Martha Lappas

Publications of Martha Lappas

• Diagnostic accuracy of maternal serum macrophage inhibitory cytokine-1 and pregnancy-associated plasma protein-a at 6-10 weeks of gestation to predict miscarriage.

  Authors: Stephen Tong, Gene-Lyn Ngian, Joseph L Onwude, Michael Permezel, Burcu Saglam, Sarah Hay, Justin C Konje, Tim H Marczylo, Gabrielle Fleming, Susan P Walker, Martha Lappas

  Obstetrics and gynecology. 05/2012; 119(5):1000-8.

  : To determine whether serum macrophage inhibitory cytokine-1, pregnancy-associated plasma protein-A (PAPP-A), anandamide, or β-human chorionic gonadotropin (hCG) measured in an asymptomatic: To determine whether serum macrophage inhibitory cytokine-1, pregnancy-associated plasma protein-A (PAPP-A), anandamide, or β-human chorionic gonadotropin (hCG) measured in an asymptomatic population in the middle of the first trimester with a viable fetus predicts subsequent miscarriage. : We undertook a prospective cohort study at Mercy Hospital for Women between 2004 and 2008. Participants (N=782) were recruited from prenatal clinics, where samples were taken from asymptomatic women at 6 0/7 to 10 6/7 weeks of gestation. We collected samples from only those women for whom we were able to obtain ultrasound evidence of a singleton with fetal cardiac activity. Serum macrophage inhibitory cytokine-1, PAPP-A, anandamide, and β-hCG concentrations were assayed. : Twenty-one (2.7%) miscarried and 761 did not. Among those who miscarried, macrophage inhibitory cytokine-1 and PAPP-A were significantly decreased at 63% (multiples of the median (MOM) 0.63, 25-75 percentiles 0.33-0.88) and 23% (MOM 0.23, 25-75 percentiles 0.12-0.48) of levels seen among those with ongoing pregnancies (P<.001 for both comparisons). In contrast, neither serum β-hCG (MOM 0.99, 25-75 percentiles 0.46-1.86) nor anandamide (MOM 1.07, 25-75 percentiles 0.87-1.19) was elevated or decreased among those who miscarried compared with those with ongoing pregnancies. At a fixed 10% false-positive rate (90% specificity), a test combining macrophage inhibitory cytokine-1 and PAPP-A yielded 63% sensitivity and a 6.6 positive likelihood ratio in predicting miscarriage. : Low serum levels of macrophage inhibitory cytokine-1 and PAPP-A measured from asymptomatic women at 6-10 weeks of gestation with viable pregnancies can predict subsequent miscarriage. These analytes are likely to have an important biological role in early pregnancy and are likely to be useful clinical biomarkers for miscarriage and other early pregnancy complications. : II.

• Increased chemerin concentrations in fetuses of obese mothers and correlation with maternal insulin sensitivity.

  Authors: Gillian Barker, Ratana Lim, Gregory E Rice, Martha Lappas

  The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians. 04/2012;

  Abstract Objective: The aim of this study was to determine the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating concentrations of chemerin in cord andAbstract Objective: The aim of this study was to determine the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating concentrations of chemerin in cord and maternal plasma, and (ii) gene expression and release of chemerin from human placenta and adipose tissue. Design: Chemerin concentrations were measured in maternal and cord plasma from 62 normal glucose tolerant women (NGT) and 69 women with GDM at the time of term elective Caesarean section. Placenta and adipose tissue expression and release of chemerin was measured from 22 NGT and 22 GDM women. Results: There was no effect of maternal obesity or GDM on maternal chemerin concentrations. Chemerin concentrations were significantly higher in cord plasma from women with maternal obesity. Cord chemerin concentrations in NGT women negatively correlated with the concentrations of maternal insulin sensitivity. There was no effect of GDM on maternal and cord chemerin concentrations, and on the release of chemerin from placenta and adipose tissue. Conclusions: At the time of term Caesarean section, pre-existing maternal obesity, and its associated insulin resistance, is associated with higher cord plasma chemerin concentrations.

• Complement C5a Regulates Pro-Labor Mediators in Human Placenta.

  Authors: Martha Lappas, Trent Woodruff, Stephen Taylor, Michael Permezel

  Biology of reproduction. 03/2012;

  Human preterm and term parturition is associated with inflammatory cascades in the uteroplacental unit. Activation of the complement cascade releases potent pro-inflammatory mediators, including theHuman preterm and term parturition is associated with inflammatory cascades in the uteroplacental unit. Activation of the complement cascade releases potent pro-inflammatory mediators, including the anaphylatoxin C5a, which exerts its biological effects through its receptors, C5AR (also known as CD88) and C5L2, official symbol GPR77. To date, there are few data available on the role of C5a and CD88 in human pregnancy, so the aim of this study was to determine the effect of C5a and CD88 on some key inflammatory pathways involved in human parturition. Placental tissue samples were obtained from normal pregnancies at the time of Caesarean section. Human placental and fetal membranes were incubated in the absence (basal control) or presence of 0.5 μg/ml (~60 nM) human recombinant C5a for 24 h. Concentrations of pro-inflammatory cytokines, prostaglandins, and 8-isoprostane (a marker of oxidative stress) were quantified by ELISA, and secretory matrix metalloproteinases (MMPs) activity by zymography. NFKB DNA binding activity and NFKBIA (IkappaB-alpha) (inhibitor of NFKB) protein degradation were analyzed by ELISA and Western blotting, respectively. In the presence of C5a, pro-inflammatory cytokines (IL6 and IL8), cyclooxygenase (COX)-2, (official symbol PTGS2) expression and subsequent prostaglandin (PGE2 and PGF(2alpha)), MMP9 enzyme production, and NFKB DNA activation were all significantly increased. The C5a-induced pro-labour responses were significantly reduced by treatment with the selective CD88 antagonist PMX53, and the NFKB inhibitor BAY 11-7082. We conclude that C5a upregulates pro-labour mediators in human gestational tissues via CD88-mediated NFKB activation.

• Hypoxanthine-xanthine oxidase (HX/XO) downregulates GLUT-1 transcription via SIRT1 resulting in decreased glucose uptake in human placenta.

  Authors: Martha Lappas, Sofianos Andrikopoulos, Michael Permezel

  The Journal of endocrinology. 01/2012;

  Appropriate fetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placentaAppropriate fetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placenta glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters in human placenta. Human placenta was incubated with in the absence or presence of 0.5 mM HX + 15 mU/ml XO (HX/XO) for 24 h. Gene and protein expression of the glucose transporters (GLUTs) were analysed by quantitative RT-PCR (qRT-PCR) and Western blotting, respectively. Glucose uptake was measured using radiolabelled (14C) glucose. HX/XO significantly decreased GLUT-1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetyl-cysteine (NAC), catalase and superoxide dismutase (SOD), or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT-1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT-1 expression by a SIRT1 dependent mechanism.

• The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry.

  Authors: Karen Oliva, Gillian Barker, Clyde Riley, Mark J Bailey, Michael Permezel, Gregory E Rice, Martha Lappas

  Journal of molecular endocrinology. 01/2012; 48(2):139-49.

  Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We usedOur aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4-5 and 5-6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

• Visfatin regulates the terminal processes of human labour and delivery via activation of the nuclear factor-κB pathway.

  Authors: Martha Lappas

  Molecular and cellular endocrinology. 08/2011; 348(1):128-34.

  The inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and preterm. Visfatin levels increase during normal human pregnancy and in infection associatedThe inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and preterm. Visfatin levels increase during normal human pregnancy and in infection associated preterm labour. The effects of visfatin in the processes of human labour and delivery, however, are not known. The aim of this study was to determine the effect of visfatin on the expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 50 ng/ml visfatin for 24 h (n=6). Inflammatory gene expression was analysed by quantitative RT-PCR (qRT-PCR), the medium was collected and cytokine, prostaglandin and 8-isoprostane (marker of oxidative stress) release was quantified by ELISA, and secretory protease activity by zymography. Visfatin significantly increased IL-6 and IL-8 gene expression and secretion, COX-2 expression and resultant prostaglandin (PG) E(2) and PGF(2α) release, and 8-isoprostane release. There was, however, no effect of visfatin on pro MMP-9 enzyme activity. These actions of visfatin were elicited via the nuclear factor-κB (NF-κB) pathway as visfatin induced the degradation of IκB-α (inhibitor of NF-κB) whilst increasing NF-κB p65 DNA binding activity. Further to this, visfatin-induced pro-labour responses were abrogated by treatment with the NF-κB inhibitor BAY 11-7082. Collectively, these data indicate that visfatin activates pro-inflammatory cytokine release and phospholipid metabolism in human placenta via activation of the NF-κB pathway. Thus, visfatin represents a novel cytokine linked to the events of human labour initiation.

• Severe early-onset preeclampsia is not associated with a change in placental catechol O-methyltransferase (COMT) expression.

  Authors: Kirsten Palmer, Burcu Saglam, Clare Whitehead, Owen Stock, Martha Lappas, Stephen Tong

  The American journal of pathology. 06/2011; 178(6):2484-8.

  The genetic deletion of catechol O-methyltransferase (COMT) in mice produces a preeclampsia-like phenotype, with mice exhibiting hypertension, proteinuria, and histological changes, consistent withThe genetic deletion of catechol O-methyltransferase (COMT) in mice produces a preeclampsia-like phenotype, with mice exhibiting hypertension, proteinuria, and histological changes, consistent with human pathological features. 2-Methoxyoestradiol, a metabolite of COMT, increases human trophoblast invasiveness in vitro under hypoxic conditions, providing further support that decreased COMT expression may have a role in preeclampsia. However, evidence confirming decreased COMT expression in human disease has been limited to small studies of placentas obtained from cases of term preeclampsia. We examined COMT expression in placentas obtained from healthy term pregnancies (n = 14), preterm normotensive pregnancies (n = 8), and pregnancies complicated by severe preterm preeclampsia (delivery at < 34 weeks' gestation; n = 22). Among our preeclamptic cohort were 10 pregnancies further complicated by HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets); and one pregnancy complicated by an eclamptic seizure. COMT expression was analyzed by RT-PCR, Western analysis, and IHC. COMT was mainly expressed in the syncytiotrophoblast. We did not find a significant difference in placental COMT expression in severe preeclampsia compared with either term or preterm normotensive cohorts. Our results suggest that severe preeclampsia may not be associated with a decrease in placental COMT expression.

• The anti-inflammatory and antioxidative effects of nicotinamide, a vitamin B(3) derivative, are elicited by FoxO3 in human gestational tissues: implications for preterm birth.

  Authors: Martha Lappas, Michael Permezel

  The Journal of nutritional biochemistry. 03/2011; 22(12):1195-201.

  The inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and at preterm. Nicotinamide, a vitamin B(3) derivative, exerts anti-inflammatory and antioxidativeThe inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and at preterm. Nicotinamide, a vitamin B(3) derivative, exerts anti-inflammatory and antioxidative properties in several cell types by interaction with various intracellular signalling proteins via modulating the activity of various transcription factors, including activation of the O subfamily of Forkhead/winged helix transcription factors (FoxO) and inhibition of nuclear factor-κB (NF-κB). The aim of this study was to determine the effect of nicotinamide on the expression of pro-labour and mediators in human placenta. The effects of nicotinamide were evaluated, over 24 h, by treating placenta with 0, 25 and 50 mM nicotinamide in the absence or presence of 10 μg/ml lipopolysaccharide (LPS). Nicotinamide treatment resulted in a significant reduction of basal and/or LPS-stimulated release and gene expression of the pro-inflammatory cytokines TNF-α, IL-6 and the chemokine IL-8, and the release of the prostaglandins PGE(2) and PGF(2)α and cyclooxygenase (COX)-2 mRNA expression. Additionally, nicotinamide treatment of human placenta resulted in attenuation of basal and LPS-induced oxidative stress, reducing 8-isoprostane release and increasing gene expression of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). There was no effect of nicotinamide on NF-κB activation. The anti-inflammatory and antioxidant actions of nicotinamide were abolished by knockdown of FoxO3 using siRNA. In conclusion, nicotinamide exerts anti-inflammatory and antioxidative effects in human placenta, in part, via activation of FoxO3. Further studies should be undertaken to define a possible implication of vitamin B(3) derivatives in the management of preterm labour and delivery.

• 2D-PAGE of ovarian cancer: analysis of soluble and insoluble fractions using medium-range immobilized pH gradients.

  Authors: Ratana Lim, Martha Lappas, Nuzhat Ahmed, Michael Permezel, Michael A Quinn, Gregory E Rice

  Biochemical and biophysical research communications. 02/2011; 406(3):408-13.

  Ovarian cancer remains a leading cause of cancer death. A comparative proteomic study was performed on normal ovarian tissue (n=5) and grade 3 ovarian tumours (n=5) to search for differentiallyOvarian cancer remains a leading cause of cancer death. A comparative proteomic study was performed on normal ovarian tissue (n=5) and grade 3 ovarian tumours (n=5) to search for differentially expressed proteins. In contrast to other studies, here we extracted proteins in soluble and insoluble protein fractions using commercial kits and also utilised three medium-range IPG strips that encompassed the broad pH range of 3-10 (pH 3-6, 5-8 and 7-10). Protein fractions were compared by 2D-PAGE and MALDI-TOF/TOF-MS. Nineteen differentially expressed proteins were identified: HSP60, Grp78, CK19, EF-Tu, MRLC2, prohibitin, Stress-70 protein, TPI and tubulin α6 were up-regulated in grade 3 tumours whereas annexin A2 and A5, antithrombin-III precursor, CBR1, GSTM2, GSTM3, RALDH1, serum albumin precursor, transthyretin precursor and vimentin were found to be down-regulated in grade 3 ovarian tumours. These proteins are associated with cytoskeleton rearrangement, cell metabolism, tumour suppression function, apoptosis and induction of host response.

• SIRT1 is a novel regulator of key pathways of human labor.

  Authors: Martha Lappas, Amberlee Mitton, Ratana Lim, Gillian Barker, Clyde Riley, Michael Permezel

  Biology of reproduction. 01/2011; 84(1):167-78.

  Human sirtuin (SIRT) 1 and SIRT2, which possess nicotinamide adenosine dinucleotide (NAD(+))-dependent deacetylase activity, exhibit anti-inflammatory actions. However, there are no data available onHuman sirtuin (SIRT) 1 and SIRT2, which possess nicotinamide adenosine dinucleotide (NAD(+))-dependent deacetylase activity, exhibit anti-inflammatory actions. However, there are no data available on SIRT1 and SIRT2 expression and regulation in human intrauterine tissues. Thus, the aim of this study was to characterize the localization and expression of SIRT1 and SIRT2 in 1) placenta and fetal membranes before and after term spontaneous labor onset, 2) prelabor fetal membranes from the supracervical site (SCS) and a distal site (DS), and 3) in response to proinflammatory stimuli. Further, the effect of SIRT activation using resveratrol and SRT1720 on prolabor mediators was also assessed. SIRT1 and SIRT2 were localized in the syncytiotrophoblast layer and the cytotrophoblasts of the placenta, amnion epithelium, trophoblast layer of the chorion, and decidual cells. Additionally, SIRT2 was found within the endothelial walls of placental vessels. SIRT2, but not SIRT1, staining was significantly lower in amnion and chorion obtained from the SCS compared to a DS. On the other hand, SIRT1, but not SIRT2, gene and/or protein expression was significantly lower in placenta, amnion, and chorion obtained after labor compared to prelabor. SIRT1 expression, but not SIRT2, was down-regulated by lipopolysaccharide (LPS) and proinflammatory cytokines TNF and IL1B. The SIRT1 activators resveratrol and SRT1720 significantly decreased LPS-induced TNF, IL6, and IL8 gene expression and release and PTGS2 mRNA expression and resultant prostaglandin (PG) E(2) and PGF(2α) release from human gestational tissues. In conclusion, SIRT1 possesses anti-inflammatory actions and thus may play a role in regulating pregnancy and parturition.

• Increased expression of alpha-enolase in cervico-vaginal fluid during labour.

  Authors: Nicole L Dellios, Martha Lappas, I Ross Young, Hannah K Palliser, Jonathan J Hirst, Karen Oliva, Mustafa Ayhan, Gregory E Rice

  European journal of obstetrics, gynecology, and reproductive biology. 11/2010; 153(1):16-22.

  The aim of this study was (i) to characterise differentially expressed proteins in cervico-vaginal fluid (CVF) at the time of preterm labour onset and (ii) to confirm these studies in human CVFThe aim of this study was (i) to characterise differentially expressed proteins in cervico-vaginal fluid (CVF) at the time of preterm labour onset and (ii) to confirm these studies in human CVF samples taken from women before and during spontaneous labour. Preterm labour was induced in sheep (n = 5) via fetal dexamethasone infusion (1 mg/24 h). CVF samples were taken prior to dexamethasone infusion (0 h), 28 h after the start of dexamethasone infusion, and immediately prior to delivery. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify differentially expressed proteins. For the human studies, paired CVF samples were taken 5-9 days before labour and during spontaneous labour onset (n = 7). There was a 4.2-fold increase in α-enolase protein expression in sheep CVF during labour. Likewise, α-enolase protein expression was significantly increased during spontaneous human labour at term. Alpha-enolase is known to be bound to neutrophils and interact in the immune response, and thus may play a role in inflammation associated with human labour.

• Rational design of immunostimulatory siRNAs.

  Authors: Michael P Gantier, Stephen Tong, Mark A Behlke, Aaron T Irving, Martha Lappas, Ulrika W Nilsson, Eicke Latz, Nigel A J McMillan, Bryan R G Williams

  Molecular therapy : the journal of the American Society of Gene Therapy. 04/2010; 18(4):785-95.

  Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on theirShort-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

• Diabetes and obesity during pregnancy alter insulin signalling and glucose transporter expression in maternal skeletal muscle and subcutaneous adipose tissue.

  Authors: Michelle Colomiere, Michael Permezel, Martha Lappas

  Journal of molecular endocrinology. 12/2009;

  Severe insulin resistance is a defining attribute of gestational diabetes mellitus (GDM). It is postulated that alterations in the insulin signalling pathway and subsequent glucose disposal is theSevere insulin resistance is a defining attribute of gestational diabetes mellitus (GDM). It is postulated that alterations in the insulin signalling pathway and subsequent glucose disposal is the underlying cause of insulin resistance in patients with GDM. The purpose of this study was to profile the insulin signalling pathway and intermediates in insulin-sensitive tissues. Subcutaneous adipose tissue and skeletal muscle were collected from normal glucose tolerant (NGT) and insulin-controlled GDM in both non-obese and obese cohorts (n=6-8 per sub-group). Expression studies of the insulin signalling pathway were performed using Western blotting and quantitative RT-PCR (qRT-PCR). This study demonstrated altered mRNA expression of insulin receptor substrate (IRS)-1, IRS-2, glucose transporter (GLUT)-1, GLUT-4 and glycogen synthase kinase (GSK)-3 isoforms genes in adipose tissue in GDM women in comparison to NGT pregnant controls. In skeletal muscle, insulin-controlled GDM was associated with decreased IRS-1, PI3-K p85alpha GLUT-1 and -4, GSK-3 isoforms and PDK-1. Both adipose tissue and skeletal muscle from women with GDM displayed decreased IRS-1 and GLUT-4 and increased PI3-K p85alpha protein expression. Both skeletal muscle and adipose tissue from obese women demonstrated lower GLUT-1 and GLUT-4 mRNA expression and diminished GLUT-4 protein expression in skeletal muscle only. Collectively, our results suggest that diabetes and obesity during pregnancy cause defects in insulin signalling transduction in adipose tissue and skeletal muscle and maybe the underlying cause of GDM.

• In response to oxidative stress, the expression of inflammatory cytokines and antioxidant enzymes are impaired in placenta, but not adipose tissue, of women with gestational diabetes.

  Authors: Martha Lappas, Amberlee Mitton, Michael Permezel

  The Journal of endocrinology. 10/2009;

  Objective: In response to oxidative stress, GDM placenta release less 8-isoprostane and TNF-alpha. The effect of oxidative stress on other cytokines and on antioxidant gene expression are unknown.Objective: In response to oxidative stress, GDM placenta release less 8-isoprostane and TNF-alpha. The effect of oxidative stress on other cytokines and on antioxidant gene expression are unknown. The aim of this study was to further explore the antioxidant status and effect of oxidative stress in GDM tissue. Design and methods: Human placenta, and subcutaneous and omental adipose tissue from women with and without GDM were exposed to hypoxanthine (HX)/xanthine oxidase (XO). Cytokine release was analysed by ELISA and cytokine and antioxidant gene expression by RT-PCR. Results: Catalase and glutathione reductase (GR) mRNA expression was higher in GDM (n=18) compared to normal (n=23) placenta. There was no difference in glutathione peroxidase (GPx) and superoxide dismutase (SOD) mRNA expression. Antioxidant gene expression was unaltered between normal (n=18) and GDM (n=10) adipose tissue. HX/XO treatment significantly stimulated cytokine release (13/16 cytokines) and cytokine mRNA expression, and decreased antioxidant gene expression (catalase and GR) in human placenta from normal pregnant women. In GDM placenta, HX/XO only significantly increased the release of 3/16 cytokines, while there was no effect on antioxidant gene expression. In normal and GDM adipose tissues, HX/XO increased pro-inflammatory cytokine and 8-isoprostane release, while there was no change in antioxidant gene expression. Conclusions: GDM placenta is characterised by increased antioxidant gene expression, and is less responsive to exogenous oxidative stress than tissues obtained from normal pregnant women. This may represent a protective or adaptive mechanism to prevent damage from further oxidative insult in utero as indicated by increased tissue antioxidant expression.

• Increased Expression of ac-FoxO1 Protein in Prelabor Fetal Membranes Overlying the Cervix: Possible Role in Human Fetal Membrane Rupture.

  Authors: Martha Lappas, Clyde Riley, Greg Rice, Michael Permezel

  Reproductive sciences (Thousand Oaks, Calif.). 04/2009;

  Forkhead box O proteins have critical roles in a number of cellular processes, including apoptosis. Acetylation and phosphorylation of forkhead box O proteins are posttranslational modifications thatForkhead box O proteins have critical roles in a number of cellular processes, including apoptosis. Acetylation and phosphorylation of forkhead box O proteins are posttranslational modifications that attenuate their transcriptional activity. As supracervical fetal membranes are characterized by increased cell death, the aim of this study was to compare the expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1 at supracervical and distal site fetal membrane. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 7) was used to analyze the protein expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1. There was no difference in forkhead box O1 and Ser-256 phosphorylated forkhead box O1 protein expression between the 2 sites. However, when compared with distal site, the intensity and extent of staining of acetylated-forkhead box O1 were greater in amnion and chorion obtained from the supracervical site. In summary, supracervical fetal membranes are characterized by increased acetylated-forkhead box O1 protein expression. Although the precise role and contribution of acetylated-forkhead box O1 in the process of human fetal membrane rupture are unknown, it has been implicated in apoptosis and/or cell cycle regulation.

• Preterm and infection-driven preterm labour: The role of peroxisome proliferator-activated receptors and retinoid X receptor.

  Authors: Sarah Holdsworth-Carson, Michael Permezel, Greg Rice, Martha Lappas

  Reproduction (Cambridge, England). 04/2009;

  Approximately 8% of births are complicated by preterm delivery. To improve neonatal outcomes, a greater understanding of preterm parturition is required. Peroxisome proliferator-activated receptorsApproximately 8% of births are complicated by preterm delivery. To improve neonatal outcomes, a greater understanding of preterm parturition is required. Peroxisome proliferator-activated receptors (PPARs) have been implicated in the regulation of term labour where they exhibit anti-inflammatory properties. We hypothesise that dysregulation of PPAR expression and activity may be associated with preterm labour and infection-associated preterm labour. The aim of this study was to compare PPARs and retinoid X receptor (RXRa) in gestational tissues from term and preterm deliveries, and from infection-associated preterm deliveries. Quantitative RT-PCR (qRT-PCR), Western blotting and activity ELISA were used to study expression and DNA binding profiles. Compared to term, preterm parturition was associated with increased expression of PPARd (mRNA and protein), PPARg (protein) and RXRa (protein) in the placenta and PPARd (mRNA and protein) and RXRa (mRNA) in the choriodecidua. There was however no change in preterm PPAR DNA binding activity compared to term. Preterm chorioamnionitis demonstrated protein degradation in the choriodecidua and was associated with a decline in mRNA of PPARa and RXRa compared to uninfected preterm cases. PPAR DNA binding activity increased in placenta (PPARd and PPARg) and decreased in amnion (PPARa and PPARg) in association with preterm chorioamnionitis. In conclusion, idiopathic preterm deliveries were associated with an increase in PPAR:RXR expression and preterm chorioamnionitis was associated with a decrease in PPAR:RXR expression and tissue-specific alterations in transcriptional activity. The data are consistent with the hypothesis that PPARs may play a role in preterm labour and infection-complicated preterm deliveries.

• Defective insulin signalling in placenta from pregnancies complicated by gestational diabetes mellitus.

  Authors: Michelle Colomiere, Michael Permezel, Clyde Riley, Gernot Desoye, Martha Lappas

  European journal of endocrinology / European Federation of Endocrine Societies. 02/2009;

  Objective: Studies in adipose tissue and skeletal muscle suggest that impaired insulin action is due to defects in the insulin signalling pathway and may play a role in the pathophysiology of insulinObjective: Studies in adipose tissue and skeletal muscle suggest that impaired insulin action is due to defects in the insulin signalling pathway and may play a role in the pathophysiology of insulin resistance associated with gestational diabetes mellitus (GDM) and obesity. The present study tested the hypothesis that endogenous expression levels in the human term placenta of insulin signalling components are altered in placental tissue from GDM women in comparison to normal controls and maternal obesity. Design and Methods: Placental tissue was collected from normal, diet-controlled GDM and insulin-controlled GDM in both non-obese and obese women (n=6-7 per group). Western blotting and quantitative RT-PCR was performed to determine the level of expression in the insulin signalling pathway. Results: There was a significant increase in insulin receptor substrate (IRS)-1 protein expression with a concurrent decrease in IRS-2 protein expression in non-obese women with insulin-controlled GDM compared to diet- controlled GDM and normal controls. A decrease in both protein and mRNA expression of Phosphatidyl-inositol-3-kianse (PI3-K) p85alpha and glucose transporter (GLUT)-4 was observed in non-obese and obese women with insulin-controlled GDM compared to normal controls. When comparing non-obese to obese patients, significant decreases in mRNA expression of insulin receptor (IR)-beta, PI3K p85alpha and GLUT-4 was found in obese patients. Conclusion: Our results suggest that post-receptor defects are present in the insulin signalling pathway in placenta of women with pregnancies complicated by diabetes and obesity. In addition, expression studies demonstrate post-receptor alterations in insulin signalling maybe under selective maternal regulation and not fetal regulation.

• Screening for biomarkers predictive of gestational diabetes mellitus.

  Authors: Harry Georgiou, Martha Lappas, George Georgiou, Adinda Marita, Valerie Bryant, Richard Hiscock, Michael Permezel, Zeinab Khalil, Gregory Rice

  Acta diabetologica. 06/2008;

  Screening for glucose intolerance during pregnancy provides an opportunity to offer management to those women diagnosed with gestational diabetes mellitus. However, there is a need to diagnoseScreening for glucose intolerance during pregnancy provides an opportunity to offer management to those women diagnosed with gestational diabetes mellitus. However, there is a need to diagnose gestational diabetes early to minimize exposure of the developing fetus to suboptimal conditions and prevent perinatal complications and their sequelae. The purpose of this study was to identify potential biomarkers for impending gestational diabetes that appear in the plasma before impaired glucose tolerance. Pregnant women were prospectively recruited to the study and blood was collected at the first antenatal visit and at the time of routine oral glucose tolerance test. Women diagnosed with gestational diabetes were matched with an equal number of normal pregnant (control) women. Biomarkers under investigation included endocrine and metabolic hormones, cytokines and chemokines, and surrogate markers of oxidative stress. Compared to controls, women with gestational diabetes exhibited elevated plasma insulin and reduced plasma adiponectin concentrations at 28 weeks gestation. Significant differences in insulin and adiponectin concentrations were also observed in plasma at 11 weeks gestation. Bivariate logistic regression analysis showed that both insulin and adiponectin are associated with subsequent development of gestational diabetes. Plasma insulin and adiponectin concentrations, when measured at 11 weeks, may be predictive of impending gestational diabetes. Further studies are warranted to determine the reliability of these biomarkers.

• Antiinflammatory effects of the cyclopentenone isoprostane 15-A(2)-IsoP in human gestational tissues.

  Authors: Martha Lappas, Michael Permezel, Sarah J Holdsworth, Guiseppe Zanoni, Alessio Porta, Greg E Rice

  Free radical biology & medicine. 07/2007; 42(12):1791-6.

  Proinflammatory prostaglandins and cytokines are involved in the initiation of human labor and delivery. Although cyclopentenone prostaglandins regulate the formation of these prolabor mediators viaProinflammatory prostaglandins and cytokines are involved in the initiation of human labor and delivery. Although cyclopentenone prostaglandins regulate the formation of these prolabor mediators via nuclear factor-kappaB (NF-kappaB) and/or peroxisome proliferator-activated receptor-gamma, recent evidence suggests that they do not exist in vivo. Cyclopentenone isoprostanes (IsoPs), which are highly reactive structural isomers of bioactive cyclopentenone prostaglandins, do exist physiologically and have been shown to inhibit the inflammatory response in macrophages. Therefore the aim of this study was to determine the effect of the synthetic cyclopentenone IosP 15-A(2)-IsoP on the expression of prolabor mediators in human gestational tissues. Human placenta and gestational membranes (n=5) were incubated in the absence or presence of 12.5, 25, and 50 microM 15-A(2)-IsoP with 10 microg/ml lipopolysaccharide (LPS). Treatment of placenta and fetal membranes with 15-A(2)-IsoP caused a dose-dependent decrease in LPS-stimulated release of the cytokines IL-1beta, IL-6, IL-8, and TNF-alpha and the prostaglandins PGE(2) and PGF(2)alpha. NF-kappaB p65 DNA binding activity was significantly inhibited by treatment with 50 microM 15-A(2)-IsoP. Collectively, these data suggest that 15-A(2)-IsoP exhibits antiinflammatory properties via antagonism of NF-kappaB activity. Cyclopentenone IsoPs may serve as negative feedback regulators of the inflammatory response in human gestational tissues.

• Effect of high oxygen on placental function in short-term explant cultures.

  Authors: Nicole G Reti, Martha Lappas, Berthold Huppertz, Clyde Riley, Mary E Wlodek, Phil Henschke, Michael Permezel, Gregory E Rice

  Cell and tissue research. 07/2007; 328(3):607-16.

  Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygenEx situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-alpha) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-alpha and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.