The effect of D-(+)-glucosamine, N-acetyl-D-glucosamine and tetraethylene glycol on the stability of oxytocin in aqueous solution.

The aim of the present study was to identify the effect of D-(+)-glucosamine, N-acetyl-D-glucosamine, tetraethyleneglycol, and the mixture of these additives on the stability of oxytocin in phosphate and acetate buffer solutions, at pH 4.5. Our findings demonstrate that tetraethyleneglycol has a destabilizing effect on oxytocin in both phosphate buffer and acetate buffer. D-(+)-Glucosamine hydrochloride had small to negligible effect at low concentrations, yielding a slight improvement lower concentrations of the additive in the presence of the buffers used, but at higher concentrations it increased the rate of degradation. N-Acetyl-D-glucosamine showed a possibly slight improvement to the stability of oxytocin. It is hypothesized that the different effect of N-acetyl-D-glucosamine compared to D-(+)-glucosamine is a consequence of the free amine group in D-(+)-glucosamine promoting a faster degradation, while the amino group is acetylated in N-acetyl-D-glucosamine and therefore no longer reactive in the same way. While it remains unclear why tetraethyleneglycol has a destabilizing effect on oxytocin, the D-(+)-glucosamine results aid in deepening our understanding of the degradation mechanism of oxytocin.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem­cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell­induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non­random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase.

BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.

Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo.

Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas.

The effect of cyclooxygenase inhibitor diclofenac on experimental murine colon carcinoma.

BACKGROUND: Cyclooxygenase-2 (Cox-2) has been found to be overexpressed in several types of human cancers and its role in tumorigenesis has been proposed. The aim of this study was to investigate the effects of the cyclooxygenase inhibitor diclofenac on the growth of murine C-26 colon carcinoma cells. MATERIALS AND METHODS: Expression of Cox-2 mRNA and protein was examined by RT-PCR analysis and immunohistochemistry, respectively. By using MTT-assay, we examined the effects of diclofenac at various concentrations on the growth of C-26 cells in vitro. The effect of diclofenac on the growth of the C-26 tumor in syngeneic mice was also investigated. RESULTS: By RT-PCR, Cox-2 mRNA was detected in C-26 cells. Cox-2 protein was localized to C-26 cells and treatment with diclofenac resulted in apoptotic cell death in a dose-dependent manner. Diclofenac administered in drinking water resulted in growth inhibition of C-26 tumor in mice and correlated with plasma levels of both PGE2 and TXB2. CONCLUSION: Our data show that diclofenac may be a potential agent for the prevention and treatment of human colon cancer.

Tumor necrosis factor-related apoptosis-inducing ligand induces apoptosis in human articular chondrocytes in vitro.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is produced by immune cells and by mediating apoptosis, TRAIL plays an important role in tumor surveillance. TRAIL binds four different membrane-bound receptors: DR4, DR5, DcR1, and DcR2. The DR4- and DR5-receptors mediate apoptosis, whereas the others do not. We demonstrated by reverse transcriptase-polymerase chain reaction and flow cytometry that, in vitro, normal human articular chondrocytes express the receptors mediating apoptosis (DR4 and DR5) and one of the decoy receptors (DcR2). Also, we demonstrated that chondrocytes were subjected to cell death within few hours after challenge with TRAIL and that cytotoxicity was dose-dependent. Treated cells had apoptotic morphology accompanied by active caspase-3 immunoreactivity. These data indicate that normal human articular chondrocytes are susceptible to TRAIL-mediated apoptosis, which otherwise is typical for transformed cells, and also that death receptors and their respective ligands may have a crucial role in cartilage generation and destruction.

Continuously infused methylene blue modulates the early cardiopulmonary response to endotoxin in awake sheep.

BACKGROUND: In endotoxemia and septic shock, enhanced generation of endogenous nitric oxide (NO) contributes to myocardial depression, hypotension, and derangement of gas exchange. We hypothesized that continuous infusion of methylene blue (MB), an inhibitor of the NO pathway, would counteract these effects in endotoxemic sheep. METHODS: Twenty-one sheep were anesthetized and instrumented for a chronic study with vascular catheters. On the day of the experiment, 18 conscious animals randomly received either an intravenous injection of MB 10 mg x kg(-1) or isotonic saline. Thirty minutes later, sheep received a 20-min intravenous infusion of Escherichia coli endotoxin 1 microg x kg(-1) and either an intravenous infusion of MB 2.5 mg x kg(-1) x h(-1) or isotonic saline, respectively, for 5 h. In addition, 3 animals were exposed to the same dose of MB alone. RESULTS: MB reduced the early endotoxin-induced declines in stroke volume, left ventricular stroke work and cardiac indices, and prevented mean arterial pressure from falling. Moreover, MB ameliorated the increases in pulmonary arterial pressure and pulmonary vascular resistance index. In addition, MB reduced the increments in venous admixture and AaPO2, decreased the falls in PaO2, SaO2, and oxygen delivery, and maintained oxygen consumption. MB also prevented the rises in body temperature and plasma nitrites and nitrates, and delayed the elevation of plasma lactate. When given alone to healthy sheep, MB transiently reduced plasma lactate and PaO2, and increased AaPO2. CONCLUSION: In ovine endotoxemia, continuously infused MB counteracts the early myocardial dysfunction and derangement of hemodynamics and gas exchange.

Infusion of methylene blue in human septic shock: a pilot, randomized, controlled study.

OBJECTIVE: To evaluate the effects of continuous infusion of methylene blue (MB), an inhibitor of the nitric oxide pathway, on hemodynamics and organ functions in human septic shock. DESIGN: Prospective, randomized, controlled, open-label, pilot study. SETTING: Multidisciplinary intensive care unit of a university hospital. PATIENTS: Twenty patients with septic shock diagnosed <24 hrs before randomization. INTERVENTIONS: Patients were randomized 1:1 to receive either MB (MB group, n = 10) or isotonic saline (control group, n = 10), adjunctive to conventional treatment. MB was administered as an intravenous bolus injection (2 mg/kg), followed 2 hrs later by infusion at stepwise increasing rates of 0.25, 0.5, 1, and 2 mg/kg/hr that were maintained for 1 hr each. During infusion, mean arterial pressure was maintained between 70 and 90 mm Hg, while attempting to reduce concurrent adrenergic support. MEASUREMENTS AND MAIN RESULTS: Hemodynamics and organ function variables were assessed over a 24-hr period, and the survival rate at day 28 was noted. Infusion of MB prevented the stroke volume and the left-ventricular stroke work indexes from falling and increased mean arterial pressure. Compared with the control group, MB reduced the requirement for norepinephrine, epinephrine, and dopamine by as much as 87%, 81%, and 40%, respectively. Oxygen delivery remained unchanged in the MB group and decreased in the control group. MB also reduced the body temperature and the plasma concentration of nitrates/nitrites. Leukocytes and organ function variables such as bilirubin, alanine aminotransferase, urea, and creatinine were not significantly affected. Platelet count decreased in both groups. Five patients treated with MB survived vs. three patients receiving conventional treatment. CONCLUSIONS: In human septic shock, continuously infused MB counteracts myocardial depression, maintains oxygen transport, and reduces concurrent adrenergic support. Infusion of MB appears to have no significant adverse effects on the selected organ function variables.

Human articular chondrocytes express functional leptin receptors.

The effects of leptin hormone are mediated by interactions with several physiological regulatory systems and the cytokine network, and by targeting cells directly. The leptin receptor is a member of the class I cytokine receptor family, and its signal transduction resembles that induced by many cytokines. We demonstrated that serially cultured human articular chondrocytes possess the leptin receptor (Ob-R), and that this receptor was present on chondrocytes in native human cartilage. In cultured chondrocytes we detected mRNA for the functional isoform of leptin receptor (Ob-Rb or Ob-R(L)), and it was revealed that ligand binding resulted in phosphorylation of signal transducers and activators of transcription, namely STAT1 and STAT5. Chondrocytes stimulated with leptin exhibited an increased proliferation and an enhanced synthesis of extracellular matrix (proteoglycans and collagen). These results indicate that leptin affects cartilage generation directly, which is a novel role for leptin in skeletal growth and development.

A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability.

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.

Interferon-gamma modulates TRAIL-mediated apoptosis in human colon carcinoma cells.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by immune cells and has been shown to play an important role in tumor surveillance due to its ability to induce apoptosis in various transformed cells. Interferon gamma (IFN-gamma), a multipotent cytokine with broad stimulatory effects on anti-tumoral immune reactions, may exert its cytotoxic activity directly on tumor cells or indirectly via stimulation of effector cells. This study was designed to determine the effect of IFN-gamma on TRAIL-mediated apoptosis in human colon carcinoma cell lines. MATERIALS AND METHODS: Cytotoxicity was assessed by MTT-assay. Expression of death receptors was measured by reverse transcriptase polymerase chain reaction. Apoptosis was assessed by caspase-8 immunoblot, DNA fragmentation and morphological studies. RESULTS: Treatment with TRAIL resulted in detectable cytotoxicity within 5 hours and was enhanced in a dose-dependent manner. When cells were pretreated with IFN-gamma, the cytotoxic effect of TRAIL increased significantly. Treated cells showed a typical apoptotic morphology that was accompanied by internucleosomal cleavage of DNA. Up-regulation of caspase-8 expression and activation were detected as a result of pretreatment with IFN-gamma and subsequent apoptosis mediated by TRAIL. CONCLUSION: Our results demonstrated that IFN-gamma sensitises human colon carcinoma cells to TRAIL-mediated apoptosis, partly by elevated caspase-8 expression.

Early events of hepatic metastasis formation in mice: role of Kupffer and NK-cells in natural and interferon-gamma-stimulated defense.

Surgical manipulation of a tumor may result in increased influx of tumor cells into the systemic and portal circulation and give rise to formation of metastases. In addition, major surgery has been reported to cause profound immunosuppression. In an attempt to increase the host-antitumor immune mechanisms following surgery we have studied the effect of preoperative administration of interferon-gamma, related to the antimetastatic effects of Kupffer cells (KC) and natural killer cells (NK-cells) in the early phase of liver metastasis formation. Colon carcinoma cells were injected into the superior mesenteric vein of syngeneic mice and after 17 days metastases were quantified by weight, number, and uptake of [125I]iododeoxyuridine. Unstimulated control mice developed 10.5 surface nodules per liver 17 days following injection of colon carcinoma cells into the superior mesenteric vein of syngeneic mice. This figure was only 2.6 in mice stimulated with a single dose of 1000 IU IFN-gamma 4 h prior to inoculation of tumor cells. Administration of GdCl3, which is reported to deplete and block the function of Kupffer cells, 24 h prior to tumor cell inoculation resulted in a 5-fold tumor mass increase relative to control. Injection of anti-asiolo-GM1 antiserum, which eliminates the hepatic NK-cells, induced a 10-fold increase in tumor mass. These results indicate an important early antimetastatic function of hepatic NK-cells and KC and that presurgical administration of IFN-gamma may be important for eliminating circulating tumor cells and inhibiting development of residual tumors.

Tumor stroma.

The accumulated evidence indicates that tumor stroma with its cells and cell products plays a much more active and important role than previously believed. Growth factors and cytokines produced by macrophages and other cells are crucial for stroma formation and angiogenesis. Lytic enzymes provided by stromal cells may be essential for invasion. TNF and other inflammatory mediators may be operative in the systemic effects of tumors, e.g. cachexia. All these effects may come about through the action of soluble substances produced by tumor cells or by more intimate interactions. There is no evidence that stromal cells are directly involved in carcinogenesis--i.e. the cellular transformation to produce the malignant cell. On the other hand, stromal cells and other components of the interstitia are instrumental in tumorigenesis--i.e. the development of a real malignant tumor from its start on the cellular or subcellular level. In one way of looking at it, the stromal cells, e.g. macrophages may be considered as "slaves", kept to carry out certain functions, synthesize essential substances e.g. growth factors that the tumor cells do not have the capacity or the degree of finely tuned machinery to produce. The objective of immunomodulation should then be to create a "slave uprising", to make the macrophages and other cells turn against their masters, stop producing growth factors and start producing harmful factors that would lead to the elimination of the malignant growth. The first target of immunomodulation in tumor disease should probably be local malignancies where no effective treatment exists today- and selected cases of metastatic prevention (181, 182).

Improvement of a cytokine (TNF-α) bioassay by serum-free target cell (WEHI 164) cultivation.

The elaboration of a sensitive bioassay for assessment of tumour necrosis factor alpha (TNF-α) in a defined medium is described. The assay is based on the cytotoxic effect of TNF-α on a target cell line, the murine fibrosarcoma WEHI 164 clone 13. Cytotoxicity was assessed by detecting the rate of tetrazolium salt reduction employing a spectrophotometer (ELISA-reader). A similar bioassay was used previously to assess TNF-α, though this was dependent on cell growth in a medium containing serum. By employing a synthetic serum replacement, the WEHI cells were adapted to growth in a defined medium which allowed both the propagation of the cell line and the assay to be performed under completely defined conditions. Thus, factors in serum that may influence the TNF-α assessment, such as growth factors, cytokines, soluble cytokine-receptors and macroglobulin, were avoided. The only protein required in this bioassay was insulin, while albumin was added as a carrier protein and to protect the cytokine against loss of biological activity during multiple freeze and thaw cycles. The present assay was optimised to achieve a high sensitivity and, by testing endogenous TNF-α originating from the macrophage-like cell line RAW in both the serum-free and serum-based assay, we found the highest sensitivity in the assay based on defined medium. The LC50 of recombinant mouse and human TNF-α were in the serum-free and serum-based assays considered to be 25 and 50 pg mL-1, respectively. The demonstration of a culture condition that enables long-term cultivation of target cells and a bioassay in a completely defined medium is in our opinion a substantial contribution to more reliable cytokine assessment.

Inhibition of establishment and growth of mouse liver metastases after treatment with interferon gamma and beta-1,3-D-glucan.

The purpose of this study was to investigate the combined antitumor effect of aminated beta-1,3-D-glucan (AG) and interferon-gamma (IFN-gamma) in an experimental liver metastasis model. Liver metastases were established by inoculation of C-26 colon carcinoma cells into the superior mesenteric vein of syngeneic mice. Treatment of mice started 24 hours after inoculation of tumor cells by daily intravenous injections of either AG, IFN-gamma, or a combination of both for a duration of 6 days. The resultant liver metastases were then quantified after an additional period of 11 days. Combination of IFN-gamma and AG inhibited the growth of liver metastases almost entirely. IFN-gamma was also very efficient, while AG alone did not exert any significant antitumor effect. These results, along with histological studies from mice receiving AG and IFN-gamma, indicated that activation and recruitment of liver macrophages may be a part of the mechanism responsible for the inhibition of metastatic growth observed in this study.

Isolation and culture of endocardial endothelial cells from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua).

This paper describes a procedure for establishing primary cultures of endocardial endothelial cells and morphologically characterizes the cultivated endothelial cells from Atlantic cod (Gadus morhua L.) and salmon (Salmo salar L.). Following incubation with collagenase and trypsin, the resulting cell suspension was seeded on substrates of fibronectin, collagen or gelatin. Cod and salmon endothelial cells attached firmly to these substrates within 24 and 5 h respectively, but did not attach to uncoated tissue-culture plastic. The contamination by non-endothelial cells was kept at a minimum by working out optimal combinations of serum concentration and growth substrate. The yield was about 3x10(6) and 1x10(6) cells per kg cod and salmon, respectively. Cultures could be maintained for several weeks but cells did not proliferate. Cultivated cod endocardial endothelial cells differed from salmon endocardial cells in having abundant cytoplasm with many vesicles and avidly taking up labelled collagen. The described method allows, for the first time, functional and morphological studies to be performed under controlled conditions on both a specialized scavenger type of endothelium from cod and a conventional flat endothelium from salmon.

Cytotoxic effect of cytokines on murine colon carcinoma cells involves TNF-mediated apoptosis.

We have studied the cytotoxic effect of TNF-alpha on C-26 murine colon carcinoma cells in vitro. Treatment with TNF-alpha alone did not result in any demonstrable cytotoxicity. However, when combined with IFN-gamma, the cytotoxic effect of TNF-alpha was enhanced in a dose-dependent manner. An agonistic TNF-R1 specific antibody and recombinant human TNF-alpha both exerted a cytotoxic effect when combined with IFN-gamma, suggesting that the cytotoxicity was mediated through the TNF-R1. The cytotoxicity was associated with production of nitric oxide without any direct involvement in the cytotoxic effect. At the ultrastructural level, treated cells displayed a typical apoptotic morphology which was not accompanied by internucleosomal cleavage of DNA as shown by conventional electrophoresis.

A sensitive serum-free colorimetric assay for the detection of cytotoxic effects of pesticides.

A new method of cell quantification in the Hybritest bioassay is described. This test is based on culturing the hybridoma cell line 1E6 in the serum-free medium RPMI-SR3 and determination of cytotoxicity by growth inhibition. By employing a tetrazolium salt (MTT), the cell number were quantified colorimetrically using an ELISA reader. This assay carried out in microtiter plates saved time, labour and expenses. It was demonstrated that the sensitivity of 1E6 to pesticide toxicity (glyphosate and cypermethrine) was considerably higher in serum-free medium than in serum-containing medium. Furthermore, it is suggested that this assay may represent an alternative to the use of living animals in toxicological experiments.

A simplified serum-free method for preparation and cultivation of human granulosa-luteal cells.

A simplified method for the preparation and long-term cultivation of granulosa-luteal cells in serum-free medium is described. The cells were harvested from women undergoing in-vitro fertilization, enriched by sedimentation and dissociated by enzymatic treatment. We demonstrated, by introducing a synthetic serum replacement (SSR2), that these primary cell cultures cultivated in monolayers on an extracellular matrix may be used in experiments exceeding 7 days with low cell loss and cell death. No adverse effect on progesterone production was found. There was a high diversity in progesterone production between cells from individual patients. After several days in culture, the cells were challenged with human chorionic gonadotrophin which revived the rapidly decreasing progesterone production. We were unable to demonstrate an increase in cell number after 7 days of cultivation when the cells were grown in medium supplemented with either serum or SSR2. The mitogens epidermal growth factor and basic fibroblast growth factor had no influence on proliferation. We also found that the present method prevents leukocyte contamination in the granulosa-luteal cell cultures. Compared with the common method based on the enrichment of granulosa-luteal cells on a density gradient (Ficoll/Percoll), this method saves time, labour and expense, in addition to augmenting purity.