The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Wang, Ji-meng [School of Medicine, Nankai University, Tianjin 300071 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Zhao, Hong-xi [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038 (China); Wang, Li [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Gao, Zhi-ying, E-mail: gaozy301@yahoo.com.cn [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Yao, Yuan-qing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China)

2013-05-10

Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

Waddlia chondrophila infects and multiplies in ovine trophoblast cells stimulating an inflammatory immune response.

Directory of Open Access Journals (Sweden)

Nick Wheelhouse

Full Text Available Waddlia chondrophila (W. chondrophila is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus. This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity.Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i. and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways.W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

Waddlia chondrophila infects and multiplies in ovine trophoblast cells stimulating an inflammatory immune response.

Science.gov (United States)

Wheelhouse, Nick; Coyle, Christopher; Barlow, Peter G; Mitchell, Stephen; Greub, Gilbert; Baszler, Tim; Rae, Mick T; Longbottom, David

2014-01-01

Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity. Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways. W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

Live cell imaging of in vitro human trophoblast syncytialization.

Science.gov (United States)

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

Downregulation of SPARC expression inhibits the invasion of human trophoblast cells in vitro.

Directory of Open Access Journals (Sweden)

Yahong Jiang

Full Text Available Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ, trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11, KISS1, insulin-like growth factor binding protein 4 (IGFBP4, collagen type I alpha 1 (COLIA1, matrix metallopeptidase 9 (MMP9, and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA, MMP1, gap junction protein alpha 1 (GJA1, et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.

Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts.

Science.gov (United States)

Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

2016-02-09

Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface.

A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells

Directory of Open Access Journals (Sweden)

Stephanie Chrysanthou

2018-04-01

Full Text Available Summary: The ten-eleven translocation (TET proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage. : TET proteins are well known for their role in pluripotency. Here, Hemberger and colleagues show that TET1 and TET2 are also critical for maintaining the epithelial integrity of trophoblast stem cells. TET1/2 ensure mitotic cell-cycle progression by stabilizing cyclin B1 and by regulating centrosome organization. These insights reveal the importance of TET proteins beyond their role in epigenome remodeling. Keywords: TET proteins, trophoblast stem cells, cell cycle, endoreduplication, self-renewal, mitosis, trophoblast giant cells, differentiation

Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators

DEFF Research Database (Denmark)

Hess, AP; Hamilton, AE; Talbi, S

2007-01-01

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important...... a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and were further treated with conditioned media (CM) from human trophoblasts (TCM) or, as a control, with conditioned media (CCM) from non-decidualized stromal cells...... regulated groups. The data demonstrate a significant induction of pro-inflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune...

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

Science.gov (United States)

Seno, Kotomi; Tanikawa, Nao; Takahashi, Hironori; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito; Shirasuna, Koumei

2018-02-15

We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O 2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). Physiological normoxia (5% O 2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O 2 . Pathophysiological hypoxia (1% O 2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O 2 -independent manner. We conclude that O 2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evidence for Differential Glycosylation of Trophoblast Cell Types*

Science.gov (United States)

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E.; Longtine, Mark S.; Schust, Danny J.; Haslam, Stuart M.; Blois, Sandra M.; Dell, Anne; Clark, Gary F.

2016-01-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3–4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2–3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2–3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. PMID:26929217

Evidence for Differential Glycosylation of Trophoblast Cell Types.

Science.gov (United States)

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E; Longtine, Mark S; Schust, Danny J; Haslam, Stuart M; Blois, Sandra M; Dell, Anne; Clark, Gary F

2016-06-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Elsevier Trophoblast Research Award lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells--focus on leptin expression.

Science.gov (United States)

Gambino, Y P; Maymó, J L; Pérez Pérez, A; Calvo, J C; Sánchez-Margalet, V; Varone, C L

2012-02-01

The steroid hormone 17β-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression. Copyright © 2012 Elsevier Ltd. All rights reserved.

Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

Directory of Open Access Journals (Sweden)

Islam M. Saadeldin

2017-01-01

Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

Directory of Open Access Journals (Sweden)

Waka Omata

Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

[Cells of immune system of mother and trophoblast cells: constructive cooperation for the sake of achievement of the joint purpose].

Science.gov (United States)

Aĭlamazian, E K; Stepanova, O I; Sel'kov, S A; Sokolov, D I

2013-01-01

In the present review modern data about change of morfo-functional properties of a trophoblast during pregnancy, and also about influence of the cytokines produced by cells of a microenvironment, including leucocytes of mother, on a functional state of trophoblast is cited. Features of interaction between trophoblast and immune cells of mother are described within physiological pregnancy and within pregnancy complicated by preeclampsia.

Gender-Dependent Survival of Allogeneic Trophoblast Stem Cells in Liver

Science.gov (United States)

Epple-Farmer, Jessica; Debeb, Bisrat G.; Smithies, Oliver; Binas, Bert

2012-01-01

In view of the well-known phenomenon of trophoblast immune privilege, trophoblast stem cells (TSCs) might be expected to be immune privileged, which could be of interest for cell or gene therapies. Yet in the ectopic sites tested so far, TSC transplants fail to show noticeable immune privilege and seem to lack physiological support. However, we show here that after portal venous injection, green fluorescent protein (GFP)-labeled TSCs survive for several months in the livers of allogeneic female but not male mice. Gonadectomy experiments revealed that this survival does not require the presence of ovarian hormones but does require the absence of testicular factors. By contrast, GFP-labeled allogeneic embryonic stem cells (ESCs) are reliably rejected; however, these same ESCs survive when mixed with unlabeled TSCs. The protective effect does not require immunological compatibility between ESCs and TSCs. Tumors were not observed in animals with either successfully engrafted TSCs or coinjected ESCs. We conclude that in a suitable hormonal context and location, ectopic TSCs can exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as a new model for studying trophoblast immunology and physiology. PMID:19523327

Decreased IL-33 Production Contributes to Trophoblast Cell Dysfunction in Pregnancies with Preeclampsia

Directory of Open Access Journals (Sweden)

Hong Chen

2018-01-01

Full Text Available Preeclampsia (PE is a life-threatening pregnancy complication which is related to aggradation of risk regarding fetal and maternal morbidity and mortality. Dysregulation of systemic inflammatory response and dysfunction of trophoblast cells have been proposed to be involved in the development and progression of PE. Some studies have demonstrated that interleukin-33 (IL-33 is an immunomodulatory cytokine that is associated with the immune regulation of tumor cells. However, little is known whether IL-33 and its receptor ST2/IL-1 R4 could regulate trophoblast cells, which are associated with the pathogenesis of PE. In this study, our target is to explore the impact of IL-33 on trophoblast cells and elucidate its underlying pathophysiological mechanisms. Placental tissues from the severe PE group (n=11 and the normotensive pregnant women’s group (n=11 were collected for the protein expression and distribution of IL-33 along with its receptor ST2/IL-1 R4 via Western blot analysis and immunohistochemistry, respectively. We discovered that the level of IL-33 was decreased in placental tissues of pregnant women with PE, while no distinction was observed in the expression of ST2/IL-1 R4. These results were further verified in villous explants which were treated with sodium nitroprusside with different concentrations, to simulate the pathological environment of PE. To investigate IL-33 effects on trophoblast cells separately, IL-33 shRNA was introduced into HTR8/SVneo cells and villi. IL-33 shRNA weakened the proliferation, migration, and invasion capacity of HTR8/SVneo cells. The migration distance of villous explants was also markedly decreased. The reduced invasion of trophoblast cells is a result of IL-33 knockdown which could be related to the decline of MMP2/9 activity and the increased utterance of TIMP1/2. Overall, our findings demonstrated that the reduction of IL-33 production was connected with the reduced functional capability of

Immunoglobulins from sera of APS patients bind HTR-8/SVneo trophoblast cell line and reduce additional mediators of cell invasion.

Science.gov (United States)

Jovanović Krivokuća, Milica; Abu Rabi, Tamara; Stefanoska, Ivana; Vrzić-Petronijević, Svetlana; Petronijević, Miloš; Vićovac, Ljiljana

2017-12-01

Immunoglobulins from sera of patients with antiphospholipid syndrome (APS) decrease trophoblast cell invasion in vitro. This study aimed to extend understanding of cellular effects of immunoglobulins from APS (aPL+) in HTR-8/SVneo cells. aPL+ IgG induced change in effector molecules important for cell invasion was investigated further. After 1h of culture 21% cells bound aPL+ IgG, as opposed to 6% in control (aPL-). This was accompanied by increase in phospho-p38 at 30min. After 24h treatment aPL+IgG decreased protein levels of integrin subunits α1 (78% of control; p<0.01), α4 (65% of control, p<0.01), α5 (76% of control; p<0.01) and β1 (80% of control; p<0.01), and secreted gal-1 (68% of control; p<0.05). ProMMP-9 was reduced to 70% of control (p<0.001). Treatment with inhibitor of p38 MAPK signaling SB202190 reversed inhibition in integrin β1 and secreted gal-1. Involvement of p38 MAPK signaling and decrease in integrin subunit α4 , proMMP-9, and secreted gal-1 in HTR-8/SVneo cells are novel and extend the list of mediators of trophoblast invasion affected by aPL. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Down-Regulation of Neuropathy Target Esterase in Preeclampsia Placenta Inhibits Human Trophoblast Cell Invasion via Modulating MMP-9 Levels

Directory of Open Access Journals (Sweden)

Ting Zhong

2018-02-01

Full Text Available Background/Aims: Neuropathy target esterase (NTE, also known as neurotoxic esterase is proven to deacylate phosphatidylcholine (PC to glycerophosphocholine as a phospholipase B. Recently; studies showed that artificial phosphatidylserine/PC microvesicles can induce preeclampsia (PE-like changes in pregnant mice. However, it is unclear whether NTE plays a key role in the pathology of PE, a pregnancy-related disease, which was characterized by deficient trophoblast invasion and reduced trophoblast-mediated remodeling of spiral arteries. The aim of this study was to investigate the expression pattern of NTE in the placenta from women with PE and normal pregnancy, and the molecular mechanism of NTE involved in the development of PE. Methods: NTE expression levels in placentas from 20 pregnant women with PE and 20 healthy pregnant women were detected using quantitative PCR and immunohistochemistry staining. The effect of NTE on trophoblast migration and invasion and the underlying mechanisms were examined in HTR-8/SVneo cell lines by transfection method. Results: NTE mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. Over-expression of NTE in HTR-8/SVneo cells significantly promoted trophoblast cells migration and invasion and was associated with increased MMP-9 levels. Conversely, shRNA-mediated down-regulation of NTE markedly inhibited the cell migration and invasion. In addition, silencing NTE reduced the MMP-9 activity and phosphorylated Erk1/2 and AKT levels. Conclusions: Our results suggest that the decreased NTE may contribute to the development of PE through impairing trophoblast invasion by down-regulating MMP-9 via the Erk1/2 and AKT signaling pathway.

MTA3 regulates CGB5 and Snail genes in trophoblast

Energy Technology Data Exchange (ETDEWEB)

Chen, Ying [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, MI 49503 (United States); Miyazaki, Jun [Department of Obstetrics and Gynecology, Fujita Health University School of Medicine, Fujita Health University, Toyoake (Japan); Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake (Japan); Nishizawa, Haruki [Department of Obstetrics and Gynecology, Fujita Health University School of Medicine, Fujita Health University, Toyoake (Japan); Kurahashi, Hiroki [Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake (Japan); Leach, Richard, E-mail: Richard.Leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group, Grand Rapids, MI 49503 (United States); Wang, Kai, E-mail: Kai.Wang@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, MI 49503 (United States)

2013-04-19

Highlights: •Impaired MTA3, raised CGB5 and Snail expression are associated with preeclampsia. •Knock-down of MTA3 causes up-regulation of CGB5 and Snail genes in BeWo cells. •MTA3 occupies CGB5 and Snail gene promoters in BeWo cells. -- Abstract: Secreted by the placental trophoblast, human chorionic gonadotropin (hCG) is an important hormone during pregnancy and is required for the maintenance of pregnancy. Previous studies have shown that dys-regulation of hCG expression is associated with preeclampsia. However, the exact relationship between altered hCG levels and development of preeclampsia is unknown. Metastasis associated protein 3 (MTA3), a chromatin remodeling protein, is abundantly expressed in the placental trophoblasts, but its function is unknown. In breast cancer, MTA3 has been shown to repress the expression of Snail and cell migration. However, whether MTA3 acts similarly in the trophoblast has not been investigated. In the present study, we examined the role of MTA3 in regulating the hCG β-subunit gene (gene name: CGB5) and Snail expression in the trophoblast cell line, BeWo, as well as its relevance to the high hCG expression levels seen in preeclampsia. First, we investigated MTA3 expression in preeclamptic placenta as compared to normal control placenta via gene expression microarray and qRT-PCR and found that MTA3 was significantly down-regulated, whereas both CGB5 and Snail were up-regulated in preeclamptic placenta. Secondly, we knocked down MTA3 gene in trophoblast cell line BeWo and found Snail and hCG were both up-regulated, suggesting that MTA3 represses Snail and hCG gene expression in trophoblasts. Next, we cloned the CGB5 and Snail promoters into the pGL3-basic vector individually and found that silencing of MTA3 by siRNA resulted in an increase of both CGB5 and Snail promoter activities. To confirm that this MTA3 inhibition is a direct effect, we performed a chromatin immune-precipitation (ChIP) assay and found that MTA3

MTA3 regulates CGB5 and Snail genes in trophoblast

International Nuclear Information System (INIS)

Chen, Ying; Miyazaki, Jun; Nishizawa, Haruki; Kurahashi, Hiroki; Leach, Richard; Wang, Kai

2013-01-01

Highlights: •Impaired MTA3, raised CGB5 and Snail expression are associated with preeclampsia. •Knock-down of MTA3 causes up-regulation of CGB5 and Snail genes in BeWo cells. •MTA3 occupies CGB5 and Snail gene promoters in BeWo cells. -- Abstract: Secreted by the placental trophoblast, human chorionic gonadotropin (hCG) is an important hormone during pregnancy and is required for the maintenance of pregnancy. Previous studies have shown that dys-regulation of hCG expression is associated with preeclampsia. However, the exact relationship between altered hCG levels and development of preeclampsia is unknown. Metastasis associated protein 3 (MTA3), a chromatin remodeling protein, is abundantly expressed in the placental trophoblasts, but its function is unknown. In breast cancer, MTA3 has been shown to repress the expression of Snail and cell migration. However, whether MTA3 acts similarly in the trophoblast has not been investigated. In the present study, we examined the role of MTA3 in regulating the hCG β-subunit gene (gene name: CGB5) and Snail expression in the trophoblast cell line, BeWo, as well as its relevance to the high hCG expression levels seen in preeclampsia. First, we investigated MTA3 expression in preeclamptic placenta as compared to normal control placenta via gene expression microarray and qRT-PCR and found that MTA3 was significantly down-regulated, whereas both CGB5 and Snail were up-regulated in preeclamptic placenta. Secondly, we knocked down MTA3 gene in trophoblast cell line BeWo and found Snail and hCG were both up-regulated, suggesting that MTA3 represses Snail and hCG gene expression in trophoblasts. Next, we cloned the CGB5 and Snail promoters into the pGL3-basic vector individually and found that silencing of MTA3 by siRNA resulted in an increase of both CGB5 and Snail promoter activities. To confirm that this MTA3 inhibition is a direct effect, we performed a chromatin immune-precipitation (ChIP) assay and found that MTA3

Synthesis and release of fatty acids by human trophoblast cells in culture

International Nuclear Information System (INIS)

Coleman, R.A.; Haynes, E.B.

1987-01-01

In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from [ 14 C]acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from [ 14 C]acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. [ 14 C]acetate was also incorporated into cellular triacylglycerol, phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with [1- 14 C]oleate; trophoblast cells then released 14 C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the 14 C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release

Reactive Oxygen Stimulation of Interleukin-6 Release in the Human Trophoblast Cell Line HTR-8/SVneo by the Trichlorethylene Metabolite S-(1,2-Dichloro)-l-Cysteine.

Science.gov (United States)

Hassan, Iman; Kumar, Anjana M; Park, Hae-Ryung; Lash, Lawrence H; Loch-Caruso, Rita

2016-09-01

Trichloroethylene (TCE) is a common environmental pollutant associated with adverse reproductive outcomes in humans. TCE intoxication occurs primarily through its biotransformation to bioactive metabolites, including S-(1,2-dichlorovinyl)-l-cysteine (DCVC). TCE induces oxidative stress and inflammation in the liver and kidney. Although the placenta is capable of xenobiotic metabolism and oxidative stress and inflammation in placenta have been associated with adverse pregnancy outcomes, TCE toxicity in the placenta remains poorly understood. We determined the effects of DCVC by using the human extravillous trophoblast cell line HTR-8/SVneo. Exposure to 10 and 20 μM DCVC for 10 h increased reactive oxygen species (ROS) as measured by carboxydichlorofluorescein fluorescence. Moreover, 10 and 20 μM DCVC increased mRNA expression and release of interleukin-6 (IL-6) after 24-h exposure, and these responses were inhibited by the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid and by treatments with antioxidants (alpha-tocopherol and deferoxamine), suggesting that DCVC-stimulated IL-6 release in HTR-8/SVneo cells is dependent on beta-lyase metabolic activation and increased generation of ROS. HTR-8/SVneo cells exhibited decreased mitochondrial membrane potential at 5, 10, and 20 μM DCVC at 5, 10, and 24 h, showing that DCVC induces mitochondrial dysfunction in HTR-8/Svneo cells. The present study demonstrates that DCVC stimulated ROS generation in the human placental cell line HTR-8/SVneo and provides new evidence of mechanistic linkage between DCVC-stimulated ROS and increase in proinflammatory cytokine IL-6. Because abnormal activation of cytokines can disrupt trophoblast functions necessary for placental development and successful pregnancy, follow-up investigations relating these findings to physiologic outcomes are warranted. © 2016 by the Society for the Study of Reproduction, Inc.

miR-520 promotes DNA-damage-induced trophoblast cell apoptosis by targeting PARP1 in recurrent spontaneous abortion (RSA).

Science.gov (United States)

Dong, Xiujuan; Yang, Long; Wang, Hui

2017-04-01

The establishment and maintenance of successful pregnancy mainly depends on trophoblast cells. Their dysfunction has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy. However, the underlying mechanisms of trophoblasts dysfunction remain unclear. DNA-damage-induced cell apoptosis has been reported to play a vital role in cell death. In this study, we identified a novel microRNA (miR-520) in RSA progression via regulating trophoblast cell apoptosis. Microarray analysis showed that miR-520 was highly expressed in villus of RSA patients. By using flow cytometry analysis, we observed miR-520 expression was correlated with human trophoblast cell apoptosis in vitro, along with decreased poly (ADP-ribose) polymerase-1 (PARP1) expression. With the analysis of clinic samples, we observed that miR-520 level was negatively correlated with PARP1 level in RSA villus. In addition, overexpression of PARP1 restored the miR-520-induced trophoblast cell apoptosis in vitro. The status of chromosome in trophoblast implied that miR-520-promoted DNA-damage-induced cell apoptosis to regulate RSA progression. These results indicated that the level of miR-520 might associate with RSA by prompting trophoblast cell apoptosis via PARP1 dependent DNA-damage pathway.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

Science.gov (United States)

Loi, Pasqualino; Galli, Cesare; Lazzari, Giovanna; Matsukawa, Kazutsugu; Fulka, Josef; Goeritz, Frank; Hildebrandt, Thomas B

2018-04-13

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

Effect of proline rich 15-deficiency on trophoblast viability and survival.

Directory of Open Access Journals (Sweden)

Katherine C Gates

Full Text Available Deviations from the normal program of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant morbidity and mortality. Proline rich 15 (PRR15 is a low molecular weight nuclear protein expressed by the trophoblast during early gestation. Lentivirus-mediated knockdown of PRR15 mRNA in ovine trophectoderm led to demise of the embryo by gestational day 15, providing compelling evidence that PRR15 expression is critical during this precarious window of development. Our objective was to determine the effect of PRR15 knockdown on trophoblast gene expression, proliferation, and survival. The first-trimester human trophoblast cell line, ACH-3P, was infected with control lentivirus or a lentivirus expressing a short hairpin (shRNA to target PRR15 mRNA for degradation, resulting in a 68% reduction in PRR15 mRNA. Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. These changes included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. Viability and proliferation decreased in PRR15-deficient cells, which was consistent with down-regulation of cell cycle-related genes CCND1 and CDK6 and an up-regulation of CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis increased significantly in the PRR15-deficient cells. Migration through a basement membrane matrix decreased and an increased population of apoptotic cells was present when treated with shRNA to target PRR15. These results suggest that PRR15 enhances trophoblast viability and survival during early implantation and placentation.

HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors

Directory of Open Access Journals (Sweden)

Maja Weber

2013-01-01

Full Text Available Introduction. JEG3 is a choriocarcinoma—and HTR8/SVneo a transformed extravillous trophoblast—cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT- is distinct from JEG3 (CDX2+ and NOTCH1+ as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo’s self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of “stemness-” associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Unsaturated fatty acids protect trophoblast cells from saturated fatty acid-induced autophagy defects.

Science.gov (United States)

Hong, Ye-Ji; Ahn, Hyo-Ju; Shin, Jongdae; Lee, Joon H; Kim, Jin-Hoi; Park, Hwan-Woo; Lee, Sung Ki

2018-02-01

Dysregulated serum fatty acids are associated with a lipotoxic placental environment, which contributes to increased pregnancy complications via altered trophoblast invasion. However, the role of saturated and unsaturated fatty acids in trophoblastic autophagy has yet to be explored. Here, we demonstrated that prolonged exposure of saturated fatty acids interferes with the invasiveness of human extravillous trophoblasts. Saturated fatty acids (but not unsaturated fatty acids) inhibited the fusion of autophagosomes and lysosomes, resulting in the formation of intracellular protein aggregates. Furthermore, when the trophoblast cells were exposed to saturated fatty acids, unsaturated fatty acids counteracted the effects of saturated fatty acids by increasing degradation of autophagic vacuoles. Saturated fatty acids reduced the levels of the matrix metalloproteinases (MMP)-2 and MMP-9, while unsaturated fatty acids maintained their levels. In conclusion, saturated fatty acids induced decreased trophoblast invasion, of which autophagy dysfunction plays a major role. Copyright © 2017 Elsevier B.V. All rights reserved.

Function of caspase-14 in trophoblast differentiation

Directory of Open Access Journals (Sweden)

Charles Adrian K

2009-09-01

Full Text Available Abstract Background Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. Methods Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. Results Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. Conclusion Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner.

Science.gov (United States)

Khera, Alisha; Vanderlelie, Jessica J; Holland, Olivia; Perkins, Anthony V

2017-06-01

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 μM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.

Effects of phytoestrogens genistein and daidzein on progesterone and estrogen (estradiol) production of human term trophoblast cells in vitro.

Science.gov (United States)

Richter, Dagmar Ulrike; Mylonas, Ioannis; Toth, Bettina; Scholz, Christoph; Briese, Volker; Friese, Klaus; Jeschke, Udo

2009-01-01

Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind on estrogen receptors alpha and beta in humans. The effects of the phytoestrogens genistein and daidzein on the production of progesterone and estrogen in isolated human term trophoblast cells in vitro were tested in this study. Cytotrophoblast cells were isolated from human term placentas. Phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast cells. Untreated cells were used as controls. After 24 h aliquots were removed and tested for progesterone and estrogen production. The production of the steroid hormones progesterone and estrogen are influenced by phytoestrogens genistein and daidzein in human term trophoblast cells. A strong inhibition effect of both phytoestrogens tested in the production of progesterone was demonstrated. In addition, a significant stimulating effect on estrogen production by genistein and daidzein was observed. Results obtained with this study show that phytoestrogens (genistein and daidzein) sufficiently reduce progesterone production in human term trophoblast cells. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that high doses of phytoestrogens at the feto-maternal interphase could play a negative role in maintenance of pregnancy. Stimulation of estrogen production by genistein and daidzein in trophoblast cells is probably due to estrogen receptor blocking effects of both phytoestrogens. Trophoblast cells seem to compensate blocking of its estrogen receptors by higher estrogen production.

Quantitative investigation of reproduction of gonosomal condensed chromatin during trophoblast cell polyploidization and endoreduplication in the east-european field vole Microtus rossiaemeridionalis

Directory of Open Access Journals (Sweden)

Bogdanova Margarita S

2003-04-01

Full Text Available Abstract Simultaneous determinations of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB have been performed in two trophoblast cell populations of the East-European field vole Microtus rossiaemeridionalis: in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two GCBs have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female. In the proliferative trophoblast cell population characterized by low ploidy levels (2–16c and in the highly polyploid population of secondary giant trophoblast cells (32–256c the total DNA content in GCB increased proportionally to the ploidy level. In individual GCBs the DNA content also rose proportionally to the ploidy level in nuclei both with one and with two GCBs in both trophoblast cell populations. Some increase in percentage of nuclei with 2–3 GCBs was shown in nuclei of the placenta junctional zone; this may be accounted for by genome multiplication via uncompleted mitoses. In nuclei of the secondary giant trophoblast cells (16–256c the number of GCBs did not exceed 2, and the fraction of nuclei with two GCBs did not increase, which suggests the polytene nature of sex chromosomes in these cells. In all classes of ploidy the DNA content in trophoblast cell nuclei with the single GCB was lower than in nuclei with two and more GCBs. This can indicate that the single GCB in many cases does not derive from fusion of two GCBs. The measurements in individual GCBs suggest that different heterochromatized regions of the X- and Y-chromosome may contribute in GCB formation.

The Elsevier Trophoblast Research Award Lecture: Importance of metzincin proteases in trophoblast biology and placental development: a focus on ADAM12.

Science.gov (United States)

Aghababaei, Mahroo; Beristain, Alexander G

2015-04-01

Placental development is a highly regulated process requiring signals from both fetal and maternal uterine compartments. Within this complex system, trophoblasts, placental cells of epithelial lineage, form the maternal-fetal interface controlling nutrient, gas and waste exchange. The commitment of progenitor villous cytotrophoblasts to differentiate into diverse trophoblast subsets is a fundamental process in placental development. Differentiation of trophoblasts into invasive stromal- and vascular-remodeling subtypes is essential for uterine arterial remodeling and placental function. Inadequate placentation, characterized by defects in trophoblast differentiation, may underlie the earliest cellular events driving pregnancy disorders such as preeclampsia and fetal growth restriction. Molecularly, invasive trophoblasts acquire characteristics defined by profound alterations in cell-cell and cell-matrix adhesion, cytoskeletal reorganization and production of proteolytic factors. To date, most studies have investigated the importance of the matrix metalloproteinases (MMPs) and their ability to efficiently remodel components of the extracellular matrix (ECM). However, it is now becoming clear that besides MMPs, other related proteases regulate trophoblast invasion via mechanisms other than ECM turnover. In this review, we will summarize the current knowledge on the regulation of trophoblast invasion by members of the metzincin family of metalloproteinases. Specifically, we will discuss the emerging roles that A Disintegrin and Metalloproteinases (ADAMs) play in placental development, with a particular focus on the ADAM subtype, ADAM12. Copyright © 2015 Elsevier Ltd. All rights reserved.

Trophoblast retrieval and isolation from the cervix: origins of cervical trophoblasts and their potential value for risk assessment of ongoing pregnancies.

Science.gov (United States)

Moser, Gerit; Drewlo, Sascha; Huppertz, Berthold; Armant, D Randall

2018-03-28

Early during human development, the trophoblast lineage differentiates to commence placentation. Where the placenta contacts the uterine decidua, extravillous trophoblast (EVT) cells differentiate and invade maternal tissues. EVT cells, identified by expression of HLA-G, invade into uterine blood vessels (endovascular EVT), as well as glands (endoglandular EVT), and open such luminal structures towards the intervillous space of the placenta. Endoglandular invasion diverts the contents of uterine glands to the intervillous space, while glands near the margin of the placenta that also contain endoglandular EVT cells open into the reproductive tract. Cells of the trophoblast lineage have thus been recovered from the uterine cavity and endocervical canal. An emerging non-invasive technology [trophoblast retrieval and isolation from the cervix (TRIC)] isolates and examines EVT cells residing in the cervix to explore their origin, biology and relationship to pregnancy and fetal status. This review explores the origins and possible uses of trophoblast cells obtained during ongoing pregnancies (weeks 5-20) by TRIC. We hypothesize that endoglandular EVT cells at the margins of the expanding placenta enter the uterine cavity and are carried together with uterine secretion products to the cervix where they can be retrieved from a Papanicolaou (Pap) smear. The advantages of TRIC for investigation of human placentation and prenatal testing will be considered. Evidence from the literature, and from archived in utero placental histological sections, is presented to support these hypotheses. We used 52 out of 80 publications that appeared between 1966 and 2017 and were found by searching the PubMed and Google Scholar databases. The studies described trophoblast invasion of uterine vessels and glands, as well as trophoblast cells residing in the reproductive tract. This was supplemented with literature on human placental health and disease. The literature describes a variety of

Glucose metabolism in cultured trophoblasts from human placenta

International Nuclear Information System (INIS)

Moe, A.J.; Farmer, D.R.; Nelson, D.M.; Smith, C.H.

1990-01-01

The development of appropriate placental trophoblast isolation and culture techniques enables the study of pathways of glucose utilization by this important cell layer in vitro. Trophoblasts from normal term placentas were isolated and cultured 24 hours and 72 hours in uncoated polystyrene culture tubes or tubes previously coated with a fibrin matrix. Trophoblasts cultured on fibrin are morphologically distinct from those cultured on plastic or other matrices and generally resemble in vivo syncytium. Cells were incubated up to 3 hours with 14 C-labeled glucose and reactions were stopped by addition of perchloric acid. 14 CO 2 production by trophoblasts increased linearly with time however the largest accumulation of label was in organic acids. Trophoblasts cultured in absence of fibrin utilized more glucose and accumulated more 14 C in metabolic products compared to cells cultured on fibrin. Glucose oxidation to CO 2 by the phosphogluconate (PG) pathway was estimated from specific yields of 14 CO 2 from [1- 14 C]-D-glucose and [6- 14 C]-D-glucose. Approximately 6% of glucose oxidation was by the PG pathway when cells were cultured on fibrin compared to approximately 1% by cells cultured in the absence of fibrin. The presence of a fibrin growth matrix appears to modulate the metabolism of glucose by trophoblast from human placenta in vitro

Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

Science.gov (United States)

Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

2016-05-01

Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Rac1/β-Catenin Signalling Pathway Contributes to Trophoblast Cell Invasion by Targeting Snail and MMP9

Directory of Open Access Journals (Sweden)

Minghua Fan

2016-03-01

Full Text Available Background/Aims: Preeclampsia is an idiopathic and serious complication during gestation in which placental trophoblast cells differentiate into several functional subtypes, including highly invasive extravillous trophoblasts (EVTs. Although the cause and pathogenesis of preeclampsia have remained unclear, numerous studies have suggested that the inadequacy of EVT invasion leads to imperfect uterine spiral artery remodelling, which plays a crucial role in the development of preeclampsia. Rac1, or Ras-related C3 botulinum toxin substrate 1, was found to be a key regulator of the migration, invasion uand apoptosis of various tumour cells. Because EVTs share similar invasive and migratory biological behaviours with malignant cells, this study aimed to determine whether the Rac1 signalling pathway affects trophoblast invasion and is thus involved in the pathogenesis of preeclampsia. Methods: We measured the activity of Rac1 and its downstream targets, β-catenin, Snail and MMP9 in placental tissues from patients experiencing a normal pregnancy and those with preeclampsia. Furthermore, we treated HTR-8/SVneo cells with a shRNA Rac1 vector and the β-catenin inhibitor IWP-2 and explored Rac1 signalling pathway activation as well as the effects of Snail and β-catenin on trophoblast invasion. Results: In placental samples from patients experiencing a normal pregnancy and those with preeclampsia, active Rac1 levels and MMP9 protein and mRNA levels were significantly decreased in term pregnancy samples compared to early pregnancy samples. Lower levels were found in preeclampsia samples than in normal term pregnancy samples, and these levels significantly declined in severe preeclampsia samples compared with mild preeclampsia samples. Further analyses demonstrated that both Rac1 shRNA and the β-catenin inhibitor significantly suppressed MMP9 and Snail activation in trophoblasts, thus impairing trophoblast invasion. Notably, silencing Rac1 down

Hepatitis C Virus Sensing by Human Trophoblasts Induces Innate Immune Responses and Recruitment of Maternal NK Cells: Potential Implications for Limiting Vertical Transmission.

Science.gov (United States)

Giugliano, Silvia; Petroff, Margaret G; Warren, Bryce D; Jasti, Susmita; Linscheid, Caitlin; Ward, Ashley; Kramer, Anita; Dobrinskikh, Evgenia; Sheiko, Melissa A; Gale, Michael; Golden-Mason, Lucy; Winn, Virginia D; Rosen, Hugo R

2015-10-15

Hepatitis C virus (HCV) is the world's most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3 and 6% with odds 90% higher in the presence of HIV coinfection. Prevention of vertical transmission is not possible because of lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for preterm delivery, perinatal mortality, and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. In this study, we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust upregulation of type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a proapoptotic response within HTR8 that could affect the morphology of the placenta. To our knowledge, for the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI as well as novel insights into mechanisms that limit vertical transmission but may paradoxically lead to virus-related pregnancy complications. Copyright © 2015 by The American Association of Immunologists, Inc.

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

Energy Technology Data Exchange (ETDEWEB)

Atay, Safinur [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Gercel-Taylor, Cicek [Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States); Kesimer, Mehmet [Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC (United States); Taylor, Douglas D., E-mail: ddtaylor@louisville.edu [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States)

2011-05-01

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

International Nuclear Information System (INIS)

Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet; Taylor, Douglas D.

2011-01-01

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm ± 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

OS041. Apolipoprotein A-I protects normal integration of the trophoblast into endothelial cellular networks in an in vitro model of preeclampsia.

Science.gov (United States)

Charlton, F; Xu, B; Makris, A; Hennessy, A; Rye, K-A

2012-07-01

Failure of the trophoblast to appropriately invade uterine spiral arteries is thought to be an initiating event in preeclampsia, a disorder associated with endothelial dysfunction. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has also been described in preeclampsia. The pro-inflammatory cytokine TNF-α inhibits trophoblast invasion of uterine endothelial cells. Previous work using an in vitro JEG-3 cell/Uterine endothelial cell co-culture model investigated the effect of apoliopoprotein A-I, the main apolipoprotein component of HDL, on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. These effects are now investigated using the human invasive trophoblast cell line HTR-8/SVneo. This study asks if apoA-I, which has established anti-inflammatory properties, can protect against the deleterious effect of TNF-α on trophoblast-endothelial cell interactions. The in vitro trophoblast-uterine endothelial cell co-culture model was used to investigate the effect of apoA-I on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. Uterine endothelial cells were pre-incubated with lipid free apoA-I (final apoA-I concentration 1 mg/mL) for 16h prior to seeding on matrigel coated plates. Tubules formed within 4h. Fluorescence-labelled HTR-8/SVneo trophoblast cells were then co-cultured with the endothelial cells±TNF-α (final concentration of 0.2ng/mL). Bright field and fluorescent images were captured after 24h. The effect of TNF-α on HTR-8/SVneo cell invasion was quantified with Image J software. Integration of HTR-8/SVneo trophoblast cells into uterine endothelial tubular networks was also imaged using live cell imaging techniques (Zeiss Axiovert). TNF-α inhibited HTR-8/SVneo (trophoblast) cell integration into endothelial tubular structures by 24.1±3.7% pintegration of trophoblast into endothelial tubular structures in the presence

Role of prostate apoptosis response 4 in translocation of GRP78 from the endoplasmic reticulum to the cell surface of trophoblastic cells.

Directory of Open Access Journals (Sweden)

Marie Cohen

Full Text Available Glucose-regulated protein 78 (GRP78 is an endoplasmic reticulum (ER molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4, a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.

In vitro effects of triiodothyronine on gene expression in mouse trophoblast cells.

Science.gov (United States)

Silva, J F; Ocarino, N M; Serakides, R

2015-01-01

The objective of the present study was to evaluate the effects of different doses of T3 (10(-4) M, 10(-7) M, 10(-9) M) on the in vitro gene expression of Tpbp, Prl3b1, VEGF, PGF, PL-1, and INFy in mouse trophoblast cells by real-time RT-PCR. Doses of 10(-7) and 10(-9) M T3 increased the mRNA levels of Tpbp, Pl3b1, VEGF, PGF, INFy and PL-1. In contrast, the dose of 10(-4) M reduced the gene expression of PL-1 and VEGF. T3 affected the gene expression of differentiation, hormonal, immune and angiogenic factors in mouse trophoblast cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of CD147 (basigin) as a mediator of trophoblast functions.

Science.gov (United States)

Lee, Cheuk-Lun; Lam, Maggie P Y; Lam, Kevin K W; Leung, Carmen O N; Pang, Ronald T K; Chu, Ivan K; Wan, Tiffany H L; Chai, Joyce; Yeung, William S B; Chiu, Philip C N

2013-11-01

Does CD147 regulate trophoblast functions in vitro? CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast. CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear. A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts. CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs. CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by si

Human placental trophoblast invasion and differentiation: a particular focus on Wnt signalling

Directory of Open Access Journals (Sweden)

Martin eKnöfler

2013-09-01

Full Text Available Wingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. Wnt signalling components also play crucial roles in murine placental development controlling trophoblast lineage determination, chorioallantoic fusion and placental branching morphogenesis. However, the role of the pathway in human placentation, trophoblast development and differentiation is only partly understood. Here, we summarize our present knowledge about Wnt signalling in the human placenta and discuss its potential role in physiological and aberrant trophoblast invasion, gestational diseases and choriocarcinoma formation. Differentiation of proliferative first trimester cytotrophoblasts into invasive extravillous trophoblasts is associated with nuclear recruitment of β-catenin and induction of Wnt-dependent T-cell factor 4 suggesting that canonical Wnt signalling could be important for the formation and function of extravillous trophoblasts. Indeed, activation of the pathway was shown to promote trophoblast invasion in different in vitro trophoblast model systems as well as trophoblast cell fusion. Methylation-mediated silencing of inhibitors of Wnt signalling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β-catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signalling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast invasion and incomplete spiral artery remodelling. Moreover, changes in Wnt signalling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, the current literature suggests a critical role of Wnt signalling in physiological and abnormal

Identification of differences in gene expression in primary cell cultures of human endometrial epithelial cells and trophoblast cells following their interaction

DEFF Research Database (Denmark)

Høgh, Mette; Islin, Henrik; Møller, Charlotte

2006-01-01

The interaction between the cell types was simulated in vitro by growing primary cell cultures of human endometrial epithelial cells and trophoblast cells together (co-culture) and separately (control cultures). Gene expression in the cell cultures was compared using the Differential Display method and confirmed...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

Science.gov (United States)

Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

2016-07-30

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

Ets-2 and p53 mediate cAMP-induced MMP-2 expression, activity and trophoblast invasion

Directory of Open Access Journals (Sweden)

Goldman Shlomit

2009-11-01

Full Text Available Abstract Background We have previously shown that Matrix metalloproteinase (MMP -2 is a key-enzyme in early trophoblast invasion and that Protein Kinase A (PKA increases MMP-2 expression and trophoblast invasion. The aim of this study was to examine MMP -2 regulation by PKA in invasive trophoblasts: JAR choriocarcinoma cell-line and 6-8 w first trimester trophoblasts. Methods The effect of Forskolin (PKA on MMP-2 expression was assessed by Northern Blot and RT-PCR. Possible transcription factors binding to consensus MMP-2 promoter sequences in response to Forskolin, were detected by EMSA binding assay and their expression assessed by western blot analysis. Antisense transfection of relevant transcription factors was performed and the inhibitory effect assessed on MMP-2 expression (RT-PCR, secretion (zymography and trophoblast invasiveness (transwell migration assay. Results We found that Forskolin increased MMP-2 mRNA in JAR cells within 24 hours, and induced binding to p53, Ets, C/EBP and AP-2. Transcription factors Ets-2, phospho- p53, C/EBP epsilon, C/EBP lambda and AP-2 alpha bound to their respective binding sequences in response to Forskolin and the expressions of these transcription factors were all elevated in Forskolin- treated cells. Inhibition of Ets-2 and p53 reduced MMP-2 expression, secretion and invasiveness of Forskolin treated cells. Conclusion MMP-2 is regulated by PKA through several binding sites and transcription factors including Ets-2, p53, C/EBP, C/EBP lambda and AP-2 alpha. Ets-2 and p53 mediate cAMP- induced trophoblast invasiveness, through regulation of MMP-2.

Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

Science.gov (United States)

Lagaye, S.; Derrien, M.; Menu, E.; Coïto, C.; Tresoldi, E.; Mauclère, P.; Scarlatti, G.; Chaouat, G.; Barré-Sinoussi, F.; Bomsel, M.

2001-01-01

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies. PMID:11312350

Sphingosine-1-phosphate promotes extravillous trophoblast cell invasion by activating MEK/ERK/MMP-2 signaling pathways via S1P/S1PR1 axis activation.

Science.gov (United States)

Yang, Weiwei; Li, Qinghua; Pan, Zhifang

2014-01-01

Successful placentation depends on the proper invasion of extravillous trophoblast (EVT) cells into maternal tissues. Previous reports demonstrated that S1P receptors are expressed in the EVT cells and S1P could regulate migration and function of trophoblast cells via S1P receptors. However, little is known about roles of S1P in the invasion of EVT cells. Our study was performed to investigate S1P effect on the invasion of EVT cells. We used the extravillous trophoblast cell line HTR8/SVneo cells to evaluate the effect. In vitro invasion assay was employed to determine the invasion of HTR8/SVneo cells induced by S1P. MMP-2 enzyme activity and relative level in the supernatants of HTR8/SVneo was assessed by gelatin zymography and western blot. Based on the above, siRNA and specific inhibitors were used for the intervention and study of potential signal pathways, and Real-time qPCR and western blot were used to test the mRNA and protein level of potential signal targets. We found that S1P could promote HTR8/SVneo cell invasion and upregulates activity and level of MMP-2. The promotion requires activation of MEK-ERK and is dependent on the axis of S1P/S1PR1. Our investigation of S1P may provide new insights into the molecular mechanisms of EVT invasion.

Steroid hormones modulate galectin-1 in the trophoblast HTR-8/SVneocell line

Directory of Open Access Journals (Sweden)

Bojić-Trbojević Žanka

2008-01-01

Full Text Available The effects of steroids on galectin-1 (gal-1 were studied in HTR-8/SVneo cells by immunocytochemistry, cell-based ELISA, the MTT proliferation test and the Matrigel TM invasion test. Dexamethasone (DEX, progesterone (PRG, and mifepristone (RU486 were used. Gal-1 was modulated in a steroid- and dose-dependent manner by DEX, which mildly but significantly stimulated production at low concentrations (0.1-10 nM, and inhibited it at 100 nM, while the effects of PRG and RU486 were opposite. HTR-8/SVneo cell invasion of Matrigel was significantly decreased in the presence of DEX and lactose. The obtained data support the proposed regulatory role of steroids in trophoblast gal-1 production.

The psychoactive compound of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC) inhibits the human trophoblast cell turnover.

Science.gov (United States)

Costa, M A; Fonseca, B M; Marques, F; Teixeira, N A; Correia-da-Silva, G

2015-08-06

The noxious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. Its consumption during gestation is associated with alterations in foetal growth, low birth weight and preterm labor. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) impairs the production of reproductive hormones and is also able to cross the placenta barrier. However, its effect on the main placental cells, the trophoblasts, are unknown. Actually, the role of THC in cell survival/death of primary human cytotrophoblasts (CTs) and syncytiotrophoblasts (STs) and in the syncytialization process remains to be explored. Here, we show that THC has a dual effect, enhancing MTT metabolism at low concentrations, whereas higher doses decreased cell viability, on both trophoblast phenotypes, though the effects on STs were more evident. THC also diminished the generation of oxidative and nitrative stress and the oxidized form of glutathione, whereas the reduced form of this tripeptide was increased, suggesting that THC prevents ST cell death due to an antioxidant effect. Moreover, this compound enhanced the mitochondrial function of STs, as observed by the increased MTT metabolism and intracellular ATP levels. These effects were independent of cannabinoid receptors activation. Besides, THC impaired CT differentiation into STs, since it decreased the expression of biochemical and morphological biomarkers of syncytialization, through a cannabinoid receptor-dependent mechanism. Together, these results suggest that THC interferes with trophoblast turnover, preventing trophoblast cell death and differentiation, and contribute to disclose the cellular mechanisms that lead to pregnancy complications in women that consume cannabis-derived drugs during gestation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1.

Science.gov (United States)

Gu, Yongzhong; Bian, Yuehong; Xu, Xiaofei; Wang, Xietong; Zuo, Changting; Meng, Jinlai; Li, Hongyan; Zhao, Shigang; Ning, Yunnan; Cao, Yongzhi; Huang, Tao; Yan, Junhao; Chen, Zi-Jiang

2016-12-01

Placenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. Expression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. Expression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3' untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. Our results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta. Copyright © 2016 Elsevier Ltd. All rights

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

Science.gov (United States)

Westwood, Melissa; Al-Saghir, Khiria; Finn-Sell, Sarah; Tan, Cherlyn; Cowley, Elizabeth; Berneau, Stéphane; Adlam, Daman; Johnstone, Edward D

2017-12-01

Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH) 2 D 3 . QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH) 2 D 3 for 48 or 72 h reduces S1PR2 (4-fold; S1P did not inhibit the migration of cells exposed to 1,25(OH) 2 D 3 (p S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα 12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Edaravone inhibits hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 expression: a possible therapeutic approach to preeclampsia.

Science.gov (United States)

Zhao, Y; Zheng, Y F; Luo, Q Q; Yan, T; Liu, X X; Han, L; Zou, L

2014-07-01

To investigate the effects of edaravone, a potent free radical scavenger used clinically, on hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 (sFlt-1) expression. A trophoblast cell line (HRT-8/SVneo) impaired by cobalt chloride (CoCl2) was used as the cell model under hypoxic conditions. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was used to measure the viability of cells exposed to CoCl2 and edaravone. The levels of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts was measured by real-time polymerase chain reaction, and the secretion of sFlt-1, VEGF, and PlGF proteins was analyzed by enzyme-linked immunosorbent assays (ELISAs). A human umbilical vein endothelial cell (HUVEC) tube-formation assay was performed to identify the effects of CoCl2 and edaravone on vascular development. CoCl2 treatment caused the loss of trophoblast viability, the formation of ROS, and sFlt-1 mRNA and protein expression in a dose-dependent manner. Pretreatment with edaravone significantly inhibited hypoxia-induced oxidative stress formation and sFlt-1 expression in trophoblasts. Neither PlGF nor VEGF mRNA or protein expression was increased by CoCl2. In the in vitro tube formation assay, edaravone showed a protective role in vascular development under hypoxic conditions. This study demonstrated that hypoxia leading to increased sFlt-1 release in trophoblasts may contribute to the placental vascular formation abnormalities observed in preeclampsia and suggested that the free radical scavenger edaravone could be a candidate for the effective treatment of preeclampsia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV-infected cat

Directory of Open Access Journals (Sweden)

Donaldson Janet R

2011-07-01

Full Text Available Abstract Background FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF and anti-inflammatory cytokines (IL-4, IL-5, and IL-10; CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor; the chemokine receptor CXCR4 (FIV co-receptor; SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. Methods We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. Results We detected IL-4, IL-5, IL-6, IL-1β, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. Conclusion Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in

Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform

Directory of Open Access Journals (Sweden)

Totey Satish M

2009-09-01

Full Text Available Abstract Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30 that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10, for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2 secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.

Expression of urokinase receptors by human trophoblast. A histochemical and ultrastructural analysis

DEFF Research Database (Denmark)

Multhaupt, H A; Mazar, A; Cines, D B

1994-01-01

BACKGROUND: Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors by troph......BACKGROUND: Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors...... at the leading edge of migrating extravillous trophoblast cells. Receptors were also abundantly expressed during the first and second trimesters of gestation by villous trophoblast, where they were located on apical villous projections and within intracellular vacuoles, a subset of which were lysosomes...

Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

Directory of Open Access Journals (Sweden)

Valeria Feinshtein

2013-09-01

Full Text Available Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD, a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp and Breast Cancer Resistance Protein (BCRP expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp for comparison. Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3 and rhodamine123(rh123. Cyclosporine A (CsA served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3 and rh123 was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-β/activin/nodal signalling inhibition.

Science.gov (United States)

Koel, Mariann; Võsa, Urmo; Krjutškov, Kaarel; Einarsdottir, Elisabet; Kere, Juha; Tapanainen, Juha; Katayama, Shintaro; Ingerpuu, Sulev; Jaks, Viljar; Stenman, Ulf-Hakan; Lundin, Karolina; Tuuri, Timo; Salumets, Andres

2017-09-01

Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-β)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-β/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

MiR-519d-3p suppresses invasion and migration of trophoblast cells via targeting MMP-2.

Directory of Open Access Journals (Sweden)

Jie Ding

Full Text Available Our study was approved by the Medical Ethics Committee of Tang Du Hospital, Fourth Military Medical University and complied strictly with national ethical guidelines. Preeclampsia (PE is a specific clinical disorder characterized by gestational hypertension and proteinuria and is a leading cause of maternal and perinatal mortality worldwide. The miR-519d-3p is upregulated in the maternal plasma of patients with PE which indicates a possible association between this microRNA and the pathogenesis of PE. No studies to date have addressed the effect of miR-519d-3p on the invasion and migration of trophoblast cells. In our study, we found that miR-519d-3p expression was elevated in placental samples from patients with PE. In vitro, overexpression of miR-519d-3p significantly inhibited trophoblast cell migration and invasion, whereas transfection of a miR-519d-3p inhibitor enhanced trophoblast cell migration and invasion. Luciferase assays confirmed that matrix metalloproteinase-2 (MMP-2 is a direct target of miR-519d-3p. Quantitative real-time PCR and western blot assays showed that overexpression of miR-519d-3p downregulated MMP-2 mRNA and protein expression. Knockdown of MMP-2 using a siRNA attenuated the increased trophoblast migration and invasion promoted by the miR-519d-3p inhibitor. In placentas from patients with PE or normal pregnancies, a negative correlation between the expression of MMP-2 and miR-519d-3p was observed using the Pearson correlation and linear regression analysis. Our present findings suggest that upregulation of miR-519d-3p may contribute to the development of PE by inhibiting trophoblast cell migration and invasion via targeting MMP-2; miR-519d-3p may represent a potential predictive and therapeutic target for PE.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

Science.gov (United States)

Okeyo, Kennedy Omondi; Kurosawa, Osamu; Yamazaki, Satoshi; Oana, Hidehiro; Kotera, Hidetoshi; Nakauchi, Hiromitsu; Washizu, Masao

2015-10-01

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 μm in pitch) with narrow mesh strands (3-5 μm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

Science.gov (United States)

Bolnick, Alan D; Bolnick, Jay M; Kohan-Ghadr, Hamid-Reza; Kilburn, Brian A; Pasalodos, Omar J; Singhal, Pankaj K; Dai, Jing; Diamond, Michael P; Armant, D Randall; Drewlo, Sascha

2017-06-01

Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB

Comparative experimental infection of Listeria monocytogenes and Listeria ivanovii in bovine trophoblasts.

Science.gov (United States)

Rocha, Cláudia E; Mol, Juliana P S; Garcia, Luize N N; Costa, Luciana F; Santos, Renato L; Paixão, Tatiane A

2017-01-01

Listeria monocytogenes is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with L. monocytogenes and L. ivanovii are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of L. monocytogenes, L. ivanovii, and L. innocua infections in bovine trophoblastic cells for up to 8 h post infection. L. monocytogenes and L. ivanovii were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although L. innocua was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to L. monocytogenes and L. ivanovii infection.

TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

Directory of Open Access Journals (Sweden)

Tien-Ling Liao

2014-09-01

Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

Is Doppler ultrasound useful for evaluating gestational trophoblastic disease?

Directory of Open Access Journals (Sweden)

Lawrence H. Lin

Full Text Available Doppler ultrasound is a non-invasive method for evaluating vascularization and is widely used in clinical practice. Gestational trophoblastic neoplasia includes a group of highly vascularized malignancies derived from placental cells. This review summarizes data found in the literature regarding the applications of Doppler ultrasound in managing patients with gestational trophoblastic neoplasia. The PubMed/Medline, Web of Science, Cochrane and LILACS databases were searched for articles published in English until 2014 using the following keywords: “Gestational trophoblastic disease AND Ultrasonography, Doppler.” Twenty-eight articles met the inclusion criteria and were separated into the 4 following groups according to the aim of the study. (1 Doppler ultrasound does not seem to be capable of differentiating partial from complete moles, but it might be useful when evaluating pregnancies in which a complete mole coexists with a normal fetus. (2 There is controversy in the role of uterine artery Doppler velocimetry in the prediction of development of gestational trophoblastic neoplasia. (3 Doppler ultrasound is a useful tool in the diagnosis of gestational trophoblastic neoplasia because abnormal myometrial vascularization and lower uterine artery Doppler indices seem to be correlated with invasive disease. (4 Lower uterine artery Doppler indices in the diagnosis of gestational trophoblastic neoplasia are associated with methotrexate resistance and might play a role in prognosis. CONCLUSION: Several studies support the importance of Doppler ultrasound in the management of patients with gestational trophoblastic neoplasia, particularly the role of Doppler velocimetry in the prediction of trophoblastic neoplasia and the chemoresistance of trophoblastic tumors. Doppler findings should be used as ancillary tools, along with human chorionic gonadotropin assessment, in the diagnosis of gestational trophoblastic neoplasia.

Is Doppler ultrasound useful for evaluating gestational trophoblastic disease?

Science.gov (United States)

Lin, Lawrence H; Bernardes, Lisandra S; Hase, Eliane A; Fushida, Koji; Francisco, Rossana P V

2015-12-01

Doppler ultrasound is a non-invasive method for evaluating vascularization and is widely used in clinical practice. Gestational trophoblastic neoplasia includes a group of highly vascularized malignancies derived from placental cells. This review summarizes data found in the literature regarding the applications of Doppler ultrasound in managing patients with gestational trophoblastic neoplasia. The PubMed/Medline, Web of Science, Cochrane and LILACS databases were searched for articles published in English until 2014 using the following keywords: "Gestational trophoblastic disease AND Ultrasonography, Doppler." Twenty-eight articles met the inclusion criteria and were separated into the 4 following groups according to the aim of the study. (1) Doppler ultrasound does not seem to be capable of differentiating partial from complete moles, but it might be useful when evaluating pregnancies in which a complete mole coexists with a normal fetus. (2) There is controversy in the role of uterine artery Doppler velocimetry in the prediction of development of gestational trophoblastic neoplasia. (3) Doppler ultrasound is a useful tool in the diagnosis of gestational trophoblastic neoplasia because abnormal myometrial vascularization and lower uterine artery Doppler indices seem to be correlated with invasive disease. (4) Lower uterine artery Doppler indices in the diagnosis of gestational trophoblastic neoplasia are associated with methotrexate resistance and might play a role in prognosis. Several studies support the importance of Doppler ultrasound in the management of patients with gestational trophoblastic neoplasia, particularly the role of Doppler velocimetry in the prediction of trophoblastic neoplasia and the chemoresistance of trophoblastic tumors. Doppler findings should be used as ancillary tools, along with human chorionic gonadotropin assessment, in the diagnosis of gestational trophoblastic neoplasia.

Placental Trophoblast Responses to Porphyromonas gingivalis Mediated by Toll-like Receptor-2 and -4

Directory of Open Access Journals (Sweden)

Banun Kusumawardani

2013-09-01

Full Text Available Trophoblast participates in preventing allorecognition and controlling pathogens that compromise fetal wellbeing. Toll-like receptors recognize conserved sequences on the pathogens surface and trigger effector cell functions. Porphyromonas gingivalis is thought to spread to the umbilical cord and cause fetal growth restriction. Objective: To characterize expression and function of TLR-2 and TLR-4 in trophoblast cells from Porphyromonas gingivalisinfected pregnant rats. Methods: Live Porphyromonas gingivalis were challenged into the maxillary first molar subgingival sulcus of female rats before and/or during pregnancy and sacrified on gestational day (GD 14 and 20. Porphyromonas gingivalis was detected by API-ZYM system in the maternal blood of the retro-orbital venous plexus and the umbilical cord. TLR-2 and TLR-4 expressions in trophoblast cells was detected by immunohistochemistry. Results: Porphyromonas gingivalis was first detected in the maternal blood and finally spread to the umbilical cord. Syncytiotrophoblast, spongitrophoblast and trophoblastic giant cell in treated groups had significantly higher expression of TLR-2 and TLR-4 than control group (p<0.05. Conclusion: Syncytiotrophoblast, spongitrophoblast and trophoblastic giant cell are able to recognize Porphyromonas gingivalis through TLR-2 and TLR-4 expression. The ligation of TLR-2 and TLR-4 promoted cytokine production and induced trophoblast cell death. These findings strengthen links between periodontal disease and fetal growth restriction.DOI: 10.14693/jdi.v20i2.150

Generation of Elf5-Cre knockin mouse strain for trophoblast-specific gene manipulation.

Science.gov (United States)

Kong, Shuangbo; Liang, Guixian; Tu, Zhaowei; Chen, Dunjin; Wang, Haibin; Lu, Jinhua

2018-04-01

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5-Cre mice, we mated Elf5-Cre mice with Rosa26 mT/mG reporter mice, and found that Elf5-Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5-Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5-Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation. © 2018 Wiley Periodicals, Inc.

The role of Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS) in proliferation, apoptosis and hormone secretion of trophoblast cells.

Science.gov (United States)

Zhao, H-D; Zhang, W-G; Sun, M-N; Duan, Q-F; Li, F-L; Li, H

2013-11-01

To investigate whether Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS), which plays an essential role in the synthesis of selenoprotein, affects proliferation, apoptosis and hormone secretion of human trophoblast cells. Human trophoblast JEG-3 cells were divided into four groups: control group, SEPSECS silenced-expression group, empty vector group and SEPSECS over-expression group. Over-expression and silenced-expression were achieved by transfection with plasmid DNA or RNA oligonucleotide, respectively. 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) and colony formation assays were performed to investigate cell proliferation, while apoptosis was tested by annexin V-FITC, PI double staining and caspases-3 activation assays, enzyme-linked immunosorbent assay (ELISA) was used to determine the level of progesterone (PG) and human chorionic gonadotropin (hCG). SEPSECS silenced-expression clearly inhibited proliferation of JEG-3 cells (p < 0.05), significantly induced cell apoptosis (p < 0.01) and reduced the production of PG and hCG (p < 0.05). On the contrary, SEPSECS over-expression significantly promoted both cell proliferation (p < 0.01) and secretion of PG and hCG (p < 0.05). SEPSECS significantly affects proliferation, apoptosis and hormone secretion of human trophoblast cells, suggesting that a potential relationship exists among SEPSECS, cell proliferation, apoptosis and hormone production of human placental trophoblast cells. Furthermore, this may provide a clue to uncover the relationship between selenium and human placental in association with an emphasis on the importance of selenium adequacy during pregnancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells

DEFF Research Database (Denmark)

Djurisic, S; Teiblum, S; Tolstrup, C K

2015-01-01

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complicati...

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation.

Directory of Open Access Journals (Sweden)

Priyadarsini Kumar

Full Text Available Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O2 and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts and a villous pathway (giving rise to multinucleated syncytiotrophoblast. Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion. Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNF�

Gestational trophoblastic neoplasia: A 6 year retrospective study

Directory of Open Access Journals (Sweden)

Sushruta Shrivastava

2014-01-01

Full Text Available Aims and Objectives: To study the clinical presentations of gestational trophoblastic neoplasia and its response to chemotherapy. Materials and Methods: This is a retrospective study of 28 women of gestational trophoblastic neoplasia evaluated over a period of 6 years from January 2004 to December 2009. Patients were evaluated on the basis of their age, number of deliveries, history of abortion or molar pregnancy, and the treatment received. All patients were scored on the basis of WHO scoring system. Patients with low risk (score /=7 received multiple agent chemotherapy with EMACO regimen. After completion of chemotherapy patients were followed for a minimum of 2 years. The response to treatment was evaluated during follow-up by clinical examination, beta hCG levels and imaging as and when required. Results: Out of 28 women only 27 could be evaluated, because 1 patient was lost to follow-up. Out of 27 patients, 18 patients (66.67% achieved complete remission with the first-line chemotherapy and additional 25.92% (7/27 achieved complete remission with second line chemotherapy resulting in complete remission of 92.5% (25/27. Conclusion: Gestational trophoblastic neoplasia is curable if patient is properly evaluated and scored. It shows good response to chemotherapy.

TNF-α inhibits trophoblast integration into endothelial cellular networks.

Science.gov (United States)

Xu, B; Nakhla, S; Makris, A; Hennessy, A

2011-03-01

Preeclampsia has been linked to shallow trophoblast invasion and failure of uterine spiral artery transformation. Interaction between trophoblast cells and maternal uterine endothelium is critically important for this remodelling. The aim of our study was to investigate the effect of TNF-α on the interactions of trophoblast-derived JEG-3 cells into capillary-like cellular networks. We have employed an in vitro trophoblast-endothelial cell co-culture model to quantify trophoblast integration into endothelial cellular networks and to investigate the effects of TNF-α. Controlled co-cultures were also treated with anti-TNF-α antibody (5 μg/ml) to specifically block the effect of TNF-α. The invasion was evaluated by performing quantitative PCR (Q-PCR) to analyse gene expression of matrix metalloproteinases-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, integrins (α(1)β(1) and α(6)β(4)), plasminogen activator inhibitor (PAI)-1, E-cadherin and VE-cadherin. JEG-3 cell integration into endothelial networks was significantly inhibited by exogenous TNF-α. The inhibition was observed in the range of 0.2-5 ng/ml, to a maximum 56% inhibition at the highest concentration. This inhibition was reversed by anti-TNF-α antibody. Q-PCR analysis showed that mRNA expression of integrins α(1)β(1) and MMP-2 was significantly decreased. VE-cadherin mRNA expression was significantly up-regulated (32-80%, p integration into maternal endothelial cellular networks, and this process involves the inhibition of MMP-2 and a failure of integrins switch from α(6)β(4) to α(1)β(1.) These molecular correlations reflect the changes identified in human preeclampsia. Copyright © 2011 Elsevier Ltd. All rights reserved.

Elsevier Trophoblast Research Award Lecture: Unique properties of decidual T cells and their role in immune regulation during human pregnancy.

Science.gov (United States)

Tilburgs, T; Claas, F H J; Scherjon, S A

2010-03-01

Maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Most studies focus on decidual NK cells and their interaction with fetal trophoblasts, whereas limited data are available on the mechanisms of fetus specific immune recognition and immune regulation by decidual T cells at the fetal-maternal interface. The aim of this review is to describe the phenotypic characteristics of decidual T cell subsets present at the fetal-maternal interface, their interaction with HLA-C expressed by fetal trophoblasts and their role in immune recognition and regulation at the fetal-maternal interface during human pregnancy. Copyright 2010 Elsevier Ltd. All rights reserved.

First-line chemotherapy in low-risk gestational trophoblastic neoplasia.

LENUS (Irish Health Repository)

Alazzam, Mo'iad

2012-01-01

This is an update of a Cochrane review that was first published in Issue 1, 2009. Gestational trophoblastic neoplasia (GTN) is a rare but curable disease arising in the fetal chorion during pregnancy. Most women with low-risk GTN will be cured by evacuation of the uterus with or without single-agent chemotherapy. However, chemotherapy regimens vary between treatment centres worldwide and the comparable benefits and risks of these different regimens are unclear.

miR-125b-1-3p inhibits trophoblast cell invasion by targeting sphingosine-1-phosphate receptor 1 in preeclampsia.

Science.gov (United States)

Li, Qinghua; Pan, Zhifang; Wang, Xuejian; Gao, Zhiqin; Ren, Chune; Yang, Weiwei

2014-10-10

Preeclampsia (PE) is the leading cause of maternal and perinatal mortality and morbidity. Understanding the molecular mechanisms underlying placentation facilitates the development of better intervention of this disease. MicroRNAs are strongly implicated in the pathogenesis of this syndrome. In current study, we found that miR-125b-1-3p was elevated in placentas derived from preeclampsia patients. Transfection of miR-125b-1-3p mimics significantly inhibited the invasiveness of human trophoblast cells, whereas miR-125b-1-3p inhibitor enhanced trophoblast cell invasion. Luciferase assays identified that S1PR1 was a novel direct target of miR-125b-1-3p in the placenta. Overexpression of S1PR1 could reverse the inhibitory effect of miR-125b-1-3p on the invasion of trophoblast cells. These findings suggested that abnormal expression of miR-125b-1-3p might contribute to the pathogenesis of preeclampsia. Copyright © 2014 Elsevier Inc. All rights reserved.

Let-7i-Induced Atg4B Suppression Is Essential for Autophagy of Placental Trophoblast in Preeclampsia.

Science.gov (United States)

Xu, Yinyan; Huang, Xinyan; Xie, Juan; Chen, Yanni; Fu, Jing; Wang, Li

2017-09-01

Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let-7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let-7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let-7i mimics suppressed the autophagic activities in human HTR-8/SVneo trophoblast cell line (HTR-8) and human placental choriocarcinoma cell line JEG-3, whereas let-7i inhibitor enhanced the activities. As a potential target of let-7i, autophagy-related 4B cysteine peptidase (Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let-7i using dual luciferase reporter assay. Furthermore, these trophoblast-like cells transfected with the let-7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let-7i mimic-reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let-7i inhibitor-induced the level of LC3II/I expression. Taken together, these findings suggest that let-7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. J. Cell. Physiol. 232: 2581-2589, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Sera of patients with recurrent miscarriages containing anti-trophoblast antibodies (ATAB) reduce hCG and progesterone production in trophoblast cells in vitro.

Science.gov (United States)

von Schönfeldt, Viktoria; Rogenhofer, Nina; Ruf, Katharina; Thaler, Christian J; Jeschke, Udo

2016-09-01

Reproductive failure including RM has been suggested to correlate with antibodies that cross react with HLA-negative syncytiotrophoblasts and we have reported that 17% of women with 2 or more miscarriages and 34% of women with 3 or more miscarriages express anti-trophoblast antibodies (ATAB). Until now, the mechanism, how ATAB interfere with pregnancy success is not known. HCG and progesterone both play fundamental roles in supporting human pregnancy. Therefore we investigated the effects of sera of RM patients containing ATAB on the hCG and progesterone production of cells of the choriocarcinoma cell line JEG-3. In vitro study to investigate effects of patient sera with and without ATAB on hCG and progesterone secretion of JEG-3 cells. The presence of ATAB was detected as described earlier. Effects of sera from ATAB positive and ATAB negative RM patients on hCG and progesterone secretion by JEG-3 cells were analysed 12 and 24h after plating. Sera of women without pregnancy pathologies served as controls. Sera of ATAB-positive RM patients significantly inhibit hCG secretion of JEG-3 cells for 12h after plating compared to sera of healthy controls (p=0.019) and significantly reduce progesterone production for 12h (p=0.046) and 24h (p=0.027) of co-culture. Sera of ATAB-negative RM patient show no significant effect on progesterone secretion. Inhibition of hCG and progesterone production might point to a mechanism, how ATAB interfere with early pregnancies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

miR-518b Enhances Human Trophoblast Cell Proliferation Through Targeting Rap1b and Activating Ras-MAPK Signal

Directory of Open Access Journals (Sweden)

Ming Liu

2018-03-01

Full Text Available Preeclampsia is a pregnancy-specific complication defined as newly onset gestational hypertension and proteinuria. Deficiency in placental development is considered as the predominant cause of preeclampsia. Our previous study found that the expression of miR-518b increased significantly in the preeclamptic placentas, indicating the potential participation of this small RNA in the occurrence of preeclampsia. In this study, data analysis using multiple databases predicted Rap1b as a candidate target of miR-518b. An evident decrease in Rap1b expression was observed in preeclamptic placentas when compared with the control placentas, which was negatively correlated with the level of miR-518b. Based on the data of in situ hybridization and immunohistochemistry showing that Rap1b exhibited similar localization with miR-518b in villous cytotrophoblast cells and column trophoblasts, we further explored their function in regulating trophoblast cell proliferation. In HTR8/SVneo cells, exogenous transfection of miR-518b reduced the expression of Rap1b, and dual-luciferase reporter assay validated Rap1b as the direct target of miR-518b. The small RNA could increase the BrdU incorporation and the ratio of cells at S phase, and enhance the phosphorylation of Raf-1 and ERK1/2. Such growth-promoting effect could be efficiently reversed by Rap1b overexpression. The data indicate that miR-518b can promote trophoblast cell proliferation via Rap1b–Ras–MAPK pathway, and the aberrant upregulation of miR-518b in preeclamptic placenta may contribute to the excessive trophoblast proliferation. The study provides new evidence to further understand the etiology of preeclampsia.

Knockdown of Heparanase Suppresses Invasion of Human Trophoblasts by Activating p38 MAPK Signaling Pathway

Directory of Open Access Journals (Sweden)

Guanglu Che

2018-01-01

Full Text Available Preeclampsia is a pregnancy-related disease with increasing maternal and perinatal morbidity and mortality worldwide. Defective trophoblast invasion is considered to be a major factor in the pathophysiological mechanism of preeclampsia. Heparanase, the only endo-β-glucuronidase in mammalian cells, has been shown to be abnormally expressed in the placenta of preeclampsia patients in our previous study. The biological role and potential mechanism of heparanase in trophoblasts remain unclear. In the present study, stably transfected HTR8/SVneo cell lines with heparanase overexpression or knockdown were constructed. The effect of heparanase on cellular proliferation, apoptosis, invasion, tube formation, and potential pathways in trophoblasts was explored. Our results showed that overexpression of heparanase promoted proliferation and invasion. Knockdown of heparanase suppressed proliferation, invasion, and tube formation but induced apoptosis. These findings reveal that downregulation of heparanase may contribute to defective placentation and plays a crucial role in the pathogenesis of preeclampsia. Furthermore, increased activation of p38 MAPK in heparanase-knockdown HTR8/SVneo cell was shown by MAPK pathway phosphorylation array and Western blotting assay. After pretreatment with 3 specific p38 MAPK inhibitors (BMS582949, SB203580, or BIRB796, inadequate invasion in heparanase-knockdown HTR8/SVneo cell was rescued. That indicates that knockdown of heparanase decreases HTR8/SVneo cell invasion through excessive activation of the p38 MAPK signaling pathway. Our study suggests that heparanase can be a potential predictive biomarker for preeclampsia at an early stage of pregnancy and represents a promising therapeutic target for the treatment of preeclampsia.

Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast.

Science.gov (United States)

Badwaik, N K; Rasweiler, J J; Muradali, F

1998-11-01

Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin.

MSX2 Induces Trophoblast Invasion in Human Placenta.

Directory of Open Access Journals (Sweden)

Hao Liang

Full Text Available Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2, a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT. However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2, vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may

[Trophoblast: conductor of the maternal immune tolerance].

Science.gov (United States)

Mesdag, V; Salzet, M; Vinatier, D

2014-11-01

Pregnancy is a temporary semi-allograft that survives for nine months. The importance of this event for the survival of the species justifies several tolerance mechanisms that are put into place at the beginning of pregnancy, some of which occur even at the time of implantation. The description of these mechanisms underlines the leadership of the trophoblast. The trophoblast is the conductor of the events, protects himself by expressing specific antigens and regulates the environment of the decidua according to the calendar of the events of the pregnancy The trophoblast and the decidual environment attract the effectors of immunity, almost all present in the decidua. The immunological atmosphere of the decidua evolves during the pregnancy modulating the level of activation of the immunological cells and adapting the level of activation to the stage of the pregnancy. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

Science.gov (United States)

Gysler, Stefan M; Mulla, Melissa J; Guerra, Marta; Brosens, Jan J; Salmon, Jane E; Chamley, Lawrence W; Abrahams, Vikki M

2016-07-01

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was

Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor.

Science.gov (United States)

Armant, D Randall; Kilburn, Brian A; Petkova, Anelia; Edwin, Samuel S; Duniec-Dmuchowski, Zophia M; Edwards, Holly J; Romero, Roberto; Leach, Richard E

2006-02-01

Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

Suppression of STAT3 Signaling by Δ9-Tetrahydrocannabinol (THC Induces Trophoblast Dysfunction

Directory of Open Access Journals (Sweden)

Xinwen Chang

2017-06-01

Full Text Available Aims: Marijuana is a widely used illicit drug and its consumption during pregnancy has been associated with adverse reproductive outcomes. The purpose of this study was to determine the effects of chronic intake of Δ9-tetrahydrocannabinol (THC, the major component of marijuana, on trophoblast function, placental development, and birth outcomes. Methods: The pathological characteristics and distribution of cannabinoid receptors in placenta were observed by immunohistochemical (IHC staining. Cell migration in response to THC was measured by transwell assays. The levels of cannabinoid receptors and Signal Transducer and Activator of Transcription 3 (STAT3 were detected by western blot. Results: We found the placenta expressed two main cannabinoid receptors, suggesting that THC induced biological responses in placental cells. Supporting this hypothesis, we observed dramatic alterations of placental morphology in marijuana users. Using THC and inhibitors of cannabinoid receptors, we demonstrated that THC impaired trophoblast cell migration and invasion partly via cannabinoid receptors. Additionally, pregnant mice injected with THC showed adverse reproductive events including reduced number of fetuses, lower maternal and placental weights. Mechanistically, STAT3 signaling pathway was involved in the THC-induced suppression of trophoblast cell motility and pregnancy outcomes. Conclusion: Our study indicates that the STAT3 signaling pathway plays a critical role in THC-induced trophoblast dysfunction.

Galectin-1 influences trophoblast immune evasion and emerges as a predictive factor for the outcome of pregnancy.

Science.gov (United States)

Tirado-González, Irene; Freitag, Nancy; Barrientos, Gabriela; Shaikly, Valerie; Nagaeva, Olga; Strand, Magnus; Kjellberg, Lennart; Klapp, Burghard F; Mincheva-Nilsson, Lucia; Cohen, Marie; Blois, Sandra M

2013-01-01

Galectin-1 (gal-1) is expressed at the feto-maternal interface and plays a role in regulating the maternal immune response against placental alloantigens, contributing to pregnancy maintenance. Both decidua and placenta contribute to gal-1 expression and may be important for the maternal immune regulation. The expression of gal-1 within the placenta is considered relevant to cell-adhesion and invasion of trophoblasts, but the role of gal-1 in the immune evasion machinery exhibited by trophoblast cells remains to be elucidated. In this study, we analyzed gal-1 expression in preimplantation human embryos and first-trimester decidua-placenta specimens and serum gal-1 levels to investigate the physiological role played by this lectin during pregnancy. The effect on human leukocyte antigen G (HLA-G) expression in response to stimulation or silencing of gal-1 was also determined in the human invasive, proliferative extravillous cytotrophoblast 65 (HIPEC65) cell line. Compared with normal pregnant women, circulating gal-1 levels were significantly decreased in patients who subsequently suffered a miscarriage. Human embryos undergoing preimplantation development expressed gal-1 on the trophectoderm and inner cell mass. Furthermore, our in vitro experiments showed that exogenous gal-1 positively regulated the membrane-bound HLA-G isoforms (HLA-G1 and G2) in HIPEC65 cells, whereas endogenous gal-1 also induced expression of the soluble isoforms (HLA-G5 and -G6). Our results suggest that gal-1 plays a key role in pregnancy maternal immune regulation by modulating HLA-G expression on trophoblast cells. Circulating gal-1 levels could serve as a predictive factor for pregnancy success in early human gestation.

The lncRNA TUG1 modulates proliferation in trophoblast cells via epigenetic suppression of RND3.

Science.gov (United States)

Xu, Yetao; Ge, Zhiping; Zhang, Erbao; Zuo, Qing; Huang, Shiyun; Yang, Nana; Wu, Dan; Zhang, Yuanyuan; Chen, Yanzi; Xu, Haoqin; Huang, Huan; Jiang, Zhiyan; Sun, Lizhou

2017-10-12

Due to limited treatment options, pre-eclampsia (PE) is associated with fetal perinatal and maternal morbidity and mortality. During the causes of PE, failure of uterine spiral artery remodeling which might be related to functioning abnormally of trophoblast cells, result in the occurrence and progression of PE. Recently, abnormal expression of long non-coding RNAs (lncRNAs), as imperative regulators involved in human diseases progression (included PE), which has been indicated by increasing evidence. In this research, we found that TUG1, a lncRNA, was markedly reduced in placental samples from patients with PE. Loss-function assays indicated that knockdown TUG1 significantly affected cell proliferation, apoptosis, migration and network formation in vitro. RNA-seq revealed that TUG1 could affect abundant genes, and then explore the function and regulatory mechanism of TUG1 in trophoblast cells. Furthermore, RNA immunoprecipitation and chromatin immunoprecipitation assays validated that TUG1 can epigenetically inhibit the level of RND3 through binding to EZH2, thus promoting PE development. Therefore, via illuminating the TUG1 mechanisms underlying PE development and progression, our findings might furnish a prospective therapeutic strategy for PE intervention.

PP042. Anti-hypertensive drugs hydralazine, clonidine and labetalol improve trophoblast integration into endothelial cellular networks in vitro.

Science.gov (United States)

Xu, B; Charlton, F; Makris, A; Hennessy, A

2012-07-01

Preeclampsia is an exaggerated maternal inflammatory state with over-expression of placental soluble fms-like tyrosine kinase 1 (sFlt-1). It is also associated with shallow trophoblast invasion and inadequate transformation of uterine spiral arteries. Antihypertensive drugs administrated in preeclampsia to control blood pressure have been reported to regulate placental and circulating cytokine production from women with preeclampsia. Whether they could modulate the interaction between trophoblast and endothelial cells are not investigated. This study is to examine the effect of pharmacological dose of anti-hypertensive hydralazine, clonidine and labetalol on trophoblast cell integration into inflammatory TNF-a pre-exposed endothelial cellular networks. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose (0.5ng/ml) inflammatory TNF-a or TNF-a plus sFlt-1 (100ng/ml) for 24hours. These cells were labelled with red fluorescence and seeded on a 24-well culture plate coated with Matrigel. Endothelial tubular structures appeared within 4hours. Green fluorescent-labelled HTR-8/SVneo trophoblast cells were then co-cultured with endothelial cells, with (or without) hydralazine (10μg/ml), clonidine (1.0μg/ml) or labetalol (0.5μg/ml). Red and green fluorescent images were captured after 24hours. Drug effect on HTR-8 cells integration into endothelial cellular networks was quantified by Image Analysis software. The conditioned media were also collected to measure concentrations of free VEGF, PLGF and sFlt-1 by ELISA. When HTR-8/SVneo trophoblast cells were co-cultured with TNF-a pre-incubated endothelial cells, hydralazine and clonidine can significantly increase the trophoblast integration into endothelial cellular networks. This increase was not seen if co-cultured with normal endothelial cells (without TNF-a pre-incubation) or with TNF-a plus sFlt-1 treated endothelial cells. Labetalol could increase the HTR-8

Syndecan-1 Acts as an Important Regulator of CXCL1 Expression and Cellular Interaction of Human Endometrial Stromal and Trophoblast Cells

Directory of Open Access Journals (Sweden)

Dunja Maria Baston-Buest

2017-01-01

Full Text Available Successful implantation of the embryo into the human receptive endometrium is substantial for the establishment of a healthy pregnancy. This study focusses on the role of Syndecan-1 at the embryo-maternal interface, the multitasking coreceptor influencing ligand concentration, release and receptor presentation, and cellular morphology. CXC motif ligand 1, being involved in chemotaxis and angiogenesis during implantation, is of special interest as a ligand of Syndecan-1. Human endometrial stromal cells with and without Syndecan-1 knock-down were decidualized and treated with specific inhibitors to evaluate signaling pathways regulating CXC ligand 1 expression. Western blot analyses of MAPK and Wnt members were performed, followed by analysis of spheroid interactions between human endometrial cells and extravillous trophoblast cells. By mimicking embryo contact using IL-1β, we showed less ERK and c-Jun activation by depletion of Syndecan-1 and less Frizzled 4 production as part of the canonical Wnt pathway. Additionally, more beta-catenin was phosphorylated and therefore degraded after depletion of Syndecan-1. Secretion of CXC motif ligand 1 depends on MEK-1 with respect to Syndecan-1. Regarding the interaction of endometrial and trophoblast cells, the spheroid center-to-center distances were smaller after depletion of Syndecan-1. Therefore, Syndecan-1 seems to affect signaling processes relevant to signaling and intercellular interaction at the trophoblast-decidual interface.

Suppression of STAT3 Signaling by Δ9-Tetrahydrocannabinol (THC) Induces Trophoblast Dysfunction.

Science.gov (United States)

Chang, Xinwen; Bian, Yiding; He, Qizhi; Yao, Julei; Zhu, Jingping; Wu, Jinting; Wang, Kai; Duan, Tao

2017-01-01

Marijuana is a widely used illicit drug and its consumption during pregnancy has been associated with adverse reproductive outcomes. The purpose of this study was to determine the effects of chronic intake of Δ9-tetrahydrocannabinol (THC), the major component of marijuana, on trophoblast function, placental development, and birth outcomes. The pathological characteristics and distribution of cannabinoid receptors in placenta were observed by immunohistochemical (IHC) staining. Cell migration in response to THC was measured by transwell assays. The levels of cannabinoid receptors and Signal Transducer and Activator of Transcription 3 (STAT3) were detected by western blot. We found the placenta expressed two main cannabinoid receptors, suggesting that THC induced biological responses in placental cells. Supporting this hypothesis, we observed dramatic alterations of placental morphology in marijuana users. Using THC and inhibitors of cannabinoid receptors, we demonstrated that THC impaired trophoblast cell migration and invasion partly via cannabinoid receptors. Additionally, pregnant mice injected with THC showed adverse reproductive events including reduced number of fetuses, lower maternal and placental weights. Mechanistically, STAT3 signaling pathway was involved in the THC-induced suppression of trophoblast cell motility and pregnancy outcomes. Our study indicates that the STAT3 signaling pathway plays a critical role in THC-induced trophoblast dysfunction. © 2017 The Author(s). Published by S. Karger AG, Basel.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

Science.gov (United States)

Djurisic, S; Teiblum, S; Tolstrup, C K; Christiansen, O B; Hviid, T V F

2015-03-01

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood

DEFF Research Database (Denmark)

Hviid, T V; Sørensen, S; Morling, N

1999-01-01

Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against...... a folate-binding protein, which recognizes trophoblast in normal tissues, in conjunction with immunomagnetic cell sorting was investigated. Verification of the presence of fetal DNA in the sorted samples was done by detection of fetal/paternal-specific short tandem repeat (STR) alleles using polymerase...... on peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably due to the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope...

The role of invasive trophoblast in implantation and placentation of primates

Science.gov (United States)

Carter, Anthony M.; Enders, Allen C.; Pijnenborg, Robert

2015-01-01

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human. PMID:25602074

The role of invasive trophoblast in implantation and placentation of primates.

Science.gov (United States)

Carter, Anthony M; Enders, Allen C; Pijnenborg, Robert

2015-03-05

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

miR-346 and miR-582-3p-regulated EG-VEGF expression and trophoblast invasion via matrix metalloproteinases 2 and 9.

Science.gov (United States)

Su, Mei-Tsz; Tsai, Pei-Yin; Tsai, Hui-Ling; Chen, Yi-Chi; Kuo, Pao-Lin

2017-03-01

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an important regulator for embryo implantation and placental development, and is clinically associated with several obstetric disorders related to insufficient or inappropriate trophoblast invasion, such as recurrent abortion, preeclampsia, and intrauterine fetal growth restriction. This study was performed to identify the microRNAs targeting EG-VEGF, and evaluate the regulatory effect on trophoblast biology. miR-346 and miR-582-3p were initially identified via bioinformatic tools, and their specific binding sites on the EG-VEGF 3'UTR were further confirmed using dual luciferase and a co-transfection assays. miR-346 and miR-582-3p were demonstrated not only to suppress EG-VEGF expression, but also inhibit trophoblast invasion and migration in the JAR and HTR-8/SVneo cell lines. We further evaluated the effect of microRNAs in HTR-8/SVneo cells coexpressing EG-VEGF and miR-346 or miR-582-3p on matrix metalloproteinase (MMP 2 and MMP 9) and the tissue inhibitors of metalloproteinase (TIMP 1 and TIMP 2) using RT-PCR, western blotting and gelatin zymography. TIMP 1 and TIMP 2 were not affected by the two microRNAs, whereas the expressions and activities of MMP 2 and MMP 9 were significantly downregulated, which in turn inhibited the invasion ability of trophoblasts. In conclusion, miR-346 and miR-582-3p regulate EG-VEGF-induced trophoblast invasion through repressing MMP 2 and MMP 9, and may become novel diagnostic biomarkers or therapeutic targets for EG-VEGF-related obstetric disorders. © 2016 BioFactors, 43(2):210-219, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Pregnancy outcomes after chemotherapy for trophoblastic neoplasia.

Science.gov (United States)

Garcia, Mila Trementosa; Lin, Lawrence Hsu; Fushida, Koji; Francisco, Rossana Pulcineli Vieira; Zugaib, Marcelo

2016-12-01

The successful development of chemotherapy enabled a fertilitysparing treatment for patients with trophoblastic neoplasia. After disease remission, the outcome of a subsequent pregnancy becomes a great concern for these women. To analyze existing studies in the literature that describe the reproductive outcomes of patients with trophoblastic neoplasia treated with chemotherapy. Systematic review was performed searching for articles on Medline/ Pubmed, Lilacs and Cochrane Library databases, using the terms "gestational trophoblastic disease" and "pregnancy outcome". A total of 18 articles were included. No evidence of decreased fertility after chemotherapy for trophoblastic neoplasia was observed. The abortion rates in patients who conceived within 6 months after chemotherapy was higher compared to those who waited longer. Some studies showed increased rates of stillbirth and repeat hydatidiform moles. Only one work showed increased congenital abnormalities. The pregnancies conceived after chemotherapy for trophoblastic neoplasia should be followed with clinical surveillance due to higher rates of some pregnancy complications. However, studies in the literature provide reassuring data about reproductive outcomes of these patients.

Gestational Trophoblastic Disease—Health Professional Version

Science.gov (United States)

Gestational trophoblastic disease (GTD) is a broad term encompassing both benign and malignant growths arising from products of conception in the uterus. GTDs contain paternal chromosomes and are placental in origin. Find evidence-based information on gestational trophoblastic disease treatment.

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

Directory of Open Access Journals (Sweden)

Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

Directory of Open Access Journals (Sweden)

Noura Abd Ellah

Full Text Available Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1. Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

Alteration of Pituitary Tumor Transforming Gene-1 Regulates Trophoblast Invasion via the Integrin/Rho-Family Signaling Pathway.

Directory of Open Access Journals (Sweden)

Seung Mook Lim

Full Text Available Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1 was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1. Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05. Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4, ITGA5, and integrin beta 1 (ITGB1; otherwise, RhoA expression was significantly decreased (p < 0.05. Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development.

Pregnancy outcomes after chemotherapy for trophoblastic neoplasia

Directory of Open Access Journals (Sweden)

MILA TREMENTOSA GARCIA

Full Text Available SUMMARY Introduction The successful development of chemotherapy enabled a fertilitysparing treatment for patients with trophoblastic neoplasia. After disease remission, the outcome of a subsequent pregnancy becomes a great concern for these women. Objective To analyze existing studies in the literature that describe the reproductive outcomes of patients with trophoblastic neoplasia treated with chemotherapy. Method Systematic review was performed searching for articles on Medline/ Pubmed, Lilacs and Cochrane Library databases, using the terms “gestational trophoblastic disease” and “pregnancy outcome”. Results A total of 18 articles were included. No evidence of decreased fertility after chemotherapy for trophoblastic neoplasia was observed. The abortion rates in patients who conceived within 6 months after chemotherapy was higher compared to those who waited longer. Some studies showed increased rates of stillbirth and repeat hydatidiform moles. Only one work showed increased congenital abnormalities. Conclusion The pregnancies conceived after chemotherapy for trophoblastic neoplasia should be followed with clinical surveillance due to higher rates of some pregnancy complications. However, studies in the literature provide reassuring data about reproductive outcomes of these patients.

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication.

Science.gov (United States)

Aagaard, Kjersti M; Lahon, Anismrita; Suter, Melissa A; Arya, Ravi P; Seferovic, Maxim D; Vogt, Megan B; Hu, Min; Stossi, Fabio; Mancini, Michael A; Harris, R Alan; Kahr, Maike; Eppes, Catherine; Rac, Martha; Belfort, Michael A; Park, Chun Shik; Lacorazza, Daniel; Rico-Hesse, Rebecca

2017-01-27

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Immunolocalization of progesterone receptors in binucleate trophoblast cells of the buffalo placenta (Bubalus bubalis

Directory of Open Access Journals (Sweden)

Carlos Eduardo Ambrósio

2007-06-01

Full Text Available The binucleate trophoblast cells (CTBs of the water buffalo placenta (Bubalus bubalis were studied with emphasis on the presence of progesterone receptor. Placentomal tissues from 27 buffalos (2-10 months of pregnancy were processed and embedded in paraplast (Paraplast Embedding Media – Paraplast Plus to locate the progesterone receptors using the immunohistochemistry technique. The immunohistochemical reaction for progesterone receptor through monoclonal antibody PgR Ab2 showed staining of CTBs, caruncular epithelial and estromal cells and blood vessel estromal pericitos present in the placentome throughout the entire gestational period analyzed. These results indicate the production of progesterone with autocrine and paracrine action in the placentome growth, differentiation and functional regulation.

Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta.

Science.gov (United States)

Belkacemi, Louiza; Gariépy, Gilles; Mounier, Catherine; Simoneau, Lucie; Lafond, Julie

2004-01-01

During pregnancy, the calcium (Ca(2+)) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca(2+) transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca(2+) in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca(2+) availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.

ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

Directory of Open Access Journals (Sweden)

Hwa J Choi

Full Text Available The Hypoxia-inducible Factor (HIF family of transcriptional regulators coordinates the expression of dozens of genes in response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental cells; therefore, trophoblast stem cells (TSCs are used to study mouse placental development. Consistent with a requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe differentiation defects and fail to form trophoblast giant cells (TGCs in vitro. Interestingly, differentiating TSCs induce HIF activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated syncytiotropholasts (SynTs and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2 signaling by ECM composition is responsible for this effect. In the absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is critical for TGC fate determination. HIF-2α ensures TSC cytoskeletal integrity and promotes invasive TGC formation by interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1-an enzyme that regulates microtubule and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.

A Rare Gestational Trophoblastic Disease: Placental Site Trophoblastic Tumor

Directory of Open Access Journals (Sweden)

Senem Yaman Tunç

2016-12-01

PSTT is a rare tumor. In contrast to other trophoblastic tumors, PSTT produces a small amount of ß-HCG and it is relatively insensitive to chemotherapy. Adjuvant chemotherapy is suggested to follow surgical treatment in the cases with metastasis.

Effects of vitamin C, vitamin E, and molecular hydrogen on the placental function in trophoblast cells.

Science.gov (United States)

Guan, Zhong; Li, Huai-Fang; Guo, Li-Li; Yang, Xiang

2015-08-01

This study aimed to investigate the effects of three different antioxidants, namely vitamin C, vitamin E, and molecular hydrogen, on cytotrophoblasts in vitro. Two trophoblast cell lines, JAR and JEG-3, were exposed to different concentrations of vitamin C (0, 25, 50, 100, 500, 1,000, 5,000 μmol/L), vitamin E (0, 25, 50, 100, 500, 1,000, 5,000 μmol/L), and molecular hydrogen (0, 25, 50, 100, 500 μmol/L) for 48 h. The cell viability was detected using the MTS assay. The secretion of human chorionic gonadotropin (hCG) and the tumor necrosis factor-α (TNF-α) were assessed and the expression of TNF-α mRNA was observed by real-time RT-PCR. Cell viability was significantly suppressed by 500 μmol/L vitamins C and E (P 0.05). The expression of TNF-α was increased by 100 μmol/L vitamin C and 50 μmol/L vitamins E, separately or combined (P vitamin C and E, separately or combined. High levels of antioxidant vitamins C and E may have significant detrimental effects on placental function, as reflected by decreased cell viability and secretion of hCG; and placental immunity, as reflected by increased production of TNF-a. Meanwhile hydrogen showed no such effects on cell proliferation and TNF-α expression, but it could affect the level of hCG, indicating hydrogen as a potential candidate of antioxidant in the management of preeclampsia (PE) should be further studied.

[Potential role of the angiogenic factor "EG-VEGF" in gestational trophoblastic diseases].

Science.gov (United States)

Boufettal, H; Feige, J-J; Benharouga, M; Aboussaouira, T; Nadifi, S; Mahdaoui, S; Samouh, N; Alfaidy, N

2013-10-01

Gestational trophoblastic disease (MGT) includes a wide spectrum of pathologies of the placenta, ranging from benign precancerous lesions, with gestational trophoblastic tumors. Metastases are the leading causes of death as a result of this tumor. They represent a major problem for obstetrics and for the public health system. To date, there is no predictor of the progression of molar pregnancies to gestational trophoblastic tumor (GTT). Only an unfavorable plasma hCG monitoring after evacuation of hydatidiform mole is used to diagnose a TTG. The causes of the development of this cancer are still poorly understood. Increasing data in the literature suggests a close association between the development of this tumor and poor placental vascularization during the first trimester of pregnancy. The development of the human placenta depends on a coordination between the trophoblast and endothelial cells. A disruption in the expression of angiogenic factors could contribute to uterine or extra-uterine tissue invasion by extravillous trophoblast, contributing to the development of TTG. This review sheds lights on the phenomenon of angiogenesis during normal and abnormal placentation, especially during the MGT and reports preliminary finding concerning, the variability of expression of "Endocrine Gland-Derived Vascular Endothelial Growth Factor" (EG-VEGF), a specific placental angiogenic factor, in normal and molar placentas, and the potential role of differentiated expressions of the main placental angiogenic factors in the scalability of hydatidiform moles towards a recovery or towards the development of gestational trophoblastic tumor. Deciphering the mechanisms by which the angiogenic factor influences these processes will help understand the pathophysiology of MGT and to create opportunities for early diagnosis and treatment of the latter. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Imaging and Clinical Data of Placental Site Trophoblastic Tumor: A Case Report

International Nuclear Information System (INIS)

Niknejadi, Maryam; Ahmadi, Firoozeh; Akhbari, Farnaz

2016-01-01

Placental site trophoblastic tumor (PSTT) is a very rare variant of gestational trophoblastic tumor. It can occur after normal termination of pregnancy or spontaneous abortion and ectopic or molar pregnancy. There is a wide range of clinical manifestations from a benign condition to an aggressive disease with fatal outcome. One of the most important characteristics of PSTT, unlike other forms of gestational trophoblastic diseases (GTD) is the presence of low beta-subunit of human chorionic gonadotropin (β-hCG) levels because it is a neoplastic proliferation of intermediate trophoblastic cells. However, human placental lactogen (hPL) is increased on histologic section and in the serum of patients too. We present a case of PSTT and discuss the differential diagnosis in order to further familiarize physicians with the diagnosis and treatment of this disease. It has a varied clinical spectrum and usually presents with irregular vaginal bleeding or amenorrhea. Diagnosis is confirmed by dilatation and curettage (D and C) and hysterectomy. Because chemotherapy is not effective, surgery is the cornerstone of treatment. This case is presented because it is a rare neoplasm with different treatments and it should be differentiated from molar pregnancy

Localization of chondromodulin-I at the feto-maternal interface and its inhibitory actions on trophoblast invasion in vitro

Directory of Open Access Journals (Sweden)

Kondo Jun

2011-08-01

Full Text Available Abstract Background Chondromodulin-I (ChM-I is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro. Results Northern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation. Conclusions This study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.

Diagnostics of trophoblast disease and the control of therapeutic efficiency based on definition of chorionic gonadotrapin and trophoblastic beta1-glycoprotein

International Nuclear Information System (INIS)

Yugrinova, L.G.; Olefirenko, G.A.; Dotsenko, Yu.S.

1982-01-01

A comparative estimation of markers of trophoblast disease, chorionic gonadotropin (CG) and trophoblastic beta 1 -glycoprotein defined by different methods is given. It is found that diagnostics and control of therapeutic efficiency for patients having trophoblast disease based on CG definition are possible. In 86% of cases the clinical diagnosis was confirmed by the immunoradioautography, and in 33% - by the immunodiffusion method. The dependence of marker detection frequency on the therapeutic efficiency is found

Blocking Epidermal Growth Factor Receptor Signaling in HTR-8/SVneo First Trimester Trophoblast Cells Results in Dephosphorylation of PKBα/AKT and Induces Apoptosis

Directory of Open Access Journals (Sweden)

J. Bolnick

2011-01-01

Full Text Available We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.

Uterine Rupture Due to Invasive Metastatic Gestational Trophoblastic Neoplasm

Science.gov (United States)

Bruner, David I.; Pritchard, Amy M.; Clarke, Jonathan

2013-01-01

While complete molar pregnancies are rare, they are wrought with a host of potential complications to include invasive gestational trophoblastic neoplasia. Persistent gestational trophoblastic disease following molar pregnancy is a potentially fatal complication that must be recognized early and treated aggressively for both immediate and long-term recovery. We present the case of a 21-year-old woman with abdominal pain and presyncope 1 month after a molar pregnancy with a subsequent uterine rupture due to invasive gestational trophoblastic neoplasm. We will discuss the complications of molar pregnancies including the risks and management of invasive, metastatic gestational trophoblastic neoplasia. PMID:24106538

Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation.

Science.gov (United States)

Malhotra, Sudha Saryu; Gupta, Satish Kumar

2017-01-01

Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

Science.gov (United States)

Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

2017-01-01

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.

Application of monoclonal antibodies against trophoblastic cells to study female infertility

Czech Academy of Sciences Publication Activity Database

Sedláková, Alena; Elzeinová, Fatima; Bukovský, A.; Madar, J.; Ulčová-Gallová, Z.; Pěknicová, Jana

2004-01-01

Roč. 51, č. 6 (2004), s. 482-483 ISSN 1046-7408. [European congress of reproductive immunology. Plzeň, 30.06.2004-03.07.2004] R&D Projects: GA MŠk LN00B030 Keywords : trophoblast * monoclonal antibody * ELISA Subject RIV: EC - Immunology Impact factor: 1.808, year: 2004

Exposure of human JEG-3 cell line to TCDD alters progesterone secretion but does not act on their viability and apoptosis

Energy Technology Data Exchange (ETDEWEB)

Augustowska, K.; Gregoraszczuk, E.L. [Jagiellonian Univ., Krakow (Poland)

2004-09-15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are lipophilic and difficult to metabolize. Any environmental exposure of living organisms to these congeners results in their accumulation in fat tissue and bioconcentration in humans via the food chain. TCDD acts as an endocrine disrupter to alter differentiation and function of the reproductive system. Therefore, these compounds represent a serious health risk, especially to the fetus and infants, whose enzymatic and metabolic systems are not yet mature. Our previous data showed high accumulation of TCDD in cultured human placental tissue which caused a decrease in hormone secretion. However, the mechanism of this action is still unclear. JEG-3 cell line from malignant placental tissue has been used as an in vitro model for investigation of the effects of xenobiotics on placenta toxicity. These cells are morphologically similar to their origin, the trophoblast of the normal first trimester placenta, and produce many peptides and steroid hormones found in normal trophoblast cells, such as hCG, GhRH, progesterone. The aim of the present study was firstly, to show dose- and time-dependent effects of TCDD on progesterone production by JEG-3 cells and secondly, to examine mechanism of its action on cell viability and apoptosis.

Uterine Rupture Due to Invasive Metastatic Gestational Trophoblastic Neoplasm

Directory of Open Access Journals (Sweden)

David I Bruner

2013-09-01

Full Text Available While complete molar pregnancies are rare, they are wrought with a host of potential complications to include invasive gestational trophoblastic neoplasia. Persistent gestational trophoblastic disease following molar pregnancy is a potentially fatal complication that must be recognized early and treated aggressively for both immediate and long-term recovery. We present the case of a 21-year-old woman with abdominal pain and presyncope 1 month after a molar pregnancy with a subsequent uterine rupture due to invasive gestational trophoblastic neoplasm. We will discuss the complications of molar pregnancies including the risks and management of invasive, metastatic gestational trophoblastic neoplasia. [West J Emerg Med. 2013;14(5:444–447.

Proportion hyperglycosylated hCG: a new test for discriminating gestational trophoblastic diseases.

Science.gov (United States)

Cole, Laurence A

2014-11-01

Hyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG with large oligosaccharide side chains. Although hCG is produced by syncytiotrophoblast cells, hyperglycosylated hCG marks cytotrophoblast cell. Hyperglycosylated hCG signals placental implantation. Total hCG in serum and urine is measured by the Siemens Immulite hCG pregnancy test; the result is in milli-international unit per milliliter. Hyperglycosylated hCG is determined by the B152 microtiter plate assay; the result is in nanogram per milliliter. Hyperglycosylated hCG results can be converted to milli-international unit per milliliter equivalents by multiplying by 11. The test measures proportion hyperglycosylated hCG, hyperglycosylated hCG / total hCG. Proportion hyperglycosylated hCG marks cases intent on developing persistent hydatidiform mole (68% detection at 17% false detection). Proportion hyperglycosylated hCG also marks persistent hydatidiform mole (100% detection at 5.1% false detection). Proportion hyperglycosylated hCG distinguishes choriocarcinoma and gestational trophoblastic neoplasm cases, absolutely discriminating aggressive cases and minimally aggressive cases. Proportion hyperglycosylated hCG identifies quiescent gestational trophoblastic disease cases. It recognizes quiescent cases that become persistent disease (100% detection at 0% false positive). Proportion hyperglycosylated hCG is an invaluable test for discriminating gestational trophoblastic diseases.

Inhibiting trophoblast PAR-1 overexpression suppresses sFlt-1-induced anti-angiogenesis and abnormal vascular remodeling: a possible therapeutic approach for preeclampsia.

Science.gov (United States)

Zhao, Yin; Zheng, YanFang; Liu, XiaoXia; Luo, QingQing; Wu, Di; Liu, XiaoPing; Zou, Li

2018-03-01

Is it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta? Inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia. PAR-1 is high-affinity receptor of thrombin. Thrombin increases sFlt-1 secretion in trophoblast via the activation of PAR-1. It is reported that the expression of both thrombin and PAR-1 expression are increased in placentas of preeclampsia patients compared with normal placentas. Trophoblast cells were transfected with PAR-1 short hairpin RNA (shRNA) or PAR-1 overexpression plasmids in vitro. Tube formation assays and a villus-decidua co-culture system were used to study the effect of PAR-1 inhibition on placental angiogenesis and vascular remodeling, respectively. Placentas from rats with preeclampsia were transfected with PAR-1 shRNA to confirm the effect of inhibiting PAR-1 overexpression in placenta. The trophoblast cell line HTR-8/SVneo was transfected with PAR-1 shRNA or PAR-1 overexpression plasmids. After 48 h, supernatant was collected and the level of sFlt-1 secretion was measured by ELISA. Human umbilical cord epithelial cells and a villus-decidua co-culture system were treated with conditioned media to study the effect of PAR-1 inhibition on tube formation and villi vascular remodeling. A preeclampsia rat model was established by intraperitoneal injection of L-NAME. Plasmids were injected into the placenta of the preeclampsia rats and systolic blood pressure was measured on Days 15 and 19. The effect of different treatments was evaluated by proteinuria, placental weights, fetal weights and fetal numbers in study and control groups. The level of serum sFlt-1 in rats with preeclampsia was also measured. Changes in the placenta microvessels were studied by histopathological staining. PAR-1 shRNA inhibited PAR-1 expression and

Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro

Directory of Open Access Journals (Sweden)

Xiangguo eWang

2016-02-01

Full Text Available Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER, and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2 in goat trophoblast cells (GTCs and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm, a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA, a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line.

Science.gov (United States)

Park, Hae-Ryung; Loch-Caruso, Rita

2014-11-15

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

Gestational Trophoblastic Disease -Choriocarcinoma

International Nuclear Information System (INIS)

Bozik, M.

2011-01-01

Gestational trophoblastic tumors are a group of a diseases from the benign hydatidiform mole, through the invasive mole to the highly malignant form of a choriocarcinoma. Choriocarcinoma is a rare tumor and it is the most malignant and aggressive neoplasm of all the gestational trophoblastic diseases. It grows rapidly and metastasizes to the lung, liver, and, less frequently, to the brain. The author presents the case of a 26-year-old woman who is indicated to the CT examination for suspected brain tumor based on the previous examinations. The patient was diagnosed with metastatic choriocarcinoma to the brain, kidney and adrenal gland on the basis of an anamnesis by her husband, a high value of beta-hCG and a gynecological examination. (author)

The clinicopathological features of intermediate trophoblastic tumor in the pineal region

Directory of Open Access Journals (Sweden)

ZHANG Yun-xiang

2012-08-01

Full Text Available Objective To evaluate the clinicopathological features of intermediate trophoblastic tumor (ITT in the pineal region. Methods A retrospective study was performed to analyse the diagnostic and therapeutic process of 1 case with ITT in the pineal region. The specimen obtained from the surgery was dealt with common tissue processing mode and cut into slices. HE staining was performed to observe histophathological features. Immunohistochemical staining (SP two-step method was performed to analyse the expression of tumor markers. Related literatures were reviewed. Results A 6-year old boy with clinical manifestations of penis enlargement and rapid growth for more than one year, presented a mass in his pineal region through MRI. The tumor was surgically excised after it is refractory to 10 times experimental radiotherapy as germinoma. The level of β-human chorionic gonadotropin ( β-hCG in his postoperative blood was decreased to normal, but gradually increased, once again followed to normal after three times chemotherapy. Patient was normal almost postoperative 6 months later by follow -up. Pathological examination showed sheets necrosis with multiple calcification and scattered fresh blood cells, epithelioid tumor cells with solid growth pattern. The tumor cells were atypical mononuclear cells with relative uniform (between heterotypic cells and partially surrounding and invasing the vascular walls. The cytoplasm of tumor cells was eosinophilic or clear, the nucleus was round or irregular in shape and some with intranuclear pseudoinclusions, and its mitotic figures were rarely seen under light microscopy. The tumor cells showed strong positive for AE1/AE3, cell adhesion molecules 5.2 (CAM5.2, human placental lactogen (hPL, octamer-binding transcription factor 3/4 (Oct3/4, epidermal growth factor receptor (EGFR and E-cadherin. P53 was also expressed. The positive rate of Ki-67 was about 10%, and β-hCG was expressed in the extremely tumor cells. The

Effects of 60Co administration on early placental cells

International Nuclear Information System (INIS)

Honda, Jin

1979-01-01

The effects of 60 Co administration on early placental cells were studied. Placental tissue and embryo obtained by induced abortion (6 - 13 weeks gestational age) were placed in the minimal essential medium (MEM) and irradiated with various doses of 60 Co. After irradiation, the villi were cultured in a CO 2 incubater at 37 0 C. Cell growth process was observed every day with the phase-contrast microscope. Between 1 and 5 days epitheloid cells were dominant, but from about 7th day on fibroblastic cells dominated the culture. In placental tissue irradiated with 100, 200, 500 rad, fibroblastic cells began to grow earlier than in non-treated. Over 3000 rad 60 Co inhibited the growth of cells and a culture was impossible. For each dose, the tissue was incubated for various periods of time, exposed to tritiated thymidine for the last hour and autoradiogram was prepared by the dipping method. The labeling index of irradiated trophoblasts showed a significant decrease compared with controls. A chromosome study was made in irradiated in vitro cell lines of fetus and placenta. There was no significant difference between the two cell lines concerning the frequency of chromosome aberration, which tended to increase as the chromosome becomes longer. It is concluded that the trophoblast is highly radiosensitive and that irradiation early in pregnancy may damage DNA synthesis in the trophoblast, and induce abortion. (author)

Possible Involvement of Human Mast Cells in the Establishment of Pregnancy via Killer Cell Ig-Like Receptor 2DL4.

Science.gov (United States)

Ueshima, Chiyuki; Kataoka, Tatsuki R; Hirata, Masahiro; Sugimoto, Akihiko; Iemura, Yoshiki; Minamiguchi, Sachiko; Nomura, Takashi; Haga, Hironori

2018-06-01

The involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Gestational trophoblastic disease: experience at a tertiary care hospital of sindh

International Nuclear Information System (INIS)

Khaskheli, M.; Imdad, A.; Baloch, S.

2007-01-01

To determine the frequency, clinical presentation and management outcomes of Gestational Trophoblastic Disease (GTD). The case records of all the gestational trophoblastic cases during study period were analyzed regarding their illness history, clinical examination, investigations, treatment and follow-up. The main outcomes were measured in terms of duration, antecedent pregnancy, investigations, treatment and the follow-up. There were a total of 1030 obstetric admissions during the study period, which included 23 cases of trophoblastic disease. Hence, frequency of GTD was 1 per 45 live births. Of these 23 cases, 19 (82.6%) patients had hydatidiform mole and 4 patients had malignant trophoblastic disease. Eight patients (34.7%) received chemotherapy while rest of the patients had suction evacuation and follow-up. Among all patients, 21 (91.3%) fully recovered and 2 (8.69%) died because of extensive disease; metastasis extending upto brain. Frequency of trophoblastic disease was high in this series compared to world and national literature. Therefore, emphasis should be on the early diagnosis of disease as proper management in the early stages strongly influences the outcome of disease. Suction evacuation and follow-up are ideal treatments for benign trophoblastic disease. (author)

Gestational trophoblastic disease: experience at a tertiary care hospital of sindh

Energy Technology Data Exchange (ETDEWEB)

Khaskheli, M; Imdad, A; Baloch, S [Liaquat Univ. of Medical and Health Sciences, Jamshoro (Pakistan). Dept. of Obstetrics and Gynaecology

2007-02-15

To determine the frequency, clinical presentation and management outcomes of Gestational Trophoblastic Disease (GTD). The case records of all the gestational trophoblastic cases during study period were analyzed regarding their illness history, clinical examination, investigations, treatment and follow-up. The main outcomes were measured in terms of duration, antecedent pregnancy, investigations, treatment and the follow-up. There were a total of 1030 obstetric admissions during the study period, which included 23 cases of trophoblastic disease. Hence, frequency of GTD was 1 per 45 live births. Of these 23 cases, 19 (82.6%) patients had hydatidiform mole and 4 patients had malignant trophoblastic disease. Eight patients (34.7%) received chemotherapy while rest of the patients had suction evacuation and follow-up. Among all patients, 21 (91.3%) fully recovered and 2 (8.69%) died because of extensive disease; metastasis extending upto brain. Frequency of trophoblastic disease was high in this series compared to world and national literature. Therefore, emphasis should be on the early diagnosis of disease as proper management in the early stages strongly influences the outcome of disease. Suction evacuation and follow-up are ideal treatments for benign trophoblastic disease. (author)

Trophoblastic progranulin expression is upregulated in cases of fetal growth restriction and preeclampsia.

Science.gov (United States)

Stubert, Johannes; Schattenberg, Florian; Richter, Dagmar-Ulrike; Dieterich, Max; Briese, Volker

2012-05-13

The expression of the anti-inflammatory glycoprotein progranulin and the hypoxia-induced transcription factor 1α (HIF-1α) in the villous trophoblast was compared between placentae from patients with preeclampsia (PE), fetal growth restriction (FGR), and normal controls. Matched pairs analysis of third trimester placentae specimens (mean gestational age 36+2) was performed by semiquantitative measurements of the immunohistochemical staining intensities for progranulin and HIF-1α expression (PE n=13, FGR n=9 and controls n=11). Further, placental progranulin mRNA expression was analyzed by qRT-PCR on term placentae (n=3 for each group). Compared to controls, villous trophoblast revealed a significantly higher expression of progranulin in cases of PE (Pprogranulin protein was not accompanied by an increase of the progranulin mRNA in term placentae. Increased expression of progranulin protein in villous trophoblast cells in cases of PE and FGR may result from disturbed placental development and, therefore, may be of pathogenetic importance. The increase was correlated to HIF-1α expression. Further evaluation of this potential mechanism of regulation is required.

Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

Science.gov (United States)

Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

2016-02-01

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. © 2015 AlphaMed Press.

Gadd45 α expression in preeclampsia placenta and the effect of Gadd45 α on trophoblast HTR8/Svneo

Directory of Open Access Journals (Sweden)

Li Wang

2016-01-01

Full Text Available Objective: To study the expression of Gadd45 α in preeclampsia placenta and the regulating effect of Gadd45 α knockdown on trophoblast HTR8/Svneo. Methods: Preeclampsia placenta tissue and normal placenta tissue were collected, and mRNA contents and protein contents of Gadd45 α were detected by fluorescent quantitative PCR and Western blotting respectively; trophoblast cells HTR8/Svneo were cultured and after transfection of Gadd45 α siRNA, cell invasion ability and expression of invasion-assiotiated molecules were detected. Results: mRNA content and protein content of Gadd45 α in preeclampsia placenta tissue were higher than those in normal placenta tissue; after transfection of Gadd45 α siRNA, mRNA content and protein content of Gadd45 α in HTR8/Svneo cells significantly decreased, and the number of invasive cells as well as expression of MMP1, MMP2, MMP3 and MMP9 significantly increased. Conclusion: The expression of Gadd45 α in preeclampsia placenta abnormally increases; inhibting the expression of Gadd45 α in trophoblasts HTR8/Svneo can promote invasion and increase the expression of MMPs molecules.

Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts.

Science.gov (United States)

Hemmings, D G; Kilani, R; Nykiforuk, C; Preiksaitis, J; Guilbert, L J

1998-06-01

Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.

Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin.

Science.gov (United States)

Lecuit, Marc; Nelson, D Michael; Smith, Steve D; Khun, Huot; Huerre, Michel; Vacher-Lavenu, Marie-Cécile; Gordon, Jeffrey I; Cossart, Pascale

2004-04-20

Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. We used immunohistochemistry to examine the location of L. monocytogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L. monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium. Because epidemiological studies indicate that the bacterial surface protein, internalin (InlA), may play a role in human fetoplacental listeriosis, we investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found on the basal and apical plasma membranes of syncytiotrophoblasts and in villous cytotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InlA+ or InlA- strains of L. monocytogenes, and to L. innocua expressing or not InlA. Quantitative assays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InlA-E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces histopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InlA-E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans is also exploited by L. monocytogenes to target and cross the placental barrier. Such a ligand-receptor interaction allowing a pathogen to specifically cross the placental villous trophoblast barrier has not been reported previously.

IL-27 Activates Human Trophoblasts to Express IP-10 and IL-6: Implications in the Immunopathophysiology of Preeclampsia

Directory of Open Access Journals (Sweden)

Nanlin Yin

2014-01-01

Full Text Available Purpose. To investigate the effects of IL-27 on human trophoblasts and the underlying regulatory signaling mechanisms in preeclampsia. Methods. The expression of IL-27 and IL-27 receptor (WSX-1 was studied in the placenta or sera from patients with preeclampsia. In vitro, we investigated the effects of IL-27 alone or in combination with inflammatory cytokine tumor necrosis factor (TNF-α on the proinflammatory activation of human trophoblast cells (HTR-8/SVneo and the underlying intracellular signaling molecules. Results. The expression of IL-27 and IL-27 receptor α (WSX-1 was significantly elevated in the trophoblastic cells from the placenta of patients with preeclampsia compared with control specimens. In vitro, IL-27 could induce the expression of inflammatory factors IFN-γ-inducible protein 10 (CXCL10/IP-10 and IL-6 in trophoblasts, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-α on the release of IP-10 and IL-6. Furthermore, the production of IP-10 and IL-6 stimulated by IL-27 was differentially regulated by intracellular activation of phosphatidylinositol 3-OH kinase-AKT, p38MAPK, and JAK/STAT pathways. Conclusions. These results provide a new insight into the IL-27-activated immunopathological effects mediated by distinct intracellular signal transduction molecules in preeclampsia.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

International Nuclear Information System (INIS)

Park, Hae-Ryung; Loch-Caruso, Rita

2014-01-01

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47 for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. - Highlights: • BDE-47 stimulated ARE reporter activity and GSH production. • BDE-47 resulted in differential

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

Energy Technology Data Exchange (ETDEWEB)

Park, Hae-Ryung, E-mail: heaven@umich.edu; Loch-Caruso, Rita

2014-11-15

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47 for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. - Highlights: • BDE-47 stimulated ARE reporter activity and GSH production. • BDE-47 resulted in differential

MR imaging of gestational trophoblastic tumor: role of gadolinium enhancement

International Nuclear Information System (INIS)

Choi, Si Young; Byun, Jae Young; Kim, Bum Su; Yun, Young Hyun; Mun, Kyung Mi; Park, Kyung Sin; Kim, Byung Kee; Bae, Seog Nyeon; Shinn, Kyung Sub.

1997-01-01

The purpose of this study is to investigate the role of gadolinium enhanced MR imaging in the evaluation of gestational trophoblastic tumors (invasive mole and choriocarcinoma). Pre-enhanced T1-and T2-weighted images and gadolinium enhanced T1-weighted images of 34 gestational trophoblastic tumors (15 choriocarcinomas, 19 invasive moles) were retrospectively evaluated and enhancement patterns were analyzed. Morphologica differences and structural characteristics were analyzed by the evaluation of tumor margin, patterns of hemorrhagic necroses, the development of intratumoral vascularity, and molar villi. Graded scores of MR findings between pre- and gadolinium enhanced images were based on the following criteria : 1) visualization of tumor margin 2) distinction between tumor necrosis and zone of trophoblastic proliferation ; and 3) molar villi. Statistical differences between graded scores of pre- and post-enhanced images were analyzed. Gadolinium enhanced MR imaging was helpful for the visualization of tumor characteristics in gestational trophoblastic tumors and in differential diagnosis between invasive mole and choriocarcinoma. (author). 16 refs., 4 tabs., 4 figs

Human parvovirus B19 antibodies induce altered membrane protein expression and apoptosis of BeWo trophoblasts.

Science.gov (United States)

Tzang, Bor-Show; Chiang, Szu-Yi; Chan, Hsu-Chin; Liu, Chung-Hsien; Hsu, Tsai-Ching

2016-11-01

Human parvovirus B19 (B19) is harmful during pregnancy since it can be vertically transmitted to the developing fetus. In addition, the anti‑B19 antibodies induced by B19 infection are believed to have a cytopathic role in B19 transmission; however, knowledge regarding the effects of anti‑B19 antibodies during pregnancy is limited. To investigate the possible roles of anti‑B19 antibodies during pregnancy, the present study examined the effects of anti‑B19‑VP1 unique region (VP1u), anti‑B19‑VP2 and anti‑B19‑nonstructural protein 1 (NS1) immunoglobulin G (IgG) antibodies on BeWo trophoblasts. Briefly, BeWo trophoblasts were incubated with purified IgG against B19‑VP1u, B19‑VP2 and B19‑NS1. Subsequently, the expression of surface proteins and apoptotic molecules were assessed in BeWo trophoblasts using flow cytometry, ELISA and western blotting. The expression levels of human leukocyte antigen (HLA)‑G were significantly increased on BeWo trophoblasts treated with rabbit anti‑B19‑VP1u IgG, and were unchanged in those treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, as compared with the control group. Furthermore, the expression levels of globoside (P blood group antigen) and cluster of differentiation (CD)29 (β1 integrin) were significantly increased in BeWo trophoblasts treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, whereas only CD29 was also significantly increased in cells treated with anti‑B19‑VP1u IgG. In addition, the number of cells at sub‑G1 phase; caspase‑3 activity; and the expression of intrinsic and extrinsic apoptotic molecules, including Fas‑associated death domain protein, activated caspase‑8, activated caspase‑3, B‑cell lymphoma 2‑associated X protein, cytochrome c, apoptotic peptidase activating factor 1 and activated caspase‑9, were significantly increased in BeWo trophoblasts treated with anti‑B19‑VP1u and anti‑B19‑NS1 IgG. In conclusion, the present

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

Science.gov (United States)

Yang, Xiaohua; Haghiac, Maricela; Glazebrook, Patricia; Minium, Judi; Catalano, Patrick M; Hauguel-de Mouzon, Sylvie

2015-09-01

What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing

In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

Science.gov (United States)

Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

2016-01-01

Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

Characterization of fetal cells from the maternal circulation by microarray gene expression analysis - Could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

DEFF Research Database (Denmark)

Hatt, Lotte; Brinch, Marie; Singh, Ripudaman

2014-01-01

stem cell microarray analysis. Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105......Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type......(s) should be targeted. Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using...

Role of ultrasound in the diagnosis and management of gestational trophoblastic disease in Rural health facilities- A case report

International Nuclear Information System (INIS)

Bach, J.F.H.

2015-01-01

Gestational trophoblastic disease (GTD) is a rare kind of proliferative disorder of trophoblastic cells which develops from the placenta in early pregnancy. It can be benign, premalignant or malignant. Molar pregnancy, also known as Hydatidiform Mole, is a form of benign GTD. The complete hydatidiform mole (CHM) sub-type which limited to endometrium is most common. It has excellent prognosis if early appropriate diagnosis and management are done. A well performed ultrasound(US) play a primordial role in the diagnosis of maternal health disorders during routine prenatal care. This helps in avoiding complications and consequently aids in achieving the objectives of the Millennium Development Goals (MDGs) in Rwanda. To understand the definition of Gestational trophoblastic disease(GTD) and to recognize key diagnostic findings of complete molar pregnancy on ultrasound and appropriate management in maternal follow up. Review the differential diagnosis for ultrasound findings seen with GTD and other modalities Ultrasound is the first modality to be used in all rural health facilities for diagnosis of suspected Gestational trophoblastic disease (GTD) for better results. It is available and free of radiation

Early embryonic demise: no evidence of abnormal spiral artery transformation or trophoblast invasion.

Science.gov (United States)

Ball, E; Robson, S C; Ayis, S; Lyall, F; Bulmer, J N

2006-03-01

Invasion by extravillous trophoblast of uterine decidua and myometrium and the associated spiral artery 'transformation' are essential for the development of normal pregnancy. Small pilot studies of placental bed and basal plate tissues from miscarriages have suggested that impaired interstitial and endovascular trophoblast invasion may play a role in the pathogenesis of miscarriage. The hypothesis that early miscarriage is associated with reduced extravillous trophoblast invasion and spiral artery transformation was tested in a large series of placental bed biopsies containing decidua and myometrium and at least one spiral artery from early, karyotyped embryonic miscarriages (trophoblast (cytokeratin), myometrium and spiral artery medial smooth muscle (desmin), and endothelium (von Willebrand factor). Trophoblast invasion and individual features of spiral artery transformation were assessed histologically in spiral arteries of miscarriages (n = 176) and controls (n = 246) and analysed statistically using a logistic regression model. Trophoblast invasion of uterine tissues and spiral artery transformation did not differ between euploid and aneuploid early miscarriage and also did not differ significantly from normal pregnancy. These findings suggest that failed trophoblast invasion and spiral artery transformation do not have a pivotal role in the pathogenesis of early miscarriage.

Gestational Trophoblastic Disease: A Multimodality Imaging Approach with Impact on Diagnosis and Management

International Nuclear Information System (INIS)

Dhanda, S.; Ramani, S.; Dhanda, S.; Ramani, S.; Thakur, M.

2014-01-01

Gestational trophoblastic disease is a condition of uncertain etiology, comprised of hydatiform mole (complete and partial), invasive mole, choriocarcinoma, and placental site trophoblastic tumor. It arises from abnormal proliferation of trophoblastic tissue. Early diagnosis of gestational trophoblastic disease and its potential complications is important for timely and successful management of the condition with preservation of fertility. Initial diagnosis is based on a multimodality approach: encompassing clinical features, serial quantitative β-hCG titers, and pelvic ultrasonography. Pelvic magnetic resonance imaging (MRI) is sometimes used as a problem-solving tool to assess the depth of myometrial invasion and extra uterine disease spread in equivocal and complicated cases. Chest radiography, body computed tomography (CT), and brain MRI have been recommended as investigative tools for overall disease staging. Angiography has a role in management of disease complications and metastases. Efficacy of PET (positron emission tomography) and PET/CT in the evaluation of recurrent or metastatic disease has not been adequately investigated yet. This paper discusses the imaging features of gestational trophoblastic disease on various imaging modalities and the role of different imaging techniques in the diagnosis and management of this entity. 1. Introduction Gestational trophoblastic disease (GTD) refers to an abnormal trophoblastic proliferation composed of a broad spectrum of lesions ranging from benign, albeit pre malignant hydatiform mole (complete and partial), through to the aggressive invasive mole, choriocarcinoma

Heat shock cognate protein 70 contributes to Brucella invasion into trophoblast giant cells that cause infectious abortion

Directory of Open Access Journals (Sweden)

Furuoka Hidefumi

2008-12-01

Full Text Available Abstract Background The cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells. Results We observed internalization and intracellular growth of B. abortus in cultured TG cells. A monoclonal antibody that inhibits bacterial internalization was isolated and this reacted with heat shock cognate protein 70 (Hsc70. Depletion and over expression of Hsc70 in TG cells inhibited and promoted bacterial internalization, respectively. IFN-γ receptor was expressed in TG cells and IFN-γ treatment enhanced the uptake of bacteria by TG cells. Administering the anti-Hsc70 antibody to pregnant mice served to prevent infectious abortion. Conclusion B. abortus infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.

Dysregulated DNA Methyltransferase 3A Upregulates IGFBP5 to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia.

Science.gov (United States)

Jia, Yuanhui; Li, Ting; Huang, Xiaojie; Xu, Xianghong; Zhou, Xinyao; Jia, Linyan; Zhu, Jingping; Xie, Dandan; Wang, Kai; Zhou, Qian; Jin, Liping; Zhang, Jiqin; Duan, Tao

2017-02-01

Preeclampsia is a unique multiple system disorder during human pregnancy, which affects ≈5% to 8% of pregnancies. Its risks and complications have become the major causes of maternal and fetal morbidity and mortality. Although abnormal placentation to which DNA methylation dysregulation is always linked is speculated to be one of the reasons causing preeclampsia, the underlying mechanisms still remain elusive to date. Here we revealed that aberrant DNA methyltransferase 3A (DNMT3A) plays a critical role in preeclampsia. Our results show that the expression and localization of DNMT3A are dysregulated in preeclamptic placenta. Moreover, knockdown of DNMT3A obviously inhibits trophoblast cell migration and invasion. Mechanistically, IGFBP5 (insulin-like growth factor-binding protein 5), known as a suppressor, is upregulated by decreased DNMT3A because of promoter hypomethylation. Importantly, IGFBP5 downregulation can rescue the defects caused by DNMT3A knockdown, thereby, consolidating the significance of IGFBP5 in the downstream of DNMT3A in trophoblast. Furthermore, we detected low promoter methylation and high protein expression of IGFBP5 in the clinical samples of preeclamptic placenta. Collectively, our study suggests that dysregulation of DNMT3A and IGFBP5 is relevant to preeclampsia. Thus, we propose that DNMT3A and IGFBP5 can serve as potential markers and targets for the clinical diagnosis and therapy of preeclampsia. © 2017 American Heart Association, Inc.

Efficacy of NETDC (New England Trophoblastic Disease Center prognostic index score to predict gestational trophoblastic tumor from hydatidiform mole

Directory of Open Access Journals (Sweden)

Khrismawan Khrismawan

2004-03-01

Full Text Available A prospective longitudinal analytic study assessing the efficacy of NETDC (New England Trophoblastic Disease Center prognostic index score in predicting malignancy after hydatidiform mole had been performed. Of the parameter evaluated; age of patients, type of hydatidiform mole, uterine enlargement, serum hCG level, lutein cyst, and presence of complicating factors were significant risk factors for malignancy after hydatidiform mole were evacuated (p<0.032. The study were done on 50 women diagnosed with hydatidiform mole with 1 year observation (January 2001-December 2002 at the Department of Obstetrics and Gynecology, Mohammad Hoesin Hospital, Palembang. The results showed that the NETDC prognostic index score predicted malignancy in 50% of high risk group and 10% in low risk group (p<0.05. This showed a higher number than that found by the WHO (19%-30%. The risk for incidence of  malignancy after hydatidiform mole in the high risk group is 9.0 times higher compared to that of the low risk group (CI: 1.769-45.786. (Med J Indones 2004; 13: 40-6 Keywords: New England Trophoblastic Disease Center (NETDC, gestational trophoblastic tumor, hydatidiform mole, high and low risk

Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts

Science.gov (United States)

Tuuli, Methodius G.; Longtine, Mark S.; Shin, Joong Sik; Lawrence, Russell; Inder, Terrie; Michael Nelson, D.

2012-01-01

The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus. PMID:22374759

Drugs Approved for Gestational Trophoblastic Disease

Science.gov (United States)

This page lists cancer drugs approved by the Food and Drug Administration (FDA) for gestational trophoblastic disease. The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

Cytotoxicant-induced trophoblast dysfunction and abnormal pregnancy outcomes: role of zinc and metallothionein.

Science.gov (United States)

McAleer, Mary Frances; Tuan, Rocky S

2004-12-01

Normal trophoblast function, including implantation, hormone production, and formation of the selectively permeable maternofetal barrier, is essential for the establishment and maintenance of the fetoplacental unit and proper fetal development. Maternal cytotoxicant exposure causes the destruction of these cells, especially the terminally differentiated syncytiotrophoblasts, and results in a myriad of poor pregnancy outcomes. These outcomes range from intrauterine growth retardation and malformation to spontaneous abortion or stillbirth. There is recent evidence that the metal-binding protein, metallothionein, is involved in the protection of human trophoblastic cells from heavy metal-induced and severe oxidative stress-induced apoptosis. Metallothionein, with its unique biochemical structure, can both bind essential metal ions, such as the transcription modulator zinc, and yet allow their ready displacement by toxic nonessential metal ions or damaging free radicals. These properties suggest that metallothionein may be responsible not only for sequestering the cytotoxic agents, but also for altering signal transduction in the affected cells. Here, we review several identified causes of adverse pregnancy outcomes (specifically, prenatal exposure to cigarette smoke and alcohol, gestational infection, and exposure to environmental contaminants), discuss the role of zinc in modulating the cellular response to these toxic insults, and then propose how metallothionein may function to mediate this protective response. Published 2005 Wiley-Liss, Inc.

Estrogen regulates progesterone production by human placental trophoblast cells in culture

International Nuclear Information System (INIS)

Grimes, R.W.

1990-01-01

We have suggested that estrogen regulates placental low-density lipoprotein (LDL) uptake and thus progesterone (P 4 ) production during primate pregnancy based on results obtained in antiestrogen-treated baboons. The objectives of the present study, were to determine whether estrogen is also important to regulation of P 4 formation by the human placenta, and whether effects of estrogen were mediated by availability of cholesterol substrate via the LDL, de novo, or deesterification pathways. Term human placenta were dispersed in 0.125% trypsin, cytotrophoblasts were purified via a 70-5% Percoll gradient, incubated 72 h in DMEM with 10% FBS to stimulate formation of syncytia, then incubated an additional 48 h with estradiol (E2). In Experiment 1, 1 μg/ml E 2 and 500 μg/MI LDL-protein, stimulated P 4 (P 2 increased LDL uptake. Scatchard analysis indicated that trophoblast uptake of [ 125 I]LDL (ng/mg cell protein) was 50% greater (P 2 (mean ± SE, 638 +/- 23; n = 6) than DMEM in the presence of antiestrogen MER-25. Moreover, uptake and degradation of LDL, and cellular content of free and esterified cholesterol, increased in a dose-dependent manner with 0.1 to 1000 ng/ml E 2 . These results suggest that estrogen regulates placental cell uptake of LDL and thus availability of cholesterol for P 4 biosynthesis during human pregnancy. In Experiment 2, E 2 Stimulated P 4 formation (ng/mg cell protein/48 h) from a control level of 194 ± 25 to 357 ± 62, in the absence of LDL. Under these conditions, cholesterol for P 4 biosynthesis must have been derived from de novo synthesis and/or deesterification of cholesteryl ester stores

Regulation of pregnancy-associated plasma protein A2 (PAPPA2 in a human placental trophoblast cell line (BeWo

Directory of Open Access Journals (Sweden)

Christians Julian K

2011-04-01

Full Text Available Abstract Background Pregnancy-associated plasma protein A2 (PAPPA2 is an insulin-like growth factor-binding protein (IGFBP protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA. Methods BeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2, oxidative stress (20 microM hydrogen peroxide, forskolin (10 microM and 100 microM, TGF-beta (10 and 50 ng/mL, TNF-alpha (100 ng/mL, IL-1beta (100 ng/mL or PGE2 (1 microM. We used quantitative RT-PCR (qRT-PCR to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay. Results Hypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression. Conclusion Our study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha, contribute to the upregulation of PAPPA2. Hypoxia, known to occur in

RISK FACTORS FOR GESTATIONAL TROPHOBLASTIC NEOPLASIA: A CASE CONTROL STUDY IN A TERTIARY HOSPITAL

Directory of Open Access Journals (Sweden)

Hema Sreedharan Nair

2016-10-01

Full Text Available BACKGROUND Gestational trophoblastic disease is a spectrum of proliferative abnormalities of the trophoblast. GTD represents a benign form of the disease while GTN is the malignant often metastatic lesion. 75-80 per cent of patients initially diagnosed as GTD will follow a benign course after dilatation and curettage. 15-20 per cent develop locally invasive disease and 3-5 per cent develop metastatic lesions. The study aims to assess the proportion of gestational trophoblastic neoplasia among women with gestational trophoblastic disease and identify the risk factors for chemotherapy in gestational trophoblastic neoplasia. MATERIALS AND METHODS This is a case-control study conducted in a tertiary hospital during a 5-year period. Cases are gestational trophoblastic neoplasia diagnosed by either rising beta-HCG levels or plateauing beta-HCG levels or by histological evidence of choriocarcinoma. Controls are cases of gestational trophoblastic disease post evacuation with normal HCG regression at 8 weeks. There were 306 controls and 57 cases. RESULTS Tabulated and analysed using SPSS package. Of the 363 patients of gestational trophoblastic disease, 57 (15.7% needed chemotherapy. 98.2% belonged to the age group of 20-35 years. 63% had gestational age of more than 12 weeks, 56.1% had pre-evacuation HCG of more than 40,000. 15.7% needed combination therapy. CONCLUSION 1. 83.1% of patients belonged to age group of 20-30 years. 2. Blood group distribution of patients with gestational trophoblastic disease did not show any significance. 3. 15.7% of total patients were diagnosed to have gestational trophoblastic neoplasia that necessitated chemotherapy. 4. When uterine size was more than 12 weeks, a statistically significant number of patients needed chemotherapy compared to non-chemotherapy group. 5. When BHCG values were more than 40,000, a statistically significant number of patients needed chemotherapy. 6. A risk score of seven or more was found to

Interaction between dendritic cells and natural killer cells during pregnancy in mice.

Science.gov (United States)

Blois, Sandra M; Barrientos, Gabriela; Garcia, Mariana G; Orsal, Arif S; Tometten, Mareike; Cordo-Russo, Rosalia I; Klapp, Burghard F; Santoni, Angela; Fernández, Nelson; Terness, Peter; Arck, Petra C

2008-07-01

A complex regulation of innate and adaptive immune responses at the maternal fetal interface promotes tolerance of trophoblast cells carrying paternally derived antigens. Such regulatory functions involve uterine dendritic cells (uDC) and natural killer (uNK) cells. The existence of a NK and DC "cross talk" has been revealed in various experimental settings; its biological significance ranging from cooperative stimulation to cell lysis. Little is known about the presence or role of NK and DC cross talk at the maternal fetal interface. The present study shows that mouse NK and DC interactions are subject to modulation by trophoblast cells in vitro. This interaction promotes a tolerogenic microenvironment characterized by downregulation of the expression of activation markers on uNK cells and uDC and dominance of Th2 cytokines. NK and DC interactions would also influence uterine cell proliferation and this process would be strongly modulated by trophoblast-derived signals. Indeed; while low proliferation rates were observed upon regular coculture allowing direct contact between uterine cells and trophoblasts, incubation in a transwell culture system markedly increased uterine cell proliferation suggesting that soluble factors are key mediators in the molecular "dialog" between the mother and the conceptus during the establishment of mouse pregnancy. Our data further reveal that the regulatory functions of trophoblast cells associated with tolerance induction are impaired in high abortion murine matings. Interestingly, we observed that secretion of interleukin-12p70 by uDC is dramatically abrogated in the presence of uNK cells. Taken together, our results provide the first evidence that a delicate balance of interactions involving NK cells, DC, and trophoblasts at the mouse maternal fetal interface supports a successful pregnancy outcome.

Gestational Trophoblastic Disease: A Multimodality Imaging Approach with Impact on Diagnosis and Management

Directory of Open Access Journals (Sweden)

Sunita Dhanda

2014-01-01

Full Text Available Gestational trophoblastic disease is a condition of uncertain etiology, comprised of hydatiform mole (complete and partial, invasive mole, choriocarcinoma, and placental site trophoblastic tumor. It arises from abnormal proliferation of trophoblastic tissue. Early diagnosis of gestational trophoblastic disease and its potential complications is important for timely and successful management of the condition with preservation of fertility. Initial diagnosis is based on a multimodality approach: encompassing clinical features, serial quantitative β-hCG titers, and pelvic ultrasonography. Pelvic magnetic resonance imaging (MRI is sometimes used as a problem-solving tool to assess the depth of myometrial invasion and extrauterine disease spread in equivocal and complicated cases. Chest radiography, body computed tomography (CT, and brain MRI have been recommended as investigative tools for overall disease staging. Angiography has a role in management of disease complications and metastases. Efficacy of PET (positron emission tomography and PET/CT in the evaluation of recurrent or metastatic disease has not been adequately investigated yet. This paper discusses the imaging features of gestational trophoblastic disease on various imaging modalities and the role of different imaging techniques in the diagnosis and management of this entity.

Involvement of reactive oxygen species in brominated diphenyl ether-47-induced inflammatory cytokine release from human extravillous trophoblasts in vitro

International Nuclear Information System (INIS)

Park, Hae-Ryung; Kamau, Patricia W.; Loch-Caruso, Rita

2014-01-01

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardant compounds. Brominated diphenyl ether (BDE)-47 is one of the most prevalent PBDE congeners found in human breast milk, serum and placenta. Despite the presence of PBDEs in human placenta, effects of PBDEs on placental cell function are poorly understood. The present study investigated BDE-47-induced reactive oxygen species (ROS) formation and its role in BDE-47-stimulated proinflammatory cytokine release in a first trimester human extravillous trophoblast cell line, HTR-8/SVneo. Exposure of HTR-8/SVneo cells for 4 h to 20 μM BDE-47 increased ROS generation 1.7 fold as measured by the dichlorofluorescein (DCF) assay. Likewise, superoxide anion production increased approximately 5 fold at 10 and 15 μM and 9 fold at 20 μM BDE-47 with a 1-h exposure, as measured by cytochrome c reduction. BDE-47 (10, 15 and 20 μM) decreased the mitochondrial membrane potential by 47–64.5% at 4, 8 and 24 h as assessed with the fluorescent probe Rh123. Treatment with 15 and 20 μM BDE-47 stimulated cellular release and mRNA expression of IL-6 and IL-8 after 12 and 24-h exposures: the greatest increases were a 35-fold increased mRNA expression at 12 h and a 12-fold increased protein concentration at 24 h for IL-6. Antioxidant treatments (deferoxamine mesylate, (±)α-tocopherol, or tempol) suppressed BDE-47-stimulated IL-6 release by 54.1%, 56.3% and 37.7%, respectively, implicating a role for ROS in the regulation of inflammatory pathways in HTR-8/SVneo cells. Solvent (DMSO) controls exhibited statistically significantly decreased responses compared with non-treated controls for IL-6 release and IL-8 mRNA expression, but these responses were not consistent across experiments and times. Nonetheless, it is possible that DMSO (used to dissolve BDE-47) may have attenuated the stimulatory actions of BDE-47 on cytokine responses. Because abnormal activation of proinflammatory responses can disrupt trophoblast functions

Involvement of reactive oxygen species in brominated diphenyl ether-47-induced inflammatory cytokine release from human extravillous trophoblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Park, Hae-Ryung, E-mail: heaven@umich.edu; Kamau, Patricia W.; Loch-Caruso, Rita

2014-01-15

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardant compounds. Brominated diphenyl ether (BDE)-47 is one of the most prevalent PBDE congeners found in human breast milk, serum and placenta. Despite the presence of PBDEs in human placenta, effects of PBDEs on placental cell function are poorly understood. The present study investigated BDE-47-induced reactive oxygen species (ROS) formation and its role in BDE-47-stimulated proinflammatory cytokine release in a first trimester human extravillous trophoblast cell line, HTR-8/SVneo. Exposure of HTR-8/SVneo cells for 4 h to 20 μM BDE-47 increased ROS generation 1.7 fold as measured by the dichlorofluorescein (DCF) assay. Likewise, superoxide anion production increased approximately 5 fold at 10 and 15 μM and 9 fold at 20 μM BDE-47 with a 1-h exposure, as measured by cytochrome c reduction. BDE-47 (10, 15 and 20 μM) decreased the mitochondrial membrane potential by 47–64.5% at 4, 8 and 24 h as assessed with the fluorescent probe Rh123. Treatment with 15 and 20 μM BDE-47 stimulated cellular release and mRNA expression of IL-6 and IL-8 after 12 and 24-h exposures: the greatest increases were a 35-fold increased mRNA expression at 12 h and a 12-fold increased protein concentration at 24 h for IL-6. Antioxidant treatments (deferoxamine mesylate, (±)α-tocopherol, or tempol) suppressed BDE-47-stimulated IL-6 release by 54.1%, 56.3% and 37.7%, respectively, implicating a role for ROS in the regulation of inflammatory pathways in HTR-8/SVneo cells. Solvent (DMSO) controls exhibited statistically significantly decreased responses compared with non-treated controls for IL-6 release and IL-8 mRNA expression, but these responses were not consistent across experiments and times. Nonetheless, it is possible that DMSO (used to dissolve BDE-47) may have attenuated the stimulatory actions of BDE-47 on cytokine responses. Because abnormal activation of proinflammatory responses can disrupt trophoblast functions

The role of surgery in the management of gestational trophoblastic neoplasia.

Science.gov (United States)

Doll, Kemi M; Soper, John T

2013-07-01

Although sensitive human chorionic gonadotropin assays and advances in chemotherapy have assumed primary importance in the management of gestational trophoblastic neoplasia, surgery remains important in the overall care of these patients. Management of molar pregnancies consists of surgical evacuation and subsequent monitoring. Hysterectomy decreases the risk of post-molar trophoblastic disease in appropriate patients and, when incorporated to primary management of gestational trophoblastic neoplasia, can decrease the chemotherapy requirements of patients with low-risk disease. In patients with high-risk disease, surgical intervention is frequently required to control complications of disease or as therapy to stabilize patients during chemotherapy. Hysterectomy, thoracotomy, or other extirpative procedures may be integrated into the management of patients with chemorefractory disease. Interventional procedures are useful adjuncts to control bleeding from metastases.

Quality of life of gestational trophoblastic neoplasia survivors: a study of patients at the Philippine General Hospital trophoblastic disease section.

Science.gov (United States)

Cagayan, M Stephanie Fay S; Llarena, Raquel T

2010-01-01

To evaluate the quality of life (QOL) of patients who were diagnosed with gestational trophoblastic neoplasia (GTN) at the Philippine General Hospital Trophoblastic Disease Section and who were in remission at the time of this study. A cross-sectional descriptive study designed to measure the QOL of all patients diagnosed as having GTN in remission and following up at the Philippine General Hospital Trophoblastic Disease Outpatient Clinic from May-August 2008 (N = 46). This study used the short form 12-question (SF-12) survey forms to evaluate the QOL of patients diagnosed with GTN. Scores from the SF-12 were analyzed using Pearson's correlation. Statistical significance was assumed for p values educational level and physical functioning. A negative correlation was found between the stage of GTN and patients' general health. In conclusion, the survivors' age, educational level and type of treatment had impact on the QOL among GTN survivors in terms of physical functioning. No relationship was established between the demographic variables and mental status. SF-12 appears to be a reliable instrument, suggesting its potential in measuring health status in GTN survivors. Age, educational attainment and type of treatment were shown to have an impact on the QOL of the surviving GTN patients.

Gestational trophoblastic disease following complete hydatidiform ...

African Journals Online (AJOL)

Gestational trophoblastic disease following complete hydatidiform mole in Mulago Hospital, Kampala, Uganda. ... The main outcome measures were pre- and post-evacuation serum hCG levels and complications associated with oral methotrexate use. Results : The prevalence of CHM was 3.42 per 1,000 deliveries.

EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

Directory of Open Access Journals (Sweden)

Kim Suk

2009-12-01

Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

The STOX1 genotype associated with pre-eclampsia leads to a reduction of trophoblast invasion by alpha-T-catenin upregulation

NARCIS (Netherlands)

van Dijk, Marie; van Bezu, Jan; van Abel, Daan; Dunk, Caroline; Blankenstein, Marinus A.; Oudejans, Cees B. M.; Lye, Stephen J.

2010-01-01

By using complementary in vitro and ex vivo approaches, we show that the risk allele (Y153H) of the pre-eclampsia susceptibility gene STOX1 negatively regulates trophoblast invasion by upregulation of the cell-cell adhesion protein alpha-T-catenin (CTNNA3). This is effectuated at the crucial

Disturbed Placental Imprinting in Preeclampsia Leads to Altered Expression of DLX5, a Human-Specific Early Trophoblast Marker.

Science.gov (United States)

Zadora, Julianna; Singh, Manvendra; Herse, Florian; Przybyl, Lukasz; Haase, Nadine; Golic, Michaela; Yung, Hong Wa; Huppertz, Berthold; Cartwright, Judith E; Whitley, Guy; Johnsen, Guro M; Levi, Giovanni; Isbruch, Annette; Schulz, Herbert; Luft, Friedrich C; Müller, Dominik N; Staff, Anne Cathrine; Hurst, Laurence D; Dechend, Ralf; Izsvák, Zsuzsanna

2017-11-07

Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5 , with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5 high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in

Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics

OpenAIRE

Hou, Shuang; Chen, Jie-Fu; Song, Min; Zhu, Yazhen; Jan, Yu Jen; Chen, Szu Hao; Weng, Tzu-Hua; Ling, Dean-An; Chen, Shang-Fu; Ro, Tracy; Liang, An-Jou; Lee, Tom; Jin, Helen; Li, Man; Liu, Lian

2017-01-01

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to...

Feasibility of central co-ordinated EMA/CO for gestational trophoblastic disease in the Netherlands

NARCIS (Netherlands)

van der Houwen, Clasien; Rietbroek, Ron C.; Lok, Christianne A. R.; ten Kate-Booij, Marianne J.; Lammes, Frits B.; Ansink, Anca C.

2004-01-01

In the Netherlands, high risk gestational trophoblastic disease (GTD) patients are treated in different referral hospitals with a national working party on trophoblastic tumours having a co-ordinating function. Our purpose was to evaluate whether this policy is a satisfactory alternative to complete

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Persistent trophoblast disease following partial molar pregnancy.

NARCIS (Netherlands)

Wielsma, S.; Kerkmeijer, L.G.W.; Bekkers, R.L.M.; Pyman, J.; Tan, J.; Quinn, M.

2006-01-01

OBJECTIVE: Human chorionic gonadotrophin (hCG) follow-up data were analysed retrospectively in all patients registered in the Hydatidiform Mole Registry at the Royal Women's Hospital, Melbourne from January 1992 to January 2001 to determine the risk of persistent trophoblast disease following

Clinical and radiological correlations in patients with gestational trophoblastic disease

Directory of Open Access Journals (Sweden)

Lana de Lourdes Aguiar Lima

Full Text Available Abstract Gestational trophoblastic disease is an abnormality of pregnancy that encompasses a group of diseases that differ from each other in their propensity for regression, invasion, metastasis, and recurrence. In the past, it was common for patients with molar pregnancy to present with marked symptoms: copious bleeding; theca lutein cysts; uterus larger than appropriate for gestational age; early preeclampsia; hyperemesis gravidarum; and hyperthyroidism. Currently, with early diagnosis made by ultrasound, most patients are diagnosed while the disease is still in the asymptomatic phase. In cases of progression to trophoblastic neoplasia, staging-typically with Doppler flow studies of the pelvis and chest X-ray, although occasionally with computed tomography or magnetic resonance imaging-is critical to the choice of an appropriate antineoplastic therapy regimen. Because it is an unusual and serious disease that affects women of reproductive age, as well as because its appropriate treatment results in high cure rates, it is crucial that radiologists be familiar with gestational trophoblastic disease, in order to facilitate its early diagnosis and to ensure appropriate follow-up imaging.

Clinical and radiological correlations in patients with gestational trophoblastic disease

International Nuclear Information System (INIS)

Lima, Lana de Lourdes Aguiar; Parente, Raphael Camara Medeiros; Amim Junior, Joffre; Rezende Filho, Jorge Fonte de; Montenegro, Carlos Antonio Barbosa; Braga, Antonio; Maesta, Izildinha

2016-01-01

Gestational trophoblastic disease is an abnormality of pregnancy that encompasses a group of diseases that differ from each other in their propensity for regression, invasion, metastasis, and recurrence. In the past, it was common for patients with molar pregnancy to present with marked symptoms: copious bleeding; theca lutein cysts; uterus larger than appropriate for gestational age; early preeclampsia; hyperemesis gravidarum; and hyperthyroidism. Currently, with early diagnosis made by ultrasound, most patients are diagnosed while the disease is still in the asymptomatic phase. In cases of progression to trophoblastic neoplasia, staging-typically with Doppler flow studies of the pelvis and chest X-ray, although occasionally with computed tomography or magnetic resonance imaging-is critical to the choice of an appropriate antineoplastic therapy regimen. Because it is an unusual and serious disease that affects women of reproductive age, as well as because its appropriate treatment results in high cure rates, it is crucial that radiologists be familiar with gestational trophoblastic disease, in order to facilitate its early diagnosis and to ensure appropriate follow-up imaging. (author)

Clinical and radiological correlations in patients with gestational trophoblastic disease

Energy Technology Data Exchange (ETDEWEB)

Lima, Lana de Lourdes Aguiar; Parente, Raphael Camara Medeiros; Amim Junior, Joffre; Rezende Filho, Jorge Fonte de; Montenegro, Carlos Antonio Barbosa; Braga, Antonio, E-mail: lanalima@hotmail.com [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil); Maesta, Izildinha [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Botucatu, SP (Brazil). Faculdade de Medicina

2016-07-15

Gestational trophoblastic disease is an abnormality of pregnancy that encompasses a group of diseases that differ from each other in their propensity for regression, invasion, metastasis, and recurrence. In the past, it was common for patients with molar pregnancy to present with marked symptoms: copious bleeding; theca lutein cysts; uterus larger than appropriate for gestational age; early preeclampsia; hyperemesis gravidarum; and hyperthyroidism. Currently, with early diagnosis made by ultrasound, most patients are diagnosed while the disease is still in the asymptomatic phase. In cases of progression to trophoblastic neoplasia, staging-typically with Doppler flow studies of the pelvis and chest X-ray, although occasionally with computed tomography or magnetic resonance imaging-is critical to the choice of an appropriate antineoplastic therapy regimen. Because it is an unusual and serious disease that affects women of reproductive age, as well as because its appropriate treatment results in high cure rates, it is crucial that radiologists be familiar with gestational trophoblastic disease, in order to facilitate its early diagnosis and to ensure appropriate follow-up imaging. (author)

Trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine induces lipid peroxidation-associated apoptosis via the intrinsic and extrinsic apoptosis pathways in a first-trimester placental cell line.

Science.gov (United States)

Elkin, Elana R; Harris, Sean M; Loch-Caruso, Rita

2018-01-01

Trichloroethylene (TCE), a prevalent environmental contaminant, is a potent renal and hepatic toxicant through metabolites such as S-(1, 2-dichlorovinyl)-l-cysteine (DCVC). However, effects of TCE on other target organs such as the placenta have been minimally explored. Because elevated apoptosis and lipid peroxidation in placenta have been observed in pregnancy morbidities involving poor placentation, we evaluated the effects of DCVC exposure on apoptosis and lipid peroxidation in a human extravillous trophoblast cell line, HTR-8/SVneo. We exposed the cells in vitro to 10-100μM DCVC for various time points up to 24h. Following exposure, we measured apoptosis using flow cytometry, caspase activity using luminescence assays, gene expression using qRT-PCR, and lipid peroxidation using a malondialdehyde quantification assay. DCVC significantly increased apoptosis in time- and concentration-dependent manners (p<0.05). DCVC also significantly stimulated caspase 3, 7, 8 and 9 activities after 12h (p<0.05), suggesting that DCVC stimulates the activation of both the intrinsic and extrinsic apoptotic signaling pathways simultaneously. Pre-treatment with the tBID inhibitor Bl-6C9 partially reduced DCVC-stimulated caspase 3 and 7 activity, signifying crosstalk between the two pathways. Additionally, DCVC treatment increased lipid peroxidation in a concentration-dependent manner. Co-treatment with the antioxidant peroxyl radical scavenger (±)-α-tocopherol attenuated caspase 3 and 7 activity, suggesting that lipid peroxidation mediates DCVC-induced apoptosis in extravillous trophoblasts. Our findings suggest that DCVC-induced apoptosis and lipid peroxidation in extravillous trophoblasts could contribute to poor placentation if similar effects occur in vivo in response to TCE exposure, indicating that further studies into this mechanism are warranted. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of invasive trophoblast in implantation and placentation of primates

DEFF Research Database (Denmark)

Carter, Anthony Michael; Enders, Allen C; Pijnenborg, Robert

2015-01-01

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more...... invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma...

Quebec Trophoblastic Disease Registry: how to make an easy-to-use dynamic database.

Science.gov (United States)

Sauthier, Philippe; Breguet, Magali; Rozenholc, Alexandre; Sauthier, Michaël

2015-05-01

To create an easy-to-use dynamic database designed specifically for the Quebec Trophoblastic Disease Registry (RMTQ). It is now well established that much of the success in managing trophoblastic diseases comes from the development of national and regional reference centers. Computerized databases allow the optimal use of data stored in these centers. We have created an electronic data registration system by producing a database using FileMaker Pro 12. It uses 11 external tables associated with a unique identification number for each patient. Each table allows specific data to be recorded, incorporating demographics, diagnosis, automated staging, laboratory values, pathological diagnosis, and imaging parameters. From January 1, 2009, to December 31, 2013, we used our database to register 311 patients with 380 diseases and have seen a 39.2% increase in registrations each year between 2009 and 2012. This database allows the automatic generation of semilogarithmic curves, which take into account β-hCG values as a function of time, complete with graphic markers for applied treatments (chemotherapy, radiotherapy, or surgery). It generates a summary sheet for a synthetic vision in real time. We have created, at a low cost, an easy-to-use database specific to trophoblastic diseases that dynamically integrates staging and monitoring. We propose a 10-step procedure for a successful trophoblastic database. It improves patient care, research, and education on trophoblastic diseases in Quebec and leads to an opportunity for collaboration on a national Canadian registry.

HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

Directory of Open Access Journals (Sweden)

Martin Zydek

2014-03-01

Full Text Available Human cytomegalovirus (HCMV is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs, the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs. Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb, TRL345, reactive with glycoprotein B (gB, but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

Comparative studies of placentation and immunology in non-human primates suggest a scenario for the evolution of deep trophoblast invasion and an explanation for human pregnancy disorders

DEFF Research Database (Denmark)

Carter, Anthony Michael

2011-01-01

in the orangutan and became polymorphic in the lineage leading to gorilla, bonobo, chimpanzee, and human. Interaction between HLA-C1 and HLA-C2 on the surface of trophoblast and killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer cells are important regulators of trophoblast invasion....... Evolution of this system in great apes may have been one prerequisite for deep trophoblast invasion but seems to have come at a price. The evidence now suggests that certain combinations of maternal genotype for KIRs and fetal genotype for HLA-C imply an increased risk of preeclampsia, fetal growth...... restriction, and recurrent abortion. The fetal genotype is in part derived from the father providing an explanation for the paternal contribution to reproductive disorders....

Roles of the insulinlike growth factor family in nonpregnant human endometrium and at the decidual: trophoblast interface.

Science.gov (United States)

Giudice, L C; Irwin, J C

1999-01-01

The insulinlike growth factor (IGF) family is believed to be important in endometrial development during the menstrual cycle and in the process of implantation. The mitogenic, differentiative, and antiapoptotic properties of the IGFs and their binding proteins, as well as their spatial and temporal expression in cycling endometrium, suggest that they may participate in endometrial growth, differentiation, apoptosis, and perhaps angiogenesis. IGFBP proteases, which increase IGF bioavailability, have been localized to endometrial stromal cells and to the human cytotrophoblast and likely play important roles in endometrial, decidual, and trophoblast physiology. IGFBP-1 is a major protein product of nonpregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. Recent evidence suggests that it may have a role in the process of shallow implantation in the clinical disorder of preclampsia. In contrast to knowledge about the roles of IGF peptides, IGFBP proteases, and IGFBPs in normal endometrial development and early human pregnancy, little information is available regarding this family in abnormal endometrial development, in occult endometrial defects, and in uterine receptivity and nonreceptivity.

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

IFPA Meeting 2010 Workshops Report II: Placental pathology; trophoblast invasion; fetal sex; parasites and the placenta; decidua and embryonic or fetal loss; trophoblast differentiation and syncytialisation.

Science.gov (United States)

Al-Khan, A; Aye, I L; Barsoum, I; Borbely, A; Cebral, E; Cerchi, G; Clifton, V L; Collins, S; Cotechini, T; Davey, A; Flores-Martin, J; Fournier, T; Franchi, A M; Fretes, R E; Graham, C H; Godbole, G; Hansson, S R; Headley, P L; Ibarra, C; Jawerbaum, A; Kemmerling, U; Kudo, Y; Lala, P K; Lassance, L; Lewis, R M; Menkhorst, E; Morris, C; Nobuzane, T; Ramos, G; Rote, N; Saffery, R; Salafia, C; Sarr, D; Schneider, H; Sibley, C; Singh, A T; Sivasubramaniyam, T S; Soares, M J; Vaughan, O; Zamudio, S; Lash, G E

2011-03-01

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb.4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sonographic image of cervix epithelioid trophoblastic tumor coexisting with mucinous adenocarcinoma in a postmenopausal woman: A case report.

Science.gov (United States)

Zhu, Yi; Zhang, Guo-Nan; Zhang, Rui-Bo; Shi, Yu; Wang, Deng-Feng; He, Rong

2017-09-01

Epithelioid trophoblastic tumor (ETT) is a distinctive but rare gestational trophoblastic neoplasia (GTN) composed of chorionic-type intermediate trophoblast cells. Approximately 50% ETT arose from the uterine cervix or lower uterine segment following a previous pregnancy with vaginal bleeding. With its unusual ability to simulate an invasive epithelioid neoplasm, ETT frequently poses a diagnostic challenge, especially involving the uterine cervix. We herein report the case of a 60-year-old female with persistent vaginal bleeding and middle-level elevation of serum human chorionic gonadotropin (hCG). Ultrasound revealed a 3.0 × 2.7 cm well-circumscribed, strongly echogenic lesion in the cervix, with a peripheral pattern of Doppler signals. The enhanced pattern by contrast-enhanced ultrasound displayed strong peripheral enhancement accompanied with globular appearance, then centripetal filling completely, and fading away rapidly. The final pathological diagnosis was ETT accompanying mucinous adenocarcinoma. Due to the pre-operative evaluation of a presumed IB2 cervix mucinous adenocarcinoma, the patient was treated with 2 courses of neoadjuvant chemotherapy followed by radical hysterectomy. The patient is currently disease-free for the past 1 year. This case report demonstrates that sonographic image of tumor shapes and blood flow could be helpful in differentiating ETT from another GTN and enable more accurate diagnosis before treatment.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

Directory of Open Access Journals (Sweden)

Rafaela J. da Silva

2017-07-01

Full Text Available Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain, whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Science.gov (United States)

da Silva, Rafaela J; Gomes, Angelica O; Franco, Priscila S; Pereira, Ariane S; Milian, Iliana C B; Ribeiro, Mayara; Fiorenzani, Paolo; Dos Santos, Maria C; Mineo, José R; da Silva, Neide M; Ferro, Eloisa A V; de Freitas Barbosa, Bellisa

2017-01-01

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and

Minimally-aggressive gestational trophoblastic neoplasms.

Science.gov (United States)

Cole, Laurence A

2012-04-01

We have previously defined a new syndrome "Minimally-aggressive gestational trophoblastic neoplasms" in which choriocarcinoma or persistent hydatidiform mole has a minimal growth rate and becomes chemorefractory. Previously we described a new treatment protocol, waiting for hCG rise to >3000 mIU/ml and disease becomes more advanced, then using combination chemotherapy. Initially we found this treatment successful in 8 of 8 cases, here we find this protocol appropriate in a further 16 cases. Initially we used hyperglycosylated hCG, a limited availability test, to identify this syndrome. Here we propose also using hCG doubling rate to detect this syndrome. Minimally aggressive gestational trophoblastic disease can be detected by chemotherapy resistance or low hyperglycosylated hCG, disease by hyperglycosylated hCG and by hCG doubling test. All were recommended to hold off further chemotherapy until hCG >3000mIU/ml. One case died prior to the start of the study, one case withdrew because of a lung nodule and one withdrew refusing the suggested combination chemotherapy. The remaining 16 women were all successfully treated. A total of 8 plus 16 or 24 of 24 women were successfully treated using the proposed protocol, holding back on chemotherapy until hCG >3000mIU/ml. Copyright © 2011 Elsevier Inc. All rights reserved.

Extravillous trophoblast invasion in placenta accreta is associated with differential local expression of angiogenic and growth factors: a cross-sectional study.

Science.gov (United States)

Duzyj, C M; Buhimschi, I A; Laky, C A; Cozzini, G; Zhao, G; Wehrum, M; Buhimschi, C S

2018-02-22

Placenta accreta is clinically associated with maternal uterine scar. Our objective was to investigate the biochemical contribution of maternal scarring to hyperinvasive trophoblast. We hypothesised that trophoblast over-invasion in placenta accreta is associated with aberrant invasion-site signalling of growth and angiogenic factors known to be involved in wound healing and promotion of cell invasion through the epithelial to mesenchymal cellular programme. Cross-sectional series. Yale-New Haven Hospital. Women with histologically confirmed normal and abnormal placentation. Placental invasion site tissue sections were immunostained for endoglin and other angiogenic regulators, and transforming growth factor β (TGFβ) proteins. Maternal serum endoglin, and the vascular endothelial growth factor (VEGF) mediators hypoxia-inducible factor-1α (HIF1α) and endostatin, were assessed using immunoassay. Differences in median H-score by immunostaining and in mean serum level by immunoassay. By immunostaining, placenta accreta samples demonstrated intervillous endoglin shedding and increased trophoblast expression of its cleavage protein matrix metalloproteinase-14. Absent decidual HIF1α and endostatin were observed in areas of VEGF upregulation. TGFβ1 was present in myocytes but not in collagen bundles into which accreta trophoblast invaded. Maternal serum endoglin decreased in praevia and accreta when corrected for gestational age. Angiogenic and growth factors at the placental invasion site are altered in accreta, both by decidual absence and within myometrial scar. We postulate this promotes the invasive phenotype of placenta accreta by activating hyperinvasive trophoblast and by dysregulating placental vascular remodelling. Yale Department of Obstetrics, Gynecology and Reproductive Sciences funds. Placenta accreta histology shows dysregulation of angiogenic and growth factors. © 2018 Royal College of Obstetricians and Gynaecologists.

Indomethacin inhibits the uptake of 22sodium by ovine trophoblastic tissue in vitro

International Nuclear Information System (INIS)

Lewis, G.S.

1986-01-01

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of 22sodium ([22Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37 degrees C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin greater than or equal to 0.2 mM reduced (P less than .01) trophoblastic release (ng/micrograms DNA) of PGF2 alpha from 205 +/- 71.2 to less than or equal to 3.3 +/- 0.2, reduced PGFM from 0.7 +/- 0.1 to less than or equal to 0.17 +/- 0.01, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [22Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [22Na], which is an indirect measure of the transport of water across epithelia, from 3680 +/- 1118 to 934 +/- 248 cpm/micrograms DNA (P less than .03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts

Lack of a Y-Chromosomal Complement in the Majority of Gestational Trophoblastic Neoplasms

Directory of Open Access Journals (Sweden)

Kai Lee Yap

2010-01-01

Full Text Available Gestational trophoblastic neoplasms (GTNs are a rare group of neoplastic diseases composed of choriocarcinomas, placental site trophoblastic tumors (PSTTs and epithelioid trophoblastic tumors (ETTs. Since these tumors are derivatives of fetal trophoblastic tissue, approximately 50% of GTN cases are expected to originate from a male conceptus and carry a Y-chromosomal complement according to a balanced sex ratio. To investigate this hypothesis, we carried out a comprehensive analysis by genotyping a relatively large sample size of 51 GTN cases using three independent sex chromosome genetic markers; Amelogenin, Protein Kinase and Zinc Finger have X and Y homologues that are distinguishable by their PCR product size. We found that all cases contained the X-chromosomal complement while only five (10% of 51 tumors harbored the Y-chromosomal complement. Specifically, Y-chromosomal signals were detected in one (5% of 19 choriocarcinomas, one (7% of 15 PSTTs and three (18% of 17 ETTs. The histopathological features of those with a Y-chromosome were similar to those without. Our results demonstrate the presence of a Y-chromosomal complement in GTNs, albeit a low 10% of cases. This shortfall of Y-chromosomal complements in GTNs may reinforce the notion that the majority of GTNs are derived from previous molar gestations.

Formaldehyde Crosses the Human Placenta and Affects Human Trophoblast Differentiation and Hormonal Functions.

Directory of Open Access Journals (Sweden)

Guillaume Pidoux

Full Text Available The chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST, which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT. Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused cotyledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2. Moreover, we show that formaldehyde-exposed trophoblasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac, an antioxidant.

Tripolar acytokinetic mitosis and formation of feto-maternal syncytia in the bovine placentome: different modes of the generation of multinuclear cells.

Science.gov (United States)

Klisch, K; Pfarrer, C; Schuler, G; Hoffmann, B; Leiser, R

1999-08-01

The vast majority of trophoblast giant cells in the ruminant placenta are binuclear and are believed to derive from mononuclear trophoblastic cells by a single acytokinetic mitosis. There is no satisfactory explanation for the generation of the small proportion of trophoblast giant cells with one, three, or more nuclei. In this light-and electronmicroscopic study of bovine placentomal tissue from the second half of gestation, developmental stages of the trophoblast giant cells are investigated. Large mitotic figures indicate mitotic polyploidization, which is proposed to be due to two subsequent acytokinetic mitoses. Tripolar mitoses offer an explanation for the development of trinucleate trophoblast giant cells. Measurements of nuclear volumes in a series of semithin sections revealed that three size classes of trophoblast giant cells occur. The approximately doubling of nuclear volume between each class is thought to reflect different levels of DNA content that result from polyploidization in this cell type. Although trinuclear feto-maternal hybrid cells are the standard outcome of the fusion of binuclear trophoblast giant cells with uterine epithelial cells, some syncytia with at least five nuclei were observed in the uterine epithelium.

Differential effects of concomitant use of vitamins C and E on trophoblast apoptosis and autophagy between normoxia and hypoxia-reoxygenation.

Directory of Open Access Journals (Sweden)

Tai-Ho Hung

2010-08-01

Full Text Available Concomitant supplementation of vitamins C and E during pregnancy has been reportedly associated with low birth weight, the premature rupture of membranes and fetal loss or perinatal death in women at risk for preeclampsia; however, the cause is unknown. We surmise that hypoxia-reoxygenation (HR within the intervillous space due to abnormal placentation is the mechanism and hypothesize that concomitant administration of aforementioned vitamin antioxidants detrimentally affects trophoblast cells during HR.Using villous explants, concomitant administration of 50 microM of vitamins C and E was observed to reduce apoptotic and autophagic changes in the trophoblast layer at normoxia (8% oxygen but to cause more prominent apoptosis and autophagy during HR. Furthermore, increased levels of Bcl-2 and Bcl-xL in association with a decrease in the autophagy-related protein LC3-II were noted in cytotrophoblastic cells treated with vitamins C and E under standard culture conditions. In contrast, vitamin treatment decreased Bcl-2 and Bcl-xL as well as increased mitochondrial Bak and cytosolic LC3-II in cytotrophoblasts subjected to HR.Our results indicate that concomitant administration of vitamins C and E has differential effects on the changes of apoptosis, autophagy and the expression of Bcl-2 family of proteins in the trophoblasts between normoxia and HR. These changes may probably lead to the impairment of placental function and suboptimal growth of the fetus.

lessons from a hybrid epithelioid trophoblastic tumor and chorio

African Journals Online (AJOL)

(36,900 mIU/ml) and examination showed a bleeding cervical mass. An initial ... characterized by abnormal proliferation of placental trophoblasts and include .... MRI is invaluable to assess extra uterine disease spread and complications.

Gestational trophoblastic neoplasms

International Nuclear Information System (INIS)

Demas, B.E.; Hricak, H.; Braga, C.

1988-01-01

Twenty-four women with suspected gestational trophoblastic neoplasms were evaluated prospectively to identify imaging algorithms optimal for treatment planning. All underwent chest radiography, chest CT, hepatic and cranial CT or MR imaging, and pelvic MR imaging. Ten also underwent pelvic CT, 13 pelvic US. The most sensitive imaging combination was chest CT, hepatic and cranial CT or MR imaging, and pelvic MR imaging. However, correct assignment to ACOG therapeutic categories was achieved by means of history, physical examination, beta subunit of human chorionic gonadotropin measurements, and chest radiography in 81% of patients. Hepatic and cranial imaging defined the need for radiation therapy. Chest CT was needed only when chest radiographs were negative. Pelvic imaging aided diagnosis but did not assist in treatment planning

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice.

Science.gov (United States)

Blois, Sandra M; Tirado-González, Irene; Wu, Julie; Barrientos, Gabriela; Johnson, Briana; Warren, James; Freitag, Nancy; Klapp, Burghard F; Irmak, Ster; Ergun, Suleyman; Dveskler, Gabriela S

2012-06-01

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

Science.gov (United States)

Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

2017-06-01

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Human papillomavirus infects placental trophoblast and Hofbauer cells, but appears not to play a causal role in miscarriage and preterm labor

DEFF Research Database (Denmark)

Ambühl, Lea M.M.; Leonhard, Anne K.; Widen Zakhary, Carina

2017-01-01

INTRODUCTION: Recently, an association between human papillomavirus (HPV) infection and both spontaneous abortion and spontaneous preterm delivery was suggested. However, the reported HPV prevalence in pregnant women varies considerably and reliable conclusions are difficult. We aimed to investig......INTRODUCTION: Recently, an association between human papillomavirus (HPV) infection and both spontaneous abortion and spontaneous preterm delivery was suggested. However, the reported HPV prevalence in pregnant women varies considerably and reliable conclusions are difficult. We aimed...... (n=103), spontaneous preterm delivery (n=69), elective abortion (n=54), and spontaneous abortion (n=44). Moreover, HPV cellular target was identified by the use of in situ hybridization. RESULTS: HPV prevalence in placental tissue was 8.7% in full-term deliveries, 8.8% in spontaneous preterm...... deliveries, 10.9% in spontaneous abortions, and 20.4% in elective abortions. 12 different HPV-types were detected and placental HPV infection was associated to a disease history of cervical cancer. HPV DNA was identified in trophoblast cells, cells of the placental villi mesenchyme including Hofbauer cells...

The invasive phenotype of placenta accreta extravillous trophoblasts associates with loss of E-cadherin.

Science.gov (United States)

Duzyj, C M; Buhimschi, I A; Motawea, H; Laky, C A; Cozzini, G; Zhao, G; Funai, E F; Buhimschi, C S

2015-06-01

Epithelial-to-mesenchymal transition (EMT) is a process of molecular and phenotypic epithelial cell alteration promoting invasiveness. Loss of E-cadherin (E-CAD), a transmembrane protein involved in cell adhesion, is a marker of EMT. Proteolysis into N- and C-terminus fragments by ADAM10 and presenilin-1 (PSEN-1) generates soluble (sE-CAD) and transcriptionally active forms. We studied the protein expression patterns of E-CAD in the serum and placenta of women with histologically-confirmed over-invasive placentation. The patterns of expression and levels of sE-CAD were analyzed by Western blot, immunoassay, and immunoprecipitation. Tissue immunostaining for E-CAD, cytokeratin-7 (epithelial marker), vimentin (mesenchymal marker), ADAM10, PSEN-1 and β-catenin expression were investigated in parallel. N-terminus cleaved 80 kDa sE-CAD fragments were present in serum of pregnant women with gestational age regulation of the circulatory levels. Women with advanced trophoblast invasion did not display circulatory levels of sE-CAD different from those of women with normal placentation. Histologically, extravillous trophoblasts (EVT) closer to the placental-myometrial interface demonstrated less E-CAD staining than those found deeper in the myometrium. These cells expressed both vimentin and cytokeratin, an additional feature of EMT. EVT of placentas with advanced invasion displayed intracellular E-CAD C-terminus immunoreactivity predominating over that of the extracellular N-terminus, a pattern consistent with preferential PSEN-1 processing. Local processing of E-CAD may be an important molecular mechanism controlling the invasive phenotype of accreta EVT. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preeclampsia: novel insights from global RNA profiling of trophoblast subpopulations.

Science.gov (United States)

Gormley, Matthew; Ona, Katherine; Kapidzic, Mirhan; Garrido-Gomez, Tamara; Zdravkovic, Tamara; Fisher, Susan J

2017-08-01

The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non-protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type-specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta.

Science.gov (United States)

Simon, Bailey; Bucher, Matthew; Maloyan, Alina

2017-09-27

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Radiation sensitivity of Merkel cell carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

Science.gov (United States)

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Deep trophoblast invasion and spiral artery remodelling in the placental bed of the lowland gorilla

DEFF Research Database (Denmark)

Pijnenborg, R; Vercruysse, L; Carter, Anthony Michael

2011-01-01

In contrast to baboon or rhesus macaque, trophoblast invasion in the human placental bed occurs by the interstitial as well as the endovascular route and reaches as deep as the inner myometrium. We here describe two rare specimens of gorilla placenta. In the light of recent findings in the chimpa......In contrast to baboon or rhesus macaque, trophoblast invasion in the human placental bed occurs by the interstitial as well as the endovascular route and reaches as deep as the inner myometrium. We here describe two rare specimens of gorilla placenta. In the light of recent findings...... in the chimpanzee, we postulated the occurrence of deep invasion in gorilla pregnancy. Tissues were processed for histology (PAS, orcein), lectin staining (Ulex europaeus agglutinin 1) and immunohistochemistry (cytokeratin 7/17, α-actin). A specimen of young but undetermined gestational age included deep placental...... no definite conclusions about the origin of the intramural trophoblast and the time-course of spiral artery invasion. A different late second trimester placenta specimen showed scattered extravillous trophoblast in the basal plate and underlying decidua, as well as a remodelled spiral artery containing...

Monoclonal antibodies against human trophoblast in female infertility

Czech Academy of Sciences Publication Activity Database

Sedláková, Alena; Elzeinová, Fatima; Bukovský, A.; Madar, J.; Ulčová-Gallová, Z.; Pěknicová, Jana

2005-01-01

Roč. 54, č. 3 (2005), s. 159 ISSN 0271-7352. [European Congress of Reproductive Immunology /3./. 05.09.11-05.09.15, Essex] R&D Projects: GA MZd(CZ) NR7838 Institutional research plan: CEZ:AV0Z50520514 Keywords : monoclonal antibodies * female infertility * trophoblast Subject RIV: EB - Genetics ; Molecular Biology

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

Science.gov (United States)

Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

2008-01-01

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. PMID:18974299

Adiponectin inhibits insulin function in primary trophoblasts by PPARα-mediated ceramide synthesis.

Science.gov (United States)

Aye, Irving L M H; Gao, Xiaoli; Weintraub, Susan T; Jansson, Thomas; Powell, Theresa L

2014-04-01

Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability.

CLO : The cell line ontology

NARCIS (Netherlands)

Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

2014-01-01

Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

A five - year review of gestational trophoblastic diseases in Port ...

African Journals Online (AJOL)

Methods: A retrospective analysis of women treated for gestational ... persistent trophoblastic disease while 8(21.1%) had chemotherapy for choriocarcinoma. ... as well as proper counseling of patients treated on the benefits of follow up visits.

Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

Science.gov (United States)

Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

2017-01-01

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

Comparative studies of placentation and immunology in non-human primates suggest a scenario for the evolution of deep trophoblast invasion and an explanation for human pregnancy disorders.

Science.gov (United States)

Carter, Anthony M

2011-04-01

Deep trophoblast invasion in the placental bed has been considered the hallmark of human pregnancy. It occurs by two routes, interstitial and endovascular, and results in transformation of the walls of the spiral arteries as they traverse the decidua and the inner third of the myometrium. Disturbances in this process are associated with reproductive disorders such preeclampsia. In contrast, trophoblast invasion in Old World monkeys occurs only by the endovascular route and seldom reaches the myometrium. Recently, it was shown that this pattern is maintained in gibbons, but that the human arrangement also occurs in chimpanzee and gorilla. There is an interesting parallel with results from placental immunology regarding the evolution of the major histocompatability complex class I antigen HLA-C and its cognate receptors. HLA-C is not present in Old World monkeys or gibbons. It emerged in the orangutan and became polymorphic in the lineage leading to gorilla, bonobo, chimpanzee, and human. Interaction between HLA-C1 and HLA-C2 on the surface of trophoblast and killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer cells are important regulators of trophoblast invasion. Evolution of this system in great apes may have been one prerequisite for deep trophoblast invasion but seems to have come at a price. The evidence now suggests that certain combinations of maternal genotype for KIRs and fetal genotype for HLA-C imply an increased risk of preeclampsia, fetal growth restriction, and recurrent abortion. The fetal genotype is in part derived from the father providing an explanation for the paternal contribution to reproductive disorders.

TROPHOBLASTIC β1 – GLYCOPROTEIN SYNTHESIS IN SEROPOSITIVE PREGNANT WOMEN

Directory of Open Access Journals (Sweden)

R. N. Bogdanovich

2005-01-01

Full Text Available Abstract. The level of trophoblastic β1 – glycoprotein (SP–1 was determined in the blood sera of 200 healthy pregnant women and 184 women with threatened abortions in term till 20 weeks of pregnancy. In group of women experiencing recurrent abortions in 38 % cases antibodies to chorionic gonadotropin, in 39,5 % cases antibodies to phospholipids, in 25,5 % – antibodies to tireoglobulin were revealed in significant amounts. In 20,65 % lupus anticoagulant was found. The majority of women in this group had changes in homeostasis. The presence of autoantibodies during pregnancy is the unfavourable factor in the development of placental insufficiency. This is proved by the decreased secretion of trophoblastic β1 – glycoprotein – a marker of the fetal part of placenta. (Med. Immunol., 2005, vol.7, № 1, pp. 85588

Chemotherapy for resistant or recurrent gestational trophoblastic neoplasia.

LENUS (Irish Health Repository)

Alazzam, Mo'iad

2012-12-01

Gestational trophoblastic neoplasia (GTN) is a highly curable group of pregnancy-related tumours; however, approximately 25% of GTN tumours will be resistant to, or will relapse after, initial chemotherapy. These resistant and relapsed lesions will require salvage chemotherapy with or without surgery. Various salvage regimens are used worldwide. It is unclear which regimens are the most effective and the least toxic.

[Gestational trophoblastic diseases in cesarean scar: an analysis of 20 cases].

Science.gov (United States)

Zhang, Ge'er; Pan, Zimin

2017-05-25

To analyze the clinical features, diagnosis and treatment of gestational trophoblastic diseases in cesarean scar. Clinical data of three cases of gestational trophoblastic diseases in cesarean scar diagnosed in Women's Hospital, Zhejiang University School of Medicine during December 2011 and December 2016 were collected. And literature search was performed in Wanfang data, VIP, CNKI, PubMed, ISI Web of Knowledge and EMbase database. A total of 20 cases of gestational trophoblastic diseases were included in the analysis. Clinical features were mainly abnormal vaginal bleeding after menopause, artificial abortion or medical abortion, which might be accompanied by abdominal pain. Serum β-human chorionic gonadotropin (β-hCG) levels were increased in 19 patients. The sonographic features were increase of uterine volume, honeycomb-like abnormal intrauterine echo (or described as multiple cystic dark area, multiple anechoic area and multiple liquid dark area) or heterogeneity echo conglomeration, and no clear bound with muscular layer in some cases. There were abundant blood flow signals inside or around the lesions. The ultrasonography indicated that the lesions were located in the anterior side of the uterine isthmus with the involvement of cesarean section scar. In 12 cases with lesions in cesarean scar shown by preliminary diagnosis, 9 underwent uterine artery embolization (UAE) for pretreatment; the blood loss greater than 1500 mL was observed in only one case without UAE; no patient received hysterectomy. In 8 patients whose lesions were not shown in cesarean scar, only one case received UAE pretreatment, and hysterectomy was performed in 3 cases due to blood loss greater than 1500 mL. Two cases were lost in follow-up and no death was reported in remaining 18 cases. The serum β-hCG levels returned to normal or satisfactory level during the follow-up in 17 cases with increased β-hCG levels before treatment and no recurrence was observed. The misdiagnosis rate and

Deep trophoblast invasion and spiral artery remodelling in the placental bed of the chimpanzee

DEFF Research Database (Denmark)

Pijnenborg, R; Vercruysse, L; Carter, Anthony Michael

2011-01-01

Deep trophoblast invasion is usually considered to be a unique feature of human placentation as compared to other primates. Because of the occasional occurrence of preeclampsia in great apes, which in the human is associated with impaired deep invasion, this uniqueness may be questioned. The avai......Deep trophoblast invasion is usually considered to be a unique feature of human placentation as compared to other primates. Because of the occasional occurrence of preeclampsia in great apes, which in the human is associated with impaired deep invasion, this uniqueness may be questioned...

Gestational trophoblastic disease in Abuth Zaria, Nigeria: A 5‑year ...

African Journals Online (AJOL)

Gestational trophoblastic disease in Abuth Zaria, Nigeria: A 5‑year review. ... Abimbola O. Kolawole, John K. Nwajagu, Adekunle O. Oguntayo, Marliya S. ... The data obtained were expressed in percentages, means, and standard deviations.

Pregnancy immunology: decidual immune cells.

Science.gov (United States)

Sanguansermsri, Donruedee; Pongcharoen, Sutatip

2008-01-01

Human pregnancy is a complex process. Placental development depends on the function of secretory molecules produced by placental trophoblast cells as well as by maternal uterine immune cells within the decidua. These decidual immune cells are T cells, natural killer cells, macrophages and dendritic cells. The interactions between the trophoblast cells and the maternal immune cells have an impact on the outcome of the pregnancy. Knowledge about the phenotypes and functions of the maternal immune cells in normal and pathological pregnancies including recurrent spontaneous abortions, preeclampsia and hydatidiform moles may improve our understanding of the immunobiology of the normal pregnancy as a whole and may provide approaches for improving the treatment of pathological pregnancies.

The Gestational Trophoblastic Diseases: A Ten Year Retrospective Study

Directory of Open Access Journals (Sweden)

Razieh Mohammadjafari

2010-01-01

Full Text Available Background: Gestational trophoblastic disease (GTD defines a heterogenenous group ofinterrelated lesions that arise from the trophoblastic epithelium of the placenta. There are severalhistologically distinct types of GTD: hydatiform mole (complete or partial, persistant/invasivegestational trophoblastic neoplasia (GTN, choriocarcinoma and placenta site trophoblastictumors. The aim of this study was to determine the frequency and risk factors of GTD amongwomen admitted to Imam Khomeini Hospital in Ahvaz, Iran.Materials and Methods: This was a cross-sectional study conducted at Imam KhomeiniHospital in Ahvaz, Iran. All hospital records related to GTD (132 from 1996 until 2006 werereviewed. Demographic and histo-pathologic characteristics were extracted. Chi-square andFisher-exact tests were used to analyze all variables. P ≤ 0.05 was considered statisticallysignificant. SPSS, version 11 was used for statistical analysis.Results: The mean age of patients was 27.6 years. Most patients who presented with GTDwere of ages 18-35 years (71.3%. There was no relationship between age and hydatiformmole during the reproductive years. There were 28 (18.9% patients over the age 40, of which18 (15.90% of these had a complete hydatiform mole. Within this group, 9 (6.8% changedto a persistent mole. There was a significant relationship between age over 40 and completemole (p<0.02. The percentage of patients with blood groups A and O was the same (37.9%.There was a significant relationship between blood groups (O+ and A+ and complete mole(p<0.05.Conclusion: The most common age range for hydatiform mole was 18-35 years. Women overthe age of 40 had a more complete hydatiform mole, which is similar to the other countries.Age and blood group are two risk factors for hydatiform mole.

A Resource for the Transcriptional Signature of Bona Fide Trophoblast Stem Cells and Analysis of Their Embryonic Persistence

Directory of Open Access Journals (Sweden)

Georg Kuales

2015-01-01

Full Text Available Trophoblast stem cells (TSCs represent the multipotent progenitors that give rise to the different cells of the embryonic portion of the placenta. Here, we analysed the expression of key TSC transcription factors Cdx2, Eomes, and Elf5 in the early developing placenta of mouse embryos and in cultured TSCs and reveal surprising heterogeneity in protein levels. We analysed persistence of TSCs in the early placenta and find that TSCs remain in the chorionic hinge until E9.5 and are lost shortly afterwards. To define the transcriptional signature of bona fide TSCs, we used inducible gain- and loss-of-function alleles of Eomes or Cdx2, and EomesGFP, to manipulate and monitor the core maintenance factors of TSCs, followed by genome-wide expression profiling. Combinatorial analysis of resulting expression profiles allowed for defining novel TSC marker genes that might functionally contribute to the maintenance of the TSC state. Analyses by qRT-PCR and in situ hybridisation validated novel TSC- and chorion-specific marker genes, such as Bok/Mtd, Cldn26, Duox2, Duoxa2, Nr0b1, and Sox21. Thus, these expression data provide a valuable resource for the transcriptional signature of bona fide and early differentiating TSCs and may contribute to an increased understanding of the transcriptional circuitries that maintain and/or establish stemness of TSCs.

Human papillomavirus infects placental trophoblast and Hofbauer cells, but appears not to play a causal role in miscarriage and preterm labor.

Science.gov (United States)

Ambühl, Lea M M; Leonhard, Anne K; Widen Zakhary, Carina; Jørgensen, Annemette; Blaakaer, Jan; Dybkaer, Karen; Baandrup, Ulrik; Uldbjerg, Niels; Sørensen, Suzette

2017-10-01

Recently, an association between human papillomavirus infection and both spontaneous abortion and spontaneous preterm delivery was suggested. However, the reported human papillomavirus prevalence in pregnant women varies considerably and reliable conclusions are difficult. We aimed to investigate human papillomavirus infection in placental tissue of a Danish study cohort. Furthermore, we studied the cellular localization of human papillomavirus. In this prospective case-control study, placental tissue was analyzed for human papillomavirus infection by nested PCR in the following four study groups: full-term delivery (n = 103), spontaneous preterm delivery (n = 69), elective abortion (n = 54), and spontaneous abortion (n = 44). Moreover, human papillomavirus cellular target was identified using in situ hybridization. Human papillomavirus prevalence in placental tissue was 8.7% in full-term deliveries, 8.8% in spontaneous preterm deliveries, 10.9% in spontaneous abortions, and 20.4% in elective abortions. Twelve different human papillomavirus types were detected, and placental human papillomavirus infection was associated to a disease history of cervical cancer. Human papillomavirus DNA was identified in trophoblast cells, cells of the placental villi mesenchyme including Hofbauer cells, and in parts of the encasing endometrium. Placental human papillomavirus infections are not likely to constitute a risk factor for spontaneous preterm labor or spontaneous abortions in the Danish population, although an effect of human papillomavirus DNA in placental cells cannot be excluded. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

OpenAIRE

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

Syncytin is involved in breast cancer-endothelial cell fusions

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

2006-01-01

Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

Rare Presentation of Metastatic Cystic Trophoblastic Tumor in a Patient Without Prior Chemotherapy

Directory of Open Access Journals (Sweden)

Michael L. Wang

2017-07-01

Full Text Available Cystic trophoblastic tumor (CTT is a rare testicular germ cell tumor (GCT predominantly seen in post-chemotherapy patients. It is prognostically similar to teratoma and requires no additional chemotherapy in the absence of a nonteratomatous GCT component. We report a case of metastatic CTT in a patient with primary testicular teratoma without prior chemotherapy. Retroperitoneal lymph node metastases contained teratoma, embryonal carcinoma, and CTT. The CTT was β-hCG positive and SALL4 negative by immunohistochemistry (IHC. CTT can arise in metastatic testicular GCT in treatment naïve patients. An important differential diagnosis is choriocarcinoma due to treatment implications, and SALL4 IHC may help.

Gestational trophoblastic disease with hyperthyroidism: Anesthetic management

Directory of Open Access Journals (Sweden)

Puneet Khanna

2012-01-01

Full Text Available The coexistence of hyperthyroidism with gestational trophoblastic disease is a known albeit rare clinical condition. We herein report the successful anesthetic management of such a case in our institute. There are only few case reports in literature of this association. Often, the diagnosis of hyperthyroid state is retrospective one, as it can be missed in the emergency scenario of patient requiring molar evacuation. This case report highlights the perioperative management and optimization of hyperthyroid state prior to surgical evacuation of the invasive hydatidiform mole.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

The Cellosaurus, a Cell-Line Knowledge Resource

Science.gov (United States)

Bairoch, Amos

2018-01-01

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

1983-11-01

Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus FIV infected cat

Science.gov (United States)

FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection m...

Activation of PAR-1/NADPH Oxidase/ROS Signaling Pathways is Crucial for the Thrombin-Induced sFlt-1 Production in Extravillous Trophoblasts: Possible Involvement in the Pathogenesis of Preeclampsia

Directory of Open Access Journals (Sweden)

Qi-tao Huang

2015-03-01

Full Text Available Backgrounds/Aims: Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1 expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT. Methods: An EVT cell line (HRT-8/SVneo was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS production were determined by DCFH-DA. Results: Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Conclusions: Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future.

Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

Science.gov (United States)

Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

2015-01-01

Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

Directory of Open Access Journals (Sweden)

Yufen Xie

2014-11-01

Full Text Available Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC, elevated O2 and mitochondrial function are necessary to placental lineages at the maternal–placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4 enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2 initiated the most TSC differentiation after 24 h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos, and high pyruvate kinase M2 (glycolysis despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2 > 0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

The risk of persistent trophoblastic disease after hydatidiform mole classified by morphology and ploidy

DEFF Research Database (Denmark)

Niemann, Isa; Hansen, Estrid S; Sunde, Lone

2007-01-01

classifications, and compared the ability of the two classifications to discriminate between patients with and without a substantial risk of persistent trophoblastic disease. METHODS: 294 cases of consecutively collected hydropic placentas clinically suspected of hydatidiform mole made the basis......OBJECTIVE: Hydatidiform mole can be classified by histopathologic characteristics and by genetic constitutions and most complete moles are diploid, whereas most partial moles are triploid. We investigated the concordance between these two classifications, characterized moles with conflicting......-molar miscarriage, 20 were triploids, 2 were diploid androgenetic and 2 were diploid biparental. In 23% of the conceptuses, the histopathologic and genetic classifications were conflicting. 5% of the patients with hydropic placentas classified as partial mole encountered persistent trophoblastic disease; however...

Activation of PPAR{gamma} by Human Cytomegalovirus for de novo Replication Impairs Migration and Invasiveness of Cytotrophoblast from Early Placenta

DEFF Research Database (Denmark)

Rauwel, Benjamin; Mariamé, Bernard; Martin, Hélène

2010-01-01

, as assessed by using well-established in vitro models of invasive trophoblast i.e. primary cultures of EVCT isolated from first trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation, placentation...... and chromatin immunoprecipitation assays. Due to the key role of PPARgamma in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARgamma human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness...

Increased placental trophoblast inclusions in placenta accreta.

Science.gov (United States)

Adler, E; Madankumar, R; Rosner, M; Reznik, S E

2014-12-01

Trophoblast inclusions (TIs) are often found in placentas of genetically abnormal gestations. Although best documented in placentas from molar pregnancies and chromosomal aneuploidy, TIs are also associated with more subtle genetic abnormalities, and possibly autism. Less than 3% of non-aneuploid, non-accreta placentas have TIs. We hypothesize that placental genetics may play a role in the development of placenta accreta and aim to study TIs as a potential surrogate indicator of abnormal placental genetics. Forty cases of placenta accreta in the third trimester were identified in a search of the medical records at one institution. Forty two third trimester control placentas were identified by a review of consecutively received single gestation placentas with no known genetic abnormalities and no diagnosis of placenta accreta. Forty percent of cases with placenta accreta demonstrated TIs compared to 2.4% of controls. More invasive placenta accretas (increta and percreta) were more likely to demonstrate TIs than accreta (47% versus 20%). Prior cesarean delivery was more likely in accreta patients than controls (67% versus 9.5%). Placenta accreta is thought to be the result of damage to the endometrium predisposing to abnormal decidualization and invasive trophoblast growth into the myometrium. However, the etiology of accreta is incompletely understood with accreta frequently occurring in women without predisposing factors and failing to occur in predisposed patients. This study has shown that TIs are present at increased rates in cases of PA. Further studies are needed to discern what underlying pathogenic mechanisms are in common between abnormal placentation and the formation of TIs. Published by Elsevier Ltd.

Monitoring cell line identity in collections of human induced pluripotent stem cells

Directory of Open Access Journals (Sweden)

Raquel Sarafian

2018-04-01

Full Text Available The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Keywords: Induced pluripotent stem cells, Genotyping, Cell line identification, Short tandem repeats, Quality control

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

Directory of Open Access Journals (Sweden)

Barré-Sinoussi Françoise

2009-05-01

Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

Establishment of cell lines with rat spermatogonial stem cell characteristics

NARCIS (Netherlands)

van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

2002-01-01

Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

Science.gov (United States)

Norris, J S; Kohler, P O

1978-01-01

Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

Directory of Open Access Journals (Sweden)

Tozeren Aydin

2006-11-01

Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

Characterization of stem-like cells in a new astroblastoma cell line

Energy Technology Data Exchange (ETDEWEB)

Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

2017-03-15

Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

ACCRETA COMPLICATING COMPLETE PLACENTA PREVIA IS CHARACTERIZED BY REDUCED SYSTEMIC LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND EPITHELIAL-TO-MESENCHYMAL TRANSITION OF THE INVASIVE TROPHOBLAST

Science.gov (United States)

Wehrum, Mark J.; Buhimschi, Irina A.; Salafia, Carolyn; Thung, Stephen; Bahtiyar, Mert O.; Werner, Erica F.; Campbell, Katherine H.; Laky, Christine; Sfakianaki, Anna K.; Zhao, Guomao; Funai, Edmund F.; Buhimschi, Catalin S.

2011-01-01

OBJECTIVE To characterize serum angiogenic factor profile of women with complete placenta previa and determine if invasive trophoblast differentiation characteristic of accreta, increta or percreta shares features of epitehelial-mesenchymal-transition (EMT). STUDY DESIGN We analyzed gestational age matched serum samples from 90 pregnant women with either complete placenta previa (n=45) or uncomplicated pregnancies (n=45). Vascular-endothelial-growth-factor (VEGF), placental-growth-factor (PlGF) and soluble fms-like-tyrosine-kinase-1 (sFlt-1) were immunoassayed. VEGF and phosphotyrosine (P-Tyr) immunoreactivity was surveyed in histological specimens relative to expression of vimentin and cytokeratin-7. RESULTS Women with previa and invasive placentation [accreta (n=5); increta (n=6); percreta (n=2)] had lower systemic VEGF (invasive previa: median [IQR]: 0.8[0.02–3.4] vs. control: 6.5[2.7–10.5] pg/mL, P=0.02). VEGF and P-Tyr immunostaining predominated in the invasive extravillous trophoblasts (EVT) which co-expressed vimentin and cytokeratin-7, a EMT feature and tumor-like cell phenotype. CONCLUSIONS Lower systemic free VEGF and a switch of the interstitial EVT to a metastable cell phenotype characterize placenta previa with excessive myometrial invasion. PMID:21316642

Cell fusion induced by ionizing radiation in various cell lines

International Nuclear Information System (INIS)

Khair, M.B.

1994-07-01

Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

Directory of Open Access Journals (Sweden)

Hee-Jung Choi

Full Text Available In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation

OpenAIRE

Harris, L. K.; Keogh, R. J.; Wareing, M.; Baker, P. N.; Cartwright, J. E.; Whitley, G. S.; Aplin, J. D.

2007-01-01

BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic ...

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

Science.gov (United States)

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Application of DNA fingerprints for cell-line individualization.

Science.gov (United States)

Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

1990-09-01

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

Directory of Open Access Journals (Sweden)

Hans eKlingemann

2016-03-01

Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

Susceptibility testing of fish cell lines for virus isolation

DEFF Research Database (Denmark)

Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

2009-01-01

and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

Science.gov (United States)

Qin, J-Z; Xin, H; Nickoloff, B J

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

International Nuclear Information System (INIS)

Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

2010-01-01

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

Science.gov (United States)

Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Pre-evacuation hCG glycoforms in uneventful complete hydatidiform mole and persistent trophoblastic disease.

NARCIS (Netherlands)

Thomas, C.M.G.; Kerkmeijer, L.G.W.; Ariaens, H.J.; Steen, R. van der; Massuger, L.F.A.G.; Sweep, F.C.

2010-01-01

OBJECTIVE: To investigate whether the glycoform distribution patterns of human chorionic gonadotropin (hCG) obtained by chromatofocusing in pre-evacuation serum are different for patients who will eventually develop into persistent trophoblastic disease in case of complete hydatidiform mole

Review: hCG, Preeclampsia and Regulatory T cells

OpenAIRE

Norris, Wendy; Nevers, Tania; Sharma, Surendra; Kalkunte, Satyan

2011-01-01

Human chorionic gonadotropin (hCG) is crucial for successful pregnancy. Its many functions include angiogenesis and immune regulation. Despite years of research, the etiology of preeclampsia remains unknown. Marked by insufficient trophoblast invasion and poor spiral artery remodeling, preeclampsia has also been linked to immune dysregulation. Here we discuss the roles of hCG in the context of endovascular cross-talk between trophoblasts and endothelial cells and immune tolerance. We propose ...

Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

Energy Technology Data Exchange (ETDEWEB)

Henderson, R.F.; Waide, J.J.; Lechner, J.F.

1995-12-01

A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

International Nuclear Information System (INIS)

Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

2003-01-01

Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

Directory of Open Access Journals (Sweden)

Ellen Melaleuca Menkhorst

Full Text Available Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT invade through the differentiated uterine endometrium (the decidua to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8 M, medroxyprogesterone acetate (10(-7 M and cAMP (0.5 mM for 14 days. Conditioned media (CM was collected on day 2 (non-decidualized CM and 14 (decidualized CM of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1, dipeptidyl peptidase 1 (DPP1/cathepsin C and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

Science.gov (United States)

The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

International Nuclear Information System (INIS)

Playle, L.C.

2001-03-01

The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

Science.gov (United States)

Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognosis of patients treated with whole brain radiation therapy for metastatic gestational trophoblastic disease

International Nuclear Information System (INIS)

Schechter, Naomi R.; Mychalczak, Borys; Jones, Walter; Spriggs, David

1996-01-01

Purpose/Objective: To evaluate the effect of multiple treatment and disease related variables on the local control and survival of patients receiving whole brain radiation therapy for metastatic gestational trophoblastic disease. Materials and Methods: Between November 1967 and December 1994, 21 patients were treated at our institution for gestational trophoblastic disease metastatic to the brain. 29% ((6(21))) were diagnosed with their brain metastases before the onset of chemotherapy (early group). 79% ((15(21))) developed their brain metastases during or after the administration of first-line chemotherapy (late group). All patients were treated with whole brain radiation therapy. The total dose ranged from 200 cGy to 3600 cGy (median 2200 cGy). Sixteen patients (76%) received concurrent systemic chemotherapy. None of the patients received intrathecal chemotherapy as a component of their initial treatment. Survival and local control were calculated from the date of diagnosis of brain metastases. Follow-up ranged from 11 months to 170 months with a median of 77 months. Results: The median overall survival was 21 months, with 2- and 5-year actuarial survivals of 46% and 31%, respectively. Neither survival nor local control was significantly affected by age at diagnosis of brain metastases (<35 vs. ≥35 years), time of presentation of brain metastases (early vs. late), or use of concurrent chemotherapy. The total dose of radiation (<2200 cGy vs. ≥2200 cGy) significantly affected initial local control, but not survival. The 5-year actuarial local control of the initial brain metastases with ≥2200 cGy was 91%, as compared to 24% with <2200 cGy (p=0.05). Survival was significantly affected by control of disease at extracranial sites. The 2- and 5-year actuarial survivals of the 9 patients whose disease was controlled at extracranial sites were 100% and 83%, respectively, as compared to 8% and 0% for the 12 whose extracranial disease was not controlled (p=0

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

Science.gov (United States)

Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

THP-1 cell line: an in vitro cell model for immune modulation approach.

Science.gov (United States)

Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

Differential heat shock response of primary human cell cultures and established cell lines

DEFF Research Database (Denmark)

Richter, W W; Issinger, O G

1986-01-01

degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

Radiation response of haematopoietic cell lines of human origin

International Nuclear Information System (INIS)

Lehnert, S.; Rybka, W.B.; Suissa, S.; Giambattisto, D.

1986-01-01

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radio-sensitivity of the cells varied widely, the D 0 s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivities. (author)

Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

Directory of Open Access Journals (Sweden)

Ashley R.P. Hinson

2013-07-01

Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

Science.gov (United States)

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Radiation sensitivity of human lung cancer cell lines

International Nuclear Information System (INIS)

Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

1989-01-01

X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

Generation of genome-modified Drosophila cell lines using SwAP.

Science.gov (United States)

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

Elsevier Trophoblast Research Award lecture: The multifaceted role of Nodal signaling during mammalian reproduction.

Science.gov (United States)

Park, C B; Dufort, D

2011-03-01

Nodal, a secreted signaling protein in the transforming growth factor-beta (TGF-β) superfamily, has established roles in vertebrate development. However, components of the Nodal signaling pathway are also expressed at the maternal-fetal interface and have been implicated in many processes of mammalian reproduction. Emerging evidence indicates that Nodal and its extracellular inhibitor Lefty are expressed in the uterus and complex interactions between the two proteins mediate menstruation, decidualization and embryo implantation. Furthermore, several studies have shown that Nodal from both fetal and maternal sources may regulate trophoblast cell fate and facilitate placentation as both embryonic and uterine-specific Nodal knockout mouse strains exhibit disrupted placenta morphology. Here we review the established and prospective roles of Nodal signaling in facilitating successful pregnancy, including recent evidence supporting a potential link to parturition and preterm birth. Copyright © 2011 Elsevier Ltd. All rights reserved.

Peptidomic analysis of human cell lines

Science.gov (United States)

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

2011-01-01

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

The transcriptional diversity of 25 Drosophila cell lines

Energy Technology Data Exchange (ETDEWEB)

Cherbas, Lucy [Indiana Univ., Bloomington, IN (United States); Willingham, Aarron [Affymetrix Inc., Santa Clara, CA (United States); Zhang, Dayu [Indiana Univ., Bloomington, IN (United States); Yang, Li [University of Connecticut Health Center, Farmington, Connecticut (United States); Zou, Yi [Indiana Univ., Bloomington, IN (United States); Eads, Brian D. [Indiana Univ., Bloomington, IN (United States); Carlson, Joseph W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Landolin, Jane M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kapranov, Philipp [Affymetrix Inc., Santa Clara, CA (United States); Dumais, Jacqueline [Affymetrix Inc., Santa Clara, CA (United States); Samsonova, Anastasia [Harvard Medical School, Boston, MA (United States); Choi, Jeong-Hyeon [Indiana Univ., Bloomington, IN (United States); Roberts, Johnny [Indiana Univ., Bloomington, IN (United States); Davis, Carrie A. [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Tang, Haixu [Indiana Univ., Bloomington, IN (United States); van Baren, Marijke J. [Washington Univ., St. Louis, MO (United States); Ghosh, Srinka [Affymetrix Inc., Santa Clara, CA (United States); Dobin, Alexander [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Bell, Kim [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Lin, Wei [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Langton, Laura [Washington Univ., St. Louis, MO (United States); Duff, Michael O. [University of Connecticut Health Center, Farmington, Connecticut (United States); Tenney, Aaron E. [Washington Univ., St. Louis, MO (United States); Zaleski, Chris [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Brent, Michael R. [Washington Univ., St. Louis, MO (United States); Hoskins, Roger A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kaufman, Thomas C. [Indiana University, Bloomington, Indiana (United States); Andrews, Justen [Indiana University, Bloomington, Indiana (United States); Graveley, Brenton R. [University of Connecticut Health Center, Farmington, Connecticut (United States); Perrimon, Norbert [Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Institute, Boston, MA (United States); Celniker, Susan E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gingeras, Thomas R. [Affymetrix Inc., Santa Clara, CA (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Cherbas, Peter [Indiana Univ., Bloomington, IN (United States)

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25

Investigation of the selenium metabolism in cancer cell lines

DEFF Research Database (Denmark)

Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

2011-01-01

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

Science.gov (United States)

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Menadione inhibits MIBG uptake in two neuroendocrine cell lines

NARCIS (Netherlands)

Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

1997-01-01

In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

Directory of Open Access Journals (Sweden)

Ismail Noorliza M

2010-09-01

Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

The curative effect of a second curettage in persistent trophoblastic disease: a retrospective cohort survey.

NARCIS (Netherlands)

Trommel, N.E. van; Massuger, L.F.A.G.; Verheijen, R.; Sweep, C.G.J.; Thomas, C.M.G.

2005-01-01

OBJECTIVE: To assess the curative effect of a second curettage in patients with low-risk Persistent Trophoblastic Disease (PTD) after molar pregnancy. METHODS: A retrospective cohort survey was performed on 2122 patients registered with the Dutch Central Registry for Hydatidiform Moles between 1987

Establishment and characterization of rat portal myofibroblast cell lines.

Directory of Open Access Journals (Sweden)

Michel Fausther

Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

International Nuclear Information System (INIS)

Medina, D.; Oborn, C.J.; Li, M.L.; Bissell, M.J.

1987-01-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of β-casein mRNA in the presence or absence of prolactin. The inducibility of β-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types

9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

International Nuclear Information System (INIS)

Heaton, D.

1992-06-01

The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D 0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D 0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

Energy Technology Data Exchange (ETDEWEB)

Heaton, D. [Rush Univ. Medical Center, Chicago, IL (United States). Therapeutic Radiology; Mustafi, R. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology; Schwartz, J.L. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)

1992-06-01

The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

Science.gov (United States)

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

International Nuclear Information System (INIS)

Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

2003-01-01

Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

Regulation of estradiol and progesterone production by CRH-R1 and -R2 is through divergent signaling pathways in cultured human placental trophoblasts.

Science.gov (United States)

Gao, Lu; Tao, Yi; Hu, Tianxiao; Liu, Weina; Xu, Chen; Liu, Jie; You, Xingji; Gu, Hang; Ni, Xin

2012-10-01

CRH and its related peptides urocortins (UCN) have been identified in placenta and implicated to play pivotal roles in the regulation of pregnancy and parturition in humans. The objectives of present study were to investigate the effects of endogenous CRH and its related peptides in the regulation of steroid production in placenta. Placental trophoblasts were isolated from term placenta tissues and cultured for 72 h. Estradiol (E(2)) and progesterone (P(4)) contents in culture media were determined by radioimmunoassay. Treatment of cultured trophoblasts with CRH or UCNI antibody showed decreased E(2), whereas increased P(4) production. Treatment of cells with CRH receptor type 1 antagonist antalarmin or CRH receptor type 2 (CRH-R2) antagonist astressin-2b also decreased E(2) but increased P(4) production. Knockdown of CRH receptor type 1 or CRH-R2 cells showed a decrease in E(2) production and an increase in P(4) production. In CRH-R2 knockdown cells, CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C-β3. Adenylyl cyclase and protein kinase A inhibitors blocked CRH-induced increased E(2) production but not decreased P(4) production. PLC inhibitor U73122 and protein kinase C inhibitor chelerythrine blocked the effects of CRH on E(2) and P(4) production in CRH-R2 knockdown cells. UCNIII, the specific CRH-R2 agonist, stimulated GTP-bound Gαi protein and phosphorylated phospholipase C-β3 expression. Both U73122 and chelerythrine blocked UCNIII-induced increased E(2) production and decreased P(4) production. We suggest that CRH and its related peptides might be involved in changes in the progesterone to estrogen ratio during human pregnancy.

The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis

International Nuclear Information System (INIS)

Blazquez, Alba G.; Briz, Oscar; Gonzalez-Sanchez, Ester; Perez, Maria J.; Ghanem, Carolina I.; Marin, Jose J.G.

2014-01-01

Acetaminophen is used as first-choice drug for pain relief during pregnancy. Here we have investigated the effect of acetaminophen at subtoxic doses on the expression of ABC export pumps in trophoblast cells and its functional repercussion on the placental barrier during maternal cholestasis. The incubation of human choriocarcinoma cells (JAr, JEG-3 and BeWo) with acetaminophen for 48 h resulted in no significant changes in the expression and/or activity of MDR1 and MRPs. In contrast, in JEG-3 cells, BCRP mRNA, protein, and transport activity were reduced. In rat placenta, collected at term, acetaminophen administration for the last three days of pregnancy resulted in enhanced mRNA, but not protein, levels of Mrp1 and Bcrp. In fact, a decrease in Bcrp protein was found. Using in situ perfused rat placenta, a reduction in the Bcrp-dependent fetal-to-maternal bile acid transport after treating the dams with acetaminophen was found. Complete biliary obstruction in pregnant rats induced a significant bile acid accumulation in fetal serum and tissues, which was further enhanced when the mothers were treated with acetaminophen. This drug induced increased ROS production in JEG-3 cells and decreased the total glutathione content in rat placenta. Moreover, the NRF2 pathway was activated in JEG-3 cells as shown by an increase in nuclear NRF2 levels and an up-regulation of NRF2 target genes, NQO1 and HMOX-1, which was not observed in rat placenta. In conclusion, acetaminophen induces in placenta oxidative stress and a down-regulation of BCRP/Bcrp, which may impair the placental barrier to bile acids during maternal cholestasis. - Highlights: • Acetaminophen induces changes in placental BCRP expression in vitro. • This drug reduces the ability of placental cells to export BCRP substrates. • Acetaminophen induces changes in Bcrp expression in rat placenta. • Placental barrier to bile acids is impaired in rats treated with this drug

The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis

Energy Technology Data Exchange (ETDEWEB)

Blazquez, Alba G., E-mail: albamgb@usal.es [Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca (Spain); CIBERehd, Instituto de Salud Carlos III, Madrid (Spain); Briz, Oscar, E-mail: obriz@usal.es [Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca (Spain); CIBERehd, Instituto de Salud Carlos III, Madrid (Spain); Gonzalez-Sanchez, Ester, E-mail: u60343@usal.es [Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca (Spain); Perez, Maria J., E-mail: mjperez@usal.es [Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca (Spain); University Hospital of Salamanca, IECSCYL-IBSAL, Salamanca (Spain); CIBERehd, Instituto de Salud Carlos III, Madrid (Spain); Ghanem, Carolina I., E-mail: cghanem@ffyb.uba.ar [Instituto de Investigaciones Farmacologicas, Facultad de Farmacia y Bioquimica, CONICET, Universidad de Buenos Aires, Buenos Aires (Argentina); Marin, Jose J.G., E-mail: jjgmarin@usal.es [Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca (Spain); CIBERehd, Instituto de Salud Carlos III, Madrid (Spain)

2014-05-15

Acetaminophen is used as first-choice drug for pain relief during pregnancy. Here we have investigated the effect of acetaminophen at subtoxic doses on the expression of ABC export pumps in trophoblast cells and its functional repercussion on the placental barrier during maternal cholestasis. The incubation of human choriocarcinoma cells (JAr, JEG-3 and BeWo) with acetaminophen for 48 h resulted in no significant changes in the expression and/or activity of MDR1 and MRPs. In contrast, in JEG-3 cells, BCRP mRNA, protein, and transport activity were reduced. In rat placenta, collected at term, acetaminophen administration for the last three days of pregnancy resulted in enhanced mRNA, but not protein, levels of Mrp1 and Bcrp. In fact, a decrease in Bcrp protein was found. Using in situ perfused rat placenta, a reduction in the Bcrp-dependent fetal-to-maternal bile acid transport after treating the dams with acetaminophen was found. Complete biliary obstruction in pregnant rats induced a significant bile acid accumulation in fetal serum and tissues, which was further enhanced when the mothers were treated with acetaminophen. This drug induced increased ROS production in JEG-3 cells and decreased the total glutathione content in rat placenta. Moreover, the NRF2 pathway was activated in JEG-3 cells as shown by an increase in nuclear NRF2 levels and an up-regulation of NRF2 target genes, NQO1 and HMOX-1, which was not observed in rat placenta. In conclusion, acetaminophen induces in placenta oxidative stress and a down-regulation of BCRP/Bcrp, which may impair the placental barrier to bile acids during maternal cholestasis. - Highlights: • Acetaminophen induces changes in placental BCRP expression in vitro. • This drug reduces the ability of placental cells to export BCRP substrates. • Acetaminophen induces changes in Bcrp expression in rat placenta. • Placental barrier to bile acids is impaired in rats treated with this drug.

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

DEFF Research Database (Denmark)

Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M

1998-01-01

receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

Co-dominant expression of the HLA-G gene and various forms of alternatively spliced HLA-G mRNA in human first trimester trophoblast

DEFF Research Database (Denmark)

Hviid, T V; Møller, C; Sørensen, S

1998-01-01

imprinting of the HLA-G locus could have implications for the interaction in the feto-maternal relationship. Restriction Fragment Length Polymorphism (RFLP), allele-specific amplification and Single Strand Conformation Polymorphism (SSCP) analysis followed by DNA sequencing were performed on Reverse...... Transcription (RT) Polymerase Chain Reaction (PCR) products of HLA-G mRNA to examine the expression of maternal and paternal alleles. Our results demonstrate that HLA-G is co-dominantly expressed in first trimester trophoblast cells. A "new" non-synonymous base substitution in exon 4 was detected. We also...

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

Science.gov (United States)

Ma, Hailun; Galvin, Teresa A; Glasner, Dustin R; Shaheduzzaman, Syed; Khan, Arifa S

2014-06-01

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell

Notch signaling activation in human embryonic stem cells is required for embryonic but not trophoblastic lineage commitment

OpenAIRE

Yu, Xiaobing; Zou, Jizhong; Ye, Zhaohui; Hammond, Holly; Chen, Guibin; Tokunaga, Akinori; Mali, Prashant; Li, Yue-Ming; Civin, Curt; Gaiano, Nicholas; Cheng, Linzhao

2008-01-01

The Notch signaling pathway plays important roles in cell fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell fate choices in human embryonic stem (hES) cells. Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hES cell lines. We report here that activation of Notch signaling is required for undifferentiated hES cells to form the pr...

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

International Nuclear Information System (INIS)

Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A.

1991-01-01

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links

Lining cells on normal human vertebral bone surfaces

International Nuclear Information System (INIS)

Henning, C.B.; Lloyd, E.L.

1982-01-01

Thoracic vertebrae from two individuals with no bone disease were studied with the electron microscope to determine cell morphology in relation to bone mineral. The work was undertaken to determine if cell morphology or spatial relationships between the bone lining cells and bone mineral could account for the relative infrequency of bone tumors which arise at this site following radium intake, when compared with other sites, such as the head of the femur. Cells lining the vertebral mineral were found to be generally rounded in appearance with varied numbers of cytoplasmic granules, and they appeared to have a high density per unit of surface area. These features contrasted with the single layer of flattened cells characteristic of the bone lining cells of the femur. A tentative discussion of the reasons for the relative infrequency of tumors in the vertebrae following radium acquisition is presented

Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

Directory of Open Access Journals (Sweden)

Khordadmehr, M.,

2014-05-01

Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

Molecular characterization of breast cancer cell lines through multiple omic approaches.

Science.gov (United States)

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy.

Directory of Open Access Journals (Sweden)

Grace Truong

Full Text Available Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT cells, modifying their bioactivity on endothelial cells (EC. Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE and spontaneous preterm birth (SPTB. HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen on exosome release was quantified using nanocrystals (Qdot® coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™ was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

Science.gov (United States)

Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

2013-12-05

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. Published by Elsevier Ireland Ltd.

Nuclear vlimata and aneuploidy in embryonic cells is caused by meiosis. Behaviour and properties of meiotic cells

OpenAIRE

Logothetou-Rella, H.

1995-01-01

This study demonstrates that human embryonic cells divide by meiosis. The use of trophoblastic tissue cells (early embryo) and amniotic cells (late embryo) exhibited the following characteristic events of meiosis: nuclear (NVs) and nucleolar (NuVs) vlimata formation; NV invasion in host cells; extrusion of chromosomes; nuclear fusion; metaphase fusion; hybrid cell formation; nuclear, nucleolar and cytoplasmic bridges, chromosomal transfer, variablesized nuc...

Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

Science.gov (United States)

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-01-01

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

Science.gov (United States)

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-06-27

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

Establishment and conventional cytogenetic characterization of three gastric cancer cell lines.

Science.gov (United States)

Leal, Mariana Ferreira; Martins do Nascimento, José Luiz; da Silva, Carla Elvira Araújo; Vita Lamarão, Maria Fernanda; Calcagno, Danielle Queiroz; Khayat, André Salim; Assumpção, Paulo Pimentel; Cabral, Isabel Rosa; de Arruda Cardoso Smith, Marília; Burbano, Rommel Rodríguez

2009-11-01

Gastric cancer is the fourth most frequent type of cancer and the second most frequent cause of cancer mortality worldwide. Only a modest number of gastric carcinoma cell lines have been isolated thus far. Here we describe the establishment and cytogenetic characterization of three new gastric cancer cell lines obtained from primary gastric adenocarcinoma (ACP02 and ACP03) and cancerous ascitic fluid (AGP01) of individuals from northern Brazil. ACP02, ACP03, and AGP01 cell lines are presently in the 60th passage. The cell lines grew in a disorganized single layer with some agglomerations and heterogeneous divisions (bipolar and multipolar). All cell lines exhibited a composite karyotype with several clonal chromosome alterations. Trisomy 8 was the most frequent alteration. Chromosome 8 aneusomy was confirmed by fluorescence in situ hybridization. All cell lines also exhibited trisomy 7 and deletion of chromosome arm 17p. These results suggest that, although frequent chromosome alterations are commonly observed due to culture process, the ACP02, ACP03, and AGP01 cell lines and primary gastric cancer from individuals of northern Brazil share genetic alterations, supporting use of these cell lines as a model of gastric carcinogenesis in this population.

DNA fingerprinting of the NCI-60 cell line panel.

Science.gov (United States)

Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

2009-04-01

The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

Science.gov (United States)

Touzard, Eve; Reinaud, Pierrette; Dubois, Olivier; Guyader-Joly, Catherine; Humblot, Patrice; Ponsart, Claire; Charpigny, Gilles

2013-10-01

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

Science.gov (United States)

Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

2017-04-01

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Cellular radiosensitivity of small-cell lung cancer cell lines

DEFF Research Database (Denmark)

Krarup, M; Poulsen, H S; Spang-Thomsen, M

1997-01-01

PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

Science.gov (United States)

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

Science.gov (United States)

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Science.gov (United States)

Hamilton, Gerhard

2014-01-01

Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

NARCIS (Netherlands)

KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

Directory of Open Access Journals (Sweden)

Kazuya Takahashi

Full Text Available Prognosis of childhood acute lymphoblastic leukemia (ALL has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ, a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+ ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ, a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19 ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19 ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19 ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good

Cellular radiosensitivity of small-cell lung cancer cell lines

International Nuclear Information System (INIS)

Krarup, Marianne; Poulsen, Hans Skovgaard; Spang-Thomsen, Mogens

1997-01-01

Purpose: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Methods and Materials: Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D 0 ), the capacity for sublethal damage repair (D q ), and the extrapolation number (n). Values for α and β were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF 2 ). Results: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. Conclusion: The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy

Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

Science.gov (United States)

Morales-Prieto, D M; Ospina-Prieto, S; Schmidt, A; Chaiwangyen, W; Markert, U R

2014-02-01

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans. Copyright © 2013. Published by Elsevier Ltd.

Fraction against Human Cancer Cell Lines

African Journals Online (AJOL)

fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and ... The morphology of the HepG2 cell nucleus was investigated by Hoechst 33342, ..... Gong F, Liang Y, Xie P, Chau F. Information theory.

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera).

Science.gov (United States)

Goodman, Cynthia L; Ringbauer, Joseph A; Li, Yao-Fa; Lincoln, Tamra Reall; Stanley, David

2017-05-01

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

Bifenthrin activates homotypic aggregation in human T-cell lines.

Science.gov (United States)

Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

2006-03-01

Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

Establishment, immortalisation and characterisation of pteropid bat cell lines.

Directory of Open Access Journals (Sweden)

Gary Crameri

Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Endogenous WNT Signals Mediate BMP-Induced and Spontaneous Differentiation of Epiblast Stem Cells and Human Embryonic Stem Cells

Directory of Open Access Journals (Sweden)

Dorota Kurek

2015-01-01

Full Text Available Therapeutic application of human embryonic stem cells (hESCs requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs. WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

Science.gov (United States)

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

Endoarterial pulmonary metastasis of malignant trophoblast associated with a term intrauterine pregnancy.

Science.gov (United States)

Carlson, J A; Day, T G; Kuhns, J G; Howell, R S; Masterson, B J

1984-02-01

A previously healthy gravida 4, para 3, developed preclampsia and progressive dyspnea at the 37th gestational week and had bilateral pulmonary infiltrates on chest roentgenogram. She delivered a healthy, term, male infant with a normal appearing placenta. Postpartum, her respiratory status gradually worsened. A lung biopsy on the 20th postpartum day revealed intravascular trophoblasts, diffuse arteriolar thrombosis with pulmonary infarction, and subacute interstitial pneumonitis. Combination chemotherapy was instituted, but the patient died from respiratory insufficiency.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

Science.gov (United States)

Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

2013-08-01

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Obstetric ultrasound aids prompt referral of gestational trophoblastic disease in marginalized populations on the Thailand-Myanmar border

NARCIS (Netherlands)

McGregor, Kathryn; Min, Aung Myat; Karunkonkowit, Noaeni; Keereechareon, Suporn; Tyrosvoutis, Mary Ellen; Tun, Nay Win; Rijken, Marcus J.; Hoogenboom, Gabie; Boel, Machteld; Chotivanich, Kesinee; Nosten, François; McGready, Rose

2017-01-01

Background: The use of obstetric ultrasound in the diagnosis of gestational trophoblastic disease (GTD) in high-income settings is well established, leading to prompt management and high survival rates. Evidence from low-income settings suggests ultrasound is essential in identifying complicated

LET effects on normal and radiosensitive cell lines

International Nuclear Information System (INIS)

Geard, C.R.; Travisano, M.

1986-01-01

Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

Myricetin suppresses invasion and promotes cell death in human placental choriocarcinoma cells through induction of oxidative stress.

Science.gov (United States)

Yang, Changwon; Lim, Whasun; Bazer, Fuller W; Song, Gwonhwa

2017-07-28

Myricetin is a bioactive compound found in a variety of vegetables and fruits, and its anti-cancer effects are well known. In this study, we confirmed that myricetin reduced proliferation of two choriocarcinoma cell lines (JAR and JEG-3) and also promoted apoptosis and regulated cell cycle progression in a dose-dependent manner in JAR and JEG-3 cells. In addition, we found that invasive and pro-angiogenic properties of malignant JAR and JEG-3 trophoblast cells were attenuated by myricetin treatment via MAPK and PI3K/AKT signaling pathways. In addition, we found that ROS production, lipid peroxidation, glutathione depletion, and loss of mitochondrial membrane potentials were enhanced in JAR and JEG-3 cells treated with myricetin. Moreover, myricetin augmented cytosolic Ca 2+ release from the endoplasmic reticulum associated with modulation of ER stress in JAR and JEG-3 cells. Our results also revealed that myricetin had synergistic antiproliferative effects with current chemotherapeutics, etoposide and cisplatin, on choriocarcinoma cells. Collectively, results of the present study provide strong evidence for the potential of myricetin to be an effective therapeutic for the prevention of human placental choriocarcinomas. Copyright © 2017 Elsevier B.V. All rights reserved.

Radiation of different human melanoma cell lines increased expression of RHOB. Level of this tumor suppressor gene in different cell lines

International Nuclear Information System (INIS)

Notcovich, C.; Molinari, B.; Duran, H.; Delgado González, D.; Sánchez Crespo, R.

2013-01-01

Previous results of our group show that a correlation exists between intrinsic radiosensitivity of human melanoma cells and cell death by apoptosis. RhoB is a small GTPase that regulates cytoskeletal organization. Besides, is related to the process of apoptosis in cells exposed to DNA damage as radiation. Also, RhoB levels decrease in a wide variety of tumors with the tumor stage, being considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. The aim of this study was to analyze the expression of RhoB in different human melanoma cell lines in relation to melanocytes, and evaluate the effect of gamma radiation on the expression of RhoB. We used the A375, SB2 and Meljcell lines, and the derived from melanocytes Pig1. It was found for all three tumor lines RhoB expression levels significantly lower than those of Pig1 (p <0.05), as assessed by semiquantitative RT-PCR . When tumor cells were irradiated to a dose of 2Gyinduction was observed at 3 hours RhoB irradiation. RhoB expression increased in all lines relative to non-irradiated control, showing a greater induction ( p< 0.05) for the more radiosensitive line SB2, consistent with apoptosis in response to radiation. The results allow for the first time in melanoma demonstrate that RhoB, as well as in other tumor types, has a lower expression in tumor cells than their normal counterparts. Moreover, induction in the expression of RhoB in irradiated cells may be associated with the process of radiation-induced apoptosis. The modulation of RhoB could be a new tool to sensitize radioresistant melanoma. (author)

Induction of apoptosis by opium in some tumor cell lines.

Science.gov (United States)

Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

2016-09-30

The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

International Nuclear Information System (INIS)

Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

2011-01-01

Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

Biological Therapy Following Chemotherapy and Peripheral Stem Cell Transplantation in Treating Patients With Cancer

Science.gov (United States)

2013-03-25

Breast Cancer; Chronic Myeloproliferative Disorders; Gestational Trophoblastic Tumor; Kidney Cancer; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Neuroblastoma; Ovarian Cancer; Sarcoma; Testicular Germ Cell Tumor

Sensory hair cell regeneration in the zebrafish lateral line.

Science.gov (United States)

Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

Science.gov (United States)

Lush, Mark E.; Piotrowski, Tatjana

2014-01-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation

International Nuclear Information System (INIS)

Raaphorst, G.P.; Bussey, A.; Heller, D.P.; Ng, C.E.

1994-01-01

Two human melanoma cell lines, one radioresistant (Sk-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD. 24 refs., 5 figs., 1 tab

Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

DEFF Research Database (Denmark)

Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

1993-01-01

A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc......-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had...

Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

Science.gov (United States)

de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

2017-03-01

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. © 2015 John Wiley & Sons Ltd.

Recombinant protein production from stable mammalian cell lines and pools.

Science.gov (United States)

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Gerhard Hamilton

2014-03-01

Full Text Available Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4 inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose-Polymerase 1 (PARP1 inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS.

Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

Directory of Open Access Journals (Sweden)

Tobias Hannes

2015-01-01

Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.