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.

UK Dental Medicines Advisory Service: questions asked by dentists - part 2: drugs used for medical emergencies in dental practice.

The UK Dental Medicines Advisory Service (UKDMAS) provides advice to dentists and other dental healthcare professionals concerning the use of medicines and medical devices in dentistry. The commonly asked questions posed to the UKDMAS concerning the prescribing and administering of dental emergency drugs in dental practice are discussed, with answers supplemented by relevant information from clinicians. These include the drugs that need to be stocked in the emergency drugs kit in dental practice, their formats and storage, and the restrictions on which members of the dental team can administer the drugs.

SLC20A1 Is Involved in Urinary Tract and Urorectal Development.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

Proprietary non-animal stabilized hyaluronic acid/dextranomer gel (NASHA/Dx) for endoscopic treatment of grade IV vesicoureteral reflux: Long-term observational study.

BACKGROUND: Since 1993, children aged >1 year with persistent grade III-V vesicoureteral reflux (VUR) and febrile urinary tract infections (UTIs) attending Uppsala University Hospital have undergone endoscopic injection with proprietary non-animal stabilized hyaluronic acid/dextranomer gel (NASHA/Dx; Deflux®). OBJECTIVE: Investigate long-term incidence of UTI, bladder dysfunction, ureteral reimplantation and overall clinical findings following endoscopic injection of NASHA/Dx. STUDY DESIGN: Children with grade IV VUR diagnosed by voiding cystourethrogram (VCUG) and dilating VUR persisting for >1 year were included in this study. 15-25 years after endoscopic treatment, patients' hospital charts were studied. Information on bladder function and UTIs was obtained via questionnaire, 8-18 years after endoscopic treatment. RESULTS: 185 patients (69 boys, 116 girls) were included in the study; 237 grade IV VUR ureters were treated. All study patients were diagnosed with VUR after a febrile UTI (i.e. pyelonephritis). According to the last voiding cystourethrogram, 69% of ureters showed a positive response (VUR grade 0-I), 7% had VUR grade II and 23% had VUR grade ≥ III. 46 patients (25%) required ureteral reimplantation during follow-up. Among patients treated during the second 5-year period compared with the first (1998-2003 versus 1993-1998), there was a significant decrease in the rate of ureteral reimplantation (31% vs 16%; p = 0.0365). This difference may be attributable to developments over time in the injection technique. UTIs occurred in 30 patients (21% of the evaluable population): 28 females and 2 males. Febrile UTIs were reported in 14 patients (10%), all females. Forty-nine patients (34%) had bladder problems (e.g. underactivity, overactivity, incontinence). Five patients underwent ureteral reimplantation 'late', 6-10 years after the last endoscopic injection. In one male patient, calcification around the NASHA/Dx implantation site was observed during routine examination 2 years after endoscopic treatment; no intervention was required. No safety issues were observed in the remaining 97% of the study population. CONCLUSIONS: This study represents the longest published follow-up of Grade IV VUR patients undergoing endoscopic treatment. Three-quarters of patients did not need ureteral reimplantation. Optimal injection technique and higher injection volume were associated with a reduced ureteral reimplantation rate. Treatment with NASHA/Dx was durable and well tolerated: long-term risks of UTI, bladder dysfunction and recurrent VUR were low.

Further support linking the 22q11.2 microduplication to an increased risk of bladder exstrophy and highlighting LZTR1 as a candidate gene.

BACKGROUND: The bladder exstrophy-epispadias complex (BEEC) is a congenital malformation of the bladder and urethra. The underlying causes of this malformation are still largely unknown; however, aside from environment, genetics is thought to play an essential role. The recurrent 22q11.2 microduplication is the most persistently detected genetic aberration found in BEEC cases. METHODS: We performed array comparative genomic hybridization (array-CGH) analysis of 76 Swedish BEEC patients. Statistical analysis was performed on current dataset pooled with previously published data on the 22q11.2 microduplication in BEEC patients. We performed massive parallel sequencing (MPS) of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication followed by functional studies. RESULTS: We identified three additional cases with the 22q11.2 microduplication. Pooling data from this study with previously published reports showed a statistically significant enrichment of the 22q11.2 microduplication in BEEC patients (2.61% in cases vs. 0.08% in controls; OR = 32.6; p = 8.7 × 10-4 ). MPS of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication identified a novel variant in LZTR1 (p.Ser698Phe) in one patient. Functional evaluation of the LZTR1 p.Ser698Phe variant in live NIH 3T3 cells showed that the concentration and cytoplasmic mobility differ between the Lztr1wt and Lztr1mut , indicating a potential functional effect of the LZTR1mut . CONCLUSION: Our study further emphasizes the involvement of the 22q11.2 region in BEEC development and highlights LZTR1 as a candidate gene underlying the urogenital malformation.

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.

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.

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.

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.

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.

Pellino 1 is a novel regulator of TNF and TLR signalling in human myometrial and amnion cells.

Preterm birth is the primary cause of neonatal deaths and morbidities. Pathological processes causally linked to preterm birth are inflammation and infection. Pellino-1 (Peli1) has previously been found to regulate the inflammatory response in non-gestational tissues in response to toll-like receptor (TLR) ligands and pro-inflammatory cytokines. The aims of this study were to determine the effect of labor on Peli1 expression in myometrium and fetal membranes, and the effect of Peli1 silencing by siRNA (siPELI1) on the production of pro-inflammatory and pro-labor mediators. The expression of Peli1 was found to be higher in myometrium and fetal membranes with term labor, compared to non-laboring samples. Peli1 mRNA and protein expression was also higher in amnion from women with preterm histological chorioamnionitis. In human primary myometrial cells, siPELI1 transfected cells showed a decrease in pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2) and adhesion molecule ICAM1 when in the presence of pro-inflammatory cytokine TNF, TLR2/6 ligand fsl-1, TLR5 ligand flagellin, and TLR3 ligand poly(I:C). Similarly in primary amnion cells, siPELI1 transfected cells decreased IL1B-induced expression and secretion of IL6 and CXCL8. In siPELI1 transfected myometrial cells, there was a decrease in prostaglandin PGF2α and its receptor, PTGFR mRNA expression when treated with TNF. There was a decrease in NF-κB RELA transcriptional activity in siPELI1 transfected cells in the presence of TNF, fsl-1 and flagellin, but not poly(I:C). Our study suggests a novel role for Peli1 in regulating pro-inflammatory and pro-labor mediators through TNF and TLR signalling.

Evaluation of the ISL1 gene in the pathogenesis of bladder exstrophy in a Swedish cohort.

Bladder exstrophy is a congenital closure defect of the urinary bladder with a profound effect on morbidity. Although the malformation is usually sporadic, a genetic background is supported by an increased recurrence risk in relatives, higher concordance rates in monozygotic twins and several associated chromosomal aberrations. Recently, the ISL1 gene was presented as a candidate gene for bladder exstrophy and epispadias complex (BEEC) development in two different studies. In our study, we screened for genetic variants in the ISL1 gene in DNA from 125 Swedish patients using Sanger sequencing and array-CGH analysis. In addition, we evaluated ISL1 expression in RNA of human bladder during embryonic and fetal weeks 5-10 relative to that in lung tissue (week 9). In total, 21 single-nucleotide variants were identified, including a potentially novel missense variant, c.137C>G p.(Ala46Gly), substituting a conserved amino acid. This variant was inherited from an unaffected mother. No structural variants were identified. RNA sequencing revealed ISL1 mRNA expression during the critical time frame of human bladder development. In conclusion, we did not detect any known or likely pathogenic variants in the ISL1 gene in 125 Swedish BEEC patients, indicating that variation in the ISL1 gene is not a common genetic mechanism of BEEC development in the Swedish population.

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.

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.

TLR2, TLR3 and TLR5 regulation of pro-inflammatory and pro-labour mediators in human primary myometrial cells.

Preterm birth continues to be a significant global health care issue, due to our lack of understanding of the mechanisms that drive human labour and delivery. Toll-like receptors (TLRs) are essential in triggering an inflammatory response in human gestational tissues, leading to the production of pro-inflammatory and pro-labour mediators, and thus preterm birth. The aims of this study were to determine whether the adaptor molecules associated with TLR2, TLR3 and TLR5 signalling are involved in human myometrium. Primary human myometrial cells were transfected with siRNA against TIRAP, IRAK1, IRAK4, TAK1and stimulated with bacterial product fsl-1 (TLR2); TRIF, TRADD, TRAF6, RIP1, TAK1 and stimulated with dsRNA viral analogue poly(I:C) (TLR3); IRAK1, IRAK4, TAK1 and stimulated with bacterial product flagellin (TLR5), and assayed for production of pro-inflammatory and pro-labour mediators. Cells transfected with TIRAP, IRAK1, IRAK4 or TAK1 all showed a decrease in fsl-1-induced expression of cytokines (IL-1α, IL-1ß, IL-6), chemokines (GRO-α, IL-8, MCP-1), adhesion molecule ICAM-1, cyclooxygenase (COX)-2 mRNA and release of PGF2α and MMP-9 expression. Cells transfected with TRIF, TRAF6, RIP1 or TAK1 all decreased production of poly(I:C)-induced IL-1α, IL-1ß, IL-6, GRO-α, IL-8, MCP-1, ICAM-1 and MMP-9 expression. Cells transfected with IRAK1, IRAK4 or TAK1 all showed decreased expression of flagellin-induced cytokine and chemokine expression, ICAM-1 and MMP-9 expression. Lastly, transfection with these siRNAs decreased fsl-1, poly(I:C) and flagellin-induced NF-κB transcriptional activity. Our study signifies that these adaptor molecules are necessary for the proper production of cytokines, chemokines and pro-labour mediators after TLR ligation.

Hepatitis A virus cellular receptor 2 (HAVCR2) is decreased with viral infection and regulates pro-labour mediators OA.

PROBLEM: Intrauterine infection caused by viral infection has been implicated to contribute to preterm birth. Hepatitis A virus cellular receptor 2 (HAVCR2) regulates inflammation in non-gestational tissues in response to viral infection. METHOD OF STUDY: The aims of this study were to determine the effect of: (i) viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on HAVCR2 expression; and (ii) HAVCR2 silencing by siRNA (siHAVCR2) in primary amnion and myometrial cells on poly(I:C)-induced inflammation. RESULTS: In human foetal membranes and myometrium, HAVCR2 mRNA and protein expression was decreased when exposed to poly(I:C). Treatment of primary amnion and myometrial cells with poly(I:C) significantly increased the expression and release of pro-inflammatory cytokines TNF, IL1A, IL1B and IL6; the expression of chemokines CXCL8 and CCL2; the expression and secretion of adhesion molecules ICAM1 and VCAM1; and PTGS2 and PTGFR mRNA expression and the release of prostaglandin PGF2α . This increase was significantly augmented in cells transfected with siHAVCR2. Furthermore, mRNA expression of anti-inflammatory cytokines IL4 and IL10 was significantly decreased. CONCLUSION: Collectively, our data suggest that HAVCR2 regulates cytokines, chemokines, prostaglandins and cell adhesion molecules in the presence of viral infection. This suggests a potential for HAVCR2 activators as therapeutics for the management of preterm birth associated with viral infections.

Placental Pim-1 expression is increased in obesity and regulates cytokine- and toll-like receptor-mediated inflammation.

INTRODUCTION: Obesity is a growing epidemic, and as a consequence the number of obese pregnancies has also increased. Pregnancy is characterised by maternal and placental inflammation which is intensified with maternal obesity. The proviral integration site for Moloney murine leukemic virus (Pim)-1 protein is a serine/threonine kinase involved in a wide range of inflammatory diseases. In relation to obesity, however, its role has not been elucidated in human placenta. The aims were to determine the placental expression of Pim-1 with pre-existing maternal obesity and its role in regulating placental inflammation associated with obesity. METHODS: Human placenta was obtained at the time of term Caesarean section from lean and pre-existing obese pregnant women to determine the effect of maternal obesity on Pim-1 expression. To determine the effect of Pim-1 on the inflammatory response induced by bacterial endotoxin LPS and pro-inflammatory cytokines TNF-α or IL-1ß, the chemical inhibitor SMI-4a and siRNA were used. RESULTS: Pim-1 protein and mRNA expression was significantly increased in placenta of obese women. SMI-4a significantly suppressed the expression and release of pro-inflammatory cytokine IL-6 and chemokines GRO-α and MCP-1 when stimulated with LPS or TNF-α in placenta. Primary trophoblast cells transfected with Pim-1 siRNA had decreased expression and release of pro-inflammatory cytokines IL-1ß, IL-6, chemokines GRO-α and MCP-1, when stimulated with LPS, TNF-α or IL-1ß. DISCUSSION: The findings from this study implicate Pim-1 may contribute to placental inflammation in pregnancies complicated by maternal obesity. Thus, therapeutic targets for Pim-1 may improve fetal outcomes complicated by obese pregnancies.

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.

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.

Resveratrol ameliorates the chemical and microbial induction of inflammation and insulin resistance in human placenta, adipose tissue and skeletal muscle.

Gestational diabetes mellitus (GDM), which complicates up to 20% of all pregnancies, is associated with low-grade maternal inflammation and peripheral insulin resistance. Sterile inflammation and infection are key mediators of this inflammation and peripheral insulin resistance. Resveratrol, a stilbene-type phytophenol, has been implicated to exert beneficial properties including potent anti-inflammatory and antidiabetic effects in non-pregnant humans and experimental animal models of GDM. However, studies showing the effects of resveratrol on inflammation and insulin resistance associated with GDM in human tissues have been limited. In this study, human placenta, adipose (omental and subcutaneous) tissue and skeletal muscle were stimulated with pro-inflammatory cytokines TNF-α and IL-1ß, the bacterial product lipopolysaccharide (LPS) and the synthetic viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) to induce a GDM-like model. Treatment with resveratrol significantly reduced the expression and secretion of pro-inflammatory cytokines IL-6, IL-1α, IL-1ß and pro-inflammatory chemokines IL-8 and MCP-1 in human placenta and omental and subcutaneous adipose tissue. Resveratrol also significantly restored the defects in the insulin signalling pathway and glucose uptake induced by TNF-α, LPS and poly(I:C). Collectively, these findings suggest that resveratrol reduces inflammation and insulin resistance induced by chemical and microbial products. Resveratrol may be a useful preventative therapeutic for pregnancies complicated by inflammation and insulin resistance, like GDM.