Placental inflammation in pre-eclampsia by Nod-like receptor protein (NLRP)3 inflammasome activation in trophoblasts.

Pre-eclampsia is associated with increased levels of cholesterol and uric acid and an inflamed placenta expressing danger-sensing pattern recognition receptors (PRRs). Crystalline cholesterol and uric acid activate the PRR Nod-like receptor protein (NLRP)3 inflammasome to release interleukin (IL)-1ß and result in vigorous inflammation. We aimed to characterize crystal-induced NLRP3 activation in placental inflammation and examine its role in pre-eclampsia. We confirmed that serum total cholesterol and uric acid were elevated in pre-eclamptic compared to healthy pregnancies and correlated positively to high sensitivity C-reactive protein (hsCRP) and the pre-eclampsia marker soluble fms-like tyrosine kinase-1 (sFlt-1). The NLRP3 inflammasome pathway components (NLRP3, caspase-1, IL-1ß) and priming factors [complement component 5a (C5a) and terminal complement complex (TCC)] were co-expressed by the syncytiotrophoblast layer which covers the placental surface and interacts with maternal blood. The expression of IL-1ß and TCC was increased significantly and C5a-positive regions in the syncytiotrophoblast layer appeared more frequent in pre-eclamptic compared to normal pregnancies. In-vitro activation of placental explants and trophoblasts confirmed NLRP3 inflammasome pathway functionality by complement-primed crystal-induced release of IL-1ß. This study confirms crystal-induced NLRP3 inflammasome activation located at the syncytiotrophoblast layer as a mechanism of placental inflammation and suggests contribution of enhanced NLRP3 activation to the harmful placental inflammation in pre-eclampsia.

Toll-like receptor profiling of seven trophoblast cell lines warrants caution for translation to primary trophoblasts.

INTRODUCTION: Excessive placental inflammation is associated with pregnancy complications. Toll-like receptors (TLRs) are sensors for danger signals from infections and damaged tissue and initiate inflammation. Trophoblasts in the placenta broadly express TLRs. Trophoblast cell lines are used as surrogates for primary trophoblasts for in vitro studies, but the inflammatory translatability of trophoblast cell lines warrants examination. We aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines and compare this to primary trophoblasts. METHODS: The five choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P, and the two SV40 transfected trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts (n = 6). TLR1-10 gene expression was analyzed by RT-qPCR. Cells were stimulated by specific TLR1-9 ligands for 24 h and cytokine release was measured by a 10-plex immunoassay. RESULTS: All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. In contrast, SV40 transfected cell lines showed restricted TLR gene expression, but SGHPL-5 cells displayed significantly increased levels of interleukin (IL)-6, IL-8, IL-12 and vascular endothelial growth factor A after TLR3 and/or TLR4 activation (P < 0.01), while TLR2 activation increased IL-6 and IL-8 levels (P < 0.05). HTR8/SVneo cells responded to TLR3 activation by increased IL-6 and interferon (IFN)-γ (P < 0.05). The SGHPL-5 TLR profile most closely resembled primary trophoblast. DISCUSSION: The characterized trophoblast cell line TLR profiles serve as a reference and warrant caution when selecting trophoblast cell lines as in vitro models for immune responses in primary trophoblasts.

Meta-analysis of the human leukocyte antigen-G (HLA-G) 14 bp insertion/deletion polymorphism as a risk factor for preeclampsia.

The non-classical major histocompatibility complex, human leukocyte antigen (HLA)-G, plays an important role in pregnancy. HLA-G mediates proper interaction between maternal immune cells and fetal trophoblasts invading the uterine wall, to ensure successful placental development and function. Several HLA-G gene variants have been shown to be associated with development of preeclampsia (PE), but the reported associations of the HLA-G 14 base pair (bp) insertion/deletion (I/D) polymorphism (rs66554220) with PE are inconsistent. In this meta-analysis of HLA-G 14 bp I/D in each member of the family triad, we estimated risk (odds ratio [OR], 95% confidence interval) of associations with PE based on nine published offspring, nine mother and three father case-control studies. No significant increased risk associations between PE and HLA-G 14 bp I/D were detected in any of the family triad members (offspring: OR = 1.08-1.21, P = 0.57-0.74; mothers: OR = 1.11-1.28, P = 0.07-0.44; fathers: OR = 1.09-1.65, P = 0.07-0.70). Of the 20 comparisons performed, 14 (70%) were non-heterogeneous and seven of these had zero heterogeneity (I(2) = 0%). Sensitivity treatment confirmed robustness for the overall lack of association for HLA-G 14 bp I/D. In subgroup analysis, significant association between HLA-G 14 bp I/D and PE was shown in offspring from primipara (OR = 1.66-1.95, P = 0.04) and European Caucasian pregnancies (OR = 1.37-2.03, P = 0.02-0.03). However, heterogeneity and sensitivity tests suggest that further investigation is needed to determine if HLA-G 14 bp I/D is involved in trophoblast HLA-G expression and PE development in these subgroups.

Cytomegalovirus antibody status at 17-18 weeks of gestation and pre-eclampsia: a case-control study of pregnant women in Norway.

OBJECTIVE: To assess the association between maternal cytomegalovirus (CMV) antibodies in mid-pregnancy and pre-eclampsia. DESIGN: Nested case-control study. SETTING: Pregnancies registered in the Norwegian Mother and Child Cohort Study (MoBa): a large population-based pregnancy cohort (1999-2006). SAMPLE: A cohort of 1500 women with pre-eclampsia and 1000 healthy pregnant women. METHODS: Plasma samples and pregnancy-related information were provided by the MoBa. Antibody status (CMV IgG and CMV IgM) and levels (CMV IgG) at 17-18 weeks of gestation were determined by enzyme-linked immunosorbent assay (ELISA). MAIN OUTCOME MEASURE: A diagnosis of pre-eclampsia, as defined in the Medical Birth Registry of Norway. RESULTS: There was no evidence of an effect of CMV IgG seropositivity on the likelihood of developing pre-eclampsia, and CMV IgG antibody levels among women who were seropositive did not differ between groups. Adjusted for maternal age, parity and smoking, the odds ratio for pre-eclampsia in women seropositive for CMV IgG was 0.89 (95% CI 0.74-1.05; P = 0.17). The proportions of women who were seropositive for IgM did not differ between women with pre-eclampsia and women who were healthy (P = 0.98). Among nulliparous women, the proportion of women who were seropositive for CMV IgG was slightly lower among women with pre-eclampsia (53.5%) than among healthy women (59.8%) (P = 0.03). Subgroup analyses were performed for women with early or late onset pre-eclampsia, with preterm delivery and/or with neonates that were small for gestational age, but antibody status did not differ between pre-eclampsia subtypes and controls. CONCLUSIONS: The presence of maternal antibodies to CMV was not associated with pre-eclampsia in our study. The results suggest that CMV infection is unlikely to be a major cause of pre-eclampsia.

Violent behaviour in U.K. military personnel returning home after deployment.

BACKGROUND: There is growing concern about an alleged rise in violent behaviour amongst military personnel returning from deployment to Iraq and Afghanistan. The aims of this study were to determine the prevalence of violence in a sample of U.K. military personnel following homecoming from deployment in Iraq and to examine the impact of deployment-related experiences, such as combat trauma, on violence, and the role of sociodemographics and pre-enlistment antisocial behaviour. METHOD: This study used baseline data from a cohort study of a large randomly selected sample of U.K. Armed Forces personnel in service at the time of the Iraq war (2003). Regular personnel (n=4928) who had been deployed to Iraq were included. Data, collected by questionnaire, included information on deployment experiences, sociodemographic and military characteristics, pre-enlistment antisocial behaviour, post-deployment health outcomes and a self-report measure of physical violence in the weeks following return from deployment. RESULTS: Prevalence of violence was 12.6%. This was strongly associated with pre-enlistment antisocial behaviour [adjusted odds ratio (aOR) 3.6, 95% confidence interval (CI) 2.9-4.4]. After controlling for pre-enlistment antisocial behaviour, sociodemographics and military factors, violence was still strongly associated with holding a combat role (aOR 2.0, 95% CI 1.6-2.5) and having experienced multiple traumatic events on deployment (aOR for four or more traumatic events 3.7, 95% CI 2.5-5.5). Violence on homecoming was also associated with mental health problems such as post-traumatic stress disorder (aOR 4.8, 95% CI 3.2-7.2) and alcohol misuse (aOR 3.1, 95% CI 2.5-3.9). CONCLUSIONS: Experiences of combat and trauma during deployment were significantly associated with violent behaviour following homecoming in U.K. military personnel. Post-deployment mental health problems and alcohol misuse are also associated with increased violence.

OS046. Genome-wide association scans identify novel maternalsusceptibility loci for preeclampsia.

INTRODUCTION: We have successfully utilized a family-based study design to localize several positional candidate preeclampsia susceptibility genes to chromosomes 2q22(ACVR2A,LCT,LRP1B,RND3,GCA),5q (ERAP2) and 13q(TNFSF13B). We now report on our continued positional cloning efforts using an alternative genome-wide association (GWA) mapping strategy in large Caucasian case-control cohorts from Australia and Norway. OBJECTIVES: To identify maternal genetic risk loci for preeclampsia. METHODS: The unrelated Australian samples (545 cases,547 controls) were genotyped using Illumina BeadChip technology (700K loci) and have been analyzed using PLINK. All unrelated Norwegian samples were genotyped across several Illumina BeadChip substrates and consist of 847 cases (700K loci) and 638 controls. The Norwegian control samples originate from other HUNT studies pertaining to migraine (n=95,700K loci), lung cancer (n=89,370K loci) and normal brain pathology (n=454,2.5M loci). To analyze a concordant set of 2.5-3 million genotypes across all Norwegian samples we are currently using MaCH to impute those loci not directly genotyped. The Norwegian GWA data will be analyzed in SOLAR utilizing empirical kinship estimates to account for any distant relatedness. RESULTS: 1078 Australian samples (538 cases,540 controls) and 648, 175 SNPs passed our quality control metrics. Two SNP associations (rs7579169,p=3.6×10(-7); rs12711941,p=4.3×10(-7)) satisfied our genome-wide significant threshold (p<5.1×10(-7)). These SNPs reside less than 15kb downstream from the 3 terminus of the Inhibin, beta B (INHBB) gene on 2q14.2. Sequencing of the INHBB locus in our patient cohort identified a third intergenic SNP to significantly associate with preeclampsia (rs7576192,p=1.5×10(-7)). These three SNPs confer risk (OR>1.56) and are in strong linkage disequilibrium with each other (r(2)>0.9) but not with any other genotyped SNP ±200kb. The analysis of the Norwegian GWAS is underway. CONCLUSION: The Australian GWAS has identified a novel preeclampsia risk locus on chromosome 2q. The INHBB gene closest to our SNP associations is a plausible positional candidate susceptibility gene. There is a substantive body of evidence implicating inhibins, activins and other members of the TGF-ßsuperfamily to have a role in the development of preeclampsia. The biological connection between ACVR2A and INHBB leads us to speculate that our linkage-based and GWA-based study designs, respectively, have identified a key biological pathway involved in susceptibility to preeclampsia.

OS070. Shared genetic risk factors for preeclampsia and cardiovascular disease.

INTRODUCTION: There is compelling evidence to support the hypothesis that a maternal constitutional predisposition to cardiovascular disease (CVD) is a key component in development of preeclampsia. In particular, CVD and preeclampsia share pathological features such as endothelial dysfunction and inflammation, and have several metabolic abnormalities in common. In support of this hypothesis, our recent genetic dissection of the Australian preeclampsia susceptibility locus on chromosome 2q22 revealed shared novel genetic risk factors for preeclampsia and CVD-related traits. OBJECTIVES: To replicate association between our recently reported 2q22 preeclampsia risk variants and CVD-related traits in an independent Australian population based cohort. METHODS: Four independent SNPs from four genes, rs35821928 (LRP1B), rs17783344 (GCA), rs115015150 (RND3) and rs2322659 (LCT), were recently found to be significantly associated with preeclampsia susceptibility and CVD-related traits. These SNPs were genotyped in a large independent Australian cohort rich in quantitative CVD risk traits; The Western Australian Pregnancy Cohort (Raine) Study. This cohort comprises of blood samples from 1246 mothers and 1461 adolescents and clinical measures such as, but not limited to, anthropometric measures of adiposity and lipid-related measures. Genetic association analyses of these four potential preeclampsia susceptibility SNPs against the CVD-related risk traits were performed using the software package R. All statistical analyses assumed an additive model of gene action. RESULTS: Several significant associations (p<0.05) for all four SNPs with a variety of CVD-related risk traits were detected, both for the mothers and the adolescents. The LRP1B SNP was associated with HDL/cholesterol ratio, LDL cholesterol, triglycerides, skinfold measures and weight. The GCA SNP was associated with total cholesterol, HDL cholesterol, serum insulin, hemoglobin, blood glucose, BMI and skinfold measures. The RND3 SNP was associated with triglycerides and waist-hip ratio. The LCT SNP was associated with hemoglobin, blood glucose and abdominal skinfold. CONCLUSION: We have recently identified genetic variants within the LRP1B, GCA, RND3 and LCT genes to be significantly associated with preeclampsia susceptibility and CVD-related risk traits. We have now demonstrated thatthese specific genetic variants are associated with CVD-related risk traits in an independent population. Our collective findings provide substantial empirical data to support the hypothesis that genetic risk factors for preeclampsia and CVD are, at least in part, shared.

The acceptability of 'Trauma Risk Management' within the UK Armed Forces.

BACKGROUND: Trauma-support programmes may benefit employees of organizations that routinely expose their staff to traumatic events. However, in order for such programmes to be effective, staff need to find them acceptable. AIMS: To investigate whether Trauma Risk Management (TRiM), an example of such a programme, is acceptable within a military population and whether it is viewed as complementing or replacing pre-existing personnel support systems. METHODS: Semi-structured interviews were undertaken with serving Royal Navy personnel who had served on one of six ships, which had received TRiM training some 12-18 months previously. Three hundred and thirty interview transcripts were subjected to qualitative analysis and themed categories were generated. RESULTS: The majority of personnel who were aware of TRiM were supportive of its aims. TRiM was also viewed as supplementing other personnel support measures rather than replacing them. Personnel interviewed thought that TRiM practitioners needed to be carefully selected, supported by line management and to pay particular attention to the issue of confidentiality. CONCLUSIONS: The TRiM system appeared generally acceptable to military personnel and is seen to supplement rather than replace existing mechanisms. However, these data support careful selection of potential TRiM practitioners and demonstrate the need for senior management support for the system if it is to be accepted by those who might benefit from its use.

Risk factors for post-traumatic stress disorder among UK Armed Forces personnel.

BACKGROUND: There is considerable interest in understanding further the factors that increase the risk of post-traumatic stress disorder (PTSD) for military personnel. This study aimed to investigate the relative contribution of demographic variables; childhood adversity; the nature of exposure to traumatic events during deployment; appraisal of these experiences; and home-coming experiences in relation to the prevalence of PTSD 'caseness' as measured by a score of 50 on the PTSD Checklist (PCL) in UK Armed Forces personnel who have been deployed in Iraq since 2003. METHOD: Data were drawn from the first stage of a retrospective cohort study comparing UK military personnel who were deployed to the 2003 Iraq War with personnel serving in the UK Armed Forces on 31 March 2003 but who were not deployed to the initial phase of war fighting. Participants were randomly selected and invited to participate. The response rate was 61%. We have limited these analyses to 4762 regular service individuals who responded to the survey and who have been deployed in Iraq since 2003. RESULTS: Post-traumatic stress symptoms were associated with lower rank, being unmarried, having low educational attainment and a history of childhood adversity. Exposure to potentially traumatizing events, in particular being deployed to a 'forward' area in close contact with the enemy, was associated with post-traumatic stress symptoms. Appraisals of the experience as involving threat to one's own life and a perception that work in theatre was above an individual's trade and experience were strongly associated with post-traumatic stress symptoms. Low morale and poor social support within the unit and non-receipt of a home-coming brief (psycho-education) were associated with greater risk of post-traumatic stress symptoms. CONCLUSIONS: Personal appraisal of threat to life during the trauma emerged as the most important predictor of post-traumatic stress symptoms. These results also raise the possibility that there are important modifiable occupational factors such as unit morale, leadership, preparing combatants for their role in theatre which may influence an individual's risk of post-traumatic stress symptoms. Therefore interventions focused on systematic preparation of personnel for the extreme stress of combat may help to lessen the psychological impact of deployment.