Involvement of p38 MAP kinase in lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and prostaglandin E(2) in human choriodecidua.

Chorioamnionitis is implicated in the pathogenesis of preterm delivery. However, the detailed mechanisms by which infection induces preterm labor are not well understood. This study has assessed the involvement of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced pro- and anti-inflammatory cytokine and prostaglandin (PG) production in human choriodecidua. Samples of choriodecidua were collected before the onset of labor from women undergoing elective cesarean sections at term for breech presentation, previous cesarean delivery or cephalopelvic disproportion. Concentrations of TNFalpha, IL-10, PGE(2) and PGF(2)alpha in culture supernatants were measured by ELISA. Expression of COX-2 protein was analyzed by Western blotting. In human choriodecidual explants, LPS induced TNFalpha and IL-10 production in a dose- and time-dependent manner. LPS also up-regulated COX-2 expression and PG synthesis. Phosphorylations of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal kinases (JNK) were also confirmed by Western blotting. Furthermore, the effect of MAPK inhibitors was examined on LPS-induced pro- and anti-inflammatory cytokines and PG synthesis. Among the MAPK inhibitors examined, the p38 MAPK inhibitor, SB202190, significantly suppressed LPS-induced cytokine and PG production. SB202190 most profoundly suppressed the TNFalpha to IL-10 ratio, demonstrating that p38 MAPK inhibitor reduced predominantly TNFalpha other than IL-10 production. Phospho-p38 MAPK immunostaining was intense in extravillous trophoblast cells. The p38 MAPK seems to be most involved in signaling mechanisms when infection and inflammation cause preterm labor through PG synthesis. Novel therapeutic modalities targeting p38 MAPK may prevent to arrest preterm labor.

Macrophage-activating lipopeptide-2 induces cyclooxygenase-2 and prostaglandin E(2) via toll-like receptor 2 in human placental trophoblast cells.

We have examined whether toll-like receptor (TLR)2-mediated stimulation by macrophage-activating lipopeptide-2 (MALP-2), originally purified from Mycoplasma fermentans, induces cyclooxygenase (COX)-2 and prostaglandin (PG)E(2) in human placental trophoblast cells. The signaling mechanism by which MALP-2 exerts its effect has also been examined. Human placental trophoblast cells isolated from term placenta were used. TLR expression in trophoblast cells was confirmed by multiplex PCR and immunocytochemistry, and examined whether MALP-2 induces COX-2 and PGE(2) by Northern blotting, RT-PCR, Western blotting and ELISA, respectively. The activation of NF-kappaB and MAP kinases (ERK1/2 and p38) was examined by Western blotting. The effects of inhibitors of NF-kappaB, MEK1/2 and p38 on MALP-2-induced PGE(2) production were also evaluated. TLR2, TLR6 and TLR4 were expressed in human placental trophoblast cells. MALP-2 significantly induced COX-2 expression and enhanced PGE(2) production in a dose-dependent manner. MALP-2 induced the activation of NF-kappaB, ERK1/2 and p38 MAPK. Inhibitors of NF-kappaB, MEK1/2 and p38 blocked MALP-2-inducible PGE(2) production. TLR2-mediated stimulation by MALP-2 induces COX-2 and PGE(2) in human placental trophoblast cells via NF-kappaB and MAP kinases pathways.

Progesterone and progestational compounds attenuate tumor necrosis factor alpha-induced interleukin-8 production via nuclear factor kappa B inactivation in endometriotic stromal cells.

OBJECTIVE: To investigate whether and how P, dienogest (synthetic progestin), and danazol affected tumor necrosis factor alpha (TNFalpha)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. PATIENT(S): Ten patients who underwent laparoscopic surgery. INTERVENTION(S): Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. MAIN OUTCOME MEASURE(S): In the presence of TNFalpha (0.1 ng/mL) and E2 (10(-7) mol/L), the cells were cultured in medium with P (10(-6) mol/L), danazol (10(-6) mol/L), or dienogest (10(-7) mol/L). The expression of the IL-8 gene and protein was determined by Northern blotting and ELISA, respectively. Activation of nuclear factor (NF)-kappaB was evaluated by electrophoretic mobility shift assay. RESULT(S): Adding TNFalpha (0.1 ng/mL) together with E2 markedly enhanced gene and protein expression of IL-8. The up-regulation of the IL-8 gene and protein expression by TNFalpha and E2 was significantly reduced by the addition of P, dienogest, or danazol. Electrophoretic mobility shift assay revealed that incubation with TNFalpha and E2 induced NF-kappaB activation. Adding P, dienogest, or danazol attenuated NF-kappaB activation. CONCLUSION(S): The present study demonstrates for the first time that P and progestational compounds attenuate the expression of IL-8 by reducing TNFalpha-induced NF-kappaB activation in endometriotic stromal cells, suggesting a possible molecular mechanism of hormone therapy for controlling the growth of endometriosis.

Cervical Ureaplasma urealyticum colonization might be associated with increased incidence of preterm delivery in pregnant women without prophlogistic microorganisms on routine examination.

AIM: We examined whether the detection of Ureaplasma urealyticum DNA in the cervix is associated with preterm labor and delivery. METHODS: Eighty-two women (23 preterm labor cases and 59 controls) with no evidence of prophlogistic microorganisms on routine microbiologic examination were enrolled for this study. U. urealyticum colonization was examined using polymerase chain reaction of cervical swabs. RESULTS: The positivity rate of U. urealyticum DNA in preterm labor cases was significantly higher than that in the controls (87.0%vs 45.8%, P=0.0007). Women in the U. urealyticum-positive group more frequently delivered preterm compared with those in the negative group (36.2%vs 11.4%, P=0.0111). In five cases that delivered preterm with histologically confirmed chorioamnionitis, U. urealyticum DNA was detected in the placenta. CONCLUSIONS: Cervical U. urealyticum colonization might be associated with preterm labor and delivery.

Coculture with a human granulosa cell line enhanced the development of murine preimplantation embryos via SCF/c-kit system.

PURPOSE: To evaluate the effect on the coculture of murine embryos with a human ovarian granulosa tumor derived cell line (KGN cells). METHODS: We observed microscopically the growth of murine preimplantation embryos in the coculture system with KGN cells or in the presence with exogenous stem cell factor (SCF). The reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze the gene expression of SCF in KGN cells cocultured with murine embryos. RESULTS: The coculture system with KGN cells significantly increased the rate of embryo development to late blastocyst and to hatching stage. We also found that coculture with murine embryos enhanced the gene expression of SCF in KGN cells. Adding human recombinant SCF to the medium significantly enhanced embryo development to late blastocyst and hatching stage. CONCLUSIONS: KGN cells may facilitate preimplantion embryo development through SCF/c-kit paracrine system.

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion.

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.

Interleukin-1beta-induced expression of IL-6 and production of human chorionic gonadotropin in human trophoblast cells via nuclear factor-kappaB activation.

PROBLEM: The cytokine, interleukin-6 (IL-6), stimulates the production of human chorionic gonadotropine (hCG) in chorionic cells. The purpose of this study was to examine the role of nuclear factor-kappaB (NF-kappaB) during the induction of IL-6 by IL-1beta in human trophoblast cells. METHOD OF STUDY: Reverse transcription-polymerase chain reaction was used to determine the gene expressions of IL-1, IL-1 receptor (IL-1R), IL-6R and gp130 in a human choriocarcinoma cell line, BeWo. The BeWo cells were cultured for 24 hr with IL-1beta (0-10 ng/mL), IL-1beta (10 ng/mL) together with IL-1 receptor antagonist (IL-1Ra) or NF-kappaB inhibitor, N-tocyl-l-phenylalanine chloromethyl ketone (TPCK). The concentrations of IL-6 in culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The gene expressions of IL-1R, IL-6R and gp130, but not IL-1beta, were observed. The concentration of IL-6 was enhanced by IL-1beta in a dose-dependent fashion. The addition of IL-1Ra neutralized the effects of IL-1beta on IL-6 secretion. IL-1beta induced expression of phosphorylated IkappaB and the activation of NF-kappaB. The addition of TPCK reduced the IL-1beta-induced IL-6 expression. Adding IL-1beta increased beta-hCG production in a dose-dependent manner, and the addition of TPCK neutralized the effect of IL-1beta. CONCLUSION: IL-1beta may induce the IL-6 expression and hCG production in human BeWo cells via NF-kappaB.

A combination of interleukin-6 and its soluble receptor impairs sperm motility: implications in infertility associated with endometriosis.

BACKGROUND: We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid of women with endometriosis. This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor (sIL-6R) on in vitro sperm motility. METHODS: Sperm (n = 20) were cultured with IL-6 or sIL-6R, or with a combination of both. After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis system. Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined in sperm by RT-PCR analysis and western blot analysis. RESULTS: Addition of IL-6 or sIL-6R individually to the culture media had no affect on sperm motion. However, adding a combination of IL-6 and sIL-6R dose-dependently reduced the percentage of motile and rapidly moving sperm. Adding anti-IL-6R antibody abolished these adverse effects. Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R. CONCLUSIONS: A combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility. IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility.

Use of the LAP DISK (abdominal wall sealing device) in laparoscopically assisted myomectomy.

OBJECTIVE: To evaluate the efficacy of an abdominal wall sealing device (the LAP DISK) used during laparoscopically assisted myomectomy (LAM). DESIGN: Retrospective study. SETTING: Tottori University Hospital, Yonago, Japan. PATIENT(S): All 43 patients who underwent LAM using the LAP DISK. INTERVENTION(S): Ultrasonography and magnetic resonance imaging. MAIN OUTCOME MEASURE(S): Treatment strategy, operative outcome, and postoperative pregnancy rate. RESULT(S): Weight and size of the myomas removed ranged from 40-700 g (mean: 208.0 g) and 2-10 cm (mean: 5.4 cm). Mean blood loss was 42.3 mL. Half of the 18 patients who had been diagnosed with primary infertility for >2 years became pregnant without postoperative assisted reproductive techniques. CONCLUSION(S): The LAP DISK, a useful device for LAM, allows surgeons to remove myomas safely and repair uterine defects effectively while minimizing blood loss and trauma.

Hepatocyte growth factor/Met system promotes endometrial and endometriotic stromal cell invasion via autocrine and paracrine pathways.

Endometrial stromal cells reportedly have a role in the initial invasion of endometrial tissue into the peritoneum. Hepatocyte growth factor (HGF), which is a ligand for the c-met protooncogene product (Met), stimulates proliferation and invasion of a large number of cells. In this study we investigated the role of the HGF/Met system in the pathogenesis of endometriosis. HGF concentrations in the peritoneal fluid of patients with endometriosis were significantly higher than in those without endometriosis and correlated positively with revised American Society of Reproductive Medicine scores. We showed that the peritoneum and endometriotic stromal cells may be major sources of HGF in peritoneal fluid. Endometrial and endometriotic stromal cells expressed the Met receptor, which was activated by endogenous and exogenous HGF. HGF enhanced stromal cell proliferation and invasion. We also demonstrated that the HGF-stimulated stromal cell invasion was due in part to the induction of urokinase-type plasminogen activator, a member of the extracellular proteolysis system. In conclusion, the HGF/Met system is involved in the pathogenesis of endometriosis by promoting stromal cell proliferation and invasion of shed endometria and endometrial lesions via autocrine and paracrine pathways.

Gonadotropin-releasing hormone agonist treatment reduced serum interleukin-6 concentrations in patients with ovarian endometriomas.

OBJECTIVE: To determine whether serum interleukin (IL)-6 can be measured in patients with ovarian endometriomas and whether these measurements are useful in managing this disease. DESIGN: A controlled clinical study and an in vitro study. SETTING: Department of Obstetrics and Gynecology, Tottori University, Japan.Twenty-two patients with ovarian endometriomas. INTERVENTION(S): Laparoscopic cystectomy for ovarian endometriomas was performed. Gonadotropin-releasing hormone (GnRH) agonist was administered for 3 months in nine patients before laparoscopic surgery. Endometriotic stromal cells obtained from patients with endometriomas with or without GnRH agonist treatment were cultured. MAIN OUTCOME MEASURES(S): IL-6 concentrations in serum or supernatant of the cell culture were measured using ELISA. RESULTS: The serum concentration of IL-6 in patients with endometriomas was higher at the time of diagnosis than in those without endometriomas. Laparoscopic surgery significantly reduced serum levels of IL-6. Serum IL-6 concentrations also decreased after treatment with GnRH agonist. IL-6 production was attenuated in the endometriotic stromal cells obtained from patients with GnRH agonist treatment compared with patients without such treatment. CONCLUSION(S): GnRH agonist treatment may decrease IL-6 production in endometriotic cells. Measurement of serum IL-6 concentrations may be of value in managing patients with endometriomas.