Alterations in the mechanical, chemical and biocompatibility properties of low-cost polyethylene and polyester meshes after steam sterilization.

INTRODUCTION: In Africa and other Low Resource Settings (LRS), the guideline-based and thus in most cases mesh-based treatment of inguinal hernias is only feasible to a very limited extent. This has led to an increased use of low cost meshes (LCMs, mostly mosquito meshes) for patients in LRS. Most of the LCMs used are made of polyethylene or polyester, which must be sterilized before use. The aim of our investigations was to determine changes in the biocompatibility of fibroblasts as well as mechanical and chemical properties of LCMs after steam sterilization. MATERIAL AND METHODS: Two large-pored LCMs made of polyester and polyethylene in a size of 11 x 6 cm were cut and steam sterilized at 100, 121 and 134 °C. These probes and non-sterile meshes were then subjected to mechanical tensile tests in vertical and horizontal tension, chemical analyses and biocompatibility tests with human fibroblasts. All meshes were examined by stereomicroscopy, scanning electron microscopy (SEM), LDH (cytotoxicity) measurement, viability testing, pH, lactate and glycolysis determination. RESULTS: Even macroscopically, polyethylene LCMs showed massive shrinkage after steam sterilization, especially at 121 and 134 °C. While polyester meshes showed no significant changes after sterilization with regard to deformation and damage as well as tensile force and stiffness, only the unsterile polyethylene mesh and the mesh sterilized at 100 °C could be tested mechanically due to the shrinkage of the other specimen. For these meshes the tensile forces were about four times higher than for polyester LCMs. Chemical analysis showed that the typical melting point of polyester LCMs was between 254 and 269 °C. Contrary to the specifications, the polyethylene LCM did not consist of low-density polyethylene, but rather high-density polyethylene and therefore had a melting point of 137 °C, so that the marked shrinkage described above occurred. Stereomicroscopy confirmed the shrinkage of polyethylene LCMs already after sterilization at 100 °C in contrast to polyester LCMs. Surprisingly, cytotoxicity (LDH measurement) was lowest for both non-sterile LCMs, while polyethylene LCMs sterilized at 100 and 121 °C in particular showed a significant increase in cytotoxicity 48 hours after incubation with fibroblasts. Glucose metabolism showed no significant changes between sterile and non-sterile polyethylene and polyester LCMs. CONCLUSION: The process of steam sterilization significantly alters mechanical and structural properties of synthetic hernia mesh implants. Our findings do not support a use of low-cost meshes because of their unpredictable properties after steam sterilization.

An in vitro study on the biocompatibility of fibroblasts in sterile and non-sterile low-cost and commercial meshes.

INTRODUCTION: Despite several successful studies with low-cost meshes (LCM) for the treatment of inguinal hernias in India and Africa, a nationwide application has not been possible for a variety of reasons. One problem is the special preparation and sterilization of these meshes-naturally, they should comply with international standards and demands, which is often difficult to achieve in Africa. Our primary approach was to determine whether there are differences in the biocompatibility of fibroblasts between non-sterile and sterile LCMs and commercial meshes (CM). MATERIALS AND METHODS: Two polyester CMs with different pore size and a polyester LCM were examined as both sterile and non-sterile. LCM was plasma sterilized at 60 °C and steam sterilized at 134 °C. Sterile and non-sterile meshes were soaked with an antibiotic (penicillin/streptomycin) and antimycotic solution (amphotericin B). Human fibroblasts from healthy subcutaneous tissue were used. Various tests for evaluating the growth behavior and cell morphology of human fibroblasts were conducted. Semiquantitative (light microscopy) and qualitative (scanning electron microscopy) analyses were performed after 1 week and again after 12 weeks. The metabolism of fibroblasts was checked by pH measurements and glucose analyses. Biocompatibility of fibroblasts on sterile and non-sterile meshes was carried out by luminescence methods (cell viability and apoptosis) as well as calorimetric methods for proliferation determination (BrDU assay) and cytotoxicity (LDH assay). RESULTS: Light and electron microscopy revealed a moderate growth of fibroblasts on all investigated mesh types. The results of glycolysis and the pH value were within the normal range for all sterile and non-sterile meshes. In biocompatibility studies, no elevated level of apoptosis was detected. The viability measurement of mitochondrial activity of fibroblasts showed a 50% inhibition of mitochondria in all nets, with the exception of non-sterile CM, whereas mitochondrial activity was increased in the non-sterile CM. A proliferation measurement (BrdU test) revealed different growth inhibition in the sterile and non-sterile meshes. This growth inhibition was significantly stronger, particularly for non-sterile CM light meshes, than it was for the non-sterile LCM. CONCLUSION: Again, our studies show no significant differences in biocompatibility of fibroblasts between expensive and low-cost meshes. In addition, we detected fibroblast growth even in sterile meshes, independent of the mesh group. To our knowledge, the present study is the first of its kind in terms of qualitative equivalence of sterile and non-sterile in vitro mesh samples. We do not wish to create future patient studies with non-sterilized meshes saturated with antibiotics/antimycotics. However, perhaps we can prove in future studies that under semi-sterile conditions with certain LCMs, wound infection rates can be acceptable.

In-vitro examination of the biocompatibility of fibroblast cell lines on alloplastic meshes and sterilized polyester mosquito mesh.

INTRODUCTION: The use of alloplastic implants for tissue strengthening when treating hernias is an established therapy worldwide. Despite the high incidence of hernias in Africa and Asia, the implantation of costly mesh netting is not financially feasible. Because of that various investigative groups have examined the use of sterilized mosquito netting. The animal experiments as well as the clinical trials have both shown equivalent short- and long-term results. The goal of this paper is the comparison of biocompatibility of human fibroblasts on the established commercially available nets and on sterilized polyester mosquito mesh over a period of 12 weeks. MATERIALS AND METHODS: Three commercially available plastic mesh types and a gas-sterilized mosquito polyethylenterephtalate (polyester) mesh were examined. Human fibroblasts from subcutaneous healthy tissue were used. Various tests for evaluating the growth behavior and the cell morphology of human fibroblasts were conducted. The semi-quantitative (light microscopy) and qualitative (scanning electron microscopy) analyses were performed after 1 week and then again after 12 weeks. The cell proliferation and cytotoxicity of the implants were investigated with the help of the 5'-bromo-2'-deoxyuridine (BrdU)-cell proliferation test and the LDH-cytotoxicity test. The number of live cells per ml was determined with the Bürker counting chamber. In addition, analyses were made of the cell metabolism (oxidative stress) by measuring the pH value, hydrogen peroxide, and glycolysis. RESULTS: After 12 weeks, a proliferation of fibroblasts on all mesh is documented. No mesh showed a complete apoptosis of the cells. This qualitative observation could be confirmed quantitatively in a biochemical assay by marking the proliferating cells with BrdU. The biochemical analysis brought the proof that the materials used, including the polyester of the mosquito mesh, are not cytotoxic for the fibroblasts. The vitality of the cells was between 94 and 98%. The glucose metabolism as well as the pH value of the fibroblasts showed no significant differences between the tested meshes. The examination of the oxidative stress via measurement of the H2O2 concentration showed values in the normal range for the commercially alloplastic meshes and the mosquito mesh. CONCLUSIONS: Our examination showed no significant difference with regard to biocompatibility between the officially approved and cost-intensive meshes and the sterilized (autoclaved) mosquito mesh. Due to the proven strength and stability of the mosquito mesh and their proven compatibility, the implantation of the sterilized mosquito mesh in additional in vivo studies must be considered. A wide-scale and cost-effective treatment of hernias could thus be guaranteed, not only in Third World countries.

Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis.

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

A retrospective in vitro study of the impact of anti-diabetics and cardioselective pharmaceuticals on breast cancer.

BACKGROUND: In a retrospective controlled study, a tumor-protective effect, regarding breast cancer, was determined for the medicines metformin and glitazone (anti-diabetics), bisoprolol, and propranolol (cardioselective ß1 adrenoceptor antagonists). Our main goal was to provide evidence, showing the tumor-protective effects of beta-blockers and of antidiabetics via investigations in vitro. MATERIALS AND METHODS: Four different medicines were tested in cell cultures: Propranolol: 2.4 mg/ml and 0.3 mg/ml; bisoprolol: 0.1 mg/ml and 0.05 mg/ml; metformin: 7.5 mg/ml, 2.5 mg/ml, and 0.15 mg/ml; and glitazone: 2.5 mg/ml, 0.15 mg/ml, and 0.05 mg/ml. The human breast cancer cell lines MCF7 and BT20 (estrogen receptor-positive and -negative; ATCC; cell density: 5×10(5) cells/ml) were used. Both cell lines were cultured under sterile conditions in incubators at 37°C, with a humidified atmosphere of 5% CO(2). The influences of the drugs were determined through cytotoxicity and proliferation assays and performance of a hydrogen peroxide assay. Morphological observations (light microscopy) and metabolic investigations (pH value, glucose) were also performed. RESULTS: The application of the beta-blocker propranolol resulted in highly cytotoxic effects (>90%) in both cell lines. In contrast, bisoprolol did not have any effects, neither in cytotoxicity tests nor in cell proliferation assays. The anti-diabetic metformin had a higher cytotoxic influence on the BT20 than did on the MCF7 cell line. The cell proliferation of BT20 was significantly inhibited after the addition of 2.5 mg/ml metformin and of 2.5 mg/ml glitazone. The application of glitazone also resulted in an increase of hydrogen peroxide and a decrease of the pH value. CONCLUSION: The strongest cytotoxic effect was observed with propranolol suggesting that, in clinical practice, this pharmaceutical can be used in patients with breast cancer who have hypertension. A specific clinical recommendation for anti-diabetics is not yet possible.

Effects of phytoestrogen extracts isolated from flax on hormone production of trophoblast tumour cells Jeg 3 and BeWo.

UNLABELLED: AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and ß/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and ß as well as progesterone receptor expressions were also assessed. RESULTS: In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERß expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells. CONCLUSION: Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERß expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

Online monitoring of cellular metabolism in the MCF-7 carcinoma cell line treated with phytoestrogen extracts.

BACKGROUND: Phytoestrogens are naturally occurring, plant-derived, nonsteroidal phytochemicals with anticarcinogenic potential. The aim of this study was to isolate phytoestrogens from the flax root of Linum usitatissimum and to test their effect on cellular metabolism in the human mammalian carcinoma cell line MCF-7 using the Bionas 2500 analysis system. MATERIALS AND METHODS: Metabolically relevant parameters such as acidification, oxygen consumption and cell adhesion were registered continuously over 8 and 24 hours on six sensor chips in parallel at different concentrations of flax root extracts. RESULTS: The extracts from flax roots of L. usitatissimum reduced extracellular acidification, respiration and adhesion in a concentration-dependent manner. CONCLUSION: The Bionas 2500 analysis system allows multiparametric online monitoring of cellular processes and can be used to detect the mode of action of anticarcinogenic compounds in cellular metabolism.

Effects of phytoestrogen extracts isolated from flax on estradiol production and ER/PR expression in MCF7 breast cancer cells.

BACKGROUND: In this study, we tested the effects of crude extracts from flax (Linum usitatissimum) on the production of estradiol and expression of estrogen receptor (ER) and progesterone receptor (PR) in human breast cancer MCF7 cells. MATERIALS AND METHODS: Isoflavone and lignan extracts from flax plant Linum usitatissimum were obtained, using different extraction methods. Breast carcinoma cells (MCF7) were incubated with various concentrations of the isolated extracts. Untreated MCF7 cells were used as controls. Supernatants were removed at designated times and tested for estradiol with an ELISA method. Furthermore, the effect of phytoestrogen extracts on the production of ERa and ERbeta as well as on PR was examined. RESULTS AND CONCLUSION: Production of estradiol is elevated in MCF7 cells in a concentration-dependent manner after stimulation with isoflavone and lignan extracts from Linum usitatissimum. Expression of ERalpha is up-regulated after stimulation with lower concentrations of lignan extracts from flax plants, unchanged at median concentrations and down-regulated at high concentrations. Expression of ERbeta is down-regulated in a concentration-dependent manner.

Abortion is associated with increased expression of FasL in decidual leukocytes and apoptosis of extravillous trophoblasts: a role for CRH and urocortin.

Human reproduction is remarkably inefficient, with more than half of spontaneous conceptions failing to complete the first trimester. However, little is known on the molecular events that take place at the implantation site during abortion. Here, we examined the hypothesis that the expression of the proapoptotic Fas/FasL system at the implantation site is impaired in abortions. We found that, in contrast to normal pregnancy, abortive deciduas contain leukocytes that are positive for FasL and extravillous trophoblasts (EVTs), which show increased expression of Fas and increased rates of apoptosis. In addition, the neuropeptides, corticotropin-releasing hormone and urocortin, were elevated in placental material obtained from abortions. In vitro, these peptides induced the expression of FasL in decidual lymphocytes (DL) obtained from elective termination of pregnancy placentas and thus potentiated the cells' ability to induce Fas-mediated apoptosis in an EVT-based hybridoma cell line. Finally, DL from abortion sites effectively induced apoptosis of EVT without prior treatment. It is possible that these events may impede successful early placentation and thus contribute to the pathophysiology of human abortion.

Expression of the pantumour Thomsen-Friedenreich antigen in the human placenta with the diagnosis of hydatidiform mole.

BACKGROUND: The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope, Galbeta1-3GalNAc) has long been known as a pancarcinoma antigen. Specific carrier proteins of the TF-antigen are the mucins, in particular Mucin 1. Here, we present our results of immunohistochemical identification of this carbohydrate antigen in human placenta. MATERIALS AND METHODS: Paraffin-embedded placental and decidual tissues from patients with the diagnosis hydatidiform mole were incubated with different monoclonal antibodies directed against TF-epitope (CD 176, IgM) and against Mucin 1 (CD 227, IgG). RESULTS: No expression of the TF-antigen or of Mucin 1 (Muc 1) was found in the decidual tissues, but the samples of chorionic tissues were TF- and Muc 1-antigen positive. As positive control, placental samples of the first trimester were investigated. CONCLUSION: A disorder of the extravillous trophoblast cells is present in hydatidiform mole.

Expression of the glycodelin A gene and the detection of its protein in tissues and serum of ovarian carcinoma patients.

BACKGROUND: Glycodelin A (GdA), also known as placental protein 14 (PP14), has been detected in endometrial, cervical and ovarian carcinoma cells. It is suspected to be a marker of human ovarian cancer tissues. MATERIALS AND METHODS: We investigated serum, tissue and cyst fluid samples of patients with an ovarian carcinoma in contrast to patients with benign and malignant diseases such as uterine myoma, endometriosis, cervical, uterine and breast cancer, and metastases of bladder and colon carcinoma. Used methods were enzyme-immunoassay, immunohistochemistry (IHC) and polymerase chain reaction (PCR). RESULTS: In 81% of the control group the GdA-expression was negative, which was confirmed by IHC and PCR. Of the ovarian carcinoma group only 52% showed correspondence between IHC and PCR. CONCLUSION: These results indicate that determination of GdA is not sensitive or specific enough for use as a tumour marker.

Effects of phytoestrogen extracts from Linum usitatissimum on the Jeg3 human trophoblast tumour cell line.

BACKGROUND: Phytoestrogens are a diverse group of nonsteroidal plant compounds which have similar effects to endogenous estrogens in humans and have been ascribed potential anticarcinogenic activities. We tested the effects of phytoestrogen extracts from different plant organs of flax, Linum usitatissimum, on cell proliferation in trophoblast tumour cells of the cell line Jeg3. MATERIALS AND METHODS: Phytoestrogen extracts were prepared from leaves, stems and roots of L. usitatissimum using different extraction methods. The isolated phytoestrogens were identified using HPLC-MS analysis. The influence on cell proliferation (MTT test) was determined in the trophoblast tumour cells, Jeg3. RESULTS: Cell proliferation of trophoblast tumour Jeg3 cells was significantly affected by the phytoestrogens isolated from leaves, stems and roots of L. usitatissimum. Root extracts inhibited Jeg3 cell growth significantly. CONCLUSION: A cell culture model system of the human trophoblast tumour cell line, Jeg3, was established to test the effect of potential phytoestrogens on cell proliferation. It was shown that the roots of L. usitatissimum contain measurable concentrations of lignans and isoflavones.

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Development and characterization of monoclonal antibodies for the immunohistochemical detection of glycodelin A in decidual, endometrial and gynaecological tumour tissues.

AIMS: Glycodelin is a glycoprotein with a molecular weight of 28 kDa. Unusual LacdiNAc structures have been identified on glycodelin A, isolated from amniotic fluid. Three major functions of this glycoprotein have been identified. Glycodelin is an immunosuppressive molecule, a marker of morphological differentiation, and a contraceptive. Because no monoclonal antibodies for glycodelin A are commercially available, our aim was to develop and characterize three monoclonal antibodies against this glycoprotein. METHODS AND RESULTS: Glycodelin A was purified from amniotic fluid by three chromatographic steps and its purity was checked by SDS-PAGE. Antibodies were generated from immunized BALB/c mice. Three IgG1 monoclonal antibodies detecting glycodelin A were cloned. All three antibodies recognized carbohydrate structures of glycodelin A and did not cross-react with glycodelin S. They are applicable to immunohistochemistry (frozen and paraffin sections), ELISA and Western blots. CONCLUSION: The new antibodies can be used for the detection of glycodelin A in frozen and paraffin-embedded decidual and endometrial tissue. One antibody (A87-B/D2) can be used for the detection of glycodelin in endometrial and ovarian tumour tissues. Because glycodelin A is a major secretory endometrial product during the luteal phase, in early pregnancy and in gynaecological tumours, the new antibodies are, potentially, valuable tools for the study of endometrial development and tumour progression.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Flax-seed extracts with phytoestrogenic effects on a hormone receptor-positive tumour cell line.

UNLABELLED: The higher soy intake in the Asian population compared to Europeans is believed to be an essential factor for the lower incidence of hormone-dependent tumours in Asia. It has already been shown that soya beans, with their ingredients genistein and daidzein from the isoflavonoid group, have protective effects on hormone-caused diseases. Lignans are another, less investigated, group of phytoestrogens. The aim of this study was to investigate the effects of flax-seed, which is typically found in Northern European diets, on the proliferation and hormone production of an estrogen receptor (ER)-positive trophoblast tumour cell line. MATERIALS AND METHODS: Trophoblast tumour cells of the cell line Jeg3 were incubated with 2 different concentrations of the isolated crude extract of flax-seed and 7 chemically partitioned extract fractions. Untreated cells were used as controls. After 48 h of stimulation, cell proliferation was measured using the BrdU method. The concentrations of hCG and progesterone produced by the trophoblast tumour cells were measured 48 h after stimulation. Extract fractions with antiproliferative effects in the BrdU- test were analysed by HPLC-MS. RESULTS: Our study showed an inhibitory influence of some of the isolated flax-seed fractions on the Jeg3 tumour cells. Proliferation of the Jeg3 cells was decreased by flax-seed fractions I, V, VI and VII in a dose-dependent manner. Inhibition of hCG production by flax-seed extracts III, V, VI and VII was also dose-dependent. Extract fractions V and VI decreased the production of progesterone by 58% to 86%. Some extract fractions showed a stimulating effect on hormone production and cell proliferation. HPLC-MS analysis showed the presence of matairesinol and biochanin A in flax-seed fraction VI. DISCUSSION: Flax-seed seems to have similar inhibitory effects to soya on hormone production and proliferation of hormone-sensitive tumour cells. Our results showed a dose-dependent inhibition by isolated flax-seed extracts on the Jeg3 cell line. Matairesinol and biochanin A seem to be useful candidates for extended tests on other tumour cell lines and normal tissues to evaluate the potential benefit of a lignan-containing therapy in hormone-dependent diseases.

[Stimulation of HCG, estrogen and progesterone production in isolated trophoblast cells by glycodelin A or its N-glycans]. / Stimulierung der hCG-, Estrogen- und Progesteronproduktion in isolierten Trophoblastzellen durch Glycodelin A bzw. seine N-Glycane.

UNLABELLED: The immunosuppressive protein glycodelin A (GdA) is secreted by decidual tissue in high rates in the first trimester of pregnancy. GdA forms about 10 % of the total protein amount released by the decidua in the first trimester of pregnancy and has a unique glycosylation including fucosylated LacdiNAc structures. The purpose of this study was to investigate the effect of glycodelin A and its N-glycans on hCG, estrogen and progesterone release by cytotrophoblasts in vitro. Trophoblast cells were isolated from human term placenta by fragmentation of villous tissue. Trophoblast cells fuse in vitro to syncytiotrophoblast cells. Trophoblast cells were incubated with GdA and various glycans of GdA. The production of hCG and progesterone was measured after 24 and 48 hours. The production of hCG, estrogen and progesterone was increased in GdA and glycan-treated cell cultures as compared to untreated trophoblast cell cultures. CONCLUSION: HCG, estrogen and progesterone are markers for the differentiation process of cytotrophoblast cells. The results suggest that GdA with its distinct glycosylation modulates the differentiation of trophoblasts.

Glycodelin A and differentiation of first trimester trophoblast cells in vitro.

AIM: The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro. METHODS: Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen-Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid. CONCLUSIONS: The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.

[Investigations on regulation of HCG by cortisol (prednisolon) in trophoblast cells in vitro]. / Untersuchungen zur Regulation von hCG durch Kortisol (Prednisolon) in der Trophoblastzelle in vitro.

OBJECTIVE: Trophoblast cells synthesize a variety of hormones, in which hCG plays a major role. On the strength of special enzymes they are capable of catalyzing the reaction cortisol <--> cortisone. In vitro experiments showed the influence on ACTH- and cortisol secretion by CRH, ACTH and prednisolon. In this study we describe the influence of cortisol (prednisolon) on hCG production of trophoblast cells in vitro. MATERIAL AND METHODS: Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. After adjusting the cell suspension to a defined cell concentration of 1 x 10 (6) cells/ml cells were cultivated. The addition of prednisolon followed every eight hours. The samples were collected after 24 hours for a total of 96 hours also from unstimulated cultures. Culture supernatants were assayed for hCG by enzyme-immunometric methods. RESULTS: The addition of prednisolon (50 microg/ml) stimulates the concentration of hCG in a time-depending manner. CONCLUSIONS: The trophoblast cell shows an increase in the concentration of hCG after stimulation with cortisol. For the first time an influence of cortisol (prednisolon) on hCG production could be demonstrated in cultured trophoblast cells.

[Investigations on regulation of CRH, ACTH and cortisol in trophoblast cells in vitro]. / Untersuchungen zur Regulation von CRH, ACTH und Kortisol in der Trophoblastzelle in vitro.

OBJECTIVE: Trophoblast cells synthesise CRH and ACTH, which are peptide hormones. On the strength of special enzymes they are capable of catalyzing the reaction cortisol <--> cortisone. In vitro experiments should give a proof of influence to ACTH- and cortisol secretion by CRH, ACTH and prednisolon. The basal rate of cortisol secretion was examined in a long term experiment. MATERIAL AND METHODS: Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. After adjusting the cell suspension to a defined cell concentration of 1 x 10(6) cells/ml the cells were cultivated. The addition of CRH, ACTH or prednisolon followed every eight hours. The samples collected 20 or 30 minutes later, also from unstimulated cultures, were assayed for ACTH and cortisol by enzyme-immunometric methods. RESULTS: The concentration of cortisol shows a rhythmical course in long term cell cultures. The addition of CRH (500 ng/ml, 1 microg/ml) stimulates the concentration of ACTH- and cortisol in a time-depending manner. The addition of ACTH (500 ng/ml-2 microg/ml) stimulates the concentration of cortisol in a time-depending manner. The addition of prednisolon stimulates the concentration of ACTH. CONCLUSIONS: The trophoblast cell shows a rhythmical course in the concentration of cortisol. For the first time a CRH-ACTH-cortisol feedback loop could be demonstrated in cultured trophoblast cells.