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Expression and functional analysis of suppressyn in primary trophoblast cells
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Abstracts / Placenta 36 (2015) A1eA14
CMV-DNA was detected in the blood and urine of the newborn. The placenta immunostaining also showed that anti-CMV antibody was positive. Case 2: A 32-year-old woman was referred to our hospital at 32 weeks’ gestation, because the ultrasound findings of her fetus showed mild lateralventriclemegaly. Any other obvious abnormal findings were not observed. She delivered a 2450g (-1.8SD) newborn without any complications at 39 weeks’ gestation, the Apgar scores were 8/9 points. The ultrasound examination of the newborn detected expanded bilateral polyependymal cysts and dilatation of bilateral ventricles, and the fetal MRI presented similar findings. CMV-DNA was detected in the blood and urine of the newborn. The placenta immunostaining also showed that anti-CMV antibody was positive. Conclusions: We considered that the pathologic examination of placenta after birth was also useful for diagnosis in congenital CMV infection.
JPA2015-09. A CASE OF ATYPICAL CELLULAR CHORANGIOMA Akira Kohata 1, Atsushi Kasamatsu 1, Hidetaka Okada 1, Yoshiko Uemura 1, Sachiko Minamiguchi 2. 1 Kansai medical university hospital, Japan; 2 Kyouto medical university hospital, Japan Atypical Cellular Chorangioma is rare case. This case is with the blood transfusion syndrome between mother and fetus. Mother is 29years old and pregnancy 29weeks and 5days. When the patient came our hospital, Fetus was no problem. Pregnancy 30weeks and 1day, we found fetus hydrops and placental edema. Because uterine contraction increase in same day and fetus was breech presentation, we did emergency caesarean section. Neonatal was 1414g and Apgar score was 6/7 and newborn’s Hb was 4.0/dl. Mother’s blood examination shows 4.7% of HbF, so we diagnosis the blood transfusion syndrome between mother and fetus. Placental weight is 700g with edema .There is no chorioamnionitis, so we think the edema came from culatory disturbance. We found 5mm node which tissue proliferation increases and Ki-67 was high grade positive and CD31 was positive but no invasion around tissue. So pathological diagnosis was atypical cellular chorangioma. Atypical cellular chorangioma is benign but some report say complication of angioma in baby or mother. We did detailed examination and found that they didn’t have angioma either. It’ s not clear there is relation between atypical cellular chorangioma and blood transfusion syndrome between mother and fetus, but some report says relation of chorangioma and blood transfusion syndrome between mother and fetus. We need to collect more case of atypical cellular chorangioma and study relation between atypical cellular chorangioma and blood transfusion syndrome between mother and fetus.
JPA2015-11. AGE AND HMGB1 INDUCES INFLAMMATORY RESPONSE IN HUMAN TROPHOBLAST CELL LINE Kotomi Seno 1, Koumei Shirasuna 1, Narumi Shimamura 1, Shogo Akihide Ohkuchi 2, Hisataka Iwata 1, Takehito Shiratsuki 1, Kuwayama 1. 1 Tokyo University of Agriculture, Japan; 2 Jichi Medical University, Japan The placenta is a vital organ for establishing and maintaining pregnancy. Preeclampsia, a pregnancy-specific hypertensive syndrome, complicates 5% of all pregnancies worldwide. Pathophysiological changes of preeclampsia include inflammation and immune cell activation. Indeed, a generalized inflammatory response by cytokines such as interleukin (IL)-6 and tumor necrosis factor-a (TNFa), have been considered to be the main pathological features of preeclampsia. Recently, it has been reported that the levels of advanced glycation end products (AGE) and high mobility group box1 (HMGB1) associated with the induction of sterile inflammation are increased in the peripheral blood of women of preeclampsia compared with normal pregnant women. Therefore, in the present study we investigated the effects of AGE and HMGB1 on inflammatory response in human trophoblast cell line Sw.71. At first, Sw.71 trophoblast cells were incubated with or without AGE or HMGB1. Treatment of AGE or HMGB1 stimulated
the expression of NF-kB and clearly increased secretion of inflammatory cytokines including IL-6, IL-8 and CCL2. Also, AGE or HMGB1 increased the expression of these receptors RAGE or TLR-4. In addition, AGE or HMGB1 stimulated the production of reactive oxygen species (ROS) in placental cells. Treatment of NF-kB inhibitor, ROS scavenger NAC, RAGE antagonist, and TLR-4 inhibitor clearly reduced IL-6 secretion. These results suggest that AGE or HMGB1 induce NF-kB and ROS production via RAGE and TLR4, resulting the increase of inflammatory IL-6 secretion in human trophoblast cell lines.
JPA2015-12. EXPRESSION AND FUNCTIONAL ANALYSIS OF SUPPRESSYN IN PRIMARY TROPHOBLAST CELLS Jun Sugimoto 1, Tadatsugu Kinjo 2, Danny Schust 3, Takaya Oda 1, Yoichi Aoki 2, Jinno Yoshihiro 1. 1 Department of Human Molecular Biology, Graduate School of Medical Science, University of the Ryukyus, Japan; 2 Department of Obstetrics and Gynecology, Graduate School of Medical Science, University of the Ryukyus, Japan; 3 Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Japan Cell-cell fusion is central to trophoblast syncytialization. A well-characterized human endogenous retrovirus (HERV)-derived pro-fusogenic mediator, syncytin-1, may be balanced by the actions of a novel HERVderived protein, suppressyn. Suppressyn has been shown to block cell-cell fusion in the trophoblast cell lines Bewo and HTR8. Here, we have expanded the study of suppressyn ex vivo to an analysis in primary trophoblast cells. After optimizing primary trophoblast cell isolation and culture to a syncytialized state after 96 hours in vitro, we analyzed the expression of several fusion-related genes, including suppressyn. Fusionrelated gene expression mimicked that reported previously, although in primary cells, suppressyn was expressed exclusively in non-fused cells prior to differentiation. Treatment of primary trophoblast cells immediately after isolation with a suppressyn-specific siRNA decreased the proportion of non-fused cells at 96 hours when compared to cells treated with a scrambled control. Our results support an in vivo role for suppressyn in normal placental development.
JPA2015-13. POSSIBLE ROLE OF CD59 IN SPIRAL ARTERY REMODELING. Masashi Ueda 1, Akihito Horie 1, Yukiyasu Sato 2, Koh Suginami 1, Hirohiko Tani 1, Yumiko Miyazaki 1, Asuka Okumiya 1, Ikuo Konishi 1. 1 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, Japan; 2 Department of Obstetrics and Gynecology, Otsu Red Cross Hospital, Japan Objective: Human extravillous trophoblast (EVT) invades maternal decidua and spiral artery. Arterial invasion of endovascular EVT (eEVT) causes structural remodeling of spiral artery, called “spiral artery remodeling”. During the remodeling, eEVT makes direct contact with complement components contained in the maternal blood. Since eEVT is foreign to maternal immunity, the complement can be activated by eEVT. Nevertheless, it is not clear why eEVT is not eliminated by the activation of complement. It is well known that activation of complement leads to cell lysis and death through the formation of membrane attack complex (MAC) on the cell surface. CD59 is an 18-20 kDa glycosylphosphatidylinositol-anchored protein that restricts the formation of MAC. In the present study, we investigated expression and function of CD59 on EVT. Method: Tissue samples at early pregnant implantation sites were collected from therapeutic hysterectomies. These samples were subjected to immunohistochemistry by CD59. Placental tissues were aseptically obtained from legal abortions of normal early pregnancies and were subjected to villous explant cultures to isolate primary EVT. Using all the samples was approved by the Ethics Committee of Kyoto University Hospital. Isolated EVT, human immortalized EVT cell line (Swan71), and human choriocarcinoma cell lines (BeWo, JAR and JEG) were subjected to RTPCR, immunocytochemistry and flow cytometry to examine CD59
Abstracts / Placenta 36 (2015) A1eA14
CMV-DNA was detected in the blood and urine of the newborn. The placenta immunostaining also showed that anti-CMV antibody was positive. Case 2: A 32-year-old woman was referred to our hospital at 32 weeks’ gestation, because the ultrasound findings of her fetus showed mild lateralventriclemegaly. Any other obvious abnormal findings were not observed. She delivered a 2450g (-1.8SD) newborn without any complications at 39 weeks’ gestation, the Apgar scores were 8/9 points. The ultrasound examination of the newborn detected expanded bilateral polyependymal cysts and dilatation of bilateral ventricles, and the fetal MRI presented similar findings. CMV-DNA was detected in the blood and urine of the newborn. The placenta immunostaining also showed that anti-CMV antibody was positive. Conclusions: We considered that the pathologic examination of placenta after birth was also useful for diagnosis in congenital CMV infection.
JPA2015-09. A CASE OF ATYPICAL CELLULAR CHORANGIOMA Akira Kohata 1, Atsushi Kasamatsu 1, Hidetaka Okada 1, Yoshiko Uemura 1, Sachiko Minamiguchi 2. 1 Kansai medical university hospital, Japan; 2 Kyouto medical university hospital, Japan Atypical Cellular Chorangioma is rare case. This case is with the blood transfusion syndrome between mother and fetus. Mother is 29years old and pregnancy 29weeks and 5days. When the patient came our hospital, Fetus was no problem. Pregnancy 30weeks and 1day, we found fetus hydrops and placental edema. Because uterine contraction increase in same day and fetus was breech presentation, we did emergency caesarean section. Neonatal was 1414g and Apgar score was 6/7 and newborn’s Hb was 4.0/dl. Mother’s blood examination shows 4.7% of HbF, so we diagnosis the blood transfusion syndrome between mother and fetus. Placental weight is 700g with edema .There is no chorioamnionitis, so we think the edema came from culatory disturbance. We found 5mm node which tissue proliferation increases and Ki-67 was high grade positive and CD31 was positive but no invasion around tissue. So pathological diagnosis was atypical cellular chorangioma. Atypical cellular chorangioma is benign but some report say complication of angioma in baby or mother. We did detailed examination and found that they didn’t have angioma either. It’ s not clear there is relation between atypical cellular chorangioma and blood transfusion syndrome between mother and fetus, but some report says relation of chorangioma and blood transfusion syndrome between mother and fetus. We need to collect more case of atypical cellular chorangioma and study relation between atypical cellular chorangioma and blood transfusion syndrome between mother and fetus.
JPA2015-11. AGE AND HMGB1 INDUCES INFLAMMATORY RESPONSE IN HUMAN TROPHOBLAST CELL LINE Kotomi Seno 1, Koumei Shirasuna 1, Narumi Shimamura 1, Shogo Akihide Ohkuchi 2, Hisataka Iwata 1, Takehito Shiratsuki 1, Kuwayama 1. 1 Tokyo University of Agriculture, Japan; 2 Jichi Medical University, Japan The placenta is a vital organ for establishing and maintaining pregnancy. Preeclampsia, a pregnancy-specific hypertensive syndrome, complicates 5% of all pregnancies worldwide. Pathophysiological changes of preeclampsia include inflammation and immune cell activation. Indeed, a generalized inflammatory response by cytokines such as interleukin (IL)-6 and tumor necrosis factor-a (TNFa), have been considered to be the main pathological features of preeclampsia. Recently, it has been reported that the levels of advanced glycation end products (AGE) and high mobility group box1 (HMGB1) associated with the induction of sterile inflammation are increased in the peripheral blood of women of preeclampsia compared with normal pregnant women. Therefore, in the present study we investigated the effects of AGE and HMGB1 on inflammatory response in human trophoblast cell line Sw.71. At first, Sw.71 trophoblast cells were incubated with or without AGE or HMGB1. Treatment of AGE or HMGB1 stimulated
the expression of NF-kB and clearly increased secretion of inflammatory cytokines including IL-6, IL-8 and CCL2. Also, AGE or HMGB1 increased the expression of these receptors RAGE or TLR-4. In addition, AGE or HMGB1 stimulated the production of reactive oxygen species (ROS) in placental cells. Treatment of NF-kB inhibitor, ROS scavenger NAC, RAGE antagonist, and TLR-4 inhibitor clearly reduced IL-6 secretion. These results suggest that AGE or HMGB1 induce NF-kB and ROS production via RAGE and TLR4, resulting the increase of inflammatory IL-6 secretion in human trophoblast cell lines.
JPA2015-12. EXPRESSION AND FUNCTIONAL ANALYSIS OF SUPPRESSYN IN PRIMARY TROPHOBLAST CELLS Jun Sugimoto 1, Tadatsugu Kinjo 2, Danny Schust 3, Takaya Oda 1, Yoichi Aoki 2, Jinno Yoshihiro 1. 1 Department of Human Molecular Biology, Graduate School of Medical Science, University of the Ryukyus, Japan; 2 Department of Obstetrics and Gynecology, Graduate School of Medical Science, University of the Ryukyus, Japan; 3 Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Japan Cell-cell fusion is central to trophoblast syncytialization. A well-characterized human endogenous retrovirus (HERV)-derived pro-fusogenic mediator, syncytin-1, may be balanced by the actions of a novel HERVderived protein, suppressyn. Suppressyn has been shown to block cell-cell fusion in the trophoblast cell lines Bewo and HTR8. Here, we have expanded the study of suppressyn ex vivo to an analysis in primary trophoblast cells. After optimizing primary trophoblast cell isolation and culture to a syncytialized state after 96 hours in vitro, we analyzed the expression of several fusion-related genes, including suppressyn. Fusionrelated gene expression mimicked that reported previously, although in primary cells, suppressyn was expressed exclusively in non-fused cells prior to differentiation. Treatment of primary trophoblast cells immediately after isolation with a suppressyn-specific siRNA decreased the proportion of non-fused cells at 96 hours when compared to cells treated with a scrambled control. Our results support an in vivo role for suppressyn in normal placental development.
JPA2015-13. POSSIBLE ROLE OF CD59 IN SPIRAL ARTERY REMODELING. Masashi Ueda 1, Akihito Horie 1, Yukiyasu Sato 2, Koh Suginami 1, Hirohiko Tani 1, Yumiko Miyazaki 1, Asuka Okumiya 1, Ikuo Konishi 1. 1 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, Japan; 2 Department of Obstetrics and Gynecology, Otsu Red Cross Hospital, Japan Objective: Human extravillous trophoblast (EVT) invades maternal decidua and spiral artery. Arterial invasion of endovascular EVT (eEVT) causes structural remodeling of spiral artery, called “spiral artery remodeling”. During the remodeling, eEVT makes direct contact with complement components contained in the maternal blood. Since eEVT is foreign to maternal immunity, the complement can be activated by eEVT. Nevertheless, it is not clear why eEVT is not eliminated by the activation of complement. It is well known that activation of complement leads to cell lysis and death through the formation of membrane attack complex (MAC) on the cell surface. CD59 is an 18-20 kDa glycosylphosphatidylinositol-anchored protein that restricts the formation of MAC. In the present study, we investigated expression and function of CD59 on EVT. Method: Tissue samples at early pregnant implantation sites were collected from therapeutic hysterectomies. These samples were subjected to immunohistochemistry by CD59. Placental tissues were aseptically obtained from legal abortions of normal early pregnancies and were subjected to villous explant cultures to isolate primary EVT. Using all the samples was approved by the Ethics Committee of Kyoto University Hospital. Isolated EVT, human immortalized EVT cell line (Swan71), and human choriocarcinoma cell lines (BeWo, JAR and JEG) were subjected to RTPCR, immunocytochemistry and flow cytometry to examine CD59