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Exposure of primary human trophoblast cells to cytokines induces surface and soluble chemokine receptors (CCRs)
58
MONDAY,
Conclusions: Coadministration of tetracycline HCl with the contraceptive patch did not affect the pharmacokinetics of 17d-NGM or EE. Based on these data, tetracycline is not expected to affect the contraceptive efficacy of EVRA”. Treatment with EVRA”, with or without tetracycline HCl, was safe and well tolerated.
FC1.23 DAILY FEATURED
STUDIES
SEPTEMBER
FC1.23.03 DEVELOPMENT-DEPENDENT GLUCOCORTICOID METABOLISM IN HUMAN OVARIAN GRANULOSA CELLS P.Y.K.Yon& J.Thong, ‘R.Andrew, ‘B.Walker, S.G.Hillier; University of Edinburgh, Centre For Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, ‘Molecular Medicine Centre, Crewe Road, Edinburgh EH4 2XU, United Kingdom
I
FC1.23.01 EXPOSURE OF PRIMARY HUMAN TROPHOBLAST CELLS TO CYTOKINES INDUCES SURFACE AND SOLUBLE CHEMOKINE RECEPTORS (CCRs) S. Vassiliadis (l), E. Koumantakis (2), I. Athanassakis (1). (1) Dept. Biology, University of Crete, Heraklion, Greece. (2) Dept. OBIGYN, University Hospital, Heraklion, Greece. Objectives: We examined the ability of IFN71 and IL-4 to induce CCR3 or CXCR4 on mature human trophoblast cells from term placenta since expression of CCRs on trophoblast cells may provide a valid mechanism for the in utero transmission of HIV. Study Methods: We defined the constitutive and inducible expression of surface CCR3 and CXCR4 on primary human trophoblasts during short periods of cell culture by immunofluorescence after exposure to IFN-71 and IL-4. In addition, released CCR activity was also monitored by ELISA. Results: Kinetic experiments show that the constitutive expression of both CCR3 and CXCR4 reach a peak of expression after 6 hours of culture, whereas by 24 hours they have almost disappeared. In the presence of IFN-71, CCR3 increases in expression after 4 hours of incubation, reaching highest levels at 24 hours of culture, whereas CXCR4 is kept at lower levels as compared to non-treated cells. In the presence of IL-4, CCR3 declines from 2 to 8 hours of culture to increase again at 24 hours by 50%. Under the IL-4 stimulus, CXCR4 shows a peak of expression at 8 hours of culture. Interestingly, we detect soluble CCR activity in the culture supernatants of trophoblast cells, which follows an inversely proportional pattern of that corresponding to surface expression. Conclusions: Surface induced expression of CCRs on trophoblast cells has a fast turnover rate between 2 and 8 hours of culture. The released CCR activity, however, may account for the development of an inhibitory mechanism against viral transmission by absorbing virions in the extracellular matrix.
FC1.23.02 RECURRENT VENOUS THROMBOEMBOLISM DURING HORMONE REPLACEMENT THERAPY. RESULTS OF THE ESTROGEN IN VENOUS THROMBOEMBOLISM TRIAL E. Ovintad, E. Hoibraaten, H. Arnesen, S. Larsen, E. Wickstrom, PM Sandset, Ullevaal University Hospital, dept of Hematology, Oslo, Norway. Objectives: The aim of the study was to determine if hormone replacement therapy (HRT) alters the risk of venous thromboembolism (VTE) in high risk women; i.e. women with previous VTE. Study Methods: Randomized, double-blinded, and placebo-controlled clinical trial with a stratified design combined with a double-triangular sequential design. 140 females with previously verified VTE, were randomized to either 2 mg estradiol plus lmg norethisteron acetate one tablet daily (n=71), or placebo (n=60). The primary outcome was the occurrence of deep venous thrombosis or pulmonary embolism. Results: 8 women in the hormone group and one woman in the placebo group experienced VTE. The study was terminated early based on the results of circumstantial evidence emerging during the trial. The final analysis gave a median unbiased estimate of the incidence of VTE of 2.2% and 10.6% in the placebo group and HRT group respectively (p=O.O4). In the hormone group, four women experienced deep venous thrombosis, three pulmonary embolism and one cerebral sinus vein thrombosis. All these events happened within 220 days after inclusion. The one patient in the placebo group suffered pulmonary embolism at day 413. Conclusions: In women with previous VTE, HRT increases the risk of recurrent VTE and should be avoided. This study confirms the result of the epidemiological studies showing a small increased risk of VTE on HRT, which might be generalized to most women
Objectives: llfl hydroxysteroid dehydrogenase (llflHSD), which is responsible for the bidirectional conversion between cortisol (F) and its inactive form, cortisone (E), has been implicated in the regulation of ovarian folliculogenesis and oogenesis. Expression of its two known isomers (llflHSD1, llflHSD2) by human granulosa cells (GC) has been shown to be developmentally regulated. Immature GC (IGC) almost exclusively express llgHSD2 mRNA while luteinised GC (LGC) express predominantly llflHSD1 mRNA. The aims of this study were: (1) to confirm the switch in 1lgHSD expression from type 2 to type 1 by measuring the interconversion between E and F in IGC and LGC, and follicular fluid levels of E and F, and (2) to determine if 1 lgHSD1 reductase activity (E to F conversion) is gonadotrophically regulated in vitro. Methods: LGC were aspirated from periovulatory follicles in IVF patients during oocyte recovery, 35h after injection of hCG. IGC were collected from antral follicles (3.16mm) in women undergoing oophorectomy for benign gynaecological conditions. Cells (l-5 x 10’) were incubated for 4h at 37°C $n medium 199 containing 50pmol of E and F, which included O.lpCi [ HI-E or F, followed by steroid extraction, thin-layer chromatography, and spectrometry to determine % conversion between E and F. For objective (2), IGC was incubated with FSH and LH for 48h before the 4h incubation described above. Experiments in triplicate. Results: Results are mean&EM values. % conversion of E to F by LGC (n=7) was 36.3*3.7%, compared with only 0.6*0.4% for IGC (n=5) (p
FC1.23.04 DIFFERENTIALLY EXPRESSED GENES IN NORMAL HUMAN MAMMARY TISSUE DURING TAMOXlFEN THERAPY (LUD 5646). I. L. Gebrim, Federal University SBo Paula Brazil, Rua Pedro de Toledo 781, 4 th floor, SBo Paula, Brazil, 04039032. Introduction: Tamoxifen (TAM) an orally effective, synthetic antiestrogen used first in women with metastatic breast cancer disease has become an essential part of any therapeutic strategy for the control and prevention of breast cancer. In order to identify TAM responsive genes we performed cDNA arrays using total RNA isolated from normal breast tissue from patients treated with placebo or TAM (20 mgiday) for 4 weeks. The cDNA fragments used on membranes were generated by the Human Cancer Genome Project, funded by the Ludwig Institute for Cancer Research and FAPESP (Fonda@0 de Amparo ?IPesquisa do Estado de SBo Paula, BRAZIL). This is a program for human gene discovery and human coding region compilation based on a novel concept for the high throughput sequencing of human open reading frames. The project consists entirely on the sequencing and analysis of short cDNA fragments generated preferentially from the central coding portions of expressed human genes obtained from tumors including breast, ovarian and cervical cancer. The fragments are termed ORESTES (Open Reading frame ESTs) and are generated using a strategy developed and patented by the Ludwig Institute. Within the project it is aimed to assemble the data, together with existing data in GenBank and
*
MONDAY,
Conclusions: Coadministration of tetracycline HCl with the contraceptive patch did not affect the pharmacokinetics of 17d-NGM or EE. Based on these data, tetracycline is not expected to affect the contraceptive efficacy of EVRA”. Treatment with EVRA”, with or without tetracycline HCl, was safe and well tolerated.
FC1.23 DAILY FEATURED
STUDIES
SEPTEMBER
FC1.23.03 DEVELOPMENT-DEPENDENT GLUCOCORTICOID METABOLISM IN HUMAN OVARIAN GRANULOSA CELLS P.Y.K.Yon& J.Thong, ‘R.Andrew, ‘B.Walker, S.G.Hillier; University of Edinburgh, Centre For Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, ‘Molecular Medicine Centre, Crewe Road, Edinburgh EH4 2XU, United Kingdom
I
FC1.23.01 EXPOSURE OF PRIMARY HUMAN TROPHOBLAST CELLS TO CYTOKINES INDUCES SURFACE AND SOLUBLE CHEMOKINE RECEPTORS (CCRs) S. Vassiliadis (l), E. Koumantakis (2), I. Athanassakis (1). (1) Dept. Biology, University of Crete, Heraklion, Greece. (2) Dept. OBIGYN, University Hospital, Heraklion, Greece. Objectives: We examined the ability of IFN71 and IL-4 to induce CCR3 or CXCR4 on mature human trophoblast cells from term placenta since expression of CCRs on trophoblast cells may provide a valid mechanism for the in utero transmission of HIV. Study Methods: We defined the constitutive and inducible expression of surface CCR3 and CXCR4 on primary human trophoblasts during short periods of cell culture by immunofluorescence after exposure to IFN-71 and IL-4. In addition, released CCR activity was also monitored by ELISA. Results: Kinetic experiments show that the constitutive expression of both CCR3 and CXCR4 reach a peak of expression after 6 hours of culture, whereas by 24 hours they have almost disappeared. In the presence of IFN-71, CCR3 increases in expression after 4 hours of incubation, reaching highest levels at 24 hours of culture, whereas CXCR4 is kept at lower levels as compared to non-treated cells. In the presence of IL-4, CCR3 declines from 2 to 8 hours of culture to increase again at 24 hours by 50%. Under the IL-4 stimulus, CXCR4 shows a peak of expression at 8 hours of culture. Interestingly, we detect soluble CCR activity in the culture supernatants of trophoblast cells, which follows an inversely proportional pattern of that corresponding to surface expression. Conclusions: Surface induced expression of CCRs on trophoblast cells has a fast turnover rate between 2 and 8 hours of culture. The released CCR activity, however, may account for the development of an inhibitory mechanism against viral transmission by absorbing virions in the extracellular matrix.
FC1.23.02 RECURRENT VENOUS THROMBOEMBOLISM DURING HORMONE REPLACEMENT THERAPY. RESULTS OF THE ESTROGEN IN VENOUS THROMBOEMBOLISM TRIAL E. Ovintad, E. Hoibraaten, H. Arnesen, S. Larsen, E. Wickstrom, PM Sandset, Ullevaal University Hospital, dept of Hematology, Oslo, Norway. Objectives: The aim of the study was to determine if hormone replacement therapy (HRT) alters the risk of venous thromboembolism (VTE) in high risk women; i.e. women with previous VTE. Study Methods: Randomized, double-blinded, and placebo-controlled clinical trial with a stratified design combined with a double-triangular sequential design. 140 females with previously verified VTE, were randomized to either 2 mg estradiol plus lmg norethisteron acetate one tablet daily (n=71), or placebo (n=60). The primary outcome was the occurrence of deep venous thrombosis or pulmonary embolism. Results: 8 women in the hormone group and one woman in the placebo group experienced VTE. The study was terminated early based on the results of circumstantial evidence emerging during the trial. The final analysis gave a median unbiased estimate of the incidence of VTE of 2.2% and 10.6% in the placebo group and HRT group respectively (p=O.O4). In the hormone group, four women experienced deep venous thrombosis, three pulmonary embolism and one cerebral sinus vein thrombosis. All these events happened within 220 days after inclusion. The one patient in the placebo group suffered pulmonary embolism at day 413. Conclusions: In women with previous VTE, HRT increases the risk of recurrent VTE and should be avoided. This study confirms the result of the epidemiological studies showing a small increased risk of VTE on HRT, which might be generalized to most women
Objectives: llfl hydroxysteroid dehydrogenase (llflHSD), which is responsible for the bidirectional conversion between cortisol (F) and its inactive form, cortisone (E), has been implicated in the regulation of ovarian folliculogenesis and oogenesis. Expression of its two known isomers (llflHSD1, llflHSD2) by human granulosa cells (GC) has been shown to be developmentally regulated. Immature GC (IGC) almost exclusively express llgHSD2 mRNA while luteinised GC (LGC) express predominantly llflHSD1 mRNA. The aims of this study were: (1) to confirm the switch in 1lgHSD expression from type 2 to type 1 by measuring the interconversion between E and F in IGC and LGC, and follicular fluid levels of E and F, and (2) to determine if 1 lgHSD1 reductase activity (E to F conversion) is gonadotrophically regulated in vitro. Methods: LGC were aspirated from periovulatory follicles in IVF patients during oocyte recovery, 35h after injection of hCG. IGC were collected from antral follicles (3.16mm) in women undergoing oophorectomy for benign gynaecological conditions. Cells (l-5 x 10’) were incubated for 4h at 37°C $n medium 199 containing 50pmol of E and F, which included O.lpCi [ HI-E or F, followed by steroid extraction, thin-layer chromatography, and spectrometry to determine % conversion between E and F. For objective (2), IGC was incubated with FSH and LH for 48h before the 4h incubation described above. Experiments in triplicate. Results: Results are mean&EM values. % conversion of E to F by LGC (n=7) was 36.3*3.7%, compared with only 0.6*0.4% for IGC (n=5) (p
FC1.23.04 DIFFERENTIALLY EXPRESSED GENES IN NORMAL HUMAN MAMMARY TISSUE DURING TAMOXlFEN THERAPY (LUD 5646). I. L. Gebrim, Federal University SBo Paula Brazil, Rua Pedro de Toledo 781, 4 th floor, SBo Paula, Brazil, 04039032. Introduction: Tamoxifen (TAM) an orally effective, synthetic antiestrogen used first in women with metastatic breast cancer disease has become an essential part of any therapeutic strategy for the control and prevention of breast cancer. In order to identify TAM responsive genes we performed cDNA arrays using total RNA isolated from normal breast tissue from patients treated with placebo or TAM (20 mgiday) for 4 weeks. The cDNA fragments used on membranes were generated by the Human Cancer Genome Project, funded by the Ludwig Institute for Cancer Research and FAPESP (Fonda@0 de Amparo ?IPesquisa do Estado de SBo Paula, BRAZIL). This is a program for human gene discovery and human coding region compilation based on a novel concept for the high throughput sequencing of human open reading frames. The project consists entirely on the sequencing and analysis of short cDNA fragments generated preferentially from the central coding portions of expressed human genes obtained from tumors including breast, ovarian and cervical cancer. The fragments are termed ORESTES (Open Reading frame ESTs) and are generated using a strategy developed and patented by the Ludwig Institute. Within the project it is aimed to assemble the data, together with existing data in GenBank and
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