosynthesis by amnion-derived AV3 cells is accompanied by increased prostaglandin H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged. PGHS-2 promoter-reporter constructs (-891/+9 and 5' deletions thereof) were prepared. The regions containing concensus nuclear factor kappaB (NF-kappaB) elements (-447/-438 and -222/-213) did not enhance promoter activity. Elements associated with both basal and TNF-alpha-stimulated expression lie between bases -52 and -203. Site-directed mutagenesis of nuclear factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of the -203/+9 construct by over 95 per cent. Electrophoretic mobility-shift assays using oligonucleotides derived from these sites demonstrated formation of specific DNA-protein complexes. Both NF-IL6 and CRE unlabelled oligonucleotides inhibited complex formation with the NF-IL6 oligonucleotide probe. Unlabelled CRE oligonucleotide also effectively inhibited formation of the complex with the CRE probe, but reduced effectiveness was observed when the NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensus NF-kappaB oligonucleotide failed to compete for either probe. TNF-alpha treatment did not increase levels of these complexes. Thus NF-kappaB does not enhance basal or TNF-alpha-responsive PGHS-2 transcription in amnion-derived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regulating PGHS-2 expression in these cells is indicated, but requires further clarification.

Amnion↗
PubMed2000

Cytokine-induced coordinate expression of enzymes of prostaglandin biosynthesis and metabolism: 15-hydroxyprostaglandin dehydrogenase.

The effects of pro-inflammatory cytokines on 15-hydroxyprostaglandin dehydrogenase (PGDH) expression and prostaglandin (PG) metabolizing activity were investigated in placental tissue. Treatment of trophoblast cells in primary culture with interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) resulted in decreased prostaglandin metabolizing activity. This was assessed both by determining the ratio of production of prostaglandins to their metabolites, and by Northern blot analysis of PGDH mRNA abundance. Dexamethasone had similar inhibitory effect. We hypothesize that proinflammatory cytokines act to enhance prostaglandin actions through coordinated effects on activities of enzymes of both prostaglandin biosynthesis and metabolism, perhaps in concert with effects on receptor function.

Blotting, Northern↗
PubMed2000

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways.

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

Amnion↗
PubMed2000

NF-IL6 and CRE elements principally account for both basal and interleukin-1 beta-induced transcriptional activity of the proximal 528bp of the PGHS-2 promoter in amnion-derived AV3 cells: evidence for involvement of C/EBP beta.

Prostaglandin H synthase (PGHS)-2 promoter fragments (-528 to +9 bp and 5' unidirectional deletions thereof) were cloned upstream of the chloramphenicol acetyl-transferase (CAT) reporter gene. These were transfected into amnion-derived AV3 cells. The region, -528 to -203, which includes NF-kappa B sites, had little influence on CAT expression. The region, -203 and -52, however, was responsible for most of the basal promoter activity and also conferred responsiveness to interleukin (IL)-1 beta (>3-times basal). Point mutations of NF-IL6 and cAMP response element (CRE) in this region reduced both basal and IL-1 beta-stimulated production of CAT; dual mutation eliminated IL-1 beta responsiveness. Factors in nuclear extracts from control or IL-1 beta-stimulated AV3 cells specifically complexed the NF-IL6 and CRE sequences. However, the NF-IL6 and CRE oligonucleotides cross-competed, suggesting a common factor. C/EBP beta was identified by supershift assay as interacting with both sequences. To a lesser extent C/EBP alpha and delta also interacted with the NF-IL6 site. However, CRE binding protein (CREB), was absent from the complex with the CRE. In conclusion, NF-IL6 and CRE elements principally account in AV3 amnion cells for basal and IL-1 beta-inducible transcriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-kappa B elements play no substantial role. C/EBPs, particularly C/EBPbeta, are implicated in control of PGHS-2 transcription through the NF-IL6 and CRE sites.

Amnion↗
PubMed2000

Intercellular adhesion molecule-1 (ICAM-1) in cervicovaginal fluid of women presenting with preterm labor: predictive value for preterm delivery.

PROBLEM: Clinically useful tests for the prediction and diagnosis of preterm labor and delivery remain to be established. We have hypothesized that soluble intercellular adhesion molecule-1 (sICAM-1) in the cervicovaginal fluid of women with preterm labor may be a useful diagnostic tool. METHOD OF STUDY: The cervicovaginal fluid of 103 women between 24(0) and 33(6) weeks gestation with preterm contractions and intact membranes was assayed for sICAM-1. RESULTS: Elevated sICAM-1 concentrations predicted short intervals to delivery (area under receiver operator characteristic (ROC) curves, 0.70-0.72 for delivery within 3, 7 and 10 days), with high specificity. Characteristics for delivery within 3 days at a 3 ng/mL threshold for a positive test were sensitivity 33.3%, specificity 98.9%, and positive and negative predictive values of 75.0% and 93.9%, respectively. Predictive ability was independent of and complementary to that of fetal fibronectin (fFN). CONCLUSIONS: Measurement of sICAM-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor, particularly in conjunction with fFN testing.

Adult↗
PubMed2000

Does leptin exhibit cytokine-like properties in tissues of pregnancy?

PROBLEM: To determine whether leptin exhibits cytokine-like properties in gestational tissues in light of its homologies with the class I family of cytokines. METHOD OF STUDY: WISH and JEG3 cells, and amnion and choriodecidua explants, were treated inflammatory modulators (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha] and bacterial lipopolysaccharide [LPS]) and leptin production was measured by immunoassay. Other agents known to regulate adipocyte leptin production were also tested for comparative purposes. In addition, WISH cells, JAR cells and placental explants were treated with leptin to assess its effects on production of IL-8, IL-6 and prostaglandin E2 (PGE2). RESULTS: Leptin production by all cells and tissues studied was unaffected by treatment with IL-1beta (2.5 ng/mL), TNF-alpha (25 ng/mL) and LPS (2.5 microg/mL). Dexamethasone stimulated leptin production over two-fold by WISH and JEG3 cells, whereas insulin also stimulated a two-fold increase in leptin production in JEG3 cells. IL-6 production by JAR cells and placental explants was stimulated (two- to three-fold) by leptin (300 ng/mL). PGE2 production was unaffected. CONCLUSIONS: Leptin derived from gestational tissues is unlikely to play a role in inflammatory reactions within the placenta, but may regulate placental cytokine production. The physiological significance of amnion-derived leptin remains to be established.

Amnion↗
PubMed2000

A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines.

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V(max) of 19.9 nmol/min/mg of protein and a mean K(m) of 652.5 microM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V(max) was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K(m), 175. 4 +/- 24.2 microM (CV = 14.5%), was significantly lower (P <.0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V(max) and 8 and 35% for K(m), respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2. 6 nmol/min/mg of protein, respectively, at 200 microM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.

Cell Line↗
PubMed1999

15-Deoxy-Delta(12,14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells.

Apoptosis has been described in placental (trophoblast) tissues during both normal and abnormal pregnancies. We have studied the effects of the cyclopentenone prostaglandins (PGs) on trophoblast cell death using JEG3 choriocarcinoma cells. PGJ(2), Delta(12)PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)) (10 microM) significantly reduced mitochondrial activity (MTT assay) over 16 h by 17.4 +/- 4.7%, 28 +/- 9.3%, and 62.5 +/- 2.8%, respectively (mean +/- sem), while PGA(2) and PGD(2) had no effect. The synthetic PPAR-gamma ligand ciglitizone (12.5 microM) had a potency similar to 15dPGJ(2) (69 +/- 3% reduction). Morphological examination of cultures treated with PGJ(2) and its derivatives revealed the presence of numerous cells with dense, pyknotic nuclei, a hallmark of apoptosis. FACS analysis revealed an abundance (approximately 40%) of apoptotic cells after 16-h treatment with 15dPGJ(2) (10 microM). The caspase inhibitor ZVAD-fmk (5 microM) significantly diminished the apoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2). JEG3 cells expressed PPAR-gamma mRNA by Northern analysis. These novel findings imply a role for PPAR-gamma ligands in various processes associated with pregnancy and parturition.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Cytokine abundance in placental tissues: evidence of inflammatory activation in gestational membranes with term and preterm parturition.

OBJECTIVES: This study of the changes in cytokine concentrations in gestational tissues from women with term and preterm labor was undertaken to assess the extent of inflammatory activation associated with spontaneous labor and delivery. STUDY DESIGN: Extracts of amniotic, chorionic-decidual, and placental tissues from women delivered at term before labor (n = 15), at term after labor (n = 15), and preterm (n = 31) were assayed for interleukin 1beta, interleukin 6, and interleukin 8. RESULTS: In amniotic tissues of women delivered by spontaneous labor at term the median interleukin-6, interleukin-8, and interleukin-1beta concentrations were 3.8 to 5.4 times those of tissues from women delivered at term without labor (P <.05, Mann-Whitney U test). Interleukin-6 and interleukin-8 concentrations were also significantly increased (3. 3-4 times) in chorionic-decidual tissues. Marked increases (approximately 3-6 times) in the concentrations of all 3 cytokines were observed in both amniotic and chorionic-decidual tissues from women with preterm deliveries with respect to those from women with term deliveries after labor. Cytokine concentrations were significantly correlated within amniotic tissues from both women with term delivery after labor and women with preterm delivery and also in preterm chorionic-decidual tissues but not preterm placental tissues. Concentrations of cytokines in the tissues of women delivered preterm were not significantly affected by mode of delivery, treatment with antibiotics, or twin birth. In preterm tissues with evidence of intrauterine infection only amniotic interleukin-1beta concentrations were significantly elevated (P <. 05). Little or no labor-related change in cytokine concentrations was seen within placental tissues. CONCLUSIONS: Increased cytokine abundance in gestational membranes associated with labor supports the view that an inflammatory process is involved in both term and preterm labor. This process does not, however, appear to be evident in the villous placenta.

Cytokines↗
PubMed1999

Key enzymes of prostaglandin biosynthesis and metabolism. Coordinate regulation of expression by cytokines in gestational tissues: a review.

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.

Chorioamnionitis↗
PubMed1999

Conformationally altered beta 2-glycoprotein I is the antigen for anti-cardiolipin autoantibodies.

Anti-cardiolipin autoantibodies (aCL) induce thrombosis and recurrent fetal death. These antibodies require a 'cofactor', identified as beta 2-glycoprotein I (beta 2-GPI), to bind phospholipids. We show here that aCL can bind beta 2-GPI in the absence of phospholipid. Binding of aCL to beta 2-GPI is dependent upon the beta 2-GPI being immobilized on an appropriate surface including cardiolipin, irradiated polystyrene and nitrocellulose membrane. This effect cannot be explained by increased antigen density of beta 2-GPI immobilized on these surfaces. Rather, conformational changes that occur following the interaction of beta 2-GPI with phospholipid render this protein antigenic to aCL. Liquid-phase beta 2-GPI was not antigenic for aCL. Thus, aCL cannot bind circulating beta 2-GPI. These findings may explain why patients with aCL can remain healthy for many years but then undergo episodes of thrombosis or fetal loss without changes in their circulating aCL profile, as the triggering event for these pathologies can be predicted to be one that renders beta 2-GPI antigenic for aCL.

Antibodies, Anticardiolipin↗