Antioxidant gene expression in preeclamptic placentae: a preliminary investigation.

Oxidative stress has been implicated in the pathogenesis of preeclampsia. This study measured the relative mRNA expression of antioxidant proteins glutathione peroxidase 1 and 4, glutathione reductase, thioredoxin 1 and 2, thioredoxin reductase 1, thioredoxin peroxidase 3 and superoxide dismutase 1 and 2 in preeclamptic and non-preeclamptic placentae. Quantitative real-time PCR was conducted on placental mRNA isolated from preeclamptic and control patients. Cycle threshold numbers and fold differences were calculated as a measure of linear product amplification and used for comparison. The mRNA expression of glutathione reductase was significantly reduced (fold difference 0.41, p<0.05) in preeclamptic placenta when compared to controls while the expression of thioredoxin peroxidase 3 was significantly increased (fold difference 3.25, p<0.001) in the preeclamptic placentae. No significant difference in expression was observed for glutathione peroxidase 1 and 4, thioredoxin 1 and 2, thioredoxin reductase 1 and superoxide dismutase 1 and 2. These results suggest that it is the abnormal oxidative insult associated with preeclampsia not mRNA expression of antioxidant proteins that may be responsible for reduced antioxidant enzyme activity in preeclamptic placentae.

Chronic hypoxia in vivo reduces placental oxidative stress.

Decreased placental oxygenation and increased oxidative stress are implicated in the development of preeclampsia. Oxidative stress arises from imbalance between pro-versus anti-oxidants and can lead to biological oxidation and apoptosis. Because pregnant women living at high altitude (3100 m, HA) have lowered arterial PO2 and an increased incidence of preeclampsia, we hypothesized that HA placentas would have decreased anti-oxidant enzyme activity, increased oxidative stress (lipid peroxidation, protein oxidation and nitration) and greater trophoblast apoptosis than low-altitude (LA) placentas. We measured enzymatic activities, lipid and protein oxidation and co-factor concentrations by spectrophotometric techniques and ELISA in 12 LA and 18 HA placentas. Immunohistochemistry (IHC) was used to evaluate nitrated proteins and specific markers of apoptosis (activated caspase 3 and M30). Superoxide dismutase activity was marginally lower (p=0.05), while glutathione peroxidase activity (p<0.05), thioredoxin concentrations (p<0.005) and thioredoxin reductase activity p<0.01 were all reduced in HA placentas. Decreased anti-oxidant activity was not associated with increased oxidative stress: lipid peroxide content and protein carbonyl formation were lower at HA (p<0.01). We found greater nitrotyrosine residues in the syncytiotrophoblast at 3100 m (p<0.05), but apoptosis did not differ between altitudes. Our data suggest that hypoxia does not increase placental oxidative stress in vivo. Nitrative stress may be a consequence of hypoxia but does not appear to contribute to increased apoptosis. Lowered placental concentrations of anti-oxidants may contribute to the susceptibility of women living at HA to the development of preeclampsia, but are unlikely to be etiological.

The association between third trimester multivitamin/mineral supplements and gestational length in uncomplicated pregnancies.

BACKGROUND: Widespread use of maternal micronutrient supplements have been correlated to gestational length and outcome in women predisposed to pre-eclampsia and preterm birth. However, research is yet to be conducted examining the influence of micronutrient supplements on outcomes at term in uncomplicated pregnancies. AIM: To analyse the relationship between third trimester micronutrient supplementation and gestation length at birth, demographics and maternal birthing outcomes in well women at term in a South East Queensland representative population. METHODS: This research retrospectively analysed existing data pertaining to 427 uncomplicated, pregnancies birthing at the Gold Coast and Logan Hospitals using information gathered through the Environments for Healthy Living Study and Queensland perinatal data collection. Data were analysed using SPSS v20 by Chi square, ANOVA and regression analysis. FINDINGS: Women in the third trimester taking individual zinc, folic acid or iron supplements in combination with a multivitamin were twice as likely to birth beyond 41 completed weeks (AOR 2.054, 95% CI 1.310-7.383, p=0.038) then those who did not take any supplement when controlled for established confounders. Non supplement users were found to experience a lower rate of post dates labour and requirements for induction (AOR 0.483, 95% CI 0.278-0.840, p=0.01). CONCLUSION: Length of gestation demonstrates significant associations with micronutrient supplementation practices. Well women consuming third trimester individual micronutrient supplements in addition to multivitamins experienced a longer gestation at term compared to women taking no micronutrients, increasing their risk for postdates induction of labour.

IFPA meeting 2015 workshop report I: placental mitochondrial function, transport systems and epigenetics.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.

Increased anti-oxidant enzyme activity and biological oxidation in placentae of pregnancies complicated by maternal asthma.

Our previous work has demonstrated that alterations in placental function are associated with changes in fetal development in pregnancies complicated by asthma. The pathophysiology of asthma in adults and children and intrauterine growth restriction during pregnancy are associated with oxidative stress. Based on this information, we examined whether placental anti-oxidant pathways and markers of biological oxidation were altered in pregnancies complicated by asthma. Anti-oxidant enzyme activities of superoxide dismutase, glutathione peroxidase and thioredoxin reductase, thioredoxin concentrations, lipid and protein oxidation levels were measured in placentae of pregnancies complicated by asthma and compared to uncomplicated, non-asthmatic pregnancies. Placental tissue homogenates of pregnancies complicated by asthma demonstrated significantly increased levels of lipid peroxidation (25.7+/-1.8 micromol/mg protein versus 12.1+/-1.6 micromol/mg protein, P=0.008) and protein carbonyl concentrations (414.6+/-51.4 units/mg protein versus 222.3+/-32.6 units/mg protein, P=0.0032) when compared to non-asthmatic controls. The activities of the anti-oxidant proteins superoxide dismutase (2.17+/-0.09 units/mg protein versus 1.67+/-0.09 units/mg protein, P=0.014) and thioredoxin reductase (54.0+/-6.9 units/mg protein versus 28.7+/-6.0 units/mg protein, P=0.009) were significantly increased in the presence of maternal asthma. Placental thioredoxin levels (102.9+/-5.3 ng/mg protein versus 92.9+/-8.6 ng/mg protein, P=0.37) and glutathione peroxidase activity (27.3+/-2.2 mmol/min/mg protein versus 28.3+/-2.2 mmol/min/mg, P=0.83) were not significantly different in pregnancies complicated by asthma and non-asthmatic pregnancies. There was no effect of fetal sex, asthma severity or treatment for asthma on these pathways. Maternal asthma during pregnancy is associated with increased placental enzymatic anti-oxidant capacity and also increased protein oxidation suggesting there is a compensatory increase in anti-oxidant activity in response to increased oxidative stress.

Increased biological oxidation and reduced anti-oxidant enzyme activity in pre-eclamptic placentae.

Oxidative stress occurs when cellular levels of reactive oxygen species exceed anti-oxidant capabilities and has been implicated in the pathogenesis of pre-eclampsia. In this study we have examined the tissue levels of endogenous anti-oxidant proteins (superoxide dismutase, glutathione peroxidase, thioredoxin reductase and thioredoxin) and the level of lipid and protein oxidation in placental samples from normal and pre-eclamptic pregnancies. Pre-eclamptic tissue homogenates demonstrated significantly increased levels of lipid peroxidation (20.68 +/- 7.811 microM protein versus 5.33 +/- 4.03 microM/mg protein, P < 0.001) and a trended increase in protein carbonyl concentration (248.1 +/- 97.71 units/mg protein versus 209.7 +/- 82.6 U/mg protein) when compared to controls. The levels and activities of the anti-oxidant proteins superoxide dismutase (2.48 +/- 0.6 U/mg protein versus 2.02 +/- 0.51 U/mg protein, P <0.02), thioredoxin reductase (19.25 +/- 9.81 U/mg protein versus 13.02 +/- 5.66 U/mg protein,P = 0.02), thioredoxin (107.00 +/- 18.11 ng/mg protein versus 91.12 +/- 21.18 ng/mg protein, P = 0.02) and glutathione peroxidase (17.33 +/- 6.63 mmol/min/mg protein versus 11.50 +/- 3.11 mmol/min/mg, P < 0.02) were all found to be significantly reduced when comparing pre-eclamptic placental tissue homogenates to gestational age-matched control placentae from non-pre-eclamptic pregnancies. The results of this study demonstrate a decreased enzymatic anti-oxidant capacity and increased oxidation in placental tissue from pre-eclamptic women, which may contribute to the pathogenesis of this complex disorder.

Corticotropin releasing hormone-binding protein (CRH-BP): plasma levels decrease during the third trimester of normal human pregnancy.

In pregnancy, maternal plasma corticotropin releasing hormone (CRH) concentrations rise substantially in the third trimester and fall rapidly post-partum. A binding protein (BP) specific for CRH exists in the human circulation which inactivates CRH, thus possibly explaining why maternal ACTH does not rise outside normal limits throughout gestation. We here describe the measurement of CRH-BP directly in plasma during human pregnancy using a radioimmunoassay that is not affected by the presence of the high plasma levels of CRH that occur at this time. In 119 healthy non-pregnant individuals, mean CRH-BP levels were 4.46 nmol/L +/- 1.0 (SD), with a wide range of 1.81-7.24 nmol/L. Plasma CRH-BP in 34 pregnant women randomly sampled during the first and second trimesters also averaged 4.46 nmol/L +/- 1.54, with individual values ranging from 1.59-7.51 nmol/L and there was no correlation of CRH-BP levels with gestational age. In a group of 14 women sampled sequentially throughout the third trimester, plasma CRH-BP averaged 4.56 nmol/L +/- 1.70 at 30-35 weeks gestation and fell dramatically to 1.84 nmol/L +/- 0.43 at weeks 38-40 (P < 0.001). The post partum recovery in CRH-BP levels occurred within 48 hours of delivery. These results indicate that there is an increase in the availability of free, potentially bioactive CRH at term to stimulate the release of ACTH from the maternal pituitary and/or to act at a peripheral, non-pituitary CRH receptor(s).

Processing of procorticotropin-releasing hormone (pro-CRH): molecular forms of CRH in normal and preeclamptic pregnancy.

This study examined the different molecular forms of CRH in normal and preeclampsia maternal plasma and protease-blocked placental extracts using antibodies to different regions of the CRH precursor, pro-CRH. In the absence of protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 19-kDa pro-CRH, its approximately 8-kDa intermediate metabolite, pro-CRH125-194, its approximately 2.8-kDa midportion fragment, pro-CRH125-151, and 4.75-kDa CRH1-41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing was more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, proCRH125-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR, the messenger ribonucleic acid levels of CRH precursor in preeclampsia placentas were 1.7-fold higher than those in normal placentas (37.83 +/- 3.48 vs. 21.83 +/- 2.59 attomoles/microg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH1-41 cross-reactivity (4.72 +/- 1.22 pmol/g) than normal term placentas (1.52 +/- 0.39 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. The content of pro-CRH(125+/-151)-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placentas (4.23 +/- 1.39 pmol/g) than in those from normal term pregnancies (1.44 +/- 0.32 pmol/g; P < 0.01). Sequential plasma samples from 10 women with normal pregnancy and 5 women with preeclampsia were assayed for pro-CRH(125-151)- and CRH(1-41)-immunoreactive species In normal pregnancy, maternal plasma CRH(1-41) immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH(1-41) levels at each gestational age point were higher than those at the equivalent stage of normal pregnancy. In contrast, the levels of pro-CRH(125-151)-immunoreactive species remained barely detectable throughout normal and preeclamptic pregnancy. Both pro-CRH and CRH(1-41), but not pro-CRH(125-151), were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue from degrading enzymes during extraction and show that most of the CRH in the human placenta exists as unprocessed pro-CRH, with very little in the form of CRH(1-41) except in preeclampsia. Our studies using maternal plasma indicate that CRH(1-41) is the only one of the pro-CRH fragments studied to be maintained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.

Corticotrophin-releasing hormone-binding protein in human fetal plasma.

During pregnancy maternal plasma corticotrophin-releasing hormone (CRH) levels rise 1000-fold whilst fetal plasma levels are often 100-fold higher than the concentrations seen in normal non-pregnant human plasma. Despite these high CRH levels neither the maternal nor fetal pituitary releases excessive amounts of ACTH. A specific CRH-binding protein (CRHBP) exists in the maternal circulation which is able to bind and inactivate the ACTH releasing activity of CRH. In this study we have used a specific CRHBP radioimmunoassay to determine the level of CRHBP in fetal and maternal plasma samples. Fetal samples were collected by cordocentesis between 20 and 33 weeks gestation and matched maternal samples were taken by venepuncture at the same time. In a second study, plasma samples were collected from 8 women at fortnightly intervals from week 20 to term, at labour and post-partum. A fetal sample, taken from the umbilical vein, was collected immediately post-delivery. The mean maternal CRHBP concentration for the samples collected between 20 and 33 weeks (n = 23) was 8.12 nmol/l and the fetal level was 8.62 nmol/l. Data from the second study showed that at term the maternal CRHBP concentration decreased significantly (P < 0.025) to 6.32 nmol/l. The fetal CRHBP level also decreased significantly (P < 0.001) at term to a level of 5.84 nmol/l. The CRHBP in both fetal and maternal plasma was shown to be functional by 125I-CRH binding and gel permeation chromatography. The capacity of maternal and fetal plasma to bind 125I-CRH decreased at term in agreement with the quantitation of plasma CRHBP by radioimmunoassay.

Plasma measurements of corticotrophin-releasing hormone-binding protein in normal and abnormal human pregnancy.

Using corticotrophin-releasing hormone-binding protein (CRHBP) purified from human plasma and a 25 amino acid peptide corresponding to the C-terminus of CRHBP we have been able to produce rabbit polyclonal antisera specific for CRHBP. This has allowed the development of a radioimmunoassay which is able to detect CRHBP specifically in human plasma regardless of the presence of endogenous CRH. We have used this assay to estimate the level of CRHBP in non-pregnant human plasma to be approximately 20 nmol/l with a range of 9.1-40.6 nmol/l. We have also examined sequential plasma samples taken from 84 normal pregnant women at fortnightly intervals from 16 weeks gestation through to term. Four women were also sampled during labour and the first week postpartum. The median plasma level of CRHBP at week 16 of normal pregnancy was 21.59 nmol/l, levels rose slightly during the early part of the third trimester (26.76 nmol/l at week 30, (P < 0.01) and fell markedly towards term (19.72 nmol/l, P < 0.01) with only 8.70 nmol/l at labour. CRHBP levels returned to normal non-pregnant levels within 48 h of parturition suggesting a role for the fetoplacental unit in CRHBP production. In eight pregnancies complicated by diabetes, CRHBP levels at each gestational age were similar to those recorded for normal pregnancy. However, in pregnancies complicated by pre-term labour (n = 9) and pre-eclampsia (n = 7), plasma CRHBP levels were significantly reduced (P < 0.01).

Corticotrophin-releasing hormone (CRH)-binding protein interference with CRH antibody binding: implications for direct CRH immunoassay.

Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding protein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6.4 nmol/l) purified from human plasma, CRH-BP diluent alone, normal human plasma (containing 5.8 nmol endogenous CRH-BP/l) and normal sheep plasma (containing no CRH-BP) on the binding of 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; with 5 nmol CRH-BP/l, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and isolation of corticotrophin-releasing hormone-positive cells from the human placenta.

During human pregnancy, plasma corticotrophin-releasing hormone (CRH) levels rise from undetectable amounts prior to 20 weeks gestation to reach a peak near term, with an exponential rise during the final 5 weeks of gestation. Within hours of parturition plasma levels fall and rapidly return to undetectable baseline measurements. The appearance of CRH in maternal plasma has been attributed to the placental production and subsequent release into the maternal circulation of this hormone. Previous studies have shown that human placental extracts contain a CRH-like peptide and this has been reinforced by the observation of CRH mRNA in placental tissue. Initial attempts to identify the site of production using immunocytochemistry have led to conflicting results. This study attempts to clarify this situation by using a variety of highly specific anti-CRH antibodies to show the cellular expression of placental CRH. Intense CRH staining was observed in the syncytial trophoblast layer in first trimester and term chorionic villi, whilst the underlying cytotrophoblast appeared to be negative. The fetal membranes also contained CRH immunoreactivity with the cytotrophoblast cells in the chorionic membrane displaying the most intense staining. CRH immunoactivity was also observed in the amnion and in some cells in the decidua. As a model of cellular CRH expression, cytotrophoblast cells from term chorionic membrane were isolated and shown to be positive for CRH.

Immunocytochemical localization of thioredoxin in human trophoblast and decidua.

An immunocytochemical investigation into the expression of thioredoxin in human reproductive tissues was performed using monoclonal antibodies produced against recombinant human thioredoxin. First trimester and term human placental villi, decidua and term fetal membranes were examined for thioredoxin content and cellular localization. In first trimester tissue strong thioredoxin staining was observed in the underlying cytotrophoblast cells and in the stromal cells present in the decidua, but not in the syncytiotrophoblast surrounding the chorionic villi. In term placental villi very little thioredoxin was observed. Term fetal membranes proved to be a rich source of thioredoxin, the most intense staining was seen in the cytotrophoblast cells in the chorionic membrane, with the amnion and decidua also showing positive immunoreactivity. The potential role/s that thioredoxin may play within the placental bed is considered.

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells.

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.

Corticotrophin-releasing hormone receptor type 1: generation and characterization of polyclonal antipeptide antibodies and their localization in pituitary cells and cortical neurones in vitro.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced antipeptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

Selenium supplementation protects trophoblast cells from mitochondrial oxidative stress.

INTRODUCTION: Oxidative stress plays an important role in the pathogenesis of preeclampsia, a placental disorder affecting approximately 7% of pregnancies. Trophoblast cells are susceptible to oxidative stress which causes increased cell death and placental turnover. In this study, inhibitors of the mitochondrial respiratory chain were utilised to induce oxidative stress and the effect that selenium supplementation had on trophoblast viability was investigated. METHODS: Trophoblast cells (BeWo, JEG-3 and Swan-71) were treated with Na Selenite (100 nM) or Selenomethionine (500 nM) to increase the biological activity of antioxidants Glutathione Peroxidase and Thioredoxin Reductase. The cells were then oxidatively stressed with the addition of increasing doses of Antimycin C and Rotenone and the Resazurin end point assay was used to assess cellular activity. RESULTS: There was a significant dose dependent decrease in the cellular activity in BeWo, JEG-3 and Swan-71 when treated for 4 h with increasing concentrations of Antimycin (40-320 µM) and Rotenone (100-800 nM). Prior incubation with Na Selenite and Selenomethionine was able to protect trophoblast cells from oxidative stress at Rotenone concentrations of 200 and 400 nM (P < 0.001) and Antimycin concentrations of 80-240 µM (P < 0.001). DISCUSSION: These data suggest that selenoproteins such as Glutathione Peroxidase and Thioredoxin Reductase have an important role in protecting trophoblast mitochondria from oxidative stress. CONCLUSIONS: This study emphasises the importance of maintaining an adequate selenium supply during pregnancy and especially in pregnancies complicated by conditions such as preeclampsia.

Alterations in acetylcholine, PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide.

The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1-100 µM) or LPS (1-100 ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100 µM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100 ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100 µM), stretch-induced release for pseudomonal LPS (≥ 10 ng/mL) and both basal and stimulated release for E. coli LPS (≥ 10 ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100 µM) or LPS (100 ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.

Selenium supplementation protects trophoblast cells from oxidative stress.

Oxidative stress is a key feature in the pathogenesis of pre-eclampsia and antioxidants have been proposed as a potential therapy in the treatment of this important complication of pregnancy. In this report selenium supplementation was used to up-regulate the antioxidant enzymes glutathione peroxidase and thioredoxin reductase and the protective effect that this had on cellular metabolism during oxidative stress was examined. Bewo and Jeg-3 trophoblast cells were supplemented with organic and inorganic forms of selenium and 3 forms of peroxide in a range of doses were utilised to generate oxidative stress. Thioredoxin reductase and glutathione peroxidase activity were maximally expressed after supplementation with 100 nM NaSe and 500 nM SeMethionine. Application of H2O2 in the range of 200-400 µM for 24h resulted in significant (p<0.001) inhibition of cellular activity, an effect negated by Se supplementation. Tert-butyl H2O2 and cumene H2O2 concentrations between 30 and 50 uM similarly inhibited cellular activity and this could be significantly (p<0.001) reversed by Se supplementation. Auranofin, a specific inhibitor of thioredoxin reductase and glutathione peroxidase was used to prove that the protective effect generated by Se supplementation was due to up regulation of these enzymes. These studies provide direct evidence that selenium supplementation can up-regulate endogenous antioxidant systems and protects trophoblast cells from oxidative stress. This may inform the development of future therapies for pre-eclampsia and emphasises the importance of selenium adequacy during pregnancy.

Selenium and preeclampsia: A global perspective.

Preeclampsia is a complex multisystem disorder of pregnancy where oxidative stress plays an important aetiological role. The role of selenium in the synthesis of endogenous antioxidants is well documented, and a significant reduction in selenium has been reported in preeclamptic women. The objective of this study was to map global selenium status and preeclampsia incidence. This study identified peer reviewed journal articles reporting national preeclampsia incidence (%) and matched these with reported values of selenium intake and plasma/serum selenium concentrations (µg/L). Matched data were obtained for 45 regions, reporting 6456,570 births, spanning Europe, Asia, Australasia, Africa, North and South America. Increasing plasma selenium concentration was found to be correlated with a reduction in preeclampsia incidence (Pearson's r=-0.604, P<0.0001). Countries with a reported serum/plasma selenium level of ⩾95µg/L were considered selenium sufficient and a significant reduction in preeclampsia incidence for countries above this value (P=0.0007) was noted. Significant reductions in preeclampsia incidence were found to coincide with increases in plasma/serum selenium concentration in the New Zealand (P=0.0003) and Finland (0.0028) populations following Government intervention. This study supports the hypothesis that selenium supplementation may be beneficial in reducing oxidative stress in women at risk of preeclampsia.