The effect of DDE and dieldrin and their parental substances (DDT and aldrin) on PGE2 and PGF2α release from bovine endometrial explants collected during 120 to 180 days of the gestational period.

Dieldrin and DDE are environmental metabolites of the organochlorine pesticides aldrin and DDT, respectively. During pregnancy, these chemicals can quickly infiltrate through the placental barrier, accumulate in amniotic fluid and fetus, and act as endocrine disruptors (EDs). The aim of this study was to investigate the effect of DDE and dieldrin and their parental substances at concentrations of 1 and 10 ng/ml on secretion of PGE2 and PGF2α from bovine endometrial explants (120-150 and 151-180 days of pregnancy) after 24 hr of incubation with EDs. The mRNA expression of COX2, PGES and PGFS and the concentrations of PGE2 and PGF2α were measured. EDs did not affect (p>0.05) COX2 gene expression, but DDT and DDE decreased (p⟨0.05) PGES expression and PGE2 secretion in the explants from 120-150 days of pregnancy. Depending on the dose, DDT and DDE increased (p⟨0.05) PGFS expression and PGF2α secretion from the explants from 120-150 days and decreased PGF2α secretion (p⟨0.05) from the explants from 151-180 days of pregnancy. Aldrin and dieldrin decreased (p⟨0.05) PGFS expression and PGF2α secretion from all explants. In summary, EDs disrupt the secretion of PGE2 and PGF2α by influencing the gene expression of PGES and PGFS.

Urocortins exhibit differential effects on PGE2 and PGF2α output via CRHR2 in human myometrium.

Urocortins (UCNs), belonging to corticotropin-releasing hormone (CRH) family, exert their function via CRH receptor type 1 (CRHR1) and 2 (CRHR2). Our previous studies have demonstrated that CRH acts on CRHR1 to potentiate prostaglandins (PGs) output induced by inflammatory stimuli in myometrial cells. In the present study, we sought to investigate the effects of UCNs on prostaglandin (PG) output via CRHR2 in cultured human uterine smooth muscle cells (HUSMCs) from pregnant women at term. We found that UCN and UCN 3 treatment promoted PGE2 and PGF2α secretion in a dose-dependent manner. In contrast, UCN2 dose-dependently inhibited PGE2 and PGF2α secretion. Their effects were reversed by CRHR2 antagonist and CRHR2 siRNA. Mechanically, we showed that UCN and UCN3 suppressed cAMP production and led to Gi activation while UCN2 stimulated cAMP production and activated Gs signaling. Further, UCN and UCN3 but not UCN2 activated NF-κB and MAPK signaling pathways through Gi signaling. UCN and UCN3 stimulation of PGs secretion were dependent on Gi/adenylyl cyclase (AC)/cAMP, NF-κB and MAPK signaling pathways. UCN2 suppression of PGs output was through Gs/AC/cAMP signaling pathways. Our data suggest that UCN, UCN2 and UCN3 can finely regulate PGs secretion via CRHR2, which facilitates the functional status of the uterus during pregnancy.

Effects of non-esterified fatty acids on relative abundance of prostaglandin E2 and F2α synthesis-related mRNA transcripts and protein in endometrial cells of cattle in vitro.

Cows nearing parturition have a negative energy balance (NEB), which is closely associated with lesser fertility. The NEB results in greater fat mobilisation and production of a large amount of non-esterified fatty acid (NEFA). Prostaglandins (PG), especially prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), have important functions in regulating reproductive function. There, however, is little known about how the synthesis and release of PG are affected by NEFA. In this study, there was a focus on effects of NEFA on PG secretion as well as relative abundances of mRNA transcript and protein for PG synthetases and PG receptors in bovine endometrial (BEND) cells. Proliferation rate of BEND cells decreased in a concentration-dependent manner as NEFA increased in the media. The concentrations of PGE2 and PGF2α in NEFA treatment groups also decreased, while the ratio of PGE2/PGF2α and the relative abundances of proteins and mRNA that regulate PG synthesis and PG receptor mRNA transcripts and protein were greater as the NEFA concentration increased. Collectively, when there were large NEFA concentrations in the medium, there was a lesser release of PGE2 and PGF2α, however, there was a greater ratio of PGE2/PGF2α and relative abundances of mRNA transcripts and protein for PG synthetases and PG receptors in BEND cells, which changed the internal milieu and physiological function of the uterus with possible effects on fertility after calving. These findings provide important information that will help for further investigation of associations between NEB and fertility in dairy cows during the non-lactation to lactation-transition period.

Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals.

OBJECTIVE: To evaluate the effects of a hypoenergetic diet (HD)associated with açaí pulp consumption on oxidative stress, antioxidant status and inflammatory biomarkers in overweight, dyslipidemic individuals. RESEARCH METHODS & PROCEDURES: A randomized, double-blind, placebo-controlled clinical trial was conducted for 90 days. The study began with a 30-day run-in period, during which the intervention was exclusively a HD. Following this period, volunteers were randomized into 2 groups, and 200 g of either açaí pulp or placebo were added to the HD for 60 days. Anthropometric measurements, arterial pressure, oxidative stress and antioxidant status biomarkers, inflammatory and biochemical biomarkers were evaluated. RESULTS: Sixty-nine volunteers completed the clinical trial, 30 of which were in the HD + açaí group and 39 in HD + placebo group. Plasma 8-isoprostane concentrations significantly reduced 60 days after the intervention in the açaí group (p = 0.000), and there was a significant difference between the groups (açaí versus placebo; p = 0.037). Regarding inflammatory status parameters, a significant reduction in IL-6 was observed in the HD + açaí group (p = 0.042), and IFN-γ decreased significantly in both groups, HD + açaí (p = 0.001) and HD + placebo (p = 0.008); there were, however, no differences between the groups. Lipid profile parameters and blood glucose levels did not show change, regardless of nutritional intervention. CONCLUSION: The addition of açaí to a HD, for 60 days, reduced oxidative stress and improved inflammation in overweight, dyslipidemic individuals.

Endometrial stromal cell miR-29c-3p regulates uterine contraction.

Uterine peristalsis plays a vital role in fertility and female reproductive health. Although uterine peristalsis is thought to be correlated with some hormones and uterine pathologies, the physiological mechanisms underlying uterine peristalsis remain not quite clear. This study aimed to identify changes in miRNA in the endometrium of patients with abnormally high-frequency (hyper-) and low-frequency (hypo-) peristalsis to clarify whether miRNAs regulate uterine peristalsis. We used a miRNA microarray and RT-qPCR to identify changes in miRNA in endometrial tissue, a collagen gel contraction assay on co-cultured human endometrial stromal cells (ESCs) to analyze how the altered regulation of miRNAs influences uterine smooth muscle (USM) contraction, Western blots and other assays to elucidate the potential mechanisms involved. We found that among several differentially regulated miRNAs, miR-29c-3p was overexpressed in endometrial samples from patients with hypoperistalsis; oxytocin receptor (OXTR) expression was low in endometrial samples from patients with hypoperistalsis. Bioinformatic analysis and luciferase assays indicated that OXTR is a target of miR-29c-3p, which attenuates its expression. Additionally, downregulation of miR-29c-3p in ESC cultures increased the expression of aldo-keto reductase family 1, member C3 (AKR1C3) and increased the release of prostaglandin F2 alpha (PGF2α). Co-cultured ESCs overexpressing miR-29c-3p reduced USM cell contractions; the opposite tendency was found when ESCs were transfected with a miR-29c-3p inhibitor. To conclude, miR-29c-3p in endometrial cells regulates uterine contractility by attenuating the expression of OXTR and reducing PGF2α release.

Cooperative effects of sequential PGF2α and IL-1ß on IL-6 and COX-2 expression in human myometrial cells†.

The change from the state of pregnancy to the state of parturition, which we call uterine transitioning, requires the actions of inflammatory mediators and results in an activated uterus capable of performing the physiology of labor. Interleukin (IL)-1ß and prostaglandin (PG)F2α are two key mediators implicated in preparing the uterus for labor by regulating the expression of uterine activation proteins (UAPs) and proinflammatory cytokines and chemokines. To investigate this process, primary human myometrial smooth muscle cells (HMSMC) isolated from the lower segment of women undergoing elective cesarean sections at term (not in labor) were used to test the inflammatory cytokine and UAP outputs induced by PGF2α and IL-1ß alone or in sequential combinations. PGF2α and IL-1ß regulate mRNA abundance of the PGF2α receptor FP, the IL-1 receptor system, interleukin 6, and other UAPs (OXTR, COX2), driving positive feedback interactions to further amplify their own proinflammatory effects. Sequential stimulation of HMSMC by PGF2α and IL-1ß in either order results in amplified upregulation of IL-6 and COX-2 mRNA and protein, compared to their effects individually. These profound increases were unique to myometrium and not observed with stimulation of human fetal membrane explants. These results suggest that PGF2α and IL-1ß act cooperatively upstream in the birth cascade to maximize amplification of IL-6 and COX-2, to build inflammatory load and thereby promote uterine transition. Targeting PGF2α or IL-1ß, their actions, or intermediates (e.g. IL-6) would be an effective therapeutic intervention for preterm birth prevention or delay.

The Effects of Once-Weekly Dulaglutide and Insulin Glargine on Glucose Fluctuation in Poorly Oral-Antidiabetic Controlled Patients with Type 2 Diabetes Mellitus.

Aim. To compare the effects of once-weekly Dulaglutide with once-daily glargine in poorly oral-antidiabetic controlled patients with type 2 diabetes mellitus (T2DM). Method. A total of 25 patients with T2DM admitted into Department of Endocrinology from December 2012 to August 2013 were randomly assigned into two groups: Dulaglutide group (n = 16) and glargine group (n = 9). All patients received either Dulaglutide or glargine treatments for 52 weeks. Continuous glucose monitoring systems (CGMS) were applied to them for two 72 h periods at before and after the treatment each. Patient general clinical data were collected and analyzed. Result. Fast blood glucose (FBG) of the glargine group declined more significantly than the Dulaglutide group after treatment (p < 0.05). The mean blood glucose (MBG), standard deviation of blood glucose (SDBG), mean amplitude of glycemic excursion (MAGE) within a day, the largest amplitude of glycemic excursion (LAGE), M-value, absolute means of daily difference (MODD) of glycemic excursion, the percentage of time (≤2.8 mmol/L, ≤3.9 mmol/L, ≥10.0 mmol/L, ≥13.9 mmol/L, 3.9-7.8 mmol/L, and 9-10.0 mmol/L), maximum glycemic value, and minimum glycemic value were similar between the two groups (p > 0.05). The incidence of hypoglycemia was also similar between the two groups (p > 0.05). Though serum levels of TNF-α, IL-6, and 8-PGF2α all decreased, significant reduction was found in TNF-α and 8-PGF2α. TNF-α was only significantly reduced in the Dulaglutide group, while 8-PGF2α was seen in both groups. Conclusion. For T2DM patients with poorly controlled oral antidiabetic drugs, once-weekly Dulaglutide not only has the same effect on glucose fluctuation as once-daily glargine but also significantly reduced TNF-α and 8-PGF2α after a 52 week treatment protocol. This trial is registered with ClinicalTrials.gov NCT01648582.

Identification of SMAD3 as a Novel Mediator of Inflammation in Human Myometrium In Vitro.

Preterm birth remains the primary cause of early neonatal death and is a major determinant for long-term health consequences. Aberrant intrauterine inflammation and infection are known to augment the synthesis of proinflammatory cytokines and induce uterine contractions, which can subsequently lead to preterm birth. The transforming growth factor-ß (TGF-ß) superfamily members regulate numerous cellular processes through the activation of intracellular mediators known as mothers against decapentaplegic homolog (SMADs). Studies in nongestational tissues have shown that SMAD3 plays a role in immune regulation and inflammation; however, its role in human labour remains unknown. Thus, the present study aimed at (i) characterising the expression of SMAD3 in the human myometrium; (ii) determining the effect of bacterial and viral products and proinflammatory cytokines on SMAD3 transcriptional activity in primary human myometrial cells; and (iii) investigating the effect of SMAD3 siRNA knockdown on the production of prolabour mediators in primary human myometrial cells. Phosphorylated (i.e., active) SMAD3 protein expression was lower in the myometrium after spontaneous term labour compared to the myometrium from nonlabouring women. Using a luciferase assay, the proinflammatory cytokines IL-1ß and TNF, and viral analogue polyinosinic : polycytidylic acid (poly(I : C)) significantly reduced SMAD3 transcriptional activity in human primary myometrial cells. Loss-of-function studies found that SMAD3 knockdown in myometrial cells significantly increased IL-1ß- and poly(I : C)-induced proinflammatory cytokines (IL-1A, IL-6), chemokines (IL-8, MCP-1), the adhesion molecule ICAM-1, COX-2 mRNA expression, and subsequent PGF2α release. In conclusion, SMAD3 deficiency is associated with increased production of proinflammatory and prolabour mediators in the human myometrium.

A role for trypanosomatid aldo-keto reductases in methylglyoxal, prostaglandin and isoprostane metabolism.

Trypanosomatid parasites are the infectious agents causing Chagas disease, visceral and cutaneous leishmaniasis and human African trypanosomiasis. Recent work of others has implicated an aldo-keto reductase (AKR) in the susceptibility and resistance of Trypanosoma cruzi to benznidazole, a drug used to treat Chagas disease. Here, we show that TcAKR and homologues in the related parasites Trypanosoma brucei and Leishmania donovani do not reductively activate monocyclic (benznidazole, nifurtimox and fexinidazole) or bicyclic nitro-drugs such as PA-824. Rather, these enzymes metabolise a variety of toxic ketoaldehydes, such as glyoxal and methylglyoxal, suggesting a role in cellular defence against chemical stress. UPLC-QToF/MS analysis of benznidazole bioactivation by T. cruzi cell lysates confirms previous reports identifying numerous drug metabolites, including a dihydro-dihydroxy intermediate that can dissociate to form N-benzyl-2-guanidinoacetamide and glyoxal, a toxic DNA-glycating and cross-linking agent. Thus, we propose that TcAKR contributes to benznidazole resistance by the removal of toxic glyoxal. In addition, three of the four enzymes studied here display activity as prostaglandin F2α synthases, despite the fact that there are no credible cyclooxygenases in these parasites to account for formation of the precursor PGH2 from arachidonic acid. Our studies suggest that arachidonic acid is first converted non-enzymatically in parasite lysates to (PGH2-like) regioisomers by free radical-mediated peroxidation and that AKRs convert these lipid peroxides into isoprostanes, including prostaglandin F2α and 8-iso-prostaglandin F2α.

Strategies to decrease oxidative stress biomarker levels in human medical conditions: A meta-analysis on 8-iso-prostaglandin F2α.

The widespread detection of elevated oxidative stress levels in many medical conditions has led to numerous efforts to design interventions to reduce its effects. Efforts have been wide-ranging, from dietary changes to administration of antioxidants, supplements, e.g., omega-3-fatty acids, and many medications. However, there is still no systemic assessment of the efficacy of treatments for oxidative stress reduction across a variety of medical conditions. The goal of this meta-analysis is, by combining multiple studies, to quantitate the change in the levels of the popular oxidative stress biomarker 8-iso-prostaglandin F2α (8-iso-PGF2α) after a variety of treatment strategies in human populations. Nearly 350 unique publications with 180 distinct strategies were included in the analysis. For each strategy, the difference between pre- or placebo and post-treatment levels calculated using Hedges' g value of effect. In general, administration of antibiotics, antihyperlipidemic agents, or changes in lifestyle (g = - 0.63, - 0.54, and 0.56) had the largest effect. Administration of supplements, antioxidants, or changes in diet (g = - 0.09, - 0.28, - 0.12) had small quantitative effects. To fully interpret the effectiveness of these treatments, comparisons to the increase in g value for each medical condition is required. For example, antioxidants in populations with coronary artery disease (CAD) reduce the 8-iso-PGF2α levels by g = - 0.34 ±â€¯0.1, which is quantitatively considered a small effect. However, CAD populations, in comparison to healthy populations, have an increase in 8-iso-PGF2α levels by g = 0.38 ±â€¯0.04; therefore, the overall reduction of 8-iso-PGF2α levels is ≈ 90% by this treatment in this specific medical condition. In conclusion, 8-iso-PGF2α levels can be reduced not only by antioxidants but by many other strategies. Not all strategies are equally effective at reducing 8-iso-PGF2α levels. In addition, the effectiveness of any strategy can be assessed only in relation to the medical condition investigated.

Anti-Inflammatory Potential of In Vitro Cultures of the White Button Mushroom, Agaricus bisporus (Agaricomycetes), in Caco-2 Cells.

Agaricus bisporus (white button mushroom) is one of the most popular culinary-medicinal mushrooms worldwide. This species has for decades been the subject of numerous scientific studies. The aim of this study was to examine the pro- or anti-inflammatory properties of A. bisporus and biomass extracts from in vitro cultures growing in Oddoux medium enriched with α-linolenic acid in colon epithelial Caco-2 cells activated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α. Incubation of Caco-2 cells with A. bisporus extracts resulted in decreased expression of cyclooxygenase-2 and prostaglandin F2α receptor compared with the LPS- and/or TNF-α-activated cells, whereas the expression of nuclear factor (erythroid-derived 2)-like 2 increased after incubation. Interleukin-6 level decreased significantly in Caco-2 cells after supplementation with mushroom extracts. The amounts of monoun-saturated and polyunsaturated fatty acids differed significantly in Caco-2 cell membranes after supplementation with A. bisporus extracts. Our findings suggest the presence of anti-inflammatory and antioxidant properties of A. bisporus biomass extracts from in vitro cultures.

A20, an essential component of the ubiquitin-editing protein complex, is a negative regulator of inflammation in human myometrium and foetal membranes.

STUDY QUESTION: Does A20 regulate mediators involved in the terminal processes of human labour in primary myometrial and amnion cells? SUMMARY ANSWER: A20 is a nuclear factor-kappa B (NF-κB) responsive gene that acts as a negative regulator of NF-κB-induced expression of pro-labour mediators. WHAT IS KNOWN ALREADY: Inflammation is commonly implicated in spontaneous preterm birth and the processes involved in rupture of foetal membranes and uterine contractions. In myometrium and foetal membranes, the pro-inflammatory transcription factor NF-κB regulates the transcription of pro-labour mediators in response to inflammatory stimuli. In non-gestational tissues, A20 is widely recognised as an anti-inflammatory protein that inhibits inflammation-induced NF-κB signalling. STUDY DESIGN, SIZE, DURATION: Primary human amnion and myometrial cells were used to determine the effect of pro-inflammatory mediators on A20 expression and the effect of A20 siRNA on the expression and secretion of pro-labour mediators. The expression of A20 was assessed in myometrium and foetal membranes from non-labouring and labouring women at preterm and or term (n = 8 or nine samples per group). PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of pro-inflammatory mediators and of A20 siRNA in cell cultures were determined by quantitative RT-PCR (qRT-PCR), western blots, immunoassays, gelatin zymography and luciferase assays. A20 expression in tissue samples was assessed by qRT-PCR. Statistical significance was ascribed to a P value < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: In primary cells isolated from myometrium and or amnion, the pro-inflammatory cytokines IL1B and TNF, the bacterial products flagellin and fsl-1, and the viral double stranded RNA analogue poly(I:C) significantly increased A20 mRNA expression via NF-κB. A20 siRNA studies in primary myometrial and amnion cells demonstrated an augmentation of inflammation-induced expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL1, CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1), contraction-associated proteins (PTGS2, PTGFR, PGF2α) and the extracellular matrix degrading enzyme MMP9, as well as NF-κB activation. Inhibition of NF-κB activity significant attenuated inflammation-induced expression of pro-labour mediators in A20 siRNA transfected cells. Finally, A20 mRNA expression was decreased in myometrium and foetal membranes with labour, and in foetal membranes with chorioamnionitis. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The conclusions of this study are solely reliant on the data from in vitro experiments using cells isolated from myometrium and amnion. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study raise the possibility that targeting A20 may be a therapeutic approach to reduce inflammation associated with spontaneous preterm birth. STUDY FUNDING AND COMPETING INTEREST(S): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. There are no competing interests.

Effects of preimplantation factor on interleukin-6 and prostaglandin F2α and E2 in the bovine endometrium.

Preimplantation factor (PIF) is a pregnancy specific peptide with immune modulatory properties exerted on the human endometrium. Viable bovine embryos secrete PIF, but its effect on the bovine endometrial immune response is unknown, both in native and inflammatory stimulated endometrial tissue. An ex vivo bovine endometrial tissue culture model was used with lipopolysaccharide (LPS) as an inflammatory stimulant. The effect of synthetic PIF (sPIF) was assessed, in three separate experiments, on the secretion or mRNA expression of essential prostaglandins and cytokines. Radioimmunoassays were used to assess prostaglandin secretion and ELISA for IL-6 secretion from endometrial explants. mRNA expression of IL6 and IL8 was analysed from endometrial explants with real-time PCR. Synthetic PIF reduced native IL-6 secretion from explants when pre-treated for 24 h. There was no effect of sPIF on IL-6 secretion from LPS challenged explants; however, sPIF increased IL6 mRNA expression when challenged with 500 ng/mL LPS. There was no effect of sPIF on prostaglandin secretion or mRNA expression of IL8. Therefore, sPIF is able to modulate the native IL-6 production pathway in the bovine endometrium, yet demonstrates no effect on prostaglandin secretion or IL8 expression. Unlike in human studies, effects of sPIF were minimal, thus sPIF is not an effective modulator of the immune targets investigated in the bovine endometrium.

Associations of self-reported smoking, cotinine levels and epigenetic smoking indicators with oxidative stress among older adults: a population-based study.

Tobacco smoking and oxidative stress (OS) are both related to a wide spectrum of adverse age-related health outcomes, but their association is not yet well-established. We examined the associations of self-reported smoking indicators, serum cotinine levels and smoking-related DNA methylation biomarkers with two urinary proxy markers of OS, 8-isoprostane (8-iso) and 8-hydroxy-2'-deoxyguanosine (8-oxodG), in two independent subsets of older adults recruited in Germany (discovery set: n = 978, validation set: n = 531). We obtained DNA methylation profiles in whole blood samples by Illumina Human Methylation450K Beadchip and measured the urinary levels of both OS markers using commercial ELISA kits. After controlling for potential confounders, current smoking, cumulative smoking exposure (pack-years) and serum cotinine levels (ng/ml) were strongly associated with 8-iso levels (p values <0.0001, 0.004 and 0.001, respectively). Of 151 previously identified smoking-related CpG sites, 71 loci were associated with 8-iso levels after correction for multiple testing (FDR < 0.05) in the validation phase and were designated as loci related to 8-iso levels defined OS. In addition, serum cotinine levels, cumulative smoking exposure and a smoking index (SI) based on the 71 identified loci manifested monotonic associations with 8-iso levels. However, we did not observe any associations between these smoking indicators and 8-oxodG levels. In conclusion, this study suggests that smoking-related epigenetic alterations are closely correlated with smoking-induced OS. The identified CpG sites could potentially be prognostic epigenetic markers of OS and OS-related health outcomes. Our findings and the underlying mechanisms should be followed up in further, preferably longitudinal studies.

TRADD, TRAF2, RIP1 and TAK1 are required for TNF-α-induced pro-labour mediators in human primary myometrial cells.

PROBLEM: TNF-α plays a central role in the processes of human labour and delivery. This study sought to determine the role of the adaptor proteins TNFR1-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), receptor interacting protein 1 (RIP1) and transforming growth factor beta-activated kinase 1 (TAK1) in TNF-α-induced formation of pro-labour mediators. METHOD OF STUDY: Human primary myometrial cells were transfected with siRNA against TRADD (siTRADD), TRAF2 (siTRAF2), RIP1 (siRIP1) or TAK1 (siTAK1), treated with TNF-α, and assayed for pro-inflammatory mediators expression. RESULTS: siTRADD, siTRAF2, siRIP1 and siTAK1 significantly decreased TNF-α-induced IL-1α, IL-1ß, IL-6, IL-8, MCP-1 mRNA expression and release of IL-6, IL-8 and MCP-1; and cyclooxygenase (COX)-2 expression and release of prostaglandin PGF2α . There was a significant attenuation of TNF-α-induced expression of adhesion molecules ICAM-1 and VCAM-1 mRNA with siTRADD, siTRAF2 or siRIP1. siTRADD and siRIP1 significantly attenuated TNF-α-induced MMP-9 mRNA expression and release and nuclear factor κB (NF-κB) transcriptional activity. There was a significant increase in TNF-α-induced sVCAM-1 release, MMP-9 mRNA expression and NF-κB activity with siTAK1. CONCLUSION: TRADD, TRAF2, RIP1 and TAK1 are involved in TNF-α signalling in human myometrium. Further studies are required to determine whether inhibition of these proteins can prevent preterm birth.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating ß-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective ß-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on ß-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating ß-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3ß-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO.

Reinterpreting the best biomarker of oxidative stress: The 8-iso-prostaglandin F2α/prostaglandin F2α ratio shows complex origins of lipid peroxidation biomarkers in animal models.

Oxidative stress is elevated in numerous environmental exposures and diseases. Millions of dollars have been spent to try to ameliorate this damaging process using anti-oxidant therapies. Currently, the best accepted biomarker of oxidative stress is the lipid oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α), which has been measured in over a thousand human and animal studies. 8-iso-PGF2α generation has been exclusively attributed to nonenzymatic chemical lipid peroxidation (CLP). However, 8-iso-PGF2α can also be produced enzymatically by prostaglandin-endoperoxide synthases (PGHS) in vivo. When failing to account for PGHS-dependent generation, 8-iso-PGF2α cannot be interpreted as a selective biomarker of oxidative stress. We investigated the formation of 8-iso-PGF2α in rats exposed to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2α/PGF2α ratio to quantitatively determine the source(s) of 8-iso-PGF2α. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6±19.4% of measured 8-iso-PGF2α, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2α (86.6±8.0% of total). In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS (59.5±7.0) and CLP (40.5±14.0%). In conclusion, significant generation of 8-iso-PGF2α occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The 8-iso-PGF2α/PGF2α ratio accurately determines the source of 8-iso-PGF2α and provides an absolute measure of oxidative stress in vivo.

Untangling the Web: Toxic and Protective Effects of Neuroinflammation and PGE2 Signaling in Alzheimer's Disease.

The neuroinflammatory response has received increasing attention as a key factor in the pathogenesis of Alzheimer's disease (AD). Microglia, the innate immune cells and resident phagocytes of the brain, respond to accumulating Aß peptides by generating a nonresolving inflammatory response. While this response can clear Aß peptides from the nervous system in some settings, its failure to do so in AD accelerates synaptic injury, neuronal loss, and cognitive decline. The complex molecular components of this response are beginning to be unraveled, with identification of both damaging and protective roles for individual components of the neuroinflammatory response. Even within one molecular pathway, contrasting effects are often present. As one example, recent studies of the inflammatory cyclooxygenase-prostaglandin pathway have revealed both beneficial and detrimental effects dependent on the disease context, cell type, and downstream signaling pathway. Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenases, are associated with reduced AD risk when taken by cognitively normal populations, but additional clinical and mouse model studies have added complexities and caveats to this finding. Downstream of cyclooxygenase activity, prostaglandin E2 signaling exerts both damaging pro-inflammatory and protective anti-inflammatory effects through actions of specific E-prostanoid G-protein coupled receptors on specific cell types. These complexities underscore the need for careful study of individual components of the neuroinflammatory response to better understand their contribution to AD pathogenesis and progression.

Defective secretion of Prostaglandin F2α during development of idiopathic persistent corpus luteum in mares.

Five mares that developed idiopathic persistent corpus luteum (PCL) were compared with 5 mares with apparently normal interovulatory intervals (IOIs). Progesterone (P4) and a metabolite of prostaglandin F2α (PGFM) were assayed daily beginning on the day of ovulation (Day 0). Transition between the end of an initial progressive P4 increase and the beginning of a gradual decrease in P4 occurred on mean Day 6. The gradual decrease in P4 between Days 6 and 12 was less (approached significance, P < 0.06) in the PCL group than in the IOI group. The P4 concentration on Day 12 (before luteolysis in IOI group) was greater (P < 0.05) in the PCL group than in the IOI group. In a post hoc comparison, an interaction (P < 0.04) of group by day for Days 4 to 7 indicated that the end of the progressive increase in P4 was temporally associated with a transient increase in concentration of PGFM in IOI mares but not in PCL mares. Complete luteolysis (P4 < 1 ng/mL) occurred in the IOI mares on Days 13 to 15. Partial luteolysis (mean P4 decrease, 62%) occurred in 3 of the 5 PCL mares. Normalization to the day at the end of the most pronounced P4 decrease in the IOI mares and in the 3 PCL mares with partial luteolysis resulted in a day-by-group interaction (P < 0.05) for PGFM concentration. The interaction was partly from lower PGFM concentration on the day at the end of the pronounced P4 decrease in the 3 PCL mares than in the IOI mares. The peak of a transient PGFM increase and the day at the end of the most pronounced decrease in P4 were synchronized in each IOI mare but not in any of the 3 PCL mares. In the other 2 PCL mares, partial luteolysis did not occur, and a transient increase in PGFM was not apparent. Results tentatively indicated that the relationship between P4 and PGFM may be altered as early as Day 6 in PCL mares and supported the hypothesis that prostaglandin F2α secretion is defective in mares with idiopathic PCL.

Differentially Expressed Genes in Endometrium and Corpus Luteum of Holstein Cows Selected for High and Low Fertility Are Enriched for Sequence Variants Associated with Fertility.

Despite the importance of fertility in humans and livestock, there has been little success dissecting the genetic basis of fertility. Our hypothesis was that genes differentially expressed in the endometrium and corpus luteum on Day 13 of the estrous cycle between cows with either good or poor genetic merit for fertility would be enriched for genetic variants associated with fertility. We combined a unique genetic model of fertility (cattle that have been selected for high and low fertility and show substantial difference in fertility) with gene expression data from these cattle and genome-wide association study (GWAS) results in ∼20,000 cattle to identify quantitative trait loci (QTL) regions and sequence variants associated with genetic variation in fertility. Two hundred and forty-five QTL regions and 17 sequence variants associated primarily with prostaglandin F2alpha, steroidogenesis, mRNA processing, energy status, and immune-related processes were identified. Ninety-three of the QTL regions were validated by two independent GWAS, with signals for fertility detected primarily on chromosomes 18, 5, 7, 8, and 29. Plausible causative mutations were identified, including one missense variant significantly associated with fertility and predicted to affect the protein function of EIF4EBP3. The results of this study enhance our understanding of 1) the contribution of the endometrium and corpus luteum transcriptome to phenotypic fertility differences and 2) the genetic architecture of fertility in dairy cattle. Including these variants in predictions of genomic breeding values may improve the rate of genetic gain for this critical trait.