Copy number variants suggest different molecular pathways for the pathogenesis of bladder exstrophy.

Bladder exstrophy is a rare congenital malformation leaving the urinary bladder open in the midline of the abdomen at birth. There is a clear genetic background with chromosome aberrations, but so far, no consistent findings apart from 22q11-duplications detected in about 2%-3% of all patients. Some genes are implicated like the LZTR1, ISL1, CELSR3, and the WNT3 genes, but most are not explained molecularly. We have performed chromosomal microarray analysis on a cohort of 140 persons born with bladder exstrophy to look for submicroscopic chromosomal deletions and duplications. Pathogenic or possibly pathogenic microdeletions or duplications were found in 16 patients (11.4%) and further 9 with unknown significance. Two findings were in regions linked to known syndromes, two findings involved the same gene (MCC), and all other findings were unique. A closer analysis suggests a few gene networks that are involved in the pathogenesis of bladder exstrophy; the WNT-signaling pathway, the chromosome 22q11 region, the RIT2 and POU families, and involvement of the Golgi apparatus. Bladder exstrophy is a rare malformation and is reported to be associated with several chromosome aberrations. Our data suggest involvement of some specific molecular pathways.

The immunoproteasome inhibitor ONX-0914 regulates inflammation and expression of contraction associated proteins in myometrium.

There are currently no effective treatments to prevent spontaneous preterm labor. The precise upstream biochemical pathways that regulate the transition between uterine quiescence during pregnancy and contractility during labor remain unclear. It is well known however that intrauterine inflammation, including infection, is commonly associated with preterm labor. In this study, we identified the immunoproteasome subunit low-molecular-mass protein (LMP)7 mRNA expression to be significantly upregulated in laboring human myometrium. Silencing LMP7 using siRNA-targeted knockdown of LMP7 and its inhibitor ONX-0914 in human myometrial cells and tissues decreased proinflammatory cytokines (IL-6), cell chemotaxis (CXCL8, CCL2 expression, and THP-1 migration), cell to cell adhesion (ICAM1 expression and myometrial adhesion), contraction-associated proteins (PTGS2, FP, PGE2, and PGF2α), as well as suppressing contractions in myometrial cells and in myometrial tissues obtained from laboring women. In addition, LMP7 silencing reduced NF-κB RelA activity. ONX-0914 alleviated inflammation (CCL3, CXCL1, PTGS2, and IL-6) in myometrium, placenta, fetal brain, amniotic fluid, and maternal serum induced by LPS in pregnant mice. Collectively, our data suggest a novel role for ONX-014 to suppress uterine activation and contractility associated with preterm labor.

Unifying Children's Surgery and Anesthesia Stakeholders Across Institutions and Clinical Disciplines: Challenges and Solutions from Uganda.

BACKGROUND: There is a significant unmet need for children's surgical care in low- and middle-income countries (LMICs). Multidisciplinary collaboration is required to advance the surgical and anesthesia care of children's surgical conditions such as congenital conditions, cancer and injuries. Nonetheless, there are limited examples of this process from LMICs. We describe the development and 3-year outcomes following a 2015 stakeholders' meeting in Uganda to catalyze multidisciplinary and multi-institutional collaboration. METHODS: The stakeholders' meeting was a daylong conference held in Kampala with local, regional and international collaborators in attendance. Multiple clinical specialties including surgical subspecialists, pediatric anesthesia, perioperative nursing, pediatric oncology and neonatology were represented. Key thematic areas including infrastructure, training and workforce retention, service delivery, and research and advocacy were addressed, and short-term objectives were agreed upon. We reported the 3-year outcomes following the meeting by thematic area. RESULTS: The Pediatric Surgical Foundation was developed following the meeting to formalize coordination between institutions. Through international collaborations, operating room capacity has increased. A pediatric general surgery fellowship has expanded at Mulago and Mbarara hospitals supplemented by an international fellowship in multiple disciplines. Coordinated outreach camps have continued to assist with training and service delivery in rural regional hospitals. CONCLUSION: Collaborations between disciplines, both within LMICs and with international partners, are required to advance children's surgery. The unification of stakeholders across clinical disciplines and institutional partnerships can facilitate increased children's surgical capacity. Such a process may prove useful in other LMICs with a wide range of children's surgery stakeholders.

Bromodomain protein BRD4 is increased in human placentas from women with early-onset preeclampsia.

Preeclampsia affects 5% of all pregnancies and is a serious disorder of pregnancy, characterised by high maternal blood pressure, placental hypoxia, fluid retention (oedema) and proteinuria. Women with preeclampsia are associated with exaggerated levels of pro-inflammatory cytokines, chemokines and anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFLT1). Studies in non-gestational tissues have described the bromodomain (BRD) and extraterminal family of proteins, in particular BRD4 to play a critical role in propagating inflammation and is currently a therapeutic target for treating cancer, lung inflammation and asthma. The aims of this study were to: (i) determine the effect of severe early-onset preeclampsia on placental BRD4 expression; (ii) the effect of loss of BRD4 function by siRNA-targeted knockdown or with the BRD inhibitor JQ1 in human primary trophoblast cells and human umbilical vein endothelial cells (HUVECs) on TNF-stimulated production of pro-inflammatory mediators, cell adhesion molecules and anti-angiogenic markers and (iii) the effect of BRD4 suppression on placental sFLT1 secretion under hypoxia conditions and in preeclampic placenta. BRD4 mRNA expression was significantly increased (sevenfold) in severe early-onset preeclampsia placenta. BRD4 silencing resulted in a significant reduction in TNF-induced IL6, CXCL8, CCL2, CXCL1 and sFLT1-e15a mRNA expression and IL6, CXCL8, CCL2, CXCL1 and sFLT1 secretion in primary trophoblast and HUVECs. Additionally, JQ1 treatment significantly reduced placental sFLT1 secretion under hypoxic conditions and in preterm preeclamptic placenta. In conclusion, these findings suggest BRD4 may play a central role in propagating inflammation and endothelial dysfunction associated with the pathophysiology of early-onset preeclampsia.

IRF5 is increased in labouring myometrium and regulates pro-labour mediators.

Preterm birth continues to be the leading cause of neonatal mortality and morbidities that can extend into adult life. Few treatment options stem from our incomplete understanding of the mechanisms of human labour and delivery. Activation of the inflammatory response in gestational tissues by inflammation and/or infection leads to the production of pro-inflammatory and pro-labour mediators, thus preterm birth. Interferon regulatory factor 5 (IRF5) has recently emerged as an important pro-inflammatory transcription factor involved in acute and chronic inflammation. The aims of this study were to determine the expression of IRF5 in human myometrium from labouring and non-labouring women, and whether IRF5 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by pro-inflammatory cytokines or toll-like receptor (TLR) ligands. IRF5 mRNA and protein expression was significantly higher in human myometrium after spontaneous term labour, compared to non-labouring tissues. IRF5 mRNA expression was also significantly higher in primary myometrial cells treated with the pro-inflammatory cytokines IL1B or TNF. In primary myometrial cells, IRF5 knockdown by siRNA (siIRF5) was associated with significantly decreased expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1) and contraction-associated proteins PTGS2, PGF2α and PTGFR when in the presence of IL1B, TNF, fsl-1 (TLR2/6 ligand) or flagellin (TLR5 ligand). siIRF5-transfected cells also displayed decreased NF-κB RELA transcriptional activity in the presence of these preterm birth mediators. Our study suggests a novel role for IRF5 in the regulation of the inflammatory response in human myometrium.

PARK7 regulates inflammation-induced pro-labour mediators in myometrial and amnion cells.

Preterm birth is a prevalent cause of neonatal deaths worldwide. Inflammation has been implicated in spontaneous preterm birth involved in the processes of uterine contractility and membrane rupture. Parkinson protein 7 (PARK7) has been found to play an inflammatory role in non-gestational tissues. The aims of this study were to determine the expression of PARK7 in myometrium and fetal membranes with respect to term labour onset and to elucidate the effect of PARK7 silencing in primary myometrium and amnion cells on pro-inflammatory and pro-labour mediators. PARK7 mRNA expression was higher in term myometrium and fetal membranes from women in labour compared to non-labouring samples and in amnion from preterm deliveries with chorioamnionitis. In human primary myometrial cells transfected with PARK7 siRNA (siPARK7), there was a significant decrease in IL1B, TNF, fsl-1 and poly(I:C)-induced expression of pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2), adhesion molecule ICAM1, prostaglandin PGF2α and its receptor PTGFR. Similarly, amnion cells transfected with siPARK7 displayed a decrease in IL1B-induced expression of IL6, CXCL8 and ICAM1. In myometrial cells transfected with siPARK7, there was a significant reduction of NF-κB RELA transcriptional activity when stimulated with fsl-1, flagellin and poly(I:C), but not with IL1B or TNF. Collectively, our novel data describe a role for PARK7 in regulating inflammation-induced pro-inflammatory and pro-labour mediators in human myometrial and amnion cells.

Placental Ras Regulates Inflammation Associated with Maternal Obesity.

Heightened placental inflammation and dysfunction are commonly associated in pregnant obese women compared to their pregnant lean counterparts. The small GTPase superfamily members known as the rat sarcoma viral oncogene homolog (Ras) proteins, in particular, the K-Ras and H-Ras isoforms, have been implicated to regulate inflammation. The aims were to determine the placental Ras expression and activity with maternal obesity and its role in regulating placental inflammation. Human placenta was obtained at term Caesarean section from lean and obese pregnant women to determine the effect of maternal obesity on Ras protein expression and activity. To determine the effect of Ras on inflammation induced by bacterial endotoxin LPS and proinflammatory cytokines TNF-α or IL-1ß, the chemical inhibitor lonafarnib (total Ras inhibitor) and siRNA (siKRAS and siHRAS) were used. Total Ras protein expression together with combined K-Ras and H-Ras activity was significantly increased in the placenta of obese pregnant women and when stimulated with LPS, IL-1ß, or TNF-α. Lonafarnib significantly suppressed LPS-, IL-1ß-, or TNF-α-induced IL-6, IL-8, MCP-1, and GRO-α expression and secretion in placental tissue. Primary trophoblast cells transfected with siKRAS or siHRAS demonstrated only K-Ras silencing significantly decreased IL-1ß-, TNF-α-, or LPS-induced IL-6, IL-8, and MCP-1 expression and secretion. Furthermore, siKRAS significantly reduced downstream ERK-1/2 activation induced by LPS. In trophoblast cells, ERK-1/2 signalling is required for IL-6, IL-8, MCP-1, and GRO-α secretion. These studies implicate a role for K-Ras in regulating inflammation in human placenta. Suppressing overactive placental K-Ras function may prevent adverse fetal outcomes complicated by maternal obesity.

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.

SMAD7 regulates proinflammatory and prolabor mediators in amnion and myometrium.

Preterm birth continues to be a significant public health problem. Infection (bacterial and or viral) and inflammation, by stimulating proinflammatory cytokines, adhesion molecules, and matrix metalloproteinase 9 (MMP9), play a central role in the rupture of membranes and myometrial contractions. SMAD7 has been implicated in regulating the inflammatory response; however, no studies have been performed with regard to human labor. In this study, we determined the effect of spontaneous human labor and prolabor mediators on SMAD7 expression in myometrium and fetal membranes. Functional studies were employed to investigate the effect of siRNA knockdown of SMAD7 (siSMAD7) in regulating infection and inflammation-induced prolabor mediators. SMAD7 mRNA and protein expression were significantly higher with spontaneous term labor, compared to no labor, in myometrium and fetal membranes. SMAD7 expression was also significantly higher in amnion from women with preterm chorioamnionitis. The proinflammatory cytokines IL1B and TNF, the bacterial product fsl-1, and the viral dsRNA analog poly(I:C) significantly increased SMAD7 in myometrial cells and amnion cells. In myometrial cells, siSMAD7 cells significantly decreased cytokine (IL6) and chemokine (CXCL1, CXCL8, CCL2 are also known as GRO-alpha, interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1)) production induced by IL1B, TNF, and fsl-1. There was also a decrease in the expression of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in siSMAD7 cells, and MMP9 expression. In amnion, siSMAD7 cells treated with IL1B also decreased cytokine and chemokine production, ICAM1 and MMP9 expression. In conclusion, we report a proinflammatory role for SMAD7 in human gestational tissues, with SMAD7 silencing attenuating the inflammatory response.

Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro.

STUDY QUESTION: Does proviral integration site for Moloney murine leukaemic virus (PIM)1 kinase play a role in regulating the inflammatory processes of human labour and delivery? SUMMARY ANSWER: PIM1 kinase plays a critical role in foetal membranes in regulating pro-inflammatory and pro-labour mediators. WHAT IS KNOWN ALREADY: Infection and inflammation have strong causal links to preterm delivery by stimulating pro-inflammatory cytokines and collagen degrading enzymes, which can lead to rupture of membranes. PIM1 has been shown to have a role in immune regulation and inflammation in non-gestational tissues; however, its role has not been explored in the field of human labour. STUDY DESIGN, SIZE, DURATION: PIM1 expression was analysed in myometrium and/or foetal membranes obtained at term and preterm (n = 8-9 patients per group). Foetal membranes, freshly isolated amnion cells and primary myometrial cells were used to investigate the effect of PIM1 inhibition on pro-labour mediators (n = 5 patients per treatment group). PARTICIPANTS/MATERIALS, SETTING AND METHODS: Foetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and from preterm pre-labour rupture of membranes (PPROM) (n = 9 per group). Amnion was collected from women with and without preterm chorioamnionitis (n = 8 per group). Expression of PIM1 kinase was determined by qRT-PCR and western blotting. To determine the effect of PIM1 kinase inhibition on the expression of pro-inflammatory and pro-labour mediators induced by bacterial products lipopolysaccharide (LPS) (10 µg/ml) and flagellin (1 µg/ml) and pro-inflammatory cytokine tumour necrosis factor (TNF) (10 ng/ml), chemical inhibitors SMI-4a (20 µM) and AZD1208 (50 µM) were used in foetal membrane explants and siRNA against PIM1 was used in primary amnion cells. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: PIM1 expression was significantly increased in foetal membranes after spontaneous term labour compared to no labour at term and in amnion with preterm chorioamnionitis compared to preterm with no chorioamnionitis. There was no change in PIM1 expression with preterm labour or PPROM compared to preterm with no labour or PPROM. In human foetal membranes, PIM1 inhibitors SMI-4a and AZD1208 significantly decreased the expression of pro-inflammatory cytokine interleukin-6 (IL6) and chemokines CXCL8 and CCL2 mRNA and release, prostaglandin prostaglandin F2α (PGF2α) release, adhesion molecule intercellular adhesion molecule 1 mRNA expression and release, and oxidative stress marker 8-isoprostane release after stimulation with either LPS or flagellin. Primary amnion cells transfected with PIM1 siRNA also showed decreased expression of IL6, CXCL8 and CCL2, PTGS2 mRNA and PGF2α release, and matrix metalloproteinase-9 (MMP9) expression, when stimulated with TNF. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The conclusions were drawn from in vitro experiments using foetal membrane explants and primary cells isolated from amnion. Animal models are necessary to determine whether PIM1 kinase inhibitors can prevent spontaneous preterm birth in vivo. WIDER IMPLICATIONS OF THE FINDINGS: PIM1 kinase inhibitors may provide a novel therapeutic approach for preventing spontaneous preterm birth. STUDY FUNDING/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. The authors have no conflict of interest.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery.

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.

Genome-wide association study and mouse expression data identify a highly conserved 32 kb intergenic region between WNT3 and WNT9b as possible susceptibility locus for isolated classic exstrophy of the bladder.

Bladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 × 10(-5); follow-up: P = 0.0025; combined: 1.09 × 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway.

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor.

Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.

Serious complications in male infant circumcisions in Scandinavia indicate that this always be performed as a hospital-based procedure.

AIM: More than 7000 male circumcisions are performed in Scandinavia every year. Circumcision is regulated in different ways in Sweden and Denmark and Norway. This study reviewed and described factors associated with complications of circumcision in infant boys in Scandinavia over the last two decades and discussed how these complications could be avoided. METHODS: Data on significant complications following circumcision on boys under the age of one in Scandinavia over the last 20 years were collected. Information was retrieved from testimonies of circumcisers, witnesses, medical records and verdicts. A systematic review was performed of fatal cases in the literature. RESULTS: We found that 32 cases had been reported to the health authorities in the three countries, and we identified a total of 74 complications in these cases. These included four boys with severe bleeding and circulatory shock, which ended in the death of one boy. Other less serious complications may have occurred in other boys, but not been reported. CONCLUSION: Complications following male circumcision in Scandinavia were relatively rare, but serious complications did occur. Based on the analyses of the severe cases, we argue that circumcision should only be performed at hospitals with 24-hour emergency departments.

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3(hi) regulatory T cells.

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen-specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE-labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T-cell responses. High LPS RGPE increased IFN-γ(+) Th1 and IL-4(+) Th2 effector cell induction and consistently decreased CD4(+) Foxp3(hi) Treg-cell induction. IL-10-producing T-cell frequency was unaltered, but IL-10 secretion was increased by high LPS RGPE. RGPE-stimulation of TLR-transfected cell lines revealed that high LPS pollen also contained a TLR2-ligand, and both batches a TLR9-ligand. Beta-1,3-glucans were detected in large and small MW fractions and were also T-cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2- into a Th1-response. Instead, proinflammatory responses are exacerbated and Foxp3(hi) Treg-cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.

FOXM1 is lower in human fetal membranes after spontaneous preterm labour and delivery.

Spontaneous preterm birth is usually associated with infection, inflammation or both. Forkhead box (FOX) M1 (FOXM1), a member of the FOX family of transcription factors, has been associated with inflammation. The aim of this study was to determine whether FOXM1 regulates the expression and release of pro-labour mediators in human gestational tissues. FOXM1 mRNA and protein expression were determined in fetal membranes from women at (1) preterm no labour: Caesarean section with no labour and (2) preterm labour: after spontaneous labour and delivery. Primary amnion cells were utilised to investigate the effect of small interfering RNA (siRNA)-mediated gene silencing of FOXM1 on pro-labour mediators. Spontaneous preterm labour decreased FOXM1 gene and nuclear protein expression. FOXM1 silencing in primary amnion cells increased interleukin (IL)-1ß-induced pro-inflammatory cytokines (IL-6 and IL-8 mRNA expression and secretion), cyclooxygenase (COX)-2 expression and subsequent prostaglandin (PG)E2 and PGF2α release as well as gene expression and secretion of the matrix-degrading enzyme matrix metalloproteinase 9 (MMP-9). In conclusion, spontaneous preterm labour is associated with decreased FOXM1 expression in fetal membranes.

A novel role for FOXO3 in human labor: increased expression in laboring myometrium, and regulation of proinflammatory and prolabor mediators in pregnant human myometrial cells.

Preterm birth is the leading factor causing neonatal mortality and morbidity. Inflammation plays a central role in stimulating uterine contractility, which is responsible for approximately one-third of all preterm births. Recent studies have shown that the transcription factor Forkhead box O3 (FOXO3) regulates inflammation in nongestational tissues such as adipocytes and hepatocytes. Thus, in this study, we sought to determine the effect of 1) human term labor on myometrial FOXO3 expression and 2) FOXO3 inhibition and FOXO3 overexpression on proinflammatory and prolabor mediators in human myometrial cells. Higher FOXO3 gene and protein expression were detected in myometrium obtained from women in labor when compared to samples taken from nonlaboring women. Myometrial cells were isolated from pregnant human myometrium, and FOXO3 silencing was achieved using siRNA and overexpression using a cDNA clone. We found that the loss of FOXO3 in myometrial cells was associated with a significant decrease in IL1B-induced IL6 and IL8 expression and production, cyclooxygenase ([COX]-2, official symbol PTGS2) expression and subsequent prostaglandin (PGE2 and PGF2alpha) release, and matrix metalloproteinase 9 (MMP9) and mRNA expression and activity. Conversely, FOXO3 overexpression increased cytokine expression and secretion, prostaglandin production, and MMP9 expression in myometrial cells treated with IL1B. In summary, we have identified FOXO3 as an upstream mediator of inflammation in human myometrium. Thus, FOXO3 may present an alternative therapeutic target for preventing preterm birth and its associated morbidity and mortality.

SIRT6 is decreased with preterm labor and regulates key terminal effector pathways of human labor in fetal membranes.

Preterm birth is a major determinant of neonatal mortality and morbidity, affecting approximately one-third of preterm births as a result of prelabor rupturing of membranes. Infection and inflammation have strong causal links to preterm delivery, resulting in the activation of nuclear factor-kappaB (NFKB) and its downstream targets. Human sirtuin (SIRT) 6, which has ADP-ribosyl transferase and deacetylase activity, exhibits anti-inflammatory actions. The aims of this study were to determine the effect of 1) human preterm labor on SIRT6 expression in human gestational tissue and 2) the effect in primary amnion cells of SIRT6 inhibition, using small interfering RNA (siRNA) on prolabor mediators. To determine the effect of human preterm labor on SIRT6 expression, human fetal membranes were collected from women at preterm at the time of Cesarean section (no labor; n = 9) and from women after spontaneous labor and delivery (n = 9). SIRT6 mRNA and protein expression were significantly lower in fetal membranes after spontaneous preterm labor. Transfection of primary amnion cells with SIRT6 siRNA was associated with an increase in IL-1beta-induced proinflammatory cytokine gene expression and release (IL6, IL8, TNF [TNF-alpha]), cyclooxygenase ([COX]-2; official symbol PTGS2) expression and subsequent prostaglandin (PGE(2) and PGF(2alpha)) release, and MMP9 gene expression and release of pro-MMP9. To determine whether SIRT6 affects NFKB transcriptional activity, primary amnion cells were transfected with NFKB tagged with luciferase and stimulated with IL1B. As expected, IL1B induced NFKB transcriptional activity. However, when cells were also cotransfected with a vector expressing SIRT6, there was a decrease in NFKB transcriptional activity. In conclusion, SIRT6 plays a role in regulating the terminal effector pathways of human labor and delivery via the NFKB pathway.

Dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta, foetal membranes and myometrium.

A tenet of contemporary obstetrics is that a significant proportion of preterm births involve bacterial infection. Bacterial endotoxin induces pro-inflammatory cytokines, prostaglandins and proteases via the pro-inflammatory pathway nuclear factor-κB (NF-κB), which plays a key role in initiating uterine contractions and rupture of foetal membranes. In non-gestational tissues, the phytophenols curcumin, naringenin and apigenin exert anti-inflammatory properties via inhibition of NF-κB. The aim of this study was to determine whether these treatments regulate pro-inflammatory and pro-labour mediators in human gestational tissues. Placenta, foetal membranes and myometrium were treated with curcumin, naringenin and apigenin in the presence of lipopolysaccharide (LPS) or interleukin (IL)-1ß. In placenta and foetal membranes, all treatments significantly reduced LPS-stimulated release and gene expression of pro-inflammatory cytokines IL-6 and IL-8; placenta decreased cyclooxygenase (COX-2) mRNA expression, subsequent release of prostaglandins PGE2 and PGF2α and expression and activity of matrix-degrading enzyme matrix metalloproteinase (MMP)-9. In myometrial cells, all treatments attenuated IL-1ß-induced COX-2 expression, release of PGE2 and PGF2α and expression and activity of MMP-9. Although naringenin significantly attenuated IL-1ß-induced IL-6 and IL-8 mRNA expression and release, there was no effect of curcumin and apigenin. LPS-stimulated release of 8-isoprostane, a marker of oxidative stress, was attenuated by all treatments. NF-κB p65 DNA-binding activity was also decreased using these treatments. In conclusion, curcumin, naringenin and apigenin exert anti-inflammatory properties in human gestational tissues by inhibiting the transcriptional activity of NF-κB. Further studies should be undertaken to define a possible implication of these natural spices in the management of preterm labour and delivery.