Biomedical subjects

M D Mitchell

Publications and source records attributed to M D Mitchell.

393 records · Page 22Linked to original sources

PubMedDate not supplied

Prostanoid stimulation of cytokine production in an amnion-derived cell line: evidence of a feed-forward mechanism with implications for term and preterm labor.

OBJECTIVE: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. METHODS: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. RESULTS: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. CONCLUSION: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMedDate not supplied

Differential regulation in human amnion epithelial and fibroblast cells of prostaglandin E(2) production and prostaglandin H synthase-2 mRNA expression by dexamethasone but not tumour necrosis factor-alpha.

Previous studies have identified both pro-inflammatory cytokines and glucocorticoids as positive regulators of amnion prostaglandin (PG) biosynthesis. The stimulatory effects of dexamethasone (Dex), a glucocorticoid agonist, on prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression and PG biosynthesis in amnion have been attributed to an atypical response by the mesenchymal cells of the amnion. The objective of this study was to confirm previous findings concerning cell type-dependant Dex-induced upregulation of PGHS-2 mRNA expression and PG production using separated amnion cell populations, in comparison with the effects of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). Amnion cells from placentae delivered at term by caesarian section were isolated by tryptic digestion and epithelial cells were then separated from mesenchymal cells by differential absorption onto plastic. After 24-72 h, the two cell populations were passaged and sub-cultured. Cells were treated with Dex (10(-9)-10(-6) m) or TNF-alpha (0.1-50 ng/ml) or media alone. Thereafter, PGE(2)production was determined and PGHS-2 mRNA content analysed by a competitive quantitative RT-PCR method established and validated for this study. PGE(2)production in fibroblast-enriched cultures was increased to 310+/-41 per cent (mean+/-sem, n=4 wells per treatment point) of control in the presence of 10(-8) m Dex. Conversely, PGE(2)production in Dex-treated amnion epithelial cells was decreased to 67+/-24 per cent of control. Altered PGE(2)biosynthesis was accompanied by the upregulation of PGHS-2 mRNA in amnion fibroblasts but not in epithelial cells. TNF-alpha increased PG output and PGHS-2 expression independent of cell type. Glucocorticoids therefore appear to have opposing effects on PG biosynthesis in the two major cellular components of the human amnion.

Amnion↗

PubMedDate not supplied

Cytosolic phospholipase A(2)and 15-hydroxyprostaglandin dehydrogenase mRNA expression in murine uterine and gestational tissues during late pregnancy.

We evaluated the changes in mRNA expression of cytosolic phospholipase A(2)(cPLA(2)) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in intrauterine and gestational tissues during mid-late murine pregnancy. Tissues (decidual caps, fetal membranes, and placentae, uterus, and cervix) were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation. Total RNA was isolated and evaluated for cPLA(2)and PGDH expression by northern blot analysis normalized to GAPDH expression. Expression of mRNA for cPLA(2)increased in the placentae and decidual caps on day 18 and 19 pm, respectively. There was also increased expression for PGDH mRNA in the placenta and fetal membranes at the later stages of pregnancy. The tissue specific differences in expression of cPLA(2)and PGDH suggest that changes in enzymatic regulation of PG production and degradation may be crucial for the initiation of labour.

Animals↗

PubMedDate not supplied

Regulation of prostaglandin biosynthesis in dispersed choriodecidual cells in culture.

We have evaluated the prostaglandin (PG) production and PG biosynthetic gene expression in a choriodecidual dispersed cell culture system. Cells dispersed from human choriodecidual membranes by dispase and trypsin digestion were evaluated after 1,3,5 and 7 days of culture for basal and tumour necrosis factor alpha (F-alpha) stimulated PGE2 production. The highest rates of production (P < 0.05) were obtained with cells treated after 3 days of culture, (3.7 +/- 1) x 10(2) pg PGE2 per 16 h per microg total cellular protein (mean +/- SEM), which was 3.9 times basal rate after 3 days culture. In choriodecidual cells treated after 3 days in culture, expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNAwas similarly responsive toTNF-alpha (3.9 times basal within 3 h of 30 ng/ml TNF-alpha) while there was little effect on PGHS-1 or cytosolic phospholipase A2 expression. Hence, the dispersed choriodecidual cell culture system described retainsTNF-alpha responsive PG biosynthetic capacity which is at least in part upregulated via increased expression of PGHS-2 mRNA.

Cells, Cultured↗

PubMedDate not supplied

Efficacy and specificity of non-steroidal anti-inflammatory drugs for the inhibition of cytokine-stimulated prostaglandin E(2) secretion by amnion-derived WISH cells.

Prostaglandin H synthase-2 (PGHS-II) specific inhibitors have been proposed as a potential treatment in the prevention of preterm birth. We examined the efficacy of PGHS inhibitors on basal and cytokine-stimulated prostaglandin (PG) production by the amnion-like WISH cell line. WISH cells were treated with interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha in the presence or absence of indomethacin, etodolac, 5,5-dimethy-3-(3-fluorophenyl)-4-(4-methlysulphonyl) phenyl-2 (5H)-furanone (DFU) or nimesulide (1.6-1000 nM) for 16 h. PG production was then measured using radioimmunoassay. Nimesulide and DFU were the most selective non-steroidal anti-inflammatory drugs (NSAIDs) of IL-beta-stimulated PG production in these studies with an a IC(50)(basal)/IC(50)(stimulated) ratio of, respectively, 142.2 and 113.8, followed by etodolac (25.3) and indomethacin (2.2). Similar results were obtained when cells were stimulated with TNF-alpha. The results of this study suggest that PGHS-II-selective NSAIDs may be effective in the prevention of cytokine-driven amnion PG production associated with preterm labour.

Amnion↗

PubMedDate not supplied

Evidence for a common mechanism of action of interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor on prostaglandin production in human chorion cells.

PROBLEM: Although chorion produces prostaglandins in response to interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and epidermal growth factor (EGF), little attention has been given to the mechanisms of action of prostaglandin biosynthesis in this tissue. METHODS: IL-1 beta, TNF alpha, and EGF induced a concentration-related stimulation of prostaglandin E2 (PGE2) biosynthesis in human chorion cells, and this stimulation was enhanced by the addition of exogenous arachidonic acid. RESULTS: Protein synthesis inhibition with cycloheximide or actinomycin D resulted in complete abrogation of the stimulation of PGE2 production by IL-1 beta, TNF alpha, and EGF. Finally, all three stimulants induced a more rapid recovery of PGE2 production in chorion cells after acetylsalicylic acid pretreatment than controls. CONCLUSIONS: It is suggested that IL-1 beta, TNF alpha, and EGF all act to stimulate human chorion PGE2 production primarily via induction of prostaglandin endoperoxide synthase activity.

Aspirin↗

PubMedDate not supplied

Cyclosporin A attenuates increased prostaglandin and thromboxane production in response to various stimuli in human decidua.

PROBLEM: This study was undertaken to evaluate the effects of cyclosporin A on prostanoid (prostaglandin and thromboxane) production by human decidua. METHOD: Decidual cells were isolated from term placentae obtained at elective cesarean section before the onset of labor. Cells were grown to confluence and then incubated for 16 h with cyclosporin A (0.1-100 ng/ml) in the presence and absence of interleukin 1 beta (IL-1 beta, 10 ng/ml), phorbol 12-myristate 13-acetate (PMA, 10(-7) M) and ionomycin (0.5 micron). Prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) were measured by radioimmunoassay, and cellular protein was determined. RESULTS: IL-1 beta, PMA, and ionomycin all stimulated decidual PGE2 and TXB2 production as expected. However, these stimulatory actions were attenuated by 20% when cells were coincubated with cyclosporin A (100 ng/ml). All concentrations of cyclosporin A tested were within the therapeutic range. CONCLUSIONS: Our results indicate that cyclosporin A does not stimulate decidual prostanoid production and is probably unrelated to preterm labor and delivery in allograft recipients.

Cells, Cultured↗

PubMedDate not supplied

Revolution through genomics in investigative and discovery toxicology.

The remarkable technologic and methodologic advances spurred on by the Human Genome Project are being applied throughout the life sciences. In the field of toxicology, high-resolution assays now make it possible to discover virtually all the differences in gene expression brought on by exposure to a particular xenobiotic. There are 2 principal approaches used to build a catalog of changes in gene expression: hybridization microarrays and gel-based methods, such as differential display and AFLP-based mRNA finger-printing. The power of such approaches is exemplified by the identification of more than 300 genes that differ in expression level by at least 2-fold in response to the nongenotoxic rodent liver carcinogen phenobarbital.

Animals↗

PubMedDate not supplied

Mechanism of cytokine stimulation of prostaglandin biosynthesis in human decidua.

OBJECTIVE: The purpose of this study was to determine the mechanism of action of the cytokines tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) on the stimulation of prostaglandin (PG) production in human decidua. METHODS: Decidual cells from term placentas were grown in culture until confluent. Incubations were performed with TNF alpha or IL-1 beta, and with cycloheximide, actinomycin D, arachidonic acid, or acetylsalicylic acid (ASA). Prostaglandin E2 was measured by radioimmunoassay and cellular protein determined. RESULTS: The concentration-related stimulation of decidual PGE2 production by IL-1 beta and TNF alpha was completely abrogated by cycloheximide and actinomycin D treatment. Although arachidonic acid alone stimulated decidual PGE2 biosynthesis, the addition of IL-1 beta or TNF alpha consistently augmented this effect. Both cytokines induced recovery of PGE2 biosynthesis from ASA pretreatment more rapidly than controls. CONCLUSIONS: Interleukin-1 beta and TNF alpha both act on decidual PG biosynthesis in a manner requiring new protein synthesis. In combination with our other results, this suggests that IL-1 beta and TNF alpha act to induce PG endoperoxide synthase activity.

Cycloheximide↗

PubMedDate not supplied

Tumor necrosis factor-alpha inhibits ovulation and steroidogenesis, but not prostaglandin production in the perfused rat ovary.

OBJECTIVE: We tested the null hypothesis that tumor necrosis factor-alpha (TNF-alpha) does not decrease ovulation, estradiol and progesterone production, or prostaglandin (PG) E2, PGF2alpha, or 6 keto-PGF1alpha production in the open bursa rat ovarian perfusion model. METHODS: Experimental animals were controlled for age, weight, litter, and pregnant mare's serum gonadotropin (PMSG) aliquot. Female Sprague-Dawley rats, 26-27 days old, were injected with 25 IU PMSG. Forty-eight hours later, the right ovary was dissected, the bursa removed, and the specimen placed in the perfusion chamber with defined media. Luteinizing hormone and isobutylmethylxanthine were given as an ovulatory trigger. Test perfusions also received TNF-alpha in 0.8-nmol/L, -pmol/L, and -fmol/L doses. Samples were collected at 0, 1, 3, 5, 7, 10, and 20 hours. Ovulations were counted at 20 hours. Steroids and PGs were measured. RESULTS: The addition of TNF-alpha to the rat ovarian perfusion model resulted in a dose-dependent decrease in ovulations (mean +/- standard deviation): 16.14 +/- 6.2 in controls (n = 7) versus 2.38 +/- 3.4 with TNF-alpha 0.8 nmol/L (n = 7), and 4.3 +/- 1.5 with TNF-alpha 0.8 pmol/L (n = 3), both P < .001. Tumor necrosis factor-alpha also inhibited estradiol (P < .005) and progesterone production (P < .05) throughout, but produced no significant changes in PG production. CONCLUSIONS: Tumor necrosis factor-alpha inhibits ovulation in a dose-dependent fashion, and inhibits estradiol and progesterone production without altering PG production in the open bursa rat ovarian perfusion model.

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

Regulation of activin A, inhibin A, and follistatin production in human amnion and choriodecidual explants by inflammatory mediators.

OBJECTIVE: To determine the effects of inflammatory mediators on the production of activin A, inhibin A, and the binding protein follistatin in term amnion and choriodecidual tissues. METHODS: The effects of interleukin-1 beta (IL-1 beta; 1 ng/mL), tumor necrosis factor-alpha (TNF-alpha; 10 ng/mL), and bacterial lipopolysaccharide (LPS; 5 microg/mL) on production rates of activin A, inhibin A, and follistatin by term choriodecidual and amnion membranes in explant culture were determined using specific enzyme-linked immunoabsorbent assays. RESULTS: All explants (n = 6 placentas) produced detectable amounts of activin A, inhibin A, and follistatin under basal conditions; choriodecidual production rates were more than tenfold higher than amnion rates. In amnion explants, activin A production was stimulated by IL-1 beta and TNF-alpha to 450 +/- 155.4% and 531 +/- 170.8% of control, respectively (mean +/- standard error of the mean; P <.05 by analysis of variance), whereas production of inhibin and follistatin was stimulated to a much more modest extent. Similar responses were observed in the choriodecidual explants. Lipopolysaccharide had no significant effect on amnion activin A production, but stimulated choriodecidual production to 290 +/- 34% of control. Lipopolysaccharide exerted only limited effects on inhibin A and follistatin production. CONCLUSIONS: Treatment with proinflammatory mediators resulted in a preferential increase in activin A production compared with that of inhibin A or follistatin. These findings suggest that inflammation of the gestational membranes could result in increased local activin A production and bioactivity.

Activins↗

PubMedDate not supplied

Inflammatory cytokine mRNA in human gestational tissues: implications for term and preterm labor.

OBJECTIVE: To determine if inflammatory cytokine mRNA in gestational tissues is present only in the setting of infection-associated preterm labor or under several other clinical conditions. METHODS: Human gestational tissues were collected from 51 women experiencing 1) term cesarean delivery without labor, 2) normal term vaginal delivery, 3) preterm cesarean delivery without labor, 4) preterm vaginal delivery without chorioamnionitis, and 5) preterm vaginal delivery with concomitant chorioamnionitis. Decidua, chorion, and amnion were isolated, total RNA from each tissue was extracted, and the presence of inflammatory cytokine mRNA was determined by polymerase chain reaction. Interleukin (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor-alpha mRNA was detected using specific oligonucleotide primers. RESULTS: Interleukin-1 beta mRNA was rarely found in tissues preterm without labor but was readily detected in both maternal and fetal tissues after labor, regardless of gestational age. Interleukin-6 mRNA was rare in tissues from the nonlaboring patient but was found in almost all tissues after labor. Interleukin-8 mRNA was detected in all tissues at term, both in nonlaboring and laboring patients. Tumor necrosis factor-alpha mRNA was detected in only 20-50% of tissues after labor, and was rarely detected in the absence of labor. CONCLUSIONS: Inflammatory cytokine mRNA is commonly expressed in human gestational tissues after normal labor and preterm labor with or without associated intrauterine infection. There was no difference in the pattern of expression of mRNA inflammatory cytokine in women who did or did not have clinically evident intrauterine infection.

Adolescent↗

PubMedDate not supplied

Elevated concentrations of prorenin and renin in amniotic fluid of women with chorioamnionitis.

OBJECTIVE: The objective of this study was to determine the concentrations of prorenin and renin in amniotic fluid of women under various physiologic and pathologic conditions. METHODS: Amniotic fluid was collected from women with chorioamnionitis treated in the Labor and Delivery Unit, University of Utah Medical Center, and from gestation-matched control women with preterm labor who were delivered within 1 week and later than 1 week and at term in labor or not in labor. Prorenin and renin concentrations were measured in these fluids by a direct immunoradiometric assay. RESULTS: Large amounts of prorenin and renin were detected in all samples with 85% to 95% in the form of prorenin. Concentrations of both prorenin and renin were significantly higher in the amniotic fluid of women with chorioamnionitis than in the amniotic fluid of control women. There were no other significant differences between the groups. CONCLUSIONS: Our data indicate that amniotic fluid concentrations of renin and prorenin are elevated in women with clinically evident chorioamnionitis. We suggest that these elevations reflect a possible role for renin and prorenin in the pathophysiologic process of intrauterine infection-associated preterm labor.

Amniotic Fluid↗

PubMedDate not supplied

Expression of interleukin-10 in human gestational tissues.

OBJECTIVE: To determine production of interleukin-10 (IL-10) by interleukin-1 beta (IL-1 beta) in cultured decidual, chorion, and amnion cells and whether IL-10 is produced in gestational tissues under the setting of infection-associated preterm labor. METHODS: Decidual, chorion, and amnion cells were isolated from term placentas and grown in primary culture. The cells were incubated with various concentrations of IL-1 beta and then culture supernatants were assayed for IL-10 by enzyme-linked immunosorbent assay. In subsequent studies, gestational membranes were isolated from a normal-term pregnancy and a preterm pregnancy complicated by chorioamnionitis. Tissues were evaluated for IL-10 expression by immunohistology and in situ hybridization. Human gestational tissues were collected from 38 women experiencing: 1) term cesarean delivery without labor; 2) normal-term vaginal delivery; 3) preterm cesarean delivery without labor; 4) preterm vaginal delivery without chorioamnionitis; and 5) preterm vaginal delivery with concomitant chorioamnionitis. Amnion, chorion, and decidua were isolated, total RNA from each tissue was extracted, and the presence of IL-10 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Decidual cells in culture produced IL-10 in response to IL-1 beta, but chorion and amnion cells produced no IL-10 protein. In vivo protein expression by immunohistology showed that most protein was detected within decidua while cells within amnion and chorion rarely had detectable IL-10 protein. In vivo RT-PCR samples demonstrated the strongest IL-10 mRNA signal from decidua samples, although IL-10 mRNA was also noted in chorion and amnion of placentas obtained after preterm labor. CONCLUSION: Maternal decidual cells can potentially produce IL-10, but fetal membranes (amnion and chorion) appear to have limited capabilities to produce IL-10. The relative inability of fetal tissues to produce IL-10 may play an important role in the pathophysiology of infection-associated preterm labor.

Amnion↗

PubMedDate not supplied

Ceramide stimulates prostaglandin production by human amnion and decidual cells.

OBJECTIVE: To determine whether ceramide regulates prostaglandin (PG) production by cultured human amnion cells and decidual cells independently of interleukin-1 beta (IL-1 beta). METHODS: Cells were grown in monolayer culture and then incubated with varying concentrations of ceramide, IL-1 beta, ceramide in the presence and absence of IL-1, and control media. Production of PGE2 was determined using a specific radioimmunoassay. RESULTS: Ceramide induced a significant concentration-dependent increase in PGE2 production by amnion cells and decidual cells. However, PGE2 production induced by IL-1 beta was significantly more than with ceramide alone, and there was no potentiation of PGE2 production with coincubation of ceramide and IL-1 beta. CONCLUSION: We suggest that term human amnion cells and decidual cells are responsive to ceramide independent of IL-1 beta and that generation of these substances in response to an infection in the uterus may lead to increased PG production by human gestational tissues, indicating that there are several mechanisms leading to PG production by these cells.

Amnion↗