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The effects of lipopolysaccharide on fetoplacental artery production of interleukin-10
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491 THE EFFECTS OF LIPOPOLYSACCHARIDE ON FETOPLACENTAL AR- TERY PRODUCTION OF INTERLEUKIN-10 BOBBY HOWARD 1 , ANDREA FISK 1 , JAMES WRIGHT 1 , CHRISTINE KOVAC 1 , NATHAN HOELDTKE 2 , PETER NAPOLITANO 1 , 1 Madigan Army Medical Center, Tacoma, WA 2 Tripler Army Medical Center, Honolulu, HI OBJECTIVE: To determine the effects of lipopolysaccharide (LPS) on fetoplacental artery production of interleukin-10 (IL-10). STUDY DESIGN: Placentas were obtained from normal pregnancies following elective repeat cesarean delivery at term prior to the onset of labor. Three chorionic plate arteries were then dissected from each of these placentas. Arteries were incubated in Dulbecco’s Modified Eagles Media alone, media plus LPS (50 ng/mL) at initiation of incubation (immediate LPS group) or media plus LPS (50 ng/mL) added after 48 hours of incubation (delayed LPS group). Samples of the tissue culture media were collected and assayed for IL-10 by immunoassay (Roche). Statistical analysis was performed using analysis of variance with a P-value < 0.05 considered significant. Results are reported as mean ± SEM. RESULTS: Following incubation with LPS for eight hours, the IL-10 level in the immediate LPS group (1064 ± 3 pg/mL/mg tissue, P < 0.001) and delayed LPS group (2064 ± 253 pg/mL/mg tissue, P < 0.001) was significantly higher than the IL-10 level in the corresponding sample from the control group (74 ± 58 pg/mL/mg tissue and 82 ± 10 pg/mL/mg tissue). Following incubation with LPS for twenty-four hours, the IL-10 level was also higher in the immediate LPS group (1648 ± 177 pg/mL/mg tissue, P < 0.001) and delayed LPS group (1961 ± 334 pg/mL/mg tissue, P < 0.001) than the corresponding IL-10 level in controls (85 ± 50 pg/mL/mg tissue and 157 ± 81 pg/mL/mg tissue). CONCLUSION: Interleukin-10 production by fetoplacental arteries is increased markedly following exposure to LPS in this in vitro model. This pilot study characterizes the IL-10 response to LPS in this model and provides baseline values for further evaluation of potential interventions for fetal inflammatory states. 492 ROLE OF SRC FAMILY KINASES DURING TYROSINE PHOSPHO- RYLATION OF PHOSPHOLIPASE C-g1 DURING PHASIC MYOMETRIAL CONTRACTIONS MARK PHILLIPPE 1 , DANIEL ENGLE 1 , LEIGH SWEET 1 , 1 University of Vermont College of Medicine, Obstetrics and Gynecology, Burlington, VT OBJECTIVE: The intracellular signaling events during spontaneous myometrial contractions are yet to be defined. Previous reports have suggested that activation of PLC-c1 in response to tyrosine phosphorylation plays an important role. These studies sought to characterize the Src kinase(s) involved in PLC-c1 activation. STUDY DESIGN: Uterus and other tissues were obtained from proestrus/ estrus Sprague-Dawley rats. For in vitro contraction studies, longitudinal uterine strips were suspended in 3 mL tissue baths containing Earle’s balanced salt solution at 37 C aerated with 95% O2/5% CO2. Inhibition of specific Src kinase isoforms was performed using PP1 (Src inhibitor), Damnacanthal (Lck inhibitor), and Piceatannol (Syk inhibitor) followed by inhibition of PLC-c1 dephosphorylation using potassium bisperoxo(1,10 phenanthroline) oxovanadate (bpV(phen)). Western blots were performed using antibodies to phosphotyrosine-PLC-c1, total PLC-c1, Src and Lck kinase proteins. RESULTS: Spontaneous contractions of non-pregnant rat uterine strips were significantly suppressed in response to 50-100 lM PP1 and Damn compared to strips treated with vehicle alone (p < 0.05). BpV(phen) enhanced PLC-c1 activation leading to increased phasic contractions in control strips; PP1 and Damn inhibited this response. In contrast, Pice had no effect on spontaneous contractions and did not inhibit bpV(phen) stimulation. Western blots confirmed significant PP1 and Damn inhibition of phos- photyrosine-PLC-c1 (p < 0.05); whereas Pice had no effect. Additional Western blots confirmed robust expression of Src and Lck kinase proteins in uterus compared to other rat tissues. CONCLUSION: Spontaneous uterine contractions are associated with PLC- c1 activation in response to tyrosine-phosphorylation. These studies have confirmed that at least two Src kinases (Src and Lck) are expressed in rat uterine tissue, modulate phosphotyrosine-PLC-c1 levels, and regulate contractile activity. (Funded by NIH HD-28506). 493 CENTRAL NEUROPEPTIDE-Y (NPY) STIMULATES PUTATIVE HYPOTHA- LAMIC APPETITE CENTERS (C-FOS EXPRESSION) IN THE NEAR TERM OVINE FETUS MOSTAFA EL-HADDAD 1 , MICHAEL ROSS 2 , 1 University of California, Los Angeles, Perinatal Medicine, Rancho Palos Verdes, CA 2 University of California, Los Angeles, Harbor-UCLA Medical Center, Torrance, CA OBJECTIVE: NPY, a potent appetite stimulant, is synthesized in the adult arcuate hypothalamic nucleus (ARC) of many species and released into the paraventricular nucleus (PVN) to stimulate food intake. In the near term ovine fetus, central NPY stimulates oral sucrose ingestion. However, it is unknown whether this response is specific to appetite mechanisms, or results from nonspecific stimulation of dipsogenic neurons. C-Fos is a protein which represents the expression of the immediate-early gene and has been used as a marker of neuronal activity. We hypothesized that NPY stimulated fetal ingestion is secondary to activation of functional, orexic pathways. STUDY DESIGN: Time-dated pregnant ewes and fetuses (n = 4) were prepared with fetal vascular and intracerebroventricular (ICV) catheters and studied at 133 ± 1 days gestation. After five days of postoperative recovery, ovine fetuses received ICV injections of either NPY (0.5 mg/kg, 1ml; n = 2) or artificial cerebrospinal fluid (1ml; n = 2). Fetuses were monitored for 90 min after which animals were killed and fetal brains were perfused for subsequent C-Fos staining. RESULTS: ICV injection of NPY markedly increased fetal hypothalamic C-Fos staining in putative hypothalamic appetite centers (arcuate 72 ± 8 vs. 20 ± 10; PVN 65 ± 5 vs. 11 ± 6; perifornical 53 ± 10 vs. 1 ± 0; lateral hypothalamus 97 ± 29 vs. 1 ± 0; all P < 0.05) as compared to aCSF treated fetuses. There was no stimulation of C-FOS in the dipsogenic neurons. CONCLUSION: As evidenced by C-Fos activation, putative appetite centers are responsive to NPY in the near term ovine fetus. Together with the demonstration of NPY stimulation of fetal ingestive behavior, these results indicate that appetite mechanisms are functional in the near term ovine fetus. The wide stimulation of appetite centers throughout the hypothalamus may extend into neonatal period and explain upregulated neonatal appetite and the augmented nutrient intake necessary for newborn growth. 494 THE REDUCTION OF ADIPONECTIN LEVELS DURING THE IMMEDIATE POSTPARTUM CANNOT BE EXPLAINED BY PLACENTAL OR FETAL PRODUCTION SHALI MAZAKI TOVI 1 , HANNAH KANETY 2 , EYAL SCHIFF 1 , RINA HEMI 2 , CLARA PARIENTE 2 , ANN SHOHAM 1 , EYAL SIVAN 1 , 1 Sheba medical center, ObGyn, Tel Hashomer Israel 2 Institute of Endocrinology, Sheba medical center, Tel Hashomer Israel OBJECTIVE: Adiponectin is an adipocyte-derived plasma protein with insulin-sensitizing and anti-atherosclerotic properties found abundantly in cord blood. The aim of this study was to examine maternal adiponectin levels and to establish a possible connection between these levels and placental or fetal production. STUDY DESIGN: The study was comprised of three studies. Study 1 - blood was obtained from 23 women one day prior to and four days after delivery. Study 2ÿseventeen blood samples were obtained from mothers and their newborns at delivery. Blood samples were tested for adiponectin and leptin. Study 3ÿhuman placental tissues were tested for adiponectin mRNA by RT-PCR. RESULTS: Study 1- Maternal adiponectin levels before and after delivery were 22.2 ± 7.0 and 17.5 ± 6.8 g/ml respectively (P < 0.03). A similar decline was measured for leptin, known to be expressed in the placenta, (27.9 ± 13.4 and 13.2 ± 8.0 ng/ml, respectively, P < 0.001). No correlation was found between adiponectin levels and maternal characteristics. Study 2ÿCord blood adiponectin levels were significantly higher when compared with maternal adiponectin levels (61.1 ± 19.0 vs. 22.7 ± 6.8 g/ml, P < 0.001). No correlation was found between cord blood and maternal adiponectin (r = 0.3, P < 0.2). Study 3ÿthere was no expression of adiponectin mRNA in human placental tissues. CONCLUSION: Our findings demonstrate that adiponectin levels are relatively high in the serum of women in the third trimester and decline after delivery. These findings can not be explained either by placental production or by transport from fetal compartments. December 2003 Am J Obstet Gynecol S194 SMFM Abstracts
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Transcript of The effects of lipopolysaccharide on fetoplacental artery production of interleukin-10
491
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493 CENTRAL NEUROPEPTIDE-Y (NPY) STIMULATES PUTATIVE HYPOTHA-LAMIC APPETITE CENTERS (C-FOS EXPRESSION) IN THE NEAR TERMOVINE FETUS MOSTAFA EL-HADDAD1, MICHAEL ROSS2, 1University ofCalifornia, Los Angeles, Perinatal Medicine, Rancho Palos Verdes, CA2University of California, Los Angeles, Harbor-UCLA Medical Center,Torrance, CA
OBJECTIVE: NPY, a potent appetite stimulant, is synthesized in the adultarcuate hypothalamic nucleus (ARC) of many species and released into theparaventricular nucleus (PVN) to stimulate food intake. In the near term ovinefetus, central NPY stimulates oral sucrose ingestion. However, it is unknownwhether this response is specific to appetite mechanisms, or results fromnonspecific stimulation of dipsogenic neurons. C-Fos is a protein whichrepresents the expression of the immediate-early gene and has been used asa marker of neuronal activity. We hypothesized that NPY stimulated fetalingestion is secondary to activation of functional, orexic pathways.
STUDY DESIGN: Time-dated pregnant ewes and fetuses (n = 4) wereprepared with fetal vascular and intracerebroventricular (ICV) catheters andstudied at 133 ± 1 days gestation. After five days of postoperative recovery, ovinefetuses received ICV injections of eitherNPY (0.5mg/kg, 1ml; n = 2) or artificialcerebrospinal fluid (1ml; n = 2). Fetuses were monitored for 90 min after whichanimals were killed and fetal brains were perfused for subsequent C-Fos staining.
RESULTS: ICV injection of NPY markedly increased fetal hypothalamicC-Fos staining in putative hypothalamic appetite centers (arcuate 72 ± 8 vs.20 ± 10; PVN 65 ± 5 vs. 11 ± 6; perifornical 53 ± 10 vs. 1 ± 0; lateralhypothalamus 97 ± 29 vs. 1 ± 0; all P < 0.05) as compared to aCSF treatedfetuses. There was no stimulation of C-FOS in the dipsogenic neurons.
CONCLUSION: As evidenced by C-Fos activation, putative appetite centersare responsive to NPY in the near term ovine fetus. Together with thedemonstration of NPY stimulation of fetal ingestive behavior, these resultsindicate that appetite mechanisms are functional in the near term ovine fetus.The wide stimulation of appetite centers throughout the hypothalamus mayextend into neonatal period and explain upregulated neonatal appetite and theaugmented nutrient intake necessary for newborn growth.
December 2003Am J Obstet Gynecol
S194 SMFM Abstracts
THE EFFECTS OF LIPOPOLYSACCHARIDE ON FETOPLACENTAL AR-TERY PRODUCTION OF INTERLEUKIN-10 BOBBY HOWARD1, ANDREAFISK1, JAMES WRIGHT1, CHRISTINE KOVAC1, NATHAN HOELDTKE2,PETER NAPOLITANO1, 1Madigan Army Medical Center, Tacoma, WA2Tripler Army Medical Center, Honolulu, HI
OBJECTIVE: To determine the effects of lipopolysaccharide (LPS) onfetoplacental artery production of interleukin-10 (IL-10).
STUDY DESIGN: Placentas were obtained from normal pregnanciesfollowing elective repeat cesarean delivery at term prior to the onset of labor.Three chorionic plate arteries were then dissected from each of these placentas.Arteries were incubated in Dulbecco’s Modified Eagles Media alone, media plusLPS (50 ng/mL) at initiation of incubation (immediate LPS group) or mediaplus LPS (50 ng/mL) added after 48 hours of incubation (delayed LPS group).Samples of the tissue culture media were collected and assayed for IL-10 byimmunoassay (Roche). Statistical analysis was performed using analysis ofvariance with a P-value < 0.05 considered significant. Results are reported asmean ± SEM.
RESULTS: Following incubation with LPS for eight hours, the IL-10 level inthe immediate LPS group (1064 ± 3 pg/mL/mg tissue, P < 0.001) and delayedLPS group (2064 ± 253 pg/mL/mg tissue, P < 0.001) was significantly higherthan the IL-10 level in the corresponding sample from the control group(74 ± 58 pg/mL/mg tissue and 82 ± 10 pg/mL/mg tissue). Followingincubation with LPS for twenty-four hours, the IL-10 level was also higher inthe immediate LPS group (1648 ± 177 pg/mL/mg tissue, P < 0.001) anddelayed LPS group (1961 ± 334 pg/mL/mg tissue, P < 0.001) than thecorresponding IL-10 level in controls (85 ± 50 pg/mL/mg tissue and157 ± 81 pg/mL/mg tissue).
CONCLUSION: Interleukin-10 production by fetoplacental arteries isincreased markedly following exposure to LPS in this in vitro model. This pilotstudy characterizes the IL-10 response to LPS in this model and providesbaseline values for further evaluation of potential interventions for fetalinflammatory states.
ROLE OF SRC FAMILY KINASES DURING TYROSINE PHOSPHO-RYLATION OF PHOSPHOLIPASE C-g1 DURING PHASIC MYOMETRIALCONTRACTIONS MARK PHILLIPPE1, DANIEL ENGLE1, LEIGH SWEET1,1University of Vermont College of Medicine, Obstetrics and Gynecology,Burlington, VT
OBJECTIVE: The intracellular signaling events during spontaneousmyometrial contractions are yet to be defined. Previous reports have suggestedthat activation of PLC-c1 in response to tyrosine phosphorylation plays animportant role. These studies sought to characterize the Src kinase(s) involvedin PLC-c1 activation.
STUDY DESIGN: Uterus and other tissues were obtained from proestrus/estrus Sprague-Dawley rats. For in vitro contraction studies, longitudinal uterinestrips were suspended in 3 mL tissue baths containing Earle’s balanced saltsolution at 37 C aerated with 95%O2/5% CO2. Inhibition of specific Src kinaseisoforms was performed using PP1 (Src inhibitor), Damnacanthal (Lckinhibitor), and Piceatannol (Syk inhibitor) followed by inhibition of PLC-c1dephosphorylation using potassium bisperoxo(1,10 phenanthroline)oxovanadate (bpV(phen)). Western blots were performed using antibodiesto phosphotyrosine-PLC-c1, total PLC-c1, Src and Lck kinase proteins.
RESULTS: Spontaneous contractions of non-pregnant rat uterine stripswere significantly suppressed in response to 50-100 lM PP1 and Damncompared to strips treated with vehicle alone (p < 0.05). BpV(phen) enhancedPLC-c1 activation leading to increased phasic contractions in control strips; PP1and Damn inhibited this response. In contrast, Pice had no effect onspontaneous contractions and did not inhibit bpV(phen) stimulation.Western blots confirmed significant PP1 and Damn inhibition of phos-photyrosine-PLC-c1 (p < 0.05); whereas Pice had no effect. Additional Westernblots confirmed robust expression of Src and Lck kinase proteins in uteruscompared to other rat tissues.
CONCLUSION: Spontaneous uterine contractions are associated with PLC-c1 activation in response to tyrosine-phosphorylation. These studies haveconfirmed that at least two Src kinases (Src and Lck) are expressed in rat uterinetissue, modulate phosphotyrosine-PLC-c1 levels, and regulate contractileactivity. (Funded by NIH HD-28506).
494 THE REDUCTION OF ADIPONECTIN LEVELS DURING THE IMMEDIATEPOSTPARTUM CANNOT BE EXPLAINED BY PLACENTAL OR FETALPRODUCTION SHALI MAZAKI TOVI1, HANNAH KANETY2, EYALSCHIFF1, RINA HEMI2, CLARA PARIENTE2, ANN SHOHAM1, EYALSIVAN1, 1Sheba medical center, ObGyn, Tel Hashomer Israel 2Institute ofEndocrinology, Sheba medical center, Tel Hashomer Israel
OBJECTIVE: Adiponectin is an adipocyte-derived plasma protein withinsulin-sensitizing and anti-atherosclerotic properties found abundantly in cordblood. The aim of this study was to examine maternal adiponectin levels and toestablish a possible connection between these levels and placental or fetalproduction.
STUDY DESIGN: The study was comprised of three studies. Study 1 - bloodwas obtained from 23 women one day prior to and four days after delivery. Study2�seventeen blood samples were obtained frommothers and their newborns atdelivery. Blood samples were tested for adiponectin and leptin. Study 3�humanplacental tissues were tested for adiponectin mRNA by RT-PCR.
RESULTS: Study 1- Maternal adiponectin levels before and after deliverywere 22.2 ± 7.0 and 17.5 ± 6.8 g/ml respectively (P < 0.03). A similar declinewas measured for leptin, known to be expressed in the placenta, (27.9 ± 13.4and 13.2 ± 8.0 ng/ml, respectively, P < 0.001). No correlation was foundbetween adiponectin levels and maternal characteristics. Study 2�Cord bloodadiponectin levels were significantly higher when compared with maternaladiponectin levels (61.1 ± 19.0 vs. 22.7 ± 6.8 g/ml, P < 0.001). No correlationwas found between cord blood and maternal adiponectin (r = 0.3, P < 0.2).Study 3�there was no expression of adiponectin mRNA in human placentaltissues.
CONCLUSION: Our findings demonstrate that adiponectin levels arerelatively high in the serum of women in the third trimester and decline afterdelivery. These findings can not be explained either by placental production orby transport from fetal compartments.
