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P46. Dysregulated level of novel circulating autoantibodies in preeclampsia
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 209–258
Netherlands, b University Medical Center Groningen, Department of Pathology and Medical Biology, Division of Medical Biology, Immunoendocrinology, Groningen, The Netherlands, c University Hospital Jena, Department of Obstetrics, Placenta-Lab, Jena, Germany) Introduction: Syncytiotrophoblast extracellular membrane vesicles (STBEV) are thought to be involved in the pathogenesis of preeclampsia (PE). They might play a role in directing the immune response and monocyte activation in PE. Objectives: We hypothesize, that STBEV from preeclamptic placentae induced an altered activation pattern of peripheral immune cells compared to normal STBEV. Methods: STBEV were gathered after 2 hours of ex vivo placenta perfusion and isolated from perfusion suspension by centrifugation (microvesicles, 30 min 19.000 g) or ultra-centrifugation (exosomes, 70 min 100.000 g). Peripheral blood of healthy non-pregnant women was stimulated for 24 hours with either microvesicles or exosomes. Distribution and activation of immune cell subsets were analyzed via flow-cytometry. Monocytes and granulocytes were stained for monocytes (CD115, CD14, CD16), granulocytes (CD66b) and the activation marker CD11b. Lymphocyte subsets were stained for T-cells, NK-cells and NKT-cells (CD3, CD8, CD16, CD56); T-helper cells (Th1 – Tbet, Th2 – CD294, Th17 – RoRgt); regulatory T-cells (FoxP3) and markers for activation (CD69), memory function (CD45Ro) and cytotoxicity (Perforin, GranzymeB, GranzymeK). Results: Normal STBEV upregulated the number of intermediate monocytes (CD14++CD16+) and activated monocytes and granulocytes, while monocyte maturation and activation was reduced with PE STBEV. Also, normal STBEV promoted a Th1 phenotype whereas PE STBEV supported a Th2/Th17 phenotype. Cytotoxicity was induced in memory T-cells, NKT-cells and CD16+CD56++ NK-cells by normal STBEV. PE STBEV induced reduced cytotoxicity, especially in regulatory immune cell subsets like CD8+ memory Treg- and CD16+CD56++ NK-cells. The effects of normal placentae were induced by both STBEV types, still exosomes showed a more prominent impact. Although PE STBEV in general led to a reduced immune activation compared to normal STBEV, PE microvesicles showed increased activation abilities. Conclusion: Incubation with normal STBEV led to activation of monocytes, granulocytes, memory T-cells, NKT-cells and CD16+CD56++ NK-cells. PE STBEV showed reduced abilities to activate these cell types, especially Treg- and NK-cells. We suggest that this reduction of regulatory immune subsets supports an impairment of the immune system as seen for example in PE. However, PE STBEV does not induce the increased activation of monocytes apparent in PE. Concluding, our data show that placental exosomes have a more prominent effect in healthy pregnancy, while the deregulatory effect of microvesicles increases in PE. doi:10.1016/j.preghy.2015.07.097
P45. Target cells of pregnancy-associated extracellular vesicles Árpád Ferenc Kovács a, Nóra Fekete a, Bálint Alasztics b, József Gábor Joó b, Mária Prosszer b, Edit Buzás a, János Rigó Jr. b, Éva Pállinger a (a Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary , b Semmelweis University, 1st Department of Obstetrics and Gynaecology, Budapest, Hungary ) Introduction: Extracellular vesicles (EVs) produced by living cells are abundant in body fluids. They have a significant role in the intercellular cross-talk. The cellular origin of circulating EVs is characteristic for pregnancy complications and may determine their regulatory effects on maternal immune system.
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Objectives: The aim of our study was identification of the target cells of circulating EVs isolated from the blood plasma of both healthy pregnant women and patients with pregnancy complications. The cell binding ability of isolated EVs of preeclamptic patients and pregnant women with gestational hypertension or chronic hypertension were compared. Methods: EVs were isolated by differential centrifugation from ACD anticoagulated plasma samples (6 samples per group). Multicolor flow cytometry was used for the recognition of EV-lymphocyte interaction. PKH26 stained isolated EVs were incubated with immunophenotyped human peripheral blood lymphocytes. T cell subsets were defined on the basis of their CD3, CD4 and CD8 expressions, while B lymphocytes were identified by CD19 labelling. Results: Although circulating EVs bound to T lymphocytes in all groups, their binding ability to T cell subsets differed substantially. EVs isolated from healthy pregnant women or from the plasma of women in the chronic hypertension group, showed more pronounced binding to Th than to Tc cells. In contrast, EVs isolated from preeclamptic patients had preferential binding to Tc cells. Equal binding to Tc and Th cells could be detected in the case of gestational hypertension. Although we could detect the binding of circulating EVs to B cells in each group, the most characteristic binding was observed in preeclampsia. Conclusion: We identified T and B lymphocytes as target cells of pregnancy-associated circulating EVs. The differences in the EV binding may have been caused by both the diverse surface molecular patterns of EVs and the functionality associated immunophenotypes of circulating lymphocytes. Although we have to further characterise the molecular relationships between EVs and lymphocytes, our findings indicate that pregnancy-associated EVs have a role in the systemic regulation of maternal immune system. doi:10.1016/j.preghy.2015.07.098
P46. Dysregulated level of novel circulating autoantibodies in preeclampsia Kjartan Moe a, Harald Heidecke b, Ralf Dechend c, Anne Cathrine Staff a (a University of Oslo, Institute for Clinical Medicine, Oslo, Norway, b Celltrend GmbH, Luckenwalde, Germany, c The Charitè, Franz-Vollhard Clinic, HELIOS Clinic, Berlin, Germany) Introduction: The contributions of the T-cell population and Natural Killer cells have been extensively studied in relation to pregnancy and adverse pregnancy outcomes. Less attention has been given to the B-cell population although maternal levels of circulating autoantibodies against the angiotensin II type 1 (AT1)-receptor and antiphospholipid antibodies (aPLs) have been shown to be associated with preeclampsia. Members of the PAR (protease activated receptor) family and VEGF (vascular endothelial growth factor) family have previously been shown to be implicated in preeclampsia. Objectives: The aim of this exploratory study was to study levels of IgG autoantibodies against these families of PAR and VEGF proteins in pregnant women with and without preeclampsia. Methods: We developed novel immunoassays detecting levels of IgG autoantibodies against PAR-1, PAR-2, PlGF (Placental Growth Factor), VEGF-A, VEGF-B, VEGF-receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2). Results: We found that levels of autoantibodies against PAR-1, PAR-2, VEGF-A, VEGF-B, PlGF, VEGFR-1 and VEGFR-2 were lower (p < 0.05) in preeclamptic pregnancies (n = 42) compared to normotensive pregnancies (n = 46). Clinical features associated with augmented risk for the preeclampsia syndrome (such as primigravidity, primiparity, obesity and a small for gestational age fetus) were also associated with lower levels of the autoantibodies we investigated, although not always reaching statistical significance.
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Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 209–258
Conclusion: We speculate that these autoantibodies may play a protective role in the development of preeclampsia and are involved in the dysregulation of the PAR and VEGF pathways. doi:10.1016/j.preghy.2015.07.099
P47. First trimester urine and serum metabolomics to predict preeclampsia and gestational hypertension Marie Austdal a, Line Tangerås b, Ragnhild Skråstad c, Kjell Salvesen d, Rigmor Austgulen e, Tone Bathen f, Ann-Charlotte Iversen g (a Norwegian University of Science and Technology and St. Olavs Hospital, Department of Circulation and Medical Imaging, Trondheim, Norway , b Norwegian University of Science and Technology and St. Olavs Hospital, Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway, c Norwegian University of Science and Technology and St. Olavs Hospital, Department of Laboratory Medicine, Children’s and Women’s Health, Trondheim, Norway, d St. Olavs Hospital, National Centre for Fetal Medicine, Trondheim, Norway, e Norwegian University of Science and Technology (NTNU), Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway, f Norwegian University of Science and Technology, Department of Circulation and Medical Imaging, Trondheim, Norway, g Norwegian University of Science and Technology, Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway) Introduction: New methods are required for improved prediction of preeclampsia. Metabolomics is the study of low molecular weight metabolites in tissues or biofluids. Objectives: To evaluate early prediction of preeclampsia and gestational hypertension by 1H nuclear magnetic resonance (NMR) metabolomics. Methods: 1H NMR spectra of urine and serum samples from 599 women with medium-to high risk of preeclampsia in weeks 11–14 of pregnancy were analyzed by principal component analysis and partial least squares discriminant analysis. Variable selection was
applied on the metabolic profiles to find the best markers of prediction. Preeclampsia developed in 26 of the women and gestational hypertension in 21 women. Selected metabolites were combined with maternal characteristics in a logistic regression model. Results: Using urine metabolomic profiles, preeclampsia could be predicted at 51.3% sensitivity, gestational hypertension at 40% sensitivity and both combined at 37% sensitivity at 10% false positive rate (FPR). Increased creatinine and decreased hippurate were the main discriminating metabolites in urine. Using serum metabolomic profiles, preeclampsia could be predicted at 15% sensitivity, gestational hypertension at 33% sensitivity and both combined at 30% sensitivity at 10% FPR. Women who later developed preeclampsia or gestational hypertension had increased serum VLDL and decreased HDL. Combining maternal markers (age >35 or <20, and mean arterial blood pressure) and selected urinary metabolite ratios (hippurate and creatinine) in a logistic regression model, preeclampsia could be predicted with a sensitivity of 42% at 10% FPR. Conclusion: First trimester metabolomic profiles in urine and serum can independently predict hypertensive disorders of pregnancy. A panel of metabolites measured in urine in the first trimester may improve prediction rates for preeclampsia in combination with maternal biophysical markers. doi:10.1016/j.preghy.2015.07.100
P48. Possible laboratory markers and anthropometric women with preeclampsia – Preliminary results Natine Fuzihara Rosa, Stephany Risnic Chvaicer, Ana Paula de Almeida Righi, Claudia Valéria Chagas Siqueira, Diego Gomes Ferreira, Maria Renata Lopes Natale Poltronieri, Rogerio Gomes dos Reis Guidoni, Sergio Floriano Toledo, Leda Ferraz, Patricia Lopes Andrade, Francisco Lazaro Pereira Sousa, Vivian Macedo Gomes Marçal (UNILUS – Lusiada Foundation, 1st Department of Obstetrics and Gynaecology, Santos, Brazil) Introduction: Preeclampsia (PE) affects 5–8% of pregnant women, it is a multi-systemic damage which obesity is a predisposing factor and may be recognized by anthropometric measurements. The increased adiposity results in tissue necrosis and release of fatty acids, promoting metabolic changes known as lipotoxicity that
Netherlands, b University Medical Center Groningen, Department of Pathology and Medical Biology, Division of Medical Biology, Immunoendocrinology, Groningen, The Netherlands, c University Hospital Jena, Department of Obstetrics, Placenta-Lab, Jena, Germany) Introduction: Syncytiotrophoblast extracellular membrane vesicles (STBEV) are thought to be involved in the pathogenesis of preeclampsia (PE). They might play a role in directing the immune response and monocyte activation in PE. Objectives: We hypothesize, that STBEV from preeclamptic placentae induced an altered activation pattern of peripheral immune cells compared to normal STBEV. Methods: STBEV were gathered after 2 hours of ex vivo placenta perfusion and isolated from perfusion suspension by centrifugation (microvesicles, 30 min 19.000 g) or ultra-centrifugation (exosomes, 70 min 100.000 g). Peripheral blood of healthy non-pregnant women was stimulated for 24 hours with either microvesicles or exosomes. Distribution and activation of immune cell subsets were analyzed via flow-cytometry. Monocytes and granulocytes were stained for monocytes (CD115, CD14, CD16), granulocytes (CD66b) and the activation marker CD11b. Lymphocyte subsets were stained for T-cells, NK-cells and NKT-cells (CD3, CD8, CD16, CD56); T-helper cells (Th1 – Tbet, Th2 – CD294, Th17 – RoRgt); regulatory T-cells (FoxP3) and markers for activation (CD69), memory function (CD45Ro) and cytotoxicity (Perforin, GranzymeB, GranzymeK). Results: Normal STBEV upregulated the number of intermediate monocytes (CD14++CD16+) and activated monocytes and granulocytes, while monocyte maturation and activation was reduced with PE STBEV. Also, normal STBEV promoted a Th1 phenotype whereas PE STBEV supported a Th2/Th17 phenotype. Cytotoxicity was induced in memory T-cells, NKT-cells and CD16+CD56++ NK-cells by normal STBEV. PE STBEV induced reduced cytotoxicity, especially in regulatory immune cell subsets like CD8+ memory Treg- and CD16+CD56++ NK-cells. The effects of normal placentae were induced by both STBEV types, still exosomes showed a more prominent impact. Although PE STBEV in general led to a reduced immune activation compared to normal STBEV, PE microvesicles showed increased activation abilities. Conclusion: Incubation with normal STBEV led to activation of monocytes, granulocytes, memory T-cells, NKT-cells and CD16+CD56++ NK-cells. PE STBEV showed reduced abilities to activate these cell types, especially Treg- and NK-cells. We suggest that this reduction of regulatory immune subsets supports an impairment of the immune system as seen for example in PE. However, PE STBEV does not induce the increased activation of monocytes apparent in PE. Concluding, our data show that placental exosomes have a more prominent effect in healthy pregnancy, while the deregulatory effect of microvesicles increases in PE. doi:10.1016/j.preghy.2015.07.097
P45. Target cells of pregnancy-associated extracellular vesicles Árpád Ferenc Kovács a, Nóra Fekete a, Bálint Alasztics b, József Gábor Joó b, Mária Prosszer b, Edit Buzás a, János Rigó Jr. b, Éva Pállinger a (a Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary , b Semmelweis University, 1st Department of Obstetrics and Gynaecology, Budapest, Hungary ) Introduction: Extracellular vesicles (EVs) produced by living cells are abundant in body fluids. They have a significant role in the intercellular cross-talk. The cellular origin of circulating EVs is characteristic for pregnancy complications and may determine their regulatory effects on maternal immune system.
247
Objectives: The aim of our study was identification of the target cells of circulating EVs isolated from the blood plasma of both healthy pregnant women and patients with pregnancy complications. The cell binding ability of isolated EVs of preeclamptic patients and pregnant women with gestational hypertension or chronic hypertension were compared. Methods: EVs were isolated by differential centrifugation from ACD anticoagulated plasma samples (6 samples per group). Multicolor flow cytometry was used for the recognition of EV-lymphocyte interaction. PKH26 stained isolated EVs were incubated with immunophenotyped human peripheral blood lymphocytes. T cell subsets were defined on the basis of their CD3, CD4 and CD8 expressions, while B lymphocytes were identified by CD19 labelling. Results: Although circulating EVs bound to T lymphocytes in all groups, their binding ability to T cell subsets differed substantially. EVs isolated from healthy pregnant women or from the plasma of women in the chronic hypertension group, showed more pronounced binding to Th than to Tc cells. In contrast, EVs isolated from preeclamptic patients had preferential binding to Tc cells. Equal binding to Tc and Th cells could be detected in the case of gestational hypertension. Although we could detect the binding of circulating EVs to B cells in each group, the most characteristic binding was observed in preeclampsia. Conclusion: We identified T and B lymphocytes as target cells of pregnancy-associated circulating EVs. The differences in the EV binding may have been caused by both the diverse surface molecular patterns of EVs and the functionality associated immunophenotypes of circulating lymphocytes. Although we have to further characterise the molecular relationships between EVs and lymphocytes, our findings indicate that pregnancy-associated EVs have a role in the systemic regulation of maternal immune system. doi:10.1016/j.preghy.2015.07.098
P46. Dysregulated level of novel circulating autoantibodies in preeclampsia Kjartan Moe a, Harald Heidecke b, Ralf Dechend c, Anne Cathrine Staff a (a University of Oslo, Institute for Clinical Medicine, Oslo, Norway, b Celltrend GmbH, Luckenwalde, Germany, c The Charitè, Franz-Vollhard Clinic, HELIOS Clinic, Berlin, Germany) Introduction: The contributions of the T-cell population and Natural Killer cells have been extensively studied in relation to pregnancy and adverse pregnancy outcomes. Less attention has been given to the B-cell population although maternal levels of circulating autoantibodies against the angiotensin II type 1 (AT1)-receptor and antiphospholipid antibodies (aPLs) have been shown to be associated with preeclampsia. Members of the PAR (protease activated receptor) family and VEGF (vascular endothelial growth factor) family have previously been shown to be implicated in preeclampsia. Objectives: The aim of this exploratory study was to study levels of IgG autoantibodies against these families of PAR and VEGF proteins in pregnant women with and without preeclampsia. Methods: We developed novel immunoassays detecting levels of IgG autoantibodies against PAR-1, PAR-2, PlGF (Placental Growth Factor), VEGF-A, VEGF-B, VEGF-receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2). Results: We found that levels of autoantibodies against PAR-1, PAR-2, VEGF-A, VEGF-B, PlGF, VEGFR-1 and VEGFR-2 were lower (p < 0.05) in preeclamptic pregnancies (n = 42) compared to normotensive pregnancies (n = 46). Clinical features associated with augmented risk for the preeclampsia syndrome (such as primigravidity, primiparity, obesity and a small for gestational age fetus) were also associated with lower levels of the autoantibodies we investigated, although not always reaching statistical significance.
248
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 209–258
Conclusion: We speculate that these autoantibodies may play a protective role in the development of preeclampsia and are involved in the dysregulation of the PAR and VEGF pathways. doi:10.1016/j.preghy.2015.07.099
P47. First trimester urine and serum metabolomics to predict preeclampsia and gestational hypertension Marie Austdal a, Line Tangerås b, Ragnhild Skråstad c, Kjell Salvesen d, Rigmor Austgulen e, Tone Bathen f, Ann-Charlotte Iversen g (a Norwegian University of Science and Technology and St. Olavs Hospital, Department of Circulation and Medical Imaging, Trondheim, Norway , b Norwegian University of Science and Technology and St. Olavs Hospital, Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway, c Norwegian University of Science and Technology and St. Olavs Hospital, Department of Laboratory Medicine, Children’s and Women’s Health, Trondheim, Norway, d St. Olavs Hospital, National Centre for Fetal Medicine, Trondheim, Norway, e Norwegian University of Science and Technology (NTNU), Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway, f Norwegian University of Science and Technology, Department of Circulation and Medical Imaging, Trondheim, Norway, g Norwegian University of Science and Technology, Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Trondheim, Norway) Introduction: New methods are required for improved prediction of preeclampsia. Metabolomics is the study of low molecular weight metabolites in tissues or biofluids. Objectives: To evaluate early prediction of preeclampsia and gestational hypertension by 1H nuclear magnetic resonance (NMR) metabolomics. Methods: 1H NMR spectra of urine and serum samples from 599 women with medium-to high risk of preeclampsia in weeks 11–14 of pregnancy were analyzed by principal component analysis and partial least squares discriminant analysis. Variable selection was
applied on the metabolic profiles to find the best markers of prediction. Preeclampsia developed in 26 of the women and gestational hypertension in 21 women. Selected metabolites were combined with maternal characteristics in a logistic regression model. Results: Using urine metabolomic profiles, preeclampsia could be predicted at 51.3% sensitivity, gestational hypertension at 40% sensitivity and both combined at 37% sensitivity at 10% false positive rate (FPR). Increased creatinine and decreased hippurate were the main discriminating metabolites in urine. Using serum metabolomic profiles, preeclampsia could be predicted at 15% sensitivity, gestational hypertension at 33% sensitivity and both combined at 30% sensitivity at 10% FPR. Women who later developed preeclampsia or gestational hypertension had increased serum VLDL and decreased HDL. Combining maternal markers (age >35 or <20, and mean arterial blood pressure) and selected urinary metabolite ratios (hippurate and creatinine) in a logistic regression model, preeclampsia could be predicted with a sensitivity of 42% at 10% FPR. Conclusion: First trimester metabolomic profiles in urine and serum can independently predict hypertensive disorders of pregnancy. A panel of metabolites measured in urine in the first trimester may improve prediction rates for preeclampsia in combination with maternal biophysical markers. doi:10.1016/j.preghy.2015.07.100
P48. Possible laboratory markers and anthropometric women with preeclampsia – Preliminary results Natine Fuzihara Rosa, Stephany Risnic Chvaicer, Ana Paula de Almeida Righi, Claudia Valéria Chagas Siqueira, Diego Gomes Ferreira, Maria Renata Lopes Natale Poltronieri, Rogerio Gomes dos Reis Guidoni, Sergio Floriano Toledo, Leda Ferraz, Patricia Lopes Andrade, Francisco Lazaro Pereira Sousa, Vivian Macedo Gomes Marçal (UNILUS – Lusiada Foundation, 1st Department of Obstetrics and Gynaecology, Santos, Brazil) Introduction: Preeclampsia (PE) affects 5–8% of pregnant women, it is a multi-systemic damage which obesity is a predisposing factor and may be recognized by anthropometric measurements. The increased adiposity results in tissue necrosis and release of fatty acids, promoting metabolic changes known as lipotoxicity that