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P22 Elevated levels of pentraxin 3 in HELLP syndrome and preeclampsia: GenPE study
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Poster Presentations / Pregnancy Hypertension 1, Supplement 1 (2010) S43–S76
infection with C. pneumoniae and CMV and immune response in normal and preeclampsia complicated pregnancies. The first noteworthy finding of this work has shown that preeclampsia was associated with increased C. pneumonia genomic DNA loads compared with normal pregnancy controls and had higher anti-CMV IgG seropositivity than in women with normotensive interuterine growth restriction and normal pregnancy controls. Additionally, data synthesis revealed that IgG seropositivity of C. pneumonia and CMV was more prevalent among women with preeclampsia than normal pregnancy controls. The second finding of this work observed that early onset preeclampsia had increased Toll like receptor (TLR)-2 and -4 mRNA and protein expressions, elevated mRNA expressions of cryopyrin, NF-κB subunits and IL-1β, as well as increased TNF-α: IL-10 and IL-6: IL-10 ratios compared with normal pregnancy controls. Third, through a comprehensive review of published literatures and in combination of our study results, data suggested that TLR2 (Arg753Gln) and TLR4 co-segregating (Asp299Gly and Thr399Ile) gene polymorphisms seems in susceptibility to development of early onset preeclampsia. Our research findings indicated that TLR- microbial with C. pneumonia and CMV interactions play an important role in maternal inflammatory syndroms in preeclampsia. This work may contribute for identification of novel anti-inflammatory targets for preeclampsia treatment, eventually leading to improved health care for both mom and fetus.
P21 Therapeutic effects of maternal melatonin on hemic/reperfusion-induced oxidative cerebral damage in neonatal rats Fumiaki Hamada 1 , Kazushi Watanabe 2 , Akihiko Wakatsuki 2 , Ryuhei Nagai 3 , Koichi Shinohara 2 , Yoshihiro Hayashi 3 , Rina Imamura 3 , Takao Fukaya 3 . 1 Kochi Prefectural Hata-Kenmin Hospital, Kochi, Japan; 3 Aichi Medical School, Japan; 4 Kochi Medical School, Japan Background: We previously demonstrated that prophylactic administration of melatonin to pregnant rats can protect against ischemia/reperfusion (I/R)-induced oxidative cerebral damage in the fetal rats. However, the effects of maternal administration of melatonin after an ischemic episode on the brains of neonatal rats exposed to oxidative stress in utero have not been evaluated. Objectives: The purpose of this present study was to investigate whether maternal administration of melatonin after an ischemic episode can prevent oxidative cerebral damage in neonatal rats. Methods: The utero-ovarian arteries were occluded bilaterally for 30 min in female Wistar rats on day 16 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation. Melatonin solution (10 mg/kg) or vehicle was injected intraperitoneally at post-reperfusion hr 0, 1, 3, 6, and 12. After surgery, melatonin solution (20μg/ml) or vehicle was administered freely via drinking water to vaginal delivery. Control rats underwent a sham operation. We collected brain tissue from neonatal rats that were delivered naturally and measured the respiratory control index (RCI) as indicators of mitochondrial respiratory activity. Histological evaluation was performed on the Cornu Ammonis (CA1) and Cornu Ammonis (CA3) regions of the hippocampus. Results: I/R significantly reduced the RCI, but melatonin administration at post-reperfusion hr 0 or 1 reversed I/R-induced reductions in the RCI. In contrast, melatonin administration at post-reperfusion hr 3 to 12 had no protective effect. Histological analysis revealed a decrease in the ratio of normal to whole pyramidal cell number in the CA1 and CA3 regions in the I/R group. While melatonin administration within 3hr protected against degeneration, administration 6hr after reperfusion failed to protect. Conclusions: These results suggest that maternal administration of melatonin within 1hr after an ischemic/oxidative episode can prevent I/Rinduced oxidative cerebral damage in neonatal rats.
P22 Elevated levels of pentraxin 3 in HELLP syndrome and preeclampsia: GenPE study Norma Serrano 1 , Luis Alfonso Diaz 1 , Carolina Paez 1 , Ricardo Ortiz 1 , Monica Beltran 1 , Alvaro Monterrosa 2 , Clara Meza 3 , German Monsalve 4 , Enrique Sanin 5 , Wilmar Saldarriaga 6 . 1 Universidad Autonoma de Bucaramanga UNAB, Santander, Colombia; 2 Universidad de Cartagena, Colombia; 3 CES, Colombia; 4 Clinica el Prado, Colombia; 5 Universidad Pontificia Bolivariana, Colombia; 6 Universidad del Valle, Colombia Background: Few small studies have suggested to Pentraxin 3 (PTX3), an inflammatory marker, to be associated with preeclampsia. However, there is considerable uncertainty whether this associated is real or an artifact.
Aims: To investigate the effect of PTX3 levels on preeclampsia and HELLP in a large case-control collection. Methods: GenPE is a Colombian large, multi-centre, ongoing case-control study designed to investigate genetic and non-genetic risk factors for PE (www.genpe.org). The current report includes 286 PE alone, 264 PE with HELLP and 281 healthy pregnancies (controls). All participants signed an informed consent document approved by the Ethical Review Board from the Universidad Autónoma de Bucaramanga. Plasma PTX3 was measured using ELISA technique (Alexis Biochemical, San Diego, USA). Odds ratios with 95% confidence interval (CI) using logistic regression were obtained to measure the association between PTX3 concentrations with the risk of PE with or without HELLP syndrome. Results: PTX3 levels were moderately elevated among preeclamptic women (7.62 ng/mL, IQR 8.13; p=0.0014) and significantly higher in patients with HELLP syndrome (8.49 ng/mL, IQR 10.21; p<0.0001) compared to normal pregnancies (5.45 ng/mL, IQR 6.08). After adjustment for potential confounders the odds ratio of PE without HELLP for women in top vs. bottom quartile compared to healthy pregnant women was 4.95 (95%CI: 2.37- 10.32). For a similar quartile comparison, the odds ratio of HELLP was of 9.36 (95%CI 3.72-23.52) compared to healthy pregnant women. P-value for a trend effect across quartiles was p<0.001 in both comparisons. Conclusion: Higher than average levels of PTX3 were strongly associated with preeclampsia. This effect was almost twice as high for women with HELLP. To minimise possible biases that could explain this association (reverse causation and residual confounding) prospective observational studies and genetic studies using Mendelian Randomization analysis are needed.
P23 Development of workflow for quantitative urinary proteomics using TMT™ labelling for potential biomarker discovery Hiten D. Mistry 1 , Andrew J. Weston 2 , Malcolm A. Ward 3 , Lucy C. Chappell 1 , Kate Bramham 1 , Lucilla Poston 1 . 1 Maternal & Fetal Research Unit, Division of Reproduction & Endocrinology, King’s College London, UK; 2 NIHR-Biomedical Research Centre, Institute of Psychiatry, King’s College London, UK; 3 KCL Proteomics Facility, Institute of Psychiatry, King’s College London, UK Background: Proteomics is a rapidly advancing technique which gives functional insight into gene expression in living organisms. Urine is an ideal medium for study as it is readily available, easily obtained, less complex than other bodily fluids and potentially a rich source of biomarkers. Aims: 1) To make a qualitative assessment of urine protein profiles from healthy non-pregnant (NP), 15 and 20 week healthy pregnant and pre-eclamptic (PE) women; 2) to develop a method of relative protein quantification, employing an isobaric labelling approach using Tandem Mass Tags (TMT™), suitable for potential biomarker discovery. Methods: Urine (1.8 ml) was collected following written informed consent from NP, 15 and 20 weeks’ gestation and clinically diagnosed PE women. Protein recovery was optimised using acetone precipitation and immunodepletion techniques. Qualitative profiles were carried out using SDS-PAGE on 3 individual urine samples from each group. Two selected gel regions from each group were also profiled using LC/MS/MS. Additionally, protein from 2 urine samples (15 weeks’ and PE) were subjected to intact TMT™ duplex labelling, gel-based trypsin fractionation and LC/MS/MS. ScaffoldQ+ was used to identify and quantify the relative protein levels. Results: Sufficient protein concentrations were obtained from the volumes used. Visual inspection of the gels identified differential presence between groups. ScaffoldQ+ confirmed 82 proteins with high confidence and a set of proteins identified only in PE urine. TMT™ reporters were identified from peptides of intact labelled proteins, allowing quantification of selected components of the proteome. Conclusions: We have successfully optimised a workflow for urine with varying protein concentrations with the potential for multiplex quantification of up to 6 samples, reducing error and analysis times in biomarker discovery experiments. Proteins with differential levels between groups have been identified, which could be targeted in subsequent work using selective reaction monitoring MS.
Poster Presentations / Pregnancy Hypertension 1, Supplement 1 (2010) S43–S76
infection with C. pneumoniae and CMV and immune response in normal and preeclampsia complicated pregnancies. The first noteworthy finding of this work has shown that preeclampsia was associated with increased C. pneumonia genomic DNA loads compared with normal pregnancy controls and had higher anti-CMV IgG seropositivity than in women with normotensive interuterine growth restriction and normal pregnancy controls. Additionally, data synthesis revealed that IgG seropositivity of C. pneumonia and CMV was more prevalent among women with preeclampsia than normal pregnancy controls. The second finding of this work observed that early onset preeclampsia had increased Toll like receptor (TLR)-2 and -4 mRNA and protein expressions, elevated mRNA expressions of cryopyrin, NF-κB subunits and IL-1β, as well as increased TNF-α: IL-10 and IL-6: IL-10 ratios compared with normal pregnancy controls. Third, through a comprehensive review of published literatures and in combination of our study results, data suggested that TLR2 (Arg753Gln) and TLR4 co-segregating (Asp299Gly and Thr399Ile) gene polymorphisms seems in susceptibility to development of early onset preeclampsia. Our research findings indicated that TLR- microbial with C. pneumonia and CMV interactions play an important role in maternal inflammatory syndroms in preeclampsia. This work may contribute for identification of novel anti-inflammatory targets for preeclampsia treatment, eventually leading to improved health care for both mom and fetus.
P21 Therapeutic effects of maternal melatonin on hemic/reperfusion-induced oxidative cerebral damage in neonatal rats Fumiaki Hamada 1 , Kazushi Watanabe 2 , Akihiko Wakatsuki 2 , Ryuhei Nagai 3 , Koichi Shinohara 2 , Yoshihiro Hayashi 3 , Rina Imamura 3 , Takao Fukaya 3 . 1 Kochi Prefectural Hata-Kenmin Hospital, Kochi, Japan; 3 Aichi Medical School, Japan; 4 Kochi Medical School, Japan Background: We previously demonstrated that prophylactic administration of melatonin to pregnant rats can protect against ischemia/reperfusion (I/R)-induced oxidative cerebral damage in the fetal rats. However, the effects of maternal administration of melatonin after an ischemic episode on the brains of neonatal rats exposed to oxidative stress in utero have not been evaluated. Objectives: The purpose of this present study was to investigate whether maternal administration of melatonin after an ischemic episode can prevent oxidative cerebral damage in neonatal rats. Methods: The utero-ovarian arteries were occluded bilaterally for 30 min in female Wistar rats on day 16 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation. Melatonin solution (10 mg/kg) or vehicle was injected intraperitoneally at post-reperfusion hr 0, 1, 3, 6, and 12. After surgery, melatonin solution (20μg/ml) or vehicle was administered freely via drinking water to vaginal delivery. Control rats underwent a sham operation. We collected brain tissue from neonatal rats that were delivered naturally and measured the respiratory control index (RCI) as indicators of mitochondrial respiratory activity. Histological evaluation was performed on the Cornu Ammonis (CA1) and Cornu Ammonis (CA3) regions of the hippocampus. Results: I/R significantly reduced the RCI, but melatonin administration at post-reperfusion hr 0 or 1 reversed I/R-induced reductions in the RCI. In contrast, melatonin administration at post-reperfusion hr 3 to 12 had no protective effect. Histological analysis revealed a decrease in the ratio of normal to whole pyramidal cell number in the CA1 and CA3 regions in the I/R group. While melatonin administration within 3hr protected against degeneration, administration 6hr after reperfusion failed to protect. Conclusions: These results suggest that maternal administration of melatonin within 1hr after an ischemic/oxidative episode can prevent I/Rinduced oxidative cerebral damage in neonatal rats.
P22 Elevated levels of pentraxin 3 in HELLP syndrome and preeclampsia: GenPE study Norma Serrano 1 , Luis Alfonso Diaz 1 , Carolina Paez 1 , Ricardo Ortiz 1 , Monica Beltran 1 , Alvaro Monterrosa 2 , Clara Meza 3 , German Monsalve 4 , Enrique Sanin 5 , Wilmar Saldarriaga 6 . 1 Universidad Autonoma de Bucaramanga UNAB, Santander, Colombia; 2 Universidad de Cartagena, Colombia; 3 CES, Colombia; 4 Clinica el Prado, Colombia; 5 Universidad Pontificia Bolivariana, Colombia; 6 Universidad del Valle, Colombia Background: Few small studies have suggested to Pentraxin 3 (PTX3), an inflammatory marker, to be associated with preeclampsia. However, there is considerable uncertainty whether this associated is real or an artifact.
Aims: To investigate the effect of PTX3 levels on preeclampsia and HELLP in a large case-control collection. Methods: GenPE is a Colombian large, multi-centre, ongoing case-control study designed to investigate genetic and non-genetic risk factors for PE (www.genpe.org). The current report includes 286 PE alone, 264 PE with HELLP and 281 healthy pregnancies (controls). All participants signed an informed consent document approved by the Ethical Review Board from the Universidad Autónoma de Bucaramanga. Plasma PTX3 was measured using ELISA technique (Alexis Biochemical, San Diego, USA). Odds ratios with 95% confidence interval (CI) using logistic regression were obtained to measure the association between PTX3 concentrations with the risk of PE with or without HELLP syndrome. Results: PTX3 levels were moderately elevated among preeclamptic women (7.62 ng/mL, IQR 8.13; p=0.0014) and significantly higher in patients with HELLP syndrome (8.49 ng/mL, IQR 10.21; p<0.0001) compared to normal pregnancies (5.45 ng/mL, IQR 6.08). After adjustment for potential confounders the odds ratio of PE without HELLP for women in top vs. bottom quartile compared to healthy pregnant women was 4.95 (95%CI: 2.37- 10.32). For a similar quartile comparison, the odds ratio of HELLP was of 9.36 (95%CI 3.72-23.52) compared to healthy pregnant women. P-value for a trend effect across quartiles was p<0.001 in both comparisons. Conclusion: Higher than average levels of PTX3 were strongly associated with preeclampsia. This effect was almost twice as high for women with HELLP. To minimise possible biases that could explain this association (reverse causation and residual confounding) prospective observational studies and genetic studies using Mendelian Randomization analysis are needed.
P23 Development of workflow for quantitative urinary proteomics using TMT™ labelling for potential biomarker discovery Hiten D. Mistry 1 , Andrew J. Weston 2 , Malcolm A. Ward 3 , Lucy C. Chappell 1 , Kate Bramham 1 , Lucilla Poston 1 . 1 Maternal & Fetal Research Unit, Division of Reproduction & Endocrinology, King’s College London, UK; 2 NIHR-Biomedical Research Centre, Institute of Psychiatry, King’s College London, UK; 3 KCL Proteomics Facility, Institute of Psychiatry, King’s College London, UK Background: Proteomics is a rapidly advancing technique which gives functional insight into gene expression in living organisms. Urine is an ideal medium for study as it is readily available, easily obtained, less complex than other bodily fluids and potentially a rich source of biomarkers. Aims: 1) To make a qualitative assessment of urine protein profiles from healthy non-pregnant (NP), 15 and 20 week healthy pregnant and pre-eclamptic (PE) women; 2) to develop a method of relative protein quantification, employing an isobaric labelling approach using Tandem Mass Tags (TMT™), suitable for potential biomarker discovery. Methods: Urine (1.8 ml) was collected following written informed consent from NP, 15 and 20 weeks’ gestation and clinically diagnosed PE women. Protein recovery was optimised using acetone precipitation and immunodepletion techniques. Qualitative profiles were carried out using SDS-PAGE on 3 individual urine samples from each group. Two selected gel regions from each group were also profiled using LC/MS/MS. Additionally, protein from 2 urine samples (15 weeks’ and PE) were subjected to intact TMT™ duplex labelling, gel-based trypsin fractionation and LC/MS/MS. ScaffoldQ+ was used to identify and quantify the relative protein levels. Results: Sufficient protein concentrations were obtained from the volumes used. Visual inspection of the gels identified differential presence between groups. ScaffoldQ+ confirmed 82 proteins with high confidence and a set of proteins identified only in PE urine. TMT™ reporters were identified from peptides of intact labelled proteins, allowing quantification of selected components of the proteome. Conclusions: We have successfully optimised a workflow for urine with varying protein concentrations with the potential for multiplex quantification of up to 6 samples, reducing error and analysis times in biomarker discovery experiments. Proteins with differential levels between groups have been identified, which could be targeted in subsequent work using selective reaction monitoring MS.